Chronic inflammation is linked to various diseases, including cancer, diabetes mellitus, obesity, and hypertension. The critical question—how is chronic inflammation linked to acute inflammation and are there any physiological counterparts to chronic inflammation , 2. AnswIn a simplistic approach, it is possible to divide acute inflammation into the two phases: the onset and the resolution (termination of inflammation and return to homeostasis) , 4.Chronic inflammation for various reasons lacks a complete resolution phase—it never ends. There are different reasons for it such as prolonged contact with infection or irritants and the presence of cells that continuously secrete inflammatory mediators. During chronic inflammation, anti-inflammatory cytokines are released continuously—along with pro-inflammatory cytokines . So, wheAccording to recent studies, there is a long-lasting immune post-resolution phase even after acute inflammation, which might be essential for immune tolerance . It can There are various examples of the transition to immune suppression during chronic inflammation.One such example is the capability of essential inducers of inflammation  to function as pro-resolvers—they promote factors necessary for anti-inflammatory and immunosuppressive responses  . InflammAnother example is that one of the primary inflammatory mediators—IL-6 is needed for regenerative and protective processes in the body. For instance, in mice, IL-6 was essential for liver regeneration, gut barrier repair, and the suppression of inflammation in the kidney and pancreas .The transition of inflammation to immune suppression can also occur at the cellular level. As an illustration, macrophages shut down the generation of pro-inflammatory mediators and activate a transcriptional program resulting in the release of anti-inflammatory cytokines  .Other recent studies have shown that long-term activated dendritic cells (DCs) significantly changed their profile toward a non-functional, tumor-promoting, and anti-inflammatory phenotype . Such DCThese mechanisms are involved in endotoxin tolerance—attenuated production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), IL-6, and IFN-γ, and increased production of anti-inflammatory cytokines such as IL-10 and TGFβ in response to repeated exposure to LPS (lipopolysaccharide) or a gram-negative infection , 15.Above-mentioned enzymes are interconnected as arginase enzymatic activity might be mandatory for the subsequent IDO1 upregulation. Arginine is actively metabolized by arginase to produce urea and l-ornithine. Polyamine spermidine is generated downstream of the decarboxylation of l-ornithine. Spermidine can promote IDO1 phosphorylation and signaling events in DCs, possibly via direct activation of the Src kinase, which has IDO1-phosphorylating activity .There is much evidence that low-grade inflammation is significant for maintaining immune tolerance in immune-privileged sites. A successful pregnancy requires fine-tuning the level of inflammation. Either the increase or the decrease in the level of inflammatory mediators leads to negative consequences . For insAccording to recent studies, it might be proposed that chronic moderate antigen stimulation might be necessary for successful immune tolerance in pregnancy as the repeated LPS exposure leads to placental endotoxin tolerance .Pregnancy, especially implantation, evokes an inflammatory reaction, which includes the upregulation of inflammatory cytokines —they are critical mediators of a healthy pregnancy. Besides, various leukocytes are found in the decidua, including maternal natural killer (NK) cells, DCs, macrophages, and lymphocytes .The specific subset of NK cells, which constitutes about 50–90% of total lymphoid cells in the uterus, plays a vital role at the fetal-maternal interface during the first trimester of the pregnancy .HLA-G molecules are considered to be crucial for the immunological tolerance of the fetus by the mother. At the same time, they may be involved in the active secretion of pro-inflammatory cytokines , 22. AnoKieffer et al.  have shoDue to the possible tolerogenic role of low-intensity inflammation, anti-inflammatory therapy can reverse the level of immune tolerance factors. As an example of this in mice, prednisolone, known as an immune suppressor, disrupted the expected expansion of CD4+ T cells in early pregnancy, inhibiting the generation of both suppressive Treg cells and effector T cells .Other anti-inflammatory substances, nonsteroidal anti-inflammatory drugs (NSAIDs), are known for their serious fetal side effects. NSAIDs inhibit the production of prostaglandins, which are essential for successful embryonic implantation . ProstagAccording to the concept of the necessity of low-grade physiological inflammation for maintaining immune tolerance, every immune-privileged organ should contain the source of inflammatory factors.In the central nervous system, glial cells, along with the neurons, can act as the source of inflammatory factors in a steady-state . A certaBasal IL-6 and IFN-γ levels in the healthy brain are also linked to the maintenance of brain homeostasis . IFN-γ iInterestingly, cytokines that are associated with the induction of neuroinflammation in some cases can exert a protective effect. For instance, the administration of IL-12 (which amplifies Th1 polarization) during the early phases of experimental autoimmune encephalomyelitis  suppressed the disease through the induction of IFN-γ , 37. IL-Brain NK cells control the inflammation by killing pro-inflammatory microglial cells, which are activated within minutes of ischemia onset .NK cells are a major lymphocyte subpopulation within the rat testis . SertoliAccording to modern approaches, the traditional labels of pro- and anti-inflammatory are too simplistic to describe cytokine actions. Genetic ablation of IL-17  declined gut epithelial cell proliferation and exacerbated dextran sulfate sodium (DSS)-induced murine colitis . OverallGenerally, in recent years we have been faced with a revision of the typical inflammatory role of inflammatory factors , 45. ForDiet and gut flora metabolites promote the ability of DCs to control T cell responses in the gut and other sites  , 49. ThuIn contrast to younger individuals, older individuals have elevated levels of inflammatory cytokines, especially IL-6 and TNF-α , 52. FatThe increased basal level of inflammatory cytokines can mediate increased immune tolerance. That might be the reason why older persons have higher autoimmunity but a lower prevalence of autoimmune diseases . The priYoung individuals have decreased basal levels of various inflammatory cytokines—this may explain a reduced immunological tolerance, and, consequently, higher susceptibility to autoimmune diseases, which is observed in young people . ProgresAs we have previously shown, constitutive slightly elevated cytokine levels may be linked to immune tolerance. These are the same cytokines whose levels rise significantly during inflammatory and autoimmune disorders . The main difference between these two conditions is the cytokine concentration—slightly elevated in immune tolerance and significantly elevated during exacerbations.According to the latest research, a very low cytokine level is sufficient to develop a specific effect. For example, binding of as few as 4 IL-6 molecules per cell seemed to result in statistically significant bioactivity, whereas binding of 16 IL-6 molecules triggered extensive cellular responses .The cytokine profile of physiological fluids related to immune-privileged organs  is of significant interest. It may reflect the constitutively increased content of cytokines in the tissues of these organs.For instance, the GM-CSF level in plasma of healthy adults is about 2.5 pg/ml while the GM-CSF level in tears (this fluid is related to ocular immune privilege) of healthy subjects is about 30 pg/ml , 60. TheIn other physiological fluids related to immune-privileged sites, we can also observe a slightly increased level of specific pro-inflammatory cytokines: for example, in amniotic fluid, CSF , and semen –68. In tPerhaps, IL-1β, IL-6, and GM-CSF might be some of the most critical cytokines for tolerance induction. These cytokines are also involved in the induction of immune-suppressive myeloid-derived suppressor cells (MDSCs) by various cancer cell lines .As already mentioned, there is an increased level of some inflammatory cytokines in immune-privileged sites, compared with blood plasma. Inflammatory cytokines are also critical at the beginning of the resolution phase of inflammation.Can we talk about physiologic low-grade inflammation, which is linked to immune tolerance, as the physiologic counterpart of chronic inflammation (or para-inflammation)? , 70.There might be various consequences of this hypothesis—for example, in violation of the diversity of microbiota antigen stimulation —autoimmune lesions may occur . AnotherThus, the maintenance of chronic low-grade inflammation might be the universal mechanism of immune tolerance. The basement of such physiologic low-grade inflammation might be in naturally increased pro-inflammatory cytokine secretion. This process can be observed both in disease  and in health (immune-privileged sites) .It is well-known that chronic exposure to antigens may cause not only immune tolerance but also cause an excessive immune reaction, including autoimmunity , 72–74. The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Coronaviruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), are highly transmissible pathogens, and they can be lethal in humans. Bats are believed to be the original sources of these lethal pathogens. In December 2019, a cluster of pneumonia cases was reported in Wuhan and a novel coronavirus  has been linked to the causative agent. Early studies indicated that the new virus was genetically similar to a bat betacoronavirus, providing evidence for bat origin of the third human-infecting coronavirus in less than 20 years. We here highlight the potential for viral spillover from animals to humans and discuss evolutionary mechanisms leading to the outbreaks.Coronavirinae in the family Coronaviridae in the order Nidovirales. Coronavirinae is further divided into four genera, Alpha-, Beta-, Gamma-, and Deltacoronavirus, based on their phylogenetic relationships and genomic structures [Since the identification of the first coronavirus – infectious bronchitis virus (IBV) isolated from birds  – many cructures .Alpha-), OC43, HKU1, SARS-CoV, and MERS-CoV (Beta-), whereas only SARS- and MERS-CoV have caused large worldwide outbreaks with fatality, others usually cause mild upper-respiratory tract illnesses. A novel coronavirus was identified in a pneumonia patient in Wuhan on January 9 of this year represents the seventh human-infecting coronaviruses.Coronaviruses occasionally jump across host barriers, often with lethal consequences. The alpha- and betacoronaviruses only infect mammals and usually cause respiratory illness in humans and gastroenteritis in animals. Gamma- and deltacoronaviruses mainly infect birds, and no human infection has been reported. Six coronaviruses known to infect humans are 229E, NL63 (genus Betacoronavirus and caused the first confirmed case of Middle East Respiratory Syndrome (MERS) in Saudi Arabia in 2012. Over 2000 MERS-related infections have been reported as of 2019 with a ∼34% fatality rate (https://www.who.int/).Severe acute respiratory syndrome  first emerged in Guangdong province, China in 2002  and quicPaguma larvata) [Rhinolophus) in 2005, pointing to a bat origin of SARS-CoV [Bats are notorious for carrying many emerging or re-emerging viruses. Epidemiological investigations have shown that almost all SARS patients have a history of animal exposure prior to the disease. SARS-CoV and anti-SARS-CoV antibodies were first found in the masked palm civet (larvata) . HoweverSARS-CoV . This viSARS-CoV . AdditioSARS-CoV , furtherViruses isolated from MERS patients were found to have close contact with dromedary camels, suggesting the animal origin . PhylogeIt is worthy of notice that in 2016 swine acute diarrhea syndrome was found in Guangdong province, China, with a mortality up to 90% for piglets. The causative pathogen was identified as a new coronavirus, called swine acute diarrhea syndrome coronavirus (SADS-CoV), shared 95% genomic identity with bat alphacoronavirus HKU2 , suggesthttps://www.who.int/).On December 31, 2019, a cluster of pneumonia cases was reported in the city of Wuhan in Hubei province, China. Laboratory tests identified a new coronavirus, excluding the possibility of a variety of pathogens including SARS-CoV, MERS-CoV, influenza virus, avian influenza virus, adenovirus, and other common respiratory pathogens. As of February 11, 2020, a total of 43,103 confirmed novel coronavirus (term hereafter as 2019-nCoV) cases have been reported globally, with 42,708 from China and 1017 deaths  database (https://www.gisaid.org), by the Chinese Center for Disease Control and Prevention and others. Early published studies showed that the 2019-nCoV had 96.2% similarity to a bat betacoronavirus [Sarbecovirus of the Betacoronavirus. It has been proved that the new virus uses the same cell entry receptor – ACE2 as SARS-CoV, although it only had 79.5% genome sequence similarity to SARS-CoV [Several genome sequences of the 2019-nCoV have been released in the onavirus , thus clSARS-CoV ,17. By JSARS-CoV .Betacoronavirus, based on the published genetic background and phylogenetics for purposes of clarity [Alpha- and Betacoronavirus [A simplified phylogeny is shown in Fig.  clarity . The 201 clarity , which ionavirus ,19. Howehttps://bigd.big.ac.cn/ncov/#genome). Most of changes in ORF8 and S appeared in the last half of January 2020, while ORF1ab and ORF7a were from the first half. Same nonsynonymous changes were found in the S and ORF8 of a familial cluster case reported from Guangdong, i.e. L84S (Leu replaced by Ser at amino acid position 84) in three members and H49Y (His by Tyr) in two, indicating viral evolution may have occurred. However due to the limited sequence release and no functional changes given at present, adaptive evolution cannot be confirmed with these changes. Thus close monitoring of the virus's mutation, evolution, and adaptation is needed.Similar to SARS-CoV at the early stage , the 201Recombination of viruses occurs when two different parent viruses co-infect the same host cell and interact during replication, generating descendant viruses that have genes from both parental viruses. Recombination has often been associated with the expansion of the host range, increases in virulence, the evasion of host immune system and the evolution of resistance to antiviral treatments . Both poAs expected, recombination events were also involved in the emergence and evolution of MERS-CoV ,24. Due Given the high prevalence of diverse SARSr-CoVs and MERSr-CoVs carried by different animals, and frequent contact histories revealed by people infected with the animal viruses, as proposed in a widely accepted hypothesis Fig.  on the gFrequent human-animal contact is the major cause for viral cross-species transmission. Next-generation sequencing is a highly efficient method for rapid identification of microorganisms and for surveillance of pathogens for infectious diseases . Animal


Purpose: Some epidemiological studies and animal studies have reported a relationship between leukocyte telomere length (LTL) and bone mineral density (BMD). However, the causality underlying the purported relationship has not been determined. Here we performed a two-sample MR analysis to test the causal link between telomere length and BMD.Results: Our research suggested no causal link of LTL and BMD using IVW method. The weighted median, MR-Egger regression and MR.RAPS method yielded a similar pattern of effects. MR-Egger intercept test demonstrated our results were not influenced by pleiotropy. Heterogeneities among the genetic variants on heel estimated BMD and TB-BMD vanished after excluding rs6028466. “Leave-one-out” sensitivity analysis confirmed the stability of our results.Conclusion: Our MR analysis did not support causal effect of telomere length on BMD.2 statistics and “leave-one-out” sensitivity analysis were performed to evaluate the horizontal pleiotropy, heterogeneities and stability of these genetic variants on BMD.Methods: We utilized 5 independent SNPs robustly associated with LTL as instrument variables. The outcome results were obtained from GWAS summary data of BMD. The two-sample MR analysis was conducted using IVW, weighted median, MR-Egger regression and MR.RAPS method. MR-Egger intercept test, Cochran’s Q test and I The incidence of aging-related disorders has dramatically increased in modern society, owing to the development of healthcare and the changes of socio-economy and lifestyle . OsteopoTelomeres, capping and protecting the termini of eukaryotic chromosomes from fusion and degradation, are the dynamic nucleoprotein-DNA complexes, which consist of hexameric nucleotide sequences (TTAGGG) repeats and associated protective proteins , 11. It Mendelian randomization is a technique, using germline genetic variants as instrument variables (IV) for exposure to study the causal relation between the exposure phenotype and the outcome phenotype. In order to obtain unbiased estimates, MR need to fulfill three key assumptions: IV1) genetic variants used in analysis should be significantly associated with the exposure; IV2) genetic variants extracted as instrument variables for exposure are independent of confounding factors that are associated with the selected exposure and outcome; and IV3) the genetic variants affects the outcome only through the exposure and not via other biological pathways  We selected 16 SNPs as instrumental variables to investigate causal relationships between LTL and BMD in European ancestry . After pWe chose 5 independent SNPs associated with LTL in European ancestry to perform the MR analysis for the causal link between LTL and FN-BMD, LS-BMD, FA-BMD, heel estimated BMD, TB-BMD and TB-BMD (age over 60). The effect of all the 5 SNPs on the outcome GWAS was present, and no palindromic SNPs was found. Among these SNPs, only rs6028466 was nominally associated with reduced level of FN-BMD, heel estimated BMD and TB-BMD . None of the 5 SNPs was significantly associated with BMD outcomes at the Bonferroni-corrected significance threshold (p < 0.002) , as shown in In two-sample MR analysis, the decrease of LTL did not have a causal link with the level of FN-BMD, LS-BMD, FA-BMD, heel estimated BMD, TB-BMD and TB-BMD (age over 60) basing on IVW, WM, MR-Egger regression and MR.RAPS methods, as shown in The MR-Egger regression results showed that the horizontal pleiotropy would not bias the causal effect of LTL on FN-BMD , LS-BMD , FA-BMD , heel estimated BMD , TB-BMD  and TB-BMD (age over 60)  2 statistics showed that there was not statistically significant heterogeneity among the effects of individual LTL-associated SNPs on FN-BMD, LS-BMD, FA-BMD and TB-BMD (age over 60) outcomes. However, obvious heterogeneities existed in the effect of LTL-associated SNPs on heel estimated BMD  and TB-BMD  Cochran’s statistics test and IIn the sensitivity analyses, we observed a consistent no causal association between genetically predicted LTL and level of BMD using the “leave-one-out” method. It suggested the stability of our results Previous publications reported that the alterations of telomere length as well as telomere dysfunction played important roles in age-related impaired homeostasis of bone cells which promoted osteoporosis . In thisIn the two-sample MR analysis, we selected SNPs with genome-wide significance and independent inheritance (without any LD) as IVs to detect the causal link between TLT and BMD. After excluding the SNPs associated with phenotypes related to BMD, there were 5 genetic variants finally included as instrumental variables for further MR analysis. F statistic used to assess the instrument-exposure association was much greater than 10, hinting the small possibility of weak instrumental variables bias . To makeOur research was the first MR analysis on this topic, it contained several important strengths. First, the causal association of LTL and BMD was not distorted by many confounding factors. It was mitigated by using genetic variants as proxies in MR analysis. Second, four methods of MR analysis on six groups of BMD GWAS summary data were performed to draw a conclusion. Third, to avoid pleiotropic effects, we eliminated SNPs which was recognized associated with confounding factors through searching the Phenoscanner database and no pleiotropic effects was detected by MR-Egger regression method. Fourth, sensitivity analysis was performed and we found the conclusion was of stability. Last, to reduce potential bias, the GWAS summary data we drew for LTL and BMDs was from European descent individuals (except for TB-BMD (age over 60): 86% European ancestry) and adjusted for many common-seen factors, such as: sex, age, height and weight. However, some limitations of our MR analysis need to be considered. First, although selected SNPs were significantly associated with LTL in their respective GWASs, yet 2 SNPs (rs7675998 and rs6028466) did not achieve genome-wide significance with TLT in Mangino et al. study, from which we acquired summary data . BesidesIn this study, we aimed to determine the causal role of telomere length in level of BMD and osteoporosis by using two-sample MR analysis. However, the results of our research did not provide evidence to support our hypothesis. These findings suggested measures to influence telomere length may have no beneficial effects on BMD and telomere length can not be an indicator to monitor bone mineral density. Updated MR analysis is warranted to revalidate our findings when more and better IVs for LTL and sex-stratified GWAS summary data for BMD are available. Moreover, other factors that may play causal roles in osteoporosis still need to be identified and determined to develop strategies for monitoring, preventing and overcoming osteoporosis.http://www.gefos.org/) or got them from the IEU GWAS database (https://gwas.mrcieu.ac.uk/). Each study included was approved by their institutional ethics review committees, all participants provided written informed consent. Each SNP was tested for association with BMD, adjusting for many common-seen components, such as: sex, age, height and weight etc.To obtain a more comprehensive and reliable conclusion of the causal link between telomere length and BMD, we downloaded several publicly available GWAS summary statistics of BMD from the GEnetic Factors for OSteoporosis Consortium , Lumbar Spine bone mineral density , and Forearm bone mineral density  were downloaded from GEFOS, it is the largest GWAS on DXA-measured BMD to date .Summary GWAS data of ultrasound-derived heel estimated BMD on 426,824 European participants  were got from IEU GWAS database .A meta-analysis comprising 56,284 individuals of European ancestry was performed to investigate the genetic determinants of Total Body-bone mineral density (TB-BMD) . The metTo validate the causal link of telomere length and BMD in the elderly, GWAS summary statistics of TB-BMD (age over 60) on 22,504 mixed participants were downloaded from the GEFOS website. Most participants were European ancestry (86%) .https://www.ebi.ac.uk/gwas/) using the genome-wide significance threshold 5×10−8, including the seven SNPs from the largest GWAS for LTL of Europeans ancestry  [We used the 16 single-nucleotide polymorphisms (SNPs) previously utilized by Haycock et al. as IVs to investigate the causal relationships between telomere length and specific diseases . A serie., 2013) . To supp., 2013) . Each SNIn a standard two-sample MR study, it is important to ensure that the instrumental SNPs for the exposure are not in linkage disequilibrium (LD), since instrumental SNPs in strong LD may cause biased results. In this study, we performed the clumping process with the European samples from the 1000 genomes project to estimate LD between SNPs. The SNPs would be extracted from 1000 genomes data, and LD calculated between them. Amongst those pairs of SNPs that had LD R-square above the specified threshold (R-square = 0.001), only the SNP with the lower P-value would be retained. In the clumping process, we set the window size = 10,000 kb.http://www.phenoscanner.medschl.cam.ac.uk/). When a SNP was associated with another phenotype other than telomere length, we checked whether the associated phenotype was associated with BMD by conducting a literature search. A SNP was excluded from the analysis if it was significantly associated with any phenotype which was a risk factor for osteoporosis or low BMD after Bonferroni correction . Factors significantly associated with osteoporosis or BMD include rheumatoid arthritis, celiac disease, mean corpuscular hemoglobin, red blood cell count, body fat percentage, premature menopause, cognitive impairment and anemia etc. [2(n-k-1)/k(1-R2), R2: variance of exposure explained by selected instrumental variables, n: sample size, k: number of instrumental variables). If F statistic was much greater than 10 for the instrument-exposure association, the possibility of weak instrumental variables bias was small [According to the assumptions of MR analysis, the selected instrumental SNPs should affect the outcome only through the exposure and not via other biological pathways . To explore potential violations of this assumption, each instrumental SNPs was queried against the Phenoscanner database  to estimate the causal associations between LTL and BMD using four methods, which included inverse variance weighting (IVW), weighted median (WM), MR-Egger regression and Robust Adjusted Profile Score (MR.RAPS) method. The IVW method with fixed effect used a meta-analysis approach combining Wald estimates for each SNP  to get the overall estimates of the effect of LTL on BMD . If sign2 statistics to identify whether there was a higher heterogeneity between causal effects estimated using the variants individually than that would be expected by chance. In our analysis, a P-value of <0.05 or an I2 value of >50% would be regarded as significant heterogeneity. Additionally, we performed sensitivity analyses to evaluate the reliability of the association between genetically predicted LTL and risk of low BMD. We performed “leave-one-out” validation analyses, where the MR was performed again but leaving out each SNP in turn, to identify if a single SNP was driving the association.We conducted the MR-Egger regression to assess the potential pleiotropic effects of the SNPs used as IVs. The intercept term in MR Egger regression could be a useful indication of whether directional horizontal pleiotropy was driving the results of a MR analysis . We usedAll data sources were de-identified and publicly available, and thus, no ethical committee approval was required.All statistical analyses were conducted using R version 3.6.3 , the Two-Sample MR package  and STATAll data generated or analyzed during this study are included in this published article and its supplementary information filesSupplementary FiguresSupplementary Table 1Supplementary Table 2Supplementary Table 3


As of 1st April 2020, COVID-19, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had infected over 800,000 people and caused over 40,000 deaths in 205 countries and territories. COVID-19 has had its heaviest toll on Europe, United States and China. As of 1st of April 2020, the number of confirmed COVID-19 cases in Africa was relatively low, with the highest number registered by South Africa, which had reported 1,380 confirmed cases. On the same date , Africa had reported 5,999 confirmed cases, of which 3,838  occurred in South Africa, Algeria, Egypt, Morocco and Tunisia, with the remaining 2,071 cases distributed unevenly across the other African countries. We speculate that while African nations are currently experiencing much lower rates of COVID-19 relative to other continents, their significantly lower testing rates may grossly underestimate incidence rates. Failure to grasp the true picture may mean crucial windows of opportunity shut unutilized, while limited resources are not deployed to maximum effect. In the absence of extensive testing data, an overestimation of spread may lead to disproportionate measures being taken, causing avoidable strain on livelihoods and economies. Here, based on the African situation, we discuss COVID-19 diagnostic challenges and how they may blunt responses.Since its emergence in Wuhan, China in December 2019, novel Coronavirus disease - 2019 (COVID-19) has rapidly spread worldwide, achieving pandemic status on 11 This is routinely done by carrying out nucleic acid amplification tests (NAAT). Currently, RT-PCR detection of unique sequences of the viral genome is the gold standard for COVID-19 testing where the N, E, S and RdRP (RNA-dependent RNA polymerase) genes are targeted. Sample handling should be carried out in a BSL-2 biosafety cabinet under strict adherence to personal protective equipment (PPE) guidelines. However, RT-PCR is labor intensive, severely constraining the capacity for quick turnaround times from sample collection to results transmission. In many contexts, getting results takes days . As a coIn addition to suspect case diagnosis, widespread COVID-19 testing is critical for disease monitoring and surveillance. Such testing is recommended so as to meet the following objectives :a)Monitor disease trends in contexts of rapid person-person virus spread.b)Quickly identify cases in countries/regions where the virus is not circulating.c)Generate epidemiological data for risk assessment regionally, nationally or globally.d)Generate epidemiological data to guide government responses in terms of policy, resource mobilization and distribution.However, how extensively governments rollout testing for surveillance purposes is dependent on each country’s prevailing circumstances and preferred COVID-19 control strategy. For instance, aggressive, widespread testing in South Korea early in their epidemic, including the deployment of drive-through testing . While iThe benefits of large-scale COVID-19 testing have been demonstrated in several countries. However, most low-middle income countries (LMICs), including the majority of African countries, lack capacity for large scale testing. These countries are facing numerous challenges in their efforts to diagnose suspect cases, trace contacts for further testing and roll out surveillance testing. For instance, as a consequence of inadequate testing capacity, at the time of preparing this review Kenya had carried out 2,563 tests only, of which 122 returned positive . While KA major diagnostic challenge stems from the nature of RT-PCR tests, the gold standard in COVID-19 testing. COVID-19 RT-PCR test kits are expensive, making the cost of large-scale testing prohibitive for LMICs. Additionally, the test requires expensive equipment, including PCR machines and adequately equipped BSL-2 labs. These factors, and the need for highly trained personnel, mean that in most African countries, very few centers have the capacity to run COVID-19 tests. Furthermore, completing a COVID-19 RT-PCR test is a multi-step process that begins with sample collection, often at a point of care that is far away from the country’s test center(s). The sample must then be carefully packaged and transported to the distant testing site under conditions that protect sample integrity. At the lab, laborious sample preparation, including RNA extraction is done, followed by reaction mix preparation and RT-PCR . This mu COVID-19 diagnostic challenges are not unique to African countries and LMICs. Consequently, numerous private and public institutions have developed rapid diagnostic tests (RDTs) aimed at speeding and expanding testing, crucial factors in the struggle to slow COVID-19 spread. RDTs, which are largely based on immunoassays, may be direct, through detection of SARS-CoV-2 antigens or indirect, through detection of anti-SARS-CoV-2 antibodies . AdvantaThe COVID-19 diagnostic challenges faced by African nations highlight long-running diagnostic challenges for a wide range of diseases. Part of our group’s research has been the development of RDTs and POCDs for various diseases, including placental malaria and bacterial infections. We contend that an effective means of achieving mass testing at the required scale, is to fund the development COVID-19 RDTs locally, to meet local demand – bearing in mind that the knowledge and relevant local and international collaborations are already in place. Such solutions should then be aggressively deployed for points of care and home use, particularly in rural settings. In fact, this strategy is being used by Senegal, which together with UK collaborators, is developing an affordable COVID-19 RDT (expected to cost $1 per test) for home use in African countries . SimilarEvidently, COVID-19 testing by RT-PCR is not applicable in most parts of Africa considering that vast populations live in rural settings with poor transport and communication infrastructure. RT-PCR requires expensive equipment, skilled personnel, reagents and reliable power supplies. Additionally, the long turnaround time of 4-24 hours (and days in some contexts) , may disDifferent strategies exist for point of care testing, all with inherent merits and demerits: 1) Use of antibody testing. As the body mounts an immune response, antibodies against SARS-CoV-2 antigens are generated. These antibodies may serve as indicators of infection. However, since detectable antibodies may lag behind the appearance of clinical symptoms, most infections will be missed, causing a high rate of false negatives. Conversely, persistence of antibodies after virus elimination from the body may result in a high rate of false positives. Moreover, a high cross reactivity of SARS-CoV-2 and SARS-CoV S protein against plasma samples from 15 patients has been observed, which may impact test interpretation , are curOther near point of care tests include radiological imaging with Chest CT-scan. This approach has been shown to be highly sensitive (at 97%) but poorly specific (at 48%) in a Chinese study  and limited available personnel and implementation for these technologies. The authors describe the need for funding local development and production of RDTs to detect COVID-19 in order to achieve widespread availability and testing. The present form of the manuscript should be published given its valuable contributions for scientific review and policy but could be further improved with minor changes described below:et al., 2020; Rothan & Byrareddy, 2020).” However, current research  indicates that many cases are asymptomatic. It would be better to qualify the statement so as not to imply that asymptomatic infection is rare. In section “SARS-CoV-2 transmission and pathogenesis”, the authors indicate that “…almost all infections become symptomatic by day 11  but the review does not highlight how this might be done and could benefit from examples of success overcoming these challenges in the diagnostics or other fields. https://www.ecdc.europa.eu/en/publications-data/overview-rapid-test-situation-covid-19-diagnosis-eueea or the PDF itself:https://www.ecdc.europa.eu/sites/default/files/documents/Overview-rapid-test-situation-for-COVID-19-diagnosis-EU-EEA.pdf The “ECDC: An overview of the rapid test situation for COVID-19 diagnosis in the EU/EEA. 2020” link currently goes to the wrong location but should go to the website:Is the review written in accessible language?YesAre all factual statements correct and adequately supported by citations?YesAre the conclusions drawn appropriate in the context of the current research literature?YesIs the topic of the review discussed comprehensively in the context of the current literature?YesReviewer Expertise:development of point-of-care diagnostics for infectious diseases, microfluidics, biosensors,I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The present review by the authors provides important information on diagnostic challenges facing African countries in their combat against the ongoing COVID-19 pandemic. The paper reviews the history of the COVID-19 and traces its spread to Africa, details the mechanisms of transmission and pathogenesis of the disease in a concise and easy-to-understand style, and provides an in-depth examination of the diagnostic challenges Africa faces in assessing the full scale of the pandemic in the continent. The authors contend that most African countries lack the capacity to administer mass screening to ascertain the extent of the disease spread, and call for support toward the development of homegrown RDTs and POCTs as a strategy to achieve mass screening of COVID-19 in Africa. Overall, the paper succeeds in presenting a concise review of COVID-19 situation in Africa, identifies diagnostic challenges and suggests POCTs as a widely deployable testing approach which, if implemented for mass screening, can be effective for monitoring the spread of the ongoing pandemic. Hence, I find the paper to be of reasonable priority in view of the current situation, and recommend its publication even in the present form. I believe that paper contains scientifically valuable information and would be an important resource for scientists as well as policy makers in the battle against the ongoing scourge not only in Africa but worldwide.The authors ought to mention the impact of mass screening/diagnostics on the existing weak health infrastructure in Africa. The success story from South Korea arose out of a highly coordinated system of diagnosis, isolation and treatment, but this may not be possible with the underdeveloped healthcare infrastructure in Africa. One concern would be that identifying positives without appropriate follow up measures can lead to public fear and stigmatization, which might undermine the intended purpose of mass screening.Poor research funding has always been cited as a major hindrance to the development of homegrown scientific solutions in Africa. The authors ought to comment on the challenges facing the suggested development of RDT/POC in Africa, most notably, lack of financial support and political goodwill. Specific to the present COVID-19 case, would it be faster and cheaper importing the diagnostic tools, as is already being done by some countries?COVID-19 point of care testing strategies” section before “Surmounting COVID-19 diagnostic challenges” section, for consistency with the conclusion.The authors may wish to put ““Are Africa’s diagnostic challenges blunting response effectiveness?” which appears in the title. It would be nice to reassess and address the question by providing an answer to it in the conclusion part.The authors ought to provide a clear-cut answer to the questionIt would be informative to provide a list of some of the RDTs/POCTs available for rapid deployment or, alternatively, cite this paper (access via the URL below) which provides an updated list of POCTs.  https://aasopenresearch.org/articles/3-8/v1The link for the reference below is not correct. Please check.   However, in revising the article for publication, the authors may wish to consider the following minor comments/corrections: Ref: ECDC: An overview of the rapid test situation for COVID-19 diagnosis in the EU/EEA. 2020. The link provided in the manuscript opens up this page:https://www.medrxiv.org/content/10.1101/2020.03.18.20038059v1Is the review written in accessible language?YesAre all factual statements correct and adequately supported by citations?YesAre the conclusions drawn appropriate in the context of the current research literature?YesIs the topic of the review discussed comprehensively in the context of the current literature?YesReviewer Expertise:Bioengineering, Biotechnology, Biomechanics, MicrofluidicsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


The excessive use or abuse of pesticides and veterinary drugs leads to residues in food, which can threaten human health. Therefore, there is an extremely urgent need for multi-analyte analysis techniques for the detection of pesticide and veterinary drug residues, which can be applied as screening techniques for food safety monitoring and detection. Recent developments related to rapid multi-residue detection methods for pesticide and veterinary drug residues are reviewed herein. Methods based on different recognition elements or the inherent characteristics of pesticides and veterinary drugs are described in detail. The preparation and application of three broadly specific recognition elements—antibodies, aptamers, and molecular imprinted polymers—are summarized. Furthermore, enzymatic inhibition-based sensors, near-infrared spectroscopy, and SERS spectroscopy based on the inherent characteristics are also discussed. The aim of this review is to provide a useful reference for the further development of rapid multi-analyte analysis of pesticide and veterinary drug residues. Pesticides and veterinary drugs are indispensable for increasing food production, as well as improving animal breeding and aquaculture . PesticiThe traditional methods for pesticide and veterinary drug residues determination are usually based on instrumental techniques such as gas chromatography (GC), high performance liquid chromatography (HPLC), or chromatographic methods coupled with mass spectrometry (MS) detectors . These mCommon rapid methods are capable of detecting a single target with high specificity , whereasIn this review, two strategies of rapid multi-residue methods are addressed for the measurement of pesticides and veterinary drugs . The firThe accurate determination of trace target analytes in complex food matrices is a major challenge for the development of rapid detection methods . The useAntibodies have long been the most common recognition elements used in rapid detection methods. Antibody-based approaches, such as the enzyme-linked immunosorbent assay (ELISA), have been utilized as alternatives to the routine techniques for the detection of pesticide and veterinary drug residues in food and environmental samples. For multi-residue detection, there are four ways to obtain broadly specific antibodies. Generic antibodies prepared by “general-structure” immunogens are one kind of broadly specific antibodies A. Broad-Generic antibodies are generated by immunization with “general-structure” haptens, which exhibit the common features of an entire class of related analytes , as showO,O-diethyl thiophosphate moiety and a benzene ring were designed by two separate research groups [O,O-diethyl O-(3-carboxyphenyl) phosphorothioate with different carboxy groups in the meta position of the benzene ring, in order to prepare immunogens or coating antigens. A broadly specific monoclonal antibody was generated, which showed high and uniform sensitivity to seven O,O-diethyl OPs and six O,O-dimethyl OPs. These trial-and-error based hapten design methods are often unpredictable, time-consuming, and expensive. This is because of the required simple chemical structure comparisons at the two-dimensional (2D) level when the haptens are designed. With the development of computer-assisted molecular modeling, the influences of the hapten structures and spacers on steric and electronic properties can be investigated and evaluated at the three-dimensional (3D) level [O,O-diethyl Ops. Molecular modeling can also assist immunochemists design the most appropriate hapten for other pesticides and veterinary drugs. The generic antibodies of sulfonamides (SAs) and fluoroquinolones (FQs) with a wide recognition profile have also been developed using molecular modeling and theoretical tools.To solve this problem, several strategies have been developed to obtain the desired antibodies . Taking h groups ,18,19. Sh groups  synthesiD) level . The potD) level . With thD) level . Xu et aD) level  used 2D 50 values were 0.067–0.139 μg/L, with cross-reactivity of 48.2–418.7% for the five antibacterial synergists under optimal conditions. Due to the icELISA requiring enzyme-labeled secondary antibodies and complicated procedures, the 5C4 antibody was also used to develop a direct competitive ELISA (dcELISA) method [50 values were 0.208–9.24 μg/L, that is, 1.36–31.16 fold higher than those of icELISA. However, the dcELISA method required lower testing costs and less time for the detection of multiple antibacterial synergists in real samples. For improvement of the sensitivity of ELISA, chemiluminescence enzyme immunoassays have been developed with luminol as a chemiluminescence substrate. Zeng et al. [Various immunochemical methods can be applied for the detection of pesticide and veterinary drugs. ELISA involving antibodies combined with enzymatic markers has taken a leading position in immunoassay methods . Due to ) method . The IC5g et al.  utilizedIn addition to ELISA methods, lateral flow immunoassay (LFIA) is another conventional immunochemical method ,32, whicGeneric antibodies have successfully been used in multi-analyte assays for pesticide and veterinary drug residues. However, the desired generic immunogen used to produce generic antibodies with broad specificity and high affinity are difficult to design. Meanwhile, the generic antibody cannot have a consistent affinity for all of the analytes, which leads to different relative responses to the target analytes in the detection methods. In addition, the developed assays cannot distinguish each analyte respectively when the analytes are mixed, as is typically the case in real samples. Furthermore, generic antibodies can be produced by the same class of analytes with a general structure. Thus, broad specificity antibodies against different classes of target analytes cannot be obtained using this approach.In order to broaden the recognition spectrum of antibodies, multi-hapten strategies have been used to produce various antibodies in one experimental animal. By using this approach, broadly specific antibodies against different classes of pesticide and veterinary drug residues can be produced. A multi-hapten immunogen can be obtained by two approaches: (1) Several different haptens are coupled to one carrier protein simultaneously ; or (2) O,O-dimethyl thiophosphate hapten and O,O-diethyl phosphate hapten) to BSA. A broadly specific polyclonal antibody showed selectivity to eleven organothiophosphate pesticides and five organophosphate pesticides [By using the first approach, four multi-hapten antigens for parathion have been conjugated to bovine serum albumin (BSA) to generate a broadly specific antibody. The sensitivity of ELISA decreased, due to the increase of structural complexity. However, the specificity of other target analytes was broadened in this immunoassay . Furthersticides . Using tsticides . The obtsticides .Broad-spectrum antibodies have also been used to develop immunoassays for high-throughput screening of pesticide and veterinary drug residues. An icELISA method was developed with polyclonal antibodies against dichlorvos and paraoxon . The LODCompared with generic antibodies, broad-spectrum antibodies have broader recognition spectra and do not require the design of a generic antigen. However, due to antigenic competition and the low coupling ratio of each antigen in multi-hapten strategies, lower affinities to analytes are obtained than that in antibodies derived from a single immunogen. The higher total number of hapten molecules on the surface of a multi-hapten immunogen may not be advantageous for lymph cells to recognize the carrier protein, which would result in poor induction of the immune system . MeanwhiBispecific antibodies contain two different antigen-binding sites, which can detect two distinct residues  Figure C. BispecSeveral multi-analyte immunoassays for the screening of pesticide and veterinary drug residues have been developed using bispecific antibodies. A bispecific antibody-based icELISA method was developed for the detection of 5-morpholinomethyl-3-amino-2-oxazolidone and leucomalachite green in aquatic products . The LODBispecific antibodies can realize the simultaneous analysis of two different classes of pesticide and veterinary drug residues. However, bispecific antibodies are difficult to obtain. Due to the co-dominant expression of immunoglobulin genes, ten different antibody species were obtained with only one contributing to the desired bispecificity. This drawback led to the production of a large percentage of non-functional antibodies . TherefoIn addition to generating the above three kinds of broadly specific antibodies, an easy way to realize the multi-analyte analysis of residues is to incorporate numerous specific antibodies into a single test. Individual antibodies or antigens could be distinguished by position or by labeling with different labels. Through the flexible combination of antibodies, analytes with very different structures can be detected simultaneously. Moreover, generic antibodies, broad-spectrum antibodies, and bispecific antibodies can also be utilized in combination to develop multi-analyte analyses, which can further broaden the recognition spectrum of antibodies. In contrast to multi-analyte assays using the above three kinds of broadly specific antibodies, assays based on multi-antibodies can distinguish each analyte semi-quantitatively or quantitatively. Single-label and multi-label strategies have been applied in multi-analyte assays.As different antibodies can be coated on different zones of biochips or lateral flow strips, single-label strategies can be carried out in LFIA or biochip microarray assays to realize multi-analyte analysis. Han et al.  developeMulti-label strategies have a wider range of applications, compared with single-label strategies. In addition to LFIA and biochip microarray assays, ELISA and fluorescence-linked immunosorbent assay (FLISA) can also realize quantitative analysis for each analyte by using a multi-label strategy. Yu et al.  developeMulti-analyte analysis based on multi-antibodies can realize high-throughput, highly efficient, rapid, and quantitative analyses. However, multiplex antibodies and different labels result in an inevitable increase of detection cost. Meanwhile, it is not easy to obtain good sensitivity to different targets in multi-analyte assays. The ideal ratio of mixed antibodies should be carefully optimized. Moreover, cross-reactions with other analogues should be avoided when using mixed antibodies.Aptamers serve as an ideal alternative to an antibody for the analysis of pesticide and veterinary drug residues . AptamerAptamers usually have high specificity for target molecules which allow them to only be used for detecting specific targets. As it is impossible to select aptamers for all of the analytes, being able to generate aptamers that can recognize a group of molecules with a common structure would be significant for the detection of residues . By ratiBroad-spectrum aptamers can be derived from conventional SELEX procedures, with a little modification, by changing the single target to multiplex targets . By usinVarious aptasensors have been developed to screen pesticides and veterinary drugs. For example, the above OP broad-spectrum aptamer SS2-55 has been used in colorimetric, electrochemical, and SERS assays . AuNPs cBroadly specific aptamers can also be obtained by aptamer truncation besides selection strategy. The nucleotides contributing to target binding are designed and reserved. Therefore, broadly specific aptamers obtained by truncation usually contain relatively few bases. For example, a shortened 8-mer broadly specific aptamer with high affinity to four different TCs has been truncated from an original 76-mer aptamer . The 8-mTruncated broadly specific aptamers can be flexibly applied in the design of aptasensors. Zhou et al.  utilizedAptamers can be easily synthesized and obtained after the sequences of the selected aptamers have been released in the literatures. Therefore, multi-analyte analyses of residues with multi-aptamers have attracted significant interests, with respect to constructing rapid and high-throughput screening methods. Multiple-label strategies and multiple-channel strategies are commonly used in multi-analyte analyses.2+ and Pb2+) which could generate distinct differential pulse voltammetry peaks were used as signal tracers. The LODs for KAN and streptomycin were 74.50 and 36.45 pM, respectively. In addition to being labeled with different signal traces, novel magnetic tripartite DNA assembly nanostructure probes have been constructed for the detection of KAN, aflatoxin M1, and 17β-estradiol [By using a multiple-label strategy, multi-analyte analysis can be realized within a single system. Different fluorophores ,79, metastradiol . CombineWhen a higher number of target analytes needs to be detected, multiple-channel strategies, which feature multiplex channels or reaction positions, present advantages. Cheng et al.  developeMIPs have been identified as another substitute for antibodies, due to their advantages of low cost, high chemical stabilities, and specific recognition . The con−12 and 6.81 × 10−11 M for trichlorfon and acephate, respectively. Furthermore, another molecule sharing a common structure with OP pesticides, diethylphosphonoacetic acid, has been used as a template to synthesize MIPs [−13 and 2.05 × 10−14 mol/L, respectively. Furthermore, surface plasmon resonance sensor fabricated molecular imprinting nanofilms were developed for the detection of three pesticides [Broadly specific MIPs have been used as recognition elements in biomimetic ELISA, electrochemical assays, and SERS assays for multi-residue detection. MIPs were used as an antibody substitute to develop a biomimetic ELISA method for the detection of trichlorfon and acephate. A 4-(Dimethoxyphosphorothioylamino) butanoic acid sharing a common structure with OPs pesticides was used as a template molecule . The LODize MIPs . The obtsticides . The LODAside from the above mentioned multi-residue detection methods with different recognition elements, enzymatic inhibition-based sensors, NIR spectroscopy, and SERS spectroscopy have also been used for the multi-analyte analysis of pesticide and veterinary drug residues, with the development of bio-neuroinformatics technology, spectroscopy technology, and nanotechnology. These detection methods were mainly developed based on the inherent characteristics of pesticides and veterinary drugs. Therefore, these methods can realize non-destructive, rapid, low cost, and environmentally friendly detection without the use of recognition elements. Enzymatic inhibition-based biosensors have been successfully applied in multi-residue screening and detection, based on the enzyme inhibition characteristics of pesticide residues. Due to the specific functional groups and chemical structures of pesticides and veterinary drugs, different absorption bands and spectral intensities are shown in the NIR or SERS spectrum. Thus, enzyme-based sensors, NIR spectroscopy, and SERS spectroscopy have become promising detection techniques for the multi-analyte analysis of residues.The activities of cholinesterases, acetylcholinesterase(AChE), and butyrylcholinesterase can be inhibited by OP and carbamate (CM) pesticides . Based o−3 mM.AChE can hydrolyze certain substrates to colored products, while the color development can be decreased in the presence of OP or CM pesticides . TherefoDrosophila melanogaster AChE B394 and B394, were used to modify the biosensors. The combined responses of the two pesticides were modeled by two different ANNs. Both pesticides could be quantified with low errors from a direct measurement step. Moreover, one-step AChE disposable biosensors based on genetically modified enzymes and ANNs has been developed to determine mixtures of three OP residues [Drosophila melanogaster  were used to modify the biosensor array. The biosensor system successfully identified and quantified mixtures of chlorpyriphos oxon, chlorfenvinphos, and azinphosmethyl oxon.The above detection methods can be used for the rapid pre-screening of OP or CM pesticides, in order to discriminate whether the sample contains pesticides or not. No qualitative or quantitative information about individual pesticides in the complex matrix can be obtained. Therefore, multi-sensor arrays combined with artificial neural networks (ANNs) have been developed for multi-residues quantitative analysis. Different types of native or genetically engineered AChEs portrayed different sensitivities to OP or CM pesticides, which were used to modify the multi-biosensors. Combined with the data processing of ANNs, the pesticide mixtures could be discriminated . An AChEresidues . Two AChEnzymatic inhibition based multi-residue detection methods have been applied in the rapid pre-screening of OP and CM pesticides. However, there are still some problems which need to be solved. One is how to retain the original catalytic activity of AChE after immobilization. Therefore, the immobilization surface should be well-designed. The second is the issue of the re-activation of an enzymatic layer complicating the operation of a biosensor. Therefore, a disposable biosensor with cheap production cost is desirable for field conditions. The third is a selectivity problem. It is difficult to determine which kinds of OPs inhibit the AChE activity in a particular sample. Although multi-sensor arrays combined with ANNs can provide a partial solution this problem. Due to the needs of different AChEs and the complex processing of ANNs, the practicality and operability need to be further improved.NIR spectroscopy methods have been recognized as a non-destructive evaluation approach for the quality control of intact food . Aside fTypically, NIR absorption spectra are collected and the spectral information is analyzed for the assessment of residues. Jamshidi et al.  applied S-AWLSGSD-RC-LWPLSR model was established with eight characteristic wavelengths, having correlation coefficients of cross-validation of 0.757 and 0.898 for chlorpyrifos and imidacloprid, respectively [However, the spectra used in the above methods were collected from a small portion of the tested samples, which cannot guarantee the data accuracy and representativeness. Therefore, hyperspectral imaging technology has been used, in combination with NIR spectroscopy methods. Hyperspectral imaging integrated spectroscopy and imaging can be used to obtain both spectral and spatial information from samples . An NIR-ectively . Moreoveectively . The besectively . The calNIR spectroscopy methods are capable of realizing multi-residue detection in intact food. In addition to the characteristic of non-destructive, NIR spectroscopy has the advantages of no sample preparation needed, fast response time, environmental friendliness, and low operating cost. Therefore, NIR spectroscopy methods have a great application potential in online monitoring and field detection.SERS is a promising method, combining Raman spectroscopy and nanotechnology for the rapid and non-invasive detection of samples. The chemical bonds and vibrational properties of functional groups are represented by Raman spectral bands, providing a fingerprint for target analytes . With th−3, 10−3, and 10−4 ng/cm2. A nanoporous cellulose paper-based AuNRs SERS substrate was constructed for the detection of three pesticides [−2, respectively. Chen et al. [2, respectively.The sensitivity of SERS is influenced by the molecular intrinsic vibration of the targets and the interaction between targets and the SERS substrate. Pesticides and veterinary drugs with certain thiol or amine functional groups can bind strongly to Au and Ag substrates . Therefosticides . The LODn et al.  developeSERS has demonstrated excellent sensitivity, simple sampling, and rapid test speed for the multi-residues detection of pesticide and veterinary drug residues. With facile design, SERS substrates with the advantages of flexibility, excellent adaptability, cost-efficiency, and suitability for large-scale production can be constructed for non-destructive practical applications. With the development of nanotechnology and the improvement of instruments, SERS can provide a routine and cost-effective detection method for the multi-analyte analysis of pesticide and veterinary drug residues.The residues of pesticides and veterinary drugs threaten human health. Therefore, several screening assays have been developed for the rapid multi-residue detections of pesticides and veterinary drugs. Different broadly specific recognition elements have been prepared and applied for simultaneous analysis. In addition, enzymatic inhibition-based sensors, NIR spectroscopy, and SERS spectroscopy, based on the inherent characteristics of pesticides and veterinary drugs, can also realize multi-analyte analysis. With the development of genetic engineering and computer modeling technology, recognition elements with the desired broad specificity and high affinity to multiplex targets can be designed and obtained. Meanwhile, the design of novel portable instruments and the synthesis of novel functional materials serve to promote the development of new detection methods with more target analytes and higher sensitivities. With the combination of rapid multi-analyte analysis methods and traditional instruments, efficient and accurate detection systems which feature simplified test processes and reduced determination time can be built.


Background: Recent randomized clinical trials have examined the efficacy of different combinations of systemic and local treatment approaches for metastatic hormone-sensitive prostate cancer (mHSPC). We compared the efficacy of these combined regimens in order to identify the optimal therapy for specific patient subgroups.Methods: The treatments were abiraterone (ABI), apalutamide (APA), docetaxel (DOC), enzalutamide (ENZ), and radiotherapy (RT) combined with androgen-deprivation therapy (ADT). Five electronic databases were searched up to May 7, 2020 for relevant trials. The risk of bias in the included trials was evaluated with the Cochrane tool. The hazard ratio (HR) with 95% confidence interval (CI) was determined for the included trials and indirect comparisons were performed using the R software.Results: In total, 10 randomized, controlled trials with 11,194 patients were included in the meta-analysis. ADT + RT was superior to ADT monotherapy in terms of overall survival  and conferred a survival benefit in a subgroup of low-volume patients . Combined systemic treatments were significantly superior to ADT monotherapy in comparisons of survival and prostate-specific antigen response, including in the high-volume subgroup; meanwhile, in the low-volume subgroup only ADT + ENZ  showed a significant clinical benefit. In the Gleason score <8 subgroup, all combined systemic treatments were superior to ADT monotherapy, but the results were only significant for ADT + APA  and ADT + DOC . In the Gleason score ≥8 subgroup, ADT monotherapy was inferior  to combined treatments. In a ranking of performed comparisons, ADT + ENZ was the optimal regimen, although this was non-significant. Combined therapies also demonstrated superiority in quality-of-life indicators such as time to skeletal events and pain progression.Conclusion: ADT + radiotherapy led to superior outcomes in mHSPC patients with low-volume disease. While all combined systemic regimens confer a survival advantage over ADT monotherapy, the optimal treatment approach for certain mHSPC patient subgroups remains to be determined. Prostate cancer (PC) is the most common cancer type in senior males worldwide . In the The therapeutic armamentarium for metastatic hormone-sensitive (mHSPC) patients has rapidly expanded with the development of novel treatments such as an androgen synthesis inhibitor, chemotherapy, and next-generation antiandrogen drugs, which have greatly improved the efficacy of ADT. The STAMPEDE trial demonstrated that combining docetaxel (DOC) or abiraterone (ABI) with ADT conferred a clinically meaningful survival advantage in patients with mHSPC , 3. MeanTo address this issue, in this study we indirectly compared different combined treatment approaches , ADT + ENZ, ADT + RT, and ADT monotherapy) in order to identify an optimal treatment strategy for prolonging survival and improving quality of life (QoL) in mHSPC patients. We also performed subgroup analyses of patients with different disease volumes and Gleason scores.clinicaltrials.gov) and gray literature  were searched to identify all relevant publications up to May 7, 2020. The complete search strategy is described in The systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines, and the protocol was registered a priori in the PROSPERO database. Computerized literature databases  were retained; retrospective, prospective, and non-randomized studies were excluded.We focused on the following eight interventions: ADT monotherapy, ADT + APA, ADT + AAP, ADT + DOC, ADT + ENZ, and ADT + RT.Outcomes of interest were overall survival (OS), prostate-specific antigen progression-free survival (PSA-PFS), time to symptomatic skeletal events (SSE), time to pain progression, and time to chemotherapy. OS was defined as the time from randomization to death for any reason. PSA-PFS was measured as the interval from randomization to the earliest event of PSA progression according to the PC Working Group 2 criteria . Time toOnly articles published in English were included. Titles and abstracts were screened for initial study inclusion. The full texts of potential articles were thoroughly examined when the title and abstract were insufficient to determine whether the study met the inclusion criteria. In instances where there was more than one publication resulting from the same clinical trial, we selected the most complete publication for analysis. The detailed PRISMA flow graph of the inclusion process is presented in Two investigators independently extracted the data from the included studies. The following information was obtained from the publications: author, year of publication, sample size, interventions, and main outcome measures defined in our inclusion criteria. We also extracted subgroup outcome data from each study . As in the CHAARTED study, high-volume disease was defined as the presence of visceral metastases or ≥4 bone lesions with ≥1 lesion beyond the vertebral bodies and pelvis .The methodologic quality of each study was assessed with the Cochrane Collaboration tool by two independent researchers. Aspects such as randomization, allocation, and blinding processes, outcome reporting, and other types of bias were assessed, with each one assigned a grade of low, high, or uncertain risk based on the result of the quality evaluation. Any disagreement was resolved by discussion with a third reviewer until a consensus was reached.p < 0.05 was considered statistically significant. Based on the deviance information criterion (DIC) value, a fixed-, or random-effects model was applied to the Bayesian framework model for data analysis. The convergence of the models was assessed with Brooks-Gelman-Rubin, trace, and density plots. A funnel plot was used to assess publication bias. Rank probabilities were calculated to determine the hierarchy of treatments. The “mtc.anohe” command in the “gemtc” package was used to evaluate global heterogeneity, which was documented with the variance parameter I2; a value >50% was considered indicative of significant heterogeneity. The geometry of the treatment network was established using the R software. Inconsistency was assessed if any closed loop existed in the network. The following comparisons of treatments were performed: ADT monotherapy, ADT + APA, ADT + AAP, ADT + DOC, ADT + ENZ, and ADT + RT for OS and PSA-PFS; ADT + APA, ADT + AAP, ADT + ENZ, and ADT monotherapy for time to SSE and time to pain progression; and ADT + APA, ADT + AAP, and ADT monotherapy for time to chemotherapy.Indirect comparisons were performed based on the Bayesian framework model using the “gemtc” package of the R 3.5.3 software , 14. OS The literature search returned of 639 records. After removing duplicates, 516 publications were retained for title and abstract screening, and 15 were selected for full-text assessment. Ultimately, 10 RCTs with 11,194 participants were included in our analysis. The baseline characteristics of the included studies are listed in ADT monotherapy, ADT + APA, ADT + AAP, ADT + DOC, ADT + ENZ, and ADT + RT were compared in our study. The treatment network is shown in The results of indirect comparisons are shown in Nine of the 10 trials were included in the analysis of PSA-PFS. ADT + AAP , ADT + APA , ADT + DOC , ADT + ENZ , and ADT + RT  conferred a survival benefit over ADT monotherapy. ADT + APA was the most effective combined treatment regimen (83.17%) whereas ADT + RT (84.20%) ranked last. Results of indirect comparisons are shown in We compared two health-related QoL outcomes; the results of indirect comparisons are shown in The four regimens  were compared in terms of time to pain progression; all three combined therapies were found to be superior to ADT monotherapy but only ADT + AAP showed a statistically significant difference . ADT + AAP was the highest-ranking regimen (79.90%), although all indirect comparisons yielded non-significant results.Two trials (TITAN and LATITUDE) were included in the analysis. Indirect comparisons revealed that both ADT + AAP  and ADT + APA  prolonged the time to chemotherapy compared to ADT monotherapy . ADT + AEight of the 10 trials were included in this subgroup analysis; the ARCHES and STAMPEDE-ABI arm were excluded because eligible outcome data were lacking. The results of indirect comparisons are shown in In the high-volume disease subgroup, ADT + AAP , ADT + APA , ADT + DOC , and ADT + ENZ  prolonged OS compared to ADT monotherapy, whereas ADT + RT  did not demonstrate a survival advantage. Ranking results revealed that ADT + AAP was the best therapeutic option (62.28%) for the high-volume subgroup. No statistically significant differences were observed in the comparisons of the included combined systemic therapies, while ADT + RT was inferior to the other regimens in this subgroup.In the low-volume disease subgroup, all combined treatments showed an OS benefit over ADT monotherapy, but statistical significance was only observed for ADT + ENZ  and ADT + RT . The ranking results revealed that ADT + ENZ was the best option (78.90%) for low-volume disease, although the benefit was non-significant. With the exception of ADT + ENZ vs. ADT + DOC , none of the comparisons between the combined therapies yielded significant results.The ENZAMET, TITAN, GETUG-AFU-15, CHAARTED, STAMPEDE-DOC arm, LATITUDE, and STAMPEDE-RT arm trials were included in this subgroup analysis; ARCHES and STAMPEDE-ABI arm trials were excluded because relevant outcome data were lacking. The results of indirect comparisons are shown in In patients with a Gleason score <8, ADT + APA  and ADT + DOC  showed superiority over ADT monotherapy; similar trends were observed for ADT + AAP  and ADT + ENZ , although the differences were non-significant. ADT + RT  was inferior to ADT monotherapy but without statistical significance. ADT + AAP (56.04%) was the highest-ranking combined treatment regimen.In patients with a Gleason score ≥8, all of the combined treatments were superior to ADT monotherapy; however, the differences were not statistically significant. ADT + AAP (32.98%) ranked highest  among the combined regimens.Only two comparisons of combined therapies  yielded statistically significant results, both in the Gleason <8 subgroup.I2-value of 35.54%, indicating that there was no significant heterogeneity. The full results of the convergence, inconsistency, publication bias, and heterogeneity analyses can be found in The potential scale reduction factor was limited to 1, reflecting good convergence in this analysis. The funnel plot of the included trials was near-symmetrical, indicating that there was no obvious publication bias. Given the lack of a closed loop in the network graph, the inconsistency assessment was not applicable to our study. The heterogeneity analysis for the whole network generated an The present study analyzed 10 high-quality, large-scale clinical trials involving 11,194 patients. All of the combined treatment regimens improved OS compared to ADT monotherapy, with ADT + ENZ ranking highest .post-hoc analyses of several large-scale clinical trials  trials were lower than the current standard of care; in the latter study, the RT was 36 Gy in 6 fractions or 55 Gy in 20 fractions, which are lower than the 57-Gy regimen that was shown to be inferior in the CHHiP trial . AnotherIn summary, ADT + RT demonstrated superiority over ADT monotherapy in our analysis of OS and the low-volume subgroup of mHSPC patients. Furthermore, ADT + RT showed comparable efficacy to most combined systemic treatment regimens in the low-volume subgroup. The combined systemic therapies showed a significant advantage over ADT monotherapy in all comparisons performed in this study, with ADT + ENZ identified as the optimal treatment in most cases. Based on limited data, we also showed that patients receiving combined therapies experienced less of a decline in QoL compared to those treated with ADT monotherapy. Based on these findings, the selection of an appropriate treatment approach for mHSPC patients by the physician should be made based on discussions regarding potential toxicities as well as the duration and cost of treatment.All datasets generated for this study are included in the article/YW and ZW: study concept and design. YW and HG: acquisition of data and analysis and interpretation of data. YW: drafting of the manuscript. JT, HW, ZH, and ZW: critical revision of the manuscript. YW and JW: statistical analysis. ZW: obtaining funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Introduction: Both lumbar endoscopic unilateral laminotomy bilateral decompression (LE-ULBD) and minimally invasive transforaminal lumbar interbody fusion (MI-TLIF) have been used to treat one-level lumbar spinal stenosis (LSS) with degenerative spondylolisthesis, while the differences of the clinical outcomes are still uncertain.Methods: Among 60 consecutive patients included, 24 surgeries were performed by LE-ULBD and 36 surgeries were performed by MI-TLIF. Patient demographics, operation characteristics and complications were recorded. Sagittal parameters, including slip percentage (SP) and slip angle (SA) were compared. The visual analog scale (VAS) score, the Oswestry Disability Index (ODI) score, and Macnab criteria were used to evaluate the clinical outcomes. Follow-up examinations were conducted at 3, 6, 12, and 24 months postoperatively.Results: The estimated blood loss, time to ambulation and length of hospitalization of the LE-ULBD group were shorter than the MI-TLIF group. Preoperative and final follow-up SP of the LE-ULBD group was of no significant difference, while final follow-up SP of the MI-TLIF group was significantly improved compared with preoperative SP. The postoperative mean VAS and ODI scores decreased significantly in both LE-ULBD group and MI-TLIF group. According to the modified Macnab criteria, the outcomes rated as excellent/good rate were 95.8 and 97.2%, respectively, in both LE-ULBD group and MI-TLIF group. Intraoperative complication rate of the LE-ULBD and the MI-TLIF group were 4.2 and 0%, respectively. One case of intraoperative epineurium injury was observed in the LE-ULBD group. Postoperative complication rate of the LE-ULBD and the MI-TLIF group were 0 and 5.6%, respectively. One case with transient urinary retention and one case with pleural effusion were observed in the MI-TLIF group.Conclusion: Both LE-ULBD and MI-TLIF are safe and effective to treat one-level LSS with degenerative spondylolisthesis. Lumbar spinal stenosis (LSS) with degenerative spondylolisthesis is an important cause of low back pain . The optEven though both decompression alone and decompression plus instrumented fusion has been performed to treat patients of LSS with degenerative spondylolisthesis, it is controversial about the necessity of instrumented fusion after decompression , 8, 12. The present study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the ethics committee of Tongji Medical College, Huazhong University of Science and Technology. Written informed consent was obtained from the individuals for the publication of any potentially identifiable images or data included in this article.This retrospective study included 60 patients  of LSS with degenerative spondylolisthesis, who underwent LE-ULBD or MI-TLIF in our department between January 2016 and December 2017. An informed consent has been obtained from each patient included. In these patients, 24 surgeries were performed by LE-ULBD and the other 36 surgeries were performed by MI-TLIF.Inclusion criteria were as follows: patients with typical symptoms, such as leg pain, numbness, motor weakness, neurogenic claudication or radiculopathy; lateral, flexion, and extension radiographs indicating grade 1 degenerative spondylolisthesis; computed tomography (CT) and magnetic resonance imaging (MRI) indicating one-level LSS with grade 1 degenerative spondylolisthesis, in agreement with clinical symptoms and signs; a history of failed conservative treatment for more than 3 months or progressive neurological symptoms; and follow-up for at least 24 months. Exclusion criteria were as follows: patients with radiographic confirmation of isthmic spondylolisthesis; lumbar pathologies requiring surgery at two or more levels; previous lumbar fusion surgery; and tumors, infections, or other lesions , 11, 17.Decision-making criteria: One-level LSS with grade 1 degenerative spondylolisthesis could be treated by either LE-ULBD and MI-TLIF. Patients without facet joint diastasis, significant motion on flexion and extension radiographs, were thought as the surgical indication for LE-ULBD . HoweverAll the surgeries were performed by the senior author. Both LE-ULBD and MI-TLIF were performed under general anesthesia with the patient in the prone position.via the incision to assist the insertion of the working sheath and the endoscopic surgical system . All the subsequent procedures were performed under constant irrigation with excellent endoscopic visualization. The inferior edge of the cranial lamina and the base of the spinous process of the ipsilateral side were removed by the endoscopic burr (Spinendos), enabling access into the spinal canal. Once the epidural space was entered, undercutting of the contralateral cranial lamina was performed. Then the ipsilateral and contralateral ligamentum flavum was identified and removed piecemeal with endoscopic punches and forceps. To minimize dural and epineural injury, the contralateral ligamentum flavum can be removed at first, then the ipsilateral ligamentum flavum is removed. Subsequently, ipsilateral and contralateral medial facetectomy was performed to decompress the lateral recess and ensure adequate decompression of the traversing nerve root. Once the traversing nerve root was decompressed, it was reflected medially using a blunt dissector. The bipolar radiofrequency electrocoagulator  was used for hemostasis, soft-tissue clearance and adhesion release. Prior to surgery completion, we ensured there was no significant dural sac damage or active bleeding. No drainages were required. These procedures were presented in Posteroanterior and lateral fluoroscopy were used to locate the interlaminar space at the surgery segment. A 10 mm skin incision was lateral to the outer border of the interlaminar window. Soft tissue expanders were applied Posteroanterior and lateral fluoroscopy were used to locate the pedicles of the surgery level. The Wiltse approach was undertaken through a paramedian skin incision. A Quadrant tubular dilator  was used for unilateral facet exposure. Facetectomy was performed on the ipsilateral side in order to visualize the transforaminal disc space. Laminectomy and lateral recess decompression were performed to decompress the spinal canal. Besides, the tubular retractor could be angled medially to complete a more extensive decompression of central canal stenosis and the contralateral side. Ligamentum flavum was adequately resected to expose the ipsilateral traversing and exiting nerve roots. A standard discectomy and endplates removal were performed. The autogenous and allogeneic  bone graft was placed anteriorly and contralateral to the annulotomy, then an intervertebral cage filled with autogenous and allogeneic bone graft was placed. In addition, unilateral pedicle screws were placed ipsilateral to the approach, and contralateral pedicle screws were placed through a contralateral incision. Rods were sized appropriately and tunneled through the paramedian incisions. The incisions were irrigated and closed in layers with drainages kept for no more than 48 h. A representative case is shown in Patient demographic, past medical history, symptom and signs were collected and summarized. The operation time, estimated blood loss, time to ambulation, length of hospitalization, intraoperative and postoperative complications were recorded. The healthcare cost was also recorded.Follow-up examinations were conducted at 3, 6, 12, and 24 months postoperatively. Plain radiography was performed preoperatively and postoperatively at follow-up time points. Preoperative and final follow-up sagittal parameters, including slip percentage (SP) and slip angle (SA) were compared . SP was p < 0.05 was considered statistically significant.All data are presented as mean ± standard deviation. SPSS 22.0  was used to perform the statistical analyses. GraphPad Prism 6  was used to generate plots. Non-parametric data was analyzed by Mann-Whitney U test or Wilcoxon signed-rank test. A Twenty-four patients were included in the LE-ULBD group, and the other 36 patients were included in the MI-TLIF group. The clinical characteristics of both groups are summarized in P = 0.183). The estimated blood loss, time to ambulation and length of hospitalization of the LE-ULBD group were shorter than the MI-TLIF group . Final follow-up SP of the LE-ULBD group was of no significant difference with preoperative SP, while final follow-up SP of the MI-TLIF group was significantly improved compared with preoperative SP. Final follow-up SA was 8 ± 3 in the LE-ULBD group, 8 ± 4 in the MI-TLIF group (P = 0.827). Final follow-up SA of both groups were of no significant difference with preoperative SA . One case of intraoperative epineurium injury was observed in the LE-ULBD group. No nerve injury, dural injury, or cauda equina syndrome was observed in the MI-TLIF group. Postoperative complication rate of the LE-ULBD and the MI-TLIF group were 0 and 5.6%, respectively (P = 0.244). One case with transient urinary retention and one case with pleural effusion were observed in the MI-TLIF group. No reoperation was observed within 90 days or during 24 months of follow-up.Intraoperative and postoperative complications of LE-ULBD and MI-TLIF group were also compared . IntraopThe healthcare cost of both groups was listed in Even though traditional decompression plus instrumented fusion has been performed to treat patients of LSS with degenerative spondylolisthesis, extensive detaching the paraspinal muscles from the spinous processes and lamina may cause increased intraoperative blood loss, postoperative pain and weakness secondary to muscle denervation. Besides, supraspinous and interspinous ligaments injury and extensive facetectomy may cause iatrogenic spinal instability, requiring additional posterior fixation for stabilization . TherefoDuring LE-ULBD, laminotomy and foraminotomy could be safely performed under excellent endoscopic visualization to guarantee complete decompression, minimize surgical trauma, and prevent iatrogenic spinal instability –15. Howevia a unilateral approach with minimize traumatization to the paraspinal musculoligamentous structures; to ensure the sufficient decompression of the lateral recess and foramen under excellent endoscopic visualization to minimize neurological injury; and to preserve the stability of the spine with minimized foraminotomy (The advantages of LE-ULBD were to perform bilateral decompression minotomy , 13–15. MI-TLIF has been demonstrated to be a safe option for lumbar fusion with minimized iatrogenic traumatization to the paraspinal musculoligamentous structures , 25. MI-Even though there are growing evidences suggest that decompression alone is of excellent clinical outcome for LSS with degenerative spondylolisthesis, it is still controversial for the necessity of arthrodesis , 27–30. There are some limitations to the present study. Firstly, it is a retrospective, non-randomized controlled cohort study with a small sample size and short follow-up period. There also may be selection bias, as surgeons determined whether decompression alone or decompress plus instrumented fusion should be performed. Further prospective, randomized, controlled studies, with larger sample sizes and longer follow-up periods should be conducted to determine the optimal surgical management for patients of LSS with degenerative spondylolisthesis. As a result, further studies should be conducted to compare the clinical outcomes of LE-ULBD and MI-TLIF for LSS with degenerative spondylolisthesis. Besides, preexisting adjacent level degeneration was not evaluated and compared in the present study.Both LE-ULBD and MI-TLIF are safe and effective to treat one-level LSS with degenerative spondylolisthesis. Although limited by small sample size, short follow-up period, and patients lost to follow-up, this study validates the safety of LE-ULBD as an alternative treatment for one-level LSS with degenerative spondylolisthesis.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Tongji Medical College, Huazhong University of Science and Technology. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individuals for the publication of any potentially identifiable images or data included in this article.WH and CY designed the study. WH, BW, WK, and QW collected, assembled, and analyzed the data. Project planning was performed by BW, WK, QX, XW, YZ, and SL. WH wrote the manuscript. All authors read, edited, and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lead, a known neurotoxicant, has previously received attention in Parkinson’s disease (PD) research, but epidemiologic studies have been limited in sample size and findings are equivocal. We generated two methylation-based biomarkers for cumulative tibia and patella bone-measured lead exposure in 1528 PD patients and 1169 controls. PD status was associated with increased levels of the DNAm biomarker for tibia-lead levels. We estimated a meta-OR for PD of 1.89 per unit DNAm tibia-lead increase . The current study supports the notion that chronic and long-term lead exposure tracked via DNAm may contribute to PD pathogenesis. Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by gradual loss of dopaminergic neurons in the substantia nigra. PD etiology is complex and multi-factorial, likely involving a combination of genetic, aging, and environmental components. Lead, a known neurotoxicant, has previously received attention in PD, but findings in epidemiologic studies have been equivocal.Lead passes through the blood–brain barrier and enters neurons and glia through calcium channels, affecting neurotransmitter release, energy metabolism, and inducing reactive oxygen species . Long-teClearly, bone-lead-measurement is the gold-standard for long-term lead exposure, given that the half-life of lead in the tibia is approximately 20 years . HoweverFor our analysis, we used genome-wide DNA methylation (DNAm) data from two publicly available PD studies (Gene Expression Omnibus (GEO) accession numbers GSE145361 (SGPD), GSE72774 and GSE72776 (PEG)). DNAm was measured with the Human Methylation 450 k BeadChip in whole blood samples.The System Genomics of Parkinson’s Disease (SGPD) is a consortium of three studies from across Australia and New Zealand . The datThe Parkinson’s Environment and Genes (PEG) study is population-based study from three agricultural counties of Central California . GEO datWe generated two epigenetic biomarkers for cumulative lead exposure (tibia and patella), developed in the Normative Aging Study (NAS). The epigenetic biosensors of patella and tibia are linear combinations of 59 and 138 CpGs, respectively, identified with site-by-site analysis and combined via machine-learning algorithms trained on K x-ray fluorescence (KXRF) in-vivo measures of bone-lead . To deteQ, and calculated a meta OR using a fixed-effects model with weights based on precision.In order to quantify the relationship between the DNAm lead and PD, we used logistic regression to estimate odds ratios (ORs) and 95% CIs for PD. The OR is a measure of association that compares the odds of PD based on exposure, with an OR greater than 1 indicating that the odds of PD increase with exposure. Regression diagnostics showed that model assumptions were met. Based on data availability from GEO, we controlled for age (estimated with the Horvath DNAmAge in SGPD ), sex, aCharacteristics of SGPD and PEG participants can be found in Table Q indicated no significant heterogeneity study results, p = 0.25). Specifically, with minimal adjustment , we estimated in SGPD, an OR for PD of 2.06 per unit DNAm tibia-lead increase  and in PEG of 1.60 , with a meta-OR of 1.89 . Further adjustment for blood cell composition, mean methylation by sample, and smoking status (PEG only), resulted in a meta OR of 1.52 . The DNAm biomarker for patella-lead levels was inversely associated with PD in SGPD ), but not in PEG  (heterogeneity between study results p = 0.006). Results for both DNAm markers stratified by sex can be found in Additional file PD status was strongly associated with the DNAm biomarker for tibia-lead levels Table  in both In two large population-based PD studies, we used DNAm to estimate cumulative, bone-lead levels by applying an externally developed epigenetic (DNAm) biosensor , and fouWe found no association between DNAm patella-lead and PD in PEG, and even an inverse-association in the SGPD study. Interestingly, one of the previous epidemiologic studies also only observed tibia- but not patella-lead measurements to be associated with PD . This isThe inordinate advantage of our study is the availability of DNAm-lead biomarkers, which also allows for future applications to all PD studies, or more generally studies of age-related disease, with methylation data and larger-scale systematic research into the long-term neurotoxicity of lead. The technology and methodology to track many health-related factors through methylation is rapidly developing. However, currently the DNAm-lead biomarkers have two limitations in our opinion, first, that they were developed in an all-male cohort, and second, that the specificity has not yet been validated beyond the initial study . Future Additional file 1: Box plot figures of the DNAm lead-biomarkers by PD and stratified by study.Additional file 2: Table showing DNAm estimated cumulative lead exposure and PD risk, stratified by sex.Additional file 3: Table showing DNAm estimated cumulative lead exposure and PD risk in PEG, with and without controlling for smoking.