The serum levels of M-CSF, TNF-α, TGF-β, and IL-6 were measured by an enzyme-linked immunosorbent assay, and their temporal changes and correlation between such changes were analyzed. The post-/pretreatment ratio of M-CSF levels was associated with anorexia after 2 weeks and fatigue after 2, 4, and 12 weeks. The M-CSF level in the RJ group was higher than that in the placebo group at the same timepoints. The TNF-α level in the RJ group was lower than that in the placebo group between 6 and 12 weeks, and the TGF-β level in the RJ group was higher than that in the placebo group; however, contrasting findings were detected after 12 weeks. Additionally, the M-CSF level was significantly correlated with the TGF-β level after 4 weeks and IL-6 level after 8 and 10 weeks. Among TNF-α, TGF-β, and IL-6, the post-/pretreatment ratio of TGF-β after 12 weeks was associated with TKI-induced anorexia, and the ratios after 10 and 12 weeks were associated with fatigue. Our results demonstrated that an oral intake of RJ suppressed anorexia and fatigue via complex mechanisms associated with inflammation-related factors, such as M-CSF and TGF-β in renal cell carcinoma patients treated with TKIs. In addition, we newly found that such RJ-related effects were dependent on the treatment duration.Inflammation is a common adverse event of anti-cancer therapy. Royal jelly (RJ) modulates inflammation by regulating the levels of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, and interleukin (IL)-6 produced by macrophages. Macrophage colony stimulating factor (M-CSF) is a crucial regulator of macrophage activities, and we hypothesized that RJ alters M-CSF levels. In this randomized controlled trial, we investigated the association between M-CSF and adverse events in renal cell carcinoma patients treated with tyrosine kinase inhibitors (TKIs) after an oral intake of RJ ( Chronic inflammation is closely associated with the pathogenesis of various types of chronic diseases, and many investigators have paid special attention to the preventive effects of a variety of nutraceuticals that regulate disease-related inflammation ,2. AdditRenal cell carcinoma (RCC) is one of the major types of urological cancer, and metastatic RCC is recognized as a fatal disease. At present, various types of anti-cancer agents are commonly used for patients with advanced/metastatic RCC, and tyrosine kinase inhibitors (TKIs) are the most prevalent first-line therapy . ConversRoyal jelly (RJ) is secreted by the hypopharyngeal and mandibular glands of worker honeybees and is considered safe for human consumption and good for overall health. The biological effects of RJ on macrophage activity are not fully understood; however, several components of RJ, such as 10-hydroxy-trans-2-decanoic acid (10H2DA) and adenosine N1-oxide, have been reported to affect the activation and function of macrophages both in vivo and in vitro ,18. AddiWe previously reported that an oral intake of RJ suppresses anorexia and fatigue in RCC patients treated with TKIs . HoweverThe study protocol was approved by the Human Ethics Review Committee of Nagasaki University Hospital . In the original protocol, we tried to recruit 60 patients in this clinical study. However, unfortunately, it failed to reach the goal. All experiments complied with the principles embodied within the Declaration of Helsinki and patients provided written informed consent to participate in all aspects of the study.p = 0.446) [The study population and protocol were similar to those of our previous reports ,19. Brie= 0.446) ,19. In s= 0.446) ,19. The p < 0.05.Results are expressed as the median and interquartile range. The Mann–Whitney U test was used to compare the continuous variables, and Pearson’s correlation coefficient (r) was used to evaluate the relationship between these variables. The coefficient of variation was calculated as a ratio of the standard deviation to the mean. All the statistical analyses were two-sided and were performed on a personal computer using the statistical package StatView for Windows . Significance was set at n = 17) and RJ group (n = 16) regarding age (p = 0.101), sex (p = 0.909), Eastern Cooperative Oncology Group performance status (p = 0.598), grade (p = 0.425), pT stage (p = 0.201), lymph node metastases (p = 0.881), and distant organ metastases (p = 0.325). Additionally, no significant difference was observed between the placebo and RJ group regarding the type of TKI administered (p = 0.539) [Our study population consisted of 23 males and 10 females, and the median age at the time of treatment was 68 (range: 54–79) years. With regard to pathological features, 81.8% (27/33 patients) and 72.7% (24/33 patients) were diagnosed with high-grade disease (Fuhrman grade 3 and 4) and high pT stage (pT3 and 4), respectively. Finally, no significant differences were observed between the placebo (= 0.539) .p = 0.014 and 0.007, respectively). Conversely, no significant difference was detected in the ratio of M-CSF between 6 and 10 weeks; however, a remarkable difference was observed at 12 weeks , but not for the other timepoints, including 4 and 12 weeks.Similarly, the ratios at 2, 4, and 12 weeks in patients without fatigue were higher than those experiencing fatigue  [p = 0.014; p = 0.013) than that in the placebo group at 12 weeks [Although the post-/pretreatment ratio of serum TNF-α levels between the placebo and RJ group demonstrated no significant difference at 2 and 4 weeks, the ratio in the RJ group was significantly lower than that in the placebo group at 6, 8, and 10 weeks (p = 0.020) than that in patients without fatigue . In addition, with regards to fatigue, the ratio of TGF-β at 8 weeks demonstrated a similar trend; however, this difference was not significant  but not for the other timepoints, including 12 weeks . Another study showed that two weeks of RJ administration led to changes in the serum levels of IL-1β, IL-10, and TNF-α in a rat model of colitis . Moreovep < 0.05) than in the control [Our results showed that the oral intake of RJ affected serum TNF-α levels. Unfortunately, there is no report regarding the influence of RJ on the serum level of TNF-α in human subjects. However, in a rat colitis model, the serum level of TNF-α was significantly lower in the RJ treatment group -irradiated human skin fibroblasts . In addiIt is not yet clear whether there is a direct correlation between M-CSF and TNF-α, TGF-β, or IL-6. Histologically, activated macrophages are divided into two phenotypes, namely pro-inflammatory (M1) and anti-inflammatory (M2) macrophages, and it has been established that M-CSF induces macrophage polarization toward the anti-inflammatory (M2) phenotype. M2 macrophages secrete anti-inflammatory cytokines, including TGF-β . In lineIn the present study, we found that an oral intake of RJ decreased post-/pretreatment ratio of serum TNF-α levels after 6 weeks of treatment. However, a direct significant correlation was not found between TNF-α and M-CSF levels for all treatment periods. TNF-α is secreted from M1 macrophages polarized by granulocyte-M-CSF . This fiThis study has several major limitations. Firstly, the number of patients included was relatively small. However, we think that bias was minimal because we employed a double-blind, randomized study design. Secondly, there were differences in the patients’ backgrounds. There is a possibility that the pharmacological effects of TKIs could influence the changes in inflammation-related factors, although we confirmed that there is no significant correlation between the post-/pretreatment ratio of the serum levels of M-CSF, TNF-α, TGF-β, or IL-6 and relative dose intensity in our study population. TKIs are still one of the major treatments for RCC patients, and many investigators pay special attention to cost, safety, and new treatment strategies using TKIs ,33,34. IAn oral intake of RJ modulates serum M-CSF levels in RCC patients treated with TKIs, and these changes in M-CSF are associated with TKI-induced anorexia and fatigue. Temporal changes in M-CSF, TNF-α, TGF-β, and IL-6 levels after RJ intake are dependent on the treatment duration. RJ inhibits TKI-induced anorexia and fatigue via complex mechanisms associated with M-CSF and TGF-β, which also vary with the treatment duration. Although changes in serum M-CSF levels caused by RJ positively correlated with those of IL-6, the clinical significance of this correlation is not clear. Further long-term studies with larger study populations are required to clarify the relationship between RJ and chronic inflammation-related symptoms in RCC patients.


The somatic effects of empire can be found in Tim Winton’s “pneumatic materialism”, an aesthetic preoccupation in his novels with moments of anoxia, or the deprivation of oxygen to the brain. This essay will consider how Winton's novel engage with pneumatic materialism in response to questions of uneven development traditionally associated with the Global South, thereby disrupting clear South–North distinctions. By blurring his concerns across the North–South divide, Winton shows a willingness to think of empire as a series of relations that are not bound by national or territorial borders so much as by substances in the air. He does this, I argue, in his use of the breath. In visual depictions of the Global South, Australia and New Zealand provide a boot-shaped, antipodean exception to the otherwise stable Brandt Line. Yet these countries, too, have their subalterns, disrupting the complacency of a “developed” North all too willing to make uneven development a problem of “over there”. As work by López , PrashadThis essay will disrupt the visual clarity of the Brandt line by considering how Winton’s novels engage with problems “in the air” that refuse to adhere to strict formulas of development and economic security. I argue Winton uses a breath-related formalism – here theorized as a pneumatic materialism – to show how uneven development, traditionally associated with the Global South, might be brought to bear when considering how issues of class, race, environment and mental health affect Western Australia, the region of Winton’s concern. By showing how a conceptual North–South divide functions internally to a nation in the Global North, Winton shows a willingness to think of empire as a series of relations that are not bound by national or territorial borders. Winton’s geographies are more local and more diffuse: aerial, they permeate the bodies of his characters, which are rendered porous, in turn, through this relation with air. Winton enacts this bodily porosity, I argue, in his use of breath.Winton’s novels betray an aesthetic preoccupation with moments of breathlessness, especially when it leads to a deprivation of oxygen to the brain. While characters have very different encounters with cerebral hypoxia , each crisis combines a scientific illustration of anoxic effects with a spiritual meditation on the afterlife. Breath provides Winton with the formal means to keep his conflation of hallucination, based on natural science, and revelation, based on divine inspiration, rigorously indeterminate. Winton uses this formal indeterminacy to respond to conditions as various as the occlusion of Indigenous Australians from Australia’s colonial history, the consequences of poaching fish and mining the land for asbestos and bauxite, and the erotic attachment to (auto) asphyxiation.inter alia, Fish from Cloudstreet, Clare Barker explains:As a trope, a narrative device, disability enables postcolonial writers to tell vivid stories about colonialism and its aftermath, stories that resonate outward from a character’s disabled body to address ‘damage,’ inequality, and power and its abuses in the postcolonial world. , 100Winton’s anoxic crises do engender damaged bodies, indexing a “postcolonial politics” of air in the wider field of disability studies  to develop, in this “middle space of witness”, what I call “pneumatic materialism”: a spiritual materialism, which is used variously as narrative technique, theme and plot device.Breath, in Winton’s texts, is a theologico-scientific witness” , 108. WiPace McCredden, I will argue that the consequences of Winton’s juxtaposition of the ordinary with the sacred is not a meaning-making extension “beyond the human”  across these divergent marked occlusions would be useful for future “intersectional” responses to Winton’s work. My argument is that In some ways the speed of this theological introduction, however frustrating, allows me to present an interesting aspect of Winton’s limited scientific interest: the materialist interests within his writing seem bent on refuting the post-Cartesian dualism that he himself seems to endorse. But the concern with breath in Winton’s novels is not simply a regression to a simpler pre-scientific respiratory theory. Rather, Winton’s particular form of theological realism begins from the observation of natural phenomena, and precisely the machinic qualities of the breath, from which he extrapolates theological implications.oeuvre. It appears in Cloudstreet  or the playing of a musical instrument (Breath). Even when this framing device is not included, breath features as the impulse behind certain character movements (Dirt Music). At the same time, Winton’s signature device, the vignette, might, in its impressionistic brevity, also be understood as breath’s translation into aesthetic form. The vignette is the contracted presentation of a life, impression or scene. It may demand more than a single physiological breath, but it functions as a “breath-unit”, or a discrete conceptual image. Indeed, the vignette requires brevity in order that it might impress upon the reader this unity of expression. Breath figures not simply as poetic function: Winton’s formal concern with breath extends from, and is underpinned by, an observable interest in the effect breath may have on perception, particularly those distortions, permanent or temporary, that accompany cerebral anoxia. Breath also gives rise to important plot turns, in moments of hallucination.Pneumatic materialism develops over the course of Winton’s udstreet  as a strrt Music  addresse  implied in the phrase “hands over”. “Handing over” suggests that something remains in death. Death itself is rendered inconclusive, not because of a certain resurrection, but because this middle spirit must be recognized and received in order to complete the act of handing over. So, while the narrative exceeds the terms of Fish Lamb’s death, it does so without fully recognizing the “handing over” of Fish’s spirit to death or resurrection.Rambo meditates on the moment in John 19:30 when Jesus bows his head and, at the moment of his death, “hands over his spirit  I wasn’t drowning yet. I hadn’t started to inhale any water. I mean, people say that drowning is an easy death. I think that was an old romantic thing that’s … I think it would be horrible. It’d be like suffocating. , while Winton imagines comparable experiences: he likens drowning to suffocation. Certainly, feelings of asphyxiation in Winton’s work are often treated in much the same way as liquid immersion.Dirt Music, written before the interview, and Breath, written after, Winton develops metaphoric connections between drowning and suffocation. At the same time, the experience is marked by an aesthetic appreciation of such moments when he sees bubbles and thinks about beauty. Despite an immediate threat of drowning, perhaps even because of it, Winton is thrown into an aesthetic contemplation of the water. Like Fish, in his dual roles as narrator and character, Winton’s moment of crisis is also a moment of pleasant reflection. Hallucination plays an important role in the embodied aesthetic experience of this moment, as Winton indicates earlier in the interview: “You jump in the water and just … It was like a hallucinatory experience, you know? Fish, sharks, dolphins, seals and weird noises, like something out of a Kubrick movie”.Drowning, in its medical definition, “is the process of expiring respiratory impairment from submersion/immersion in liquid”  into moments that would otherwise be meaningless (“apparent abyss”). If “Kubrick’s labouring astronauts are most eloquent when they don’t speak … their every breath says all we need to know about their situation”, how then does breath create a fragile mesh of contingency across Winton’s own novels?  a poetics that draws on hallucination and the vignette to (2) thread together a materialist theology that is (3) fragile and risky. In what follows, I want to recognize the breath-devices that elucidate the formal role played by breath, hallucination and vignette in Cloudstreet, a house on Cloud Street shared by two families, the Pickles and the Lambs, is the object around which the story will turn: the industrious Lambs putting their indolent landlords, the Pickles, to shame with their determination to make the best of the property; the Pickles, unable to sell the house as a stipulation of its inheritance, similarly tied to their situation because of almost unremitting poverty. Critics have noted its parallels to Australian settler origins. The story is told through a series of third person vignettes, though these vignettes do gesture towards free indirect discourse. This gesture becomes somewhat more overt by the time one reaches the end of the novel, when we find out that the novel’s impersonal third person narrator has been Fish Lamb, the unnamed “overripe man’s body” that jumps into the water in the novel’s unmarked preface. Though the reader would be forgiven for not noticing it, the novel that follows is the trace of this unnamed man’s “having known the story for just a moment”. For it works through, in more or less chronological order, how these two families come together for the picnic that brackets the beginning and end of the novel. A catalyst for the Lambs is the death and rebirth of Fish Lamb in an accident with a net while the family is fishing in the local river. As a result of the cerebral anoxia Fish suffers from “drinking in the river”, he sustains permanent brain damage. Yet, as we find out in the novel’s closing, as Fish returns to the river, the narrator has been Fish all along, but only consciously so “for those seconds it takes to die, as long as it takes to drink the river, as long as it took to tell you all this”. A spiritual Fish who reacquaints himself with his brain-damaged animate body as he drowns: “drinking his way into the tumble past the dim panic of muscle and nerve into a queer and bursting fullness”.Cloudstreet the ambivalence between spiritual and material pneuma registers as an opposition of two discourses, exemplified when the Lambs take Fish to a doctor, or “the quack” . Pike begins to play after waking from a dream, where “I’m hanging limp in a faint green light while all the heat ebbs from my chest and the life begins to leach out of me” (7). This drowning moment is interrupted by “someone at the surface, swimming down” (8). The impression that they will “drag me clear, blow air into me hot as blood” is dispelled when he recognizes his own face on the would-be saviour: “my own mouth opens. A chain of shining bubbles leaks forth but I do not understand” (8). The blown air, “hot as blood”, echoes references to the “hot breath” of Luther Fox and Georgie Jutland in their respective efforts to aspirate each other at the end of Dirt Music, and the problematically (un)named “blackfella” of Cloudstreet (208), to insist that connections to the land must be routed through Indigenous cultures. Quick’s encounter with the “blackfella” leads him back to the neighbourhood of Cloudstreet, which is just one of the reasons why Peter Mathews identifies the character as “a liminal prophet, corporeal but seeming to possess an occasional otherworldliness, to remind the inhabitants of Cloudstreet about the importance of that place to the two families” . The references to Wittenoom have prepared us to imagine that the immediate consequences of these mining claims (“someone gonna kick us off sooner later”) gesture to other, as yet unimagined, harms. In Breath the details of Eva’s death  by autoerotic asphyxiation have been covered over for reasons of shame: “I couldn’t say. I would risk setting off the rolling mass of trouble inside me. I choked it down. At quite some cost” (Winton It may be that Winton draws on this reference, like the characters Menzies and Axle in amilies” , 2. Afte” Winton , 236. IfAnoxic moments in Winton cohere into a novelistic infrastructure. It is an infrastructure that relies on a pneumatic materialism, wherein spirituality is sourced through a materialist understanding of how respiration affects human perception. Identifying pneumatic materialism in Winton’s work has wider implications than as a simple aesthetic response to his form: it develops a network of transversal approaches to Australia’s Global South. When the Black Lives Matter movement took the shocking, final, physiological confession of Eric Garner, “I can’t breathe”, as a rallying cry for Black oppression in the United States, it challenged the so-called Global North to interrogate what constitutes “breathable life”.


Clinicians may be less inclined to consider a diagnosis of cryptococcal meningitis in people without HIV infection or transplant-related immunosuppression. This may lead to a delay in diagnosis particularly if disseminated cryptococcal disease mimics cerebral septic emboli in injection drug use (IDU) leading to a search for endocarditis or other infectious sources. Though, IDU has been described as a potential risk for disseminated cryptococcal disease.We present two cases of cryptococcal meningitis in IDU without HIV or other obvious immune deficits. Both patients presented with at least 2 weeks of headache and blurred vision. They developed central nervous system (CNS) vasculitis, one of which mimicked septic cerebral emboli, but both resulted with poor neurologic outcomes.IDU likely induces an underappreciated immune deficit and is a risk factor for developing cryptococcal meningitis. This diagnosis, which can mimic cerebral septic emboli through involvement of a CNS vasculitis, should be considered in the setting of IDU. While physicians may be familiar with the presentation of cryptococcal meningitis in people living with HIV, clinicians may be less inclined to consider the diagnosis in other patient populations leading to a delay in diagnosis. Appreciation of non-HIV risk factors for cryptococcal disease may be helpful for physicians, with risk for cryptococcal disease described in organ transplant recipients, patients with hematologic malignancies, sarcoid, liver disease, or receiving immunosuppressive agents , 2. A feCryptococcus neoformans. Interestingly, initial CSF cultures from the prior LP remained without growth. He was treated with liposomal amphotericin and flucytosine and required serial lumber punctures with subsequent placement of a lumbar drain to manage his CSF pressures. CSF cultures cleared within 4 days of therapy, but due to lack of clinical improvement steroids were added after 10 days. He improved, completed 6 weeks of induction therapy with liposomal amphotericin and flucytosine, steroids were tapered and a VP shunt placed. He was discharged on fluconazole 400 mg daily for consolidation therapy. Outpatient, fluconazole was reduced to 200 mg daily for maintenance therapy 3 weeks later.A 26-year-old male with reported IDU of heroin presented with 2 weeks of headache, blurred vision, and confusion with nonsensical speech without fevers. Lumbar puncture revealed a CSF WBC count of 423 cells/μL, 64% lymphocytes, with RBC 6 cells/μL and a CSF glucose of 26 mg/dL (serum glucose of 139 mg/dL). HIV, hepatitis testing and serum cryptococcal Ag titer returned negative at that time , RBC 126 cells/μL with a CSF glucose of 14 mg/dL. Cryptococcal Ag remained positive at 1:160, but no organisms were isolated on these or multiple subsequent CSF cultures. He had his VP shunt removed and was again treated with 6 weeks of liposomal amphotericin and flucytosine with methylprednisolone added for 7 days followed by a steroid taper due to concern for a Post-Infectious Inflammatory Response Syndrome (PIIRS). CSF pressures were also managed by serial lumbar punctures and a ventricular drain followed by repeat VP shunt placement. Despite these efforts, he clinically deteriorated with worsening neurologic function and evidence of cerebral vasospasm of the right middle cerebral artery (MCA) and anterior cerebral artery (ACA) on transcranial doppler exam. A repeat MRI brain was obtained that revealed large bilateral subacute ACA territory infarcts and an infarct in the left MCA territory. After 6 weeks of treatment he was transitioned to consolidation therapy with fluconazole 800 mg daily but remains with profound neurologic deficits.A 30-year-old male with ongoing IDU and chronic HCV presented with 4 weeks of headache, blurry vision, hearing loss, and gait imbalance. The patient reported a couple of ED visits at an outside hospital the few weeks prior but was sent home each time. CT brain revealed a posterior fossa tumor versus abscess with hydrocephalus. A ventricular drain was placed to manage his hydrocephalus with CSF revealing WBC 18 cells/μL (51% segmented cells), RBC 195 cells/μL and a CSF glucose of 52 mg/dL in context of serum glucose of 119 mg/dL. Cultures were without growth. MRI brain was obtained which revealed multiple acute infarctions in cerebrum, brainstem, and cerebellum likely secondary to septic embolic, but also with basilar leptomeningitis (see Fig. Cryptococcal meningitis can be a diagnostic challenge as illustrated by our case series. We note in our cases serum cryptococcal Ag titers may be negative and ventricular CSF may not be diagnostic. The need for lumbar CSF, which may be discordant with ventricular CSF results, to diagnose basilar meningitis such as cryptococcus has been noted in prior case series . In our Clinicians should consider cryptococcal infection as a diagnostic consideration in IDU with cerebral disease due to the apparent increased risk of disseminated disease in this cohort. While meningitis caused by Candida or Mucor organisms seen in IDU might be due to direct bloodstream inoculation, the mechanism of infection is not entirely clear with cryptococcal meningitis but we are unaware of evidence to suggest a mechanism of infection aside from inhalation , 10. TheAn increased incidence and severity of all types of infections has long been noted in IDUs, suggesting impaired immunity . AlteratMore cases of cryptococcosis are expected in the IDU population with the ongoing opioid epidemic. We urge clinicians to consider a diagnosis of cryptococcal disease in IDUs, which may present as similarly to septic cerebral emboli due to CNS vasculitis or with other atypical manifestations. IDU appears to be a significant risk factor for development of disseminated cryptococcal infection.


The coronavirus disease 2019 (COVID‐19) pandemic has led to a public health crisis and global panic. This infectious disease is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Digital polymerase chain reaction (dPCR), which is an emerging nucleic acid amplification technology that allows absolute quantification of nucleic acids, plays an important role in the detection of SARS‐CoV‐2. In this review, we introduce the principle and advantages of dPCR, and review the applications of dPCR in the COVID‐19 pandemic, including detection of low copy number viruses, measurement of the viral load, preparation of reference materials, monitoring of virus concentration in the environment, detection of viral mutations, and evaluation of anti‐SARS‐CoV‐2 drugs. We also discuss the challenges of dPCR in clinical practice. The coronavirus disease 2019 (COVID‐19) pandemic has led to a public health crisis and global panic. Digital polymerase chain reaction (dPCR), which is an emerging nucleic acid amplification technology that allows absolute quantification of nucleic acids, plays an important role in the detection of the pathogen of COVID‐19. In this review, the authors introduce the principle and advantages of dPCR, review the applications of dPCR in the COVID‐19 pandemic, and discuss the challenges of dPCR in clinical practice. Viral nucleic acid testing is the main method for diagnosing COVID‐19, and reverse transcriptase quantitative polymerase chain reaction (RT‐qPCR) is the most widely employed technology in the clinic.Here, we introduce the principle and advantages of dPCR, review the applications of dPCR in COVID‐19 pandemic, and discuss the challenges of dPCR in COVID‐19 pandemic and relevant clinical applications.22.1PCR is a molecular technique for exponential amplification of a specific segment of DNA, which was developed by Kary Mullis in the 1980s.Taking the SARS‐CoV‐2 detection based on droplet‐based dPCR as an example, we show the workflow of dPCR in Figure 2.2Absolute quantification: In qPCR, the fluorescence of the amplified target molecules is monitored after each amplification cycle. The copy number of target molecules is quantified by mapping the relation between the cycle threshold (Ct) of the fluorescence amplification curve with a standard curve generated from a reference material. The amplification curves are usually vulnerable to inadequate amplification efficiency and sample quality, inhibitors, and system error. In contrast, dPCR divides the PCR reaction mixture into tens of thousands or even millions of reaction units, and the fluorescence of each unit is detected at the end of amplification. The copy number of target molecules is calculated by the fraction of the reaction units containing the target molecules, according to the Poisson distribution. Consequently, dPCR is less sensitive to inhibitors than qPCR, and copy number calculation does not rely on reference materials. Therefore, dPCR is eligible to provide more reliable and accurate quantification results than qPCR. For instance, Hindson et al.Higher sensitivity: As aforementioned, dPCR partitions the reaction mixture into tens of thousands of subreactions. Therefore, the number of nontarget DNA is decreased by a few orders of magnitude in each subreaction that contains the targets . As a result, the nonspecific interference exerted on targets decreases by several orders of magnitude, which benefits the valid amplification of the targets. Hence, dPCR is equipped with high sensitivity and is able to precisely quantify rare targets. For example, in the detection of rare BRAF V600E mutation, dPCR and qPCR could detect 0.001% and 1% mutant fraction, respectively.Better stability: Discretization also separates the amplification inhibitors in the reaction mixture into many individual reaction units, which reduces the total number of inhibitors in each unit and thus improves the resistance to inhibitors. In the detection of human cytomegalovirus, dPCR showed better inhibitor tolerance than qPCR. Moreover, even though inhibitors may influence PCR amplification in a dPCR assay, the affected subreactions can still be correctly identified because their fluorescence intensity is still significantly higher than background fluorescence.Higher precision: qPCR can routinely resolve a twofold difference in copy number in a single assay because the template doubles at each cycle and the fluorescence intensity is twofold higher after a cycle. In dPCR, it is theoretically possible to detect a difference in one target molecule through discretizing the PCR reaction mixture, and increasing the number of the partitions can improve the precision. For example, in a study of quantitative detection of human cytomegalovirus, the coefficient of variation (CV) of dPCR was lower than qPCR: the CV was fourfold and 1.5‐fold lower for dPCR compared with qPCR at the viral copy number of 10,000 copies/mL and 1000 copies/mL, respectively.33.1Although RT‐qPCR is currently the gold standard for the diagnosis of COVID‐19, a high false‐negative rate of RT‐qPCR tests for SARS‐CoV‐2 has been reported.3.2Many types of clinical samples can be used in COVID‐19 testing, including nasal swabs, throat swabs, sputum, blood, urine, stool, and bronchoalveolar lavage fluid. Selecting an appropriate type of sample is crucial for accurate clinical diagnosis. To increase the positive detection rate of COVID‐19 testing, it is necessary to evaluate the viral load of each type of sample and choose the sample type with the highest viral load when possible. As dPCR allows absolute quantification, it plays an important role in the quantification of viral loads in clinical samples of COVID‐19 patients. For example, Yu et al.3.3Safe handling of samples from suspected COVID‐19 cases is critical to protect medical laboratory personnel from infection. Inactivation of the virus is performed by most laboratories to ensure that the samples are safe to handle at a reduced biosafety level. However, the inactivation treatment may affect the quality of the sample and lead to false‐negative results. Therefore, it is necessary to understand the specific impact of sample pre‐treatment on the test results. Chen et al.3.4In clinical management and treatment, it is necessary to monitor the progress of the patient's illness. For most viruses, a higher viral load is associated with more severe illness. Therefore, it is important to study the relationship between viral load and the severity of COVID‐19. The high sensitivity and high precision of dPCR make it capable of monitoring changes in the viral load in patients. Yu et al.3.5Nucleic acid reference materials are used to evaluate the performance of detection methods and construct standard curves for RT‐qPCR. Reference materials also serve as a positive control for the detection of SARS‐CoV‐2 nucleic acid. dPCR enables absolute quantification of nucleic acids, thus it has been used to characterize nucleic acid reference materials. For example, van Kasteren et al.3.6Understanding the modes of transmission of SARS‐CoV‐2 and its distribution in the environment are critical to the formulation of disease prevention and control strategies. A detection method with high sensitivity and high tolerance to inhibitors is needed to accurately test samples collected in diverse environments. Liu et al.3.7△PRRA) in Vero‐E6‐propagated isolates, human organoids, experimentally infected hamsters, and COVID‐19 patients. They found that SARS‐CoV‐2△PRRA naturally existed in COVID‐19 patients and was transmissible. However, these variants only appeared with very low frequency in COVID‐19 patients, indicating that the wild type with the PRRA insertion was more competitive than SARS‐CoV‐2△PRRA. This study demonstrated that dPCR is suitable for the detection of known SARS‐CoV‐2 mutations, especially for some low‐frequency mutations.RNA viruses have high mutation rates, which are correlated with virulence and evolvability. Viral mutation research is essential for evaluating viral drug resistance and understanding the mechanisms related to immune escape and pathogenesis. Viral mutations can be identified by next‐generation sequencing, but the detection of known genetic mutations in laboratory samples and clinical samples requires a more sensitive technology. Wong et al.3.8Ct value to indicate the change in the viral load. Since dPCR allows quantification without a standard curve, and it is highly precise, the authors believe that dPCR is more suitable for evaluating the effectiveness of anti‐SARS‐CoV‐2 drugs than RT‐qPCR.A reduction in the viral load is an important indicator of the effectiveness of antiviral drugs. For example, in a study of remdesivir use in adult patients with severe COVID‐19, Wang et al.4As an emerging technology for absolute quantification of target nucleic acids, dPCR is highly sensitive and specific to low‐abundance DNA and highly resistant to amplification inhibitors. Thus, dPCR can be used for detection of low copy number viruses, measurement of the viral load, preparation of reference materials, monitoring of virus concentration in the environment, detection of viral mutations, and evaluation of anti‐SARS‐CoV‐2 drugs.Although the advantages and clinical application prospects of dPCR have been demonstrated in many studies, some challenges need to be overcome so that dPCR can be widely used in different types of medical units, including primary hospitals and epidemic areas: (1) the sample throughput of the available dPCR systems needs to be improved to fully meet the requirement of mass screening for COVID‐19; (2) a highly automated digital PCR system needs to be developed to simplify the operation process of dPCR and reduce hands‐on time; (3) the cost of dPCR instruments and reagents needs to be reduced; (4) clinical laboratory standards and guidelines of dPCR are required to assure the quality of the results; (5) multicenter evaluation is required to test the performance of dPCR‐based SARS‐CoV‐2 nucleic acid detection kits, and the detection kits need to be approved by regulatory agencies such as China's National Medical Products Administration (NMPA) and the United States Food and Drug Administration (FDA). With the joint efforts of researchers and the industry, dPCR can be widely used in laboratories or hospitals in the near future to assist scientific research and solve clinical problems.The authors declare no conflict of interest.


Inflammatory demyelinating disorders of the central nervous system are debilitating conditions of the young adult, here we focus on multiple sclerosis (MS) and neuro-Behçet disease (NBD). MS is an autoimmune disorder of the central nervous system. NBD, a neurological manifestation of an idiopathic chronic relapsing multisystem inflammatory disease, the behçet disease. The diagnosis of MS and NBD relies on clinical symptoms, magnetic resonance imaging and laboratory tests. At first onset, clinical and imaging similarities between the two disorders may occur, making differential diagnosis challenging and delaying appropriate management. Aiming to identify additional discriminating biomarker patterns, we measured and compared gene expression of a broad panel of selected genes in blood and cerebrospinal fluid (CSF) cells of patients suffering from NBD, MS and non inflammatory neurological disorders (NIND). To reach this aim, bivariate and multivariate analysis were applied. The Principal Analysis Component (PCA) highlighted distinct profiles between NBD, MS, and controls. Transcription factors foxp3 in the blood along with IL-4, IL-10, and IL-17 expressions were the parameters that are the main contributor to the segregation between MS and NBD clustering. Moreover, parameters related to cellular activation and inflammatory cytokines within the CSF clearly differentiate between the two inflammatory diseases and the controls. We proceeded to ROC analysis in order to identify the most distinctive parameters between both inflammatory neurological disorders. The latter analysis suggested that IL-17, CD73 in the blood as well as IL-1β and IL-10 in the CSF were the most discriminating parameters between MS and NBD. We conclude that combined multi-dimensional analysis in blood and CSF suggests distinct mechanisms governing the pathophysiology of these two neuro-inflammatory disorders. Multiple sclerosis (MS) and neurological Behçet disease (NBD) are two inflammatory recurrent diseases affecting the central nervous system (CNS). MS is defined as an immune-mediated neuroinflammatory neurodegenerative disorder of the CNS, characterized by pleocytosis and T-cell infiltration in the cerebrospinal fluid (CSF) . NBD, meDespite these similarities, these two pathologies exhibited different cranial MRI lesions. Indeed, NBD patients present lesions in the brainstem extending to the diencephalic structure and basal ganglia region, while MS patients present lesions mainly localized in periventricular areas . Serum aHowever, there are overlaps in the clinical feature characteristics of these two diseases, particularly at the early stages. Therefore, this may result in a misleading diagnosis on the basis of clinical and radiological criteria. Due to the crucial role of the immunological response in the pathophysiology of these two diseases affecting the CNS, a better characterization of cellular markers would be of considerable clinical relevance and may help guide clinicians in choosing the appropriate treatment.So far, comparative immunological studies between NBD and the relapsing-remitting (RR) form of MS (RRMS) have shown commonalities and differences in profiles at the immunological level. In the context of the Th1 response pathway, the predominantly secreted cytokines are mainly IFN-γ, IL-2, TNF-α, and IL-12 . A compaTh2 and T regulatory cells’ implication in the CSF of neurological disorders have been poorly investigated. The cytokine signature produced by Th2 cells are IL-4, IL-5, IL-13, and GATA3, which is the major regulator of Th2 differentiation. Studies on Th2 populations in the CSF have shown comparable expressions of GATA-3 and IL-4 between NBD, MS, and the control group . The immThe previous investigations of cellular markers associated with these two disorders were mainly focused on one or two features. In our study, we aimed to analyze simultaneously multiple parameters that would better distinguish the two CNS disorders at the first stage. The expression levels of both blood and CSF genes related to the Th1, Th2, Th17, and Treg populations have been compared, using multivariate and univariate statistical analysis. Our findings expand the current understanding of the immune process mediating the two neurological disorders  and emphasize new potential cellular biomarkers that would enable a more accurate and specific diagnosis.Patients with neurological diseases were enrolled from the Neurology department of Charles Nicolle hospital and the National Institute of neurology Mongi Ben Hamida  from 2017 to 2020. For this study, all recruited patients were sampled during the first clinical episode of acute disease prior to any immunomodulatory treatment. After a period of clinical follow up to validate the final diagnosis, 66 subjects were included, and they were selected according to clinical examination, magnetic resonance imaging (MRI), and CSF laboratory data . A total of 21 MS patients had RR form and fulfilled the revised McDonald Criteria . All MS Peripheral blood mononuclear cells (PBMCs) were purified from blood patients by Ficoll technique and stored in Trizol reagent. CSF cells were also resuspended in an RLT buffer supplemented with 2% of beta mercapto-ethanol. Cells from blood and CSF were stored at −80°C for RNA extraction using an RNA isolation kit (Qiagen) under RNase-free conditions, according to the manufacturer’s protocol. The quality and quantity of RNA were assessed using agarose gel electrophoresis and NanoDrop analysis. RNase-free DNase I treated RNA (Qiagen) was reverse transcribed using High-capacity cDNA reverse transcription kit (Applied Biosystems). This reaction was performed with the following parameters: 1 μg RNA for 25°C for 10 min, 37°C for 2 h, and 85°C for 5 min. Quantitative real-time PCR was carried out using the Applied Biosystems ABI PRIZM 7500 Real-Time PCR System. The cycling protocol was set as follows: 15 s at 95°C and 1 min at 60°C for 40 cycles. Each 20 μl reaction contained 10 μL of 2× Sybr Green, 0.5 μL of each primer , and 5 μL of cDNA. Relative quantification of mRNA levels was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous reference. Samples were run in duplicate in three independent experiments. In this study, we used targeting genes encoding the Th1 , Th2 , Th17 , inflammation , T reg , and the purinergic axis . HPLC-purified oligonucleotides primer sequences are reported in T-tests for continuous variables.Demographic characteristics of patients and controls were compared with contingency tables for categorical variables and non-parametric Z-score normalization and centering by mean) prior to principal component analysis (PCA)  . Scaled is (PCA) . The hieis (PCA) . The preis (PCA) . Then, aWe compared PBMCs and CSF cells’ mRNA expression data of patients with the RR form of MS disease and NBD with the parenchymal syndrome and a control group including subjects with non-inflammatory neurological diseases (NIND).p = 0.4121). As expected, MS was more prevalent for women (17:4 vs 9:13 in NBD). RRMS patients showed an increased proportion of intrathecal immunoglobulin (Ig) synthesis , and more frequently the presence of oligoclonal bands (OCBs) compared to NBD and NIND . There was no significant difference in the blood-brain barrier (BBB) disruption among the three groups as demonstrated by the CSF to albumin ratio . The same expression trend was unexpectedly noticed for IL-1β in the blood (Box 3). In counterpart, genes related to inflammation, namely INFγ, TNF-α, and IL-6, were down-regulated in NIND PBMCs (Box 2). Moreover, gene expressions of variables associated with cellular activation  as well as variables associated with the purinergic system  were down-regulated in NIND CSF (Box 2). This differential gene expression observed in the NIND group shows a decrease of gene expression linked to inflammation and lymphocyte differentiation, in line with an absence of inflammatory damage of the CNS in this control group.As expected, RRMS patients showed increased expression of TNF-α, IL-1β, and intrathecal IgG synthesis in the CSF as compared to the other groups. Moreover, this group displayed activation of the Th1/Th2 and Th17 axes in the CSF. Interestingly, the RRMS expression profile showed a lower-expression of regulatory parameters  in the two compartments (Box 5).In accordance with the systemic nature of Behçet disease, the NBD group demonstrated an exacerbated immune response in the blood. This is illustrated by an increased expression of inflammatory markers (IL-6 and TNF-α), effector related genes , and the regulatory parameters  (Box 4). In the CSF, we distinguished an up-regulation of well-characterized immune factors IL-6, IL-10, CD39, and A2A. However, Foxp3 represents the most down-regulated parameter in NBD CSF (Box 4 and 2).p = 0.0034) and NIND (p = 0.0049) , CD39 , and A2A (NBD vs RRMS: p < 0.0001 NBD vs NIND: p < 0.0001)  . Similar 0.0001) . Further 0.0001) .Having described the differential expression levels, we next investigated the interdependence of the studied genes . We founAnother significant module was visualized in both CSF correlograms of RRMS and NIND groups. This module involved regulatory markers forming a positive correlation with inflammation markers IL-6 and IL-17 in RRMS and NIND groups, respectively . InteresOtherwise, the correlograms emphasized specific correlation modules for either NIND or RRMS. More precisely, we observed a positive correlation between genes related to Th1, Th17, and IL-1β immune axis in CSF of RRMS patients . RegardiWe used the area under the curve (AUC) of ROC analysis aiming to evaluate the discriminatory strength of CSF and blood parameters between the groups of diseases. Indeed, the AUC in a ROC analysis can be used to measure the quality of a diagnostic test ranging from acceptable (AUC 0.7–0.8) through to excellent (AUC 0.8–0.9) and outstanding (AUC > 0.9).The performance of each predictor in discriminating each pair of disease groups was assessed by measuring the AUC . We firsThe most relevant finding was the outperformance of blood parameters compared to those expressed in CSF to discriminate between NBD and RRMS patients . These pThe overlapping symptoms and MRI findings between MS and NBD renders diagnosis difficult at the early stage of the disease. Here, we investigated the expression genes related to the major immune axes in blood and CSF compartments of treatment-naive patients suffering from RRMS and NBD. We first performed a multivariate analysis using combined blood and CSF parameters; we observed three distinct clusters corresponding to the three studied groups.These clusters are not identified when blood PCA analysis alone is performed. Interestingly, individual CSF PCA analysis distinguishes patients from both RRMS and NBD from the NIND control group, but not from each other. Our results highlight the preponderant role, at the expression level, of IL-17 and regulatory markers IL-4, IL-10, and Foxp3 in the blood as the major contributors to the segregation of the two studied neurological disorders. In addition, by further exploring the physiopathological signaling events, we observed a fine-tuning of T-cell activation localized in the CSF of RRMS patients, in contrast to a more generalized inflammatory response in the blood and CSF of NBD patients. Finally, comparisons with the control group showed that RRMS and NBD patients are characterized by the activation of Th1 and Th17 effectors cells and the elevation of A2A receptor in the CSF compartment.In line with previous reports, NBD patients present an enhanced expression of T-bet (Th1) and RoR-γt (Th17) and a low expression of GATA 3 (Th2) . The secMore interestingly, the enhanced expression of A2A observed in the CSF of the two neuro-inflammatory diseases is consistent with previous reports that described the up-regulation of A2A on T cells, macrophages, and microglia within the inflamed CNS tissue . In addiThe pathogenesis of NBD is poorly understood and the differentiation from other neuro-inflammatory diseases based on clinical and cellular markers remains challenging at the early stage of the disease. We herein identified, for the first time, the best predictors of NBD in both PBMCs and CSF compartments. Specific gene expression indicates elevated IL-6, IL-10, CD39, and A2A in the CSF and an exacerbated immune response in the blood of NBD compared to MS and NIND. These results are in accordance with data previously reported showing increased T cell activation and inflammation in the blood of patients suffering from various manifestations of Behçet disease, expressing higher levels of IL-1β in synovial fluid compared to that in osteoarthritis patients . FurtherThe most differentially expressed transcripts in the CSF between MS and controls are, as expected, transcripts related to immunoglobulins as previously suggested . The CSFin situ lesions (The AUC of ROC analysis identified IL-17 and CD73 expressions in PBMCs as the strongest discriminant parameters, distinguishing RRMS from NBD with high accuracy. In the blood of Behçet disease patients, an elevated level of IL-17 was described more frequently in those developing neurological involvement and is considered as a marker of disease progression . However lesions . We spec lesions  whilst n lesions .In this study, we also focused on the purigenic mechanism which implicates the two ecto-enzymes CD39 and CD73. In the blood compartiment, we showed that, although up-regulated in NBD, the CD73 was significantly decreased in MS patients and correlates with Foxp3 . In lineInterestingly, the most discriminatory and outstanding CSF predictor between these two diseases is the IL-1β. This potentially argues in favor of the infiltration of inflammatory monocytes into the CNS of MS patients, since IL-1β promotes their differentiation into antigen-presenting cells to activate CD4 reactive cells . In agreThe multivariate approach on a set of key genes, used in the current study, allowed for the most comprehensive insights into expression profiles in CSF and blood of MS, NBD, and NIND. During the acute phase of the first onset of the diseases, the CSF of these two neuro-immunological disorders shows an activation of Th1, Th17, and the purinergic axes CD39/CD73. Interestingly, our model predicted blood IL-17 and CD73 expression as discriminative markers between those two pathologies.Nevertheless, we admit that this study is subject to certain limitations; indeed, the number of studied parameters could be further extended to better assess the potential role of other diagnostic markers for differentiating MS and NBD. We also acknowledge that a higher number of patients would increase the statistical power of this study. Our priority was to include patients based on strict selection criteria used during patients’ recruitment.In conclusion, the most striking finding in this study is the identification of genes that contribute to the pathophysiology of MS and NBD. We differentially characterize patients with NBD from those with MS and the controls. These clusters combined both genes expressed in blood and CSF. These data could pave the way to validation of specific mRNA signatures which could help establish differential diagnosis at early stages of the disease.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of Pasteur Institut of Tunisia. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The reviewer KH and the authors OM, KB, SB, SBS, M-RB, OS, and MB are not members of the same institution, but both their institutions are affiliated to the same university. The reviewer KH declared this shared affiliation with authors OM, KB, SB, SBS, M-RB, OS, and MB to the handling editor at time of review. The remaining 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.


Dear Editor,,,We read with interest recent articles in this journal regarding the utility of next-generation sequencing for the diagnosis bacterial meningitis.Streptococcus pneumoniae and S. suis PCRs were all negative. He was diagnosed with bacterial meningitis, and given a combination of ceftriaxone (2 g/12 h) and dexamethasone (0.4 mg/kg/12 h). His clinical condition steadily improved. His second and third CSF samples became negative by Gram stain. The other CSF parameters also improved, except the glucose, which remained low . Given this new diagnostic result of the admission CSF and because the patient had recovered clinically, the patient was given 24 million units of penicillin G for every 4 h. After day 43 of hospitalization, all CSF parameters had normalised , with a body temperature of 40 °C, a blood pressure of 140/80 mmHg and neck stiffness. Initial Gram-stain and microscopy of CSF showed Gram-positive cocci, 8449 white cells/uL with 91% neutrophils, elevated protein and low glucose level, and high lactate concentration A. Routinined low A. On dayined low B. After rmalised A. Likewirmalised C. The paS. agalactiae ), likely attributed to a combination of the high level of sequence similarities of the 16S rRNA region between them and the sequencing errors introduced by the MinION systems. Analysis of sequencing data generated during the 25, 50 and 75 min of sequencing run time also yielded the same results . Details about the MinION procedure are presented in Supplementary Materials.Additionally, MinION sequencing of complete 16S rRNA gene was retrospectively carried out on the extracted nucleic acid of the admission CSF yielded a total of 14,848 reads after 100 min of sequencing run. Of these, 11,556 reads (79%) were successfully aligned to alactiae D. The reS. pneumoniae and S. suis) or genus (Neisseria) found in the CSF samples by diagnostic work up of the clinical study  caused by S. agalactiae has been reported with increased frequency in recent years,S. agalactiae should be considered as an important differential diagnosis for patients presenting with acute CNS infections in Vietnam.In addition to CSF samples described in the present study and a recent pilot study from Korea,Owing to the unavailability of the reagents at the time of patient admission, we were not able to perform real-time diagnosis using MinION sequencing on the collected CSF samples. However, same day diagnosis is theoretically achievable, because the current workflow takes 5 – 6 h to operate. Prospective study is urgently needed to assess its translational potential in the diagnosis of bacterial meningitis.Since September 2017, a prospective observational study aiming at exploring the utility potential of next-generation sequencing in patients presenting with central nervous system (CNS) infections has been conducted in the brain infection ward of the Hospital for Tropical Diseases (HTD) in Ho Chi Minh City, Vietnam. HTD is a tertiary referral hospital for patients with infectious diseases from southern provinces of Vietnam, serving a population of >40 million. Any patient (≥16 years) with an indication for lumbar puncture was eligible for enrolment. Patient was excluded if no written informed consent was obtained. As per the study protocol, CSF, plasma and urine samples were collected at presentation alongside demographic, meta-clinical data and results of routine diagnosis. After collection, all clinical specimens were stored at −80 °C until analysis.The clinical study received approvals from the Institutional Review Board of the HTD and the Oxford Tropical Research Ethics Committee of the University of Oxford. Written informed consent was obtained from each study participant or relative (if the patient was unconsciousness).https://github.com/rrwick/Porechop). Determination of bacterial genus/species composition in the obtained reads was then carried out using Epi2Me interface , a platform for cloud-based analysis of MinION data. Overall, the whole procedure of MinION sequencing of 16S rRNA gene takes 5–6 h to complete .Sequencing of complete 16S rRNA gene was retrospectively performed using MinION Nanopore sequencer (ONT), following the manufacturer's instructions. In brief, amplification of the complete 16S rRNA gene and library preparation were carried out on extracted nucleic acid using 16S Barcoding Kit  and primers (27F 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R 5′-GGTTACCTTGTTACGACTT-3′), followed by the sequencing of the amplified product using R9.4 Flow cells (ONT). MinION reads were first basecalled using Albacore v2.1.7 (ONT), followed by demultiplexing using Porechop .


Diabetic nephropathy is a common and serious complication of diabetes mellitus (DM) and is one of the leading causes of end-stage renal disease worldwide. Although there have been many investigations on biomarkers for DN, there is no consistent conclusion about reliable biomarkers. The purpose of this study was to perform a systematic review and meta-analysis of the role of circulating retinol-binding protein 4 (RBP4) in the type 2 diabetes mellitus (T2DM) patients with kidney diseases.  We searched the PubMed, MEDLINE, EMBASE, and Web of Science databases for publications. For the 12 cross-sectional studies that we included in the review, we calculated standard mean differences (SMD) with 95% confidence intervals (CI) for continuous data when the applied scales were different. Risk of bias of included trials was assessed by using the Newcastle-Ottawa Scale. P = 0.001, SMD 1.07, 95% CI ]. The estimated glomerular filtration rate (eGFR) was negatively associated with circulating RBP4 concentrations in patients with T2DM . The albumin-to-creatinine ratio (ACR) was positively associated with circulating RBP4 concentrations in patients with T2DM .  RBP4 concentrations in the micro-, macro-, or micro+macroalbuminuria groups were significantly higher than those in the normal albuminuria group of T2DM patients [ The levels of circulating RBP4 were significantly higher both in T2DM subjects with micro/macroalbuminuria and in T2DM subjects with declined eGFR. The levels of circulating RBP4 were positively correlated with ACR but negatively correlated with eGFR. Circulating RBP4 could be a reliable biomarker for kidney diseases in T2DM. Diabetes mellitus (DM) affects more than 463 million people globally, and this number is supposed to increase to 700 million by 2045 . DiabetiRBP4 gene can elevate insulin sensitivity  and showed significant heterogeneity ] ] . However, there was no significant difference in the RBP4 levels between the normal albuminuria DM group and the non-DM group.Six studies , 30, 32,, 1.25)] . Four st, 1.25)] . CompareP = 0.0009, SMD 2.14, 95% CI ].Four trials, including 490 participants, reported the circulating RBP4 concentrations in the DM with CKD and DM without CKD groups , 27, 29.To explore the relationship between circulating RBP4 concentration and kidney diseases, we performed a correlation analysis between RBP4 and eGFR/ACR.n = 1254; Z = −0.48, 95% CI , P < 0.0001]. The summary r was -0.45, indicating moderate correlation.Seven trials –29, 32 hn = 2072; Z = 0.20, 95% CI , P = 0.001]. The summary r was 0.20, indicating low correlation.Five trials , 29, 31 P values of all factors in the analysis were greater than 0.05, indicating the absence of publication bias.Egger's regression and Begg's rank correlation analysis were performed to evaluate publication bias . The P vI2 of heterogeneity was reduced to 29% in the comparison of RBP4 concentrations in the macroalbuminuria and normal control groups . Thus, eGFR is another independent determinant for elevated circulating RBP4 levels in diabetic patients.RBP4 levels have been found to be significantly increased in a T2DM group with CKD and low eGFR compared with the non-CKD group of subjects with T2DM in this article. Levels of adipocytokines, such as adiponectin and leptin, were elevated in renal failure –37. SimiFurthermore, a strong correlation was reported between eGFR/ACR and circulating RBP4 concentrations in subjects with T2DM in our article. This is consistent with the results of the meta-analysis by Park et al.  in whichP = 0.001). However, this relationship between serum and urinary RPB4 concentrations needs to be investigated further. The present meta-analysis revealed that circulating RBP4 levels were associated with renal dysfunction related to DM, which should be further investigated experimentally.Albuminuria and renal dysfunction are the most common clinical manifestations of DN in patients with T2DM. Our meta-analysis revealed that circulating RBP4 levels were elevated only in patients with diabetic kidney diseases, rather than in simple diabetes subjects without DN. There may be two reasons to explain these differences in circulating RBP4 levels in diabetic subjects with and without kidney diseases. First, hepatocytes and adipocytes are important sites of synthesis of RBP4, whereas the kidneys are important sites of catabolism of circulating RBP4 . MaintenMoreover, many researchers have explored the role of RBP4 in DM and other diseases. Li et al.  reportedThis meta-analysis is the first to present the role of circulating RBP4 in kidney diseases in subjects with T2DM. Most of the trials included in the analysis were of high quality. Moreover, there was no publication bias in any of the comparisons. However, there were some limitations of our study. Firstly, the sample size was small, and some critical data had not been presented in the publications. For example, only one study performed ROC analysis of prediction for eGFR  and anotIn summary, the levels of circulating RBP4 were significantly higher both in T2DM subjects with micro/macroalbuminuria and in T2DM subjects with declined eGFR. The levels of circulating RBP4 were positively correlated with ACR but negatively correlated with eGFR. Circulating RBP4 could be a reliable biomarker for kidney diseases in T2DM.


Pelvic obliquity (PO) is common in individuals with cerebral palsy (CP). The prevalence of PO in a population of children with CP and its associations with scoliosis, hip displacement and asymmetric range of hip abduction were analysed.Over a 5-year period all pelvic radiographs from the Swedish surveillance programme for CP and the recorded data for scoliosis and hip abduction in children from southern Sweden at Gross Motor Function Classification System (GMFCS) levels II–V were analysed. PO and hip displacement calculated as migration percentage (MP) were measured on supine pelvic radiographs and compared with presence of scoliosis and side difference in hip abduction. Statistical analyses comprised chi-squared and binominal testing.P = 0.025), while combined supra- and infrapelvic association was more common in GMFCS level V . Isolated infrapelvic or no association was seen in 74% of children ≤10 years of age while 76% of children ≥11 years old had suprapelvic or combined supra- and infrapelvic association with PO (P < 0.001).In total, 337 children were analysed, of whom 79 had a PO of ≥5°. The proportion of children with PO increased from 16% in GMFCS level II to 34% in level V. Scoliosis combined with PO was reported in 38 children, of whom 34  had the convexity opposite to the high side of the PO. Asymmetric abduction with PO was reported in 45 children, of whom 40  had reduced abduction on the high side of the PO. Asymmetric MP of ≥5% with PO was seen in 62 children, of whom 50 had higher MP on the high side of the PO . Children in GMFCS levels II–IV more often had isolated infra-pelvic association with PO (47% versus 19% in GMFCS V, There was a strong association between the high side of the PO and the side of scoliosis, highest MP, and lowest range of abduction when PO was measured in supine position. PO was more often associated with infrapelvic factors. PO was seen in young children indicating the need for early awareness of this complication. Pelvic obliquity (PO), a common deformity in individuals with cerebral palsy (CP), is defined as an asymmetry of the pelvis in the frontal plane and can be measured clinically or radiographically in standing, sitting or lying positions. PO can result in pain, pressure ulcers and difficulties in maintaining sitting and standing postures with reduced functional abilities , 2. The The Swedish CP Follow-Up Program (CPUP) is a surveillance programme, which monitors more than 95% of the total population of children with CP in Sweden . CPUP inThe primary aim of this study was to analyse the prevalence of PO in a population of children in a hip surveillance programme at different ages and and gross motor function levels. The second aim was to analyse the associations between PO, scoliosis, hip displacement, and range of hip abduction.In CPUP, the diagnosis of CP is verified by a neuropaediatrician after the child turns 4 years old. Gross motor function is classified by the child’s physiotherapist according to the expanded and revised Gross Motor Function Classification System (GMFCS-E&R) . GMFCS-EIn this cross-sectional study, all children in GMFCS levels II-V up to 18 years of age in southern Sweden  who had a radiographic hip examination performed during a 5-year period from 1 July 2014 to 31 June 2019 were analysed. Children in GMFCS level I were not included due to limited number of participants. For children who underwent repeated radiographic examinations during the study period, the first examination was used. Children treated by varus osteotomy of the proximal femur and/or scoliosis surgery before the first examination were excluded. The PO was measured with a line joining the maximum prominence of the ischial tuberosities or through the inferior aspect of both triradiate cartilages (Hilgenreiner’s line) and the horizontal reference frame of the radiograph  and the association between the high side of the PO and the convexity of the scoliosis was analysed. In children with S-shaped scoliosis, the direction of the lower curve was recorded. PO was finally calculated related to the difference in hip abduction and MP between the hips on the high and low sides of the PO (= infra-pelvic association). The children with PO were analysed by two-sided binomial tests  with theThe study was approved by the Medical Research Ethics Committee at Lund University (LU-443-99).During the 5-year study period, 385 individuals  were examined radiographically. Of these, 38 children  had been treated by varus osteotomy, 9  for scoliosis and 1 girl had undergone both varus osteotomy and spine surgery before the first examination. These 48 children were excluded from the study, leaving 337 individuals  remaining in the analysis. Their age at examination and GMFCS distribution are presented in Table In the study cohort of 337 children, the PO ranged between 0° and 18°. The left side was higher in 106 cases, the right in 171, and 60 radiographs showed no PO Fig. . The PO The number of children with PO increased with each GMFCS level. The proportion of children with PO ≥ 5° was 16% in level II, 17% in level III, 26% in level IV and 34% in level V , moderate (n = 30) or severe (n = 20). The number of children with PO ≥ 5° and mild scoliosis was 13 (29%), moderate scoliosis 13 (43%) and severe scoliosis 12 (60%). The curve convexity was at the opposite side to the high level of the PO in 9 of the 13 hips with mild scoliosis and in all 25 hips with moderate and severe scoliosis. The proportion of children with curve convexity opposite the high side of PO was 34/38 = 89% (95% CI 80–99%).Scoliosis was reported in 95 children, graded as mild .The MP was ≥5% lower on the high side of the PO in 50 children, ± 4% in 17 children and ≥ 5% higher on the low side of PO in 12 children. The proportion of children with ≥5% lower MP on the high side of PO was 50/62 = 81% (95% CI 71–90%).Isolated combination of supra-pelvic asymmetry and PO, i.e., scoliosis with the curve deviation to the low side of PO, without higher MP or lower range of abduction on the high side of PO, was seen in 2 children. Isolated combination of infra-pelvic asymmetry with PO, i.e., higher MP or lower range of abduction on the high side without scoliosis, was seen in 30 children. Both supra- and infra-pelvic asymmetry and PO was seen in 31 children and in 16 children neither supra- nor infra-pelvic asymmetry was seen Fig. .Fig. 5VP = 0.025), while combined supra- and infra-pelvic asymmetry was more common in GMFCS level V    and the most reduced range of hip abduction.Porter et al.  analysedThe higher frequency of PO seen in the children with lower levels of motor function is consistent with the known higher frequency of scoliosis, hip dislocation, windswept position and reduced range of hip motion in children at higher GMFCS levels –14.There was no clear age trend for PO. The children excluded because of surgery were usually > 6 years of age, which might bias the results. PO was also common among children < 5 years old, supporting the importance of early follow-up of these deformities . There wn = 39) than scoliosis (n = 10) indicating that the dominance of infra-pelvic association would have been even greater if these children had been included. The children are followed in a hip surveillance programme in which several have been treated with preventive adductor–psoas tenotomy, which also may have reduced the number of POs with infra-pelvic aetiology. The fact that more young children have infra-pelvic association with PO also indicates that PO is more often initiated by asymmetric hip abduction or asymmetric hip displacement than by scoliosis. This is supported by the longitudinal study reported by Letts et al. [PO was more often seen in combination with hip displacement or asymmetric range of abduction than in combination with scoliosis. There were more children excluded due to femoral varus osteotomy (s et al. , which sPO can cause major problems, and is often difficult to treat. PO uncovers the femoral head on the high side of the obliquity which might accelerate a hip displacement. PO in combination with unbalanced scoliosis causes an unbalanced sitting position with higher pressure on the low side of the obliquity with risk of pain and decubitus ulceration. It is therefore important that PO is prevented as far as possible by symmetrical and varied positions of lying and sitting from an early age , and thaThere are several limitations to this study. The cross-sectional design means that we cannot prove any causal relationship between PO and scoliosis and infra-pelvic asymmetries. Scoliosis was assessed by clinical examination. However, this standardized assessment has shown high sensitivity, inter-rater reliability and validity compared with radiographic Cobb angle measurement . The stuIn summary, PO measured in the supine position had a strong association with scoliosis, hip displacement and asymmetric range of hip abduction, in which the high side of the PO was usually opposite to the convex side of the scoliosis, and on the same side as the largest hip displacement and the lowest range of abduction. PO was more often associated with hip displacement and/or asymmetric range of hip abduction than with scoliosis, and this association was more commonly seen in younger children, indicating that PO most often has an infra-pelvic cause. The presence of PO should be recorded in hip surveillance programmes and as far as possible prevented.


Cerebrospinal fluid (CSF) analysis is important for detecting inflammation of the nervous system and the meninges, bleeding in the area of the subarachnoid space that may not be visualized by imaging, and the spread of malignant diseases to the CSF space. In the diagnosis and differential diagnosis of neurodegenerative diseases, the importance of CSF analysis is increasing. Measuring the opening pressure of CSF in idiopathic intracranial hypertension and at spinal tap in normal pressure hydrocephalus constitute diagnostic examination procedures with therapeutic benefits.The indications and contraindications must be checked before lumbar puncture (LP) is performed, and sampling CSF requires the consent of the patient.Puncture with an atraumatic needle is associated with a lower incidence of postpuncture discomfort. The frequency of postpuncture syndrome correlates inversely with age and body mass index, and it is more common in women and patients with a history of headache. The sharp needle is preferably used in older or obese patients, also in punctures expected to be difficult.In order to avoid repeating LP, a sufficient quantity of CSF (at least 10 ml) should be collected. The CSF sample and the serum sample taken at the same time should be sent to a specialized laboratory immediately so that the emergency and basic CSF analysis program can be carried out within 2 h.The indication for LP in anticoagulant therapy should always be decided on an individual basis. The risk of interrupting anticoagulant therapy must be weighed against the increased bleeding risk of LP with anticoagulant therapy.As a quality assurance measure in CSF analysis, it is recommended that all cytological, clinical-chemical, and microbiological findings are combined in an integrated summary report and evaluated by an expert in CSF analysis.Recommendations (most important 3-5 recommendations on a glimpse):https://www.awmf.org). /uploads/tx_szleitlinien/030-141l_S1_Lumbalpunktion_und_Liquordiagnostik_2019-08.pdf). The present article is an abridged translation of the above cited guideline. The guideline has been jointly edited by the DGLN and DGN.In view of the importance and developments in CSF analysis, the S1 guideline “Lumbar puncture and cerebrospinal fluid analysis” was recently prepared by the German Society for CSF analysis and clinical neurochemistry (DGLN) and published in German in accordance with the guidelines of the AWMF ( This guideline contains basic recommendations concerning practical procedures for CSF space puncture, in particular with regard to indications and possible contraindications, information and consent, selection of the puncture needle, procedure in patients treated with anticoagulants, and thrombocytic function inhibitors, sample collection, treatment, and analysis as well as for compiling findings. The long version deals in detail with individual clinical presentations which had to be shortened for reasons of space in the present guideline. The structure, table of contents, and basic features are presented here.The present article is an abridged translation of the guideline recently published online neuritis in acute or chronic form constitute core indications for lumbar puncture (LP) Table . CSF anaBefore electively collecting CSF, the presence of clinical CSF pressure signs must be ruled out. Cranial imaging  prior to LP is needed in special cases , but is not necessary in the absence of clinical signs of increased cerebral pressure. The removal of CSF in cases of increased cerebrospinal pressure can lead to an entrapment of neuronal structures due to axial displacement of the brain and may be fatal. Examination of the ocular fundus by ophtalmoscopy is less sensitive than cross-sectional imaging as a congestive papilla may be absent despite increased intracranial pressure. The presence of a congestive papilla in the case of idiopathic intracranial hypertension does not represent a contraindication for a relief puncture.A platelet count below 50,000/μL, a quick test below 50%, an INR of more than 1.8, and a clearly pathologically activated partial thromboplastin time (aPTT) are considered contraindications for LP. When in doubt, the platelet aggregation time can be determined by apparatus or the bleeding time can be clinically determined by a scratch test.Thrombopenia below 50,000/μL is a relative contraindication and thrombopenia below 10,000/μL an absolute contraindication. In the case of thrombocyte counts below 10,000/μL, thrombocytes should always be substituted prior to LP. In the range between 10,000 and 50,000/μL an increased complication rate is to be expected. The decision for thrombocyte substitution must be made individually.Therapeutically induced coagulation disorders should - if medically justifiable - be stopped before the procedure, and their effect should be eliminated by medication if necessary Table .Table 2Patients anticoagulated with Phenprocoumon or other coumarin derivates should be transitionally switched to heparin, as this can be antagonized more rapidly. At this point we would like to refer to the S1 guideline of the DEGAM  on the subject of bridging . For the use of NOAK  such as dabigatran, rivaroxaban, apixaban, and edoxaban for the prophylaxis or therapy of thromboembolic events no systematic studies are available. Initial recommendations  considerAndexanet alfa has already been approved and is available as an antidote for bleeding caused by Factor Xa inhibitors. Approval was granted in the first half of 2019 in Germany. If discontinuation of NOAK is associated with increased thromboembolic risk, conversion to heparin (bridging) is recommended , 111.A case of a bleeding complication after LP under a double platelet inhibition with ASS  and clopidogrel has been reported . SystemaASS does not need to be discontinued for LP.Both superficial and deep inflammation of the skin or subcutis, but also inflammation of the muscle in the area of the puncture site represent a contraindication for LP.Here, the risk of the intervention must be weighed against the potential benefit.In emergency situations , which cannot be delayed, the LP can also be carried out without a declaration of consent from patients who are unable to consent. It is recommended to document this consideration in written form.The benefit of the diagnostic measure must be weighed against the additional risk of inducing premature labor. In cases of idiopathic intracranial hypertension (IIH), relief punctures with reduction in visual acuity are among the therapeutic options available, even during pregnancy , 166.Outside the scope of individual case decisions , a patient who is capable of giving consent or the legal representative of a patient who is not capable of giving consent is required for LP. The information should always be provided in written form and patients should be given sufficient time for reflection. The procedure differs depending on the indication for the puncture and is also dependent on the patient’s level of consciousness. If an appropriate reflection period cannot be adhered to for clinical reasons, this must be noted separately. In this case, the physician performing the procedure must also document the indication. The patient can waive a further reflection period in written form.Information about risk and benefit.Adverse consequences if LP is not carried out, depending on the respective suspected diagnosis.Identification of alternative diagnostic methods.Procedure of the examination.Possibility of local anesthesia. If a local anesthetic is used, possible hypersensitivity reactions must always be pointed out.Explanation of the technical aspects of the puncture:Indications of possible adverse effects.The information for the patient should include the following:It should also be pointed out that patients may need to be hospitalized and the inpatient stay extended if side effects develop. In exceptional cases it may be necessary to perform a second puncture (with blood patch); in very rare cases, surgical measures may be necessary to treat complications .Possible occurrence of a centrally caused circulatory or respiratory disorderPossible occurrence of suboccipital hemorrhage with atypical course of an arterial vessel Information on suboccipital puncture should include the alternative of other puncture routesIf a suboccipital puncture is to be performed, additional reference to this should be made:For purposes of clarification, ready-made information sheets are commercially available.The LP can be performed in an outpatient or inpatient setting after the patient has been informed in detail about benefits, procedures, and risks and after the patient has provided documented consent. In addition to the severity of the clinical presentation, patient-related factors such as age, weight, comorbidities, and coagulation status; organizational aspects such as the availability of the examination procedure; further CSF analysis; and the patient’s wishes also play a role in deciding whether the LP should be performed in an outpatient or inpatient setting. In advance, it must be checked whether special precautions must be taken to ensure that the CSF is properly processed, e.g., information about the laboratory or the laboratory courier in order to guarantee a prompt cell count or cytological processing. Preanalytics also play a role, for example, when CSF samples need to be freshly stained for microscopy. The container in which the CSF is collected and stored also affects the results: Proteins that tend to form aggregates, for example, amyloid-β1-42, are particularly highly absorbed by certain tube materials such as glass or polystyrene, resulting in false-positive results in Alzheimer’s disease diagnostics ) Several studies have shown that the concentration ratio of Aβ1-42 to Aβ1-40 (Aβ1-42/1-40)  shows muRoutinely performed basic CSF parameters: cell count, CSF differential cytology, glucose and lactate concentrations, quotient diagrams , and OCB by isoelectric focusing.Specific parameters when AD is suspected: Aβ1-42, Aβ1-40, Tau, and pTau181.Specific parameters when CJD is suspected: 14-3-3 protein, Tau, and PrPSc-aggregation assay (RT QuIC). If sporadic CJD is suspected, a stepwise approach is recommended for economic reasons, performing the PrPSc aggregation assay (RT QuIC) only when the 14-3-3 protein test is positive.To optimize a diagnosis-oriented interpretation as well as to enable inter-center comparison of the results, diagnosis-oriented interpretation algorithms are recommended, for example, the Erlangen Score .The ES combines the results of the biomarkers of amyloidosis (Aß1-42 and Aß1-42/1-40) and the biomarkers of neurodegeneration (Tau and pTau) into a five-step ordinal scale. This is particularly relevant, as common reference values for AD biomarkers are currently unavailable. Each laboratory should develop and validate its own specific reference values, which should be continuously verified in quality control schemes.In the basic CSF analysis, cell count, blood-CSF barrier function, and humoral signs of inflammation are usually normal . TherefoSimilarly, good diagnostic values can also be achieved if Nf-L is measured in serum . The com2O) in normal pressure hydrocephalus (NPH). However, characteristic fluctuations develop over the long term  [The CSF opening pressure is usually normal (< 20 cm H030/063) .Studies investigating the relevance of degeneration markers in the differential diagnosis for other dementia syndromes and gait disturbances are summarized in Table 1-42 concentrations in the CSF than healthy controls and slightly higher Aß1-42 levels than Alzheimer’s patients. Nevertheless, sensitivity and specificity are not high enough for reliable differentiation.Chen et al.  conducteIf a subarachnoid hemorrhage (SAH) is presumed according to clinical symptoms, but has not been proven by a CT or MRI scan, LP is necessary . Cytological detection of erythrophagocytosis  is most specific, also enabling chronological assessment (see Table A CSF that is not visibly hemorrhagic (erythrocytes < 1000/μL) does not exclude a SAH, particularly in cases of smaller or older hemorrhage. Both xanthochromia and erythrophagocytosis may still be lacking in acute cases  . As a ruIt is of particular relevance to distinguish lymphomatous meningitis from inflammatory lymphocytic reactions . The mosTo confirm a CSF fistula, prostaglandin D synthetase  or beta2 transferrin are measured. Both proteins are found in abundance in CSF, whereas concentrations in most other bodily fluids and tissue are very low. Levels above a given threshold indicate the presence of CSF. Under certain conditions, false-negative or -positive results may be obtained. In case of renal failure, for instance, BTP in other bodily fluids are higher than usual, leading to false-positive results . If CSF Detection of glucose is of little value to prove or rule out CSF leakage . Other C2OBMI < 30: >200 mmH2OBMI > 30: > 250 mmHFor a diagnosis of idiopathic intracranial hypertension (IIH) biochemical and cytological CSF parameters must be normal. See also the AWMF guideline “idiopathic intracranial hypertension” 030/093) . In cont3 . In co2O (5 ml/min) and 52 mm H2O (1 ml/min). The needle caliber used for LP may also affect the opening pressure (Table These values are based on the CSF opening pressure and, thus, CSF for biochemical and cell analysis should be drawn after measuring the pressure. Pressure values taken after withdrawal of CSF are not reliable. The lowering of the CSF pressure depends on the rate of withdrawal. Withdrawal of 20 ml CSF lowers the pressure between 92 mm Hre Table .Table 2If IIH is clinically suspected and no elevated pressure is measured on the initial LP, the pressure should be measured continuously to detect B and plateau waves , 182.


Surface electromyography (sEMG) has long been used in research, health care, and other fields such as ergonomics and brain-machine interfaces. In health care, sEMG has been employed to diagnose as well as to treat musculoskeletal disorders, pelvic floor dysfunction, and post-stroke motor deficits, among others. Despite the extensive literature on sEMG, the clinical community has not widely adopted it. We believe that in developing countries, such as Chile, this phenomenon may be explained by several interacting barriers. First, the socioeconomics of the country creates an environment where only high cost-effective treatments are routinely applied. Second, the majority of the sEMG literature on clinical applications has not extensively translated into decisive outcomes, which interferes with its applicability in low-income contexts. Third, clinical training on rehabilitation provides inadequate instruction on sEMG. And fourth, accessibility to equipment  may constitute another barrier, especially among developing countries. Here, we analyze socio-economic indicators of health care in Chile and comment on current literature about the use of sEMG in rehabilitation. Then we analyze the curricula of several physical therapy schools in Chile and report some estimations of the training on sEMG. Finally, we analyze the accessibility of some available sEMG devices and show that several match predefined criteria. We conclude that in developing countries, the insufficient use of sEMG in health might be explained by a shortage of evidence showing a crucial role in specific outcomes and the lack of training in rehabilitation-related careers, which interact with local socioeconomic factors that limit the application of these techniques. Since the ′40s, surface electromyography (sEMG) has been used in a variety of settings, including motor-control research, education, health care, rehabilitation, ergonomics, and human-computer interfaces, among others . Unlike The literature on sEMG is extensive, and despite the total publications counting in the thousands, the number of papers devoted to clinical applications is considerably fewer. For example, a search in PubMed with the terms “(surface EMG[Title/Abstract]) OR (sEMG[Title/Abstract]) OR (surface electromyography[Title/Abstract])” leads to 8,270 records. Adding terms such as “physiotherapy,” “stroke,” “gait,” or “back pain” leads to 60, 376, 497, 124, and 521 records, respectively. This suggests an important gap between the total literature on sEMG and the part of it dedicated to clinical applications. This gap prevents the inclusion of sEMG applications into clinical practice guidelines  but not in clinical internships. These might be substantial reasons that explain why rehabilitation professionals do not routinely use sEMG in their practice .Another barrier to the widespread use of sEMG in developing countries may simply be the accessibility  of the current EMG devices. Thus, we explore and compare the characteristics of several devices to shed some light on this topic.In developing countries, such as Chile, there are substantial barriers that impact health care access. The high inequity in income distribution determines the opportunity and quality of health care. For example, the Gini index, which measures income-distribution inequality , shows that Chile scores 44.4 , placingPublic spending on health is also low. The Domestic General Government Health Expenditure index  shows thwww.minsal.cl), there are currently 23 high-complexity (type 1), and 37 medium-high (type 2) hospitals in the country. According to our 2017 census, the current population is 17.5 million people. Considering that roughly 80% of this population is cared for by the public system, this implies that each of these centers has to take care of about 230,000 people.Additionally, high-complexity health care centers and specialists are located mostly in the capital . AccordiThese complex and intermingled factors create a scenario where health policies favor mostly high-impact and cost-effective interventions. For example, for 80 high-prevalence pathologies, the chilean government warrants access to diagnostic tools and treatments for which there is sufficient evidence of cost-effectiveness . As a paDuring the last three decades, evidence-based medicine (EBM) has encouraged the testing of many procedures and interventions that were routinely prescribed but not clinically proven . Based oIn the case of sEMG, a growing number of publications have explored its use in gait analysis , 16, musAs we discussed earlier, developing countries, such as Chile, favor high cost-effective approaches, which poses considerable obstacles in applying potentially effective but unproven tools.As we previously discussed, sEMG has been an essential tool to understand the neuromuscular system; nevertheless, it has also been long employed as a tool for therapy in the form of biofeedback  to treatOne of the main difficulties with the application of sEMG in this context is that the pooled evidence does not offer reliable supporting results, which has been reflected in clinical guidelines and, therefore, in clinical practice.For example, a 2007 meta-analysis of 13 studies on EMG-BF for post-stroke rehabilitation  showed tReasons for the failure of these meta-analysis are explained by high study heterogeneity , small sDespite being successfully used in several fields, there has been limited pooling of data or systematic reviews of EMG-BF for interventions. The lack of demonstrated effect size resulting from this is a barrier for the implementation of this tool in clinical use.Surface electromyography offers several benefits to rehabilitation professionals, nevertheless, its lack of widespread use may also be explained by insufficient training. To approach this question, we contacted 17 physical therapy (PT) schools, and eight sent us the curricula from their career. These account for 21.1% of the PT students of the country . As a means to approach the level of influence these schools exert in the local educational landscape, we report the national ranking of the schools' Universities  41). By. By41). We first counted the number of curricula in which at least one course mentioned any of the keywords. This simple approach revealed that five out of eight curricula met these criteria . These cThe total number of courses where EMG contents are present might be a loose proxy for the amount of training on this technique. Accordingly, for each curriculum with at least one EMG course, we counted the number of courses in which any of the keywords above were present. This approach resulted in a mode of one course per career mentioning any of the keywords .Finally, during the internship, the PT student is placed in a real clinical environment that shapes the repertoire of techniques that he or she will use as a professional therapist. Hence, the presence of sEMG content on these clinical internships might be crucial for the use of this technique as a future PT. We found that none of the internships and clinical oriented courses mentioned any EMG-related content in their description . Thus, sConsidering all the barriers to successfully applying sEMG to the clinical practice, a shortage of accessible EMG devices may add another barrier to its use. Here, we consider accessibility as the combination of portability, affordability, and ease of use of a particular EMG device. We define these criteria as follows:Portability: the device has a small size , can be easily carried to different locations, has internal batteries, and does not require a computer for operation.Affordable: considering that one of the main end-users of these systems may be the physical therapist, we defined this term based on the average monthly income before taxes (AMI) of a Chilean PT. We chose this parameter because, in their clinical practice, many PTs have to purchase their own equipment. The official statistics indicate that the 1st year after school, the AMI is U$740, and during the 5th year after graduation, it rises to U$1,330 . TherefoEase of use: all the necessary elements  are provided, and is compatible with smartphones or tablets (obtained from brochures or website descriptions).The results are described in We were pleased to find that at least four devices met all the predefined criteria, which provides the technical means to use sEMG directly in the office. This finding suggests that EMG device accessibility would not necessarily mean a barrier for the use of sEMG.amazon.com).Finally, another issue could be related to the cost of electrodes, which may impose another barrier. Nevertheless, for most of the applications, either disposable (~U$0.15/piece) or reusable (~U$0.40/piece) electrodes do not constitute a substantial obstacle for using sEMG  and developers , in which the patients are at the very center of their activity. Clinicians may perfectly understand patients' problems, but without help from engineers, they will not be able to solve them. On the other hand, engineers and designers that are not connected to a clinical setting may create solutions that are either too complex or too difficult to implement and do not necessarily solve practical problems. In either case, the patients' particular needs are left unmet. We think that these types of interactions are probably the best remedy to transform problems and needs into meaningful solutions and to advance the research on sEMG.In a different vein, our analysis of the curricula from Chilean PT schools, confirmed our suspicion of a lack of training in this area. In the schools with EMG training, this is taught mainly in one or two courses during the entire career, and the training takes place at the beginning of the career but not in the clinical internships, which may explain why PTs do not regularly use sEMG in their practice.Regarding the accessibility of sEMG devices, we found that at least four devices met predefined criteria of being portable, affordable, and easy to operate. This suggests that accessibility or price does not constitute in itself a barrier for the use of sEMG in the clinical practice and that the main limitations arise from political and economic characteristics of the country, from a paucity of compelling clinical indications, and from an insufficient amount of training.Finally, some of the barriers for the use of sEMG in the clinical domain may interact with each other in a circular manner, for example, the paucity of clinical evidence and the view of sEMG only as biofeedback may create insufficient pressure for both the development of health policies and for the training of rehabilitation professionals in sEMG. This insufficient training leads to an insufficient mass of professionals using sEMG, which leads to fewer grant applications and less research in the area, which leads us to the starting point. Also, there could be insufficient teaching dedicated to instrumentation or on technologies for rehabilitation. These types of courses could broaden the view on interventions and techniques that could enrich the clinical practice of rehabilitation professionals.Therefore, we think there is a need for more high-quality clinical evidence that presents an unavoidable pressure to employ this technique. There is a need for advancing the training not only in PT schools, but also in speech therapy and occupational therapy as well. A critical mass of professionals trained on these techniques backed up by sufficient clinical evidence may create the perfect scenario for the massive use of surface electromyography.https://data.worldbank.org/indicator/SI.POV.GINI,https://data.worldbank.org/indicator/SH.XPD.GHED.GD.ZS, https://www.mifuturo.cl/buscador-de-estadisticas-por-carrera.Publicly available datasets were analyzed in this study. This data can be found here: JA-R and HM-V contributed equally to the manuscript and approved the submitted version. 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.