Disruption of the blood–brain barrier (BBB) is an important pathophysiological process of anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis. A recent multi-center study showed that soluble (s) CD146 is a potential biomarker for monitoring early BBB damage and central nervous system inflammation, but little is known about sCD146 in anti-NMDAR encephalitis.Twenty-three anti-NMDAR encephalitis patients and seventeen controls with non-inflammatory neurological diseases were recruited. sCD146 and inflammatory cytokines in cerebrospinal fluid (CSF) and serum were detected by ELISA. Modified Rankin scale (mRS) scores were used to assess the neurological status of each patient. A follow-up review was completed three months after discharge.sCD146 levels in the CSF of patients with the acute stage anti-NMDAR encephalitis were significantly increased compared with controls and accompanied by increases in TNF-α, IL-6 and IL-10. CSF sCD146 was positively correlated with neuroinflammatory factors in the CSF and with mRS score. Three months after effective treatment, CSF sCD146 in patients was significantly decreased but remained significantly different compared with the controls.Our data suggested that higher expression of CSF sCD146 correlated with more serious neurological damage. Therefore, levels of CSF sCD146 may represent a promising indicator for monitoring disease and optimizing clinical treatment decisions in the early stages of anti-NMDAR encephalitis. Anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis has gradually become recognized as an autoimmune disease targeting neuronal synapses. Generally, neuropsychiatric symptoms, including cognitive dysfunction, seizure, abnormal movements, autonomic instability and hypoventilation, are the most classic clinical features to be presented –4. AlthoCD146 is an adhesion molecule which was discovered in 1987 in the plasma membrane of human melanoma cells , 6. In rIn this study, twenty-three patients who were diagnosed with anti-NMDAR encephalitis from 2018 to 2020 according to the revised anti-NMDAR encephalitis diagnosis criteria of 2016 were recruited from the Department of Neurology, The Affiliated Brain Hospital of Guangzhou Medical University, Guangzhou, China. The diagnosis of anti-NMDAR encephalitis was confirmed based on clinical manifestations and positive identification of antibodies against the NRI subunit of NMDAR in the CSF by cell-based analysis. Seventeen patients with non-inflammatory neurological diseases were selected as controls, including seven cases of Parkinson’s disease and ten cases of normal pressure hydrocephalus. Both anti-NMDAR encephalitis patients and controls were negative for the detection of common viruses and other pathogens by PCR. The study was approved by the Ethics Committee of The Affiliated Brain Hospital of Guangzhou Medical University. All participants provided informed consent before proceeding with the subsequent study. CSF samples from patients and controls were collected by lumbar puncture within three days of admission for diagnosis; blood samples were collected at the same time. CSF and blood samples were processed within 30 min of collection and centrifuged at 4000 rpm for 5 min. Supernatants of the CSF and serum samples were separated as soon as possible, transferred, numbered and stored at −80°C until they were used in ELISAs. The integrity of the BBB was evaluated by calculating the CSF to serum albumin quotient (QAlb), which represented an approximation of BBB breakdown. Modified Rankin Scale (mRS) scores were used to assess the neurological status of each patient. In addition to tumor removal, all patients were treated with first-line immunotherapy comprising intravenous methylprednisolone (1000 mg for 3–5 days), intravenous immunoglobulin (IVIg) (1 g/kg for 5 days per cycle) or plasma exchange, alone or combined , 22. ThrCommercial sandwich ELISA kits were used to detect the inflammatory cytokines tumor necrosis factor-α (TNF-α) , matrix metalloproteinase-2 (MMP-2), interleukin-6 (IL-6), IL-10 and sCD146  in the CSF and serum. Assays were performed according to the manufacturer’s instructions.Descriptive statistics were performed on demographic factors and clinical features of patients. The Kruskal-Wallis test was used to analyze the differences in TNF-α, MMP-2, IL-6, IL-10, and sCD146 levels among subgroups. Pearson’s test or Spearman’s test were used to evaluate the correlations between TNF-α, MMP-2, IL-6, IL-10, sCD146 and mRS scores. A value of P<0.05 was considered statistically significant, and all statistical analyses were conducted using SPSS version 17.0 .3, where values <6 indicate no BBB damage; 6–8, mild; 8.1–10, moderate; >10, severe damage. Mild dysfunction was detected in four patients, moderate in two and severe in four. IVIg (69.57%) and steroids (78.26%) were the major treatments.Detailed clinical features and levels of inflammatory cytokines in CSF or serum collected from patients with anti-NMDAR encephalitis (n=23) and controls with non-inflammatory neurological diseases (n=17) are presented in The expression of sCD146 in CSF of patients was notably increased in anti-NMDAR encephalitis patients in the acute stage compared with controls (P<0.001) but no difference was shown in serum sCD146 between these two groups. The QAlb and CSF MMP2 of anti-NMDAR patients were also significantly higher than those in the control group, as were the levels of CSF TNF-α, IL-10, and IL-6 (P<0.001).All patients were followed up for three months after receiving different treatments. CSF sCD146 levels were significantly decreased from 54.0±32.2 to 29.9±8.5 (ng/ml) (P=0.001), and TNF-α, IL-10 and IL-6 in CSF were also decreased significantly Table 2.As shown in There was a significant positive correlation between CSF sCD146 levels and mRS scores at the acute stage   of patients, no significant correlation was found between QAlb and CSF sCD146. Similar to our data, Wu, et al. found that only 15.21% of patients in the acute stage had elevated QAlb . PreviouIn this study, we found CSF IL-6, TNF-α and IL-10 to be increased in anti-NMDAR encephalitis patients in the acute stage. We further investigated the relationship between sCD146 and the proinflammatory cytokines IL-6 and TNF-α, and the anti-inflammatory cytokine IL-10. The results showed that all these three proinflammatory factors were positively correlated with CSF sCD146. In previous studies, abnormal elevation of sCD146 has been reported to correlate with proinflammatory factors, such as TNF-α, IL-13 and IL-17 , 27. EarThree months after effective treatment, the levels of sCD146 in CSF of the patients were significantly decreased. Eleven patients (47.8%) recovered well (mRS=1), but seven patients (30.43%) remained with mRS scores above 3 and none had an mRS score of 0, which meant that no patients had achieved a complete recovery in 3 months. Similar research by Titulaer et al.  showed tHere we have shown that the level of CSF sCD146 was positively associated with mRS in the acute stage of anti-NMDAR encephalitis. ROC curve analysis showed that CSF sCD146 ≥33.75 ng/ml can be used as an index to predict the severity of anti-NMDAR encephalitis, and the accuracy of CSF sCD146 in indicating the severity of this disease was remarkable. From this we could infer that the higher the expression of CSF sCD146, the more serious the neurological damage. Our findings implied that CSF sCD146 might hold potential in assessing the severity of anti-NMDAR encephalitis.Since the initial description of this disease in 2007 , the optFurthermore, it was reported in MS that CSF sCD146 could enhance the expression of adhesion molecules and promote the migration of leukocytes into the CNS . Our preThere were several limitations to our study. First, this was a single-center study, the nature of which may have introduced biases related to a small sample size and a shorter follow-up period. Second, we analyzed CSF and serum samples from anti-NMDAR encephalitis patients but did not conduct further study on pathogenesis.In this study, we found that the CSF sCD146 was significant increased in anti-NMDAR encephalitis patients with the acute stage, which were positively correlated with the levels of neuroinflammatory factors and mRS score. These findings suggested that the higher the expression of CSF sCD146, the more severe the neurological damage. Moreover, long-term high expression of CSF sCD146 and other inflammatory factors was accompanied by BBB dysfunction. Therefore, the levels of CSF sCD146 may represent a promising indicator for optimizing clinical treatment decisions in the early stage and disease monitoring in anti-NMDAR encephalitis.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study was approved by the Ethics Committee of The Affiliated Brain Hospital of Guangzhou Medical University. 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.This work was supported by Guangzhou Planned Project of Science and Technology (202102080073) and Natural Science Foundation of Guangdong Province .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.


Mosquitoes harbor microbial communities that play important roles in their growth, survival, reproduction, and ability to transmit human pathogens. Microbiome transplantation approaches are often used to study host-microbe interactions and identify microbial taxa and assemblages associated with health or disease. However, no such approaches have been developed to manipulate the microbiota of mosquitoes.Culex quinquefasciatus to Aedes aegypti) and within (Ae. aegypti to Ae. aegypti) species to validate the approach and determine the number of mosquitoes required to prepare donor microbiota. After the transfer, we monitored mosquito development and microbiota dynamics throughout the life cycle. Typical holometabolous lifestyle-related microbiota structures were observed, with higher dynamics of microbial structures in larval stages, including the larval water, and less diversity in adults. Microbiota diversity in recipient adults was also more similar to the microbiota diversity in donor adults.Here, we developed an approach to transfer entire microbial communities between mosquito cohorts. We undertook transfers between (This study provides the first evidence for successful microbiome transplantation in mosquitoes. Our results highlight the value of such methods for studying mosquito-microbe interactions and lay the foundation for future studies to elucidate the factors underlying microbiota acquisition, assembly, and function in mosquitoes under controlled conditions.Video AbstractThe online version contains supplementary material available at 10.1186/s40168-022-01256-5. A substantial body of evidence has emerged revealing the importance of microbiota to the biology of the animal hosts they associate with, which has stimulated broad interest in understanding the assembly of these communities. However, the daunting complexity of the microbiota present in many higher eukaryotes and the lack of conventional microbiology techniques to culture these microbes have limited our ability to address important questions in the field. As such, the mechanisms facilitating host-microbe interactions and the functional role of the microbiome as a holistic unit are poorly elucidated. Microbiota transplantation approaches are one promising technique for basic research and therapeutics, but studies employing these techniques have mainly been undertaken in mammalian systems –6, and tWolbachia, which infects the germline of Culex quinquefasciatus and two Anopheles species, but not Aedes aegypti, and is transovarially transmitted to offspring each generation [Microbiome research has expanded in mosquitoes given their medical relevance and interesting biology. As holometabolous insects, mosquitoes have distinct aquatic and terrestrial life stages, including larvae that molt through four aquatic instars before pupating on the water’s surface and emerging as terrestrial adults , 8. All neration –32.Altogether, the microbiota associated with the mosquito gut and other tissues can have profound impacts on mosquito biology by modulating larval growth and development , 33–35 aWe previously developed an approach to generate and maintain gnotobiotic mosquitoes colonized by individual bacterial taxa by sterilizing the surface of mosquito eggs and inoculating water containing bacteria-free (axenic) larvae hatched from surface-sterilized eggs under sterile conditions . SimilarCx. quinquefasciatus to Ae. aegypti) and within (Ae. aegypti to Ae. aegypti) species to validate the approach and determine the number of mosquitoes required to prepare donor microbiota. We chose to utilize these species in our experiments for two reasons. First, both Ae. aegypti and Cx. quinquefasciatus are biomedically relevant vector species that are commonly studied in the laboratory [Ae. aegypti life history [Cx. quinquefasciatus mosquitoes are readily infected by Wolbachia in the laboratory and commonly exhibit natural infections in the field [Ae. aegypti mosquitoes are generally recalcitrant to infection and are not known to harbor natural infections [Ae. aegypti and Cx. quinquefasciatus donors into Ae. aegypti recipients, we could therefore also establish whether our transplantation approach (i) recapitulated patterns of microbiota assembly and persistence previously observed in Ae. aegypti (positive control) and (ii) supported the expectation that we would not be able to successfully transfer Wolbachia from Cx. quinquefasciatus donors to Ae. aegypti recipients (negative control). Altogether, our results demonstrate the transfer of a complete microbiota from one mosquito cohort to another and lay the foundation for future studies to examine microbiota assembly and function in mosquitoes colonized by defined microbiomes with potential for exploitation for mosquito and disease control.Here, we developed an approach to transfer entire microbial communities between mosquito cohorts. We undertook transfers between  and Cx. quinquefasciatus (Houston) mosquitoes used as donors in this study were maintained in the same insectary and reared under the same conditions as described previously [Ae. aegypti colony, collected in Galveston, Texas, had been reared in the insectary for over 10 years, while the Cx. quinquefasciatus colony, collected in Houston, Texas, had been reared for 17 generations. In order to characterize microbiota diversity in both donor species, 150 individual 3- to 4-day-old sugar-fed adult females of each species were collected, surface sterilized by immersing in 70% ethanol for 5 min followed by 3- to 5-min washes in sterile 1X PBS, and stored at −20 °C for downstream sequencing. Adult mosquitoes from both species were also used to generate donor pools for microbiota transplantation. In brief, cohorts of 10, 20, 40, or 80 3- to 4-day-old adult female mosquitoes were collected, surface-sterilized in 70% ethanol for 5 min, rinsed three times in sterile 1X PBS for 5 min, and transferred individually to sterile 2-ml safe lock tubes  containing 5 mm steel beads and 500 μl of sterile 1X PBS  to 4 ml (pool of 80), were finally adjusted to a total volume of 50 ml using sterile water, and we repeated the entire process a total of three times to produce a total of 150 ml of each donor pool prior to use in downstream experiments  L1 larvae served as the recipient host for all transplantations. In brief, axenic larvae were prepared by submerging eggs in 70% ethanol for 5 min, then transferred to a solution of 3% bleach and 0.01% Decon-Quat 200V (Ab Scientific Ltd) for 3 min, followed by a wash in 70% ethanol for 5 min, and finally rinsing three times in sterile water  In order to assess successful microbiome transplantation, four sets of recipient samples were collected for sequencing: (i) 200 μl of larval water from each replicate flask, collected when larvae had molted to the third instar, (ii) third instar larvae (pools of 5) collected from the same flasks, and at least three individual sugar-fed adult females that had emerged from pupae either (iii) 3 or (iv) 6 days prior to collection. Pupae produced from replicate flasks containing the same donor pool were pooled and transferred to sterile water in a sterile plastic chamber for adult emergence. Newly emerged adults were provided 10% sucrose in sterile water ad libitum until collection as described above. Total genomic DNA was extracted from all recipient samples using a NucleoSpin Tissue Kit . Larvae and adults were surface sterilized as described above before DNA isolation.http://www.r-project.org/). In brief, differences in alpha and beta diversity between donor and recipient samples were analyzed by Kruskal-Wallis tests or Bonferroni-corrected pairwise Dunn’s tests to compare recipient samples to their respective donor pool. Differences in the proportion of rare and common taxa present in different donor pools were analyzed by pairwise Fisher’s exact tests. Finally, the significant of donor and recipient sample clustering by donor species was analyzed by permutational multivariate analysis of variance (PERMANOVA) implemented in QIIME.Subsamples from the DNA extracts were used for amplifying the V3-V4 region of the bacterial 16S rRNA gene using primers 341F (CCTACGGGNGGCWGCAG) and 805R (GACTACHVGGGTATCTAATCC) , followeAe. aegypti and Cx. quinquefasciatus), we characterized the microbiota of 40 individual adult mosquitoes by sequencing the V3-V4 regions of the 16S rRNA gene. After filtering, denoising, merging, and removing chimeras, we obtained a total of 2,544,303 reads with a median sequencing depth of 32,803 reads per sample . We obtained a total of 438,839 sequences representing 68 and 64 ASVs across the four Ae. aegypti and four Cx. quinquefasciatus donor pools we sequenced, respectively, and each pool was dominated by the same four genera detected in the 40 individuals previously sequenced from each donor species population , larvae (collected as third instars), and adults from each culture (collected 3- and 6-day postemergence). A total of 50, 40, and 108 water, larval, and adult samples were sequenced, respectively, with a total of five samples being discarded prior to downstream analyses due to low sequencing depth (< 1000 reads) , and Flavobacteriaceae  that were less abundant in input donor communities   Figs. . More spres Fig. . Wolbachals Fig. , althougals Fig.  and 7.FiAe. aegypti donors to Ae. aegypti recipients) and between  different donor and recipient species in order to assess our ability to recapitulate donor microbial diversity in adult recipients.Numerous studies describing the diversity and function of mosquito-associated microbial taxa and assemblages have been published recently, leading to the discovery of potential interactions between mosquitoes and their microbiota that could be manipulated to enhance or reduce mosquito fitness and/or vector competency (reviewed in ). HoweveOur results demonstrate the successful transfer of bacteria from donor to recipient populations of mosquitoes, with recipient adult individuals retaining the vast majority of donor bacterial diversity. Our results also demonstrate that relatively small pools of donor microbiota  are sufficient to capture taxa that are highly abundant and/or common in a given donor mosquito population. This suggests that our methods could readily be applied to study microbiota isolated from field populations, even in cases where large-scale mosquito collections are difficult. However, future work will be necessary to confirm that the patterns observed using our approach are indeed the same for donor microbiota generated from field mosquitoes that harbor microbial communities that are more diverse than those present in laboratory mosquitoes and that are not adapted to the laboratory environment , 73–78. Nasonia wasps to assess the selective pressures contributing to observed patterns of phylosymbiosis in the system [Drosophila melanogaster flies and Bombus terrestris bumblebees to identify relationships between host microbiota composition and different thermotolerance or immunity phenotypes [Ae. aegypti identifying life stage as a dominant factor shaping mosquito microbiota [These results also highlight the value of our approach for studying the underlying bases of mosquito-microbe interactions, as has been demonstrated in other animals. For example, microbiome transplantation approaches have been employed in e system  as well enotypes , 46. Recenotypes . Indeed,crobiota , 79–84. crobiota , 86. FutWolbachia, which was present in the Cx. quinquefasciatus donors used in this study, would not be successfully transferred to Ae. aegypti recipients. We also appreciate that tissue localization of bacteria and other microbes may be pertinent for the transplantation process, and that future studies should integrate complementary approaches to verify the viability of key members of the donor microbiota in recipient individuals’ posttransplantation. Here, we prepared donor microbiota from whole-body mosquitoes and similarly assessed microbiota assembly and persistence in whole-body recipient individuals. Additional work will be necessary to establish patterns of donor microbiota colonization across recipient host tissues and/or to validate methods for isolation of microbiota from specific donor and recipient tissues. Methods for long-term preservation of donor microbiota will also be necessary to facilitate long-term studies and intra- and interlaboratory comparisons of microbiota assembly across different host strains. Nevertheless, our results provide a critical first step toward the standardization of microbiome studies in the field of vector biology to include mosquito hosts colonized by defined microbiota that can be replicated within and between labs. They also provide a critical first step toward our ability to recapitulate and study field-derived microbiota in laboratory settings.Future work is also warranted to determine if the approach developed herein is relevant for other microbes such as fungi or viruses, which are also known to impact mosquito biology , 88. TheWe have successfully isolated and transplanted microbiomes from donor to recipient mosquitoes. This approach lays the foundation for future work to facilitate standardized studies of mosquito-microbe interactions, to examine host-microbe interactions, and to identify effective strategies for manipulating mosquito microbiota to control mosquito populations and mosquito-borne diseases.Additional file 1: Supplementary Table 1. Sequencing and diversity statistics for 16S rRNA gene amplicon libraries prepared from donor and recipient samples.Additional file 2: Supplementary Table 2. Taxonomic assignments and prevalence of each ASV in each sample, along with whether a particular ASV was considered ‘rare’ or ‘common’ among individuals of a particular donor species.Additional file 3: Supplementary Figure 1. Significantly negative correlation between relative abundance of ASVs belonging to the bacterial family Enterobacteriaceae and ASVs belonging to the genus Serratia across all Ae. aegypti and Cx. quinquefasciatus donor individuals .Additional file 4: Supplementary Figure 2. (Left) Relative abundance of bacterial genera in Ae. aegypti donor pools and recipient samples. Biological replicates were pooled for the bar graphs presented. Low abundance genera (<1%) are represented by the ‘Other’ category. (Right) Alpha diversity of donor pools and recipient samples, as measured by Shannon’s H index and ASV richness. Box-and-whisker plots show high, low, and median values, with lower and upper edges of each box denoting first and third quartiles, respectively. Asterisks (***) indicate samples that significantly differed from the donor pools .Additional file 5: Supplementary Figure 3. (Left) Relative abundance of bacterial genera in Cx. quinquefasciatus donor pools and recipient samples. Biological replicates were pooled for the bar graphs presented. Low abundance genera (<1%) are represented by the ‘Other’ category. (Right) Alpha diversity of donor pools and recipient samples, as measured by Shannon’s H index and ASV richness. Box-and-whisker plots show high, low, and median values, with lower and upper edges of each box denoting first and third quartiles, respectively. Asterisks (***) indicate samples that significantly differed from the donor pools .Additional file 6: Supplementary Figure 4. Average Bray-Curtis dissimilarity between (b/w) donor pools versus between a given donor pool and recipient samples. Mean values ± standard errors are shown. Asterisks (***) indicate comparisons for which the average dissimilarity between a given donor pool and group of recipient samples was significantly higher than that expected as a result of the transplantation procedure itself  .Additional file 7: Supplementary Figure 5. Significantly negative correlation between relative abundance of ASVs belonging to the bacterial family Enterobacteriaceae and ASVs belonging to the genus Serratia across all recipient Ae. aegypti adults .


This study aimed to compare between the clinical and laboratory characteristics of neurosyphilis and those of syphilis in human immunodeficiency virus (HIV) positive and explore the risk factors associated with the occurrence of neurosyphilis in the HIV infected.In-patients diagnosed with HIV and syphilis co-infection who underwent a lumbar puncture and completed cerebrospinal fluid (CSF) examination were divided into neurosyphilis group and syphilis group. The demographic characteristics, symptoms and signs, and laboratory tests of the 2 groups were comparatively analyzed. Logistic regression analysis was used to explore the risk factors associated with the occurrence of neurosyphilis.P = .010), and the patients from the neurosyphilis group were mainly infected via heterosexual contact. The proportion of serum toludine red unheated serum test (TRUST) titer ≥1:16 in the neurosyphilis group were 78.8%, which was significantly higher compared to the syphilis group (48.9%). The level of CSF white blood cell count, CSF protein, and CSF HIV viral load in the neurosyphilis group were significantly higher than those of the syphilis group. The proportion of patients with neurological symptoms and signs in the neurosyphilis group was significantly higher compared to the syphilis group (P < .001). Multivariate logistic regression analysis showed that heterosexual contact transmission route, not received antiretroviral therapy, lower CD4 cell count and higher serum TRUST titer, untreated with syphilis, and neurological symptoms and signs were risk factors associated with the occurrence of neurosyphilis.Among 81 patients, 33 patients were assigned to the neurosyphilis group, and 48 to the syphilis group. There were no significant differences in the age, gender, marital status, acquired immunodeficiency syndrome course, opportunistic infections, serum HIV viral load, and history of syphilis treatment. The difference in HIV transmission route between the 2 groups was statistically significant (The serum TRUST titer, CSF white blood cell count, CSF protein level, CSF HIV viral load, and the percentage of neurological symptoms and signs in the neurosyphilis group were higher. Heterosexual transmission route, not received antiretroviral therapy, and untreated with syphilis prompted the possibility of neurosyphilis occurrence. In recent years, it was the rapid growth of patients with syphilis infection in China. According to the report of an epidemiological survey, the incidence of syphilis with average annual growth rate of 13.37% during 2000 to 2013, was second only to tuberculosis, and hepatitis B, became an important public health problem. It was reported nationally 358,534 cases of syphilis cases in 2010, with incidence of 26.86 per 100,000 in 2010, and increased 17.02% than in 2009, listed third among B class infectious diseases of China. Syphilis did great harm to the human body, which can invade the skin mucosa, cardiovascular, nerve, bone and so on to cause multi-system damage, and can also cause abortion, stillbirth, and congenital syphilis in pregnant women. Neurosyphilis caused by TP invasion of the central nervous system (CNS), and infringe patients with meninges and blood vessels, cause nerve tissue degeneration or vascular lesions in CNS. Its clinical manifestation was complicated, the lack of specificity, and many patients were misdiagnosed or undiagnosed. Some of the symptoms included neurological impairment manifestations, which lacked specificity. HIV and TP also affected the CNS with the same transmission route among the same vulnerable population. HIV/AIDS and syphilis co-infection can increase the likelihood of developing neurosyphilis.By the end of 2017, there were 758,600 human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients living in China, and 134,500 new HIV/AIDS cases were reported in 2017, an increase of 7.09%  over 2016. Syphilis is a chronic sexually transmitted disease caused by –8 A review of syphilis showed that the incidence of neurosyphilis among HIV-infected patients was 2.1% compared to 0.6% among those without HIV/AIDS. HIV could not only directly infect CNS, but also affected CNS through the opportunistic infection or tumor caused by immunosuppression, thus complicating the clinical manifestations and diagnosis of patients and increasing the difficulty of diagnosis of neurosyphilis. This caused delaying in diagnosis and treatment of neurosyphilis infection in HIV/AIDS population. Ghanem et al suggested that rapid plasma regain test (RPR) titer ≥1:128, male, not receiving antiretroviral therapy (ART) were risk factors for developing neurosyphilis in syphilis patients with HIV infection. A study in Taiwan, China, showed that CD4 T lymphocyte counts (CD4 cell counts) ≤200 cells/μL was associated with failure of neurosyphilis treatment.In recent years, due to the increase of high risk behaviors among men who have sex with men, unprotected sexual behavior in the elderly, and casual sexual behavior among the youth, the infection rate of neurosyphilis in HIV/AIDS population is also increasing year by year.With the widespread use of antibiotics in treatment of syphilis, cardiovascular syphilis, and other serious types of syphilis have been rare. However, due to hidden incidence and atypical clinical manifestations, more and more cases of neurosyphilis reported in China, which seriously harmed public health safety. Therefore, the correct understanding of neurosyphilis is of great significance to improve healthcare workers diagnosis, treatment, and control the prevalence of the disease.To further understand the clinical characteristics of neurosyphilis in HIV/AIDS patients, and explore the risk factors associated with neurosyphilis, we undertook this retrospective study to comparatively analyze the data of HIV/AIDS patients with neurosyphilis and syphilis co-infection at 2 hospitals of infection diseases in Beijing and Hangzhou.22.1In this study, we retrospectively reviewed the medical records of all patients diagnosed with HIV/AIDS and syphilis co-infection, who underwent a lumbar puncture and completed cerebrospinal fluid (CSF) examination from January 1, 2013 to August 31, 2017at Beijing Youan Hospital, Capital Medical University in Beijing, and Hanzhou Xixi hospital in Hangzhou, which were designated infection diseases hospitals located in Beijing and Hangzhou, respectively.Treponema pallidum particle agglutination (TPPA) qualitative result, CSF pressure, CSF TRUST result, CSF TPPA result, CSF white blood cell (WBC) count, CSF protein level, CSF glucose level, neurological symptoms and signs, imaging findings examined with cranial computerized tomography, and/or magnetic resonance imaging.The following demographic and clinical data were collected from medical records: age, gender, marital status, possible HIV transmission route, CD4 cell count, HIV/AIDS course , ART status (ART-positive group included patients who had ART lasting for 3 months or more before the admission), opportunistic infections (OIs), serum HIV viral load , history of syphilis treatment, serum toludine red unheated serum test (TRUST) titer , serum Those patients were divided into HIV/AIDS with neurosyphilis group (referred to as Neurosyphilis group) and HIV/AIDS with syphilis group (referred to as Syphilis group) according to the following diagnostic and exclusion criteria.This study was approved by the Institution Ethics Committee of Beijing Youan hospital, Capital Medical University. The data were analyzed retrospectively and anonymously, the committee decided to waive the need for written informed consent from all participants.2.2 The diagnostic criteria for neurosyphilis was based on Sexually Transmitted Diseases Treatment Guidelines (2015) and Treatment guidelines of Syphilis, Gonorrhea, Genital Herpes, and Genital Chlamydia Trachomatis Infection (2014). TRUST and TPPA tests were used instead of venereal disease research laboratory (VDRL) and FTA-ABS tests since these last 2 tests were not carried out in the 2 hospitals. The criteria for the diagnosis of neurosyphilis included positive serum TRUST test, positive serum TPPA test, and one or more of the following: positive CSF TRUST test; and positive CSF TPPA test, and CSF WBC count >20 × 106/L. The criteria for the diagnosis of syphilis included positive serum TRUST test, positive serum TPPA test, negative CSF TRUST test, and one or more of the following: negative CSF TPPA test; and positive CSF TPPA test, and CSE WBC counts <20 × 106/L.The diagnosis of HIV/AIDS was based on Chinese guidelines for the diagnosis and treatment of HIV/AIDS (2018).Early syphilis included primary, secondary, and early latent syphilis. Late syphilis included late latent and syphilis of unknown duration. Patients with other concurrent CNS infections or caused by not syphilis were excluded from our study.2.3t test. Categorical data were analyzed by the chi-square test. Multivariable analysis was performed with logistic regression. The overall multivariate logistic regression analysis was built by identifying, as potential predictors, all variables that either attained a significance level of less than 0.10 in the analyses. Next, a stepwise forward selection procedure was used to obtain a parsimonious model using a significance level for entry and exit from the model of 0.05. IBM SPSS 23.0 for Windows  was used for all analyses. A 2-tailed P value <.05 was considered statistically significant.Continuous variables with normal distribution were assessed using the mean and standard deviation, or else using the median and inter quartile range. Categorical data were reported as number (n) (%). Continuous variables were analyzed using the Student 33.1A total of 81 HIV-infected patients were confirmed to have syphilis, divided 33 patients into the neurosyphilis group and 48 patients into the syphilis group. Most of the patients were middle-aged unmarried men. More than half of the patients were newly diagnosed with HIV/AIDS. Only 39 of patients (48.1%) had taken ART for more than 3 months before admission. Fifty-three of patients (65.4%) had OIs; 61 of patients (75.3%) had not been treated for syphilis. There were no statistically significant differences between the 2 groups in terms of age, gender, marital status, HIV course, ART, OIs, HIV viral load, and history of syphilis treatment (Table P = .048). The proportions of CD4 cell count ≤100 cells/μL, 100 to 200 cells/μL, 200 to 500 cells/μL, ≥500 cells/μL between the 2 groups were significant difference (P = .048). There was also a significant difference in possible HIV transmission route between the 2 groups (P = .010). Heterosexual contact was the predominant transmission route in the neurosyphilis group (45.5%), while homosexual contact was the main transmission route in the syphilis group (62.5%).However, the median CD4 cell counts of neurosyphilis group was 219 cells/μL, which was significantly higher than that of syphilis group (90 cells/μL), (3.2P = .007)  TRUST titers ≥1:16, 100% (80/80) with TPPA test positive. The proportion of serum TRUST titer ≥1:16 in the neurosyphilis group was significantly higher than that in the syphilis group (78.8% vs 48.9%), and the observed difference was statistically significant (3.3P < .001). The clinical manifestations of neurosyphilis sorted by the highest incidence included: the decline of muscle strength (7), paropsia (7), headache or dizziness (7), rash (6), abnormal ophthalmic examination (6), vomiting (4), heterophthongia (4), paresthesia (4), dysgnosia (4), hypoacusis (2), changes in consciousness (1), emotional lability (1), pathological signs (1), meningeal sign (1). Except lower percentage of incidence of fever  and higher percentage of incidence of decline of muscle strength  in the neurosyphilis group compared to the syphilis group, there was no significant difference in other manifestations between the 2 group . Among the imaging findings of neurosyphilis patients, 39.4% were normal, 36.4% with abnormalities. The abnormalities manifested as low density infection foci (15.2%), ischemic infarction (15.2%), leukodystrophy (3.0%), and encephalatrophy (3.0%).Sixty-one of the patients took imaging examination. The findings were divided into abnormal and normal categories. There was no significant difference between the 2 groups (3.56/L) and CSF protein level (786 mg/L) in the neurosyphilis group were significantly higher compared to those of the syphilis group  (P< .001). There were no significant differences in CSF pressure, CSF glucose levels, CSF chloride, and CSF red blood cells (Table P = .002).CSF WBC count , CD4 cell count <200 cells/μL , previous untreated with syphilis , and serum TRUST titer ≥1:16 . The protective factor associated with occurrence of neurosyphilis was received ART  , CSF protein (786 vs 362 mg/L), and CSF HIV viral load  of the neurosyphilis group were significantly higher than those of the syphilis group. TP could invade various organs of the body, including the CNS, cause transient or persistent inflammation, which manifested as abnormal CSF examination. CSF WBC count was an indicator used to diagnose neurosyphilis when it exceeded 20 × 106/L in HIV/AIDS patients. It was very sensitive but with low specificity. CSF proteins were usually elevated in patients with neurosyphilis; however, they neither had sensitivity nor specificity. The previous study showed that the CSF WBC count and protein were higher in symptomatic neurosyphilis patients compared to asymptomatic ones, while most of the symptomatic patients had abnormal results of CSF examination. Another study showed that CSF protein was closely related to the presence of symptoms. Syphilis infection can temporarily increase HIV replication, and neurosyphilis may amplify HIV viral load in CSF, which may be a consequence of intrathecal immune activation and subsequent amplification of HIV replication. Our results showed that the CSF WBC, CSF protein, and CSF HIV viral load in the neurosyphilis group were significantly higher compared to the syphilis group, which was consistent with the results from previous studies. Since most of the patients in our study were not treated with ART, there was a more remarkable change of these indicators between the 2 groups. There were no significant differences in CSF pressure, CSF glucose level, and CSF chloride between the 2 groups. Since no previous studies reported on these indicators, we assumed they did not significantly change in neurosyphilis patients.The levels of CSF WBC , ischemic infarction (15.2%), white matter lesions (3.0%), and brain atrophy (3.0%).The imaging findings of neurosyphilis tended to lack specificity; they varied from normal to a variety of manifestations such as abnormalities in the cerebral cortex and white matter of the brain, atrophy of the brain, meningeal enhancement, and cerebrovascular lesion. A previous study analyzed the imaging findings from 35 neurosyphilis patients, of which 32 were HIV-positive; 11 (31%) had normal imaging findings, 8 (23%) had cerebral infarction, 7 (20%) had nonspecific white matter lesions, 2 (6%) showed meningitis and off-axis enhancement suggesting meningitis. Since these 2 tests were not performed at the hospitals of the study, the CSF TRUST and CSF TPPA experiments were used as a replacement for CSF VDRL and CSF FTA-ABS. Because HIV virus could cross the serum–brain barrier, and elevate the CSF WBC and CSF protein level, the CSF WBC count> 20 × 106/L was selected as the abnormal standard according to the 2015 US guidelines. China's 2014 guidelines defined CSF protein >500 mg/L as a threshold for abnormality, while the normal range of CSF protein at our hospital was 0 to 600 mg/L. We could not define CSF protein >500 mg/L as a criterion to diagnose neurosyphilis. Since we did not find any other criteria for the diagnosis of neurosyphilis with CSF protein, to ensure the diagnostic accuracy of neurosyphilis, CSF protein was not included in the diagnostic criteria in the present study.It should be noted that the diagnosis of neurosyphilis depended on clinical manifestations, syphilis serology, and CSF examination. The diagnostic criteria slightly varied in different guidelines. The CSF VDRL experiment had high specificity but lacked sensitivity, while the CSF FTA-ABS had high sensitivity but poor specificity.There were some limitations in this study. First, since our study was a retrospective one, it was difficult to control the baseline of included patients. Some data such as HIV possible transmission route were not acquired in many patients, and some tests such as serum HIV viral load, CSF HIV viral load, and imaging examination were not performed for a high number of patients. The sample size of our study was small, only in-patients, and thus could lead to insufficient test efficiency in statistical analysis, which should be addressed by further multi-center studies. Second, the diagnostic criteria in this study was set on serum TRUST and TPPA test values with 1 or more other results of positive CSF test, and CSF TRUST and CSF TPPA tests were used as a replacement for CSF VDRL and CSF FTA-ABS. Other diagnosis guidelines favored VDRL and TPPA findings for the diagnosis of neurosyphilis. Therefore, generalizability is limited where these lab tests are performed.5Lower CD4 cell count, higher serum TRUST titer, HIV transmitted by heterosexual contact, not received ART, and untreated with syphilis, were associated with neurosyphilis among HIV/AIDS patients with syphilis. The presence of neurological symptoms and signs prompted the possibility of neurosyphilis occurrence. Lumbar puncture and CSF testing for the diagnosis of neurosyphilis is required in patients infected with HIV and syphilis.LA, YJH conceived and coordinated the study, designed, performed, and analyzed the experiments, wrote the draft. SJC, WH, LJW, SY, YJZ, DLL, and WXW carried out the data collection, data analysis, and revised the manuscript. All authors reviewed the results and approved the final version of the manuscript.Conceptualization: An Liu.Data curation: Jianhua Yu, JinChuan Shi, Hu Wan, Jianwei Li, Ying Shao, Jiangzhu Ye, Lili Dai, An Liu.Formal analysis: Jianhua Yu, JinChuan Shi, Hu Wan, Jianwei Li, Jiangzhu Ye, Lili Dai.Investigation: JinChuan Shi, Hu Wan, Jianwei Li, Ying Shao, Jiangzhu Ye, Lili Dai, An Liu.Methodology: Hu Wan, Jianwei Li, Ying Shao, Jiangzhu Ye, Lili Dai, Xiwen Wang, An Liu.Project administration: Lili Dai, Xiwen Wang, An Liu.Software: Jianhua Yu, JinChuan Shi, Xiwen Wang.Validation: Jiangzhu Ye, Xiwen Wang.Writing – original draft: Jianhua Yu.Writing – review & editing: An Liu.


The association of 12-month eGFR and allograft loss was modified by donor age (but not donor types) where a higher risk of allograft loss in recipients with lower compared with higher 12-month eGFR being most pronounced in the younger donor age groups (p < 0.01). Recipients with eGFR <30 ml/min/1.73 m2 12-months after transplantation experienced ≥2.5-fold increased risk of overall allograft loss compared to those with eGFR of >60 ml/min/1.73 m2, and the magnitude of the increased risk is most marked among recipients with younger donors. Careful deliberation of other factors including donor age when considering eGFR as a surrogate for clinical endpoints is warranted.Reduced estimated glomerular filtration rate (eGFR) at 12-months after kidney transplantation is associated with increased risk of allograft loss, but it is uncertain whether donor age and types modify this relationship. Using Australia and New Zealand registry data, multivariable Cox proportional modelling was used to examine the interactive effects between donor age, types and 12-month eGFR on overall allograft loss. We included 11,095 recipients . Recipients with lowest 12-month eGFR (<30 ml/min/1.73 m  ThereThe growing use of expanded criteria (or higher Kidney Donor Profile Index [KDPI]) donors has prompted clinicians to recognize that specific donor factors, including donor age and comorbidities, may influence short- and long-term outcomes after transplantation ‒18. ManyAll adult patients with kidney failure (aged 18 years or older) in Australia and New Zealand who had received first kidney transplants from adult living or deceased donors (aged 18 years or older) between 2000 and 2017 were included. Recipients of multiple organ allografts and those who had received prior transplants were excluded. Kidney transplant recipients with failed allografts within 12-months post-transplant and those without a recorded eGFR measurement at 12 months were excluded from the study. This study was approved by the University of Western Australia Human Research Ethics Committee (reference: 2019/RA/4/20/4584) and is reported here according to The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines .Baseline characteristics included donor factors ; recipient factors ; and transplant-related factors .2. The primary outcome of this study was overall allograft loss . The secondary outcomes were death-censored allograft loss, death with a functioning allograft and all-cause mortality .Post-transplant kidney function, especially allograft function at 12-months post-transplant, was chosen as the exposure of interest for three reasons: 1) It is one of the most important clinical outcomes identified by both patients, caregivers and healthcare professionals , 15, 22;Data are presented as number (proportion), mean (standard deviation [SD]) and median (interquartile range [IQR]) where appropriate, with comparisons between groups examined by chi‐square test, analysis of variance (ANOVA) or Kruskal-Wallis test, respectively. The associations between 12-month eGFR, primary and secondary outcomes were examined using adjusted Cox regression models. Grouped LASSO (least absolute shrinkage and selection operator) regularized logistic regression was used for variables selection . The varp < 0.01) observed for the outcome of overall allograft loss. We next constructed models evaluating the two-way interaction between categories of 12-month eGFR  and donor age, with donor age thresholds informed by restricted cubic splines  between categories of 12-month eGFR and donor age for overall allograft loss, but not for death-censored allograft loss. However, a three-way interaction between 12-month eGFR, donor age and donor type were not observed for allograft and patient outcomes.The 12-month eGFR and donor age was considered as the two-way interaction term, and 12-month eGFR, donor age and donor types were considered as the three-way interaction term. We first tested the interaction using continuous measures of 12-month eGFR and donor age, with significant interactions  and corresponding 95% confidence intervals (95% CI). The proportional hazard assumptions for all Cox regression models were examined graphically by Schoenfeld residuals with no evidence of departures from proportional hazards for allograft loss or mortality. A sensitivity analysis examining the associations between 12-month eGFR and outcomes were undertaken with the inclusion of other donor characteristics of diabetes, hypertension and smoking history in the Cox regression models. All analyses were undertaken using SAS  and STATA , with 2 and 4,085 (36.8%) had eGFR <30 and >60 ml/min/1.73 m2.Of the 12,683 first kidney transplants performed in 2000–2017, we excluded 1,588 recipients who lost their allografts within 12 months post-transplant or had no recorded 12-month eGFR, leaving a study cohort of 11,095 recipients . The mea2 were younger, less likely to have prevalent vascular disease or diabetes, and had shorter mean waiting time than recipients with 12-month eGFR ≤60 ml/min/1.73 m2. Recipients with 12-month eGFR values of >60 ml/min/1.73 m2 were more likely to have received living donor kidneys and of younger donor age compared to those with 12-month eGFR of ≤60 ml/min/1.73 m2. The proportion of kidney transplant recipients with 12-month eGFR >60 ml/min/1.73 m2 increased from 30.9% between 2000 and 2004 to 38.8% between 2013 and 2017. Conversely, the proportion of recipients with 12-month eGFR <30 ml/min/1.73 m2 reduced from 9.4% between 2000 and 2004 to 7.7% between 2013 and 2017.2 compared to recipients of donor kidneys aged >30–60 and >60 years. Conversely, approximately 17% of recipients with older donor kidneys (aged >60 years) had 12-month eGFR of <30 ml/min/1.73 m2 compared to 3% of recipients with younger donor kidneys (aged 18–30 years) (A higher proportion of recipients who received kidneys from younger donors aged 18–30 years had 12-month eGFR >60 ml/min/1.73 m0 years) .2, recipients with the lowest 12-month eGFR (<30 ml/min/1.73 m2) experienced the greatest risk of overall allograft loss ; where those with the highest eGFR at 12-months experienced a lower risk of overall allograft loss (adjusted HR 0.67 [0.62–0.74]). Compared to recipients of older donor kidneys, recipients with younger donor kidneys experienced a reduced risk of overall allograft loss.The estimates of the main model for overall allograft loss are shown in 2: HR 5.74 ; 30–60 ml/min/1.73 m2: HR 1.37 ; >60 ml/min/1.73 m2: referent). In recipients of kidneys from older donors aged >60 years, the HRs for overall allograft loss were attenuated at lower 12-month eGFR values  (eferent) .2, the adjusted HR for 12-month eGFR of <30 ml/min/1.73 m2 was 3.94  for death-censored allograft loss, 1.30  for death with a functioning allograft and 1.78  for all-cause mortality. The respective HRs for 12-month eGFR of >60 ml/min/1.73 m2 were 0.56 , 0.82  and 0.77 . These relationships were not modified by donor age.The estimates of the main models (without interaction) for death censored allograft loss, death with a functioning allograft and all-cause mortality are shown in 2 received kidneys from donors with a history of diabetes or hypertension compared to recipients with higher 12-month eGFR . This association was modified by donor age but not donor types. Recipients of younger donor kidneys with a lower 12-month eGFR value of less than 30 ml/min/1.73 m2 experienced up to 6-times greater risk of overall allograft loss compared to those with higher 12-month eGFR values. This association was attenuated in recipients with older donor kidneys.In this contemporary cohort of kidney transplant recipients, recipients with 12-month eGFR less than 30 ml/min/1.73 mObservational data shown a direct association between donor age and kidney function at 12-months and long-term allograft and patient survivals , 27‒29. 2 at 12-months , indicating that eGFR will likely remain one of the best and practical index measures for longer-term kidney allograft outcome  and therefore, kidney allograft biopsies are often required to guide clinical management . Our stude novo donor-specific anti-HLA antibody), presence of and severity of proteinuria and the development (and severity) of de novo comorbid conditions such as post-transplant diabetes and hypertension that may have influenced allograft function and allograft survival post-transplant; which were not adequately collected by the ANZDATA registry but may potentially have modified our study findings. It was determined a priori that change in eGFR would not be considered in this study given that the majority of landmark clinical studies had utilized a single time point eGFR measurement as the primary or secondary endpoint. However, our other work has shown that change in eGFR is a valuable predictor of long-term outcomes , the impact of various adverse clinical events/hospitalizations occurring during the time course of the follow-up period, lack of availability of biopsy data and changing nature of immunological risk  12-month post-transplant is associated with adverse long-term allograft outcomes but whether donor factors such as age, modify this association in unknown. Using data from the Australia and New Zealand Dialysis and Transplant (ANZDATA) registry, we have shown that the relationship between 12-month eGFR and allograft loss was modified by donor age. Even though the trend and nature of the relationships between 12-month eGFR and allograft loss were similar, the magnitudes of the risk were dissimilar among donor age subgroups.


Cerebrospinal fluid (CSF) diagnostics has emerged as a valid tool for a variety of neurological diseases. However, CSF diagnostics has been playing a subordinate role in the diagnosis of many neurological conditions. Thus, in the multitude of neuromuscular diseases in which motor neurons are affected, a CSF sample is rarely taken routinely. However, CSF diagnostics has the potential to specify the diagnosis and monitor the treatment of neuromuscular disorders. In this review, we therefore focused on a variety of neuromuscular diseases, among them amyotrophic lateral sclerosis (ALS), peripheral neuropathies, and spinal muscular atrophy (SMA), for which CSF diagnostics has emerged as a promising option for determining the disease itself and its progression. We focus on potentially valuable biomarkers among different disorders, such as neurofilaments, cytokines, other proteins, and lipids to determine their suitability, differentiating between different neurological disorders and their potential to determine early disease onset, disease progression, and treatment outcome. We further recommend novel approaches, e.g., the use of mass spectrometry as a promising alternative techniques to standard ELISA assays, potentially enhancing biomarker significance in clinical applications. Cerebrospinal fluid (CSF) diagnostics emerged as a valid tool for a variety of neurological diseases. It is routinely used to detect neuronal diseases such as acute or chronic meningitis or encephalitis and is even able to distinguish between an acute viral and a bacterial intrathecal infection ,2. FurthA literature search was performed using the Medline and Cochrane databases included in PubMed. As abundance of published articles was highly variable among the discussed neuromuscular disorders, the parameters for study selection were adapted accordingly. For ALS the terms, “ALS” or “amyotrophic lateral sclerosis” and “CSF” or “cerebrospinal fluid” were chosen as a first indication. Additionally, the terms “cytokine” or “neurofilament” or “TDP-43” were added. As for neurofilaments, the number of studies was comparably high (103 publications), so only studies conducted within the last 5 years  containing a high number of patients in the cohorts (>45 ALS patients within the cohort) were used for in-depth review. For all other terms, a search period of 15 years was chosen. We further focussed on studies using similar techniques (ELISA) enabling comparability of studies for different biomarkers. For SMA, the terms “SMA” or “spinal muscle atrophy” or “spinal muscular atrophy” and “CSF” or “cerebrospinal fluid” were chosen. Results were further filtered for studies published in the last 15 years .For the reviewed peripheral neuropathies, a smaller number of publications was found compared to the literature research on ALS, therefore specific exclusion criteria in terms of study population or publication date weren’t applicable. The terms searched for Guillain-Barré syndrome were “Guillain-Barré syndrome” or “GBS” in combination with “CSF”, “cerebrospinal fluid”, “biomarker”, “neurofilament”, “proteomics”, or “lipidomics”. For multifocal motor neuropathy, the terms “multifocal motor neuropathy” or “MMN” and “CSF”, “cerebrospinal fluid” or “biomarker” were selected. The terms used for Lyme neuroborreliosis were “neuroborreliosis” or “lnb” in combinaton with “CSF”, “cerebrospinal fluid” or “biomarker”. For all studies, only those matching the questions covered in this review were included. Literature search terms and inclusion and exclusion criteria for all neuromuscular disorders discussed in this review are further visualized in a flow diagram .ALS is a heterogeneous neurodegenerative disease affecting both upper and lower motor neurons and is marked by a progressive loss of bulbar and limb function. In 5% of all cases ALS is found to be familial. To date over 100 gene mutations and over 30 genes have been associated with different types of familial ALS . HoweverDiagnostic tools encompass neuroimaging, whereby a routine MRI is conducted. In this context, patients with ALS might show atrophy of motor cortices and degeneration of motor tracts ,26, but In clinical routine, liquid biopsies most often present as valuable tools for disease verification. Commonly, blood, plasma, saliva or CSF samples are used as liquid biopsy options. Since ALS is a neurodegenerative disorder, CSF could be the most suitable liquid biopsy option, as it constantly surrounds the brain tissue. Several studies have already focused on the evaluation of various potential biomarkers detectable in CSF for their ability to reliably differentiate between ALS, various other neurological diseases, so-called ALS mimics, and healthy cohorts ,41,42,43Cytokines comprise a large group of small, secreted proteins, including interleukins, chemokines, interferons, and tumor necrosis factor. They function as elementary mediators in inflammatory processes and are secreted by various immune cells like T and B lymphocytes, endothelial cells, mast cells, and macrophages ,45,46, eTateishi et al. investigated cytokine concentration in the CSF of patients with sporadic ALS (sALS), lower motor neuron disease (LMND), and of a control group with non-inflammatory neurological diseases (ONDs) . The ALSH36D polymorphism in the hemochromatosis gene (HFE), all patients in this study were also genotyped for H36D and C282Y HFE. Results showed that all cytokines except for IL-1 were detected. In addition, thirteen differential cytokines, among them eleven increased in ALS and two in OND, were observed . Those with the most significant p-value were IL-6, GM-CSF, IL-10, IL-2, and IL-15. The elevated values of IL-17, CCL4, G-CSF, VEGF, and CCL2 are consistent with the results of the previous study. Surprisingly, the five markers with the most significant p-values do not show any correspondence. Authors pointed out that some of these markers are involved in microglia pathways, lymphocyte activation, and cell proliferation, therefore confirming the association between ALS pathogenesis and CNS inflammation. However, for systematic analysis of the correlation between HFE variants and CSF cytokine profiles, all individuals were grouped based on their genotypes (homo- and heterozygotes were clustered as one). H63D HFE variation is highly associated with familial ALS and, similarly to C282Y HFE, causes cellular iron accumulation. Patients carrying an H63D HFE variant compared to wild type controls showed enhanced concentrations of β-2 microglobulin and CXCL8. In contrast, subjects with the C282Y HFE allele demonstrated elevated levels of IL-7, IL-12, and PDGF bb. Interestingly, CXCL8, IL-7 and IL-12 exhibited elevated levels in ALS compared to OND in the study reported by Tateishi et al., therefore the relation between HFE variants remains unclear.Another study by Mitchell et al. also applied the multiplex bead-based immunoassay for analysis of 27 cytokines and growth factors in CSF . In relaA further study by Furukawa et al. evaluated the cytokine profiles of patients with progressive muscular atrophy (PMA), ALS, and multifocal motor neuropathy (MMN) . Due to n = 40) and CSF (n = 65) samples from individuals with non-inflammatory diseases were used as controls. Here, ELISA was used exclusively as a cytokine assay. Based on results of this study, it appears that many interleukins and their inhibitors, such as IL-1β, IL-37, IL-1Ra, and IL-36 were often not detectable in all samples. A significant increase was only detected for IL-18 (p < 0.0001) and IL-18BP (p < 0.0001) in serum of sALS. The authors linked the elevated IL-18 levels to the inflammasome complex and accordingly to caspase-1 activity, which is as well increased in SOD 1 mouse model of ALS. The fact that increased caspase-1 activity is usually accompanied by an enhanced level of IL-1β, which is missing in this study, was therefore described by the authors as a more local tissue-related outcome and therefore not detectable in body fluids. The lack of detection of some cytokines and their inhibitors in serum or CSF underlines this assumption. The approach of Italiani et al. focused on specific analysis of the interleukin-1 family in ALS. In general, the IL-1 family comprises eleven cytokines, including receptor antagonists and pro- and anti-inflammatory molecules . Here, tIn conclusion, a transferable and distinct CSF or serum cytokine analysis in ALS appears to be quite challenging. Through comparison of the studies mentioned here, it becomes evident that many of the measured cytokines do not always appear to be differentially regulated. In addition, the findings by Furukawa et al.  pointed TARDBP have been found to be associated with ALS and FTLD . Nf. Nf40]. Besides the commercially available ELISA kits, Olsson et al. designed an in-house ELISA using 2 NfL monoclonal antibodies . They weAn ambitious study evaluating the comparability and reproducibility of NfL and pNfH determination via ELISA assay carried out by Oeckl et al. set out to test ALS patient and control CSF samples from 15 different centers in a multicenter study . All samBesides CSF-based studies to elucidate the concentration of NfL and pNfH, the majority of studies focus on determining plasma-derived pNfH concentrations, enabling a less invasive method to predict ALS diagnosis. One particular study we want to focus on in our review is a plasma and CSF combined study conducted by De Schaepdryver et al. The authors examined 85 ALS patients, 215 patients with various other neurological disorders , and 31 ALS mimics enlarging the so far conducted studies by a clear recommendation whether NfL and pNfH are able to distinguish between ALS and clinically highly similar disorders, such as Kennedy disease or motor neuropathy . AuthorsAdditionally, studies comparing plasma/serum and CSF levels of either NfL or pNfH came to similar conclusions ,94. HoweCombining hypotheses and results raised and determined in the aforementioned studies, NfL and especially pNfH have been developed as reproducible and sensitive prognostic markers distinguishing ALS from healthy people but also from patients suffering from different neurological disorders and ALS mimics. Still, we have to point out that factors, such as the usage of different body fluids, different cohort compositions, varying protocols for body fluid withdrawal and collection, and patient specific parameters such as mutations and autoantibodies may alter the specificity of the results. Nevertheless, all studies displayed promising and comparable results using ELISA assays for the diagnosis of ALS in large cohort studies.Peripheral neuropathies summarize a multitude of diseases that affect peripheral motoric, sensory and/or autonomic nerves. Usually, a distinction is made by the pattern found in nerve conduction studies into axonal or demyelinating neuropathies. Causes can be, for example, metabolic, toxic, inflammatory, hereditary, or paraneoplastic . Due to Guillain-Barré syndrome usually presents with an acute ascending palsy and sensory loss, often following respiratory or gastrointestinal infections . The diaThe above mentioned neurofilament light chain (NfL) and phosphorylated neurofilament heavy chain (pNfH) are intracellular proteins that are found in the CNS as well as in the peripheral nervous system. They were not only evaluated as biomarkers in ALS but also in GBS. This circumstance already suggests a possible pitfall that the differentiation of diseases might not be eased by this marker. An overview of the evaluated studies is displayed in p = 0.0135). Outcomes were measured using both the Medical Research Council grading system (MRCS) and the F-score. Patients with elevated NfH CSF levels (>0.73 ng/mL) had a worse outcome , MRCS ≤ 45 , NfH levels correlated with the final F-score  and MRCS .Petzold et al. investigated NfH CSF levels in 23 patients with GBS using a standard ELISA . The uppp = 0.019, multivariate testing p = 0.004). NfH levels above the upper normal limit of 0.73 ng/mL therefore predicted a poor outcome .In a subsequent study by Petzold et al., NfH CSF levels in 38 GBS patients were evaluated by ELISA and compared to levels of patients with other neurological diseases . There wn = 11) and AMAN (n = 11) and compared them to other neurological diseases  using a commercial sandwich ELISA [p < 0.001). A comparison between AMAN and AIDP showed a significant difference with higher pNfH levels in AMAN (p < 0.05). In AMAN, increased levels of pNfH correlated with GBS disability scale scores in the acute phase , the plateau phase , and the recovery phase .A study by Wang et al. investigated CSF pNFH levels in the GBS subgroups AIDP . Patients that showed a poor outcome at follow-up had higher NfL level than patients with a good outcome. NfL levels over 10000 ng/L had persistent disability with a median ODSS of 5.5, patients below this limit had a median ODSS of 0. NfL levels also correlated with the F-score in the acute phase . Initial Nf correlated also with the quality of life at follow-up, as measured by the PCS score .The approach of Axelson et al. included CSF sampling at the onset of the disease and a clinical follow-up 9–17 years after the disease in 18 patients . Hughes p < 0.001) increased in the neuropathy group  compared to healthy controls . No significant differences between the different diseases were found. The CSF NfL levels in both groups were unfortunately not mentioned in the paper. A correlation between clinical severity and NfL levels in CSF could not be shown.Mariotto et al. determined NfL levels in CSF and serum with an ELISA in 25 patients with different inflammatory neuropathies including 5 GBS patients . Serum Np = 0.02). GBS disability scale score correlated with CSF NfL (p = 0.005). To differentiate between NfL of central or peripheral nerval origin, a ratio of CSF NfL and serum NfL was formed. A ratio of 12.8 was used as a cutoff. Patients with a lower ratio had axonal or mixed patterns in nerve conduction studies and a slightly higher clinical affection. Patients with a higher NfL-ratio showed a demyelinating pattern.Körtvelyessy et al. investigated NfL levels in CSF and serum in 21 GBS patients using an ELISA. Levels were compared to 19 controls with non-neurological diseases . In CSF,In conclusion, Nfs seem to be a promising marker not necessarily for diagnosis of GBS, but for differentiation between subtypes and even prediction of long-term outcome especially in axonal subtypes. Further investigations are, however, needed, especially studies with larger cohorts of patients, which may potentially answer further questions, e.g., which Nf would be the more promising biomarker. A more complicated question would be whether this biomarker could identify patients at risk of severe disease course, select them for a more aggressive treatment, or ameliorate the clinical outcome.p = 3.81 × 10−8) compared to 15 controls with ONDs [p = 1.34 × 10−7). The SM cut-off for optimum sensitivity and specificity was 0.00118 nmol/μL. Elevated SM correlated negatively with conduction velocity, which is a marker of demyelination, in 12 patients. A correlation with an axonal pattern in nerve conduction studies could not be shown.Sphingomyelin is a sphingophospholipid typically found in the membrane of the Schwann cells that form the myelin sheath of peripheral nerves . Its invith ONDs . To rulep < 0.0001) compared to patients with other neurological diseases [p = 0.003), Overall Neuropathy Limitation Scale  and MRC sum score .To further investigate this novel biomarker, another study was conducted by Capodivento et al. in 2021. Twelve patients with the demyelinating subtype of GBS showed significantly elevated SM CSF levels (diseases . Some ovp = 2 × 10−9). All lipid classes were elevated, but to a different extent. Cholesteryl esters, phosphatidylcholine, and SM showed the biggest differences. The most elevated species were plasma derived. The concentration of several plasma-derived lipids correlated with the F-score. A lower relative abundancy of these plasma-derived species compared to brain-derived species correlated with clinical recovery. Plasma infiltration into the CSF seemed to play the main role.Péter et al. investigated markers of demyelination through a shotgun lipidomic mass spectrometry-based approach. A total of 19 patients with GBS were compared to 34 controls with non-demyelinating disorders . In totaThese studies show the possible value of SM as a biomarker in demyelinating GBS. Especially interesting is the correlation with clinical severity. Further investigations, especially with larger cohorts, are needed.p = 0.001). Cystatin C was significantly lower in GBS than in controls (p < 0.001) [Cystatin C is currently widely used in the clinic to assess the glomerular filtration rate  and was < 0.001) ,115. Ano< 0.001) . Those sp = 0.05). A correlation to the F-score could not be shown. Zhang et al. tested transthyretin CSF levels in 19 GBS patients with an ELISA approach [p < 0.05). A correlation to functional scores was not found. In conclusion, transthyretin as a biomarker for GBS needs further investigation due to the contradictory findings with both increased and decreased values. Because of the missing correlation to clinical scores, its value seems to be subordinate compared to Nf and SM.Transthyretin is a plasma protein predominantly synthesized in the liver. Until now, there are more than 60 mutations known to cause systemic amyloidosis . In routapproach . In compp = 0.05). Even a significant difference when compared to chronic inflammatory demyelinating polyneuropathy (CIDP) could be shown . A correlation with the F-score could not be shown.Chang et al. identified potential biomarkers through 2-dimensional gel electrophoresis and MALDI-TOF mass spectrometry in the CSF of 24 patients with GBS . Haptoglp < 0.001). A correlation with the F-score was not found. Li et al. found a 1.66-fold overrepresentation (p < 0.001) of haptoglobin in the above-mentioned study. No significant difference between AIDP and AMAN was found. In conclusion, haptoglobin seems to be a potential biomarker for GBS. A shortcoming, compared to other markers is the missing correlation to clinical severity. However, further investigations, especially due to the small cohort size, seem necessary.The aforementioned paper by Jin et al. found a significant elevation in the CSF of five GBS patients by 2-dimensional gel electrophoresis followed by MALDI-TOF mass spectrometry. A further quantification or correlation with clinical data was not performed. A similar result with related methods was found by Lehmensiek et al. . ThroughTau is a protein expressed in neurons of both the central and peripheral nervous system. Although the abnormal aggregation of tau into so-called neurofibrillar tangles is one of the major hallmarks in a variety of neurodegenerative diseases, its definite function remains elusive . Due to p < 0.01).Süssmuth et al. evaluated tau CSF levels using a sandwich ELISA in 61 patients with a multitude of neurological disorders, including five patients with GBS . Four ofp = 0.008). Wang et al. measured tau CSF levels in 43 patients with GBS using a sandwich ELISA and compared them to a group of patients with other neurological diseases [p < 0.001) was found in both the AIDP and AMAN subgroups. A correlation of tau levels in CSF was found with the clinical severity in AMAN .In the above-mentioned study by Petzold et al., CSF tau levels in 38 GBS patients were measured using ELISA. When compared to controls with ONDs, a significant difference in tau levels was not found. However, a correlation of tau levels with F-score as a measurement for the outcome was found  and non-inflammatory polyneuropathies . Sainaghi et al. tested the CSF of nine GBS patients, eight CIDP patients, and seven controls for concentrations of 50 different cytokines [p < 0.002).As mentioned above, cytokines are essential mediators in inflammatory processes and are secreted by various immune cells like T and B lymphocytes, endothelial cells, mast cells, and macrophages ,45,46 anytokines . CXCL10 The investigations of cytokines show promising results in the differentiation of GBS and CIDP, which is a highly relevant question due to its different therapeutic approaches. However, further investigations in greater cohorts seem necessary.M1 antibodies. However, the typical antibody is only present in about every second patient, so there is a need for more biomarkers. Furukawa et al. investigated the CSF levels of 28 different cytokines using a multiplex bead array assay and ELISA in 12 patients with MMN, eight with PMA, 26 with ALS, and 10 control patients with other neurological disorders [MMN is an inflammatory disease caused by a focal destruction of the myelin sheath of the lower motor neurons. Clinically, it is manifested by weakness and atrophy of the innervated muscles. MMN is an important differential diagnosis compared to motor neuron diseases like lower motor neuron predominant ALS and PMA due to its promising treatment options compared to those degenerative diseases . Typicalisorders . In PMA,Borrelia burgdorferi s. l. The spirochetal infection causes a variety of symptoms including skin manifestations, lymphocytic meningitis, and peripheral neuropathies. A diagnosis of neuroborreliosis is usually supported by an elevated cell count in CSF and a ratio of IgG anti-Borrelia antibodies greater than two in comparison between CSF and serum. It was found that the criterion of elevated CSF antibodies only has a sensitivity of 75% and a specificity of 97% [Neuroborreliosis is caused by y of 97% . Therefoy of 97% . A magneSMN1 and SMN2, encode SMN [SMN1, leading to a low abundance of SMN protein. The severeness of the SMA phenotype correlates with the number of SMN2 copies the patient has [SMN2; onasemnogene abeparvovec, a non-replicating adeno-associated virus capsid delivering a SMN1 copy; and nusinersen, which is an antisense oligonucleotide increasing the protein level of SMN by modulating the splicing of SMN2-mRNA [Spinal muscular atrophy (SMA) is an autosomal-recessively inherited disease characterized by a severe loss of lower motor neurons due to low levels of the survival motor neuron (SMN) protein . Two gencode SMN . SMA patient has . Five diient has . Currentient has : risdiplMN2-mRNA . Until nAlb). Both values are measures of an intact blood–CSF barrier [Alb values were caused by the repeated intrathecal administration of nusinersen and the repeated lumbar punctures [Alb value and total protein level in CSF are age-related [Besides protein biomarkers, there are different routine parameters often evaluated in CSF samples of SMA patients. Total protein concentration in CSF is regularly determined as well as the quotient of albumin levels in CSF and serum  are concerned as “increased”. The researchers use the cell count as an indicator of possible inflammation caused by the repeated injection of nusinersen. The number of cells is commonly measured via manual counting after Pappenheim staining is performed. In the studies included in this review, only a few patients showed an increased number of white blood cells in the CSF and the few findings were not thought to be treatment-related ,135. NevIn the past, several research groups have evaluated proteins as potential biomarkers for the progression of SMA over the course of nusinersen treatment. As reports about protein biomarker levels in response to nusinersen treatment are still quite rare, we also included proteins only investigated in a single study. We focused on research about aβ-peptides, tau protein, glial fibrillary acid protein (GFAP), and Nfs  and are a major component of the AD-plaques. In a recently published study, the protein levels of aβ 40- and aβ 42 of eight patients affected by SMA type 2 or 3 were monitored over 14 months of nusinersen treatment . While tAn additional protein known to be associated with AD progression and investigated as a potential biomarker for the monitoring of SMA treatment is the microtubule-associated protein tau . For SMAGFAP is a well-known marker for astrocytes in the CNS. Therefore, it is often used to investigate neuroinflammatory processes. GFAP level in CSF samples of 12 SMA type 1 patients were found to be significantly increased at the start of the investigation period compared to healthy controls . AlthougAs for GFAP, an age-related change in the protein levels in CSF was also reported for Nfs . In geneSurprisingly, there is currently only one study published in which the proteome in CSF of SMA patients was investigated using mass spectrometry . UnfortuHow such a study could be set up is graphically summarized in In this review, a variety of neuromuscular diseases have been discussed, involving the degeneration of motor neurons. The review focused on the utilization of CSF as a suitable bodyfluid for the detection of biomarkers, not only to identify the disease, but also to determine disease progression. The most promising biomarkers so far are Nfs, specifically the combination of NfL and pNfH for the identification and determination of disease progression in ALS, as levels measured by ELISA are significantly higher than in other neurological diseases and ALS mimics. Furthermore, this method is already routinely used in the clinic. However, it must be mentioned that all measurements are subject to strong fluctuations and that defined criteria for CSF withdrawal and ELISA assays must be formulated to ensure a reproducible detection. An interesting alternative for ALS diagnostics could be the quantification of TDP43, especially if the hyperphosphorylated form is included. To ensure robust quantification of these biomarkers, alternative methods besides the classical ELISA should be considered, especially the enrichment of pathological hyperphosphorylated forms and their absolute quantification using targeted mass spectrometry. In the group of peripheral neuropathies, a multitude of biomarkers has been investigated. In our opinion, for GBS the most promising candidates are SM for AIDP and Nf for AMAN. In Lyme neuroborreliosis and MMN, the examination of cytokines seems promising not only to ease the diagnosis of neuroborreliosis, but also to identify MMN as an important, treatable differential diagnosis to ALS. For SMA, no convenient biomarker in CSF can be reported that can be used for treatment monitoring different SMA types. Especially for SMA types 2 and 3, biomarkers for treatment monitoring are needed, as the benefit of the treatment needs to be ensured due to the immense costs of all FDA-approved drugs.