Postoperative cerebrospinal fluid (CSF) leakage following endoscopic endonasal surgery (EES) is a frequent complication. This study aims to identify potential risk factors of postoperative CSF leakage.A retrospective review of 360 patients who underwent EES was included. The associations between postoperative CSF leakage and patient demographics, medical history, tumor characteristics, and intraoperative repair techniques were analyzed; the diagnosis and repair of postoperative CSF leakage were also introduced.Postoperative CSF leakage occurred in 14 patients (3.9%), 2 of them cured by lumbar cistern drainage, 12 underwent endoscopic repair. Among these 12 cases, 3 were repaired twice, and the rest were cured the first time. During the repair surgery, insufficient embedded fat was detected in one case detected, seven with breached inner artificial dura, three had vascularized pedicle nasoseptal flap (VP-NSF) displacement, two with VP-NSF perforation, two with VP-NSF inactivation, and one with imperfect adherence to VP-NSF to the skull base. Eight cases had intracranial infections. Excluding one case who died of severe intracranial infection, the rest were cured and discharged without obvious sequelae. Multivariate analysis revealed that the suprasellar lesion, subarachnoid invasion, and intraoperative grade 3 flow CSF leakage were the risk factors of CSF leakage after operation, while the bone flap was a protective factor.Bone flap combined with VP-NSF and iodoform gauze for skull base reconstruction is recommended in high-risk patients, while postoperative lumbar cistern drain remains dispensable. Cerebrospinal fluid (CSF) leakage is a pathologic condition where CSF flows out from defects of the dural and skull base, and it can be caused by a multitude of different factors, mainly including trauma  and endoWhile several studies have reported the factors affecting postoperative CSF leakage, discussion related to the reconstruction defects is really rare. Here, we investigated the risk factors of postoperative CSF leakage through systematic analysis and discussed the defects in reconstruction technique. We hope our research could serve as a reference for the progression of EES.n = 57), pituitary adenomas (n = 264), tuberculum sellae meningioma (n = 32), and Rathke’s cysts (n = 7).A total of 360 patients who underwent EES were selected from the Department of Neurosurgery, the First Affiliated Hospital of Nanchang University, including 184 males and 176 females. Ages ranged from 4–81 years, with an average of (46 ± 14) years, including craniopharyngiomas (in situ (ISBF), the details are described in the article by Jin et al.  or a free mucosal flap was made according to the surgery approach (classic EES or expend EES). After harvesting the NSF, the posterior nasal tract was opened and the nasal septum bone flap was made; the details are described in our previous article . Wide opn et al.  with intn et al. . See Fign et al. .β-2 transferrin  Patients with clear liquid flow out from the nasal cavity after operation. CSF routine test, biochemical parameters , β-2 transferrin , and β-t protein  examinatt-test was used for comparison between the two groups. The number of cases or percentages is expressed in the classified data. Chi-square tests were used for comparison between groups, and group comparisons were made with chi-square or Fisher’s exact test in cases with a small number of expected outcomes. All independent variables thought to be of clinical significance a priori were placed into a logistic multiple regression model. P value <0.05 was considered statistically significant for all statistical tests.All statistical analyses were performed using SPSS version 26 . The continuous variables conforming to the normal distribution were expressed by mean ± standard deviation (M ± SD). An independent-sample The statistical steps were mainly divided into two steps: first, age, gender, and tumor type were included as influencing factors in the univariate and multifactor analysis, and then the univariate and multifactor analysis were done separately for different types of tumors.Of the cases, 3.9% had postoperative CSF leakage (14/360), including four cases of craniopharyngioma, eight cases of pituitary adenoma, and two cases of sellar tubercle meningioma. Intraoperative CSF leakage of grade 3 was found in 10 cases, grade 2 in 3 cases, and grade 1 in 1 case. Among these, nine cases of postoperative CSF leakage occurred within 14 days, five cases occurred within 15–30 days, and the average time was (13 ± 5) days. Among all, 2 cases were cured by lumbar drainage, and the other 12 cases were explored under endoscopy. We found that the leading cause of postoperative CSF leakage was inadequate reconstruction, including one case with insufficient embedded fat , seven cTwo of the 14 patients with postoperative CSF leakage were cured by lumbar cistern drainage (LD). Twelve patients underwent endoscopic exploration and repair. The principle of repair was to determine the causes and then repair. When the cause was determined to be insufficient embedded fat, refilling the subdural leak with fat is necessary . When faP = 0.003] or subarachnoid space invasion ; intraoperative grade 3 CSF leakage flow was the risk factor CSF leakage after EES , while bony reconstruction  was the protective factor. Tumor types, balloon support, postoperative LD, and sellar lesion were not significantly correlated with CSF leakage after the operation , and the leading causes of postoperative CSF leakage were insufficient subdural and epidural reconstruction in multilayer skull base reconstruction. The details are as follows: (1) Insufficient subdural reconstruction due to the inadequate embedded fat and inlaid artificial dura. Inlaid artificial dura is easily washed away in grade 3 CSF leakage flow from the suprasellar arachnoid cistern and even the third ventricle. (2) Insufficient epidural reconstruction, including displacement, necrosis, perforation of the VP-NSF, and imperfect adherence of VP-NSF to the skull base. Displacement of VP-NSF is always caused by improper support of the balloon and inadvertent removal of nasal packing. VP-NSF necrosis usually resulting from impaired vascular pedicle, including irregular nasoseptal flap (NSF) harvesting, high-pressure nasal packing, and sharp bone protuberances of the sphenoid sinus.Multivariate analysis revealed suprasellar lesion as a risk factor for postoperative CSF leakage. The possible reason is that the suprasellar lesion invades the suprasellar arachnoid cistern or even the floor of the third ventricle; tumor removal might result in opening of the floor of the three ventricles and the suprasellar arachnoid cistern while causing a large skull base defect, which in turn leads to intraoperative grade 3 flow CSF leakage, which leads to the occurrence of postoperative CSF leakage.P = 0.007), and revealed a significantly increased risk of postoperative CSF leakage in patients with incomplete arachnoid structures. In our data, only 4 out of 14 cases of CSF leakage did not develop subarachnoid invasion. Suprasellar tumors, especially tuberculum sellae meningiomas, were difficult to keep the arachnoid intact after lesion removal because of the tumor consistency, and even accompanied with injury to the brain tissue and perforating vessels  possibly due to a larger defect in the sellar diaphragm in patients with intraoperative grade 3 flow CSF leakage and the intraoperative opening of the suprasellar cistern. Therefore, once extensive leakage was determined, a more aggressive treatment is required to prevent postoperative CSF leakage.Grade 3 flow CSF leakage was the risk factor . For patients without CSF leakage during operation, whether to use sellar bone defect reconstruction still remained uncertain. The need for bony reconstruction in patients without intraoperative CSF leakage was still inconclusive, and many surgeons did not consider bony reconstruction as a necessary step when without intraoperative CSF leakage .As for other factors including sex, age, diabetes, and hypertension, none of the above was significantly associated with postoperative CSF leakage on adjusted analysis (The main nasal packing support used before was a foley balloon catheter. The multivariate statistical results proved that its usage did not significantly reduce the occurrence of postoperative CSF leakage. Balloon support can significantly reduce the incidence of postoperative CSF leakage for patients with grade 3 flow CSF leakage during operation . HoweverPostoperative LD was used to prevent postoperative CSF leakage based on our experience that a multilayered skull base reconstruction approach with VP-NSF and fascia lata repair alone is inadequate for patients of intraoperative grade 3 flow CSF leakage. However, the statistical results showed that LD was not associated with postoperative CSF leakage. The literature remains unclear on the benefits and risk of postoperative LD as an adjunct in repairing grade 3 flow CSF leakage. Hu et al.  advocateThis study still has some limitations. Preoperative BMI values , postopeTo summarize, tumor invasion of the subarachnoid space, suprasellar extension, intraoperative grade 3 flow CSF leakage risk factors for postoperative CSF leakage, and bony reconstruction was a protective factor for postoperative CSF leakage. Attention should be paid to patients with high-risk factors. Meanwhile, skull base reconstruction should be given great importance after tumor resection. We recommend using bone flap combined with VP-NSF and iodoform gauze for skull base reconstruction in high-risk patients, avoiding routine postoperative using LD. Patients with suspected postoperative CSF leakage should be explored and repaired promptly.


The Song of Everlasting Sorrow  from the perspective of style. Using the keyword and concordance functions of corpus software packages AntConc and ParaConc, this research focuses on how the translator’s style reveals itself in the target text (TT) as well as how the style of the source text (ST) is represented in the TT. Findings show that the translators have a preference for contracted negative verb forms and expressions such as “bit” over “little,” making the text more colloquial. In general, the rendering of the ST style tends not to be entirely faithful. A distinction between reader-centered and text-centered keywords is made. While the reader-centered ST keywords are, as expected, largely altered, the translation of text-centered ST keywords is also rewritten, contrary to expectations. Presumably, the translators tended to reduce the ambiguity of the ST, resulting in a more explicit TT. The article argues that the translators chose to rewrite the translation to make it more understandable for the target audience since it concerned a work from a source culture very different from the target culture.This article reports on a corpus-based study of the English translation of Wang Anyi’s award-winning novel,  The Song of Everlasting Sorrow by Michael Berry and Susan Chan Egan of the Chinese novel 长恨歌 [Chang Hen Ge] by Wang Anyi. A mixed-method methodology is applied using corpus techniques (AntConc and ParaConc).This study investigates how the style of a source text (ST) is represented in a target text (TT) as well as how the translator’s style reveals itself in the TT. The corpus of this research is the English translation of responding to the source text [style]”  and published in 1995. It was translated into English by Michael Berry and Susan Chan Egan as The Song of Everlasting Sorrow (2008). In 2009, the translation received an honorable mention for the Sixth Lois Roth Award for the Translation of a Literary Work, and in 2011 it was nominated for the Man Booker Prize.The novel According to Studies claim Berry and Egan’s translation is largely faithful. Some corpus-assisted studies, however, came to a different conclusion. Focusing on the application of degree adverbs, Although these studies show style changes, they only consider a specific, pre-selected group of words or only examine a small part of the novel, thus leaving much space for further discussion. The present study revisits these conclusions from the perspective of a corpus-based analysis.The keywords function of the AntConc generates a list of words by comparing a text with a reference corpus. The keyness feature shows the statistical significance of each occurrence. Usually, the list contains three types of keywords: proper nouns, indicators of the “aboutness” of a text, and style indicators , online.For this study, the ST and TT were paralleled at the sentence level using ParaConc , a softw2 The English-language reference corpus (ERC) applied to the exploration of the translation style (a result of the combination of the ST style and the translators’ style), was compiled from COCA sample data downloadable from https://www.corpusdata.org/formats.asp.3 While the sample contains texts of different genres, only works of fiction were fed into the ERC. The purpose of comparing the TT with a reference corpus justifies the selection of the fiction section of COCA as data for the ERC. Different from the aim of using the CRC to generate an ST keyword list that indicates the ST textual style, the application of the ERC was expected to generate a keyword list that contained both elements induced by the ST style transfer and those indicating the translators’ style. Since a translator’s style was “the translator’s characteristic use of language, his or her …preferred or recurring patterns of linguistic behaviour”  which was used to reveal the ST textual style, consists of four novels, selected according to the following criteria: (i) winners of the Mao Dun National Prize for Fiction, and (ii) translated into English.haviour” , p. 245,p < 0.0001 and Hardie’s Log Ratio as the keyword effect size measure with 2 as the threshold. AntConc produced a list of 192 keywords for the ST and 84 for the TT  and 严先生 (Mr. Yan). A problem is that the Chinese words 一些 [some], 有些 [some/a little], 一般 [as if/like] and 活跃 [lively] belong to more than one part-of-speech category. The word 一般, for example, can be an adverb or an adjective. These words are placed in the category in which they appear most frequently.Both ST and TT keywords included adverbs, verbs, and adjectives, but primarily nouns. The nouns (and pronouns in the ST list), which are “words that human beings would recognize” , online Ulysses showed a different style of adopting Chinese action verbs. Romeo and Juliet. Previous studies on style and translation have paid little attention to static verbs as a style indicator. Casino Royale, Different ways of categorizing keywords have been proposed to meet specific research goals . In theiWhile elements of the ST style were transferred into the TT, the translators also left their fingerprints on the TT. The translation of reader-centered ST keywords was essentially unfaithful, but a significant proportion of the text-centered ST keywords were also rewritten. The translators eliminated ambiguousness, leaving less room for other interpretations. This resulted in alterations in characterization and narration. The labels “mutation” and “modification” are taken from didn’t, couldn’t, wasn’t, wouldn’t, don’t, isn’t, doesn’t, and weren’t. Since the Chinese do not have contractions, these forms do not come from the ST. Although non-contracted forms are also used in the TT, they are significantly less frequent or equally frequent, as shown in wasn’t and weren’t occur less often than the standard forms in the TT, but this does not affect the general trend. All these contracted forms show a high effect size (over 6) compared with the “norm.” Since the contracted forms of negation are less formal or emphasized than their counterpart two-word forms, the translators’ preference of the latter suggests that they tend to opt for a more casual style of narration.First, the translators preferred the contracted forms of negation, as evidenced by the use of bit that enjoys the highest keyness in the TT keyword list apart from the contracted forms , although smaller than that of a bit of , also contrasts with its absence in the TT. This means that the translators prefer bit-constructions to little-contructions, although the latter is more widely used in American English. While bit is quite informal and mainly used in conversations, its adoption in the TT again indicates that translators tend to write in a casual style.As for the word orms see , the ananeither (94.80%), gradually (74.62%), bit (56.52%), and however (56.41%) are equivalent to the ST expressions, indicating that the TT style follows the ST to some extent. Moreover, the Chinese equivalent for the word gradually, 渐渐, and that of the mitigating adverbial phrase a bit (occurring 128 times), 有些, also features in the ST keyword list, suggesting further that the TT style is partly the result of an ST style transfer. Regarding the adjective proper, the expression proper young lady is used 17 times  as an equivalent of the Chinese word 淑媛 [proper young lady], which also shows the ST influence on the TT style. However, the word simply in the TT keyword list is largely (76.60%) applied due to the translators’ mediation and reduces the interpretation space for TT readers. This tendency is discussed in the following.Regarding the TT style, The ambiguity of the reader-centered ST words leaves readers plenty of room to develop their own interpretation. In the ST, degree adverbs such as 有点 [a little], 有些 [a little/some], 一些 [some], 一会儿 [a while], and 没什么 [no/little/few] express vague degrees or quantities; 觉着 [feel], 有着 [have], 带有 [have], as verbs of static or general meaning, are usually followed by abstract concepts that require active deciphering by the reader; 做人 [conduct oneself/be a proper person], 穿行 [go through], 无所谓 [do not care], and 活跃 [lively] are generally used figuratively; 好像 [as if/like] and 一般 [as if/like] imply uncertainty in speculation, judgment, or feelings. When these words occur, the narrator either sounds uncertain or gives only vague information. These words give the narrator and the characters, especially the male ones, a kind of feminine quality characterized by delicate perception and emotional mildness and restraint, reflecting the author’s intention to feminize Shanghai , p. 69.6 1–3 below, which show the translation of three adverbs, the keyness of which ranges from the highest to the lowest in the reader-centered keyword list  or verbs of sense or cognition  are static and do not describe observable actions. The whole fictional world seems to exist only in the minds of the characters and the narrator. Therefore, keeping this meaning of the verb in the TT may be necessary to stress the fictionality of the ST. However, the word was substituted in different contexts by English words of specific meaning, such as “crisscross,” “run,” “penetrate,” “move through,” and “shuttle back and forth.” As hard to avoid and indicates a causal relationship between the result introduced by it and the cause described or implied in the context. It also suggests that the language user’s evaluation of the result is negative  that are not under his control, leaving room for the reader’s own interpretation. The TT, on the other hand, does not imply this, and his action of stepping back looks very conscious.sometimes, carries no evaluative meaning, the counterpart of 总是, always, has a unique usage that both sometimes and 总是 lack. In some contexts, it does not literally mean that a particular action always happens but means that the action happens often. This hyperbolic use implies that the action or event is questionable or undesirable. Some occurrences of 总是 in the ST, if translated literally as always, could cause indeterminacy of meaning because it sounds as if the character or narrator were speaking hyperbolically. In this case, the translators chose other expressions or deleted the word to avoid ambiguity. It was kept if there was no possibility that the translation would be understood as a complaint about an undesirable event.It is worth noting that the translation of the frequency adverb 有时 [sometimes] contrasts with that of 总是 [always], the other frequency adverb in the ST keyword list, of which about 65% is either deleted or modified, as shown in The Song of Everlasting Sorrow”), there is little possibility that the translators were not able to decipher the polysemy of the above-mentioned Chinese words. Most likely this was for the sake of readability. In the “translators’ notes,” the two translators said that “[f]or the purpose of readability in English” and to save the challenge to the reader  that may indicate the translators’ style. Another point to be noted is that the register of the texts in the ERC, which covers both popular and serious literature, is not exactly the same as that of the TT, a novel of a serious theme. While we intended to use this reference corpus to reveal the general linguistic norm of the target language in the broad genre of fiction , future studies can build a reference corpus with texts of more comparable literariness to that of the TT to see if the results differ. We hope this preliminary research may constitute the first step toward more corpus-based studies on the translators’ style using the parallel-comparative model in the future.The original contributions presented in this study are included in the article/LM contributed in the literature review, data analysis, and references of the study. FP designed the project and the methodology. Both authors contributed to the “Discussion” section and approved the submitted version.


Evolving evidence suggests that measurement of cerebrospinal fluid (CSF) kappa free light chain (KFLC) synthesis has high diagnostic sensitivity and specificity for multiple sclerosis (MS), but its prognostic ability is less investigated. The usefulness of KFLC in predicting cognitive impairment (CI) is still unknown.In a monocentric longitudinal retrospecitve cohort study, KFLC-index  measured by latex-enhanced immunonephelometry was prospectively determined as part of the diagnostic workup in patients with early relapsing-remitting MS . The ability of KFLC-index to predict information processing speed (IPS) worsening as assessed with the symbol digit modalities test (SDMT) was investigated in univariable and multivariable models.In patients with KFLC-index>100 (n=31), 11 subjects (35.5%) showed reduced SDMT scores by ≥8 points at follow-up (mean follow-up time 7.3 ± 2.6 years), compared with their baseline scores (p=0.01). Baseline KFLC-index>100 was strongly associated with a higher hazard of SDMT score reduction at follow-up .Intrathecal KFLC synthesis has become an attractive diagnostic tool for MS. We show for the first time that in a real-world setting of early RRMS, KFLC-index predicted cognitive decline. Whether this predictive ability of KFLC-index also concerns other cognitive domains than IPS, warrants further investigations. Multiple sclerosis (MS) is a chronic, inflammatory, and demyelinating disease of the central nervous system (CNS) . In its Kappa free light chain (KFLC) index , a marker for intrathecal production of free kappa chains, has been increasingly recognized in recent years for its diagnostic –20 and phttp://www.msreg.net) (Patients with RRMS  (n=165),reg.net) . Excludereg.net)  and Hospreg.net) . Four pareg.net) . Magnetireg.net) . Data onBy an experienced MS nurse, patients completed the SDMT  within sMatched CSF and serum samples were obtained and consecutively analysed during the routine diagnostic work-up. Concentrations of KFLC in serum and CSF were measured using the N Latex FLC kappa kit, on an Atellica NEPH 630 instrument (Siemens), following the instructions by the manufacturers. The KFLC-index was calculated using the equation [(CSF KFLC/serum KFLC)/]. CSF- and serum albumin levels were analysed using the ALBT2 Reagent cassette on a cobas c module instrument (Roche). The CSF/serum albumin ratio was calculated as [CSF albumin (mg/L)/serum albumin (g/L)]. Board-certified laboratory technicians, who were blinded to the clinical status, using strict procedures for quality control and run-approval, performed the analyses. All analyses were performed at the Sahlgrenska Neurochemistry laboratory in Mölndal, Sweden.CSF-specific IgG-OCBs were determined using an in-house IEF method on 7.7% polyacrylamide gels and subsequent silver staining. Paired patient serum and CSF samples were run on adjacent lanes, and CSF-specific IgG-OCBs were defined as extra bands in the gamma-zone, which were not present in the corresponding serum sample. For quality control, a positive CSF sample with known CSF-specific OCBs was run on each gel. A cut-off value of IgG-OCB≥2 was considered positive.® ELISA kit; UmanDiagnostics AB, Umea, Sweden; Catalog # 10-7001 CE) as previously described  was measured by ELISA, as previously described  and CSF Tau (n=73). cNfL was analyzed using a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) method . Correlation between biomarkers and age was determined with the spearman’s rank correlation coefficient. In accordance with our results as well as previous reports on the prognostic value of KFLC-index -transformed continuous variables. The 4th quintile for cNfL in our cohort was 910 ng/L whereas for CSF Tau, it was 211 ng/L. Results of the other confounding covariates are presented only for the model including KFLC-index>100 as a predictive variable, as they did not substantially differ in the other models. Due to the exploratory nature of the study, no correction for multiple comparisons in the survival analyses was made. Statistical significane was assumed at p<0.05. All statistical analyses were performed with IBM SPSS version 28.0.1.0 . Figures were created in GraphPad prism version 9.1.0.Data distribution was assessed with the Shapiro-Wilks test, and presented as mean ± SD or as median and interquartile range (IQR). The Mann-Whitney U test, unpaired T test, χLC-index , 22, we LC-index , and treLC-index , 40, 41.LC-index . We adjuAll patients included in this study had given informed consent to be registered in the SMSreg. All individual data from the different sources were made anonymous to the authors by the replacement of the personal identity numbers by unique number codes for use in the present study. The study has been approved by the Swedish Ethical Review Agency (Dnr: 2020-06851).The study population included 77 RRMS patients, of which 54 (70.1%) were females, with a median (IQR) age at clinical onset of 32 (27.5-43) years. IgG-OCBs≥2 were present in 71 (92.2%) patients. Raw SDMT scores at last follow-up were reduced in 20 (26%) patients. KFLC-index, cNfL, and CSF Tau were non-normally distributed as was determined by the Shapiro-Wilks test of normality, whereas SDMT values at baseline and follow-up were normally distributed. Baseline demographic and clinical variables that significantly differed between patients with and without SDMT reduction were age, disease duration, total follow-up time, baseline EDSS and SDMT raw scores, as well as the number of T2 MRI lesions Table 1.The study population was dichotomized into patients with a high baseline KFLC-index  and those with a low baseline KFLC-index . Eleven of 31 patients (35.5%) with a high baseline KFLC-index performed worse, i.e. had a sustained reduction of the SDMT score by ≥8 points at follow-up/censoring  compared with baseline (p=0.01) Figure 1th quintile for cNfL (>910 ng/L) and CSF Tau (>211 ng/L), 30/77 (39%) and 32/73 (43.8%) respectively had increased CSF concentrations. In multivariable models, neither cNfL nor CSF Tau were significantly associated with higher hazard of SDMT reduction at follow-up  determined with lipid-specific oligoclonal IgM bands (LS-OCMB) on IPS worsening in RRMS . In factth quintile which has previously been used as cut-off value for prognostic purposes . Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.IR and JL contributed to conception and design of the study. IR organized the database, performed the statistical analysis, and wrote the first draft of the manuscript. SR wrote sections of the manuscript. IR, MA, LN, KB, HZ, and JL had major role in data acquisition. All authors contributed to the article and approved the submitted version.


Early and accurate detection of pathogens is important to improve clinical outcomes of bloodstream infections (BSI), especially in the case of drug-resistant pathogens. In this study, we aimed to develop a culture-independent digital PCR (dPCR) system for multiplex detection of major sepsiscausing gram-negative pathogens and antimicrobial resistance genes using plasma DNA from BSI patients. Our duplex dPCR system successfully detected nine targets  through five reactions within 3 hours. The minimum detection limit was 50 ag of bacterial DNA, suggesting that 1 CFU/ml of bacteria in the blood can be detected. To validate the clinical applicability, cell-free DNA samples from febrile patients were tested with our system and confirmed high consistency with conventional blood culture. This system can support early identification of some drug-resistant gram-negative pathogens, which can help improving treatment outcomes of BSI. Bacterial bloodstream infection (BSI) is a life-threatening condition and a major healthcare issue across the world. Gram-negative bacteria are the most common organisms that cause severe BSI -4 and arRapid identification of drug-resistant bacterial pathogens is essential to combat BSI. Blood culture is currently a gold standard for identifying bloodstream bacterial pathogens. With the introduction of automated blood culture systems, bacterial identification has become faster and more effective -13. Howe1417Recently, digital PCR (dPCR), the third generation PCR platform, has been developed. Its sensitivity is much higher than that of conventional PCR-based tools -22. dPCRIn this study, we aimed to develop a dPCR system for culture-independent, multiplex, and highly sensitive detection of major sepsis-causing gram-negative bacteria and the antimicrobial resistance genes, and to validate our system using the plasma DNA of BSI patients.The reference gram-negative bacterial strains and clinical isolates used in this study are listed in uidA and lacY for Escherichia coli [ompA for Acinetobacter baumannii [phoE for Klebsiella pneumoniae [ecfX for Pseudomonas aeruginosa [http://blast.ncbi.nlm.nih.gov/). Homology scores of the primer/probe sets for the resistance genes were calculated using the Clustal Omega software .The species-specific primer/probe sets for gram-negative bacteria used in dPCR were prepared by modifying the sequences which have been verified by previous studies; hia coli , ompA foaumannii , 26, phoeumoniae , and ecfruginosa Table 1)uidA and ruginosa -31. SpecdPCR experiments were performed using the QuantStudio 3D Digital PCR system  according to the manufacturer’s recommendations and the methodologies in a previous study . A finalA. baumannii ATCC 19606, E. coli ATCC 25922, K. pnuemoniae KCTC 12385, P. aeruginosa ATCC 27853, blaTEM and blaCTX-M-15 encoding E. coli cm241, blaNDM-1 encoding E. coli cm66, and blaIMP-1 encoding P. aeruginosa cmPA-1 were serially diluted from 50 pg/ μl to 5 ag/μl and applied 1 μl of each DNA to the dPCR reaction. For LOB test, cell-free DNA (cfDNA) was extracted from plasma of normal human blood  using the QIAamp Circulating Nucleic Acid Kit  and performed same dPCR using the blank cfDNA. LOB was defined as the number of positive signals measured in the cfDNA from normal blood as described elsewhere [To determine the LOD of our dPCR detection system, the genomic DNAs of lsewhere .Quantitative polymerase reaction (qPCR) was carried out using the ViiA 7 QuantStudio real time PCR system (Thermo Fisher Scientific) with the following cycle protocol: initial denaturation at 95°C for 10 min, 45 cycles of amplification at 95°C for 10 sec, 60°C for 20 sec, and a 72°C for 20 sec. The amplicon mixture contained 5 μl of TaqMan Fast Advanced Master Mix , 0.5 μl of same primer/probe sets used for dPCR and 5 μl of cfDNA from patients’ blood. All qPCR reactions were performed in triplicate.3 or a count of < 1,000 cells/mm3 with a predicted decrease to <500 cells/mm3 within the next two days as described elsewhere [Thirty whole blood samples were collected from fifteen febrile patients  showing clinical signs of neutropenic fever: body temperature ≥38.0°C and an absolute neutrophil count of <500 cells/mmlsewhere . Seven bompA for A. baumannii; ecfX for P. aeruginosa; phoE for K. pneumoniae; uidA and lacY for E. coli  genes, blaIMP and blaNDM-1  to 5 ag/μl (0.125 copies/μl), and applied to the dPCR system. The LOD was determined to be 50 ag (1.061 copies/μl) (A. baumannii DNA was 500 fg (9.031 copies/μl)  . To compopies/μl), indicat(500 ag) .A. baumannii (range 1-3), 2 for P. aeruginosa, 4.7 for K. pneumoniae (range 2-7), 3.7 for E. coliuidA gene (range 3-4), 3.7 for E. colilacY gene (range 0-10), 1.3 for blaTEM-type (range 0-3), 2 for blaCTX-M group-1, 4.7 for blaIMP-type (range 3-6), 1.3 for blaNDM (range 1-2), respectively  are as follows: 2 for ectively . When weectively .uidA primer-VIC and lacY primer-FAM probe sets clearly detected both uidA and lacY genes from E. coli  and performed dPCR using 25 pg of each bacterial genomic DNA mixture per reaction.  E. coli . For dupoE genes . For ideectively . We obsem241 DNA . In anotectively . Taken tE. coli, two for K. pnuemoniae, and one for P. aeruginosa) (E. coli and one P. aeruginosa) (E. coli (uidA and lacY genes), P. aeruginosa (ecfX), and K. pneumoniae (phoE) identified from blood samples of three patients  by duplex dPCR are illustrated in E. coli and two for P. aeruginosa), which were also consistently detected by duplex dPCR. However, of the six samples that were shown as bacteria negative by TaqMan qPCR, five were culture positive, suggesting that detection sensitivity of the TaqMan qPCR assay might be lower than that of the culture assay . In total, thirty cfDNA samples were used to test duplex dPCR system (five duplex reactions per sample). We found that 23 blood samples were culture positive (twenty for uginosa) . All culuginosa) . Examplere assay .Eight blood samples from febrile patients were detected with the antimicrobial resistance genes by the duplex dPCR . Of the Rapid identification of pathogens is essential to ensure appropriate antibiotic therapy and to improve clinical outcomes of BSI, especially in the case of drug-resistant bacterial infection -7, 9, 10E. coli, we designed a probe targeting the uidA gene, which encodes β-glucuronidase, a universal target for E. coli detection. However, due to the genetic similarity between E. coli and Shigella spp., the uidA alone cannot distinguish E. coli from Shigella spp [lacY, an E. coli-specific gene encoding lactose permease. For P. aeruginosa, since the ecfX gene has proved to be an appropriate P. aeruginosa-specific target [K. pneumoniae, we selected the phoE gene, which has been reported to be K. pneumoniae-specific [A. baumannii, the ompA gene was selected because of its specificity and sensitivity for detecting A. baumannii by real-time PCR [blaTEM-type, blaCTX-M group 1, blaIMP-type, and blaNDM-1). We confirmed the efficacy of all nine target genes using previously verified clinical isolates, indicating the accuracy of our system. The minimum detection limit of bacterial DNA by dPCR was 50 ag, which suggests that dPCR is 104 times more sensitive than qPCR (500 fg). In principle, the amount of genomic DNA in one E. coli cell is approximately 0.017 pg, suggesting that dPCR can detect 1 CFU/ml of bacteria in the blood. Given that 1-100 CFU/ml of bacteria exist in the blood of adult BSI patients [To improve sensitivity, we adopted a dPCR technique for identifying bacterial DNA and antimicrobial resistance genes. We designed dPCR primer/probe sets targeting major sepsis-causing gram-negative pathogens. For ella spp , 38. Thec target , 39, we specific . For A. time PCR , 20. We patients , 41, ourThe whole procedure of dPCR, from blood sampling to dPCR, took less than 3 hours, which fulfils the aim of this study. To avoid the total reaction time for multiple targets, we established a universal reaction condition for all the nine targets. In addition, we developed a duplex identification system for more efficient reaction, by mixing the target specific primer/probe sets with two different dyes, which enabled the detection of all nine targets by five duplex dPCR reactions.To examine the clinical applicability of this system, thirty cfDNA samples were tested with our duplex dPCR system and the results were compared with those from blood cultures. All blood culture results were consistent with those from our duplex dPCR system, suggesting high detection sensitivity of our system. Interestingly, seven culture negative samples (five patients) showed positive pathogen signals in duplex dPCR. It may be due to that the pathogens could have been inactivated by antibiotic treatment before the blood was sampled. In that case, although blood culture was negative, small amount of bacterial DNA that was released in the blood from the destroyed pathogens could be detected by dPCR. Indeed, four of the five (80%) discordant patients were sampled after the initiation of antibiotic treatment, while eight of the ten (80%) concordant patients were sampled before antibiotic treatment in this study . These rWe further compared the performance of duplex dPCR with that of TaqMan qPCR, the most commonly used PCR-based technique for bacteria detection. All the pathogens and antimicrobial resistance genes identified by TaqMan qPCR were consistently detected by our duplex dPCR assay. Six samples identified as bacteria-negative by TaqMan qPCR were defined as bacteria-positive by dPCR and five of them were positive in blood cultures suggesting that the inconsistencies would be due to higher detection sensitivity of dPCR than qPCR assay. These results are compatible with a previous report . The LODThere are several limitations in this study. First, two ESBL and two MBL genes, the antimicrobial resistance genes targeted in this study, cannot cover other important ESBL and MBL genes such as KPC gene, VIM gene, and various CTX-M subtype genes. Second, our system does not cover gram-positive pathogens and related antimicrobial resistance genes. There is no doubt that gram-positive pathogens are critically important pathogens of BSI. We plan to expand the targets of this duplex dPCR system to major pathogens across gram-positives and negatives, and also to expand the target genes for covering more clinically important antimicrobial resistance genes. Third, the number of clinical isolates, blood samples from BSI patients, and negative control samples might not large enough to support the performance of our system. Further larger scale validation will be required to verify the sensitivity, specificity and clinical utility of this technology.We developed a duplex dPCR based, culture-independent technique for identifying major BSI-causing gram-negative pathogens, and antimicrobial resistance genes in the cfDNA isolated from plasma of the patients. Our dPCR system detected nine targets  within 3 hours by five duplex dPCR reactions under universal condition. The duplex dPCR showed higher sensitivity than the TaqMan qPCR assays and blood cultures. To the best of our knowledge, this is the first dPCR system for screening major BSI-causing gram-negative bacteria, and major antimicrobial resistance genes together. This system can support early identification of some drug-resistant gram-negative pathogens, which can help improving treatment outcomes.http://jmb.or.kr.Supplementary data for this paper are available on-line only at


Surgical resection is the main method to treat pituitary adenoma. Cerebrospinal fluid leakage (CSF Leak) is the main complication after transsphenoidal surgery. The impact of postoperative CSF Leak can be predicted in advance, and preventive measures can be taken in time. Clinically, a variety of factors may affect the occurrence of postoperative CSF Leak. In this study, meta-analysis was used to investigate the risk factors of postoperative CSF Leak as a clinical reference.  The databases PubMed, Medline, Embrase, Cochrane library, CNKI, and CBM were searched for all studies on the risk factors of postoperative CSF Leak. Studies were screened and finally included. The quality of the included studies was assessed by the Newcastle-Ottawa scale. We used Revman 5.4 software to conduct the pooled effect size of every potential statistically significant factor. P = 0.20) was not a factor influencing postoperative CSF Leak, while BMI , P < 0.00001), tumor size , P = 0.02), whether a second operation was performed , P = 0.0002), and intraoperative CSF Leak , P < 0.00001) were risk factors for postoperative CSF Leak in patients. Discussion. BMI, tumor size, reoperation, and intraoperative CSF Leak are the risk factors of postoperative CSF Leak. However, not all the factors were covered in this study, it is still worth continuing to deeply investigate in this topic. 13 articles with a total of 5967 patients with pituitary adenoma and 405 cases of postoperative CSF Leak were finally included, accounting for 6.79%. All of the 13 articles had a quality score > 5, indicating good quality. Meta-analysis showed that patient age ,  Pituitary adenomas are benign tumors arising from the adenohypophyseal cells of the anterior pituitary gland, located in the saddle region in the middle of the base of the brain, and are characterized by slow growth, small local lesions, and nonmetastasis . AccordiAlthough most microadenomas are asymptomatic, when pituitary tumors cause pituitary hyperfunction or hypofunction, tumor space occupying effect, and pituitary tumor stroke, they will have a negative impact on the human body . The pitSurgical resection is the mainstay of treatment for pituitary adenomas, and endoscopic transsphenoidal surgery (ETS) has been widely used because of its less trauma and good safety, but this procedure also has complications such as bleeding, infection, cerebrospinal fluid leakage (CSF Leak), and decreased visual acuity . TransspHowever, the application of new technology cannot completely avoid the occurrence of surgical complications. The common complications of transsphenoidal surgery include cerebrospinal fluid leak, epistaxis, dysosmia, and sinusitis. CSF Leak is the main complication of transsphenoidal surgery, with an incidence rate between 0.5% and 14%, which may cause meningitis or tension pneumocephalus, increasing the risk of reoperation if no intervention is involved in time .Studying the effect of preoperative and intraoperative factors on the occurrence of postoperative CSF Leak can help to foresee the occurrence and development of CSF Leak after transsphenoidal surgery in advance and carry out prevention or treatment in time. In the meta-analysis by Zhou et al. , 34 obsePredictors/factors] AND [cerebrospinal fluid leak/CSF/Cerebrospinal fluid rhinorrhea] AND [pituitary adenoma] AND [endoscopic transnasal pituitary surgery/transsphenoidal surgery].The databases Embrase, Medline, PubMed, and Cochrane library were selected as English literature sources for this study, and CNKI and CBM were used as Chinese literature sources. The search was completed in December 2021. Search method: quick search of keywords; combination of keywords: [(1) All literatures were retrospective observational study papers, which were limited to cohort study or case-control study. There is no limit to the number of research centers. The randomized controlled study, case analysis, noncontinuous time series study, cross-sectional study, and case report study were excluded. (2) The patient was diagnosed with pituitary adenoma by transsphenoidal surgery. After the opening of nasal sphenoid sinus, the incision was made to expose the tumor and scrape off the tumor tissue. (3) CSF Leak: postoperative CSF Leak is defined as the outflow of clear fluid from the saddle or parasellar region within 1-7 days after surgery. Literatures in which the study purpose is intraoperative (non-postoperative) CSF Leak are excluded. (4) Literature review: the patients were divided into 2 groups for factor analysis: the group with postoperative CSF Leak and the group without postoperative CSF Leak, and complete data could be obtained.Two researchers independently completed the screening of literatures. After the literatures were searched by keywords, the title and abstract of the literatures were read to exclude the literatures that did not meet the requirements, and then, the full text of the literatures was obtained and read to further determine whether they were included. After this, 2 researchers performed a cross-check, and if there is a disagreement between two researchers, a third researcher is invited to step in to resolve it.After the included literatures were determined, two institutes further read the literatures; independently extracted the basic information, study characteristics, and outcome indicators of the literatures; and recorded them using excel table. If the data cannot be determined due to incomplete data, the original author should be contacted to obtain all the data; if the data can still not be obtained, the literature will be excluded. In order to facilitate the final statistical analysis, the data expressed as “%” will be converted to the actual number of cases. After completing this work, two researchers cross-checked the extracted data to resolve disagreements and determine the content of the data.Predictors), such as gender, age, BMI, tumor size, total resection or hemisection, intraoperative CSF Leak, operation time, cerebral edema, second operation, and preoperative chemotherapy. The information  of patients with this factor in different groups was obtained.The factors that may cause postoperative CSF Leak in the literatures were collected . You can see the site if you are interested.The Newcastle-Ottawa scale   was usedQ test was used to verify literature heterogeneity. P > 0.05 indicated no heterogeneity and good consistency. Fixed effects model analysis could be used to calculate OR the with Mantel-Haenszel method. If heterogeneity existed, random effects model analysis was used to calculate OR with the DerSimonian and Laird method. If fixed effects analysis was consistent with random effects analysis, sensitivity analysis result was stable. (4) Heterogeneity was investigated by subgroup analysis. (5) Funnel plot was used to show the results of publication bias analysis.(1) The analysis tool used is the Revman 5.4 software. (2) Use mean difference (MD) to report continuous variables (BMI and tumor size) and odd rate (OR) value and 95% CI to report binary variables , and use forest plot to present the results. (3) In this study, 584 relevant literatures were initially detected, 211 literatures were filtered out through repeated detection, and the remaining 373 literatures were included in the primary screening, and 13 literatures were finally included. The screening process is shown in 13 literatures were included in this study. The basic data, factors, and quality score of the literatures are shown in We present 6 excluded articles  after reading the full text, which were excluded mainly because of the following: (a) the study was for intraoperative CSF leakage, (b) no factor analysis, (c) no data available, and (d) patients with nonpituitary tumors, as shown in I2 = 46%, P = 0.14). Fixed effects model analysis showed that there was no significant difference in the proportion of patients with CSF Leak in different age groups , P = 0.20), as shown in Four literatures –13 reporI2 = 0%, P = 0.88). Fixed effects model analysis showed that the BMI of patients with postoperative CSF leakage was significantly higher than that of patients without CSF leakage , P < 0.00001), as shown in Seven literatures –20 reporI2 = 84%, P = 0.0003). Random effects model analysis showed that there was significant difference in tumor size between patients with postoperative CSF Leak and patients without CSF Leak , P = 0.02), as shown in Four literatures , 19–21 rI2 = 37%, P = 0.19). Fixed effects model analysis showed that the possibility of CSF Leak in the secondary surgery was higher , P = 0.0002), as shown in Four literatures , 21, 22 I2 = 81%, P < 0.0001). Random effects model analysis showed that the patients with intraoperative CSF Leak were more likely to have postoperative CSF Leak , P < 0.00001), as shown in Six literatures , 21, 22 In the analysis of tumor size, after the literatures were divided into 2 subgroups according to the calculation method of tumor size, there was no internal heterogeneity, which indicated that the calculation method of tumor size was the source of heterogeneity in the analysis of this factor. The results from the fixed effects model were the same as those from the random effects model in the remaining analyses, indicating that the results were stable.Publication bias analysis was not performed because few articles were included in the study .Endoscopic transsellar approach and craniotomy are the two main surgical resection methods for pituitary adenoma. The former is significantly less invasive than the latter, which is a reliable and minimally invasive surgery, but it may still produce complications including cerebrospinal fluid rhinorrhea, epistaxis, olfactory dysfunction, and sinusitis; cerebrospinal fluid leakage is one of the serious complications, which can increase the risk of postoperative intracranial infection . ThThe occurrence of CSF Leak may be related to patient's own characteristic factors, tumor characteristic factors, and surgical factors. The reports in various studies are not exactly the same. Therefore, in this meta-analysis, the CSF Leak caused by different factors was summarized and analyzed.The common factors affecting postoperative CSF leakage include age factor and body mass index (BMI) factor. In the 13 included literatures, gender was not the relevant factor of postoperative CSF Leak. Therefore, no summary analysis is conducted in this study. In this meta-analysis, there was no significant difference in the incidence of postoperative CSF Leak at different age levels, suggesting that age is not a relevant factor for the occurrence of postoperative CSF Leak. However, patients with a postoperative CSF Leak had a significantly higher BMI than those without, suggesting that patients with a higher body mass index (obesity) are more likely to have a postoperative CSF leak, consistent with the observations of some scholars , 28. TheMany studies , 20 haveOur study also found that the incidence of postoperative CSF Leak in patients undergoing reoperation was higher than that in patients undergoing the first operation ), which may be due to the formation of scar and tissue adhesion during the first operation, postoperative vascular proliferation, and tissue fibrosis, making it difficult to find the residual part of the tumor during reoperation, increasing the risk of CSF Leak by more invasive anatomical procedures during resection –33. Our ), which Some literatures reported that the type of tumor, texture of tumor, operation time, and preoperative cerebral edema were also risk factors of postoperative CSF Leak. However, there were few reported literatures, so we could not conduct a summary analysis. Although we found in this meta-analysis that age was not a risk factor for CSF Leak, it remains to be further determined because too few articles were included. In our study results, it was determined that BMI, tumor size, reoperation, and occurrence of intraoperative CSF Leak were related factors of postoperative CSF Leak, which were different from 4 related factors of tumor size, adenoma consistency, reoperation, and intraoperative CSF Leak obtained in the meta-analysis , which mI2 = 84%, P = 0.0003), but when we divided the 4 literatures into two subgroups according to different methods of tumor size assessment, the internal heterogeneity disappeared (P < 50%), indicating that the method of assessment was the source of heterogeneity. We also did not find serious publication bias in the publication bias analysis, but there were still few included literatures based on each risk factor, the coverage of risk factors was not comprehensive enough, and the risk factors for the occurrence of CSF Leak after transsphenoidal surgery for pituitary adenoma are still worthy of continued in-depth discussion.In the analysis of tumor size, we observed significant heterogeneity among the literatures (This study included 13 studies on the factors that influence CSF Leak after pituitary adenoma transsphenoidal surgery. According to the findings, BMI, tumor size, reoperation, and the presence of intraoperative CSF Leak were the risk factors for postoperative CSF leak. However, there are still few included literatures based on each risk factor, and the coverage of the risk factors is insufficient, and it is still worthwhile to continue to deeply investigate the risk factors for the occurrence of CSF Leak after pituitary adenoma transsphenoidal surgery.


Borrelia species PCR on CSF. In total, 156 patients were included who were classified as definite LNB (n = 10), possible LNB (n = 7), or non-LNB patient (n = 139) according to the criteria of the European Federation of Neurological Societies using a consensus strategy for intrathecal Borrelia-specific antibody synthesis. The seven antibody assays showed sensitivities ranging from 47.1% to 100% and specificities ranging from 95.7% to 100%. RF modeling demonstrated that the sensitivities of most antibody assays could be improved by including other parameters to the diagnostic repertoire for diagnosing LNB (range: 94.1% to 100%), although with slightly lower specificities (range: 92.8% to 96.4%). The most important parameters for LNB diagnosis are the detection of intrathecally produced Borrelia-specific antibodies, two-tier serology on serum, CSF-CXCL13, Reibergram classification, and pleocytosis. In conclusion, this study shows that LNB diagnosis is best supported using multiparameter analysis. Furthermore, a collaborative prospective study is proposed to investigate if a standardized diagnostic algorithm can be developed for improved LNB diagnosis.Laboratory diagnosis of Lyme neuroborreliosis (LNB) is challenging, and validated diagnostic algorithms are lacking. Therefore, this retrospective cross-sectional study aimed to compare the diagnostic performance of seven commercial antibody assays for LNB diagnosis. Random forest (RF) modeling was conducted to investigate whether the diagnostic performance using the antibody assays could be improved by including several routine cerebrospinal fluid (CSF) parameters , two-tier serology on serum, the CSF level of the B-cell chemokine (C-X-C motif) ligand 13 (CXCL13), and a IMPORTANCE The diagnosis of LNB is established by clinical symptoms, pleocytosis, and proof of intrathecal synthesis of Borrelia-specific antibodies. Laboratory diagnosis of LNB is challenging, and validated diagnostic algorithms are lacking. Therefore, this retrospective cross-sectional study aimed to compare the diagnostic performance of seven commercial antibody assays for LNB diagnosis. Multiparameter analysis was conducted to investigate whether the diagnostic performance using the antibody assays could be improved by including several routine (CSF) parameters. The results of this study show that LNB diagnosis is best supported using the detection of intrathecally produced Borrelia-specific antibodies, two-tier serology on serum, CSF-CXCL13, Reibergram classification, and pleocytosis. Furthermore, we propose a collaborative prospective study to investigate the potential role of constructing a diagnostic algorithm using multiparameter analysis for improved LNB diagnosis. Borrelia burgdorferisensu lato, is the most common tick-borne disease in temperate regions of the Northern Hemisphere , joints (Lyme arthritis), or heart (Lyme carditis), or cause acrodermatitis chronica atrophicans (ACA) (–Lyme borreliosis (LB), which is caused by misphere . LB is amisphere , 2. If uns (ACA) . In Eurons (ACA) –7 and ofs (ACA) –, 8. In ts (ACA) –.B. burgdorferisensu lato together with the presence of pleocytosis (≥5 leukocytes/μL) in the cerebrospinal fluid (CSF)  recommends the detection of an intrathecal immune response to id (CSF) . Proof oderation , 10. Thederation –13. For eration –, 14–18. ver time –21, the er time –, 15, 22.er time –, 24. Furnsu lato –30 and/osu lato –. A negatsu lato –, 33. A psu lato –, 35.Borrelia-specific antibody synthesis for LNB diagnostics is based on either the detection of these antibodies in CSF-serum pairs and subsequent calculation of a Borrelia-specific antibody index (AI)  ligand 13 (CXCL13)  14–, 36–38 oI) 14–36––41. Manygnostics , 38, 40.(CXCL13) , and a BIn total, 150 (13.7%) of 1,098 consecutive patients who underwent at least one LP in the predefined study period were included in the current study. Six additional LNB patients were included from outside the predefined study period, all of whom had taken part in two other studies , 44. DetBorrelia-specific antibody synthesis determined by the CSF-serum assays) needed for the classification of patients are shown in Table S2. The number of possible and definite LNB patients in this study differed slightly from that in our previous study (Borrelia-specific antibody synthesis was based on either a consensus strategy (this study) or the IDEIA results (previous study).All patients were classified as definite LNB, possible LNB, or non-LNB patient based on the EFNS guidelines  and consus study , as intrBorrelia-specific antibody synthesis according to the consensus strategy. A total of 139 (89.1%) of the 156 patients were classified as non-LNB patient. Thirty-nine (28.1%) of them had clinical symptoms suggestive of LNB, of whom 36 (92.3%) had neither pleocytosis nor intrathecal Borrelia-specific antibody synthesis according to the consensus strategy and 3 (7.7%) had pleocytosis only. For these three patients, another cause for their symptoms was found (Table S2). One hundred (71.9%) of the 139 patients classified as non-LNB patient did not have clinical symptoms suggestive of LNB. Eighty-one of them had neither pleocytosis nor intrathecal Borrelia-specific antibody synthesis according to the consensus strategy, and the remaining 19 had pleocytosis only.Ten (6.4%) of the 156 patients were classified as definite LNB patient, and 7 (4.5%) of the 156 patients were classified as possible LNB patient . Of the Borrelia species PCR result on CSF was found among definite LNB patients more often than among possible LNB and non-LNB patients, and this was significantly more often than among non-LNB patients. CSF-CXCL13 positivity was found among definite LNB patients significantly more often than among possible LNB and non-LNB patients. No significant differences were observed among the three study groups with regard to gender, age, symptom duration, and CSF-glucose levels.Borrelia-specific immunoglobulin (Ig)M, IgG, and the combined IgM and IgG results, hereafter referred to as overall Ig results, was comparable between definite and possible LNB patients. These percentages were significantly higher among those two patient groups than among non-LNB patients, except for the percentage of positive test results for Borrelia-specific IgM between definite LNB and non-LNB patients.All definite and possible LNB patients had a positive  C6 enzyme-linked immunosorbent assay (ELISA) result on serum, and this was significantly higher than that among non-LNB patients . Two-tieMeasurement of CSF and serum albumin concentrations and the subsequent calculation of the CSF/serum quotient for albumin (Q Alb) provided insight into the functionality of the blood-CSF barrier. A dysfunctional blood-CSF barrier was found among definite LNB patients more often than among possible LNB and non-LNB patients, and this was significantly more often than among non-LNB patients .Intrathecal total antibody synthesis among definite LNB patients was based on an IgM with or without an IgG response (Table S2). Intrathecal total antibody synthesis among possible LNB patients was based on a solitary IgM response and among non-LNB patients based on an IgM and/or IgG response. Intrathecal total IgM synthesis was found among definite and possible LNB patients significantly more often than among non-LNB patients . IntrathBorrelia-specific IgM, IgG, or both among definite LNB patients was significantly higher than that among non-LNB patients , a positive Borrelia-specific AI result was based on an IgG response with or without IgM (Table S2). Of the possible LNB patients with pleocytosis (n = 4), two had a positive Borrelia-specific AI result for IgM only in either one or two CSF-serum assays. Twelve (8.6%) of the 139 non-LNB patients had a positive Borrelia-specific AI result for IgM and/or IgG in a minority of the CSF-serum assays (Table S2).For all definite LNB patients, and for all possible LNB patients without pleocytosis (recomLine immunoblot (IB) on CSF, IgM was not detected at all. RecomLine IgG IB positivity using the revised interpretation criteria of the manufacturer (Table S3), which were elaborated on throughout this article, was more common among definite LNB patients than among possible LNB and non-LNB patients, although this was significant only between definite LNB and non-LNB patients  (Table S2). C6 ELISA positivity in CSF among possible LNB patients was comparable to that in serum  (P = 1.000). Among non-LNB patients, however, C6 ELISA positivity in CSF was significantly lower than that in serum  .Detection of recomLine IB positivity in CSF among definite LNB patients was comparable to that in serum  (Table S2) (P = 1.000). Among possible LNB patients, the overall Ig recomLine IB positivity in CSF was lower than that in serum  (P = 0.074). Among non-LNB patients, the overall Ig recomLine IB positivity in CSF was significantly lower than that in serum  .The overall Ig recomLine IgM IB) to 50.0% (Enzygnost IgM ELISA) (recomBead IgG assay) . The specificities of all IgM assays were 100%, except for the Enzygnost IgM ELISA for which the specificity was 99.3% (The sensitivities of the IgM assays to diagnose LNB among definite and possible LNB patients ranged from 0.0% (M ELISA) . The senG assay) . The oveG assay) . The oveas 99.3% . The speas 99.3% . The speas 99.3% .recomLine IgM IB, for which the PPV could not be calculated due to the absence of positive IgM results (recomLine IgG IB) to 100% (IDEIA and Medac ELISA) (recomLine IgM IB) to 96.5% (Enzygnost IgM ELISA)  of the Enzygnost IgM ELISA was 83.3%, and the PPVs of all other IgM assays were 100%, except for the  results . For thec ELISA) . The PPVc ELISA) . The negM ELISA) . For theM ELISA) . The NPVM ELISA) . The PPVM ELISA) .recomLine IB RF models). The sensitivities and NPVs of most RF models were higher than the upper limit of the respective 95% CIs obtained using the results of the antibody assays only, except for the C6 and the Enzygnost ELISA (Random forest (RF) modeling was performed to investigate if the diagnostic performance using the antibody assays could be improved by including the results of other parameters. The seven RF models performed comparably well, which was reflected by the areas under the curve (AUCs) that ranged from 0.970 to 0.993 . The prost ELISA  and B. IBorrelia species PCR on CSF contributed the least , the sensitivity of the seven antibody assays to diagnose LNB among definite and possible LNB patients showed the largest variation (range: 47.1% to 100%), although none of the differences were statistically significant. In general, differences in sensitivity between antibody assays can be influenced by several factors, such as the antigens present in the assay , 38, 46.ariation –31. Overriation –, 23, 24.Borrelia-specific IgM response in two possible LNB patients with pleocytosis, underlining the need for both IgM and IgG testing in LNB diagnostics, as was mentioned before  positive test results can be expected among controls as well, which leads to a lower specificity. Positive test results among non-LNB patients, which were found mainly for the IgG CSF-serum assays, indeed underline the positive correlation between the number of antigens present in the assay and the number of positive test results. Furthermore, antibody assays based on whole-cell lysates can generate false-positive test results due to the presence of cross-reactive antigens . Two nontibodies , 50. As t likely , 48.Borrelia-specific antibody synthesis or be the result of passive diffusion of these antibodies from the blood or of a traumatic LP. Of the two CSF-only assays tested in this study, the C6 ELISA performed best and might be useful as a screening assay since the NPV was 99.3%. Positive C6 ELISA results on CSF, however, should be confirmed using a CSF-serum assay and subsequent AI calculation that corrects for a dysfunctional blood-CSF barrier to prove intrathecal Borrelia-specific antibody synthesis.Considering that the use of a CSF-serum assay and the calculation of an AI is rather complicated, an assay tested on CSF only would be more convenient and preferable in routine clinical practice. The interpretation of positive test results using a CSF-only assay, however, is complicated because a positive test result can be caused by intrathecal Borrelia-specific antibody synthesis, the results of other parameters, such as an elevated CSF leucocyte count  have been taken off the market. This was partly caused by the new, more stringent quality requirements for ed costs . This deed costs  and makied costs .Borrelia-specific antibody synthesis, which, to our knowledge, has not been reported before.The main strengths of this study are the cross-sectional design  and the Borrelia-specific antibodies using a Reiber-based AI calculation, two-tier serology on serum, CSF-CXCL13, Reibergram classification, and pleocytosis. Furthermore, a collaborative prospective study is proposed to investigate if a standardized diagnostic algorithm can be developed using multiparameter analysis for improved LNB diagnosis.In conclusion, this study shows that LNB diagnostics is best supported using an approach that includes the detection of intrathecally produced Retrospectively, and regardless of their clinical presentation and age, all consecutive patients were eligible for inclusion if a CSF sample and a blood sample (drawn within 24 h of the LP) had been sent to the microbiology laboratory of the Diakonessenhuis Hospital, Utrecht, the Netherlands in the period between August 2013 and June 2016. Until the start of this study in 2017, leftover material from these patients had been stored at −20°C and/or −80°C. Prior to the start of this study, all samples had been freeze-thawed once to aliquot for use in the various assays in this study as well as for another study , which rAll CSF-serum pairs used in this study were anonymized. Approval of the local ethics committee was not necessary, as the main goal of our study was to compare various antibody assays and assess if additional parameters could improve LNB diagnostics for which leftover material can be used. We did, however, obtain approval from the hospital board. The results of this study are reported following the guidelines for diagnostic accuracy studies .Borrelia-specific antibodies. Possible LNB patients should have clinical symptoms suggestive of LNB with either pleocytosis or intrathecally produced Borrelia-specific antibodies. Clinical symptoms suggestive of LNB  were assumed to be present when a request for the detection of intrathecal Borrelia-specific antibody synthesis was done at our laboratory at the time of active disease in the past for which the IDEIA LNB IgM and IgG assay  was used. If the second and/or third criterion  was also fulfilled, another cause for the symptoms had to be excluded according to the EFNS guidelines. To minimalize the bias in patient classification by using only the IDEIA results, a consensus strategy for proof of intrathecal synthesis of Borrelia-specific antibodies was applied. This strategy entailed that intrathecal Borrelia-specific antibody synthesis was considered proven only if the majority of the antibody assays tested on CSF-serum pairs in this study showed a pathological Borrelia-specific (IgM and/or IgG) AI value .The EFNS guidelines  were useBorrelia antigens, and different quantification methods (Table S3). For five assays, referred to here as CSF-serum assays, the detection of intrathecally produced Borrelia-specific IgM and/or IgG was done by testing the CSF and serum of each patient simultaneously. Results of these CSF-serum assays were used to calculate a Borrelia-specific AI value as described below. For two assays, Borrelia-specific IgM and/or IgG antibodies were determined in CSF only and are referred to here as CSF-only assays. The seven antibody assays were performed according to the respective manufacturer’s instructions, unless otherwise specified here.Seven commercial antibody assays were selected, which were based on different techniques, different Borrelia-specific antibody synthesis was not done at the time of active disease in the past were tested in batch. CSF-serum pairs were tested only in the singular due to the limited amount of sample material, except CSF-serum pairs for which the CSF sample had an optical density (OD) value of ≥0.100, which were repeated. The second CSF-serum assay was the Borrelia ELISA Medac , referred to here as Medac ELISA, for the detection of IgM and IgG (Table S3). The third CSF-serum assay was the recomBead Borrelia 2.0 multiplex bead-assay , referred to here as the recomBead assay, for the detection of IgM and IgG (Table S3). For both the Medac ELISA and the recomBead assay, CSF-serum pairs with an equivocal AI value (1.3 ≤ AI < 1.5) should have been repeated following the manufacturers’ protocol (Table S3), but this was not done due to limited sample material. The fourth CSF-serum assay tested was the Borrelia burgdorferi SERION ELISA classic , referred to here as Serion ELISA, for the detection of IgM and IgG (Table S3). The fifth CSF-serum assay was the Enzygnost Borreliosis/IgM ELISA  and the Enzygnost Lyme link VlsE/IgG ELISA , referred to here as the Enzygnost ELISA (Table S3). CSF-serum pairs were tested using an adapted protocol we described previously (The first CSF-serum assay was the second-generation IDEIA LNB assay (Oxoid), referred to here as IDEIA, for the detection of IgM and IgG (Table S3) . As the eviously , as an erecomLine IB (Mikrogen GmbH) for the detection of IgM and IgG (Table S3). For practical reasons, we used the manufacturer’s protocol for serum to test the CSF. For testing CSF, however, we used a 1:10 dilution for IgM and a 1:20 dilution for IgG . In January 2019, the manufacturer of the recomLine IB revised the interpretation of the recomLine IgG IB (Table S3). Both the old and revised interpretation criteria were investigated in the current study , which measures total Ig (IgM and IgG) (Table S3) . The pront study , 63; howrecomBead assay was performed on a Bio-Plex 200 instrument using the Luminex xMAP technology  and the Bio-Plex Manager software version 6.1 (Bio-Rad Laboratories). The recomLine IB was performed on an Autoblot 3000 . Subsequently, recomLine IB strips were scanned and the intensity of the bands was recorded using recomScan software version 3.4 (Mikrogen GmbH).All ELISAs were performed on a Dynex DS2 automated ELISA instrument  and analyzed with the DS-Matrix software (Dynex Technologies). The To investigate the blood-CSF barrier functionality and the intrathecal total IgM and total IgG synthesis, CSF and serum concentrations of albumin, total IgM, and total IgG were determined at the start of this study by nephelometry and used to calculate the CSF/serum quotients for Q Alb, total IgM (Q IgM), and total IgG (Q IgG) as described previously . The Q ABorrelia-specific IgM and IgG AI value (Table S3). For the IDEIA, intrathecal Borrelia-specific antibody synthesis was proven if the fraction of Borrelia-specific IgM (or IgG) in the CSF as part of the total amount of IgM (or IgG) in the CSF exceeded that of serum, which is expressed by a Borrelia-specific IgM (or IgG) AI value (Table S3). Following the manufacturer’s protocol, a Borrelia-specific AI value of ≥0.3 was considered pathological, and a Borrelia-specific AI value of <0.3  was considered normal. Because the IDEIA is based on a capture ELISA, a correction for a dysfunctional blood-CSF barrier was not necessary  by Q IgM (or Q IgG) by considering the blood-CSF barrier functionality. As the interpretation criteria shown in Table S3 differed slightly between the four CSF-serum assays, the cutoff for intrathecal pathogen-specific (IgM and/or IgG) antibody synthesis as described by Reiber was used .For the CSF-serum pairs, either one of two calculation methods was used to calculate the nd Peter . These Bwas used . Thus, BBorrelia-specific antibodies in serum were determined previously using a two-tier protocol in which the C6 ELISA was used as a screening test and equivocal and positive C6 ELISA results were confirmed using the recomLine IB  (mLine IB . The recable S3) , 63; howResults from a number of other parameters, obtained at the time of active disease in the past, were retrieved from the patient and/or laboratory information system. These results included information about clinical symptoms, total protein and glucose concentrations in the CSF, and CSF leukocyte counts. For CSF samples with elevated erythrocyte counts , the CSF leukocyte count was corrected by subtracting 1 leukocyte/μL for each 1,000 erythrocytes/μL according to Reiber and Peter . For patBorrelia species PCR and two CXCL13 assays on CSF had been performed previously, of which the respective methods and results have been published  was applied  was used . We anal package . Dependi applied . The falFor each antibody assay, the sensitivity, specificity, PPV, and NPV with 95% CIs were calculated using the “epiR” package , for whiBorrelia species PCR on CSF (negative/positive). Blood-CSF barrier functionality and intrathecal total antibody synthesis were also included in the RF models for which the previously published Reibergram classification was used (n = 107/156), Reibergram area 2, a dysfunctional blood-CSF barrier without intrathecal total IgM and/or IgG synthesis of >10% (n = 25/156), Reibergram area 3, a dysfunctional blood-CSF barrier with intrathecal total IgM and/or IgG synthesis of >10% (n = 9/156), and Reibergram area 4, intrathecal total IgM and/or IgG synthesis of >10% with a normal blood-CSF barrier (n = 15/156) (Table S2). Subsequently, seven predictor variables were constructed based on one or multiple Reibergram areas  curve, which was subsequently used to calculate the AUC. For each RF model, the optimal cutoff for predicting LNB was calculated using the point on the ROC curve closest to the upper left corner, where both sensitivity and specificity are 100%, and this was determined by the square root of [(1 − sensitivity)2 + (1 − specificity)2]. Using the optimal cutoff value, the sensitivity, specificity, PPV, and NPV of each RF model were calculated. As the performance characteristics are based on predictions, 95% CIs were not calculated. For each RF model, the probability of misclassification and the relative importance of each predictor variable were calculated as described by Liaw and Wiener (For all RF models, definite and possible LNB patients were defined as cases and all non-LNB patients were defined as controls. In total, 10d Wiener . For eac


Clostridioides difficile infection (CDI). Long-term engraftment of donor-specific microbial consortia may occur in the recipient, but potential further transfer to other sites, including the vertical transmission of donor-specific strains to future generations, has not been investigated. Here, we report, for the first time, the cross-generational transmission of specific bacterial strains from an FMT donor to a pregnant patient with CDI and further to her child, born at term, 26 weeks after the FMT treatment.Fecal microbiota transplantation (FMT) effectively prevents the recurrence of A pregnant woman (gestation week 12 + 5) with CDI was treated with FMT via colonoscopy. She gave vaginal birth at term to a healthy baby. Fecal samples were collected from the feces donor, the mother , and the infant (meconium at birth and 3 and 6 months after birth). Fecal samples were profiled by deep metagenomic sequencing for strain-level analysis. The microbial transfer was monitored using single nucleotide variants in metagenomes and further compared to a collection of metagenomic samples from 651 healthy infants and 58 healthy adults.The single FMT procedure led to an uneventful and sustained clinical resolution in the patient, who experienced no further CDI-related symptoms up to 50 weeks after treatment. The gut microbiota of the patient with CDI differed considerably from the healthy donor and was characterized as low in alpha diversity and enriched for several potential pathogens. The FMT successfully normalized the patient’s gut microbiota, likely by donor microbiota transfer and engraftment. Importantly, our analysis revealed that some specific strains were transferred from the donor to the patient and then further to the infant, thus demonstrating cross-generational microbial transfer.The evidence for cross-generational strain transfer following FMT provides novel insights into the dynamics and engraftment of bacterial strains from healthy donors. The data suggests FMT treatment of pregnant women as a potential strategy to introduce beneficial strains or even bacterial consortia to infants, i.e., neonatal seeding.Video AbstractThe online version contains supplementary material available at 10.1186/s40168-022-01394-w. Clostridioides difficile infection (CDI), which accounts for approximately half a million infections per year in the USA alone  and JCM 30893 [GCF_009731575.1]) of  (v.1.1) . Reads o (v.1.1) , setting (v.1.1)  reformatg for 30 min. Thereafter, the supernatant was filtered through Costar SpinX centrifuge filters 0.2 μm at the recommended top speed of 15,000 g for 2 min until clear. The obtained fecal water was stored at − 20 °C before analysis at MS-Omics . For analysis, samples were acidified using hydrochloride acid, and deuterium-labeled internal standards were added. All samples were analyzed in a randomized order. Analysis was performed using a high polarity column  installed in a GC  coupled with a quadropole detector . The system was controlled by ChemStation (Agilent). Raw data was converted to netCDF format using Chemstation (Agilent), before the data was imported and processed in Matlab R2014b  using the PARADISe software described by Johnsen et al. /volume [μL]) of PBS  and vortexed to make a slurry. Then, samples were centrifuged three times at 16,000 n et al. .C. difficile toxin genes tcdA and tcdB were detected in the patient’s feces prior to FMT (P-pre-FMT), but not in feces samples 1 week after the FMT nor at any subsequent sampling points  and I6m (6.7 × 10−5).Following the application of the FMT, all CDI-related symptoms (including frequent soft and liquid stools) alleviated within 24 h, and no adverse effects related to the FMT were observed. The The patient pre-FMT displayed a lower species/MGS richness than the donor and all later patient sampling time points Fig. A. The inNext, we depicted the gut microbiota composition at the family level Fig. C. ComparAccompanying the microbiota shift, SCFAs also showed differential concentrations across samples Fig. S. The fecClostridium innocuum  [Clostridium symbiosum  [Clostridium clostridioforme (involved in bacteremia) [Veillonella parvula  [Flavonifractor plautii  [Ruminococcus gnavus (associated with Crohn’s disease) [We further assessed the top 50 species having the highest variance in the relative abundances in the donor and patient before and after FMT Fig. S. Many offection) , Clostrircinoma) , Clostriteremia) , Veilloningitis) , Flavoni cancer) , and Rumdisease) .We further assessed the gut microbiota dissimilarity between the patient and the actual donor or the independent donor over time at the species/MGS level Fig. A. The caTo investigate the potential contribution sources of the bacteria identified in the patient after FMT, we stratified the patient post-FMT gut microbiota into four categories, namely shared, donor-specific, recipient-specific, and newly detected Fig. . We founAlthough the donor-specific species represented the largest proportion in number, their abundances were low Fig. S and wereDue to subspecies heterogeneity in the human microbiome , the traWhen looking at the percentage of identical alleles, we could investigate the strain transfers for 120 different species, which was close to half of all the species 241) detected in the donor sample. Of these 120 species, 116 of them were also detected at the species level in one or more of the patient samples, and 99% of them (115/116 species) had strain transfer from the donor to the patient  were transferred from mother to infant, although this is mostly based on the detection of donor strains in the meconium sample. Interestingly, we were able to detect a Bifidobacterium breve donor strain across all infant samples, even though we never detected it in the mother. B. breve is associated with the breastfed microbiome [B. longum subsp. longum strains was observed in the infant. At 3 months of age, the donor strains were the dominant strain in the infant, while the maternal pre-FMT strain had become the vastly dominant strain at 6 months of age  in the infant that was not detected in the mother suggests that some strains transferred from the donor remained below the detection level in the mother but were still passed on at birth  or donor  and infant meconium (92% of donor strains). Furthermore, the finding of a donor strain (rth Fig. C. Interenor Fig. A indicatBacteroides, Dorea, Roseburia, Alistipes, and Parabacteroides are beneficial for a successful FMT, potentially due to their ability to produce short-chain fatty acids [C. difficile and may have contributed to the clinical resolutions [Regarding the CDI, we observed substantial differences in the gut microbiota composition between the patient and the healthy donor. The gut microbiota in the patient pre-FMT was characterized by low alpha diversity Fig. A and depty acids , 18, 69.olutions .We here present evidence for strain transfer from an FMT donor to a pregnant patient and further to her newborn baby. The findings substantiate the possibility that introduced exogenous strains in the gut environment during FMT treatment may propagate across generations potentially facilitating a novel approach for modulating the intestinal microbiota.Additional file 1: Fig. S1. Medical history for the patient from onset of CDI symptoms to FMT treatment and delivery of an infant. Fig. S2. The estimated genome coverage based on the Nonpareil. Fig. S3. Illustrations of the strain detection methods. Fig. S4. Detection of the C. difficile toxin producing genes with qPCR. Fig. S5. Tree plots to show the differences in the taxa relative abundances at different taxonomic levels. Fig. S6. Beta diversity of donors and patient post-FMT. Fig. S7. Short-chain fatty acids detected in the fecal samples. Fig. S8. Heatmap to show the top 50 species having the largest variance across donor and patient samples. Fig. S9. A diagram to show the division of the gut microbiota in the patient post-FMT. Fig. S10. Proportion of transferred donor-specific species. Fig. S11. Bray-Curtis distance to show the dissimilarity between the patient post-FMT and the patient pre-FMT. Fig. S12. The relative abundances of species in the patient post-FMT when divided into different potential sources. Fig. S13. Heatmap to show the donor-specific species in the patient gut after FMT. Fig. S14. Benchmarking of discriminative positions method and percentage of identical alleles method. Fig. S15. Detected donor strains in patient and infant samples across 120 different species based on the percentage of identical alleles between strains identified in the donor and in the patient or infant samples. Fig. S16. Detection of donor strain in the 14 species with one or more samples with another strain detected. Fig. S17. Changes in the number of species detected to have strain transfer from the donor.Additional file 2: Section S1. Additional details for strain transfer findings.


Cerebrospinal fluid (CSF) biomarkers of Alzheimer's disease (AD) are well‐established in research settings, but their use in routine clinical practice remains a largely unexploited potential. Here, we examined the relationship between CSF biomarkers, measured by a fully automated immunoassay platform, and brain β‐amyloid (Aβ) deposition status confirmed by amyloid positron emission tomography (PET).One hundred ninety‐nine CSF samples from clinically diagnosed AD patients enrolled in a clinical study and who underwent amyloid PET were used for the measurement of CSF biomarkers Aβ 1–40 (Aβ40), Aβ 1–42 (Aβ42), total tau (t‐Tau), and phosphorylated tau‐181 (p‐Tau181) using the LUMIPULSE system. These biomarkers and their combinations were compared to amyloid PET classification (negative or positive) using visual read assessments. Several combinations were also analyzed with a multivariable logistic regression model.Aβ42, t‐Tau, and p‐Tau181, and the ratios of Aβ42 with other biomarkers had a good diagnostic agreement with amyloid PET imaging. The multivariable logistic regression analysis showed that amyloid PET status was associated with Aβ40 and Aβ42, but other factors, such as MMSE, sex, t‐Tau, and p‐Tau181, did not significantly add information to the model.CSF biomarkers measured with the LUMIPULSE system showed good agreement with amyloid PET imaging. The ratio of Aβ42 with the other analyzed biomarkers showed a higher correlation with amyloid PET than Aβ42 alone, suggesting that the combinations of biomarkers could be useful in the diagnostic assessment in clinical research and potentially in routine clinical practice. Dementia is not one specific disease but rather a group of symptoms, including memory loss, disorientation, confrontational behavior, language problems, a variety of behavioral, and psychological symptoms, which are accompanied by signs of deterioration of different brain functions. Alzheimer's disease (AD) is the most common cause of dementia in elderly people, and amyloid plaques and neurofibrillary tangles (NFTs) arising in the brain are the hallmarks of AD. Individuals with AD typically have no apparent cognitive decline and remain asymptomatic for one to two decades during the preclinical phase in which there is elevated brain Aβ burden.AD biomarkers have been used in clinical practice and clinical trials. Regarding clinical utility, increasing evidence points out that there are some discrepancies between the diagnosis made only with clinical symptoms and the result based on AD biomarkers; hence biomarkers can be used to enhance confidence that individuals are accurately diagnosed with AD.In 2018, a taskforce organized by the National Institutes of Aging and the Alzheimer's Association (NIA‐AA) proposed a biomarker‐based diagnosis of AD based on the ATN classification system, which utilizes markers that reflect the main pathological hallmarks of AD: deposition of Aβ (Α), pathological Tau (T), and neurodegeneration (N).11C‐Pittsburgh Compound B [11C‐PIB], 18F‐florbetapir, 18F‐florbetaben, 18F‐flutemetamol, and 18F‐NAV4694) can visualize amyloid plaques with PET. Although these PET imaging techniques provide information in terms of the amount and spatial distribution of brain Aβ pathology, several factors including high cost, limited availability, use of radiation, and imaging of only a single type of pathology per scan, limit their widespread use.Molecular imaging biomarkers have become well established.On the other hand, CSF biomarkers, especially Aβ 1‐40 (Aβ40), Aβ42, total tau (t‐Tau), and phosphorylated tau‐181 (p‐Tau181), can be used in the AD field to evaluate the level of different AD pathology‐related biological analytes and are more accessible and less expensive than amyloid PET. However, these markers have not been used routinely and widely. One of the reasons is the considerable hesitation in requesting lumbar puncture (LP) for AD diagnosis in several countries such as the United States and Japan.To date, a number of studies have evaluated the relationship between CSF AD biomarkers and amyloid PET imaging, and found a strong correlation between the signal of amyloid PET ligands and the levels of CSF Aβ42, especially the ratio of Aβ42 with other AD biomarkers ,In this study, we tested LUMIPULSE assay kits using CSF samples obtained from individuals who had undergone amyloid PET imaging as part of the screening process to establish eligibility for a clinical research program. We measured CSF Aβ40, Aβ42, t‐Tau, and p‐Tau181 sequentially with their respective LUMIPULSE assay reagents, and then examined the relationship between the status of brain Aβ pathology confirmed by amyloid PET visual read and CSF Aβ40, Aβ42, t‐Tau, and p‐Tau181, and the combinations of these markers.Our cohort consisted of 199 subjects who had been enrolled in a clinical study (E2609‐G000‐301 or E2609‐G000‐302) conducted by Eisai and whose amyloid PET results and CSF samples were available.18F‐florbetapir, 18F‐florbetaben, or 18F‐flutemetamol. Amyloid PET was conducted in accordance with predefined procedures as mentioned previously.Brain Aβ deposition was visualized using CSF samples from previous clinical studies (E2609‐G000‐301 or E2609‐G000‐302) were used in this study.p < 0.05 was considered statistically significant.Subject baseline characteristics were summarized using descriptive statistics. Categorical variables were presented as frequencies and percentages. Continuous variables were summarized into median, first quartile (Q1), and third quartile (Q3). Group comparisons for continuous variables were performed using the Wilcoxon rank‐sum test. Fisher's exact test was used for categorical data. All tests were done under the nominal significance level α = 0.05. Receiver operating characteristic (ROC) analyses were performed to determine the cutoffs and the performance of individual biomarkers and their combinations for the discrimination between amyloid PET positive and amyloid PET negative subjects. Cutoffs were derived using the Youden Index. AUC differences were assessed with DeLong's test.n = 198, 99.5%) had a clinical dementia rating (CDR) global score of 0.5, and one subject had a CDR‐global score of 1. The median intervals between CSF collection and amyloid PET imaging in amyloid PET negative and positive group were 10.5 days and 8.0 days, respectively, which was not significantly different. Eighty‐three subjects (41.7%) were amyloid PET‐positive. There were no differences in age, sex, clinical stage, or race between amyloid PET‐negative and amyloid PET‐positive subjects. Likewise, the average interval between CSF collection and amyloid PET imaging of the two groups was not significantly different.Table p‐values of the statistically significant differences are presented as <0.001  or Aβ40 alone (p < 0.001). t‐Tau/Aβ42 had an AUC of 0.87 (95% CI 0.82–0.93), higher than t‐Tau or Aβ42 alone (both p < 0.01), and p‐Tau181/Aβ42 had an AUC of 0.86 (95% CI 0.81–0.92), higher than pTau181 or Aβ42 alone (both p < 0.05). There were no significant differences in AUCs between Aβ42/Aβ40, t‐Tau/Aβ42, and p‐Tau181/Aβ42 ratios and adding a third biomarker to these models or a generalized linear model of these markers did not improve their accuracy (data not shown).The ROC curves, the cutoff, the associated positive percent agreement (PPA), negative percent agreement (NPA), overall percent agreement (OPA) with amyloid PET status and area under the curve (AUC) of all biomarkers are shown in Figure Since brain autopsy, but not amyloid PET imaging, is the gold standard for the confirmation of brain Aβ deposition, we referred to PPA and NPA rather than sensitivity and specificity, to evaluate CSF biomarker performances. As displayed in Figure p < 0.01), and 0.9951 for Aβ42 . However, other factors, such as MMSE, sex, t‐Tau, and p‐Tau181, did not significantly add information to the model after adjustments for all potential confounding factors. The variance inflation factor (VIF) was calculated to evaluate the possibility of multicollinearity. VIF values ranged from 1.07 to 3.44 . Based on the cutoff points defined in Figure We used the Aβ42/Aβ40 ratio for further study and investigated the cases of discordance between CSF Aβ42/Aβ40 ratio and amyloid PET results. CSF Aβ42/Aβ40 ratio was negative in 15 (18%) of the 83amyloid PET‐positive individuals, and Aβ42/Aβ40 ratio was positive in 21 (18%) of the 116 amyloid PET‐negative individuals Fig. . These dIn our study, we determined the cutoffs for four CSF AD biomarkers  and their ratios measured on the fully automated LUMIPULSE G1200 platform to optimize their concordance with the brain Aβ pathology status determined by amyloid PET visual read. Consistent with previous reports,The multivariate logistic regression analyses indicated that amyloid PET status was significantly associated with CSF Aβ40 and Aβ42, whereas other factors, such as MMSE, sex, t‐Tau, and p‐Tau181, did not add significant information. Since t‐Tau or p‐Tau181 concentrations can be affected by non‐AD tauopathies,11C‐PIB, 18F‐florbetapir, 18F‐florbetaben, or 18F‐flutemetamol) could generate different cutoffs because of their different uptake characteristics.The agreement between amyloid PET imaging and CSF biomarkers has previously been studied.Various explanations have been given for the cases of discrepancy between amyloid PET imaging and CSF Aβ42/Aβ40 ratio test.On the other hand, 15 discordant cases with amyloid PET‐positive and CSF Aβ42/Aβ40 ratio negative were identified. Among them, only 1 case was positive for p‐Tau181 in CSF Fig. , and itsThe International Working Group (IWG) recommended that the diagnosis of AD should be based on both clinical and biomarker evidence,et al. showed that cognitively impaired African Americans with the APOEε4 genotype, a risk factor for AD, had significantly lower levels of t‐Tau and p‐Tau181 than did Caucasians with similar conditions. On the other hand, there was no significant difference in the level of Aβ42.Recently, it has been reported that racial differences are present in some biomarkers of AD.Blood‐based AD biomarkers with new high‐sensitivity assays have emerged.In this study, we found that the Aβ42 ratios with Aβ40, t‐Tau, or p‐Tau181 in CSF showed a substantial agreement with amyloid PET results and that the combinations of Aβ42 with other markers had a better correlation with the amount of amyloid PET results than Aβ42 alone. The multivariate logistic regression analyses indicated that amyloid PET status was significantly associated with CSF Aβ40 and Aβ42, but t‐Tau or p‐Tau181 did not substantively improve the predictive ability of the model, suggesting that using the Aβ42/Aβ40 ratio could predict the brain Aβ pathology more accurately and lead to an accurate diagnosis of AD. Moreover, the Aβ42/Aβ40 ratio test might detect brain Aβ pathology earlier than amyloid PET. This CSF Aβ42/Aβ40 ratio test therefore would be useful in the diagnostic assessment in clinical practice.APOE genotype information. Furthermore, because of the lack of longitudinal data, it was not possible to construct and evaluate a model for predicting disease progression. Further studies will be carried out to provide a more direct assessment of the ability of CSF biomarkers to predict disease progression in clinical studies.The strengths of this study are the simultaneous measurement of AD biomarkers in CSF with an automated system and the use of samples obtained from a multicenter population, including Caucasian and Asian participants. The limitations are the lack of a control group  or a real‐world population in clinical practice that includes etiologies other than AD, as well as the lack of quantification of Aβ deposition. Also, selecting patients who were eligible for screening in clinical trials might have introduced a bias in this biomarker analysis. Moreover, we were unable to obtain the H.N. and K.A. designed the study. S.I. and D.V. provided samples and clinical data. A.A. and W.M. measured CSF samples. H.N. performed data analysis and statistical analysis. H.N. and S.I. prepared the manuscript. All authors participated in discussions and interpretation of the data and results.H.N., A.K., A.A., W.M., and K.A. are employees of FUJIREBIO Inc. S.I. is an employee of Eisai Co., Ltd., and Eisai Inc. D.V. are employees of Eisai Inc. M.V., G.J., and I. V. are employees of Fujirebio‐Europe N.V. This study was funded by FUJIREBIO Inc.Figure S1 Distribution of biomarkers among the races. (A‐D) Beeswarm boxplots of LUMIPULSE biomarkers in amyloid PET positive group. Aβ40 (A), Aβ42 (B), t‐Tau (C), and p‐Tau181 (D). (E‐H) Beeswarm boxplots of LUMIPULSE biomarkers in amyloid PET negative group. Aβ40 (E), Aβ42 (F), t‐Tau (G), and p‐Tau181 (H). Box plots display the median values with the interquartile range (lower and upper hinge) and ± 1.5‐fold the interquartile range from the first and third quartile (lower and upper whiskers). Data were analyzed using Kruskal–Wallis rank sum test and there were no significant differences.Click here for additional data file.Figure S2 The status of each biomarker.  Correlation plots between Aβ42/Aβ40 and t‐Tau/Aβ42 (A) or between Aβ42/Aβ40 and p‐Tau181/Aβ42 (B) with superimposed linear regression lines with 95% confidence intervals. Positive or negative according to CSF ratios or amyloid PET results was evaluated in a scatterplot. The vertical dashed lines represent cutoffs for t‐Tau/Aβ42 (A) or p‐Tau181/Aβ42 (B), respectively. The horizontal dashed lines represent cutoffs for Aβ42/Aβ40. The statistical methods of Spearman's rank correlation coefficient (ρ) were used. p‐value is indicated. Solid line, Linear regression lines; open circle, amyloid PET positive and CSF Aβ42/Aβ40 ratio positive; cross, amyloid PET negative, and CSF Aβ42/Aβ40 ratio negative; siled circle, amyloid PET negative, and CSF Aβ42/Aβ40 ratio positive; triangle, amyloid PET positive, and CSF Aβ42/Aβ40 ratio negative. (C) Venn's diagrams show the performance of the biomarkers positivity in amyloid PET negative and positive groups. Numbers indicate the intersection positivity of the biomarkers.Click here for additional data file.Table S1 The biomarker status differentiated by race. Median  shown in each group. p values by Kruskal–Wallis rank sum test.Click here for additional data file.Table S2 The variance inflation factor (VIF) of each variable.Click here for additional data file.