Noninvasive or minimally invasive interventional surgery was selected, and the complications were less and had no significant impact on the quality of life of patients. Tumour patients are often accompanied by cerebrovascular diseases, metabolic diseases, and other basic diseases, which more or less adversely affect the surgical efficacy of tumour. In this paper, endovascular remobilization was used to treat tumour; the basic condition of patients before operation and the interventional operation plan were introduced. Through the analysis of clinical data and prognosis evaluation results of tumour patients receiving intravascular interventional therapy, the patients were divided into good prognosis group and poor prognosis group according to the modified Rankin scale score at discharge. The relationship between gender, age, history of hypertension, tumour width, tumour size, preoperative Hunt-Hess grade, interventional surgery method, and prognosis related to intravascular interventional therapy was explored. The results showed that intravascular interventional therapy for tumour patients can obtain a good prognosis, which provides a reference for the future preoperative assessment of treatment risk and possible prognosis and provides a theoretical basis for the formulation of treatment plan to improve prognosis. Combined tumour therapy includes surgery, radiotherapy, and chemotherapy. With the development of advanced technologies such as imaging, microcatheters, and stents, interventional therapy is providing new treatments for diseases that were previously untreatable or ineffective. Tumour therapy has entered the noninvasive or minimally invasive era. The purpose of digital subtraction angiography intravascular interventional therapy of tumour is to improve the therapeutic effect of local tumour and overcome the disadvantages of traditional treatment such as intravenous systemic chemotherapy, such as adverse reactions, radiation damage, and severe injury. Preoperative tumour interventional therapy can reduce tumour staging, improve surgical resection rate and survival rate, and reduce postoperative recurrence and metastasis. Interventional therapy for patients with advanced cancer can improve the quality of life and prolong the overall survival time of patients with cancer –3. ThereWith the development of medicine, interventional surgery has been applied more and more widely, not only in medical treatment, but also in surgery. Interventional therapy is appropriate for patients with severe bleeding due to trauma or whose tumours are unsuitable for major surgery. The intervention not only has less trauma and bleeding, but also recovers quickly. As long as there is a risk of surgery, interventional surgery is the same; different primary disease interventional complications are also different. The operation of interventional surgery has been greatly developed, so it has become a major minimally invasive surgical therapy not only applied to the back circulation but also widely applied to tumours. It has been recognized and accepted by the majority of patients for its accurate surgical efficacy, high success rate, and good prognosis. By adopting elastic yellow ring or stent-assisted embolization experience and clinical data, interventional embolization is safe and has a good curative effect of new treatments , 5, espeAs a relatively new treatment method, the prognosis of intravascular interventional therapy for IA has attracted much attention. Under the guidance of imaging equipment, doctors send a special catheter to the tumour's supplying artery and then inject embolization material through the catheter to completely embolize the tumour's supplying artery. B. Taslakian et al. studied the related factors that may affect the prognosis of invasive surgery, including the basic information of patients, such as gender, age, etc., which are thought to be associated with tumour incidence that is closely related to the basic diseases such as hypertension, tumour size, tumour related specific morphological features, such as neck width, and different ways of interventional surgery, the patient's clinical status classification, etc. . At presIn this study, clinical data of tumour patients undergoing intravascular intervention therapy in a hospital were selected, including age, smoking history, hypertension history, diabetes history, hyperlipidemia, aneurysm location and size, whether it was a wide-necked aneurysm, stent use, platelet suppression 1 day before surgery, and operation duration. All patients were diagnosed with intracranial aneurysm by CT angiography (CTA) and digital subtraction angiography (DSA). Magnetic Resonance Imaging (MRI) of the head was completed 3 days after surgery. All signed informed consent forms: patients with systemic infectious diseases, severe organ dysfunction, and other surgical contraindications; dissecting aneurysm, pseudoaneurysm and intracranial multiple aneurysm; pregnancy with intracranial aneurysm; intracranial and extracranial artery stenosis; intraoperative acute thrombosis and intraoperative aneurysm rupture. After admission, the patient was closely monitored in the neurological care unit, with absolute bed rest, appropriate sedation, and analgesia treatment, and stool was kept unapparent. Blood pressure was regulated to keep systolic pressure within 160 mmHg, while ensuring that the mean arterial pressure is higher than 90 mmHg. According to the patient's condition, drugs to relieve cerebral vasospasm, dehydration to reduce cranial pressure, lumbar puncture cerebrospinal fluid replacement, lateral ventricle drainage, and other treatments were used, and further interventional therapy was performed after preoperative examination was completed and the condition was stabilized . The preSimple core embolization, double microcatheter remobilization, or stent-assisted core-remobilization was selected according to the patient's condition, the situation of pulsar tumour, and the patient's willingness. During the operation, the patient was placed in the supine position. After tracheal intubation and general anaesthesia, the puncture site with the most obvious pulse 1.5 cm below the right inguinal ligament was selected as the puncture site. After routine skin disinfection for three times, sterile towel was covered. After percutaneous femoral artery puncture and catheter sheath placement, the catheter was guided from the femoral artery all the way up to the internal carotid artery. Microguide wires and microcatheters were used in the operation, and the appropriate working angle was selected in combination with the 3D reconstruction image during the operation. Remobilization with simple coil is to send a microcatheter to the tumour cavity under the guidance of a microguide wire and to deliver a certain number of microcoils through the microcatheter to fill according to the tumour size and shape nad width of the tumour neck and the parent artery.For relatively broad neck tumours or broad neck tumours with difficult stent placement, double microcatheter technology was adopted. Under the operation of two microcatheters, spring coils were, respectively, transported into the tumour cavity, and remobilization effect was achieved by winding the spring coils together into a basket. Stent-assisted coiling remobilization (SAC) is to perform coiling remobilization in the tumour cavity on the basis of placing stents in the tumour neck with absolute or relatively wide neck by microcatheter, so as to close the tumour neck and avoid coiling displacement . The theDuring the operation, the patient's whole body heparin was maintained, and high pressure heparin saline was administered to continuously flush the lumen to avoid thrombosis in the lumen guided by the catheter. It was expected that patients using stents would be given clopidogrel tablets 75 mg/d and enteric-coated aspirin tablets 100 mg/d 3 days before surgery. In case of emergency, preoperative disposable clopidogrel tablets 300 mg and enteric-coated aspirin tablets 300 mg were used.The above operations are performed by skilled people. 24 hours after the operation, strict ECG monitoring, and give anticerebral vasospasm, anti-inflammatory, fluid replacement, expansion, dehydration, and other treatments. After 3 days of subcutaneous injection of low molecular weight heparin sodium, patients were treated with oral clopidogrel 75 mg/d and enteric-coated aspirin 100 mg/d for 6 months and then maintained with enteric-coated aspirin 100 mg/d or clopidogrel 75 mg/d.All interventional procedures were performed by the same neurosurgeon via femoral artery approach under general anaesthesia. A complete set of thromboelastography (AA% and ADP%) was completed for all patients 1 day before surgery. Both patients received dual antiplatelet therapy (aspirin 300 mg and clopidogrel 300 mg) 2 hours before surgery. After successful general anaesthesia, heparinization was performed on the whole body, the dosage was 1 mg/Kg, and the dosage was reduced to 50% of the previous dosage 1 hour later until the reduction was maintained at 10 mg/hour. Procaine and heparin were given at the end of the operation if only coiling embolization of the tumour was performed. If stent-assisted embolization of the tumour is used intraoperatively, no heparin is obtained. After the operation, the coagulation image was examined immediately. If the coagulation result reached the indication of blood transfusion, plasma was given to improve the coagulation function. If stent-assisted embolization of tumour is used, subcutaneous anticoagulant injection of low molecular weight heparin should be used according to the preoperative inhibition of TEG platelet . If aspiIn this study, the relevant data of the included patients during outpatient, inpatient, or telephone follow-up 6 months to 1 year after surgery were collected, and the prognosis of the patients was evaluated according to the scores of the modified Rankin scale, as shown in n ≥ 40 and theoretical frequency T ≥ 5) or corrected chi-square test  or Fisher exact probability method (n < 40 or T < 1) was used. P < 0.05 was considered statistically significant. The factors with statistical significance in univariate analysis were further analyzed by multivariate analysis: logistic regression analysis; P < 0.05 was considered statistically significant [SPSS21 statistical software was used for statistical analysis. The data of normal distribution were represented by mean ± standard deviation, and the data of nonnormal distribution was represented by median ± quartile interval. The counting data are expressed by frequency and percentage. University analysis was performed on indicators that may affect prognosis, including gender, age, history of hypertension, tumour neck width, tumour diameter size, preoperative Hunt-Hess grade, and interventional surgery. The continuous variables were converted into classified variables, and Pearson chi-square test  study. The investigation and analysis of patients took into account the factors of previous pathological history, hypertension, smoking history, and diabetes. Thirty-one patients had a history of hypertension, 22 had a history of diabetes, and 21 had a history of smoking. The highest age was 62 years, the lowest age was 20 years, and the average age was 57.3 ± 10.3 years. BMI ranged from 36.3 to 16.7, with a mean value of 24.0 ± 3.3. The statistical results are shown in Sixty-three patients had a total of 68 tumours, of which 60 were treated with interventional therapy and 8 were untreated. Among them, there were 66 responsible tumours  are important indexes to reflect the geometric characteristics of tumour. The tumour neck size has a controlling effect on the eddy current velocity in the tumour cavity. Wide-neck tumours usually have larger blood flow and complex stress gradient, which affects the filling effect of spring coil . TherefoTumour diameter, tumour neck width, and multiple tumours are risk factors for recurrence of intracranial aneurysms treated by intravascular intervention. The tumours with larger diameter were more irregular, and the tamping degree of the tumour neck with larger width was lower, so recurrence was easy to occur. In addition, thrombus is more common in tumour lumen in patients with large tumour diameter, and it is difficult to completely occupy part of the space of thrombus in the process of embolization of microspring coil. With the absorption of thrombus in tumour lumen, it is easy to cause the expansion of residual lumen and relapse. Patients with multiple tumours have more tumours, more complex disease, and more difficult intravascular interventional therapy, so the prognosis is poor and the risk of recurrence is greater.P < 0.05 was considered as statistically significant.Univariate analysis showed that patients with a history of hypertension, large tumour diameter, clopidogrel resistance, and stent-assisted embolization had a higher incidence of postoperative microischemia. Age, smoking history, diabetes history, hyperlipidemia, aspirin resistance, tumour neck size, tumour location, and surgical duration were not statistically different between the two groups. The Chi-square test and Fisher's exact probability method were used in the univariate analysis, as shown in Multivariate binary logistic regression analysis was conducted for the above two statistically significant indicators, as shown in The essence of the tumour lies in the local lesion of the arterial wall of the parent artery. Both stent-assisted embolization and non-stent-assisted embolization are mainly for the filling of the tumour cavity, while the treatment of the parent artery is neglected. The application of stents can effectively increase the density of tumour embolization and reconstruct the parent artery wall to surround the tumour neck. Stents improve hemodynamics and thus promote endometrial hyperplasia to cover the neck of the tumour. Multivariate logistic regression analysis showed that stent-assisted embolization had a 4.172-fold higher risk of poor outcome than non-stent-assisted embolization. In the process of stent-assisted embolization, the control and operation of stents are complicated. Once there is poor stent adherence and resistance to antiplatelet aggregation drugs, it is easy to induce platelet aggregation, promote stent thrombosis, and increase the incidence of cerebral infarction. On the one hand, choosing the stent with appropriate radial force and avoiding stimulating endothelial proliferation can reduce the chance of stinting stenosis. On the other hand, preoperative and postoperative ant platelet therapy should not be ignored. Therefore, in clinical work, the sensitivity of patients to ant platelet drugs should be well grasped, and individualized ant platelet therapy should be achieved, so as to maximize the advantages of stent-supported therapy.Endovascular interventional therapy is a minimally invasive surgery, which has less tissue trauma and surgical impact than craniotomy clipping surgery. In this paper, endovascular embolization was used to treat tumour; the basic condition of patients before operation and the interventional operation plan were introduced. Through the analysis of clinical data and prognosis evaluation results of tumour patients receiving intravascular interventional therapy, the patients were divided into good prognosis group and poor prognosis group according to the modified Rankin scale score at discharge. The relationship between gender, age, history of hypertension, tumour width, tumour size, preoperative Hunt-Hess grade, interventional surgery method, and prognosis related to intravascular interventional therapy was explored. Conclusion: tumours >7 mm are a risk factor for poor prognosis. Age, sex, history of hypertension, tumour neck width, and preoperative Hunt-Hess grade were not independent risk factors affecting the prognosis of interventional therapy for tumours with anterior circulation rupture. The prognosis of tumour after intravascular intervention was related to the age, Hunt-Hess grade, and hypertension. The recurrence of the disease is related to the patient's age, Hunt-Hess grade, diabetes, hypertension, tumour diameter, tumour neck width, surgical methods, and embolization. Clinical attention should be paid to the influence of these factors, and corresponding measures should be taken to reduce the recurrence rate and improve the prognosis. Further studies are needed to determine whether preoperative control of blood pressure levels will reduce the incidence of microthromboembolic complications.


BRAF mutation and MSI). In contrast, changes in the stool microbiome were associated with lymph node involvement and the presence of synchronous metastases. We discovered new associations between microorganisms and CRC and clinical parameters. Our study represents a step forward in understanding the role of the microbiome and its interactions with factors involved in tumour progression, and it opens novel avenues for exploring new treatments and biomarkers.Dysbiosis of the gut microbiome may contribute to the heterogeneity of colorectal cancer from phenotypic, prognostic and response to treatment perspectives. We analysed CRC microbiome by 16S rRNA gene sequencing of paired tumour mucosa, adjacent visually normal mucosa and stool swabs of 178 patients with stage 0–IV CRC. We observed that tumour mucosa is dominated by pathogenic bacteria of oral origin and proposed a CRC tumour microbiome subtyping system. The subtypes and tumour mucosa genera were associated with prognostic clinical covariates .Long-term dysbiosis of the gut microbiome has a significant impact on colorectal cancer (CRC) progression and explains part of the observed heterogeneity of the disease. Even though the shifts in gut microbiome in the normal-adenoma-carcinoma sequence were described, the landscape of the microbiome within CRC and its associations with clinical variables remain under-explored. We performed 16S rRNA gene sequencing of paired tumour tissue, adjacent visually normal mucosa and stool swabs of 178 patients with stage 0–IV CRC to describe the tumour microbiome and its association with clinical variables. We identified new genera associated either with CRC tumour mucosa or CRC in general. The tumour mucosa was dominated by genera belonging to oral pathogens. Based on the tumour microbiome, we stratified CRC patients into three subtypes, significantly associated with prognostic factors such as tumour grade, sidedness and TNM staging,  Colorectal cancer (CRC) is the third most frequent cancer worldwide and the second leading cause of cancer mortality in Europe . It is aSeveral studies showed that bacteria adherent to colorectal adenomas or carcinomas were different from bacteria adherent to healthy gut mucosa ,11,12 duPrevious studies associating gut dysbiosis with CRC were focused on comparing the gut microbiome in the normal-adenoma-carcinoma sequence ,30,31,32The microbiota adherent to the mucosal tissue differs from the faecal microbiota in its needs for oxygen and nutrient types ,38. Thern = 8 individuals) and did not explore the clinical relevance of this similarity. Other studies associated microbiome on tumour or in stool with clinical variables  ventriosum group, Clostridiales Vadin BB60 group, Ruminococcaceae UCG-010 and an uncultured species and an Incertae Sedis genus from the Lachnospiraceae family . Ruminoclostridium 6 remained enriched also in the stool of patients with grade 2 left-sided, rectosigmoid and rectal tumours . Methanobrevibacter, Victivallis were significantly enriched in the mucosa of low-grade tumours of rectosigmoid and rectum .The mucosa of left-sided (for some including rectosigmoid/rectum) low-grade tumours was enriched in Christensenellaceae R-7 group, Bifidobacterium and Ruminococcaceae UCG-013 were increased in mucosa of the left-sided, rectosigmoid and rectal tumours . Similar associations were found for visually normal mucosa for Christensenellaceae R-7 group, Coprococcus 1, Lachnospira and Bifidobacterium . The increased abundance of the Christensenellaceae R-7 group in tumour mucosa of left-sided tumours was also validated in an independent dataset (p = 0.0047)  ( 0.0047) . When coR < 0.1) .Gemella, Campylobacter, Peptoclostridium and Parvimonas  on tumour mucosa, and increased Peptoclostridium, Escherichia-Shigella  in the adjacent visually normal mucosa. Early T stage tumours (pTis-2) were associated with an increase in Coprobacter, on tumour mucosa, increased Intestinimonas, Ruminococcaceae UCG-009, Holdemanella and Coprobacter on the adjacent visually normal tissue  and Prevotella 6  and Ruminococcaceae UCG-011  in the stool  (Patients with advanced T stages (pT 3–4) were characterised by a significant increase in abundance of he stool . We vali et al.) .Akkermansia in stool of patients with N1–2 stage tumours  and of the uncultured genus from the Erysipelotrichaceae family in the stool of patients with synchronous distant metastases , none of these associations were significant after FDR correction (Dorea in the stool of patients with N1–2 stage tumours (p = 0.00011) in an independent dataset   at the time of diagnosis was predominantly associated with changes in the stool microbiome. Except for the increased abundance of rrection . Neverth et al.) .We continued our characterisation of tumour microbial heterogeneity by performing hierarchical clustering of patients based on the relative abundance of the 57 tumour genera in the tumour mucosa samples (See Methods). Once the subtypes were identified, we performed between-subtype differential abundance analyses of microbiome profiles in all three sampling environments.Based on the tumour genera profiles, we observed three major subtypes of tumours (TMS1–TMS3), that could further be divided into two groups each . The bacFusobacterium, Lachnoclostridium, Bacteroides, Escherichia-Shigella and one uncultured genus from the family Lachnospiraceae. All tumours contain at least three of these bacteria, most tumours (78.7%) all five. These bacteria have high co-occurrence across the sampled environments .The B1 group and B2 group are represented by typical gut microbiome members. The B1 group consists of the five most common and most abundant genera . This subtype is enriched in right-sided (60.9%), grade 3 (58.7%), pT3 or pT4 stage (95.6%) tumours and is depleted of stage 0 and stage I tumours  (Selenomonas group (B4), Solobacterium and Howardella species, and Clostridium sensu stricto 1. In contrast to other subtypes, this subtype shows a significantly decreased abundance of typical faecal commensals such as Bifidobacterium, Ruminococcus 2, Anaerostipes and Coprococcus 1 on tumour mucosa  represents 26% (46) of tumours and is defined by the presence of B1–B4 microbial groups, and overall contains most of the high-grade associated genera ctively) . In addir mucosa . In stootricto 1 .Selenomonas group). This subtype can be further divided into two groups by the increased incidence of Leptotrichia, Granulicatella, Aggregatibacter and Neisseria (TMS2a) or Tyzzerella 4, Hungatella (ex-Clostridium hathewayi), Solobacterium, Pseudomonas and Porphyromonas (TMS2b). TMS2 tumours are predominantly from the left side, rectosigmoid or rectum (70.9%)  comprises 31% (55) of tumours and is defined mainly by the absence of B4 bacteria (the  (70.9%) . The mucevotella . Allopre samples .Incertae sedis from the Erysipelotrichaceae family and Tyzzerella 4 (TMS3a) or Clostridium sensu stricto 1, Ruminococcaceae UCG-013 and Incertae sedis from Lachnospiraceae family (TMS3b). Interestingly, subtype TMS3 contains all the tumours that lack Fusobacterium species (most of them in TMS3a) both in their mucosa and in the patients’ stool.Finally, the largest subtype, TMS3, represents 43% (77) of tumours and is mostly missing the B3–5 bacterial groups and most of the high-grade related species. TMS3 is characteristic by an increased proportion of grade 1 tumours (15.6%). In the TMS3 microbial subtype, right-sided and left-sided tumours are equally represented . The subp < 0.001). We then explored the estimated metabolic potential of the microbial communities specific to the tumour subtypes , reduction in lipid metabolism and xenobiotics biodegradation and metabolism . At the lower functional level, TMS1 subtype was characterised by increased peptidoglycan biosynthesis, novobiocin biosynthesis and ansamycin biosynthesis . The TMS2 subtype exhibited the highest score of xenobiotics biodegradation and metabolism. TMS3 subtype showed enhanced biosynthesis of other secondary metabolites, carbohydrate metabolism and amino acid metabolism and reduced metabolism of other amino acids .Most importantly, the subtypes differed significantly in the proportion of the oral genera with TMS1 median of 15.8%, TMS2 median of 12.3% and TMS3 median of 5.3%  that were not used for their definition. The analysis of microbial composition between sample types confirmed previously reported observations ,11,34 thSelenomonas 3, Selenomonas 4, Aggregatibacter, Johnsonella, Abiotrophia, Defluviitaleaceae UCG-011). Most importantly, we newly associate 22 genera of both oral and gut origin with CRC overall. Some of these genera contain species that are known human pathogens causing infections of mucosal or other tissues such as: periodontal disease  [Bergeyella and Actinobacillus) [B. cardium) or respiratory infections (A. hominis) [Defluviitaleaceae might influence CRC through the metabolism of butyrate [Tyzzerella 4 from the Lachnospiraceae family with CRC may be due to its increased occurrence in patients with higher cardiovascular risk (CVR) factor scores [Massilia was detected in patients with pancreatic cancer [Multiple factors make the CRC tumour niche a favourable environment for oral bacteria, in particular, for oral pathogens. Some of the bacteria can bind to specific proteins overexpressed on tumour cells ,77,78. Iibacter) ,83,84 inacillus) ,86; endohominis) ,88. For butyrate . The assr scores , which ar scores . Massilic cancer .Correlating the tumour microbiome with clinical variables of tumour progression, such as grade or stage, bears the promise of offering viable hypotheses on the role of bacteria in the progression of the disease. Currently, the associations between clinical variables and gut microbial composition in an ongoing disease are understudied, and only a few efforts addressed the topic on limited cohorts.Fusobacterium, Campylobacter and Mogibacterium, in CRC tumour mucosa [stool genera significantly associated with left location, namely decreasing tumour grade with increasing abundance, e.g., of Bifidobacterium, Ruminococcaceae UCG-010 and Victivallis in tumour mucosa; and of Porphyromonas and Lachnospiraceae UCG-005, in the stool. Bifidobacterium was previously shown to have anti-cancerogenic effects [The sample size of our study allowed for the study of the interaction of grade and tumour location, thus providing a finer estimation of the differences in the microbiome composition. We report 59 associations of 43 genera with tumour grade and/or location for all sample types studied. We confirmed previously reported high-grade tumour associations of r mucosa ,68. We n effects ,70,71,72 on tumour mucosa, in the left colon a higher grade was associated with a depletion in possibly beneficial  genera  on tumour mucosa or in the stool. We can only speculate whether the prolonged exposure of tumour mucosa to predominantly stool bacteria that is mechanistically related to tumours in a distant part of the colon (left-sided or with onset in rectosigmoid and rectum) can have potentially harmful or beneficial effects or whether any associations are mostly due to the well-known molecular differences in the right vs left-sided tumours [We also found location-dependent grade-predictive genera. Remarkably, while in the right colon a higher grade was associated with an increase in pathogenic genera  tumours ,93,94. T tumours , but the tumours .Akkermansia and Porphyromonas in the stool of patients with local metastases [Akkermansia in stool in patients with stage III–IV CRC. We noted that the occurrence of synchronous local and distant metastases was mainly associated with shifts in stool microbiome, while tumour specific variables such as grade or location were associated with changes in tumour microbiome. On one side, this observation raises the possibility of microbiome-based non-invasive metastasis diagnostics in colorectal cancer or monitoring the patients at risk. On the other hand, the alteration of the stool microbial community might only reflect changes in the overall health status in the presence of metastasis and cancer progression itself similarly to non-colonic malignancies [We confirmed a previously published increase of tastases , and newgnancies ,98,99.Pairwise analysis of the incidence of all genera across sample types helped us to assess their screening potential. On-tumour microbes with significant clinical associations and no difference in incidence across sample types are perfect candidates for stool-based screening studies or stool-based prognostic and predictive classifiers. Most of the tumour-specific genera, if present on tumour mucosa, were not identified in stool of the same patients in more than 50% of cases. Given that these genera prefer the mucosal environment over the stool, such associations are not entirely surprising. Consequently, these genera are better candidates for colonoscopy biopsy sample screening.Campylobacter, Porphyromonas, Streptococcus and decrease in Lachnospiraceae and Faecalibacterium). Some showed no significant clinical associations in stool, but their increased abundance on tumour mucosa was predictive of high pT stage (Parvimonas) or grade (Fusobacterium).We then compared how the previously suggested stool-based predictive microbial markers of CRC   behave wBRAF mutant, MSI-H, right-sided tumours. An interesting observation was that the tumour microbial subtypes differ not only in the type of the tumour genera they host but also in the count of potentially pathogenic microbiome correlated with high grade and stage and the proportion of oral pathogens within the tumour genera. Of the 10 high grade or high stage-related genera, TMS1 tumours had a median of 8 (80%), TMS2 of 6 (60%) and TMS3 of 4 (40%), differing thus in what we could call “microbial pathogens burden”. This subtyping could reflect differences in tumour biological properties linked with cancer progression: malignant tumours with active growth, cell and tissue atypia because of disruption of the mucus layer and dysregulation of local immunity provide more comfortable conditions to aggressive microbial consortia expansion and unconventional  species homing. Moreover, with respect to the bacteria-supported model of carcinogenesis, proved in animal models [ produce proteins such as gingipains [The three tumour–mucosa-based microbial subtypes we derived on patterns of similarity of the abundance of the tumour genera represent the first attempt to systematically describe microbial heterogeneity of CRC tumour environment. We were intrigued to see that compared to subtyping efforts based on gene expression ,100, alsl models , the patl models . Drewes l models  identifingipains  and RadDngipains , which cngipains  proposedngipains  successfngipains .It remains to be investigated whether the microbial subtypes could improve the prediction of patients’ survival and prognosis. We can speculate that the high microbial pathogen burden could worsen not only the tumour progression but also potentially the patient’s condition after the surgical resection and during and after the chemotherapy treatment. Given the fact that tumour-related genera reside also on visually normal mucosa, they could initiate CRC tissue dysplastic changes and malignisation. There is limited evidence of linkage between mucosal microbiota and metachronous adenomas growing demonstrated by Liu et al. . On the The probable presence of biofilm in the TMS1 subtype might make this subtype of interest to potential prevention and treatment strategies. Importantly, although TMS1 is enriched in proximal tumours, it occurs in 9.1–18.8% of left, rectosigmoid and rectal tumours. Remarkably, biofilm communities from the colon biopsies of healthy individuals were as potent in inducing colon inflammation as the biofilm communities from CRC hosts . The inhAnother clinically relevant observation is the association of left-sided high-grade tumours with the depletion in protective species rather than increase in bacterial pathogens. This suggests that antibiotic treatment of patients with distal tumours may have a detrimental effect on their prognosis. Coadministration of probiotics in this case could be highly beneficial.We believe that for certain patient populations, the inclusion of tumour mucosa sampling during colonoscopy for analysis of microbial composition could help to efficiently steer pre- and post-operative treatment decisions.n = 178 patients, we extended the current characterisation of the colorectal cancer microbiome in several directions. Thanks to the large sample size, we identified bacterial genera that were not previously associated with CRC tumour mucosa, clinical variables or with colorectal carcinoma at all. These genera should be studied in more detail to describe their mechanism of interaction with the disease.In our study, by analysis of 483 samples from By focusing on microbial community analysis, in contrast to classical microbiome-centred approaches, we were able to identify co-occurring species and three major tumour-microbial subtypes that correlate with clinical variables, such as grade, location and TNM staging. The subtypes also differ in what we describe as microbial pathogens burden—the number of pathogenic species correlated with increased grade and stage present on tumour mucosa, although the concept can be defined with respect to all three environments .An important limitation of our study is the lack of proper validation of all the results since adequate data is unavailable, hence these results must be taken cautiously. Additionally, it is well known that the gut microbial composition changes with dietary patterns and lifestyle, which could be region-based . More stHaving sampled the microbiome at three different complementary sites allowed the study of several environments leading to the definition of novel microbial categories with multiple implications. Our study shows that the associations with clinical variables found for the tumour mucosal or adjacent visually normal mucosa microbiome are rarely preserved in the microbial composition of stool and vice versa. While tumour histological grade, stage and location are reflected in the corresponding mucosal microbiome, the presence of lymph nodes or distant metastases influences mainly the stool microbiome. It seems that the mucosa and stool microbiome are complementary with respect to the modulation of their effects on disease progression. Tumour-mucosa biopsies from colonoscopy might need to be coupled with stool sampling for efficient screening or diagnostic purposes.Understanding the role of tumour-subtype specific microbial communities could lead to tailored strategies of CRC patient gut microbiome management through lifestyle and diet recommendations including probiotic and antimicrobial interventions.Our study is a step forward in understanding the role of the microbiome and its interactions with other factors involved in oncogenesis and tumour progression. Rather than providing definite answers it opens new avenues for exploring new treatments and biomarkers.


Staphylococcus aureus CRISPR/Cas9 (saCas9) with gRNA to the duplicated and essential VZV genes ORF62/71 (AAV2-62gRsaCas9) greatly reduced VZV progeny yield and cell-to-cell spread in representative epithelial cells and in lytically infected human embryonic stem cell (hESC)-derived neurons. In contrast, AAV2-62gRsaCas9 did not reduce the replication of a recombinant virus mutated in the ORF62 targeted sequence, establishing that antiviral effects were a consequence of VZV-genome targeting. Delivery to latently infected and reactivation-induced neuron cultures also greatly reduced infectious-virus production. These results demonstrate the potential of AAV-delivered genome editors to limit VZV productive replication in epithelial cells, infected human neurons, and upon reactivation. The approach could be developed into a strategy for the treatment of VZV disease and virus spread in HZ.Varicella Zoster Virus (VZV) causes Herpes Zoster (HZ), a common debilitating and complicated disease affecting up to a third of unvaccinated populations. Novel antiviral treatments for VZV reactivation and HZ are still in need. Here, we evaluated the potential of targeting the replicating and reactivating VZV genome using Clustered Regularly Interspaced Short Palindromic Repeat-Cas9 nucleases (CRISPR/Cas9) delivered by adeno-associated virus (AAV) vectors. After AAV serotype and guide RNA (gRNA) optimization, we report that a single treatment with AAV2-expressing  Varicella-zoster virus (VZV) is the human alphaherpesvirus that causes varicella (chickenpox) during primary infection and herpes zoster  when the virus reactivates from the latent state, often decades after the initial infection . WithoutHZ incidence and severity are reduced by boosting the existing VZV-specific immunity using vaccines. The first HZ licensed vaccine (used since 2005) was based on a higher dose version of the live-attenuated VZV strain used in the varicella vaccine, and it reduced HZ incidence by half and the disease burden by two-thirds . HoweverOne of the main hurdles in developing novel VZV therapeutic strategies has been the difficulty in modeling VZV pathogenesis in animal models. VZV demonstrates high human specificity, and does not fully replicate or cause disease in rodents, compared to the modeling of infections and diseases caused by the related herpes simplex virus type 1 (HSV-1) . Indeed,Staphylococcus aureus CRISPR/Cas9 (saCas9) system was exploited because it has a high specificity and small size, permitting efficient packaging into AAV together with guide RNA [In this study, the potential of gene editing to target VZV genomes as a means to prevent lytic, latent, and reactivated infections was investigated. The uide RNA ,38. VZV uide RNA ,40. Of tuide RNA ,42 that uide RNA ,44. Furtuide RNA . VZV IE6uide RNA ,46 and huide RNA ,48. We suide RNA ,51,52,53All cell lines except the NIH-registered human embryonic stem cell line Wa09 (H9) were purchased from ATCC. Cell-culture reagents were obtained from Thermo Fisher Scientific  unless otherwise noted. The Wa09 (H9) was obtained from WiCell  and differentiated into neurons as detailed previously . RetinalAll infection studies used virus or viral recombinants based on the Parent of Oka (pOka) strain, a wildtype clinical isolate that was the parent of the live attenuated varicella and zoster vaccines. Cell-associated VZV were prepared as previously described , as infehttp://n2t.net/addgene:61591; RRID:Addgene_61591 accessed on 10 January 2022) were performed as published online . Briefly, the vector was digested with BsaI and a dsDNA formed by the hybridization of the two oligonucleotides was ligated into the pX601 plasmid, where pX601 expresses saCas9 and the inserted gRNA from the same vector. A vector expressing Streptococcus Pyogenes CRISPR/Cas9 targeting the intergenic region between UL3 and UL4 was developed using the pX330 vector , as previously described [Three optimal gRNA target sequences with PAM motifs and guide length that were predicted to target saCas9 to each of ORF62 and ORF63 were selected using an online protocol from the Zhang lab  and Softescribed ,57. Prim13 viral genome copies (GC) per mL) ORF62-targeting AAV2 and a pX601 vector-only control lacking gRNA, were prepared by Penn Vector Core’s Gene Therapy Program . Lower titer (approximately 1010 GC/mL) control or ORF62-targeting AAV2 for use in preliminary experiments were made by transfecting HEK293 cells with the control pX601 (with no gRNA) or ORF62-targeting plasmid (pX601+62-1 gRNA), along with plasmids expressing AAV2 replication gene (pRC2-mi342 vector) and a helper plasmid that expresses adenoviral helper proteins  using the Xfect reagent . 6 h after transfection, media was changed to fresh DMEM with 10% FBS and cells were further incubated at 37 °C 5% CO2 for 72 h. Cells were then harvested into an AAV lysis buffer  and subjected to three freeze–thaw cycles. AAV were then purified using a published protocol in which a discontinuous 15%–25%–40%–60% iodixanol gradient was used. Virus at the interface between 40% and 60% iodixanol was harvested after centrifugation at 200,000× g for 2 h. at 18 °C [g for 20 min at 4 °C. AAV stocks were then aliquoted and stored at −80 °C until use. An aliquot of each virus produced was titered by quantifying AAV genomes using qPCR as detailed below.AAV reporter vectors of different serotypes expressing the green fluorescent protein under the chicken β-actin promoter CAG were acquired from Virovek . High titer  system (pOka DX), based on the VZV parental Oka strain (pOka) containing a self-excisable mini-F replicon ,62. The r) derived from the plasmid pEPS-kan2 . Resolving the internal kanr cassette by inducing a further recombination concurrent with the induction of ISceI expression resulted in BACs with restored mCherry-ORF23. The VZV derived from this BAC was termed VZV DR  and expressed mCherry from the ORF23 promoter and luciferase from the ORF57 promoter. Then, the ORF71 coding sequence was replaced with a PCR-amplified ampicillin resistance cassette (ampr), selecting for gain of ampicillin resistance in addition to resistance to chloramphenicol (chmR is directed by the replicon) and zeomycin resistance (directed by a cassette inserted downstream of the luciferase at ORF57). VZV derived from this new BAC (VZV DR-Δ71) contained two quantifiable reporters and one copy of the ORF62 gene. Evaluation of virus at passage 6 by Southern blotting confirmed that the virus contained a restored ORF71 derived from ORF62 gene (VZV-DR-Δ71: data not shown). The VZV BAC DR-Δ71 was then further subjected to a recombinatorial mutation of the ORF62 gRNA-targeted sequence region to derive VZV DR-62gmut mutant BACs and viruses. Two primers were used to amplify kanr from pEPS-kan2  with 2.5 μg of purified BAC DNA and 100 ng each of plasmids expressing ORF61 and ORF62 DNA under the constitutive hCMV immediate-early promoter, as detailed previously ,65. Upon6 cells in a single well) from cryopreserved, pre-titered, mitomycin C-treated aliquots of infected ARPE-19 cells. All assays were performed in triplicate. At the desired times after infection, infectious virus-associated cells were quantified by trypsinizing the monolayers and then seeding serial dilutions onto ARPE-19 monolayers grown in 6 wells seeded 24 h. prior and at 80% confluency. At 4–5 dpi, the formed plaques were counted under a fluorescent microscope, and averaged from the triplicate values. Titers were normalized to the exact titer of virus at day 0 determined from the inoculate and then normalized to parental virus VZV DR.Virus growth curves were determined as detailed previously ,65, initTM medium supplemented with StemFlexTM supplement (Thermo Fisher Scientific) and antibiotic/antimycotic. Cells were grown on 6-well dishes pre-coated with GelTrex Basement Membrane Mix . Colonies were passaged either by manual dissection or using ReLeSR™ Enzyme-free cell selection and passaging reagent , per the manufacturer’s instructions. Following either method, ROCK inhibitor Y-27632  was added to the culture media for 24 h. to increase cell survival and deplete dividing cells. Neural precursor cells (NPCs) were generated from hESC by co-culture with the PA6 mouse stromal fibroblast cell line  as detailed previously [Neuron cultures were prepared using a previously published protocol  based oneviously ,67. For For the fluorescent monitoring of VZV infections of neurons, neurospheres were seeded on coated 12-well glass bottom plates  and differentiated in neuron differentiation media for at least 14 days. Infections were initiated with PBS-washed, mitomycin-C treated VZV-infected ARPE-19 cells. Medium was changed the next day and every 2–3 days after. Latent infections were initiated with cell-free virus stocks of a previously detailed recombinant VZV (VZV66GFP) that expressed ORF66 as a GFP-ORF66 fusion ,68 LatenLive neuronal cultures were monitored using a Nikon TI fluorescent microscope with a 10× air objective (N.A. 0.30). Imaging of fixed neuron cultures followed previous detailed procedures ,23. NeurFlow cytometry was used to quantify GFP-fluorescent positive HEK293 cells in the initial evaluation of the efficiency of gRNAs to target genes and block protein expression. All flow cytometry samples were collected and analyzed using a BD FACSAria cytometer  and FlowJo software . Statistical analyses were performed using GraphPad Prism software . Where indicated, error bars represent standard deviation (SD) or standard error of the mean (SEM) as specified in the figure legends.The goal of the studies was to assess the potential of using gene editing as an anti-viral strategy targeting VZV, not only in lytic-infected cells that are permissive for VZV, but also in reactivating neuron cultures. We recently detailed a hESC-derived neuron culture system that can host a VZV model latent state that can subsequently be reactivated . Gene edThree gRNA candidates for each gene were selected based on an optimal on/off-target specificity, the presence of the PAM motif, and length. The appropriate hybridized oligonucleotides were then cloned into the AAV-based pX601 backbone plasmid  co-expreAAV capsid serotypes confer different tissue tropisms ,75,76,77These observations are consistent with previous studies reporting the ability of AAV2 and AAV5 to transduce ARPE-19 cells  and muri12 GC/mL and subsequently evaluated for their ability to prophylactically reduce lytic replication in VZV-infected ARPE-19 cells. ARPE-19 were mock treated or transduced with each AAV at 104 GC/cell and 4 days later, cultures were infected with a low dose (500 pfu per well of a 6-well plate) of mitomycin C-treated, cell-associated VZV DR or VZV DR-Δ71 (see Methods in High-titer preparations of AAV2-packaged saCas9 with (AAV-62-1gR-saCas9) or without (AAV-saCas9) the 62-1 gRNA sequence template were obtained at more than 104 GC/cell of ORF62-targeting AAV showed a dramatic reduction in the VZV reporter activity over time as compared to untreated and AAV-saCas9 controls for both viruses. This indicated that AAV-62-1gR-saCas9 was highly effective at reducing progeny virus production , VZV luciferase activity reported an increase in growth of both VZV DR and VZV DR-Δ71 over time. Strikingly and in contrast, cells pretreated with 10oduction a. The VZoduction b supportoduction c and theoduction d were vaThe AAV-62-1gR-saCas9 was designed to specifically target VZV at the ORF62/71 gene, but a well-known issue with gene editing technologies is the possibility of off-target effects. It is conceivable that a cellular factor required for VZV replication was damaged by off-target activities that would result in similar observations to those reported above. Therefore, as an additional control, two VZV recombinants in the background of VZV DR-Δ71 were derived, in which the guide RNA target sequence was mutated at 11 bases in the codons of ORF62 (VZV DR-62gmut clones #18 and #36) that maintained the same encoded open reading frame residues for ORF62. The expectation was that the substitutions of 11 bases in the Cas9 recognition region of ORF62 would render the 62-1 gRNA no longer able to recognize and cleave at this location . If the 4 GC/cell of AAV or left untreated. Four days later, the cultures were infected with 500 PFU of mitotically inhibited VZV DR-infected ARPE-19 cells. Viral growth was monitored visually at the same position in the cultures by microscopy over a 10-day period, and representative live-cell images of the foci of fluorescence in the neuron cultures at day 10 post infection are shown in the first column of The ability of AAV-62-1gR-saCas9 treatment to block lytic replication in hESC-derived neurons was then evaluated. We have previously shown that hESC-derived neuron cultures are able to support the spreading of productive VZV infections ,23,81. N4 GC/cell AAV or mock transduced, and then incubated in media without ACV. At 7 days post AAV transduction, GFP positive cells were again not observed, indicating that the AAV transduction itself did not induce VZV reactivation. A set of cultures was reactivated by treating with a combination of NGF withdrawal, antibody-mediated NGF depletion and the addition of 50 ng/mL 12-O-tetradecanoylphorbol-13-acetate (TPA), and cultures were monitored daily for GFP fluorescence indicating reactivation events for 7 days. After imaging, the neurons were dislodged, triturated, and seeded onto confluent ARPE-19 cells for infectious center assay , which can then be experimentally reactivated (to renew lytic reporter gene expression and produce virus that spreads to other neurons) . We thuser assay b.None of the neuron cultures latently infected with VZV66GFP and not subjected to reactivation stimuli expressed GFP. When such neurons were seeded on ARPE19 cells, infectious centers did not form. In contrast, the latently infected neurons receiving the reactivation stimulus developed numerous GFP-positive foci and formed multiple infectious centers when seeded onto ARPE-19 monolayers, indicating that productive reactivation had occurred . LatentlTo investigate whether the reduction in the number of productively infected neurons in reactivated cultures pre-treated with AAV-62-1gR-saCas9 was due to a reduction in the number reactivating genomes or a result of reduction of viral spread to additional neurons, SYBR-based qPCR quantification of VZV genomes was performed. There was an increase of more than 10-fold in genome copies at 21 days-post VZV infection in reactivated samples with or without AAV pretreatment compared to neurons not receiving a reactivation stimulus. While AAV-62-1gR-saCas9 pretreatment resulted in a significantly reduced level of genomes as compared to controls, levels of genomes measured were still higher than those seen in latently infected cultures not receiving any reactivation stimulus b,c. ThesThe goal of this study was to establish proof of principle that the CRISPR targeting of VZV genomes could be used as an antiviral strategy eventually leading to therapeutic applications. The results obtained here add to a growing body of knowledge in which several human DNA viruses (and RNA viruses with DNA genome intermediates) have been targeted by gene editing ,82,83,84Here, we demonstrated efficient antiviral activity using AAV-delivered gRNA targeting a duplicated gene to reduce VZV replication, spread and virus production in both lytic-infected epithelial cells and hESC derived neurons. Importantly, we further showed that it dramatically reduced viral replication upon reactivation from latency in vitro. Duplicated genes were targeted because of the increase in frequency of cleavage events as well as the potential to result in the division of the genome into two segments, each incapable of replicating if both cuts occurred in the same genome. This strategy eliminates the need for two separate vectors targeting unique region genes in order to generate dual-cleaved genomes, as performed in the study of Aubert et al., who showed that cleavage of the related HSV genome at two sites was more effective at reducing reactivation frequency than a single cleavage event . Aubert An important aspect of developing therapies from CRISPR/Cas enzymes is achieving an efficient delivery of the required gRNA and enzymes to the appropriate cell types. This is especially challenging for VZV, where the reservoir of latent viral genomes resides in ganglionic neurons throughout the peripheral nervous system. Therefore, the first steps in this project were to optimize the delivery of these molecules with a focus on human neurons. While both lentiviruses and AAV have been exploited for efficient gene delivery, current AAV vectors have the advantage in that they show little or no integration into the host genome . HoweverOur studies show that delivering AAV-62-1gR-saCas9 decreased VZV progeny infectivity dramatically in both epithelial cells and hESC-derived neurons, compared in no AAV and no gRNA (“AAV-saCas9”) controls. This was shown by the reduction of both progeny-virus plaque size and numbers. Intriguingly, VZV that survived AAV-62-1gR-saCas9 treatment generated significantly smaller plaques when seeded onto naïve VZV-permissive cells, particularly VZV derived from AAV-targeted neurons. This strongly suggests that the virus produced in AAV-62-1gR-saCas9-treated cells is damaged, most likely as a result of genome cleavage and error-prone DNA repair, resulting in indels from the activation of the dsDNA damage response. Given that ORF62 is an essential gene whose expression is required for the expression of most other VZV genes, we postulate that some of the still-replicating but impaired viruses in these plaques are damaged but still have ORF62 in frame that permits expression of an ORF62 protein with some functional mutations. The site of the gRNA target in region 1 of ORF62/71 is that which the Cas9 acted upon, because VZV with mutations in that region (“VZV DR-62gmut”) showed only a minor loss of replication efficiency compared to the parental strain, but were completely resistant to pretreatment with AAV-62-1gR-saCas9. This establishes that the Cas9 did not influence VZV replication because of off-targeting of the host genome, although we cannot exclude that off-targeting effects might have also occurred that did not affect VZV replication.One of the exciting potential applications of gene editing as an antiviral strategy is that it cannot only target productive replication of the virus, but can also damage the latent genomes and potentially prevent reactivation . TreatmeThe results presented here demonstrate the potential of the CRISPR/Cas9 strategy to reduce both epithelial manifestations of the reactivated disease and potential damage in the ganglia that could result from ganglionic spread after a reactivation event has initiated. Of course, much remains to be worked out in translating this approach to prevent zoster and VZV disease as a therapy. A possible initial application of AAV-mediated gene editing as an antiviral strategy could be for VZV-induced retinal diseases. The retina has been a prime target for advancing AAV-mediated gene therapy strategies, and there are promising results from both animal models and human studies that AAV-mediated delivery can restore vision loss for specific inherited defects . VZV rep


S. pneumoniae (SPN), including multidrug-resistant (MDR) strains. CPT fosamil is approved for clinical use in the United States (US) to treat community-acquired bacterial pneumonia (CABP). We evaluated the activity of CPT against clinical isolates of ceftriaxone-non-susceptible (CRO-NS) SPN from US medical centers. Ceftaroline (CPT) is a broad-spectrum cephalosporin with activity against 21,750 SPN isolates were consecutively collected (1 per patient) from 201 medical centers in 2008-2020 and tested for susceptibility (S) to CPT and comparator agents using CLSI broth microdilution methods. Among these isolates, 1,419 (6.5%) were CRO-NS (CRO MIC ≥2 mg/L). Other resistant subgroups analyzed included MDR  and extensively drug resistant  isolates.50/90, 0.25/0.25 mg/L); only 1 isolate had a CPT MIC ≥0.5 mg/L. The CPT-NS isolate exhibited CPT and CRO MIC values of 1 and 8 mg/L, respectively, and had multiple substitutions in the penicillin binding proteins (PBP), mainly PBP2x, when compared with reference sequences. Also, it showed 31 amino acid alterations in MurM. The most active comparator agents against CRO-NS SPN were linezolid , levofloxacin , tigecycline , and vancomycin . Overall, 20.5% of isolates were MDR and 7.9% were XDR. MDR and XDR rates decreased from 24.4% and 13.5% in 2008-2011 to 16.8% and 2.4% in 2018-2020, respectively. CPT retained potent activity against MDR  and XDR  isolates; whereas CRO exhibited limited activity against both MDR  and XDR  isolate subsets. Among CRO-NS isolates, 97.7% were MDR and 88.2% were XDR. CRO susceptibility varied from 86.9% in 2009 to 98.8% in 2019 and increased from 89.0% in the 2008-2011 period to 98.1% in the 2018-2020 period. CPT was active against 99.9% of CRO-NS SPN (MICCPT demonstrated potent and consistent activity over time (2008-2020) against a large collection of SPN from US medical centers, including CRO-NS, MDR, and XDR isolates. Helio S. Sader, MD, PhD, FIDSA, AbbVie (formerly Allergan) (Research Grant or Support)Basilea Pharmaceutica International, Ltd. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)Department of Health and Human Services Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors  Involved: Self): Research Grant or Support; Allergan  Involved: Self): Research Grant or Support; Amicrobe, Inc  Involved: Self): Research Grant or Support; Amplyx Pharma  Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc.  Involved: Self): Research Grant or Support; Astellas  Involved: Self): Research Grant or Support; Basilea  Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center  Involved: Self): Research Grant or Support; BIDMC  Involved: Self): Research Grant or Support; bioMerieux Inc.  Involved: Self): Research Grant or Support; BioVersys Ag  Involved: Self): Research Grant or Support; Bugworks  Involved: Self): Research Grant or Support; Cidara  Involved: Self): Research Grant or Support; Cipla  Involved: Self): Research Grant or Support; Contrafect  Involved: Self): Research Grant or Support; Cormedix  Involved: Self): Research Grant or Support; Crestone, Inc.  Involved: Self): Research Grant or Support; Curza  Involved: Self): Research Grant or Support; CXC7  Involved: Self): Research Grant or Support; Entasis  Involved: Self): Research Grant or Support; Fedora Pharmaceutical  Involved: Self): Research Grant or Support; Fimbrion Therapeutics  Involved: Self): Research Grant or Support; Fox Chase  Involved: Self): Research Grant or Support; GlaxoSmithKline  Involved: Self): Research Grant or Support; Guardian Therapeutics  Involved: Self): Research Grant or Support; Hardy Diagnostics  Involved: Self): Research Grant or Support; IHMA  Involved: Self): Research Grant or Support; Janssen Research & Development  Involved: Self): Research Grant or Support; Johnson & Johnson  Involved: Self): Research Grant or Support; Kaleido Biosceinces  Involved: Self): Research Grant or Support; KBP Biosciences  Involved: Self): Research Grant or Support; Luminex  Involved: Self): Research Grant or Support; Matrivax  Involved: Self): Research Grant or Support; Mayo Clinic  Involved: Self): Research Grant or Support; Medpace  Involved: Self): Research Grant or Support; Meiji Seika Pharma Co., Ltd.  Involved: Self): Research Grant or Support; Melinta  Involved: Self): Research Grant or Support; Menarini  Involved: Self): Research Grant or Support; Merck  Involved: Self): Research Grant or Support; Meridian Bioscience Inc.  Involved: Self): Research Grant or Support; Micromyx  Involved: Self): Research Grant or Support; MicuRx  Involved: Self): Research Grant or Support; N8 Medical  Involved: Self): Research Grant or Support; Nabriva  Involved: Self): Research Grant or Support; National Institutes of Health  Involved: Self): Research Grant or Support; National University of Singapore  Involved: Self): Research Grant or Support; North Bristol NHS Trust  Involved: Self): Research Grant or Support; Novome Biotechnologies  Involved: Self): Research Grant or Support; Paratek  Involved: Self): Research Grant or Support; Pfizer  Involved: Self): Research Grant or Support; Prokaryotics Inc.  Involved: Self): Research Grant or Support; QPEX Biopharma  Involved: Self): Research Grant or Support; Rhode Island Hospital  Involved: Self): Research Grant or Support; RIHML  Involved: Self): Research Grant or Support; Roche  Involved: Self): Research Grant or Support; Roivant  Involved: Self): Research Grant or Support; Salvat  Involved: Self): Research Grant or Support; Scynexis  Involved: Self): Research Grant or Support; SeLux Diagnostics  Involved: Self): Research Grant or Support; Shionogi  Involved: Self): Research Grant or Support; Specific Diagnostics  Involved: Self): Research Grant or Support; Spero  Involved: Self): Research Grant or Support; SuperTrans Medical LT  Involved: Self): Research Grant or Support; T2 Biosystems  Involved: Self): Research Grant or Support; The University of Queensland  Involved: Self): Research Grant or Support; Thermo Fisher Scientific  Involved: Self): Research Grant or Support; Tufts Medical Center  Involved: Self): Research Grant or Support; Universite de Sherbrooke  Involved: Self): Research Grant or Support; University of Iowa  Involved: Self): Research Grant or Support; University of Iowa Hospitals and Clinics  Involved: Self): Research Grant or Support; University of Wisconsin  Involved: Self): Research Grant or Support; UNT System College of Pharmacy  Involved: Self): Research Grant or Support; URMC  Involved: Self): Research Grant or Support; UT Southwestern  Involved: Self): Research Grant or Support; VenatoRx  Involved: Self): Research Grant or Support; Viosera Therapeutics  Involved: Self): Research Grant or Support; Wayne State University  Involved: Self): Research Grant or Support Cecilia G. Carvalhaes, MD, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support) S J Ryan Arends, PhD, AbbVie (formerly Allergan) (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support)


Nipple aspirate fluid (NAF) is thought to reflect the microenvironment of a developing breast cancer and therefore is considered an important biofluid to interrogate for diagnostic purposes.A meta-analysis of all English language research studies was performed, providing diagnostic information on the cytology of NAF across three databases and taking into account their quality scoring. The authors concluded that the diagnostic accuracy of NAF showed a pooled specificity of 0.97  and a sensitivity of 0.64 (95% CI 0.62–0.66). Of the patients who produced a sample across all studies, 38.97% provided an inadequate sample for cytologic analysis.In the search for superior methods of nipple fluid assessment, emerging technologies for fluid biomarker analysis must supersede the current diagnostic accuracy of NAF cytology while taking into account other important factors such cost effectiveness, reproducibility of results, user dependency, and turn-around time in the laboratory. Future work should focus on addressing these issues if cytology is to be replaced in clinical practice.


The older the age of patients, the more severe tics, and anxiety disorder significantly correlated with CTPh. CTPh may be considered as a part of tic spectrum with a substantial impact of anxiety disorder. CTPh are a late and age-related symptom of GTS.Coprolalia and echophenomena repeated in the patients’ mind (CTPh—cognitive tic-like phenomena) have been rarely recognized as part of Gilles de la Tourette syndrome (GTS) symptomatology and their assignment to tics, OCD or other psychopathologies has not been settled. The aim of the paper was to assess the incidence and clinical associations of CTPh in GTS, and to establish if CTPh belong to the tic spectrum. We performed a prospective, one-registration study on a cohort of 227 consecutive patients with GTS. CTPh were diagnosed during the interview and defined as brief, sudden, involuntary thoughts that had corresponding complex vocal tics. CTPh occurred at some point in the lives of 34 (15.0%) patients. The median age at onset of CTPh was 14.5 years (IQR: 10.5–17.5). CTPh were found more frequently in adults, with the most frequent onset in adolescence (44.1%). Four mental phenomena resembling tics were recognized: echolalia ( Diagnostic and Statistical Manual of Mental Disorders  in order to diagnose GTS [Gilles de la Tourette syndrome (GTS) is a neurodevelopmental disorder that most commonly affects children and adolescents, but in some cases persists into adulthood. According to the nose GTS , numerounose GTS . Individnose GTS . Neverthnose GTS , this kiOn the basis of current understanding, tics are sudden, rapid, recurrent, nonrhythmic motor movements or vocalizations . Even thIt is crucial to differentiate CTPh with the spectrum of obsessive-compulsive phenomena. OCD can be diagnosed when a patient reports obsessions/compulsions or both, the presence of which is time consuming or debilitating in terms of daily functioning and cannot be attributed to physiological effects of a substance or another medical condition. Obsessions are defined as recurrent, persistent thoughts, urges or impulses that are perceived by the patient as intrusive and unwanted; therefore, they are usually associated with anxiety and distress. In contrast to obsessions, CTPh are experienced as pleasant or neutral, do not lead to negative repercussions and are sequences in themselves (one type of CTPh does not connect to another phenomenon), while obsessions are often a coherent line of thoughts ,12. The Psychopathology related to GTS may be different in children and adults. Hirschritt reported that adults and adolescents were most likely to have OCD as well as mood, anxiety, eating and substance use disorders, whereas children were more likely to have ADHD . This laTo the best of our knowledge, there have been no studies that investigate CTPh in a clinical sample. The aims of this study were (i) to assess the prevalence and age at onset of CTPh in individuals with GTS; (ii) to assign this phenomenon to tics, OCD or another psychopathology; (iii) to distinguish between the clinical correlates of CTPh for children and adults. Based on available findings as well as our clinical experience, we hypothesized that (i) the prevalence of CTPh may vary depending on the age of the patients, (ii) CTPh are part of tic phenomenology and are neither part of OCD nor another psychopathology and (iii) clinical correlates of CTPh in children differ from those in adults.The cohort of individuals with GTS comprised 241 consecutive patients, who were evaluated from 2013 to 2019. In 14 patients, data on the presence of CTPh were not available, as these were young children unable to comprehend questions during the interview, resulting in a total sample size of 227 patients. In this group, the age of the patients ranged from 5 to 50 years . In total, 140 children  and 87 adults  were enrolled. The median age of tic onset was 6 years . The median duration of GTS was 4 years  in children and 18 years  in adults. Overall, 189 (83.3%) patients had at least one psychiatric comorbidity. Psychiatric comorbidities of GTS patients are presented in In total, 103 patients already received therapy at the time of examination, including behavioral therapy, tic-reducing drugs and OCD symptom-relieving medications.All the patients were recruited from one single outpatient clinic and were personally reviewed and evaluated by the same clinician, who was well experienced in tic disorders (PJ). Patients were referred to the clinic by general neurologists and psychiatrists, due to problematic diagnosis or tics refractory to treatment, or sought medical advice on their own because of troublesome tics. The study was designed as a one-time registration study, as patients were registered in the database only once, and no additional clinical data obtained in follow-up visits were included in the analysis.The patients were evaluated for the clinical diagnosis of GTS according to the Diagnostic and Statistical Manual of Mental Disorders criteria that were valid at the time of evaluation . All patients were systematically interviewed with the aid of a semi-structured interview that comprised demographic and clinical data that was gathered for all the patients. This schedule is based on the Tourette syndrome international database consortium (TIC) data entry form, developed by Freeman et al. , in whicThe prevalence of the most common comorbid disorders encountered in GTS was evaluated on the basis of the same, mentioned above, semi-structured clinical interview. Disorders that were listed in this semi-structured interview were as follows: ADHD, OCD, depression, anxiety disorder , oppositional defiant disorder, conduct disorder. The list of obsessions and compulsions included in the Yale–Brown Obsessive-Compulsive Scale (Y-BOCS) was used to establish clinical spectrum of OCD. Each patient was carefully questioned about all the symptoms included in the DSM as the diagnostic criteria of the above-mentioned comorbid disorders. The diagnoses of mental disorders that was made in psychiatric clinics before our evaluation were accepted and included into the analyses. One-third of children and adolescents, with more complex psychopathology, were assessed with the M.I.N.I. International Neuropsychiatric Interview for Children and Adolescents. Patients with severe psychiatric comorbidities were referred to psychiatrist to confirm the diagnosis. In this case, the psychiatric diagnosis was considered to be definite, and as such taken into consideration in the study analyses.Tic severity was measured using the Yale Global Tic Severity Scale (YGTSS) on the day of each patient’s evaluation . CTPh weCTPh were identified during the dialogue-based interview through an active inquiry. Due to lack of instruments validated for the assessment of CTPh, we used the clinical definition established by previous investigators , which wCTPh were defined as brief, sudden, recurring, neutral or pleasant mental acts, which had the equivalent among typically recognized complex vocal tics such as coprolalia, echolalia, palilalia and repeating of words or phrases. Complex and long-lasting thoughts, e.g., mentally jumping over telegraph poles along the roadside, drawing three-dimensional constructions in the mind or having the same tune in the mind for hours, were not classified as CTPh, as they do not have an analogous vocal tic. This type of diagnostic attitude precluded confusing CTPh with delusional disorder, ruminations in depression and obsessions, hyperquantivalent ideas or other psychiatric disorders. Importantly, none of the patients included in the study group were diagnosed with a psychotic disorder or bipolar disorder .CTPh were differentiated with obsessions and mental compulsions according to the differential diagnosis described in the introduction. For this purpose, interviews with patients covered questions about their emotional attitude towards appearing thoughts, the consequences of the thoughts, the natural form of the thoughts’ sequence, the possible aim of the thoughts, the resistance the patient made against these thoughts and if they were acceptable or not for the subject. Patients received questions on whether they had ever experienced short-lasting thoughts that appeared spontaneously in the mind. We asked the patients if they had repeated their own words or phrases in the mind two or more times in a row , if they had imitated several times in their mind words or phrases spoken by other people  or if they had repeated curse words voicelessly . We also asked the patients when those thoughts started, if they were present at evaluation and if the patients had ever experienced typical vocal coprolalia and echophenomena, to assess the relationship between CTPh and their vocal equivalent. If the patient reported the presence of at least one CTPh during the lifetime, he/she was included into CTPh+ group. The CTPh- group consisted of patients who had never experienced thoughts of this kind.We assumed that the factors that influence the lifetime prevalence of CTPh in GTS may differ from those that are related to CTPh at the time of examination. For example, a lifetime diagnosis of OCD does not necessarily contribute to the occurrence of CTPh as both symptoms could have occurred at different moments in the lifetime. In order to assign CTPh to tics or OCD phenomenology, lifetime and current comparative analyses were conducted. The lifetime comparative analysis included the group of the patients without any history of CTPh (CTPh-) and the group of the patients with a history of CTPh in the past or at the time of evaluation (CTPh+). When categorizing patients as having CTPh at evaluation, we took into account the two preceding weeks. In the current comparative analysis, we divided all the patients in two groups: CTPh current+  and CTPh current- (GTS patients without CTPh within last two weeks). As a result, CTPh current- included both patients without any history of CTPh and those with CTPh in the past but not at the time of evaluation. Moreover, in this analysis, we took into account only CTPh and OCD symptoms that were present at the time of the examination. The exact relationship could be determined only when both symptoms were present at the same time.Our plan of analysis included a comparison of the CTPh+ and CTPh- groups when considering the demographics and distribution of psychiatric comorbidities as well as premonitory urge and tic severity. Apart from analysis in the whole group, we also conducted the same comparison in adults and children. The statistical analyses were performed using STATISTICA version 13.1 , SPSS version 25 software  and R programming language. The Shapiro–Wilk test was used to assess the normality of distribution. In the case of the parametric variables, data were presented as arithmetic means and standard deviations (mean ± SD). For the non-parametric variables, we chose the median and quartiles (25:75) to present data. The categorical variables were presented as frequencies (percentages). Parametric data was compared using an independent t-test. For the nonparametric data, we used the Mann–Whitney U-test. The Chi-square test was performed for comparison of categorical data. For comparison of subgroups of patients with different CTPh, we used a test for small groups, the Fisher’s exact test.p-value had to reach <0.05 for the comparisons between the groups to be considered significantly different. In the course of the primary analyses, we isolated variables that emerged as significant and entered them into a logistic regression analysis in which the dependent variable was the presence of CTPh. Additionally, sex and age served as control variables in the multivariate model. Logistic regression analysis was also used to check whether CTPh were associated with their vocal equivalent.In both analyses, the n = 17), cognitive coprolalia (n = 16), cognitive palilalia (n = 13) and repeating words or phrases in the mind (n = 7). The presence of one CTPh occurred in 22 patients, of 2 CTPh in 5 patients and of 3 CTPh in 7 patients. The group of patients with only mental coprolalia (n = 8) was compared with the group with only mental echophenomena . These groups did not differ with regard to age, gender, YGTSS, premonitory urges, possibility of control over tics, relief after tic execution and psychiatric comorbidities. In 7 patients, mental coprolalia co-occurred with at least one echophenomena.All the patients who experienced CTPh recognized them as neutral, neither pleasant nor distressful in any way and reported them only when they had been asked for, and never spontaneously. Some individuals did not even realize they had these mental thoughts. CTPh occurred at some point in the lives of 34 (15.0%) patients. Four types of mental phenomena were evaluated: cognitive echolalia . In contrast, the presence of mental coprolalia did not increase the risk of mental echolalia  or mental palilalia .CTPh were reported in both patients who experienced their vocal equivalent as well as in those who did not present analogous vocalization. We checked whether CTPh were associated with their vocal equivalent but failed to demonstrate such relationship. Moreover, we assessed whether mental echophenomena had the tendency to occur together and demonstrated that mental palilalia and echolalia were associated with each other  were associated with CTPh. The YBOCS score that was available for 35 of 47 patients did not differentiate the CTPh+ and CTPh- groups . The age at onset of CTPh was known in 26 of all cases with the median value of 14.5 years (IQR 10.5–17.5). In 7 of the cases, CTPh started during childhood (before 11 years of age), in 15 of the cases it started during adolescence (12–18 years of age) and in 4 of the cases it started in adulthood.The prevalence of CTPh differed between adults and children/adolescents: 23.0% (20/87) and 10.0% (14/140), respectively  and coprolalia were similarly presented either in a form of vocalization or mental act. These observations indicate only a certain degree of relationship between the mental and vocal forms of the tic.We also looked for potential differences between various types of CTPh. However, we did not find any significant demographic and clinical differences between mental coprolalia and mental echophenomena, although very low numbers of comorbid psychiatric disorders and other variables taken into the comparative analysis do not allow for any final conclusions to be drawn. On the other hand, in a few cases mental coprolalia and mental echophenomena appeared in the same patients, which would be rather expected if these CTPh were to have a similar background. These statements should be verified in future studies.As we mentioned, the emotional attitude of the patients toward the experienced mental acts was neutral and not troublesome in any way, and they reported these thoughts only when they had been actively inquired, never spontaneously. That same was true for mental (silent) coprolalia. It was somehow surprising because, e.g., vocal coprolalia nearly always causes distress. Based on clinical experience, the possible explanation is that ‘loud’ coprolalia is distressful because it draws attention of other people and is socially unacceptable. If vulgar words/sentences are distorted or uttered in a low voice they are less bothersome and cause less distress for a patient. That is why we consider CTPh egosyntonic symptoms.CTPh correlated significantly with tic severity, premonitory urges and anxiety disorder. We formulated a hypothesis that particular mental phenomena reported by certain GTS patients may be a form of tic manifestation and we juxtaposed it with the theory that these symptoms are a representation of another psychopathology. Inferring from the positive correlation between CTPh and YGTSS , we consMoreover, we hypothesized that patients’ age could be a variable associated with the rate of CTPh, especially in patients below the age of 18. Age of the CTPh onset falls during adolescence in most patients, although it may occur at any time during the course of the disease. There is an evident association between the prevalence of this mental phenomenon and the age of the patients . The facWe found significant differences in correlates of CTPh in children/adolescents and adults. The older the child/adolescent patient was, the greater risk of CTPh they had. This correlation was not observed in adult group . MoreoveAnother possible consideration of CTPh character arises from the careful understanding of its definition: CTPh are pleasant or neutral, stimulating in their nature rather than regulating negative emotional states. Current research emphasizes two main basic human needs that should be taken into general consideration in describing and understanding psychiatric phenomena: the regulation of negative emotional states and reward seeking (stimulation) ,22. ImpoThe value of the performed study arises from both extending of scientific knowledge in the topic of mental phenomena in patients with GTS and the prospective clinical application of acquired data. In opposition to OCD, CTPh do not require treatment as they are perceived by patients as neutral and egosyntonic. It also seems important to pay attention to the presence of mental phenomena resembling tics, which do not fulfill the current diagnostic criteria of this hyperkinetic movements and, although unobservable, may belong to the spectrum of tic phenomenology. Our results suggest that GTS-affected patients who also have CTPh at the time of examination should be carefully evaluated for existing anxiety disorders and more severe tics. This could certainly influence the choice of appropriate treatment.There are several limitations to our study which include the following: lack of a control group; lack of any validated instrument in CTPh assessment; no confirmation of CTPh diagnosis from the another researcher who would evaluate the patient’s report independently; the established CTPh definition was limited to tic phenomenology with exclusion of overlapping, long-lasting mental phenomena from the OCD spectrum; recall bias could potentially influence the reported prevalence rate and age at onset of CTPh; the one-time registration study design could influence the rate of CTPh and psychiatric comorbidities; no validated rating scales for all adults and some patients under the age of 18 were used for the assessment of comorbidities; no rating scale was used to assess premonitory urges quantitatively; there could have been referral bias as the patients were evaluated by a neurologist and the cases with more severe psychopathology were referred to psychiatric clinics, which explains the low numbers of OCD and depression in the children group.We conclude that some complex tics may appear in two forms, vocal and mental. CTPh should presumably be considered as a part of the tic phenomenology with a crucial impact of comorbid anxiety disorder. CTPh are rather rare and late symptoms of GTS, tend to appear over the course of the disease and, hence, are an age-dependent symptom.


In patients with acute circulatory failure, fluid administration represents a first-line therapeutic intervention for improving cardiac output. However, only approximately 50% of patients respond to fluid infusion with a significant increase in cardiac output, defined as fluid responsiveness. Additionally, excessive volume expansion and associated hyperhydration have been shown to increase morbidity and mortality in critically ill patients. Thus, except for cases of obvious hypovolaemia, fluid responsiveness should be routinely tested prior to fluid administration. Static markers of cardiac preload, such as central venous pressure or pulmonary artery wedge pressure, have been shown to be poor predictors of fluid responsiveness despite their widespread use to guide fluid therapy. Dynamic tests including parameters of aortic blood flow or respiratory variability of inferior vena cava diameter provide much higher diagnostic accuracy. Nevertheless, they are also burdened with several significant limitations, reducing the reliability, or even precluding their use in many clinical scenarios. This non-systematic narrative review aims to provide an update on the novel, less employed dynamic tests of fluid responsiveness evaluation in critically ill patients. Fluid administration represents a first-line therapeutic intervention in patients with acute circulatory failure . The priThe decision to administer fluids has been traditionally based on the clinical signs of hypovolaemia, such as hypotension, oliguria, increased serum lactate, and static parameters of preload including central venous pressure or pulmonary artery wedge pressure . UnfortuDespite a very high degree of accuracy in predicting fluid responsiveness, especially in the case of PLR and PPV, all these methods have several significant limitations, making them impractical or even impossible to use reliably in certain clinical scenarios . For insOur aim was to identify all manuscripts relevant to the methods of predicting fluid responsiveness described in this article. For the purpose of this non-systematic narrative review, we performed a comprehensive electronic and manual search of the following databases: PubMed, Scopus, Web of Science, and Google Scholar, within the time frame of January 1990 to September 2021. Systematic reviews, meta-analyses, narrative reviews, randomized controlled trials, prospective and retrospective cohort studies, case reports, and letters to the editor were all included. Additional manuscripts were identified from the reference lists of screened articles. No language restriction was applied, although the information for non-English manuscripts was retrieved from the abstracts only. Only published or accepted manuscripts accessible online ahead of print were processed. Animal, cadaver, and manikin studies were excluded. The terms used included “fluid responsiveness”, “hypovolemia”, “preload”, “volume expansion”, “expiratory occlusion”, “inspiratory occlusion”, “arterial Doppler”, “vein variability”, “extrasystole”. J.H. and M.P. performed the initial search independently, while P.M. and J.K. reviewed their results and provided additional relevant studies.The end-expiratory occlusion test (EEOT) was first described in the study by Monnet et al. in 2009  as one o2 does not reliably predict fluid responsiveness during EEOT [In mechanically ventilated patients, EEOT is performed by interrupting the respiratory cycle at end expiration. This can be achieved, for instance, by using the expiratory hold function on the ventilator, routinely employed to measure intrinsic PEEP. Typically, an end expiratory hold lasting 15 s has been described in most of the studies ,38,39,40ing EEOT . In a sting EEOT . Similaring EEOT ,33,34,37ing EEOT . Neverthing EEOT  or 4% [3ing EEOT  using tring EEOT ,41. Howeing EEOT  and 7% fing EEOT . The saming EEOT  and oeso2O do not seem to influence the diagnostic accuracy of the test [The major limitation of EEOT is that it can only be performed in mechanically ventilated patients without any spontaneous breathing activity that would preclude a 15-s expiratory hold. PEEP levels between 5 and 14 cm Hthe test . The tidthe test ,35,40,41the test ,46 and wthe test . The maiAnother potentially useful dynamic parameter of fluid responsiveness is the respiratory variation of internal jugular vein (IJV) diameter, called the IJV distensibility/variability in mechanically ventilated patients, IJV collapsibility in spontaneously breathing patients, or jugular index ,51,52, amax) and lowest diameter (Dmin) values during one respiratory cycle are recorded. The measurement of anterior–posterior (AP) IJV diameter has been most commonly used [The recommended technique requires a patient to be placed either in the supine ,52 or 30nly used ,53,54,55nly used . It is oIJVV) is used, the calculation formula is modified to: On the other hand, if IJV variability   with 70 u et al. , IJV colu et al. ; howeveru et al. .The main advantage of IJVD compared to the more established distensibility of inferior vena cava lies in its lower technical difficulty and easier application, especially in patients with obesity, ascites, or intra-abdominal hypertension ,57. FurtThe respiratory variation of the inferior vena cava (IVC) diameter has been widely used as a reliable predictor of fluid responsiveness, although its diagnostic reliability has been questioned recently, especially in the cohort of spontaneously ventilating patients ,59. It hNevertheless, the hepatic vein spectral waveform may become atypical under many pathological conditions, such as a change in the flow direction (hepatic vein occlusion), irregularity (arrhythmias), mono-/biphasic shape , or D-wave dominance   Figure .In a prospective study with 44 mechanically ventilated patients in septic shock, HVF was evaluated from subcostal echocardiographic views by first scanning the IVC and then identifying the middle hepatic vein (MHV) . Pulse DThe authors concluded that none of the evaluated parameters were able to predict fluid responsiveness. However, an increase in ΔMHV D ≥ 21% during volume expansion was associated with a lack of fluid responsiveness with a sensitivity of 100%, specificity of 71%, and high ROC AUC of 0.918. Furthermore, MHV S-wave velocity significantly correlated with CO, as it increased after fluid challenge in the fluid responders but did not change in the non-responder group. Thus, HVF evaluation seems to offer an indicator for halting ongoing fluid administration and may serve as a surrogate to assess cardiac output changes. The main advantage of evaluating HVF is its lower technical difficulty and better availability in patients in whom obtaining standard TTE views might prove impossible, especially those after cardiac surgery. The major limitations represent the various concurrent pathologies affecting the hepatic blood flow pattern as described above. Further studies are warranted to validate the clinical utility of HVF assessment and its role in the fluid management of critically ill patients.Arterial Doppler measurements may provide valuable information about patients’ circulatory status and CO while being entirely non-invasive. Historically, oesophageal Doppler has been widely used to continuously monitor various haemodynamic variables including CO, which correlates closely with CO measured by a pulmonary artery catheter using the thermodilution method . OesophaAs FTc is considered to be a static parameter of preload, it was mostly used in conjunction with the passive leg raising (PLR) manoeuvre. In other studies, arterial blood flow (BF) was measured instead, obtained by using the standard formula: Nevertheless, such calculation of aortic flow requires the use of an oesophageal Doppler that may not be available in all patient settings. To overcome this limitation, the investigation of more easily accessible arteries has been explored, including the carotid, brachial, femoral, and splenic arteries. Most available data on arterial ultrasound Doppler monitoring focused on the carotid artery.In a study with 34 haemodynamically unstable patients in septic shock, Marik et al. . Unfortubrach) was investigated as a predictor of fluid responsiveness in 38 mechanically ventilated patients in a study by Garcia et al. [brach was measured prior to scheduled volume expansion and calculated using the following formula:Respiratory variability of brachial artery peak velocity  comparable to PPV. This was confirmed by another work by Brennan et al. [ΔVpeakn et al.  who founfemoral). Changes in CO were evaluated by measuring stroke volume with TTE. ΔVpeakfemoral was calculated for the PLR manoeuvre and fluid challenge as:Peak velocity of femoral artery flow and femoral velocity time integral variation have been evaluated in two studies in ICU patients with sepsis. Préau et al.  used a vfemoral ≥ 8% predicted fluid responsiveness with high accuracy (ROC AUC 0.93) and correlated with increased CO after fluid challenge; this parameter performed similarly to PPV measured by radial artery catheter. These results were subsequently replicated in the study of Luzi et al. in a mixed population of critically ill patients [A PLR-induced ΔVpeakpatients .S stands for the peak systolic velocity and D for the end-diastolic velocity. In a study by Corradi et al. [Splenic Doppler resistive index (SDRI) has also been proposed as a non-invasive parameter to detect hypovolemia. The technique is based on the ultrasound Doppler measurements obtained by placing the sample gate into the main branches of the splenic artery, about 1 cm past the splenic hilum. SDRI is calculated using the following formula: i et al. , SDRI wai et al.  showed tIn conclusion, the main advantages of evaluating arterial Doppler include the complete non-invasiveness and simplicity of the measurement, as all the arteries besides the splenic artery lie superficially and are easily examined by ultrasound, making this method especially handy in the emergency department. Regarding limitations, most published studies have focused on patients outside of the intensive care setting and data for critically ill patients are limited. Furthermore, in a large portion of available studies, CO was not monitored directly, and its eventual changes were only assumed as a consequence of intravascular volume reduction or upon clinical presentation ,66,67,68Most of the methods currently used for the evaluation of fluid responsiveness require advanced haemodynamic monitoring, which may not be readily available to all critically ill patients. Thus, to overcome such technical limitations, Vistisen et al. proposed that cardiac extrasystoles may be regarded as a preload modifying mechanism and their hemodynamic effect could be monitored using only standard electrocardiographic (ECG) and invasive arterial blood pressure (BP) measurements . This hyThe main advantage of using extrasystoles as a predictor of fluid responsiveness lies in its modest technical requirements, where only ECG and arterial BP waveform analysis are necessary. The method should be applicable and feasible in a large number of ICU patients. There are several significant limitations, however. First, the clinician is compelled to wait for an extrasystole to occur and unless the monitoring device used offers sufficient post-hoc analysis, an observer would be required to stand at the bedside, constantly checking the monitor. Thus, wider availability of automated ECG analysis software is required to make its application more convenient. Second, only patients with sinus rhythm have been evaluated so far. Third, there are no data on the effects of extrasystole timing in relation to the respiratory cycle . Lastly,The major limitation of the presented non-systematic review lies in the fact that it includes only small, non-randomized and single-centre studies, even including some lacking any reproducibility. Additionally, there has never been a direct comparison of the presented methods between them alone or to more established and widely used techniques of fluid responsiveness evaluation. Therefore, it is not clear whether any of the methods are superior to any other or which one should be used preferentially. Finally, the narrative nature of the review may lead to a selection and interpretation bias as it does not follow objective evidence-based criteria of study inclusion and evaluation used in systematic reviews.Adequate fluid therapy belongs to some of the most challenging yet also most essential skills of all intensive care practitioners as it has a paramount effect on a patient’s final clinical outcome. The evaluation of fluid responsiveness offers a means of differentiating patients in whom fluid administration will provide an increase in cardiac output; nevertheless, choosing the appropriate test with respect to its limitations, the clinical situation, as well as available equipment, may prove difficult. The presented novel methods of evaluating fluid responsiveness also have limitations and drawbacks; however, if used in a complementary fashion to the already-established techniques, they can provide additional valuable information to decide whether the patient requires fluid administration. In addition, the non-invasiveness of these methods represents a major advantage in highly vulnerable critically ill patients prone to iatrogenic complications. Most importantly, it is necessary to realize that fluid responsiveness is not per se an indication for volume administration. Fluid therapy should always be indicated based on clinical signs of hemodynamic instability and peripheral tissue hypoperfusion with regard to the individualized risk/benefit ratio.