Granular cell epulis is a rare benign tumor of the newborn. It originates from the alveolar ridge, most commonly from the maxillary alveolar ridge. Despite its striking appearance, the lesion is ultimately benign. However, immediate surgical treatment is required if there is a risk of airway obstruction or feeding difficulties. We report two cases of granular cell epulis presented at birth, the first with a large mass originating from the maxillary alveolar ridge and the second with the mass originating from the mandibular alveolar ridge. Both were successfully managed with surgical excision without complications. Histopathology of both masses confirmed the granular cell epulis diagnosis. Granular cell epulis is a rare tumor of the newborn and is also known as granular cell tumor of the newborn and Neumann’s tumor. This lesion on the gingiva was called “epulis” after the Greek for “to boil the gums” or “on the gums.” Neumann was the first to depict gingival overgrowth in 1871 . The tumPatient 1A full-term female infant was born to a gravida 4 para 1 + 2 mother at 40 weeks. At birth, she was noted to have a bulky mass originating from the upper gingiva protruding from the mouth immediately after delivery (Figure She was otherwise active, with no dysmorphic features, and had no other anomalies. Her breathing was normal, with no distress or desaturation, and she remained hemodynamically stable. Routine lab tests were normal. Maxillofacial surgery was consulted, and they decided to perform surgical resection. Excision of the mass was undertaken on day 3 of life. Post-operatively, she was extubated immediately, and the following day she was started on oral feeding and was breastfeeding well. She was discharged home in good condition, and there was no evidence of recurrence at a follow-up clinic visit.Patient 2The second case is a full-term female, a product of spontaneous vaginal delivery to a consanguineous parent with normal antenatal follow-up. All fetal US were reported as normal. Her Apgar score was 9 at both one and five minutes, cord pH was 7.35, birth weight 3400 g, length 52 cm, and head circumference 35 cm. She was appropriate for gestational age. She was noted to have a pedunculated mass originating from the lower gingiva, around 5 x 4 x 3 cm in size, firm in consistency, and lobulated (Figure Similar to case 1, excision of the mass was undertaken on day 2 of life, with no intraoperative complications. She was discharged home in a stable condition 48 hours later on full feeding. She was seen in the follow-up clinic with no evidence of recurrence.The mass biopsy of both patients revealed a circumscribed lesion lined by stratified squamous epithelium. The submucosa contained sheets of large polygonal cells with small central round nuclei and abundant eosinophilic granular cytoplasm, and the pathological diagnosis confirmed the preoperative diagnosis of granular cell epulis.Granular cell epulis is the most commonly used and recognized word for a rare neonatal benign tumor that is distinct from other granular cell tumors. Granular cell epulis appears as a lump in the mouth that originates from the alveolar ridge. It is more common in females than males and originates in the maxillary alveolar ridge. It displays no signs of malignancy or recurrence . The tumIn histopathology, large, round, and polyhedral histiocyte-like cells with small, oval-to-round nuclei and copious eosinophilic granular cytoplasm make up the bulk of the mass. The intervention of granular cell epulis depends on the size. The larger the size, which may interfere with feeding and breathing, necessitates surgical excision. Nonsurgical intervention is better for small masses as spontaneous regression may occur.Because granular cell epulis appears in newborns, the maternal hormonal impact may play a role in the progression of the lesion . WhetherGranular cell epulis is a rare soft-tissue tumor that arises from the oral cavity. Intervention depends on the size, as it may interfere with feeding and breathing. Most cases of congenital epulis can be diagnosed clinically. Radiological investigation postnatally may not be needed for uncomplicated cases. The overall prognosis of granular cell epulis is good.


Fluid administration is a cornerstone of treatment of critically ill patients. The aim of this review is to reappraise the pathophysiology of fluid therapy, considering the mechanisms related to the interplay of flow and pressure variables, the systemic response to the shock syndrome, the effects of different types of fluids administered and the concept of preload dependency responsiveness. In this context, the relationship between preload, stroke volume (SV) and fluid administration is that the volume infused has to be large enough to increase the driving pressure for venous return, and that the resulting increase in end-diastolic volume produces an increase in SV only if both ventricles are operating on the steep part of the curve. As a consequence, fluids should be given as drugs and, accordingly, the dose and the rate of administration impact on the final outcome. Titrating fluid therapy in terms of overall volume infused but also considering the type of fluid used is a key component of fluid resuscitation. A single, reliable, and feasible physiological or biochemical parameter to define the balance between the changes in SV and oxygen delivery  is still not available, making the diagnosis of acute circulatory dysfunction primarily clinical. Fluids are drugs used in patients with shock to increase the cardiac output with the aim to improve oxygen delivery to the cells. The response to fluid administration is determined by the physiological interaction of cardiac function and venous return. In septic shock, the beneficial clinical response of fluid administration is rapidly reduced after few hours and fluid titration is crucial to avoid detrimental fluid overload. The fluid challenge is a fluid bolus given at a defined quantity and rate to assess fluid responsiveness.The ideal fluid for critically ill patients does not exist; however, crystalloids should be used as first choice. Balanced crystalloid solutions may be associated with better outcomes but the evidence is still low. Albumin infusion may have a role in already fluid resuscitated patients at risk of fluid overload.Fluid administration is integrated into the complex management of pressure and flow “macro” hemodynamic variables, coupled to the “micro” local tissue flow distribution and regional metabolism. Macro-variables are managed by measuring systemic blood pressure and evaluating the global cardiac function. The critical threshold of oxygen delivery to the cells is difficult to estimate, however, several indexes and clinical signs may be considered as surrogate of that, and integrated in a decision-making process at the bedside. Fluid administration is one of the most common but also one of the most disputed interventions in the treatment of critically ill patients. Even more debated is the way how to appraise and manage the response (in terms of flow and pressure variables) to fluid administration, which ranges from a prosaic “just give fluids” to the fluid challenge, to the evaluation of fluid responsiveness before fluid administration to, finally, recent approaches based on machine learning and Artificial Intelligence aimed at personalizing its use –3.Shock occurs in many intensive care unit (ICU) patients, representing a life-threatening condition that needs both prompt recognition and treatment to provide adequate tissue perfusion and thus oxygen delivery to the cells . A large2), so that systemic oxygen requirements can be met  versus a slower infusion rate , which reflects less than the 25% percentile in FENICE cohort study  giving a distribution half-time of about 15 h. This rate may increase up to 20% or more in septic shock. Accordingly, the measured TER of albumin to the tissues  is said to be an index of ‘vascular permeability .The role of albumin for fluid therapy is still debated (reference 64). Although theoretically promising for its anti-inflammatory and anti-oxidant proprieties , and forOn the other waterside of fluid therapy, crystalloids are composed of water and electrolytes . Saline ® versus Saline (PLUS) study was a blinded RCT in 5037 adult patients expected to stay in the ICU for at least 72 h and needing fluid resuscitation , which is the gradient between PCO2 in mixed venous blood (PvCO2 measured with pulmonary artery catheter) and PCO2 in arterial blood (PaCO2): P(v-a)CO2 = PvCO2-PaCO2 [2 during shock emphasize that it is still useful to measure central venous PCO2 instead of mixed venous blood. In health, ΔPCO2 ranges between 2 and 6 mmHg.The venous-to-arterial COO2-PaCO2 . This po2 changes in a shock state are coupled with other indices of tissue perfusion. First of all, according to a modified Fick equation, ΔPCO2 is linearly linked to CO2 generation and inversely related to CO [2 and between impairment in microcirculatory perfusion and tissue PCO2 [2 may be due to either a low CO state or to an insufficient microcirculation to remove the additional CO2 in hypoperfused tissues despite an adequate CO.The pathophysiological background of the determinants of this index is rather complex but ΔPCOed to CO . Severalsue PCO2 . Accordi2 and ScVO2. In fact, an increased ΔPCO2 associated with a decreased ScVO2 is suggestive of a low CO, whereas a normal/high ScVO2 associated with an increased ΔPCO2 indicates impaired tissue perfusion. Pragmatically, a normal ∆PCO2 (< 6 mmHg) value in a shocked patient should defer from increasing the CO as first step; instead, regional blood flow may be impaired even in presence of a normal/high CO.These conditions may be further investigated by coupling the information obtained by ΔPCO2. This choice recognizes fluids as drugs that should only be used as long as the effect on CO is likely. Monitoring fluid responsiveness during the resuscitation phase of an episode of acute circulatory failure may be achieved by applying a “closed-loop” operative strategy, where the signs of tissue hypoperfusion and the findings of CCE are re-evaluated after each fluid bolus. More sophisticated tools are useful when the cardiovascular system reaches the plateau of clinical response or, earlier, when the CCE shows acute or acute-on-chronic cardiac dysfunction at the baseline examination of the patient.All these aspects may be integrated into a decision-making algorithm where the clinical signs of hypoperfusion are coupled with the CCE evaluation Fig. . The cliThe physiology of fluid administration in critically ill patients is of major importance in ICU. With a solid basis in the dynamic and complex balance between cardiovascular function and systemic response, fluids should be considered as drugs and intensivists should consider their pharmacodynamic and biochemical properties to optimize the therapy. A multimodal approach is required since single physiological or biochemical measurements able to adequately assess the balance between the CO and tissue perfusion pressure are still lacking. The assessment of response to fluid administration may be obtained by coupling the changes of different signs of tissue hypoperfusion using clinical and invasive hemodynamic monitoring, with the evaluation of cardiac function based on critical care echocardiography.


Recent studies have demonstrated that blocking the PI3Kδ signalling enzyme  can potently improve the anti-tumour T-cell response through direct inhibition of Tregs. This treatment also has a negative impact on MDSC numbers but the primary mechanism driving this effect has remained unclear.The 4T1 breast cancer mouse model was used in combination with PI-3065 to gain insights into the effect of PI3Kδ inhibition on MDSCs.PI-3065 treatment resulted in a concomitant reduction in MDSC expansion and tumour size. However, targeting Tregs independent of PI-3065 was also associated with reduced tumour volume and MDSC numbers. Surgical removal of tumours resulted in a rapid and significant decline in MDSC numbers, whilst ex vivo studies using cells from PI-3065-treated mice demonstrated no direct effect of the inhibitor on MDSC activity.Our data suggest that MDSCs are not inhibited directly by PI-3065 treatment but that their reduced recruitment and immunosuppression within the tumour microenvironment is an indirect consequence of PI3Kδ-inhibition-driven tumour control. This indicates that PI3Kδ inhibition drives tumour immunity by breaking down multiple immunosuppressive pathways through both direct mechanisms (on Treg) and indirect mechanisms, secondary to tumour control (on MDSCs). Elevated numbers of both Tregs and MDSCs occur in the peripheral blood of cancer patients, including breast cancer patients, and have been associated with cancer stage and poor prognosis . Tumour and spleen weight was measured at endpoint following excision of the tumour from the host.Adult (8-week-old) female BALB/c and BALB/c Nude (CAnN.Cg-Foxn1/Crl) mice were purchased from Charles River, Depletion of REGulatory T-cell (DEREG) mice, a strain of BALB/c mice developed using a Bacterial Artificial Chromosome (BAC) containing the xp3 gene , were brPI-3065 was provided by Genentech and administered by oral gavage at a dose of 75 mg/kg, with vehicle-treated mice given an equivalent volume of carrier solution, as described previously . Mice weSpleens were isolated from tumour-bearing mice, mechanically disaggregated and then passed through a 70-μm filter, prior to lysis of red blood cells using RBC lysis buffer (Biolegend).Cells were washed twice with PBS and stained using LIVE/DEAD Aqua (Invitrogen) according to the manufacturer’s instructions. Cells were washed twice with FACS buffer and Fc receptors blocked with anti-CD16/32 . Cells were surface stained with the following antibodies : CD11b , CD11c , Ly6G , Ly6C , CD45 , c-KIT , NK1.1 , CD19 , CD3 , CD8 , CD4 , FoxP3 .Tumours of comparable sizes were removed at day 18 post tumour injection. Mice were anaesthetised (isofluorane) and given a subcutaneous injection with the anti-inflammatory analgesic, Metacam (Boehringer Ingelheim). The tumour and the surrounding area were shaved and swabbed with Hibiscrub antibacterial wash and Surgical Spirit. Aseptic techniques were used to excise the tumour and close the wound using horizontal mattress sutures (Ethicon). Mice, placed in a heated recovery chamber until consciousness was regained, were monitored postoperative for well-being and suture strength. At day 21 (3 days post resection) and day 28 (10 days post resection), spleens were isolated from both resected mice and tumour-bearing mice at the equivalent time points and the splenic MDSC populations enumerated.+ T cells were isolated from naive spleens using a negative magnetic bead selection kit (Biolegend). MDSCs were isolated from the spleens of mice bearing 4T1 tumours that had either been treated with vehicle or PI-3065 using a negative magnetic bead selection kit (Stemcell). CD8+ T cells were labelled with Tag-It-Violet (Biolegend) prior to stimulation with plate-bound anti-CD3 10 μg/ml , 1 μg/ml soluble anti-CD28  and 30 IU/ml IL-2. MDSCs were added to the CD8+ T cells at a ratio of either 1:1 (MDSCs:CD8) or 4:1. Cocultures were left for 3 days prior to flow cytometric analysis.CD8t test was used to compare two groups or for multiple groups, one-way ANOVA with a Tukey’s multiple comparison post hoc test was performed. For non-normally distributed datasets, a Mann–Whitney t test was used to compare between groups and a Kruskal–Wallis test with Dunn’s multiple comparison post hoc test used to compare between multiple groups. P values of ≤0.05 were considered significant, with values of ≤0.01 considered highly significant. The number of mice used and replicates is detailed for each figure in the corresponding figure legend. The proliferation index used in Fig. In figures + Ly6G+ Ly6Clo) in vehicle-treated tumour-bearing animals, and to a lesser extent by expansion of M-MDSCs   Fig. . NK cellon) Fig. . PI-3065on) Fig. . Althoug+ T-cell response and altering the ratio of CD8:Tregs in favour of tumour control [The reduction in splenic MDSCs was associated with reduced Treg numbers and reduced tumour volume in PI-3065-treated mice, whilst vehicle mice exhibited a significantly elevated frequency of splenic MDSCs, high frequencies of Treg and larger tumours. These findings are in line with our previous study, which showed that administration of PI-3065 results in Treg inactivation, unleashing an anti-tumour CD8 control , 18.With these data in mind, we hypothesised that the effect of the PI3Kδ inhibitor on MDSC was mediated indirectly via effects on Treg and/or tumour growth. To test this, we first examined MDSC numbers in mice where Treg were depleted by administration of diphtheria toxin (DT) to DEREG mice . We foun+ MDSCs was similar in small (treated) and large (control) tumours (Fig. + PMN-MDSCs and M-MDSCs, corresponding with fewer overall MDSCs in the spleen (Figs. To confirm the importance of the tumour in expanding and maintaining splenic MDSCs we employed a tumour resection model in vehicle-treated, Treg-replete mice. We injected mice with tumours at day 0, and then split the mice into two groups; mice undergoing surgical tumour resection and mice whose tumours would continue to grow Fig. . We obseurs Fig. , there wen Figs. . To asseen Figs. , indicatWhilst the above data support the premise that tumours drive MDSC expansion and that inhibition of tumour growth via Treg depletion controls this expansion, they do not rule out direct effects of PI3Kδ-inhibition on MDSCs. Indeed, studies to date have suggested that MDSCs require signalling via PI3Kδ for maximal suppressive effect . In addiTo determine if targeting the PI3Kδ signalling pathway affected MDSC numbers in the absence of direct effects on Tregs or tumours, we injected tumours into BALB/C Nude mice and treated with either vehicle or PI-3065. PI-3065 treatment in the absence of T cells conferred no tumour control Fig. . Analysi+ T-cell proliferation in vitro [+ T cells from naive mice were stimulated with anti-CD3 and CD28 antibodies and co-cultured with MDSCs isolated from tumour-bearing vehicle- or PI-3065-treated mice. At a ratio of 1:1, neither vehicle- or PI-3065-derived MDSCs had a notable effect on CD8+ T-cell proliferation (Fig. + T-cell proliferation. However, there was no significant difference between MDSCs derived from either vehicle- or PI-3065-treated mice, implying no direct effect of the inhibitor on MDSC activity.We have previously demonstrated that the addition of PI-3065 to splenocytes has a direct effect on CD8in vitro , thus a ion Fig. . At a hiPrevious studies have demonstrated that both M-MDSCs and PMN-MDSCs are dependent on PI3K signalling for optimal immunosuppression . GeneticTregs and MDSCs are known to play reciprocal roles within the tumour microenvironment, with MDSCs promoting de novo induction of Treg , 11, whiFinally, given that Ali and colleagues previously demonstrated impaired immunosuppression by PI3Kδ genetically deficient MDSCs , we sougThe tumour itself is central to the immunosuppression mediated by MDSCs, as removal of the tumour dramatically reduces the peripheral MDSC population, enabling the establishment of a feedback loop favouring tumour rejection. Alleviation of immunosuppression by surgical removal of tumours has also been reported in patients with colorectal cancer , highligaj checklistSupplementary Figure 1


Recently in Japan, six workers at a chemical plant that manufactures resins developed interstitial lung diseases after being involved in loading and packing cross-linked water-soluble acrylic acid polymers (CWAAPs). The present study focused on assessing lung damage in rats caused by workplace-relevant inhalation exposure to CWAAP and investigated the molecular and cellular mechanisms involved in lung lesion development.3 of CWAAP-A for 4 h or to 15 or 40 mg/m3 of CWAAP-A for 4 h per day once per week for 2 months (9 exposures). In a separate set of experiments, male F344 rats were administered 1 mg/kg CWAAP-A or CWAAP-B by intratracheal instillation once every 2 weeks for 2 months (5 doses). Lung tissues, mediastinal lymph nodes, and bronchoalveolar lavage fluid were collected and subjected to biological and histopathological analyses.Using a whole-body inhalation exposure system, male F344 rats were exposed once to 40 or 100 mg/mA single 4-h exposure to CWAAP-A caused alveolar injury, and repeated exposures resulted in regenerative changes in the alveolar epithelium with activation of TGFβ signaling. During the recovery period after the last exposure, some alveolar lesions were partially healed, but other lesions developed into alveolitis with fibrous thickening of the alveolar septum. Rats administered CWAAP-A by intratracheal instillation developed qualitatively similar pulmonary pathology as rats exposed to CWAAP-A by inhalation. At 2 weeks after intratracheal instillation, rats administered CWAAP-B appeared to have a slightly higher degree of lung lesions compared to rats administered CWAAP-A, however, there was no difference in pulmonary lesions in the CWAAP-A and CWAAP-B exposed rats examined 18 weeks after administration of these materials.The present study reports our findings on the cellular and molecular mechanisms of pulmonary disease in rats after workplace-relevant inhalation exposure to CWAAP-A. This study also demonstrates that the lung pathogenesis of rats exposed to CWAAP-A by systemic inhalation was qualitatively similar to that of rats administered CWAAP-A by intratracheal instillation.The online version contains supplementary material available at 10.1186/s12931-023-02355-z. CWAAPs have long been used in a variety of products, including cosmetics and pharmaceuticals, because of their low potential for skin and eye irritation. However, in Japan, six workers at a chemical plant that manufactures CWAAPs recently developed interstitial lung diseases after being involved in weighing, packing, and transporting CWAAP products . Notably 22 min) , 6.3 CWAAP-A for 6 h per day, 5 days per week, for 13 weeks. Evaluation of all organs in the rats demonstrated that toxicity caused by inhalation exposure to CWAAP-A was limited to alveolar lesions in the lungs. Histopathological diagnosis of the lungs on the day after the final exposure showed that alveolar lesions, which were an inflammatory reaction, were observed in all rats exposed to 1, 3 and 10 mg/m3 CWAAP-A. The lowest observed adverse effect concentration (LOAEC) for CWAAP-A in this study was 1 mg/m3 for both sexes, and 0.3 mg/m3 was the no observed adverse effect concentration (NOAEC). It was also found that exposure to 3 and 10 mg/m3 CWAAP-A caused irreversible and progressive lung lesions [We recently reported the results of a 13-week subchronic inhalation study that was conducted with reference to the OECD guidelines for the testing of chemicals, OECD TG 413 , to obta lesions . ImportaInterstitial lung disease encompasses numerous lung disorders characterized by inflammation and scarring (fibrosis). Pulmonary fibrosis destroys lung tissue and decreases elasticity, making it difficult for the lungs to get enough oxygen. It is well known that transforming growth factor (TGF) β signaling is important in the progression of pulmonary fibrosis . TGFβ biAnother important consideration is the large number of CWAAPs with different molecular weights and degrees of cross-linking and the lack of information on their hazard to human health. Thus, establishment of a rapid and simple method to evaluate the potential risk of different CWAAPs is also very important. Therefore, this study also investigated using intratracheal instillation as an adjunct to inhalation exposure.CWAAP-A and CWAAP-B were purchased from a company which produces various polymers. The CWAAP-A was the same as the CWAAP-A used in our previous study . As showAll animal experiments were approved by the Animal Experiment Committee of the Japan Bioassay Research Center. Male F344 rats were purchased from Charles River Laboratories Japan, Inc.  and Japan SLC, Inc. . The rats were housed in an air-conditioned room under a 12 h light/12 h dark  photoperiod, and fed a general diet  and tap water ad libitum. After 1–2 weeks of quarantine and acclimation, they were treated with CWAAPs.3 in the field survey [3 was used in both the repeated exposure study (15 and 40 mg/m3) and single exposure study (40 and 100 mg/m3) . Inhalation exposure to CWAAP-A was started once the rats were 11 weeks old. A total 60 rats were used for the repeated exposure study. 6 rats in each group were sacrificed immediately after the last exposure and 6 rats in each group were sacrificed at the end of week 10. The remaining 8 rats in each group were sacrificed at the end of week 26 . The humidity (30–40%) in the chamber was adjusted using a valve linked to the humidification bottle. Once the humidity in the inhalation chamber was below 40%, the animals were placed in the chamber and exposure was initiated.3 in the 40 mg/m3 chamber and 100.4 mg/m3 in the 100 mg/m3 chamber, and the CWAAP-A MMAD and σg values were similar to those of the repeated exposure study. These data indicate that CWAAP-A exposure was stable throughout the exposure periods in both the repeated and single exposure studies.During exposure, the number of particles in each inhalation chamber was monitored by an optical particle controller , and the concentration of CWAAP-A in the inhalation chamber was measured at least twice from hour 1 to hour 3 after the start of exposure by collecting the test substance in the inhalation chamber on a fluoropolymer binder glass fiber filter . The particle size of CWAAP-A in the chamber was measured using a micro-orifice uniform deposit cascade impactor  during the second and the eighth exposures during the repeated exposure study. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (σg) were calculated by cumulative frequency distribution graphs with logarithmic probability. In addition, CWAAP-A in the inhalation chamber was collected on a 0.2 μm polycarbonate filter  and observed by scanning electron microscope (SEM) . Typical concentrations of CWAAP-A in the repeated exposure study are shown in Additional file In the repeated exposure study, rats were euthanized by exsanguination under isoflurane anesthesia immediately after the last exposure and at 2 weeks and 18 weeks after the last exposure. In the single exposure study, rats were sacrificed immediately after exposure and 3 days after exposure. BALF was collected as described below, and the lungs from which BALF was not collected were weighed and then fixed in 10% neutral phosphate-buffered formalin solution.3/h) in an environment of 50 mg/m3, a level that can be considered relatively close to the short-term exposure to high concentrations that occurred at the CWAAP manufacturing site. For comparison with the whole body inhalation studies, CWAAPs were administered once every 2 weeks over an 8 week period  . At 2 weeks and 18 weeks after the final CWAAP administration, rats were placed under isoflurane anesthesia and euthanized by exsanguination. BALF was collected as described below. The left lung was then weighed and fixed in 10% neutral phosphate buffered formalin solution.CWAAP-A and CWAAP-B were suspended in PBS, sonicated and neutralized by 1 M NaOH. The final concentration of the CWAAPs solution was 1 mg/ml. Our preliminary study suggested that a single intratracheal administration of 1.5 mg/kg would have too strong an effect on the lung, while a dose of 0.5 mg/kg would be much weaker and return to normal in a few weeks  for 10 min at 4 °C, and the supernatant was examined using an automatic analyzer .The left  or right  bronchus was tied off with a thread, and the opposite lung lobes were lavaged with 4–8 ml of saline. The total cell number in the BALF was counted using an Automated Hematology Analyzer . Cell populations on glass slides were prepared using Cytospin 4 . After May-Grunwald-Giemsa staining, the differential white blood cell count was made by visual observation. To measure LDH activity, the BALF was centrifuged at 1960 rpm . For TGFβ1 and TGFβ2 assays, the absorbance at 570 nm was also measured and subtracted as the background signal.SP-D concentrations in the BALF and plasma were determined using a Rat/Mouse SP-D kit YAMASA EIA . In this assay, the BALF was diluted 500- or 1,000-fold, and plasma was diluted 25-fold with the assay diluent included in the kit. BALF concentrations of TGFβ1 and TGFβ2 were measured by Human/Mouse/Rat/Porcine/Canine TGF-beta 1 Quantikine ELISA Kit (R&D Systems) and Mouse/Rat/Canine/Porcine TGF-beta 2 Quantikine ELISA Kit (R&D Systems). For the TGFβ2 assay, the BALF was diluted threefold with the sample diluent included in the kit. The absorbance at 450 nm was measured using a microplate reader .The content of hydroxyproline, a main component of collagen, in the lungs was measured using a hydroxyproline assay kit (Perchlorate-Free) . Small pieces of lung (about 30 mg) were homogenized in 10 volumes of distilled water using a portable power homogenizer . The homogenates were processed according the kit's instructions. The hydroxyproline signal (absorbance at 560 nm) was measured using a microplate reader  was stained with H&E for histological examination and the remaining sections were used for immunohistochemical analysis. The histopathological findings in this study for lung and mediastinal lymph node were determined after multifaceted discussions between certified pathologists from the Japanese Society of Toxicology and Pathology and certified medical pathologists from the Japanese Society of Pathology, based on the International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice (INHAND) . PatholoIt is known that the alcian blue pH 1.0 method primarily detects sulfate groups, and the alcian blue pH 2.5 method preferentially detects carboxyl groups. CWAAPs were stained blue by both staining methods. The present study used the alcian blue pH 1.0 method, which did not stain the mucus of bronchial and alveolar epithelium  for 10 min at room temperature. The slides were then successively stained with 0.8% orange G solution  for 10 min at room temperature, Ponceau  acid fuchsin  azophloxine  mixture for 40 min at room temperature, 2.5% phosphotungstic acid for 10 min at room temperature, and blue aniline solution  for 10 min at room temperature. Between each staining the slides were washed lightly with 1% acetic acid water. The slides were then processed for light microscopy.This method is a variant of PAS staining in which glycogen is degraded by pre-treatment with diastase to eliminate crossover against glycogen and improve specificity . Slides 2O after color detection, the sections were treated with citrate buffer at 98 °C for 30 min before incubation with the next primary antibody to denature the antibodies already bound to the section. This procedure was repeated for the second and then the third primary antibodies. HighDef red IHC chromogen  was used for the second coloration and Histogreen chromogen  for the third coloration. Coloration was followed by hematoxylin staining for 30–45 s. The slides were then processed for light microscopy. For immunofluorescence staining, all primary and secondary antibodies used were made into a cocktail for each staining step and used simultaneously. After the fluorescence-labeled secondary antibodies reaction, the sections were shielded with DAPI-containing encapsulant and used for imaging. The sections were observed under an optical microscope ECLIPSE Ni  or BZ-X810 . For measurement of phospho-SMAD3 and Tm4sf1 positive indices, the sham air group (n = 5) and the 40 mg/m3 group immediately after exposure (n = 7), and the sham air group (n = 5) and the 40 mg/m3 group after an 18-week recovery period (n = 8) were used for analysis. For the 40 mg/m3 exposure group, positive indexes were counted separately for multifocal lesions and normal surrounding tissue. In all animals, at least five fields of view were measured using a 40 × objective lens. More than 500 cells per individual were measured for phospho-Smad3 and 300 cells per individual were measured for Tm4sf1, and the mean value per individual was used for statistical analysis.Details of the multiple staining method have been described previously . Brieflyp < 0.05.Except in the case of incidence and integrity of histopathological lesions, the data comparisons among multiple groups were performed by one-way analysis of variance with a post-hoc test (Dunnett’s or Tukey’s multiple comparison test), using GraphPad Prism 5 . The incidences and integrity of lesions were analyzed by the chi-square test using GraphPad Prism 5 . All statistical significance was set at 3 for 4 h per day once per week for 2 months : 40 mg/m3 is the highest exposure concentration found in the workplace. No significant effects on final body weight or general condition were observed -Diastase positive staining: in the area marked hotspot  were observed in lung cross-sections . In the hotspot area, a large number of inflammatory cells were observed in the alveolar and perivascular interstitium, spreading to the subpleural area : we diagnosed this as alveolitis. Alveolitis was also observed in multifocal lesions . Alveolitis also developed in the lungs of rats exposed to 15 mg/m3 CWAAP-A. These findings indicate that the lesions observed at the end of the exposure period in rats exposed to 15 and 40 mg/m3 CWAAP-A continued to develop during the recovery period. In sharp contrast to the alveoli, in the bronchus and bronchiole regions neither epithelial cells nor the surrounding interstitium were prominently affected by CWAAP-A exposure  marker), and CD68  was used to identify the cell types in which TGFβ signaling was activated. The TGFβ signaling responder cells show brown nuclei (phospho-Smad3-positive), macrophages show green cytoplasm (CD68-positive), and AEC2 cells show red cytoplasm (LPCAT1-positive) Fig. C. The alred Fig. E. Furthered Fig. D–H. TheshighAEC2 cells as AEP cells, were consistently observed in the lesions from immediately after exposure to the end of the 18-week recovery period  were not observed in normal lungs or bronchiolo-alveolar hyperplasia  were not observed in normal lungs  exposures and intratracheal instillation  produced lung pathologies similar to those produced in our previous CWAAP-A inhalation study . In addi3 × h) should be determined [3 exposure level in the present study was 1440 mg/m3, and the cumulative dose for the for 10 mg/m3 exposure level (the highest exposure level) in the previous study was 3900 mg/m3. Despite this approximately 2.7-fold difference in overall exposure level, these two groups had similar findings and severity in histopathological parameters, especially lung lesions  and ventilator-induced lung injury secondary to mechanical ventilation, which is the only treatment for ARDS . ARDS isThe present study found that alveolitis with fibrous thickening of the alveolar septa occurs as a typical chronic lesion caused by CWAAP-A. Although most of the inflammatory lesions were restored to normal after an 18-week recovery period following the final exposure to CWAAP-A  are candidates for collagen-producing cells in pulmonary fibrosis , 46. TGFThe present study is the first to investigate the pulmonary toxicity of CWAAP-A in rats by comparison of both high-concentration intermittent inhalation exposure and intratracheal instillation. The results showed that the pathological lesions caused by intratracheally administered CWAAP-A were qualitatively similar to those caused by inhalation exposure, and changes in plasma and BALF biochemical and cytological parameters were also similar after both types of exposure protocols. These findings indicate that intratracheal instillation is useful for the initial assessment of acute and chronic toxicity of CWAAP-A. Furthermore, using intratracheal instillation a second CWAAP, CWAAP-B, was evaluated. Two weeks after the final administration of CWAAPs there was a significant increase in lung weight and higher LDH activity in the BALF of the rats administered CWAAP-B compared to CWAAP-A, suggesting that CWAAP-B may have higher pulmonary toxicity than CWAAP-A. Thus, intratracheal instillation, which is rapid, simple, and inexpensive, is effective for the initial investigation of pulmonary toxicity of different CWAAPs.The present study has a limitation. While we succeeded in clarifying the pulmonary toxicity of CWAAP-A in rats in the acute phase up to the chronic phase, since animals were evaluated only up to 26 weeks we could not clarify the end-stage pathogenesis of CWAAP-treated lungs, such as whether alveolitis with fibrous thickening progresses to pulmonary fibrosis. This may be the reason that in this study there was no evidence of progressive fibrosis with a high degree of destruction and remodeling of alveolar structures. Notably, one case of bronchiolo-alveolar hyperplasia, a pre-neoplastic lesion, was observed in one CWAAP-A-exposed rat in the inhalation study and one CWAAP-A treated rat in the intratracheal instillation study. To evaluate any lung carcinogenicity associated with inflammatory-fibrotic diseases caused by CWAAP, longer-term studies are needed.In this study, we investigated the cellular and molecular mechanisms related to rat pulmonary toxicity after exposure to CWAAP-A, which has caused occupational lung diseases in Japanese workplaces in recent years. The results showed that a single exposure to a high level of CWAAP-A caused alveolar injury in the acute phase and that high-concentration intermittent (repeated) exposures caused regenerative changes in the alveoli. After the end of exposure, during the recovery period, the alveolar lesions partially recovered to normal, but the some progressed to alveolitis with fibrous thickening. TGFβ signaling in AEC2 and AEP cells and AEP cell expansion were primary events in the pathogenesis of CWAAP-A induced disease. Moreover, we compared the lung pathology of CWAAP-A administered by systemic inhalation exposure with that of CWAAPs administered by intratracheal instillation and found that the lung pathologies were similar. The use of intratracheal instillation as an adjunct to inhalation exposure is expected to greatly accelerate testing of respirable CWAAP products, and consequently, to significantly increase our understanding of the pulmonary toxicity of CWAAP products.Additional file 1: Figure S1. Representative images of CWAAP-A, CWAAP-B and the chemical structural formula of CWAAP. Representative images of CWAAP-A (A), CWAAP-B (B) and the chemical structural formula of CWAAP (C-E). Acrylic acid polymer is a polymerized product of acrylic acid with carboxyl groups (C) and is anionic because of a large amount of carboxyl groups in the molecule. Cross-linked acrylic acid polymers (CWAAPs) have the characteristics of absorbing and retaining a large amount of water (E), as the polymer chain expands when it contains moisture compared to its dry state (D).Additional file 2: Figure S2. The three animal experimental protocols used in this study. The high-concentration intermittent (repeated) inhalation exposure study (A), the high-concentration single inhalation study (B), and the repeated intratracheal instillation study (C).Additional file 3: Figure S3. The whole-body inhalation exposure system using in this study. The direct-injection whole body inhalation system (A). Exposure concentrations of CWAAP-A in each chamber (B). Representative scanning electron microscope (SEM) images of the CWAAP-A particles in the chambers (C). Cumulative frequency distribution graphs with logarithmic probability (D). The mass median aerodynamic diameter (MMAD) and geometric standard deviation (σg) in the chambers measured during the second and eighth exposures (E). Scale bar: 20 μm (panel C).Additional file 4: Figure S4. Experimental protocol of a pilot study. Experimental protocol of a pilot study using a single intratracheal instillation of 0.5 or 1.5 mg/kg CWAAP-A and CWAAP-B (A). Total cell number (B) and LDH activity (C) in the BALF of CWAAPs-treated rats and their respective controls (PBS).Additional file 5: Figure S5. Representative images of the alcian blue staining of the normal rat lung and slides. Representative images of the alcian blue staining of the normal rat lung are shown in A. In B, CWAAP-A and CWAAP-B were placed on the slides and directly stained using Alcian blue.Additional file 6: Figure S6. Representative microscopic photographs of a normal rat lung (sham air) after the 18 week recovery period. All data are the results of high-concentration intermittent inhalation study.Additional file 7: Figure S7. Representative histopathological photographs of the bronchus and bronchiole in the rat lung after repeated inhalation exposure to CWAAP-A (40 mg/m3). All data are the results of high-concentration intermittent inhalation study.Additional file 8: Figure S8. Representative images of Tm4sf1 expression in the rat lung after repeated inhalation exposure to 40 mg/m3 CWAAP-A. All data are the results of high-concentration intermittent inhalation study.Additional file 9: Figure S9. Figure S9. Representative images of the AEC2 markers co-staining. Representative images of the AEC2 membranous marker RT2-70 co-staining with AEC2 cytoplasmic markers LPCAT1 (A)and ABCA3 (B) in the rat lung.Additional file 10: Figure S10. Sampling procedure of a single inhalation exposure study. Sampling procedure of the air inflated left lung without formalin injection (A) and comparative images of the right lung inflated by formalin and the left lung inflated by air (B).Additional file 11: Figure S11. Representative lung histopathological images of a single inhalation exposure study. Representative histopathological images of the right lung inflated by formalin and the left lung inflated by air of a rat 3 days after exposure to 100 mg/m3: the right lung and the left lung are from the same animal.Additional file 12: Table S1. List of primary antibodies used in this study.Additional file 13: Table S2. Summary of differences between hotspot and white spot.


Whole genome sequencing using the long-read Oxford Nanopore Technologies (ONT) MinION sequencer provides a cost-effective option for structural variant (SV) detection in clinical applications. Despite the advantage of using long reads, however, accurate SV calling and phasing are still challenging.We introduce Duet, an SV detection tool optimized for SV calling and phasing using ONT data. The tool uses novel features integrated from both SV signatures and single-nucleotide polymorphism signatures, which can accurately distinguish SV haplotype from a false signal. Duet was benchmarked against state-of-the-art tools on multiple ONT sequencing datasets of sequencing coverage ranging from 8× to 40×. At low sequencing coverage of 8×, Duet performs better than all other tools in SV calling, SV genotyping and SV phasing. When the sequencing coverage is higher (20× to 40×), the F1-score for SV phasing is further improved in comparison to the performance of other tools, while its performance of SV genotyping and SV calling remains higher than other tools.https://github.com/yekaizhou/duet.Duet can perform accurate SV calling, SV genotyping and SV phasing using low-coverage ONT data, making it very useful for low-coverage genomes. It has great performance when scaled to high-coverage genomes, which is adaptable to various clinical applications. Duet is open source and is available at  The importance of structural variants (SVs) in genetic disorders has been elaborated in recent studies using traditional molecular techniques –3. ThirdSV) and the proportion (pSV) of supporting reads with SV marks are low [SV and pSV between the true SV calls and the false signals, which can be observed and used for quality control. However, in low-coverage settings, it is common that there are only one or two supporting reads with SV marks for both true and false signals. Low-coverage data will generate SV candidates with low and very close pSV and NSV, which are hard to distinguish from true SVs and false signals based on this single feature. Therefore, novel supporting read signatures and novel features may add to the granularity of the quality control workflow, which is promising for boosting the performance of low-coverage SV calling.However, current SV detection tools including cuteSV , SVIM 1, NanoVar are low –14. ThisSV and NSV is high or median, but the tools lose discriminative power when the sequencing coverage is low. Moreover, this method does not distinguish the paternal (1|0) or maternal (0|1) haplotype of the heterozygous SVs (SV phasing), which is important for clinical applications. To achieve SV phasing and accurate SV genotyping in low-coverage, it is reasonable to solicit additional supporting read signatures, such as the haplotype tendencies of the reads potentially from single-nucleotide polymorphism (SNP) calling and phasing. The additional signatures serve as the raw materials to derive novel features, which provide the granularity for SV phasing and quality control in SV calling at low-coverage.Second, these tools can only distinguish the genotype of the detected SVs (SV genotyping). The genotype can be assigned homozygous (1/1) or heterozygous (0/1) if the level of pIn this paper, we introduce Duet, a tool for SV calling, genotyping, and phasing, optimized for ONT data. Instead of relying solely on the SV signatures on the reads –14, DuetThe schematic diagram of Duet is depicted in Fig. SNP calling generates heterozygous SNPs that can be used as a proxy to infer the reads’ haplotypes. We applied the ONT model of Clair3 , a fast Using the SNP-calling results, Duet applies WhatsHap  with “whIt is essential to apply a base SV caller that is sensitive even when using low-coverage data to reduce the initial false-negative rate, as a large proportion of false positives can be filtered out after SV phasing. After performance evaluation, cuteSV  with theDuet integrates the SV and SNP signatures obtained from the above modules to represent each supporting read, i.e., haplotype (HP), confidence of the haplotype prediction , and the number of SV marks (VC). For each SV call set with multiple supporting reads, several novel features are derived from the signatures, and an empirical rule-based decision path with corresponding cut-off values is derived accordingly.The decision path contains three layers:HP) with a very high average phasing confidence (μPC).First, an initial filter is applied to filter out SV calls that contain too small a proportion of phasable reads (pSV), to identify false positive SV calls (0/0) if with too low pSV, and homozygous variants (1|1) if with high pSV.The remaining SV calls will go through a subsequent characterization based on the proportion of reads containing SV marks . If these two values have no huge difference , the SV call will be classified as homozygous (1|1). Otherwise, if there is a huge difference between the total phasing confidence, the SV call will be classified as heterozygous and assigned to the haplotype with the dominant total phasing confidence (0|1 or 1|0).The further remaining SV calls with moderate proportion of reads with SV marks will be finally distinguished between being homozygous or heterozygous, where the phasing confidence is taken into meticulous consideration: Duet classifies the supporting reads into paternal group and maternal group based on their haplotype. Then the sum of the phasing confidence for each haplotype group is calculated  if it does not satisfy Eq. In the evaluation of SV calling, an SV candidate is determined as a true positive (TP) if it meets the following conditions:With the above definition, precision  is defined as:Recall  is defined as:F1 score is defined as:When considering the performance of SV genotyping, each TP SV call in SV calling gets further evaluated if its genotype is the same as the corresponding base call. If so, it is assigned to be a TP call, otherwise, it will be assigned as an FP call. All the FP calls in the SV calling evaluation procedure remain FP in SV genotyping. Each of the ground truth SVs will be evaluated and assigned TP if there is at least one TP call corresponding to it, otherwise it is assigned false negative (FN). Equations –4) are  are 4) aSwitch error rate is a standard metric to evaluate the phasing accuracy of abundant and adjacent genome variants such as SNPs. However, this metric fails to be a reasonable metric for SVs, which are large and distant . TherefoWe compared Duet against four state-of-the-art SV callers, SVIM , cuteSV The F1-scores are shown in Fig. The performance of Duet has stable improvement when the sequencing coverage is increased to 20×, and achieved up to 0.87 precision, 0.79 sensitivity, and 0.82 F1-score genome-wide for SV calling; 0.81 precision, 0.75 sensitivity, and 0.77 F1-score for SV genotyping; and 0.77 precision, 0.72 sensitivity, and 0.74 F1-score for SV phasing. Duet showed increased improvement compared to other tools in SV phasing. Duet still outperformed other tools in SV calling and genotyping and the second in place is cuteSV and LongPhase, respectively.The performance of Duet has slight improvement when the sequencing coverage is increased from 20× to 40×, and achieved up to 0.87 precision, 0.79 sensitivity, and 0.83 F1-score genome-wide for SV calling; 0.82 precision, 0.75 sensitivity, and 0.78 F1-score for SV genotyping; and 0.78 precision, 0.73 sensitivity, and 0.75 F1-score for SV phasing. Duet still has a great competing edge in SV phasing. Duet still outperformed other tools in SV calling and genotyping and the second in place is cuteSV and LongPhase, respectively.We further benchmarked Duet against the other tools at all three sequencing coverages on specific SV types including insertion and deletion. The results are shown in Fig. The runtime of Duet using 40 CPU threads ranged from 1.3 to 2.2 h at 8×, 1.6 to 2.3 h at 20×, and 2.0 to 3.6 h at 40× sequencing coverage across the three samples , and in between as heterozygous (0/1). For SV phasing, we set NSV ≥ 2 for both haplotypes as homozygous (1|1), NSV ≥ 2 for only one haplotype as heterozygous (0|1 or 1|0), and NSV < 2 for both haplotypes as false. The results are shown in Fig. To demonstrate the advantage of Duet’s novel features compared to the traditionally used features, we additionally compared Duet to using either pIn this study we developed a tool for SV calling, genotyping, and phasing, which we named Duet. We demonstrated that Duet is more accurate and powerful than other state-of-the-art tools in resolving genomic SVs, particularly those that lead to disease, when only low-coverage data is available, which is very useful in clinical settings and resource-constrained lab settings where sufficient genomic data coverage may not be available. At higher coverages, Duet’s performance in SV phasing is further improved in comparison to other competing tools used for the purpose, while maintaining a greater performance in SV genotyping and SV calling. The benchmark results of Duet further demonstrate that by incorporating SNP calling, SNP phasing, read haplotagging, the tailored decision tree is beneficial to SV detection, especially in accurate SV phasing and the quality control for low-coverage SV calling. Although Duet was optimized for ONT data, it has no restriction applying to PacBio data, albeit some thresholds need to be tuned to achieve the best potential.SV used by other tools has recovered its discriminative power to remove false signals. Duet’s current quality control method has clear improvement at low-coverage, and will generally be caught up by other state-of-the-art methods when the coverage goes higher.Starting from middle-coverage, the competing edge of Duet in SV calling becomes less significant. The main reason is that when the sequencing coverage goes higher, the feature pThe performance improvement of Duet from middle-coverage to high-coverage is moderate. All the benchmarked tools have similar situation. This is because our highly accurate truth sets are derived from multi-platform sequencing, and some genome regions are hard to map using single-strand sequencing technology, even with high coverage. Despite this objective explanation, there is possibly room to improve the performance of Duet at high-coverage data by updating the quality control method to fit both low- and high-coverage sequencing data in a coverage-dependent manner.There are still some limitations in Duet:In this study, we introduced Duet, a long-read-based tool for accurate SV calling and phasing. Duet showed promising results that the incorporation of SNP signatures will largely boost the performance of SV detection, especially when there is constraint in sequencing coverage and when there is a need for accurate SV phasing. The tool’s great adaptability and scaling performance in SV calling and phasing promise its usefulness in routine clinical applications.


In neovascular age-related macular degeneration (nAMD) the exact amount of fluid and its location on optical coherence tomography (OCT) have been defined as crucial biomarkers for disease activity and therapeutic decisions. Yet in the absence of quantitative evaluation tools, real-world care outcomes are disappointing. Artificial intelligence (AI) offers a practical option for clinicians to enhance point-of-care management by analysing OCT volumes in a short time. In this protocol we present the prospective implementation of an AI-algorithm providing automated real-time fluid quantifications in a clinical real-world setting.This is a prospective, multicentre, randomized (1:1) and double masked phase III clinical trial. Two-hundred-ninety patients with active nAMD will be randomized between a study arm using AI-supported fluid quantifications and another arm using conventional qualitative assessments, i.e. state-of-the-art disease management. The primary outcome is defined as the mean number of injections over 1 year. Change in BCVA is defined as a secondary outcome.Automated measurement of fluid volumes in all retinal compartments such as intraretinal fluid (IRF), and subretinal fluid (SRF) will serve as an objective tool for clinical investigators on which to base retreatment decisions. Compared to qualitative fluid assessment, retreatment decisions will be plausible and less prone to error or large variability. The underlying hypothesis is that fluid should be treated, while residual persistent or stable amounts of fluid may not benefit from further therapy. Reducing injection numbers without diminishing the visual benefit will increase overall patient safety and relieve the burden for healthcare providers.EudraCT-Number: 2019-003133-42 Nearly 196 million people worldwide are estimated to be affected by age-related macular degeneration (AMD). With a growing proportion of the elderly population worldwide, AMD is projected to affect up to 288 million people by 2040 .The treatment of nAMD was revolutionized by the introduction of anti-vascular endothelial growth factor (anti-VEGF) leading to a substantial initial restoring of visual acuity (VA) . Anti-VEDisease activity encouraging retreatment decisions in more individual treatment regimens are determined by vision loss and predefined morphologic disease biomarkers. Spectral-domain optical coherence tomography (SD-OCT) has been established as the leading diagnostic tool in everyday clinical routine to determine disease activity due to its ability to non-invasively visualize the retinal compartments and their respective pathological changes in a three-dimensional manner . ExudatiRecent studies found distinct structure-function correlations for specific exudative processes in nAMD. IRF has been consistently associated with severe and irreversible vision loss . The rolThe here presented prospective randomized controlled trial will be conducted to investigate the implementation of AI-supported macular fluid assessments in a real-world setting, compare AI-supported quantitative fluid measurements to conventional physician-based qualitative OCT assessments and identify the most efficient and relevant biomarker conditions which are providing highest vision gains with the lowest burden of interventions.The aim of this study is to compare treatment enriched with automated quantitative fluid volume measurement, with the conventional qualitative fluid assessment in the management of active nAMD. We introduce an optimized pro-re-nata (PRN) regimen, supported by AI fluid quantifications, in a real-world setting, with the goal to increase treatment effectiveness. Effectiveness will be evaluated by the primary outcome, a reduction in the number of retreatments. Secondary outcomes encompass BCVA assessment, morphologic or sensitivity changes in the macular retina, perfusion shifts in the choroid, retina or in neovascular lesions, fibrosis formation and improvements in quality-of-life.The goal of this optimized AI-supported PRN regimen is to guarantee patient safety and visual outcomes while lessening the treatment burden for the patients, physicians and budget holders alike.This study will be carried out in accordance with the ICH-GCP and follow the tenets of the declaration of Helsinki including current revisions.This phase III clinical trial will be prospective, multi-centred, multinational, randomized 1:1) patient and best-corrected visual acuity (BCVA)-examiner masked. The study aims to recruit two cohorts of 145 patients each, adding up to a total study population of 290 patients. Figure :1 patienPatient eligibility will be assessed during the baseline (BSL) visit and will follow the predefined inclusion , history of uncontrolled glaucoma (defined as intraocular pressure ≥25 mmHg) under IOP lowering treatment, presence of corneal decompensation, haze or scaring with an impact on BCVA will be excluded from our study population. Further, patients with any surgical treatment of the study eye within the last 3 months prior to baseline will be excluded. Previous anti-VEGF treatment of nAMD will not be considered an exclusion criterion. Therefore, patients with previous anti-VEGF treatment can still be included into the study population, if active nAMD is present.(I)Department of Ophthalmology and Optometry, Medical University of Vienna, Vienna, Austria;(II)Department of Ophthalmology, Landeskrankenhaus Horn, Lower Austria, Austria;(III)Department of Ophthalmology, Hopital de la Croix, Lyon, France;(IV)Department of Ophthalmology, Centre Hospitalier Intercommunal, Creteil, France;(V)Department of Ophthalmology, CHU Montpellier, Hopital Gui de Chauliac, Montpellier, France;(VI)Department of Ophthalmology, Rhena Clinique de Strasbourg, Strasbourg, France;(VII)Department of Ophthalmology, CHU Dijon, Dijon, France;(VIII)Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland;(IX)University Eye Clinic, San Giuseppe Hospital, IRCCS MultiMedica, Milan, Italy.The recruitment process will take place at different national and international study sites:Patients will be randomized 1:1 into an AI-supported PRN regimen with quantitative fluid volume measurement available for decision-making or a conventional PRN regimen, in which fluid is assessed by the treating physician in a qualitative manner. Patients in both study arms will undergo monthly scheduled visits at their respective study site. During every visit, patients receive a standardized ophthalmologic examination including BCVA tests using ETDRS charts, a slit-lamp biomicroscopy (SL) with fundoscopy (FD) and intraocular pressure (IOP) measurement.Morphological disease activity will be monitored with SD-OCT  or Cirrus ), colour fundus photography and additionally OCTA-Scans  if available.Blue-peak blue-laser autofluorescence (BAF), infrared autofluorescence (IRAF), infrared/blue reflectance (IR and BR) and fluorescein angiography (FA) , will be performed at baseline and at the end of the study (EOS) visit. Further a quality-of-life questionnaire (QS) will be filled out by all patients at the beginning and end of the study.At sites where available, patients will receive a microperimetry examination (MP)  and polarization-sensitive optical coherence tomography  scan at baseline, during the visit in the third month (Visit 3) and at the EOS visit. All activities performed during the visits are summarized in Table ®, Novartis); Aflibercept  or Brolucizumab .All eyes will be treated using a PRN regimen with monthly assessments. The anti-VEGF agents applied are determined by each study site according to their routine. Available agents are Ranibizumab  study arm will receive retreatments during Mo1 and/or Mo2 if more than 10 nl of SRF or IRF can be detected in the central 1 mm by an automated artificial intelligence algorithm . Patients in the qualitative study arm will receive retreatments during Mo1 and/or Mo2 if any fluid in the central 1 mm is detected by the investigator. Afterwards, retreatment will be performed based on quantitative or qualitative disease activity.SD-OCT scans from patients in quantitative study arm will be analysed by the AI-Software . Therefore, the SD-OCT volumes, obtained during the monthly visits, will be transferred into the AI database. The AI-based algorithm differentiates and divides specific pixels between the subcategories IRF, SRF, PED and normal tissue. The number of assigned pixels to each fluid type can be computed into an estimation of fluid volumes and their respective location. The amount of time attributed to this process, is dependent on the used volume of OCT data and lies between 1 to 2 min. Pseudonymized image sets are shared by the cloud and are processed by the algorithm on a central server. The clinical investigator will receive the results after completion on a web-based user-interface and a report will be printed out, which will be added to the case report form (CRF).In case that less than 10 nl SRF or IRF were present during Visit 2 and/or Visit 3 retreatment will only take place, if SRF or IRF increase over the 10 nl threshold. If IRF or SRF volumes in the central millimetre were over the 10 nl threshold during Visit 2 and Visit 3 a retreatment will be triggered by an increase of SRF or IRF over 50% compared to respective fluid volumes during Visit 3.For patients in the qualitative  study arm the physician will perform a qualitative fluid assessment, as present or absent, without giving special consideration to the exact volume or change in volume. In the case of fluid presence/detection, retreatment will be necessary according to standard guidelines in the literature , 20.For both arms, a reduction of 5 EDTRS letters or more related to any suspected neovascular activity compared to previous visits and/or new sub-retinal haemorrhage qualifies as a sign of disease activity and fulfils retreatment criteria.In order to adequately respond to the recent role of IRF fluctuations as harmful for visual acuity  and the In general, the final decision in both treatment arms remains at the discretion of the treating physician. If it is different from what is proposed in the study protocol, the reason will be indicated in the CRF.Since our study is the first proof-of-principle study using AI algorithms prospectively, sample size calculation is a difficult task. With regards to the results of the relaxed study arm in the FLUID-Study, we estimate that the mean number of injections in the study arm without AI is at least 8.5. Further we estimated that the standard deviations, in the study arms with and without AI would be 2.6 and 2.25, respectively. We base these estimations on the results obtained by Guymer et al. in the FLUID Study .The significance level  was set to 0.05. Given a power of 0.8 and a clinically relevant reduction of 10% of the injections during the course of 12 months  we will include 130 patients in each study arm . Taking a 10% dropout rate into account our sample size should encompass 145 participants in each study arm, resulting in a total sample size of 290 patients. However, this total sample size was calculated with a rather conservative clinically relevant difference. We expect an even greater difference in total number of injections between the two study arms therefore, an interim analysis is planned after half the patients (145) complete the study in order to verify that our sample size calculations are adequate.While the mean number of injections will be our primary endpoint, longitudinal changes in BCVA will be considered as a crucial secondary endpoint in order to guarantee treatment effectiveness.Longitudinal changes in secondary outcomes like quantitative fluid measurement or perfusion of the neovascular lesions, will be investigated for each study arm using a repeated measurement analysis of variance with Geisser-Greenhouse correction for sphericity (ε).p < 0.05.Nominal parameters in the quality-of-life questionnaire will be analysed using a chi²-test. Statistical significance is obtained with a Currently, best clinical practice proposes that patients with nAMD are treated under the treat and extend (T&E) or PRN regimen based on the presence or absence of IRF and/or SRF according to the physician’s discretion . The proOur study protocol is based on previously substantiated structure-function correlation in exudative processes during nAMD, which has been investigated thoroughly in the recent past . NonetheA study conducted by Michl et al., using automated fluid quantification indicated that IRF volumes diminished by over 90% between the baseline visit and the first month. SRF volumes were even reduced by over 95% . This suA quantitative measurement of fluid by an objective AI-algorithm outmanoeuvres most of the obstacles present in qualitative fluid assessment. Keenan et al. suggested that an AI-algorithm could surpass retinal specialists in accurately detecting fluid pooling, especially IRF and in difficult cases with lower fluid volumes in fewer B-scans . AI-algoWith regards to the developments during the Covid-19 pandemic, numerous healthcare systems have sought to minimize face-to-face contacts between patients and treating physicians. The concept of a “virtual” medical retina clinic has been brought up as a promising and innovative approach . In certReal-world outcomes are disappointing in comparison to visual acuity gains in clinical trials.Automated artificial intelligence (AI)-algorithms have shown robust results for fluid quantification in retrospective analysis in neovascular age-related macular degenerations (nAMD).Using AI it has become possible to provide real-time quantifications of macular fluid for more precise retreatment decisions.Automated AI-algorithms are for the first time implemented into real-world clinical practice.A prospective retreatment protocol based on the objective assessment of fluid activity instead of presence alone will introduce novel standards for optimized retreatment over long-term follow-up.Optimized treatment regimen will increase the treatment capacity of healthcare systems and therefore reduce vision loss in a real-world setting.


Objectives: To determine the prevalence and load of Ureaplasma spp. DNA in the cervical fluid of women with singleton pregnancies complicated by preterm prelabor rupture of membranes (PPROM) with respect to intra-amniotic infection, sterile intra-amniotic inflammation, and colonization of the amniotic fluid.Methods: A total of 217 women with PPROM between gestational ages 24 + 0 and 33 + 6 weeks were included in this study. Paired amniotic and cervical fluid samples were collected at the time of admission via transabdominal amniocentesis and using a Dacron polyester swab, respectively. Microbial invasion of the amniotic cavity was diagnosed using a combination of culture and molecular biology methods. Intra-amniotic inflammation was determined based on the concentration of interleukin-6 in the amniotic fluid. Based on the presence or absence of these conditions, the women were stratified into the following subgroups: intra-amniotic infection (with both), sterile intra-amniotic inflammation (with inflammation only), colonization (with microorganisms only), and negative amniotic fluid (without either). The Ureaplasma spp. DNA load in the cervical fluid was assessed using PCR.Results:Ureaplasma spp. DNA in the cervical fluid was found in 61% (133/217) of the women. Women with negative amniotic had similar prevalence of Ureaplasma spp. DNA in cervical fluid (55%) to those with sterile intra-amniotic inflammation (54%) but lower than those with intra-amniotic infection (73%) and colonization . Women with negative amniotic fluid had a lower load of Ureaplasma spp. DNA in their cervical fluid (median: 4.7 × 103 copies of DNA/ml) than those with intra-amniotic infection (median: 2.8 × 105 copies DNA/ml), sterile intra-amniotic inflammation (median: 5.3 × 104 copies DNA/ml), and colonization .Conclusion: In conclusion, in PPROM at <34 weeks, the presence of intra-amniotic infection, sterile intra-amniotic inflammation, or colonization of the amniotic fluid was associated with a higher prevalence and/or load of Ureaplasma spp. DNA in the cervical fluid than the absence of intra-amniotic complications. Preterm prelabor rupture of the membranes (PPROM) is defined as rupture of the fetal membranes with leakage of amniotic fluid before the onset of regular uterine activity before 37 weeks of gestation . PPROM iThe presence of microbial invasion of the amniotic cavity and intra-amniotic inflammation complicates approximately 23–41% and 17–58%, respectively, of pregnancies with PPROM . Based oUreaplasma spp. are the most common microorganisms recovered from the amniotic fluid obtained from PPROM pregnancies. Therefore, these low-virulence bacteria with sizes comparable to those of viruses represent the most common cause of intra-amniotic infection or colonization of the amniotic cavity in PPROM  age ≥18 years; 2) singleton pregnancy; 3) gestational ages between 24 + 0 and 33 + 6 weeks; 4) PPROM; and 5) amniocentesis to assess the intra-amniotic environment. The exclusion criteria were as follows: 1) pregnancy-related complications ; 2) chronic medical complications ; 3) congenital or chromosomal fetal abnormalities; 4) signs of fetal hypoxia; 4) significant vaginal bleeding; and 5) preterm labor with intact membranes.PPROM was diagnosed based on visual confirmation of amniotic fluid pooling in the posterior vaginal fornix by sterile speculum examination. If uncertainty about amniotic fluid leakage remained after the clinical examination, the presence of insulin-like growth factor-binding protein in the vaginal fluid was assessed .th gestational week. This subset of women with PPROM was managed actively .Body fluid samples  were collected at the time of admission before the administration of corticosteroids, antibiotics, or tocolytics. Transabdominal amniocentesis to obtain an amniotic fluid sample is a standard part of the department’s clinical management of women with PPROM to assess the intra-amniotic environment. Women with PPROM received corticosteroids to accelerate lung maturation and intravenous antibiotics. Women with intra-amniotic inflammation received clarithromycin for 7 days unless delivery occurred. Those without intra-amniotic inflammation were treated with benzylpenicillin for 7 days unless delivery occurred. In the case of penicillin allergy, women were treated with clindamycin for 7 days unless delivery occurred. Once the final results regarding microbial invasion of the amniotic cavity from cultivation or PCR were known, the attending clinician modified the women’s antibiotic therapies accordingly. Tocolysis was used only in women who developed regular uterine activity during the course of corticosteroids or within 24 h after their administration. The women were managed expectantly, except those with intra-amniotic infection  beyond the 28This study was approved by the institutional review board of the University Hospital Hradec Kralove . All study participants were Caucasian, and informed consent was obtained from all participants.Amniotic fluid samples, cervical fluid samples, and the clinical and demographic data of some women from this cohort were used in our previous studies Ultrasonography-guided transabdominal amniocentesis was performed before administration of corticosteroids, antibiotics, or tocolytics. The details of amniotic fluid sampling have been previously described .Ureaplasma spp. DNA  . The meaC. trachomatis/Ureaplasma/M. hominis-FRT kit  was used to detect DNA from Ureaplasma spp., M. hominis, and Ch. trachomatis in a single PCR tube for each fluid. The details of this procedure have been described previously  was used to detect DNA from Ureaplasma spp., M. hominis, and Ch. trachomatis in a single PCR tube for each fluid. The details of this procedure have been described previously. The load of Ureaplasma spp. (copies/ml) was determined using an absolute quantification technique with an external calibration curve. Plasmid DNA  was used to prepare a calibration curve.To assess the cervical fluid, a commercial AmpliSens® Ureaplasma spp. DNA in the cervical fluid and total bacterial DNA detection were performed using quantitative RT-PCR–BactQuant   × 100] and expressed as percentages. The PCR conditions used in the BactQuant assay were the same as those used for Ureaplasma spp.The relative abundance of actQuant . To quanepublic) . The relUreaplasma spp., M. hominis, or Ch. trachomatis in the amniotic fluid, their combination, positivity for the 16S rRNA gene in the amniotic fluid, aerobic/anaerobic cultivation of the amniotic fluid, or a combination of these parameters. Intra-amniotic inflammation was defined as a concentration of IL-6 in the amniotic fluid that was ≥745 pg/ml when measured using a lateral flow immunoassay point-of-care test .The demographic and clinical characteristics of the patients were compared using the non-parametric Kruskal–Wallis test for continuous variables and chi-square test for categorical variables, and the results are presented as medians (interquartile range [IQR]) and numbers (%), respectively. The normality of the data was tested using the Anderson–Darling test. The non-parametric Kruskal–Wallis or Mann-Whitney Overall, 217 women with singleton pregnancies and PPROM between gestational ages 24 + 0 and 33 + 6 weeks were included in the study. Intra-amniotic infection, sterile intra-amniotic inflammation, colonization of the amniotic cavity, and negative amniotic fluid were observed in 20% (44/217), 11% (24/217), 10% (21/217), and 59% (128/217) of women, respectively. The demographic and clinical data of the women with PPROM, as well as short-term neonatal outcomes are summarized in Ureaplasma spp., which were found in 59% (26/44) and 71% (15/21) of women with intra-amniotic infection and colonization of the amniotic cavity, respectively. All microbial findings in the amniotic fluid that were identified in the women with intra-amniotic infection are shown in The most common microorganisms in the amniotic fluid were Ureaplasma spp. DNA in the cervical fluid was identified in 61% (133/217) of the women. All of the women with Ureaplasma spp. DNA in their amniotic fluid (n = 41) also had Ureaplasma spp. DNA in their cervical fluid.Ureaplasma spp. DNA in the cervical fluid varied among the subgroups of women with intra-amniotic infection (73% [32/44]), sterile intra-amniotic inflammation (54% [13/24]), colonization (86% [18/21]), and negative amniotic fluid  and colonization (p = 0.008); however, they had a similar prevalence to those with sterile intra-amniotic inflammation (p = 1.00).Women with negative amniotic fluid had a lower prevalence of Ureaplasma spp. DNA in the cervical fluid varied among the subgroups , sterile intra-amniotic inflammation (p = 0.004), and colonization .Women with negative amniotic fluid had a lower load of Ureaplasma spp. in the cervical fluid between the subgroups of women with PPROM, the amount of bacterial DNA in the cervical fluid was first evaluated. No difference in the concentration of bacterial DNA was identified between the subgroups , sterile intra-amniotic inflammation (p = 0.05), or colonization (p = 0.008). After excluding the women with Ureaplasma spp. in the amniotic fluid, there was no difference .Women with negative amniotic fluid had a lower relative abundance than those with intra-amniotic infection  the presence of intra-amniotic infection and colonization was related to higher rates of Ureaplasma spp. DNA in the cervical fluid compared to those with negative amniotic fluid results; and 3) the presence of intra-amniotic infection, sterile intra-amniotic inflammation, or colonization of the amniotic fluid was associated with higher microbial loads of Ureaplasma spp. DNA in the cervical fluid compared to those with negative amniotic fluid; and 4) the presence of sterile intra-amniotic inflammation was related to a higher relative abundance of Ureaplasma spp. DNA in the cervical fluid compared to that in negative amniotic fluid.The principal findings of this study in women with PPROM before 34 weeks were as follows: 1) the total prevalence of Ureaplasma spp. are considered commensal microorganisms of the cervical/vaginal niche owing to: 1) their high prevalence in pregnant or non-pregnant women, and 2) the absence of a difference in the prevalence between women of reproductive ages with and without symptoms of infection of the urogenital tract  was found in this study. However, this observation was not in concordance with our previous study, where no difference in the load of Ureaplasma spp. DNA in the cervical fluid was identified in women with and without microbial invasion of the amniotic cavity  that were studied; and 2) the gestational ages considered during sampling .Abnormal microbiota in the cervical/vaginal niche have been shown to be associated not only with a higher frequency of at niche . Kwak etth PPROM . In linec cavity . The reaUreaplasma spp. DNA in the cervical fluid might represent a more precise tool with which to characterize the association between the composition of the cervical microbiome and intra-amniotic complications, as opposed to the assessment of the absolute number of copies of Ureaplasma spp. DNA that are present. In this study, women with PPROM and sterile intra-amniotic inflammation had an approximately 2.5-fold higher relative abundance of Ureaplasma spp. DNA compared to women with negative amniotic fluid results. This observation should be considered in terms of the pathophysiology of the development of sterile intra-amniotic inflammation in PPROM.The relative abundance of Ureaplasma spp. DNA in the cervical fluid; and 2) differences exist between PPROM and preterm labor with intact membranes with regard to the association between Ureaplasma spp. DNA in the cervical fluid and intra-amniotic complications. Collectively, these observations further support that pathophysiological pathways leading to the development of intra-amniotic complications might differ between PPROM and preterm labor with intact membranes.Taken together, the results from this study suggest that 1) the subgroup of women with PPROM and negative amniotic fluid differs from those with intra-amniotic complications in terms of frequency, loads, and relative abundance of Ureaplasma spp. DNA in the cervical fluid was evaluated with a specific PCR procedure, which allowed us to identify even very low loads of Ureaplasma spp. DNA. Third, thorough assessment of microbial invasion of the amniotic cavity, with the use of a combination of non-specific PCR (16S rRNA) followed by sequencing, specific PCR for Ureaplasma spp., Mycoplasma hominis, and Chlamydia trachomatis, and aerobic/anaerobic cultivation allowed us to precisely dissect the subgroups of women with sterile intra-amniotic inflammation.This study has several strengths. First, a relatively large cohort of women with a well-defined clinical phenotype for spontaneous preterm delivery (PPROM) was assessed. Secondly, the presence of Ureaplasma spp. DNA (in cases of intra-amniotic infection and colonization of the amniotic cavity caused by Ureaplasma spp.), it can affect the total microbial load of the Ureaplasma spp. DNA and its relative abundance in the fluid obtained from the endocervical canal. Unfortunately, the presence of this phenomenon is inevitable in PPROM and could affect the assessment of microbial loads and the relative abundance of Ureaplasma spp. DNA in the cervical fluid among subgroups of women with PPROM. On the other hand, it is highly unlikely that the presence of Ureaplasma spp. DNA in leaked amniotic fluid could affect the assessment of the prevalence of Ureaplasma spp. in the cervical fluid, since the cervical/vaginal niche is considered to be a primary source of Ureaplasma spp.This study has limitations that are worth mentioning. First, in women with PPROM, amniotic fluid leaks from the amniotic cavity through the cervix into the posterior vaginal fornix. This means that the leaking amniotic fluid can contaminate the cervical fluid. Therefore, the fluid obtained with a Dacron swab from the endocervical canal of women with PPROM should be considered not just as cervical fluid but as a compound fluid, composed of both cervical fluid and amniotic fluid components. Its composition, as well as the ratio between amniotic and cervical fluids, might vary among women with PPROM and depend on various clinical parameters and scenarios  that occur at the time of sampling. Therefore, if the leaked amniotic fluid contains Ureaplasma spp. were not assessed and considered in this study. This fact should be taken as a shortcoming of this study. However, there is evidence of the possibility of horizontal gene transfer in Ureaplasma spp., allowing them to carry markers of multiple serovars (Ureaplasma spp. DNA in the cervical fluid.Second, biovars, serovars, and other types of serovars . Thus, tUreaplasma spp. DNA in cervical fluid than in the absence of intra-amniotic complications.In conclusion, in PPROM at < 34 weeks, the presence of intra-amniotic infection, sterile intra-amniotic inflammation, or colonization of the amniotic fluid was associated with a higher prevalence and/or load of


To (i) improve the frequency of ascitic fluid sampling in decompensated cirrhotic patients; (ii) improve knowledge and confidence around ascitic fluid sample investigation and interpretation; and (iii) improve compliance with the decompensated cirrhosis bundle.Cirrhotic patients are vulnerable to infection and can present with subtle clinical signs and laboratory results, making diagnosis of infection challenging. Spontaneous bacterial peritonitis is common in these patients and associated with a high mortality. BSG and BASL guidance suggests that all patients with ascites should have an ascitic tap performed on presentation to screen for infection, yet compliance with this directive is poor, leading to delays in diagnosis and poorly targeted antibiotic use. The reasons for this include lack of knowledge, lack of confidence in performing ascitic fluid sampling and lack of clarity on the samples required.We undertook a two cycle quality improvement project utilizing PDSA methodology. We focused both on practice and knowledge. Data collection – Identification of all decompensated cirrhotic on the medical take across a month via presenting complaint and discharge letter screening. Retrospective case note review was performed using a standardized proforma. Simultaneously junior doctor knowledge and confidence was assessed via three online questionnaires. Cycle 1 – Awareness building through posters and email. Dissemination of guidance around ascitic fluid sampling. Online education session provided for remote access (during COVID-19). Cycle 2 – Developed and implemented a new online order bundle for decompensated cirrhotic patients. Further awareness building in ED and medicine.n=16, 15 and 14 respectively) and three rounds of knowledge surveys were undertaken . Ascitic tap attempts in appropriate patients rose from 50% to 83%. Requesting of (entirely) correct investigations for ascitic fluid increased minimally (67% to 75%). There was an increase in the percentage of those who classed themselves as somewhat or very confident (60 to 70% combined) regarding performance of ascitic tap . Despite this improvement in confidence, understanding of the correct testing and requesting procedure for ascitic fluid did not improve (96% incorrect). Compliance with the cirrhosis care bundle improved from 50% to 71%.Three months of data collection were performed . The approaches used here could be utilized more widely to improve practice.


Staphylococcus argenteus (SAR) is a novel species within the S. aureus (SAU) complex. SAR has been misidentified as SAU but has been increasingly reported worldwide as a pathogen. This study evaluated the prevalence of SAR causing bloodstream infection (BSI) in US centers, and the phenotypic, genotypic, and clinical outcomes associated with SAR BSI.785 SAU (326/459 MRSA/MSSA) from blood cultures (BC) of patients in 31 sites located in all 9 US Census Divisions during the SENTRY Antimicrobial Surveillance Program for 2019 were included. Isolates were screened for SAR by multiplex PCR. Isolates were subjected to MALDI-TOF and susceptibility (S) testing using the CLSI method. Isolates were subjected to genome sequencing, followed by DNA analysis.S. schweitzeri. SAR (ST2198) from patient 1 was S to all agents tested, as were the other 2 strains (ST2250 and ST1223), except for oxacillin. The latter 2 strains carried SCCmec type IV(2B) and a dfrG was also detected in the ST2250 lineage strain. All 3 strains carried multiple virulence genes.0.4% (3/785) SAR were detected and originated from Milwaukee , Houston , and Seattle . Patient 1 had a SAR recovered from BC 10 days after admission, but additional clinical information was not available. Patient 2 had end-stage renal disease and was admitted due to MRSA bacteremia/endocarditis, along with septic embolism in the lungs and brain. SAR grew from BC on day 4 and vancomycin was prescribed; the patient died on day 24. Patient 3 presented fever and chills in the 24 hours pre-admission, with a medical history significant for catheter-associated BSI. SAR was cultured from BC upon admission, and the patient received linezolid (5 days), but BCs remained positive. The central line was removed, and BCs cleared thereafter. MALDI-TOF generated highest scores for SAR for all 3 strains, but scores ≥2.0 were also obtained for SAU and mecA and various virulence genes.Low prevalence of SAR causing BSI was observed in US hospitals in 2019; however, SAR can clearly cause invasive infections. ST2250 has predominantly been detected in the USA and Canada, but this study showed distinct lineages causing BSI, including strains carrying Rafael Mendes, BS, JMI Laboratory: Grant/Research Support Lalitagauri M. Deshpande, PhD, Melinta: Grant/Research Support|Pfizer: Grant/Research Support Cecilia G. Carvalhaes, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Office for Assistant Secretary of Defense for Health Affairs: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support|Spero Therapeutics: Grant/Research Support.