Novel coronavirus 2019 (COVID-19) also known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, non-segmented positive-sense RNA virus belonging to the beta-coronaviridae family. This virus is known to cause severe bilateral pneumonia and acute respiratory distress syndrome (ARDS) which can lead to difficulty breathing requiring mechanical ventilation and intensive care unit management.A 77-year-old female with a history of hypertension and hyperlipidemia who presented as a transfer to our hospital facility with worsening fevers, cough, and respiratory distress.Chest X-rays revealed bilateral infiltrates worse at the lung bases and CT scan of the chest showed bilateral ground-glass opacities consistent with COVID-19. While our testing revealed a negative COVID-19 result at our institution, the result at a previous hospital returned a positive result.She was being treated aggressively in the intensive care unit with high dose intravenous ascorbic acid, hydroxychloroquine, and anti-interleukin-6 monoclonal antibody. She also received a loading dose of remdesivir however was unable to complete the course due to organ failure and requirement of vasopressors for hemodynamic stability.She remained critically ill and was eventually placed on comfort care as per the family's wishes and passed away.With a rapidly growing death rate and more than 200,000 confirmed cases worldwide, COVID-19 has become a global pandemic and major hit to our healthcare systems. While several companies have already begun vaccine trials and healthcare facilities have been using a wide-range of medications to treat the virus and symptoms, there is not yet an approved medication regimen for COVID-19 infections. The alarming increase in cases per day adds additional pressure to find a cure and decrease the global health burden and mortality rate. According to the World Health Organization, there are 207,855 confirmed cases and 8648 deaths from COVID-19 as of March 19, 2020 and rapidly increasing. Originating from bats like other virulent coronavirus (CoV) strains such as severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), COVID-19 has become the focus of the medical world and the pandemic of 2020.,4 We present a case of elderly female presenting with fever, cough, and shortness of breath found to be positive for COVID-19 and started on high-dose IV ascorbic acid, anti-interleukin-6, hydroxychloroquine, and remdesivir requiring high ventilator settings and eventually requiring vasopressors and continuous veno-venous hemodialysis (CVVHD).The novel coronavirus 2019 (COVID-19) also known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, non-segmented positive-sense RNA virus belonging to the beta-coronaviridae family.2A 77-year-old Middle-Eastern female with a medical history of hypertension and hyperlipidemia presented to the emergency department (ED) from a day care facility apartment where 2 people at the facility have tested positive for COVID-19 but she did not have any direct contact with these individuals. About 5 days before admission the patient developed a fever with a temperature of 102°F at home, and went to her primary medical doctor who sent her to the ED. In the ED she was found to have bilateral opacities on chest X-ray and had continued intermittent fevers with generalized weakness, cough, lethargy, and dyspnea and was sent for testing for COVID-19 then transferred to our facility for further management. In our facility, her temperature was 101.7°F, blood pressure 148/76 mm Hg, heart rate of 99 beats per minute, respiratory rate of 18 per minute, and oxygen saturation of 93% on room air. Physical exam was significant for a dry cough and bilateral rales on auscultation of the lung fields bilaterally but was unremarkable otherwise. A chest X-ray , headache (17.7%), and dyspnea (13.3%) were less commonly reported. These symptoms were also found on presentation with our patient. While the COVID-19 tests were pending, the CT scan of the chest provided valuable information as it met the trend of findings in infected patients. Wan et al obtained CT scans on all patients in their study and found bilateral involvement and multiple patchy or ground glass appearance to be the primary finding. Huang et al found similar findings where 98% of CT scans obtained had bilateral involvement and multilobular consolidations. These findings on CT scans are not unusual for a viral pneumonia. Influenza A (H1N1) was first found to cause a pandemic in 2009, a retrospective review of 92 patients by Çörtük et al found 69.6% of patients with H1N1 had bilateral patchy pneumonic infiltrates and 41.3% had bilateral ground glass opacities. While the lack of rapid testing for COVID-19 has caused a delay in diagnosis, perhaps the use of CT scans could provide an increased suspicion of COVID-19 infection leading to earlier treatment and management.COVID-19 is the cause of severe viral pneumonia rapidly leading to ARDS. In a case series of 135 patients, Wan et al reported 88.9% of patients presented with a fever and 76.5% had a cough.,9 In addition to vitamin and mineral supplements, hydroxychloroquine and azithromycin have obtained a large amount of attention for the treatment of COVID-19. Hydroxychloroquine, a well-known anti-malarial and auto-immune medication, is relatively inexpensive and has been extensively studied in the treatment for COVID-19. Studies have suggested hydroxychloroquine can interfere with glycosylation of the coronavirus receptors and increase endosomal pH thus inhibiting viral fusion and decreasing viral load.,11 Gautret et al reported a synergistic effect using hydroxychloroquine and azithromycin in viral elimination and decreasing viral load. Despite this evidence, the use of hydroxychloroquine for viral infections has been questioned. Roques et al reported a study using chloroquine in Chikungunya virus reporting cytokines were reduced causing the adaptive immune response to be delayed, exacerbating fever, and unchanged suppression of viral load. While further studies are in need to provide concrete evidence on the use of hydroxychloroquine, clinical trials from China have already shown promising results for COVID-19 and several countries around the world have begun using these medications. Tocilizumab, a recombinant humanized anti-interleukin-6 receptor monoclonal antibody, has been extensively used in auto-immune conditions such as rheumatoid arthritis. With this monoclonal antibody, interleukin-6 function is blocked and hence the differentiation of T helper cells and B cells into immunoglobulin-secreting cells are inhibited. The cytokine storm observed in patients with COVID-19 has been difficult to control and manage leading to increased mortality, tocilizumab therefore helps decrease the immune response and the resulting damage caused by cytokines.,15 While still not approved in the United States, tocilizumab has thus far shown promising results in clinical trials.Our patient presented in this case received treatment with vitamin C and zinc, both of which are known to improve the human immune system and aid in shortening the duration of and improving outcomes in respiratory infections including pneumonia.,14 It has been found effective at inhibiting viral replication in Ebola, SARS-CoV, and MERS-CoV infections.,16,17 Favipiravir, an RNA-dependent RNA polymerase inhibitor, has already obtained approval for the treatment of COVID-19 in China on February 15th, 2020. Studies have shown favipiravir inhibited RNA polymerase activity and thus prevented replication of RNA viruses like COVID-19 with minimal side effects. Remdesivir  is currently under several clinical trials and all of its side effects have not yet been defined. In our patient, within 2 days of starting remdesivir our patient had worsening renal function eventually requiring CVVHD and vasopressors thus preventing further treatment with the medication. While our patient was critically ill in the ICU, it is not known if this medication was the cause for further decompensation due to kidney injury. Further studies and clinical trials are required to fully understand the role of remdesivir and other medications in COVID-19 infected patients.Other treatments for COVID-19 have also emerged and have thus far shown promising results in ongoing clinical trials. Of these, remdesivir (GS-5734) and favipiravir (T-705) have become the center of attention. Remdesivir is an adenosine analog that incorporates into viral RNA causing premature termination.4COVID-19 is a serious infection that has led to thousands of cases of severe pneumonia, ARDS, and even deaths across the globe. As of now there are no approved treatments for this viral pandemic. While several medications have shown to be effective in clinical trials, further studies are needed to establish dosing, treatment course, and side effects of these medications. As the number of cases and deaths continue to increase in the world, the race to develop faster testing modalities to rapidly diagnose and manage these patients earlier continues to be the focus of the global healthcare system.Conceptualization: Steven Douedi, Jeffery Miskoff.Writing – original draft: Steven Douedi.Writing – review & editing: Steven Douedi, Jeffery Miskoff.


Background: Leptomeningeal metastasis (LM) has frequently been observed in patients with lung adenocarcinoma. So far, its diagnosis and disease course monitoring are still extremely difficult. Moreover, there is no effective treatment regimen for LM due to a lack knowledge on the molecular mechanism of LM. This study aimed to identify LM-related cerebrospinal fluid (CSF) miRNAs, which have potential value for diagnosing and monitoring LM and exploring the molecular mechanism.Methods: CSF miRNAs were screened and verified by microarray analysis and quantitative real-time PCR (qRT-PCR) in LM patients with lung adenocarcinoma and non-LM controls, and the diagnostic performance of candidate miRNAs was evaluated. Then, candidate miRNAs in matched CSF samples from LM patients at diagnosis, after initial therapy, at relapse, and after salvage therapy, were analyzed to assess the relationship between CSF miRNAs and LM disease course. The effect of candidate miRNAs on proliferation, invasion, and migration of lung adenocarcinoma cell lines was assessed. The targeted genes of the candidate miRNA were predicted by TargetScan, miRDB, and miRTarbase online analysis tools. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the functional categories of predicted target genes.Results: CSF miR-7975, miR-7977, and miR-7641 were screened and verified to be statistically significantly up-regulated in LM patients compared to non-LM controls. The three miRNAs, when combined, exhibited optimal diagnostic performance. Longitudinal data of CSF miR-7975 and miR-7977 correlated well with clinical courses of LM. Overexpression of miR-7977 promoted proliferation, migration, and invasion of lung adenocarcinoma cells. Moreover, 385 targeted genes of miR-7977 were predicted and were involved in various pathways related to cancer metastasis.Conclusions: This study offers insights for future research of CSF miRNAs as robust tools for diagnosing and monitoring LM. It also reveals a novel pathway for exploration of underlying mechanisms of LM. Leptomeningeal metastasis (LM) is known as a devastating cancer complication that occurs when malignant cells disseminate to leptomeninges and cerebrospinal fluid (CSF) compartments –3. AboutMicroRNAs (miRNAs) are a group of noncoding RNAs, which can degrade messenger RNA (mRNA) and inhibit protein translation based on complementation with sequence of the target mRNAs . ThroughCSF is a readily reachable body fluid and the most informative biofluid regarding changes that occur in the central nervous system (CNS) . The proIn this study, we first screened specific miRNAs by comprehensive miRNA microarray analyses in CSF samples from LM patients and non-LM controls, as well as in matched CSF samples from LM patients at diagnosis and after LM-directed therapy. Using quantitative real-time PCR (qRT-PCR), we then validated the expression of candidate miRNAs in CSF samples from independent LM patients with lung adenocarcinoma, and evaluated their diagnostic performance for discriminating LM patients from non-LM subjects. Next, we evaluated the effect of candidate miRNAs on proliferation, migration, and invasion of lung adenocarcinoma cell lines. The online analysis tools including TargetScan, miRDB, and miRTarbase were used to predict potential target genes of the candidate miRNA. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the functions of predicted target genes. The present study aimed to investigate LM-related miRNAs and their potential value in disease diagnosis and monitoring, and to explore the possible molecular mechanisms based on experimental validation and bioinformatic analysis.In total, we collected 186 CSF samples from three groups of participants: LM patients with lung adenocarcinoma, brain metastasis patients with lung adenocarcinoma, and non-cancer controls at the First Hospital of Jilin University, China, between September 2014 and December 2018. All samples were distributed to discovery and validation sets. Detailed clinical data are summarized in In the discovery phase, we performed comprehensive miRNA microarray analysis in CSF samples from 10 LM patients with lung adenocarcinoma and 10 non-LM controls (5 brain metastases from lung adenocarcinoma and 5 non-cancers). Meanwhile, matched CSF samples collected from 6 LM patients at diagnosis and after efficacious LM-directed therapy were also analyzed by miRNA microarray. MiRNAs differentially expressed in common between the two comparative models of miRNA microarray were determined as candidate miRNAs. Subsequently, candidate miRNAs were quantified in CSF samples from an independent validation cohort including 68 LM patients and 48 non-LM controls (43 brain metastases from lung adenocarcinoma and 5 non-cancers).To investigate if candidate miRNAs were involved in the disease course of LM, 22 matched CSF samples were collected and analyzed at diagnosis and after efficacious initial therapy, as well as 8 sequential CSF samples were collected and analyzed at four time points: at diagnosis, after efficacious initial therapy, at relapse, and after salvage therapy. Detailed clinical data are summarized in 2O and/or CSF glucose <2.3 mmol/L; (3) positive CSF cytology; (4) LM-related neuroimaging findings were worsening. Meanwhile, treatment-related side effects and other diseases that may lead to the above conditions should be excluded. The evaluation criteria for clinical response to LM-directed treatment were established on the improvement of neurologic symptoms/signs, CSF, and neuroimaging according to EANO–ESMO Clinical Practice Guidelines  Clinical Practice Guidelines . Recurreidelines .LM CSF samples in this study were from patients enrolled in the following clinical trials: ChiCTR-OOC-14005403, NCT03082144, NCT03101579, and NCT03507244. Written informed consents were obtained from all participants, which indicated willingness to donate their CSF samples for research. The study received approved from Institution Ethics Committee of the First Hospital of Jilin University and in accordance with the principles of the Declaration of Helsinki. Permission to access and publish patient information was obtained from each of the patients.The CSF samples from LM patients were collected at the time of diagnostic lumbar puncture or intrathecal chemotherapy. For brain metastasis patients with lung adenocarcinoma who were suspected of suffering LM but ultimately excluded, CSF samples were obtained by diagnostic lumbar puncture. The CSF samples of non-cancer controls were collected at the time of lumbar anesthesia prior to the necessary surgery. At least 2 ml of each CSF sample was stored in aliquots within 1 h after collection at −80°C until further processing. The blooded CSF samples were excluded.*60K) arrays. The microarray contains probes for 2,549 human miRNAs from miRbase v21.0. The miRNA molecules were labeled using the miRNA Complete Labeling and Hyb Kit from Agilent, following the manufacturer's standard protocol. Each slide was hybridized with 100 ng Cy3-labeled RNA using miRNA Complete Labeling and Hyb Kit  in hybridization oven at 55°C for 20 h. After hybridization, slides were washed in staining dishes  with Gene Expression Wash Buffer Kit . Then slides were scanned by Agilent Microarray Scanner , and the images were processed with Feature Extraction software 10.7  with default settings.Microarray analysis was performed using Agilent Human miRNA , were cultured in RPMI-1640 Medium  and Dulbecco's Modified Eagle's Medium , respectively, supplemented with 10% fetal bovine serum , 100 U/ml penicillin, and 100 mg/ml streptomycin  in a humidified incubator at 37°C in a 5% CO5 cells per well) overnight prior to transfection. MiRNA mimics (50 nM) and negative control (50 nM) were transfected into the cells by Lipofectamine 3000 reagent  in accordance with the manufacturer's instruction. The miRNA mimics and mimic negative control were purchased from GenePharma Co., Ltd .Cells were seeded in six-well plates , following the manufacturer's instructions and checked for an RNA integrity number to inspect RNA integration by an Agilent Bioanalyzer 2100 . For lung adenocarcinoma cells, total RNA was extracted using miRcute miRNA isolation kit  at 24 h after transfection with miRNA mimics or negative control nucleotides.C. Elegans miR-39  in CSF samples and U6  in cell lines were used for control. The relative expression level (REL) of each miRNA in CSF samples and cell lines was calculated with the 2−ΔCt and 2−ΔΔCt methods, respectively.Then miRNA was reverse transcribed with miScriptII RT Kit , and quantified by qRT-PCR using the miScriptSYBR® Green PCR Kit  and commercial primers . All samples were run in triplicate on Line Gene 9600 Plus system  using default settings. 3 per well. After incubation for another 12, 24, and 48 h, 10% Cell Counting Kit-8  diluted in normal culture medium was added to each well and incubated for an additional 4 h. The absorbance was measured spectrophotometrically at 450 nm. Each experiment was performed at least three times independently.At 24 h after transfection with miRNA mimics and negative control, cells were seeded into 96-well plates at a density of 5 × 10The changes in cell migration ability were detected by wound healing assay. Cells were seeded in six-well plates. At 24 h after transfection with miRNA mimics or negative control, wounds were made in the cell layer using a sterile micropipette tip. Then, the cells were washed with PBS buffer. The width of the wound gap was viewed under a microscope and photographed at 0, 9, and 24 h after wounding. Three replicate wells from a six-well plate were used for this experiment.5/ml) were collected and resuspended in 200 μl serum-free medium and added to the top well. Six hundred milliliter medium containing 15% FBS was added to the lower compartment. After 48 h of incubation, the cells on the upper surface of the membrane were carefully removed with a cotton bud. The cells that invaded through the PET filter membrane were fixed with cold methanol for 30 min, stained with 0.1% crystal violet for 20 min, then photographed and counted under a microscope.The invasion assay was performed with a transwell chamber inserted with a Polyester (PET) filter membrane  in 24-well plates . The top side of the filter was coated with Matrigel. At 24 h after transfection with mimics or negative controls, the cells . The criteria of cut-off were gene count ≥ 3.The functional annotation and pathway enrichment analysis including GO and KEGG pathway analysis , 16 wereT-test, Mann–Whitney test, and Wilcoxon matched-pairs signed rank test. A value of P < 0.05 was regarded as statistically significant. Receiver operating characteristic (ROC) curve analysis was used to evaluate diagnostic performance.GraphPad Prism  and SPSS  were used for all statistical analyses. The data were expressed as mean ± standard deviation (SD). Differential expression of miRNAs was analyzed by the unpaired GSE125193.To evaluate whether specific miRNA signatures were detectable in CSF samples from LM patients with lung adenocarcinoma, a microarray analysis of 2,549 human miRNAs was performed. The criterion of each mappable miRNA was chosen for further analysis based on differentially expressed gene analysis with fold change values of more than 2 and < 0.5. Based on the criteria described, 36 miRNAs upregulated and 15 miRNAs downregulated consistently in LM when compared with brain metastasis and non-cancer controls, respectively, were identified for further analysis . The rawGSE125193. As we have confirmed that the three miRNAs were significantly upregulated in LMs compared with non-LMs, the results indicate that the three miRNAs may be involved with the development of LM and, therefore, were selected for further validation. In addition, CSF miR-7641 with the largest fold-change value of 58.3 in LMs vs. non-LMs was also selected as a candidate miRNA.In order to screen specific miRNAs correlated with the development of LM, we also performed miRNA microarray in CSF samples of six LM patients at diagnosis and their matched CSF samples after efficacious LM-directed therapy. Compared with the levels at diagnosis, CSF miR-7975, miR-7977, and miR-4800-5p were significantly decreased after efficacious therapy . The rawThe robustness of the miRNA microarray data was studied by analyzing their levels using qRT-PCR in an independent validation set, which includes CSF samples from an additional 68 LM patients with lung adenocarcinoma and 48 non-LM controls. MiRNA expression data were normalized using cel-miR-39 levels in individual CSF specimens and reported as RELs. In concordance with the results presented in the discovery set, we found a significant difference for miR-7975, miR-7977, and miR-7641 between CSF samples from LM patients and non-LM controls . HoweverP < 0.0001, 95% CI 0.832–0.953), 0.867 , and 0.766 , respectively  value for miR-7975, miR-7977, and miR-7641 were 0.893  were compared in CSF samples of 22 LM patients at initial diagnosis with their matched CSF samples after efficacious LM-directed therapy. Compared with the levels at diagnosis, CSF miR-7975 and miR-7977 were significantly down-regulated in LM patients after initial efficacious therapy , while Cin vitro, we transfected the cells with 50 nM miRNA mimics or negative control nucleotides. The CCK-8 assay showed that overexpressing miR-7977 increased the proliferation of NCI-H1650 and A549 cells at 24 and 48 h, while transfection with miR-7975 did not affect cell proliferation  methods , 18. HowLM patients frequently suffer from disease relapse or progression. So far, there is still no definite biomarker for reflecting disease course of LM. Generally, if the development of the disease during different stages has the same pathologic process, ideal diagnostic biomarkers can be used for disease monitoring . A previThe identification of specific CSF miRNAs in this study not only provides a new method for diagnosing and monitoring LM but also develops a new way for exploring the mechanism of LM. The molecular mechanism of LM is still elusive, which may be the main obstacle to seeking efficacious treatment. Currently, efforts to better understand the molecular mechanism of LM have been severely limited by the difficulties in isolating tumor cells from CSF as well as characterizing them. Increasing evidence emerging from preclinical models of different tumor types clearly indicates that specific miRNAs play a functional role in different steps of the metastatic cascade . In our In addition, we found that the KEGG pathways of miR-7977 targeted genes included tight junction, gap junction, and other abnormal signaling pathways including the Wnt signaling pathway, Jak-STAT signaling pathway, and MAPK signaling pathway. One of the main patterns of LM is believed to occur via hematogenous route, implying that cancer cells reach the brain barrier and invade the space of CSF flow. Tight junctions have become a key factor in the studies on integrity of brain barrier including blood–brain barrier (BBB) and blood–cerebrospinal fluid barrier (BCSFB) during CNS metastasis. A previous study has confirmed that disruption of the tight junctions of the BBB is an important link in the development of brain metastasis , 29. Sinin vitro and bioinformatics analysis of miR-7977 were performed in this study. Research on detailed molecular mechanisms that miR-7977 promotes the occurrence of LM through regulating targeted genes is lacking. Therefore, it is required to perform future investigations with larger clinical samples and corresponding experiments.There are several limitations of this study. First, the CSF sample size was not large enough in the microarray analysis. Second, our study indicated that the specific CSF miRNAs have potential value in diagnosis and monitoring of LM. However, all the patients enrolled in this study were from our hospital. Potential drawbacks such as selection bias may occur. Further validation of the identified miRNAs in a larger independent population from multiple cancer centers is necessary before they can be put in a clinical application. Third, only experiments on biological functions in vitro and bioinformatic analysis suggested that miR-7977 plays a key role in the development of LM, which reveals a novel pathway to exploration of underlying mechanisms of LM, and provides a promising target in treating LM patients. CSF miRNAs are potentially of high worth for LM-related studies; however, much work still needs to be done to shepherd findings from scientific research into advanced clinical applications.In conclusion, we have successfully identified three LM-associated miRNAs  based on microarray screening and qRT-PCR validation. Moreover, this study, if confirmed in prospective clinical trials, indicates that these miRNA signatures could be a significant tool to diagnose and monitor LM. In addition, the experiments GSE125193.The datasets generated for this study can be found in tNCBI Gene Expression Omnibus (GEO) through accession number This study was approved by the Institutional Ethics Review Board of the First Hospital of Jilin University, Changchun, China, and was performed in accordance with the Declaration of Helsinki.ZP, GY, GZ, and YC designed the study. ZP and GY wrote the manuscript. ZP, GY, TJ, and HH performed the experiments and statistical analysis of the data. TJ and PG collected clinical samples and clinical data of patients. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recently new anti-CD20 monoclonal antibodies (mAbs) have been developed, but their efficacy and safety compared with rituximab are still controversial. We searched MEDLINE, Embase, and Cochrane Library for eligible randomized controlled trials (RCTs) that compared new anti-CD20 mAbs with rituximab in induction therapy of B-NHL. The primary outcomes are progression-free survival (PFS) and overall survival (OS), additional outcomes include event-free survival (EFS), disease-free survival (DFS), overall response rate (ORR), complete response rate (CRR) and incidences of adverse events (AEs). Time-to-event data were pooled as hazard ratios (HRs) using the generic inverse-variance method and dichotomous outcomes were pooled as odds ratios (ORs) using the Mantel–Haenszel method with their respective 95% confidence interval (CI). Eleven RCTs comprising 5261 patients with CD20+ B-NHL were included. Compared with rituximab, obinutuzumab significantly prolonged PFS , had no improvement on OS, ORR, and CRR, but increased the incidences of serious AEs . Ofatumumab was inferior to rituximab in consideration of ORR , and had no significant differences with rituximab in regard to PFS, OS and CRR. 131I-tositumomab yielded similar PFS, OS, ORR and CRR with rituximab. 90Y-ibritumomab tiuxetan increased ORR , but did not improve PFS, DFS, OS and CRR compared with rituximab. In conclusion, compared with rituximab in induction therapy of CD20+ B-NHL, obinutuzumab significantly improves PFS but with higher incidence of AEs, ofatumumab decreases ORR, 90Y-ibritumomab tiuxetan increases ORR.Rituximab combined with chemotherapy is the first-line induction therapy of CD20 positive B-cell non-Hodgkin lymphomas (CD20 Approximately 85–90% of NHL origin from B cells2. CD20 is an ideal target for therapy of B-cell non-Hodgkin lymphomas (B-NHL) due to its specific expression pattern and unique biological property3. Since its initial approval in 1997, the anti-CD20 monoclonal antibody rituximab has greatly improved the survival outcome of B-NHL patients with acceptable toxicity5. However, despite the success of rituximab in the treatment of CD20+ B-NHL, there are patients that fail to respond to initial therapy or relapse sooner.Non-Hodgkin lymphomas (NHL) are a group of heterogeneous malignant disorders arising from lymphocytes at various stages of differentiation6. Among them, ofatumumab is the most widely investigated. The drug is a type I anti-CD20 mAb generated via transgenic mouse and hybridoma technology7. The proposed advantages of ofatumumab over rituximab include enhanced binding affinity to CD20 and greater complement-dependent cytotoxicity (CDC) against target cells as verified with in vitro studies8. Ofatumumab has been approved for the treatment of relapsed and refractory chronic lymphocytic leukemia (CLL) by the United States Food and Drug Administration (FDA) and the European Medicines agency (EMA) in 2009 and 2010 respectively, but the efficacy of ofatumumab in patients with B-NHL is still being investigated and there is still no inconsistent result9. The third-generation anti-CD20 mAbs are a group of fully humanized and engineered antibodies that include obinutuzumab, ocaratuzumab and PRO1319216. Until now, obinutuzumab (GA101) has been approved for the treatment of CLL and follicular lymphoma (FL) by FDA and EMA10. Obinutuzumab is a type II anti-CD20 mAb with glycoengineered Fc region which can enhance binding affinity to the Fc receptor (FcR) on immune effector cells8. In vitro studies have proved that obinutuzumab has more potent direct cell death (DCD) and more effective antibody-dependent cellular cytotoxicity (ADCC) to target cells compared with rituximab12. Clinical trials which investigated whether obinutuzumab is superior to rituximab in the treatment of patients with other subtypes of B-NHL were performed, but the results are inconsistent. Additionally, novel agents that conjugate radioisotope to anti-CD20 mAbs, such as 131I-tositumomab (Bexxar) and 90Y-ibritumomab tiuxetan , have also been developed. The postulated advantages of these agents are that they could cause a crossfire effect and eradicate nearby tumor cells that are not targeted by antibody but are affected by radiation14. Promising results from clinical trials promote the approval of 131I-tositumomab (Bexxar) and 90Y-ibritumomab tiuxetan  by FDA for the treatment of FL in 2003 and 2002 respectively14. Although 131I-tositumomab (Bexxar) is now unavailable since the discontinued production by manufacturer in 2014, a number of well-designed trials have been performed to compare its efficacy with rituximab in patients with different subtypes of B-NHL before that14.To overcome the resistance and enhance anti-tumor activities, next generations of anti-CD20 monoclonal antibodies (mAbs) were developed. The second-generation anti-CD20 mAbs are a group of fully humanized IgG1 antibodies, including ofatumumab, veltuzumab, and ocrelizumab+ B-NHL.Above all, a lot of clinical trials have been performed to compare the efficacy and safety of multiple new anti-CD20 mAbs with rituximab for the treatment of B-NHL, but the results of these trials are inconsistent. It remains unclear that whether new anti-CD20 mAbs are superior to rituximab. This systematic review and meta-analysis aimed to assess the efficacy and safety of new anti-CD20 mAbs compared with rituximab in the induction therapy of CD20We searched MEDLINE, Embase, and Cochrane Library from inception to March 11th, 2019 with no language restriction. The search strategies for each database are presented in + B-NHL; (ii) randomly assigned patients to rituximab based therapy or other new anti-CD20 mAbs based therapy in induction therapy; (iii) reported data for at least one of the clinical outcomes, including progression-free survival (PFS), overall survival (OS), event-free survival (EFS), disease-free survival (DFS), overall response rate (ORR), complete response rate (CRR), and adverse events (AEs).Two investigators (CXL and XL) independently assessed eligibility of citations identified by the above search. Disagreements were resolved by discussion with a third investigator (SNX). Clinical trials that met the following criteria were included: (i) patients with CD20.15. For trials with multiple reports for survival outcomes, we extracted data from the report with the longest follow-up. Methodological quality of each included trials was assessed based on random sequence generation, allocation concealment, blinding, incomplete outcome data, and selective outcome reporting, following the guidelines in Cochrane handbook16. Any disagreements were resolved by consensus.Two investigators (CXL and XL) independently extracted data using predesigned data collection forms and cross-checked to reach a consensus. Data was extracted on trial characteristics, patient characteristics, dose and cycle of anti-CD20 mAbs, concomitant chemotherapy, follow-up and clinical outcomes. The hazard ratios (HRs) for survival outcomes were extracted or estimated with the methods previously established by Tierney et alThe primary outcomes of the meta-analysis are PFS and OS. The secondary outcomes are EFS, DFS, ORR, CRR, and incidences of AEs. The pooled HRs and 95% confidence intervals (CIs) for time-to-event data including PFS, OS, EFS and DFS were calculated using the generic inverse-variance method. The pooled odds ratios (ORs) and 95% CIs for dichotomous data including ORR, CRR and incidences of AEs were calculated using the Mantel–Haenszel method.2 test with a significant level at P < 0.1 and quantified with I2 statistic. Fixed-effects model was adopted for summary estimation if heterogeneity was not significant; otherwise, random-effects model was adopted. Subgroup analyses were performed to assess the influences of treatment history, disease subtype, concomitant chemotherapy, and patient characteristics on the therapeutic effect of new anti-CD20 mAbs versus rituximab. Sensitivity analysis was performed by omitting trial with number of patients less than 20.Statistical heterogeneity across trials was assessed by χP values were two sides and the threshold of significance was P < 0.05 except that for heterogeneity test. This work was reported according to preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement17.All analyses were conducted in Review Manager version 5.3 . All 32 , ACVBP , CVP , Bendamustine, DHAP , and BEAM . Concomitant chemotherapy regimens were completely same between the two arms in each trial. A total of 5261 B-NHL patients with a median age of 48–62 years were included in the analysis. Seven trials included only relapsed/refractory patients, and other four trials included only previously untreated patients. Patients demographic and baseline disease characteristics were well balanced between arms in all included trails. Detailed characteristics of the included 11 trials are summarized in Table All included trials were two-arm RCTs which compared new anti-CD20 mAb with rituximab in induction therapy of CD20+ B-NHL. Meta-analyses showed that compared with rituximab, obinutuzumab significantly prolonged PFS , OS , ORR  and CRR  , grade 3–5 IRR, all grades of neutropenia, grade 3–5 neutropenia, grade 3–5 thrombocytopenia, pyrexia, diarrhea, headache, grade 3–5 infections, chills, and insomnia. The incidences of other AEs were comparable between the two arms  subtype, female gender, white race, bulky disease, late stage, better Eastern Cooperative Oncology Group performance status (ECOG PS), intermediate international prognostic index (IPI) and when used in combination with bendamustine, However, the subgroup differences were not statistically significant , EFS , OS  and CRR  , OS , ORR  and CRR   for PFS and 1.25  for OS, the pooled ORs were 0.79  for ORR and 1.17  for CRR, which were all similar with the main analyses.Since the trial conducted by Quackenbush et al90Y-ibritumomab tiuxetan with rituximab in induction therapy of B-NHL. Survival outcomes including PFS , OS , and DFS  were comparable between ibritumomab arm and rituximab arm    induce the redistribution of CD20 into lipid rafts on cell membrane, leading to stronger C1q binding and more effective activation of complement cascade, initiating prominent CDC36. However, this binding mode trigger minimal DCD8. On the contrary, type II anti-CD20 mAbs do not induce the redistribution of CD20 into lipid rafts and do not evoke significant CDC, but they induce more potent DCD12. ADCC occurs after the interaction of the Fc portions from antibodies with the FcR expressed on effector cells 11. Modifications on the Fc portions of new anti-CD20 mAbs result in greater ADCC through enhancing its binding affinity to FcR10. Collectively, anti-CD20 mAbs exert antitumor effects through different mechanisms due to their structural variations. Compared with rituximab, modifications of new anti-CD20 mAbs significantly improved efficacy in preclinical studies.The predominant mechanisms of how anti-CD20 mAbs kill target cells include complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and direct cell death (DCD)8. Besides, the glycoengineering process caused defucosylation in the Fc region of obinutuzumab, which can enhance binding affinity to the FcR on immune effector cells and provoked more effective ADCC11. This may explain why obinutuzumab improve PFS of CD20+ NHL patients compared with rituximab. Although obinutuzumab-based therapy significantly prolonged PFS compared with rituximab-based therapy, no difference in OS was observed between the obinutuzumab arm and the rituximab arm. A possible reason is that the data of OS are still immature. The 4-year OS rates of patients with FL were as high as 92.6% in obinutuzumab arm and 90.3% in rituximab arm, the median OS had not been reached21. Prolonged follow-up is required to verify that if obinutuzumab-based therapy could benefit in OS compared with rituximab. Another possible explanation is that obinutuzumab was associated higher risk of fatal AEs compared with rituximab23.Obinutuzumab is a type II anti-CD20 mAb that does not cause the clustering of CD20 into lipid rafts on the membrane and shows more potent direct cell death (DCD) and antibody-dependent cellular cytotoxicity (ADCC)37. Preclinical studies have demonstrated that ofatumumab is more effective than rituximab in killing target cells regardless of FcR polymorphisms and the levels of CD20 expression38. But our meta-analysis failed to demonstrate any superiority of ofatumumab over rituximab in patients with relapsed CD20+ B-NHL. The lack of improvement in clinical outcomes with ofatumumab-based therapy in patients with relapsed CD20+ B-NHL may result from the resistance to anti-CD20 mAbs since only relapsed patients were included in the meta-analysis. Clinical trials aiming to compare the efficacy and safety of ofatumumab with rituximab in patients with previously untreated CD20+ B-NHL are required.Ofatumumab is a type I anti-CD20 mAb that binds to a unique epitope of CD20 and shows greater binding avidity than rituximab131I-tositumomab (Bexxar) and 90Y-ibritumomab tiuxetan , also have been developed and approved for treatments of B-NHL. These agents could cause a crossfire effect and eradicate nearby tumor cells that are not targeted by antibody but are affected by radiation14. Radioimmunotherapy with 131I-tositumomab could achieve a high response rate of 47–68% in heavily pretreated NHL patients39. However, the results of our meta-analysis did not demonstrate any significant differences in PFS, OS and response rate between the 131I-tositumomab arm and the rituximab arm due to the lack of benefits for 131I-tositumomab-based therapy in patients with relapsed DLBCL. In addition, the manufacture of tositumomab was discontinued for commercial reason in 2014 and it is unavailable now40. Another radio-labeled anti-CD20 mAb 90Y-ibritumomab tiuxetan was compared with rituximab in the therapy of relapsed B-NHL and this trial was included in our systematic review. Results showed that 90Y-ibritumomab achieved higher ORR, similar PFS, OS and CRR. According to a multicenter phase III randomized trial, consolidation therapy with 90Y-ibritumomab tiuxetan in patients with FL in first remission notably prolonged median PFS compared with no further treatment. Another randomized trial has compared consolidation therapy with 90Y-ibritumomab tiuxetan versus rituximab in FL patients that achieved CR or partial response (PR) after R-CHOP treatment. These results indicated that 90Y-ibritumomab tiuxetan was inferior to rituximab in PFS41. Therefore, although 90Y-ibritumomab tiuxetan was associated with slightly higher response rate as induction therapy for relapsed patients but with inferior PFS as consolidation therapy for patients in remission, the benefits of 90Y-ibritumomab tiuxetan over rituximab are still conflicting due to lack of enough evidence.Novel agents that conjugate radioisotope to anti-CD20 mAbs, such as 42. But serious AEs associated with the use of rituximab have already been an important consideration during clinical practice43. What’s more, the increasingly widespread and potentially prolonged use of rituximab and new CD20 mAbs poses a new challenge. Therefore, knowledge of serious AEs related to new CD20 mAbs antibodies is essential. Our results provide a clue for the awareness of AEs of new CD20 mAbs during their propagation.AE is another aspect that need to be considered for evaluating a new drug. In our meta-analysis, all the new CD20 mAbs showed higher incidences of AEs compared with rituximab. Overall, these biologics appear to be well tolerated and lead to fewer AEs compared with other more conventional therapies and chemotherapeutic regimens+ B-NHL participants with diverse racial and ethnic groups were included. Most of the included trials are with high quality and enough time of follow-up. What is more, in consideration of potential confounders such as treatment history, subtype, gender and concomitant chemotherapy, subgroup analysis was conducted to investigate the internal validity of this meta-analysis, indicating the consistency between different subgroups. All these characteristics enabled us to get a reliable result.To the best of our knowledge, this is the first systematic review and meta-analysis that integrated currently all available published data to compare the efficacy and safety of new anti-CD20 mAbs with rituximab. Totally 5261 CD20+ B-NHL including newly diagnosed or relapsed FL, DLBCL, transformed NHL, marginal zone lymphoma (MZL) and other indolent B-NHLs. Although they have lots of in common and no statistically significant subgroup differences are observed, the results need to be verified in more clinical trials focusing on a certain subtype of B-NHL. In addition, the absence of individual patient data did not allow further analysis of the benefits for survival outcomes in different subgroups based on disease subtypes, disease status, risk stratification and molecular characteristics. Another potential limitation of this study is that we pooled results from trials of different phases. The inclusion of earlier phase studies would increase the risk of investigator bias and confound the pooled analysis. Actually, most of the studies we included  are phase III trials except for a phase II trial (the GAUSS study) and a trial that did not provide information about study phase (Khouri 2015). The risk of investigator bias of the GAUSS study is low since it incorporated a blinded response assessment by an independent review facility (IRF) to better guide phase III trial planning22. Therefore, we think confounding resulted from the inclusion of earlier phase studies in our analysis is relatively small.Nevertheless, there are some limitations in this study. Firstly, data were pooled from participants with different disease subtypes and disease status. For the limit number of currently available clinical trials, we have to enrolled patients with any types of CD20131I-tositumomab yielded similar results with rituximab regarding PFS, OS, ORR and CRR but was associated with higher incidences of AEs. 90Y-ibritumomab achieved a higher ORR, similar PFS, OS and CRR, but was associated with higher incidences of AEs. These results might facilitate clinical decision making and assist in the design and interpretation of future trials.In conclusion, we have compared new CD20 mAbs with rituximab based on all current available published data by systematic review and meta-analysis. Obinutuzumab was demonstrated with a significant improvement in PFS, but no improvements in OS, ORR and CRR, and an increment in the incidences of AEs. Ofatumumab showed comparable results in PFS, OS and CRR, but a lower ORR and higher incidences of AEs. Supplementary Information.


The Global Enteric Multicenter Study (GEMS) was a 3-year case-control study that measured the burden, aetiology, and consequences of moderate-to-severe diarrhoea (MSD) in children aged 0–59 months. GEMS-1A, a 12-month follow-on study, comprised two parallel case-control studies, one assessing MSD and the other less-severe diarrhoea (LSD). In this report, we analyse the risk of death with each diarrhoea type and the specific pathogens associated with fatal outcomes.GEMS was a prospective, age-stratified, matched case-control study done at seven sites in Africa and Asia. Children aged 0–59 months with MSD seeking care at sentinel health centres were recruited along with one to three randomly selected matched community control children without diarrhoea. In the 12-month GEMS-1A follow-on study, children with LSD and matched controls, in addition to children with MSD and matched controls, were recruited at six of the seven sites; only cases of MSD and controls were enrolled at the seventh site. We compared risk of death during the period between enrolment and one follow-up household visit done about 60 days later (range 50–90 days) in children with MSD and LSD and in their respective controls. Approximately 50 pathogens were detected using, as appropriate, classic bacteriology, immunoassays, gel-based PCR and reverse transcriptase PCR, and quantitative real-time PCR (qPCR). Specimens from a subset of GEMS cases and controls were also tested by a TaqMan Array Card that compartmentalised probe-based qPCR for 32 enteropathogens.Escherichia coli, enterotoxigenic E coli encoding heat-stable toxin, enteroaggregative E coli, Shigella spp (non-dysentery cases), Aeromonas spp, Cryptosporidium spp, and Entamoeba histolytica increased risk of death. Of 61 deaths in children aged 12–59 months with non-dysenteric MSD, 31 occurred among 942 children qPCR-positive for Shigella spp and 30 deaths occurred in 1384 qPCR-negative children , showing that Shigella was strongly associated with increased risk of death.223 (2·0%) of 11 108 children with MSD and 43 (0·3%) of 16 369 matched controls died between study enrolment and the follow-up visit at about 60 days . 12 (0·4%) of 2962 children with LSD and seven (0·2%) of 4074 matched controls died during the follow-up period . Risk of death was lower in children with dysenteric MSD than in children with non-dysenteric MSD , and lower in children with LSD than in those with non-dysenteric MSD . In children younger than 24 months with MSD, infection with typical enteropathogenic Risk of death is increased following MSD and, to a lesser extent, LSD. Considering there are approximately three times more cases of LSD than MSD in the population, more deaths are expected among children with LSD than in those with MSD. Because the major attributable LSD-associated and MSD-associated pathogens are the same, implementing vaccines and rapid diagnosis and treatment interventions against these major pathogens are rational investments.Bill & Melinda Gates Foundation. Evidence before this studyEscherichia coli, or Shigella spp. However, few studies have included comprehensive investigation of a representative sample of diarrhoea cases in children in different low-income regions of the world where diarrhoeal illness remains a major public health problem.Although diarrhoeal-associated deaths have steadily decreased during the past two decades, diarrhoeal diseases remain responsible for an estimated 9% of all mortality in children younger than 5 years, accounting for about 499 000 to 525 000 annual deaths. Accurate descriptions of the major causative pathogens, determinants, and risk factors for these deaths are necessary to design improved preventive and therapeutic strategies that will have maximal impact on diarrhoeal disease, and thus, on young child survival. We searched PubMed for articles published in English, Spanish, French, or Portuguese between Jan 1, 2000, and Sept 30, 2019, using the terms “low and middle-income countries” or “developing countries” AND “diarrheal deaths” OR “diarrhea AND mortality”. We included earlier reports, and articles identified in reference lists, as appropriate. Previous aetiological studies have put the focus on specific pathogens in particular high-risk populations, including, among others, rotavirus, diarrhoeagenic Added value of this studyShigella antibiotics in accordance with WHO standard of care recommendations. About half the deaths in children aged 12–59 months with non-dysenteric MSD were positive for Shigella spp when quantitative real-time PCR (qPCR) testing was performed. Notably, three-quarters of the qPCR-positive fatal non-dysenteric MSD cases had negative cultures for Shigella spp.Data from the GEMS study showed that during the approximately 60-day follow-up period, children with MSD had an 8·5 times increased risk of death compared with control children without diarrhoea. The GEMS-1A study corroborates the MSD risk of death (nearly 12 times increase in cases compared with controls) and further showed that children with LSD had a 2·8 times increased risk of death compared with controls, although this difference was not statistically significant. Somewhat surprisingly, children with non-dysenteric MSD had a substantially higher risk of dying than did children with dysenteric MSD; notably, most children with dysentery in the GEMS and GEMS-1A studies received anti-Implications of all the available evidenceCollectively, these observations provide a strong rationale for vigorously acting upon all severities of diarrhoeal illness. Additionally, the prominence of delayed deaths occurring 14 or more days after enrolment of cases calls for a need to ensure follow-up for several weeks beyond the initial management of the acute diarrhoeal episode. The main pathogens implicated in MSD-associated or LSD-associated deaths were found to be the same, thus supporting the need to pursue common preventive and therapeutic strategies for diarrhoea as a major threat to child survival.vs 3·77 million for sub-Saharan Africa), the highest under-5 mortality was reported in sub-Saharan Africa (177 per 1000 livebirths vs 126 per 1000 livebirths in south Asia).2The fourth Millennium Development Goal (MDG4), which aimed to reduce mortality in children younger than 5 years by two-thirds by 2015 compared with the 1990 baseline, triggered efforts to improve monitoring and to identify the predominant causes of paediatric deaths.To achieve MDG4, the major causes of under-5 mortality in these two geographical regions had to be reduced substantially. Early estimates identified diarrhoeal disease as the second most common cause of under-5 mortality after pneumonia, accounting for approximately 19% of deaths.6One post-2015 challenge is to accelerate the decrease in mortality from diarrhoeal disease so that by 2030 the overall under-5 mortality will not exceed 25 per 1000 livebirths in any country worldwide .9The Global Enteric Multicenter Study (GEMS) was done in the first decade of the millennium when diarrhoeal disease constituted the second most frequent cause of under-5 mortality and there was uncertainty regarding the relative mortality contribution of specific enteric pathogens.Cryptosporidium spp, enterotoxigenic Escherichia coli encoding heat-stable toxin [ST-ETEC] with or without co-expression of heat-labile enterotoxin, and Shigella spp) and showed that children with MSD had more linear growth faltering during the follow-up period than did controls.The 3-year GEMS case-control study identified aetiological agents associated with moderate-to-severe diarrhoea (MSD), assuming that cases of this severity might be fatal if the patient could not access health care.Children with MSD in the GEMS study accounted for only 24·1% of all children with diarrhoeal disease who presented to the sentinel health centres; no data were obtained from children seeking health care who had the more common less-severe diarrhoea (LSD) and who were therefore ineligible for enrolment into the GEMS study. Accordingly, a 1-year follow-on study, GEMS-1A, was done, consisting of two parallel case-control studies, one enrolling cases of MSD and matched controls and the other enrolling cases of LSD and matched controls.Herein we report deaths in children with MSD  during 4 years of enrolment; deaths in children with MSD and LSD and in their matched controls during the two parallel GEMS-1A case-control studies; specific pathogens associated with fatal outcomes; deaths in cases and controls over time post-enrolment; and insights from verbal autopsies on potential causes of delayed diarrhoea-associated deaths.The 12-month GEMS-1A study of MSD and LSD used, with minor exceptions,Six of the original seven GEMS field sites, located in countries with moderate-to-high mortality of children younger than 5 years in Africa  and Asia  participated in the GEMS-1A study; cases of MSD only were enrolled in Kenya during GEMS-1A. Each site performed a population census and maintained a demographic surveillance system (DSS) during the case-control study to estimate the median population in each age stratum.Children aged 0–59 months belonging to the DSS population at each site who sought care at a sentinel health centre were screened for diarrhoea, defined as three or more loose stools during the previous 24 h. Each child with diarrhoea was assessed for eligibility. Episodes eligible for inclusion had to be new (onset after ≥7 diarrhoea-free days), acute (onset within the previous 7 days), and fulfil at least one of the following criteria for MSD: sunken eyes ; loss of skin turgor; intravenous hydration administered or prescribed; dysentery (visible blood in loose stools); or admission to hospital. The remaining non-MSD, new, acute diarrhoea episodes were eligible to be enrolled as LSD.Enrolment at GEMS sites began on Dec 1 (India), Dec 2 (Bangladesh), Dec 10 , and Dec 17 , 2007, Jan 31, 2008 (Kenya), and March 3, 2008 (Pakistan), and ended on March 3, 2011. GEMS-1A enrolment began on Oct 31, 2011, and ceased on Nov 14, 2012.The clinical protocols for the GEMS and GEMS1-A studies were approved by ethics committees at the University of Maryland , and at every field site. Written informed consent was obtained from the parent or primary caretaker of each participant.E coli pathotypes are included in the Study procedures for the GEMS and GEMS1-A studies have been described in detail elsewhere.Field workers made a single follow-up visit to households of cases and controls about 60 days after enrolment (window 50–90 days) to assess the child's vital status, capture interim medical events, and repeat anthropometric measurements.When deaths in enrolled children were detected, a time was arranged (respecting mourning practices) to perform a verbal autopsy using a WHO questionnaire.Sample size considerations and justifications have been described elsewhere.t test. Pair-wise comparisons were two-sided; results with p values of 0·05 or lower were considered significant. We made no imputation of missing values or adjustment for multiple comparisons. SAS version 9.4 was used for all analyses apart from Kaplan-Meier cumulative hazard curves, which were estimated using R version 3.5.3.Cumulative hazard curves from Kaplan-Meier risk of death analysis are shown for MSD cases during 4 years of enrolment. Pathogens associated with increased risk of death in MSD and LSD cases were identified using multiple Cox regression. Pathogens were included if there were at least five deaths that tested positive for the pathogen and if that pathogen was significantly (p<0·10) associated with death in crude analyses that controlled only for site. Pathogens were then eliminated from a model that adjusted for other pathogens until only those pathogens significantly (p≤0·05) associated with death remained. This model selection method was used in GEMS analyses.The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.Of the 223 deaths in children with MSD, 110 (49·3%) occurred at least 14 days after enrolment and 79 (35·4%) occurred at least 21 days after enrolment. 193 (86·5%) of 223 deaths in children with MSD and 40 (93·0%) of 43 deaths in control children occurred in the four African sites. Risk of death estimates in controls at Asian sites were smaller than those observed at African sites, with only one death observed per site in controls during the 4 years. However, in children with MSD, the Pakistan site showed a unique pattern, with 21 deaths, resulting in a high HR of 16·00 , children with MSD were more likely to die than their matched controls during the GEMS study  and GEMS-1A study , ST-ETEC, and Aeromonas spp infections were associated with increased risk of death .In Cox regression analysis, in all infants aged 0–11 months with MSD in the GEMS and GEMS-1A studies combined, typical enteropathogenic of death , as wereShigella spp infections detectable by qPCR, and qPCR data from a large random subset of cases from the GEMS studyEvidence of higher risk of death in children with non-dysenteric MSD than in those with dysenteric MSD , Table 2Shigella-positive by qPCR and 30 deaths occurred in 1384 children qPCR-negative for Shigella spp . Thus, Shigella in children with non-dysenteric MSD was strongly associated with an increased risk of death. Notably, only eight (25·8%) of these 31 fatal infections positive for Shigella spp by qPCR were also positive by culture, while 23 (74·2%) were culture negative.Among 61 deaths in children aged 12–59 months who had non-dysenteric MSD, 31 occurred in 942 children who were Altogether, 285 deaths occurred among cases or controls in the GEMS study  and GEMS-1A study , and verbal autopsy data were available for 266 93·3%; . Becausevs matched controls).A hallmark observation from the GEMS study was that children with MSD had an 8·5 times higher likelihood of a fatal outcome during the approximately 60-day follow-up period compared with control children without diarrhoea.In the combined MSD dataset from the GEMS and GEMS-1A studies, approximately half of MSD-associated deaths occurred following discharge from the enrolment sentinel health centre and more than 2 weeks after enrolment, and would not have been detected had there not been the systematic single follow-up visit to the household at about 60 days after enrolment. These observations imply that within the reference population, the burden of MSD-related deaths far exceeds deaths detected within the first days of onset of MSD episodes as observed in sentinel health centres.Children with MSD in the GEMS-1A study had a significantly higher risk of death during the 60-day follow-up period than did children with LSD, an observation that answers unequivocally the query about whether the GEMS definition of MSD truly encompasses illnesses with a greater likelihood of fatal outcome than LSD for the individual child.Higher numbers of deaths occurred among controls in African sites than in Asian sites, reflecting the higher risk of death in children younger than 5 years in sub-Saharan Africa. Yet the highest risk of death in children with MSD was in Pakistan. In view of similar access to care and treatment regimens for MSD at all three Asian sites, we explored whether host nutritional differences  might have contributed to the high risk of death in children with MSD in Pakistan. Indeed, children with MSD in Pakistan had significantly more linear growth faltering at baseline than did children with MSD in India and Bangladesh.Cryptosporidium spp) were similarly identified in the GEMS-1A study, making these robust observations over multiple years of surveillance. ST-ETEC can rapidly dehydrate young infants leading to death, unless effective rehydration is promptly instituted. tEPEC and Cryptosporidium spp induce similar attaching and effacing histopathological lesions in small intestinal mucosa characterised by intimate adherence to enterocytes, effaced microvilli, and actin polymerisation;Aeromonas spp (infants) and E histolytica and enteroaggregative E coli (toddlers).Several pathogens associated with increasing the risk of death from MSD in the GEMS study  is a clinical indication for treatment with anti-Because the occurrence of delayed (≥14 days post-enrolment) MSD-associated deaths observed in the GEMS study has been substantiated by findings in the GEMS-1A study and an increased risk of death  has been observed in children with LSD, we must ponder what is responsible for the deaths. Causal pathways that could plausibly culminate in delayed fatality include (1) dehydration worsening or recurring after discharge from the sentinel health centre, inadequate oral rehydration at home, and failure to return the child promptly to health care; (2) bacteraemia secondary to mucosal injury triggering over-stimulation of the systemic innate immune system leading to systemic inflammatory response syndrome ;Analysis of verbal autopsy data from children who died in the African and Asian sites of the GEMS study shows that the most attributed causes of death coincide with the above possibilities, even if information for the Asian sites is limited because of the scarcer number of deaths occurring there. Nevertheless, verbal autopsy, while valuable, is an imperfect tool for interpreting the cause of death at the individual level. More definitive investigative tools are needed.vs hospital-based) risk of death due to MSD and LSD.One limitation of the GEMS and GEMS-1A studies is that although rotavirus was documented as the predominant pathogen responsible for MSD and LSD morbidity, because enrolment was done at sentinel health centres in which effective rehydration therapy was available to replace water and electrolyte deficits, deaths from rotavirus dehydration were uncommon. However, without access to health care, many infants or toddlers with rotavirus diarrhoeal dehydration would surely have died. Rotavirus aside, for groups estimating global diarrhoeal disease mortality by aetiology,Shigella spp and ETEC that are currently in clinical development. Because vaccines against Cryptosporidium spp remain a distant goal,Cryptosporidium spp and safe, effective therapeutics to treat their infections. Collectively, such interventions can help to assure that by 2030 diarrhoeal disease will no longer be a major cause of death in children younger than 5 years in any resource-scarce, impoverished setting.The risk of death analyses in this study can help public health decision makers choose what investments to make among competing priorities. Our data emphasise the value of preventing or treating all diarrhoeal syndromes that bring children to health care, including LSD and MSD. Because the major attributable enteric pathogens associated with LSD and MSD are the same,


Radioisotope-guided sentinel lymph node dissection (sLND) has shown high diagnostic reliability in prostate (PCa) and other cancers. To overcome the limitations of the radioactive tracers, magnetometer-guided sLND using superparamagnetic iron oxide nanoparticles (SPIONs) has been successfully used in PCa. This prospective study  determined the diagnostic accuracy of magnetometer-guided sLND in intermediate- and high-risk PCa. Fifty intermediate- or high-risk PCa patients (prostate-specific antigen (PSA) ≥ 10 ng/mL and/or Gleason score ≥ 7; median PSA 10.8 ng/mL, IQR 7.4–19.2 ng/mL) were enrolled. After the intraprostatic SPIONs injection a day earlier, patients underwent magnetometer-guided sLND and extended lymph node dissection , with 447 sentinel lymph nodes SLNs  being identified and 966 LNs  being removed. Thirty-six percent (18/50) of patients had LN metastases . Magnetometer-guided sLND had 100% sensitivity, 97.0% specificity, 94.4% positive predictive value, 100% negative predictive value, 0.0% false negative rate, and 3.0% additional diagnostic value (LN metastases only in SLNs outside the eLND template). In vivo, one positive SLN/LN-positive patient was missed, resulting in a sensitivity of 94.4%. In conclusion, this new magnetic sentinel procedure has high accuracy for nodal staging in intermediate- and high-risk PCa. The reliability of intraoperative SLN detection using this magnetometer system requires verification in further multicentric studies. Histopathological examination or pelvic lymph node (LN) dissection (LND) is still the gold standard for LN staging in clinically localized prostate cancer (PCa). The number of removed LNs or the extent of LND directly influence the rate of detected LN-positive patients . On the 99mTc) nanocolloid and a gamma probe for intraoperative SLN detection are used for the established radioisotope-guided SLN identification in PCa patients. The diagnostic accuracy of this radioactive sentinel LN dissection (sLND) approach was determined in a systematic literature review. Twenty-one studies including 2509 patients were analyzed [Because of relevant importance for further therapy and the complication rate of extended LND (eLND), as well as the low detection rate of limited LND methods, the concept of targeted radioisotope-guided sentinel LN (SLN) identification used in other tumor entities was implemented in PCa . Conventanalyzed . The finanalyzed ,7.Because of the ionizing radiation emitted by the radioactive tracer material, the benefits of the established SLN procedure are associated with certain limitations. The dependence on radioisotopes or nuclear medicine facilities imposes restrictions on patient planning and hospital logistics. In principle, the application of this procedure is thus limited to small parts of the developed world. Moreover, surgical staff and patients are exposed to ionizing radiation.Superparamagnetic iron oxide nanoparticles (SPIONs) have received much attention in bioscientific research since the first report in the 1980s and have increasingly been used clinically in recent years. Biocompatible SPIONs with a suitable surface architecture have triggered research efforts for both cellular imaging and drug-delivery applications. One of the main features of SPIONs is the ability to show magnetization only in an applied magnetic field. SPIONs have the ability to form stable colloidal suspensions, which is crucial for in vivo biomedical applications ,11. The Based on these advantageous properties, SPIONs have been successfully applied for marking and intraoperative detection of SLNs in breast cancer to overcome the drawbacks of the radioisotope-based sentinel procedure . In a piIn view of these findings, we hypothesized that magnetometer-guided sLND based on intraoperative identification of SPION-marked SLNs would have high reliability in the identification of LN-positive PCa patients, being comparable with the radioisotope-guided sentinel procedure.To assess the diagnostic accuracy of magnetometer-guided sLND in PCa, this prospective single-center study analyzed intermediate- and high-risk PCa patients who underwent magnetometer-guided sLND and eLND, followed by radical retropubic prostatectomy. The diagnostic accuracy of magnetometer-guided sLND was determined using eLND as the reference standard.As planned, the study included 50 intermediate- or high-risk PCa patients who underwent radical retropubic prostatectomy with magnetometer-guided sLND after intraprostatic injection of SPIONs, with eLND being performed as the reference standard. In all, 966 LNs  15–23) were removed. At least one SLN was successfully detected by magnetometer-guided sLND in all patients (50/50), resulting in a detection rate of 100%. According to the ex vivo measurements of magnetic LN activity, a total of 447 SLNs were identified. The median number of detected SLNs was 9 (IQR 6–12).SLNs were also localized outside the established eLND template . LN metastases were found in 36% (18/50) of patients. In total, 43 LNs were metastasis-positive, with the median number of positive nodes (when present) being 2 (IQR 1–3). Taking eLND as the reference standard, the sensitivity of the magnetic SLN procedure was 100%, i.e., all patients with LN metastases were correctly detected as LN-positive. The magnetometer-guided sLND results had a specificity of 97.0%, positive predictive value (PPV) of 94.4%, and negative predictive value (NPV) of 100%, resulting in a false negative rate of 0.0%. sLND was shown to be of additional diagnostic value in one of the 18 LN-positive patients. In this case, sLND resulted in the detection of one LN metastasis outside the eLND template , while eLND did not reveal any metastases . n = 14).The percentage of LN-positive patients with metastases only in SLNs was 77.8% .After the successful application of the magnetometer-guided sentinel approach in breast cancer, the feasibility and safety of intraoperative detection of SPION-marked SLNs using a handheld magnetometer following intraprostatic SPIONs injection was demonstrated in PCa . CurrentOn the basis of results comparable to the radioactive marking of SLNs in breast cancer and the promising first results presented in our PCa pilot study (SentiMag Pro I) ,13,15, wIn the results presented for the SentiMag Pro II trial, which included PCa patients with an intermediate- or high-risk for the presence of lymphatic metastasis, SLNs were identified in all patients, resulting in a detection rate of 100%. This is better than in our pilot study, that included PCa patients with the same risk factors, where the magnetic technique successfully identified SLNs in only 89.5% of cases . For radIn the ex vivo analysis using the handheld magnetometer to identify SLNs, all LN-positive patients were correctly detected in the SentiMag Pro II study. However, one metastatic SLN was not detected intracorporeally using the SentiMag probe, resulting in the missing of one LN-positive patient and a sensitivity of 94.4%. This patient had a high-risk tumor with large volume  tumor–node–metastasis (TNM) classification of malignant tumors, 7th Edition), PSA 44 ng/mL), which may have obstructed the outflow of the tracer. In vivo, only one SLN showing weakly magnetic activity (11 counts) could be identified using the SentiMag probe. In ex vivo measurement, magnetic activity was detected in five of the 35 LNs removed, which, like the metastasis-positive SLN overlooked intraoperatively (50 counts), had a comparatively low activity. The positive SLN, which was not detected intraoperatively, was located in the deep presacral area, so that the limitations of the intracorporeal detection of SPION-marked SLNs described in the next but one paragraph may have become noticeable, taking into account the weak activity.z = 3.87, p < 0.001), too. The mean detection rates for the technetium-based standard and magnetic techniques alone were 96.8% (94.2–99.0%) and 97.1% (94.4–98.0%), respectively. Mean false-negative rates were 10.9% (range 6–22%) for the standard technique and 8.4% (2–22%) for the magnetic technique. Using a random-effects model, the total number of LNs removed was significantly higher with the magnetic technique (p = 0.003) [99mTc nanocolloid: <80 nm) which could result in marking of secondary landing sites, too [In the systematic literature review mentioned above, the median cumulative percentage results for sLND showed a sensitivity of 95.2% (81.8–100%) and false negative rate of 4.8% 0–18.2%), taking into account in vivo SLN identification [.2%, takites, too .® probe (~20 mm) could restrain the differentiation of SLN signals from the background signal from the injection site. In the near future, the higher resolution of novel probes using magnetic tunneling junction techniques (resolution ~4 mm) could lead to a sustainable improvement in intracorporeally SLN identification [There are various possible causes limiting the effectiveness of intracorporal detection of SPION-marked SLNs using a magnetometer. One problem of intracorporeally detection is that adipose tissue surrounding SLNs can limit the proximity of the probe to the LN, resulting in insufficient exposure of the LN. An insufficient measurement of the magnetic signal can be the result. Moreover, the presence of tissue in the vicinity of the probe tip of the magnetometer and the LN reduces the in vivo magnetic signal because of the negative magnetic susceptibility of surrounding tissue . The lim® probe ~ mm couldfication .® magnetic probe: 10 mm), which can have consequences for the identification of deeper nodes [Other disadvantages of the magnetometer-guided procedure are the need for frequent balancing of the magnetic baseline level and the requirement for use of plastic or other non-magnetic retractors during surgery . This cier nodes ,23. In tPreoperative SLN identification offers the surgeon a roadmap with solid information on individual location of draining LNs. So far, lymphoscintigraphy cannot be undertaken without a radioisotope tracer. However, preoperative SLN visualization on MRI after intraprostatic SPION injection was demonstrated recently in PCa patients and could replace the conventional lymphoscintigraphic procedure . MRI is Another important advantage for routine clinical use of the magnetic sentinel method is that this non-radioactive procedure does not require a radiation-controlled area or imaging facilities and can be safely implemented without facilities’ restrictions in large parts of the world. For example, in Japan, currently, approximately 30% of the facilities do not have a radiation-controlled area . Furthern = 4) of LN-positive cases, metastases were also found in non-SLNs. All four cases were patients with high aggressive PCa , in accordance with previous reports showing poorer outcomes for sLND with highly aggressive tumors [In the SentiMag Pro II study, in 22.2%  present in the blood as a supplemental, or perhaps an alternative, source of DNA to identify the clinically relevant cancer mutational landscape. This noninvasive approach may facilitate repeated monitoring of disease progression and treatment response. Potential applications of this noninvasive information in tumor staging, treatment, and disease prognosis are under discussion . For exaThe prospective monocentric SentiMag Pro II study  investigated the diagnostic accuracy of a novel technique for intraoperative SLN detection in PCa patients, using SPIONs and a handheld magnetometer.Fifty patients with intermediate- or high-risk PCa (European Association of Urology risk group definitions) scheduled for open radical retropubic prostatectomy and pelvic LND between February and September 2015 were included in this study . Inclusi®) used in this study is a component of the SentiMag® system . This system for marking and identifying SLNs comprises a handheld magnetometer, the SentiMag® unit, and the Sienna+® magnetic tracer. All are Conformité Européene (CE) certified as class IIa medical devices. The particles have a carboxydextran coating and a mean hydrodynamic diameter of 60 nm. Sienna+ has comparable functional properties to that of 99mTc nanocolloid, because upon interstitial injection the tracer flows through the lymph system and gets trapped in SLNs in the same manner as the radionuclide.The SPION tracer  into the prostate of patients using transrectal ultrasound guidance 24 h before surgery. Based on our examinations and those of others, the tracer was evenly spread as three deposits on both sides of the prostate in all cases, as described previously .Patients underwent magnetometer (SentiMag)-guided sLND and eLND, followed by radical retropubic prostatectomy. All cases were performed by two high-volume surgeons, who applied the same anatomical template during eLND. The eLND template included the area along the external iliac vessels, with the distal limit being the femoral canal. Proximally, eLND was carried out to, and included, the bifurcation of the common iliac artery. All lymphatic fatty tissue along the internal iliac artery and within the obturator fossa and the area dorsal to the obturator nerve was removed, as described by Weingärtner et al. . The lat®, Aesculap®; B. Braun Melsungen AG, Melsungen, Germany) were used to avoid interference with the magnetometer when detecting SLNs with the SentiMag probe. All SLNs detected by the SentiMag were removed, with each magnetically active LN being considered as an SLN. In addition, the magnetic activity of all LNs was measured ex vivo. For surgical reasons, LNs other than SLNs directly adjoining and adhering to SLNs were also removed if in situ separation was not possible. In such cases, LNs were macroscopically detected  ex vivo and the surgeon separated them from each other or from the containing fibro-fatty tissue. Thereafter, eLND was conducted to remove the remaining lymphatic fatty tissue from the above-named regions. Afterwards, LNs were macroscopically detected and separated from those containing fibro-fatty tissue by the surgeon.During sLND, all metal retractors were removed from the surgical field and polymer retractors , cut into 3 mm transverse sections, and routinely processed and embedded in paraffin, while 4–5 µm-thick sections were further cut and stained with hematoxylin-eosin.As established by our and other working groups, and in line with the results of an international sentinel consensus meeting, the diagnostic accuracy of sLND was assessed using conventional eLND in the same cohort as the reference standard ,37. CompThe outcomes used to analyze the diagnostic test accuracy were detection rate , sensitivity, specificity, PPV, NPV, false-positive, and false-negative rates, with all being measured at the patient level. False-negative cases were defined as patients with a histologically negative SLN, whilst cancer was found in other LNs. False-positive cases were defined as patients with SLNs containing metastases found outside the eLND template, while the eLND template did not reveal any metastases . Thus, tA 2 × 2 table with sLND as the index test and eLND as the reference standard was used to calculate sensitivity, specificity, NPV, and PPV. Additionally, the anatomical distributions of detected LN metastases and identified SLNs were analyzed.All subjects gave their informed consent for inclusion before they participated in the study. The protocol followed in this study was in accordance with the ethical standards of the 1964 Helsinki Declaration and its later amendments. The protocol was approved by the Medical Chamber of Lower Saxony, Germany (Bo/24/2014).After interstitial injection, SPIONs are filtered and retained in SLNs allowing intraoperative identification of SLNs using handheld magnetometers. Based on these characteristics, SPIONs have been successfully applied for marking and intraoperative detection of SLNs in breast cancer to overcome the drawbacks of the radioisotope-based sentinel procedure. In several studies including breast cancer patients, the magnetic SentiMag technique for SLN biopsy was non-inferior to the standard method. The results of this prospective clinical trial suggest that the magnetometer-guided radiation-free sentinel procedure could be a reliable replacement for the established radioisotope-based approach in PCa patients who are at intermediate- or high-risk for LN involvement, too. With the aim of detecting all LN metastases in high-risk patients, sLND should be performed in addition to eLND, because of its additional diagnostic value and the detection of LN metastases outside the extended template. The reliability of intraoperative SLN detection using the SentiMag system requires verification in further multicentric studies, including comparisons with other new magnetometer modalities , which were presented recently. In addition, the promising initial results in other tumor entities such as melanoma, vulvar, and penile cancer should also be pursued.


Significance: Our study introduces an application of deep learning to virtually generate fluorescence images to reduce the burdens of cost and time from considerable effort in sample preparation related to chemical fixation and staining.Aim: The objective of our work was to determine how successfully deep learning methods perform on fluorescence prediction that depends on structural and/or a functional relationship between input labels and output labels.Approach: We present a virtual-fluorescence-staining method based on deep neural networks (VirFluoNet) to transform co-registered images of cells into subcellular compartment-specific molecular fluorescence labels in the same field-of-view. An algorithm based on conditional generative adversarial networks was developed and trained on microscopy datasets from breast-cancer and bone-osteosarcoma cell lines: MDA-MB-231 and U2OS, respectively. Several established performance metrics—the mean absolute error (MAE), peak-signal-to-noise ratio (PSNR), and structural-similarity-index (SSIM)—as well as a novel performance metric, the tolerance level, were measured and compared for the same algorithm and input data.Results: For the MDA-MB-231 cells, F-actin signal performed the fluorescent antibody staining of vinculin prediction better than phase-contrast as an input. For the U2OS cells, satisfactory metrics of performance were archieved in comparison with ground truth. MAE is Conclusions: These findings contribute to the understanding of the utility and limitations of deep learning image-regression to predict fluorescence microscopy datasets of biological cells. We infer that predicted image labels must have either a structural and/or a functional relationship to input labels. Furthermore, the approach introduced here holds promise for modeling the internal spatial relationships between organelles and biomolecules within living cells, leading to detection and quantification of alterations from a standard training dataset. Intensity- and/or phase-based microscopy techniques such as brightfield, phase contrast, differential interference contrast, digital holography, Fourier ptychography, and optical diffraction tomography,–,Deep convolutional neural networks (DCNNs) capture nonlinear relationships between images globally and locally, resulting in significantly improved performance for image processing tasks compared with the traditional machine learning methods. In many studies of cells requiring subcellular details, fluorescence labels specific to selected biomolecules or organelles are imaged simultaneously or in rapid sequence in separate fluorescent signal channels to assign subcellular localization of biomolecules to certain organelles. Alternatively, co-registered phase contrast or other brightfield modality images are acquired to relate biomolecular localization to cell morphology and structural features. In several recent studies, applications of DCNNs to fluorescence microscopy of cells investigated the performance of these algorithms in super-resolution,Based on these results, we formulated two questions leading to null and alternative hypotheses. First, does model prediction performance depend on the image modality and subcellular labels selected for training and prediction? Second, do errors in predicted images contribute to the likelihood of misinterpreting biology based on the image predictions? To address these questions, we developed a DCNN-based computational microscopy technique employing a customized conditional generative adversarial network (cGAN) that models the relationships between optical signals acquired using any imaging modality or fluorescence channel, assuming the signals are co-registered. This technique was employed to fulfill two objectives addressing the hypotheses. First, we determined image prediction performance using several fluorescence channels and phase contrast images for training and compared prediction performance between sets of input and output optical signals and fluorescence labels for two independent datasets using different cell lines. Second, we compared predicted images with ground truth to identify signal error, evaluating the error fractions important and unimportant to biological interpretation through a novel quantitative prediction performance parameter. To further demonstrate the adaptability of the cGAN algorithm to different image prediction tasks, out-of-focus fluorescence images of cells were digitally refocused after application of a trained, end-to-end ranged autofocusing (AF) algorithm.,The results of this work support prediction of fluorescent labeling of cells from other image data. These predictions have the potential, in one application, to significantly reduce the cost and the effort in preparation of cell imaging experiments to end-users by replacing or supplementing actual labeling experiments. This first application requires a high degree of fidelity of predicted images to ground truth, within a tolerance of error that allows for accurate biological conclusions to be drawn. Here we introduce a tolerance level to quantify the fidelity of predicted images. A second application lies in removing artifacts and aberrations from existing image datasets. Here we demonstrate an AF method that improves blurred regions by reference-free metrics. Other applications are discussed, including transfer learning to ascertain the effects of a treatment on cells by direct comparison of actual fluorescence labels versus predictions from controls. Thus the described new method supports cell studies based on mixed virtual cell imaging. Because the VirFluoNet method is digital, it significantly reduces the possibility for cell damage from phototoxicity and signal degradation from photobleaching. This work also extended cGAN capabilities to the prediction of fluorescent images. We envision that the approaches of this study will be useful for prescreening large fluorescence microscopy datasets, identifying and correcting out-of-focus regions, and potentially saving time in multilabel fluorescence experiments by predicting some fluorescence label features from other labels signals. Due to the rapid development of hardware/platform  supporting machine learning applications, the algorithms of this study should become useful in an expanding number of applications. A future application of the approach may be the study of complex protein structures and modeling protein–protein or protein–organelle relationships.2The order of methods and results, below, follows the order of the first and second objectives described above. However, the ancillary goal of demonstrating refocusing of out-of-focus image data using a cGAN model is presented first, as this was an image preprocessing step contributing to high-quality training data for fluorescent image transforms, which would be described later.2.1To achieve AF as a preprocessing step for training data, we trained and tested the customized cGANs on a dataset of U2OS cells with labeled F-actin (U2OS-AF) and used the refocused images to enhance the input images before training models of Fluo-Fluo 2, 3, 4, defined below.For the first objective, we trained two cGAN models  to generate 4′,6-diamidino-2-phenylindole (DAPI) and vinculin from phase contrast images, respectively, and four cGAN models  for fluorescence prediction from another fluorescence image see . The traFor the second objective, training datasets of the PhC-Fluo 2 and Fluo-Fluo 1 models were evaluated for absolute image error . The ground truth and prediction channels were vinculin immunostaining, paired with either co-registered phase contrast or F-actin fluorescence label channels. Not all parts of the absolute image error contribute equally to image misinterpretation. The absolute image error was split into two components, spatial/area error and pixelwise intensity error, by summing segmented pixel area and intensity differences from a thresholded absolute error map. The threshold was scanned through the full map bit depth. A global minimum total weighted error was determined from the weighted sum of the two individual error terms.2.22.2.1The human breast cancer cell line MDA-MB-231 provided by Dr. Zaver Bhujwalla  was cultured on tissue culture-treated polystyrene dishes, in standard tissue culture conditions of 37°C with 5% 2.2.2High-content screening datasets of U2OS cells used in this study were previously made publicly available.2.2.3U2OS cell  raw images used here can be found in Ref. 2.3Patch images . The rationale for this index was that epifluorescence images of cell labels are qualitative in pixel intensity, to a certain tolerance, due to photobleaching and differences in experimental preparation, microscope instrument parameters, and camera settings. Therefore, small differences between pixel intensity values of predicted images and ground truth (intensity errors) are less likely to produce misinterpretations than a predicted signal where there is no true signal or the lack of a predicted signal where there is a true signal . Segmentation of absolute difference error maps so that error pixels above a certain tolerance are bright green highlights both errors. The sum of these two error terms, weighting each equally here for illustrative purposes, is minimized at a single error tolerance level: 3In this section, we describe the results from all implementations in three categories: (1) AF: refocusing out-of-focus images; fluorescence prediction from (2) phase contrast images and (3) fluorescence images, and (4) error quantification. We combined (2) and (3) into one section for comparative purposes. Although (1) was the preprocessing objective, (2) + (3) address the first objective, and (4) addresses the second objective.3.1We trained a DCNN model, named “AF model”, to predict focused images from blurred out-of-focus images. The model was trained and tested on the F-actin channel of a U2OS-AF dataset before applying the DCNN on out-of-focus images used in the Fluo-Fluo 2, 3, 4 model’s training and testing see . The modSupplementary Material). The lower MAE, higher PSNR, and closer to one SSIM values are expected to relate to better performance. In order, average scores were 0.01/0.012, 37.56/35.639, and 0.924/0.9 for MAE, PSNR, and SSIM, respectively. Scores in bold, which indicates “good” performance, demonstrate the feature enhancement from the AF model, compared with the scores on the right using input data that was not autofocused. The U2OS-AF model generated images that resemble the ground truth more closely than the input. Next, the AF model was used to perform the prediction on out-of-focus subset data chosen manually from the U2OS-CPS dataset to preserve the number of data samples. There is no ground truth for the focused images in the U2OS-CPS dataset, but we qualitatively evaluated the success of the network from observations of the level of focus versus depth frames and then used these images for training and analysis. Typical results are shown in ,,The AF model’s performance was evaluated by the MAE, PSNR, and SSIM3.2Following the training phase, the trained PhC-Fluo and Fluo-Fluo networks were evaluated in a blinded fashion using testing data separated from training data. Similarly, the Fluo-Fluo 2, 3, 4 models were implemented with the same cGAN framework. The difference between these models and Fluo-Fluo 1 is in the use of inputs that contain two fluorescently labeled subcellular compartments to predict the targeted protein. We trained and tested these models on the human U2OS-CPS cell dataset. These models can be used or edited as pretrained models with or without transfer learning on completely new types of data, thus making the proposed technique generalizable. Transforming one fluorescence channel into another can be based on one channel input-to-one channel output pairs. However, the success of model training depends on the correlation of the selected pairs, i.e., strongly correlated pairs of input–output data allow the model to learn a pixel-to-pixel transformation that is governed by the regularization of the network. This data-driven cross-modality transformation framework is effective because the input and output distributions share a high degree of mutual pixel-level information content, with an output probability distribution that is conditional upon the input data distribution.,In previous studies,Supplementary Material). The average scores are: MAE , PSNR , and SSIM , for DAPI/Hoechst, Endoplasmic reticulum, and Mitochondria label prediction, respectively. These results are comparable to those found in Ref. ,Supplementary Material). Each PMC was calculated across 96 samples for a single feature measurement. One sample is a region of cell to be taken from five fluorescent channels. Supplementary Material show the PMC coefficient of each feature measurement, distributed in cell, nuclei, and cytoplasm property groups, across 96 images from the U2OS-CPS testing dataset between the original five channels and hybrid-virtual two + three channels and their histograms, respectively. Ignoring the prefect correlation of 1 (self-correlation from inputs  only), the histograms show high correlation in feature measurement between the original channels and the hybrid-virtual channels, which demonstrates the reliability of using virtual channels for biological analysis. Extracted feature measurements including correlation, granularity, intensity, radius distribution, size and shape, and texture are shown in Method Section , and endoplasmic reticulum images and their corresponding ground truth fluorescence images in the testing dataset of U2OS-CPS  and its histogram  plus two original channels as inputs were strong, as seen in both the Pearson’s product-moment correlation coefficient matrices  deblurring of fluorescence images, (2) prediction of fluorescence signals related to an input fluorescence signal, and (3) transfer learning in which predicted and ground truth signal differences are attributable to an altered condition of the cells in the test dataset versus the training dataset. Unlike imaging techniques capable of focusing through a large depth-of-field such as holography, fluorescence microscopy lacks the capability for image propagation, which is necessary for digitally obtaining images at different axial planes. Traditional image focusing techniques such as deconvolution methods can be employed for fluorescence microscopy with varied success.lgorithm  and 4 isUsing the trained AF model, we have predicted the focused images of the testing U2OS-AF dataset, which only contains F-actin. To check the generalizability of the trained model, we applied it directly on a completely new dataset combining blurred signal from labels of Golgi apparatus + F-actin in the U2OS-CPS dataset without using transfer learning  is an image-centric and interpretation-focused way to standardize comparisons of algorithm performance for deep learning image regression across multiple developers. For example, two other recent works predict fluorescence image outputs from ground truth brightfield images serving as input to deep learning algorithms.5The presented methodology of image regression has a great potential to reduce time and cost of microscopy studies of cells. One of the advantages of using computational microscopy through DCNNs is to allow for a single well-tuned training process to transform fluorescence images of a certain fluorescence channel into their other fluorescence images. Training the dataset is a required step, but it is performed only once with less preprocessing. Recent related research has demonstrated that overall cell morphology and that of many organelles can be predicted from transmitted light imaging. In this work, we have extended the use of deep neural networks to predict subcellular localization of proteins from other proteins from co-registered images. Finally, the proposed DCNN will be a cost-effective tool for many biological studies involving protein–protein and organelle–protein relationships and will help researchers visualize the coordination of subcellular features under a variety of conditions aiding in understanding of fundamental cell behaviors.In future work, the algorithms developed here could be used to perform real-time virtual organelle self-coding for live-cell and intravital video microscopy governed by an inferencing device integrated to the microscope.Click here for additional data file.