Malignant pleural mesothelioma (MPM) is an uncommon but aggressive and treatment resistant neoplasm with low survival rates. In the last years we assisted to an exponential growth in the appreciation of mesothelioma pathobiology, leading several new treatments to be investigated both in the early stage of the disease and in the advanced setting. In particular, expectations are now high that immunotherapy will have a leading role in the next years. However, caution is required as results from phase II studies in MPM were often not replicated in larger, randomized, phase III trials. In this review, we describe the most promising emerging therapies for the treatment of MPM, discussing the biological rationale underlying their development as well as the issues surrounding clinical trial design and proper selection of patients for every treatment. Malignant pleural mesothelioma (MPM) is an uncommon and highly lethal cancer. The annual incidence of MPM ranges between 10 cases per million to 29 cases per million depending on the country and, because of the long latency period, the peak is expected in the 2020s ] and the number of these cells as determined by immunohistochemistry (IHC) represents a negative prognostic factor , 11. On than MPM .The management of MPM is complex and outcomes remain poor. For patients with early stage MPM the role of radical surgery is still a matter of debate and it should be considered only as part of a multimodal treatment . Looking at unresectable MPM, no major breakthroughs have been made since the approval of antifolate and platinum combination chemotherapy , 14. MedSteps have been made toward a best appreciation of mesothelioma biology and have been essential to identify novel molecular therapeutic targets, representing the rationale for testing multiple targeted therapies in MPM . NeverthIn this review, we describe last emerging therapies for mesothelioma, discussing the current status of knowledge in mesothelioma genetics and immune-biology, as well as the issues surrounding the conduction of high-quality trials in MPM and the selection of best patients for different treatments.Due to the anatomy, microscopically radical (R0) resection is not achievable in mesothelioma surgery and the goal of mesothelioma surgery is macroscopic complete resection (R1). Surgery alone is not curative; it is usually performed with chemotherapy and/or radiation therapy and reserved to a subset of patients with early tumor stage, epithelioid histology and good performance status.Therapeutic surgery in mesothelioma has historically involved either an extended pleurectomy-decortication (eP/D) or an extrapleural pneumonectomy (EPP) , 17. eP/In order to improve local control and ideally survival, radiotherapy can be given. New approaches of radical hemithoracic radiation using intensity-modulated techniques are being tested. Rimner et al. showed that hemithoracic intensity-modulated pleural radiation therapy (IMPRINT) after chemotherapy and P/D was safe in 27 MPM patients as part of a multimodality lung-sparing treatment, with an acceptable rate of radiation pneumonitis . Larger Recently, intrapleural therapies have been reported with the aim of improving loco-regional control of the disease by spreading drugs directly on the tumor surface. Several techniques with different rationale have been used with promising results: hypertermic intrapleural chemotherapy, photodynamic therapy (PDT), intrapleural immunotherapies [interferons (IFNs) and interleukin-2 (IL-2)], and gene therapy . HoweverIf given as neoadjuvant therapy, novel agents should have the ability to induce tumor shrinkage, increasing the possibility of a complete microscopic resection and ultimately prolonging overall survival while maintaining a good safety profile. Designing studies in this setting remains a challenging effort that requires multidisciplinary involvement . NeverthThis approach has also paved the way for testing the properties of immune check-point inhibitors (CIs). There are several ongoing neoadjuvant trials which aim to assess the immunomodulatory and pharmacodynamics effect of CIs, as monotherapy (NCT02707666), as combination of anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and anti-programmed cell death protein (PD-1) agents  and as combination of anti-programmed death-ligand 1 (PD-L1) with standard chemotherapy (NCT03228537).By assessing translational surrogates of response, these trials may represent an opportunity to look into predictive biomarkers, improving selection of candidates to CIs-treatment.CIs are also tested in the adjuvant setting (NCT02707666). From an immunological perspective, the main goal of combining surgery with adjuvant CIs is to reduce tumor induced immunosuppression . IncreasAnother approach to increase immune activation in the adjuvant setting is represented by vaccines, either protein, bacteria or cell-based. An adjuvant Wilms tumor 1 (WT1) vaccine , given with granulocyte-macrophage colony-stimulating factor (GM-CSF) and an immunologic adjuvant called montanide ISA 51 UFCH in MPM patients whose tumors expressed WT1 at IHC, had completed combined multimodality therapy and had no evidence of disease, showed a median progression-free survival (PFS) of 10.1 months (95% CI 5.5–20.8 months) and a median OS of 22.8 months (95% CI 9.1–37.6 months) with a favorable safety profile . GalinpeIn peritoneal mesothelioma, the feasibility of administering dendritic cells pulsed with an allogenic tumor cell lysate after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) is being assessed in the ongoing MESOPEC trial (NTR7060) . SecondaTo date, despite the neoadjuvant/adjuvant treatment represents a promising setting to test new therapeutic strategies, the global level of evidence is quite low and international guidelines  do not rp < 0.0001)], at the cost of an increased yet manageable toxicity   or best supportive care (BSC) after 4-6 cycles of first-line platinum-pemetrexed without progression. Data presented at the last ESMO conference showed an improvement in PFS (median 6.2 months vs. 3.2 months in the BSC arm [hazard ratio (HR) 0.42 (95% CI 0.28-0.63), requent) . Since pLurbinectedin is a new molecule that binds to the DNA minor groove in regulatory regions, inhibiting the function of oncogenic transcription factors. It also modulates the transcriptional program of monocytes and TAMs, hampering cytokine production . InvestiThese data support evaluation of the both gemcitabine as switch maintenance and lurbinectedin as second-line strategy in larger, randomized, phase III trials.The NovoTTF-100L represents another approach that has been recently investigated to improve the efficacy of chemotherapy. NovoTTF-100L is a portable Tumor Treating Fields (TTFields) delivery system. TTFields represent a non-invasive, regional treatment modality by which alternating electric fields (at a frequency of 150 kHz) are continuously administer to the local site to arrest tumor cancer cell division. In human mesothelioma cell cultures, combining TTFields with cisplatin or pemetrexed led to reduction in cell count, induction of apoptosis and reduced clonogenic potential . These aBased on the results of the prospective, single-arm, phase II STELLAR trial, the NovoTTF-100L System was approved by U.S. FDA in combination with pemetrexed plus platinum-based chemotherapy for the first-line treatment of unresectable locally advanced or metastatic MPM. NovoTTF-100L was approved under Humanitarian Device Exemption, an approval process guaranteed by the U.S. FDA which, taking into consideration the urgent need to identify more effective treatments for rare disease (such as MPM), allows medical devices to be marketed without requiring evidence of effectiveness.p = 0.0167). The control arm of this trial performed 4 months better than the historical cohort analyzed by Ceresoli et al.—the landmark study by Vogelzang et al.— pathway, via its tyrosine kinase receptors, is crucial for mesothelioma cells growth , thus reThe addition of bevacizumab to pemetrexed and cisplatin chemotherapy as first-line treatment with bevacizumab maintenance therapy in patients who did not progress showed improved overall survival. However, bevacizumab remains currently unlicensed in this setting since the MAPS trial was not a registration trial . Moreoven = 87 patients) (p = 0.91). The interim analysis of OS also showed no difference between groups   was highn groups .Nintedanib is also being currently investigated as only maintenance treatment for patients non-progressive after first line chemotherapy (NCT02863055).p = 0.096). However, PFS was not different by modified RECIST and no significant difference in OS was reported. As with bevacizumab, cediranib is not approved as first-line treatment combined with chemotherapy.Cediranib, a VEGFR and PDGFR inhibitor, added to first-line platinum-based chemotherapy, improved PFS in a randomized phase II trial . PrimaryRamucirumab is a monoclonal antibody that binds the extracellular domain of human VEGFR-2. Due to VEGF-R2 expression on macrophages, ramucirumab also inhibits macrophages and their infiltration into mesothelioma microenvironment, thereby decreasing tumor growth and proliferation . One-hunNew studies have recently provided a comprehensive genomic profiling of mesothelioma. Genomic analysis may help in detecting actionable alterations and developing more tailored and effective therapies for MPM patients . Tumor sCarriers of inherited loss-of-function mutations in BAP1 are predisposed to mesothelioma , 49, 50.In a recent paper, among 385 patients treated with platinum chemotherapy, median OS was increased for MPM patients who had inherited mutations in DNA repair and/or other tumor suppressor genes . This isBy inducing synthetic lethality of alternate DNA repair pathways, poly-ADP ribose polymerase (PARP) inhibitors have proved to be able to cause cell death in cell lines with loss of function of BAP1. This observation suggests that patients with mutations in BAP1 and in DNA repair genes might also benefit from treatment with PARP inhibitors . An enroin vitro and in vivo  and enhancer of zeste-homolog 2 (EZH2) pathway. Mesotheliomas with BAP1 loss proved to be responsive to EZH2 inhibition  in vivo . EZH inh in vivo .CDKN2A is a tumor suppressor gene frequently inactivated in mesothelioma. CDKN2A encodes the ADP-ribosylation factor  and INK4A  via alternative reading frames . By inhiThe hepatocyte growth factor (HGF), by binding to the MET receptor and activating its downstream target PI3K has been shown to enhance MPM cell proliferation, migration and invasiveness. Therefore, this pathway represents a compelling therapeutic target in this disease . HoweverThe enzyme argininosuccinate synthetase 1 (ASS1) leads to arginine biosynthesis from citrulline and is epigenetically suppressed in a high proportion of mesothelioma cell lines . Loss ofIn conclusion, despite our improved understanding of the biology of MPM, response to targeted therapies is hampered by intra-tumor heterogeneity and it is still unclear whether most of the actionable mutations constitute clonal or sub-clonal driver events. Longitudinal prospective studies, such as the TRACERx study in lung cancer , aiming Mesothelin (MSLN) is a glycoprotein with high expression in epithelioid mesothelioma and low expression in normal tissues, thereby it represents an attractive target for several therapies. A phase II trial comparing amatuximab  plus first-line chemotherapy vs. chemotherapy alone was prematurely stopped in January 2017, not because of unacceptable toxicity but because of business reasons (NCT02357147).According to a public announcement, anetumab ravtansine (an antibody-drug conjugate made by combining a human anti-MSLN antibody and the maytansinoid tubulin inhibitor DM4) also failed to improve PFS compared to vinorelbine in a randomized phase II trial for patients progressing after first-line (NCT02610140) .CRS-207 is a live, attenuated, non-virulent, Listeria monocytogenes (LADD) encoding human MSLN. After receiving two priming infusions of CRS-207, followed by pemetrexed/cisplatin chemotherapy, and CRS-207 booster infusions in a phase Ib trial, 89% (31/35) of patients had disease control; one complete response (3%) and 19 partial responses (54%) were reported. Reduction of tumor size was also observed post-CRS-207 infusion prior to chemotherapy in 11 patients and no treatment-related serious adverse events or deaths were observed. These results suggested that combining CRS-207 with traditional chemotherapy might potentially result in increased anti-tumor activity . HoweverLMB-100 is a next generation immunotoxin against MSLN that consists of a humanized fragment of the anti-MSLN Fab bound to a de-immunized Pseudomonas exotoxin (PE). This PE-fusion protein has been engineered to decrease its immunogenicity. A Phase I, open-label study to investigate the safety, pharmacokinetics, and activity of LMB-100 in relapsed MPM patients is planned to complete accrual this year (NCT02798536).Evaluating new combinations of MSLN directed therapies with checkpoint inhibitors and integrating MSLN targeting into new approaches such as adoptive T cell transfer might constitute the next step in the field, as first results have been promising .+ T cells (tumor-infiltrating lymphocytes), whereas PD-L1 expression is associated with shorter survival   , 82. Due 0.0001) . Looking 0.0001) .As shown in other types of cancer , combinip = 0.004), but these responses were not translated into delayed progression or improved survival (median PFS was 2.5 months (95% CI 2.1–4.2) with pembrolizumab and 3.4 months (95% CI 2.2-4.3) with chemotherapy, HR 1.06 (95% CI 0.73–1.53), p = 0.76). In this study long-term responders to pembrolizumab were also found, again underlining the importance of understanding which patients should receive this treatment instead of chemotherapy  PROMISE-meso randomized phase III trial (NCT02991482) comparing PD-1 inhibition with pembrolizumab to institutional choice single agent CT (gemcitabine or vinorelbine) as second-line treatment failed to show superiority of PD-1 treatment . Nearly otherapy . Data frotherapy .Similar to other cancers, there might be a subgroup of MPM patients who might obtain a larger benefit from CIs, but relevant biomarkers have not been determined yet. Tumor PD-L1 IHC expression (with a cut-off of 1%) was correlated to ORR in both groups of MAPS-2 trial   but resuTo improve response rate to CIs in MPM patients, two options may be pursued. The first one is to move CIs toward the first-line setting, where the reinvigoration of the immune system may be stronger and more efficient, and to combine them with chemotherapy, similar to what happened in non-small cell lung cancer. Results of the addition of the PD-L1 inhibitor durvalumab to cisplatin and pemetrexed were presented in form of an abstract at the 2018 World Conference on Lung Cancer , showingin vivo, which is hampered by expression of PD-L1 , lymphocyte activation gene 3 (LAG3) and inducible T cell co-stimulator (ICOS) are being investigated in mesothelioma –98. Inhiof PD-L1  and thisof PD-L1 , raisingof PD-L1 .Early phase trials also assessed the combination of anti-PD1/PDL1 agents and MSLN-directed therapies (in MSLN-positive patients). After results from a pre-clinical murine lung tumor model (CT26hMeso) demonstrated anti-PD1 enhanced LADD-induced tumor response , a phaseGrowing evidence that pro-angiogenesis factors have immunosuppressive activity has led researchers to evaluate the potentially synergistic combination of antiangiogenic agents and immunotherapy also in the treatment of MPM. VEGF signaling has been shown to attenuate the immune antitumor response by either influencing lymphocyte trafficking across endothelia to the tumor or directly inducing inhibitory immune cell subsets . SeveralFinally, similarly to certain types of chemotherapy, radiotherapy can be exploited for its ability to cause immunogenic cell death (ICD), thus priming the release of damage-associated molecular patterns (DAMPs) and tumor-associated antigens (TAAs) and inducing a systemic anti-tumor immune response, that may be further enhanced by PD-1 (pembrolizumab) or PD-L1 (avelumab) blockade .Vaccines represent another way to boost the immune system activation against the tumor. Both protein, vector and cell-based vaccines have been tested in MPM.Galinpepimut-S is a WT-1 synthetic peptide vaccine made out of molecules similar to those in the WT1 protein. After a phase II trial confirmed vaccine's safety when administered in the adjuvant setting, researchers' efforts are currently directed toward the assessment of the combination of galinpepimut-S and nivolumab (NCT04040231). It has been hypothesized that the negative influence of tumor microenvironment factors on the immune response might be mitigated by nivolumab, thus providing the opportunity for the reinvigorated immune cells, specifically sensitized against WT1 by the vaccine, to invade and destroy cancerous growth deposits.+ cytotoxic T–lymphocytes and CD4+ helper T-lymphocytes resulting in a potent and specific anti-tumor response  can be used to load DCs (Dendritic cells are antigen-presenting cells that present tumor-associated antigens (TAAs) to the immune system by trafficking from tumors to lymph nodes. They are essential in priming proliferation and activation of CD8response . Dendritresponse , 106. Hoload DCs . Becauseload DCs , 109, anload DCs . Resultsload DCs . A largeload DCs .Another promising cell-based strategy in mesothelioma is represented by adoptive T cell therapy. Data from a phase I trial investigating chimeric antigen receptor (CAR) T cell therapy targeted to the MSLN protein in 19 MPM patients progressed following standard platinum-based chemotherapy were recently reported . A singlOncolytic viral therapy represented in the last decades an emerging field of immunotherapy and a promising experimental strategy. Viruses can act by infecting cancer cells and leading to cell lysis after replication. This renders tumor-associated and viral antigens recognizable to the immune system, thus triggering antitumor immune responses (viroimmunotherapy) , 115. Onin situ”  with celecoxib (to reduce the number of immunosuppressive MDSCs) before chemotherapy, was well tolerated and appeared to improve overall survival rates , which provide access to direct intratumoral injection of virus, make MPM an ideal candidate for assessing the efficacy of oncolysis . The safal rates . Combinaal rates –120, sucIn the past two decades there was limited success in the development of novel therapies for MPM. Multiple biases in the design of clinical trials and the peculiar biological features of MPM were most probably responsible for delaying the discovery of effective therapeutic agents. Most of the previous trials attempted to readapt drugs that succeeded in other cancer types to MPM. However, they were either too small or not stratified for predictive biomarkers. Results from phase II studies were often not replicated in larger, randomized, phase III trials, pointing out that well controlled trials with appropriate size and duration are crucial to confirm the efficacy of a new agent .In the last few years, mesothelioma genetics, epigenetics, and the tumor microenvironment  have been studied more deeply and this knowledge has started to be properly applied to discover new therapies. In particular, expectations are now high that CIs and other immunotherapies will have a leading role in the future therapeutic armamentarium of MPM. Noteworthy, scientific evidence supporting the use of CIs in MPM are still incomplete, mainly based on non-randomized studies with surrogate end-points and they have not been always replicated in the real-life context. Because of the risk of cumulative toxicities and of the high cost of these drugs , validated biomarkers are urgently needed to select MPM patients who may benefit from immunotherapies. Since the “one-size fits all” approach is not recommended for immunotherapy and MPM and the efficacy of CIs is still to be established in a larger population, there is still a need for new treatments in MPM and the implementation of other targeted agents is eagerly awaited.Only a close collaboration between medical centers and industry may lead to the conduction of well-designed, biomarker-driven clinical trials. New trials should always include translational and quality of life components, in order to clarify the molecular basis of response or progression to treatments and to finally improve the degree of reliability of the possible benefit of new therapies for MPM.LC and JA wrote the manuscript and generated the figure and table. RH and DS contributed to the revisions of the manuscript. All authors approved the manuscript for publication.JA: No relationship to disclose in relation to the submitted work. Relevant financial activities outside the submitted work: Stock or Other Ownership: Amphera Consulting or Advisory Role: Bristol-Myers Squibb, MSD Oncology, Boehringer Ingelheim, Eli-Lilly, Roche Speakers Bureau: AstraZeneca. Research Funding: Genentech (Inst), Boehirnger Ingelheim (inst). Patents, Royalties, Other Intellectual Property: Patent: Tumor cell lysate for dendritic cell loading (Inst), SNP analyses for immunotherapy (Inst). The remaining 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.


A key element in containing the spread of the SARS-CoV-2 infection is quality diagnostics which is affected by several factors. We now report the comparative performance of five real-time diagnostic assays. Nasopharyngeal swab samples were obtained from persons seeking a diagnosis for SARS-CoV-2 infection in Lagos, Nigeria. The comparison was performed on the same negative, low, and high-positive sample set, with viral RNA extracted using the Qiagen Viral RNA Kit. All five assays are one-step reverse transcriptase real-time PCR assays. Testing was done according to each assay’s manufacturer instructions for use using real-time PCR platforms. 63 samples were tested using the five qPCR assays, comprising of 15 negative samples, 15 positive samples , and 33 samples with Tib MolBiol E-gene Ct value ranging from 36–41. All assays detected all high positive samples correctly. Three assays correctly identified all negative samples while two assays each failed to correctly identify one different negative sample. The consistent detection of positive samples at different Ct/Cq values gives an indication of when to repeat testing and/or establish more stringent in-house cut-off value. The varied performance of different diagnostic assays, mostly with emergency use approvals, for a novel virus is expected. Comparative assays’ performance reported may guide laboratories to determine both their repeat testing Ct/Cq range and/or cut-off value. The novel coronavirus, SARS-CoV-2 became a pandemic in the year 2020, and many nations shut down to contain its spread. A key element in containing its spread is to identify infected people, trace their contacts, and have them quarantined in treatment and isolation centers, or through self-isolation, especially if they are asymptomatic.Daily the world is gathering more information about SARS-CoV-2, and many diagnostic assays have been developed to identify those infected. These assays have varied performance in correctly identifying people infected and those not infected. Current knowledge shows the variability between released SARS-CoV-2 genomes is small, being >99% related, which would be an aberration as RNA-viruses are often prone to mutations ,2. FurthMost real-time PCR assays for detecting new infections use a variety of sample types, mostly nasal and oropharyngeal swabs. Across the world, various diagnostic assays have been developed, and over 200 different assays were submitted to the Foundation for Innovative New Diagnostics (FIND) for independent evaluation and comparison with the Tib MolBiol assays as the reference . PrelimiThe BGI assay uses ten μl of sample RNA to detects the SARS-CoV-2 ORF1 region, with an internal control detecting a human housekeeping gene . If deteThe Da An Gene assay detects two SARS-CoV-2 targets  and an i.58 copies /μL with ≥ 95% confidence [.72–0.91% [.98–8.53% [The Genesig assay detects the SARS-CoV-2 RdRP gene  using eiThe Liferiver real-time multiplex RT-PCR assay uses five μl of sample RNA to detect three SARS-CoV-2 genes, namely the ORF1ab, the N-gene and the E-gene . It furtThe Tib MolBiol method consists of two different assays detecting first the E-gene and subsequently the RdRP-gene for those positive on the first assay. The assay can use five–ten μl of sample RNA. The E-gene detects a 76bp fragment of SARS and SARS-CoV-2 E-gene . The manAs an ISO 15189:2012 accredited facility and a World Health Organization Prequalification testing facility, we are concerned with the establishment and maintenance of quality standards in laboratory workflows. We now report the findings from testing the same sample panel, obtained from persons presenting for diagnosis of SARS-CoV-2 infection in Lagos, Nigeria, on five different diagnostic assays with recommendations to assure high quality output.This retrospective study compared five real-time one-step reverse transcriptase PCR assays for detecting SARS-CoV-2. All assays were performed according to the manufacturers’ instructions.The Nigerian Institute of Medical Research (NIMR) established a drive-through test centre for SARS-CoV-2 in March 2020, where suspected persons visited for testing. This assay comparison was performed at its ISO 15189:2012 accredited Centre for Human Virology and Genomics between April–May 2020. This Centre is also a World Health Organization Prequalification evaluating laboratory. In a collaboration with China CDC, real-time PCR test kits made by BGI were received. After, the integration of NIMR into the Nigeria Centre for Disease Control (NCDC) testing network in March, two other real-time PCR test kits namely the Tib MolBiol MDx and Da An Gene assay were further received. Subsequently, a donation of the Genesig and Liferiver real-time PCR diagnostic assays was received. These five assays were used to test samples to determine their comparative performance. Results for clinical diagnosis used for the management of the clients were given out based on the kit in routine use at the time and its manufacturer’s instructions for use.Samples received at the laboratory for SARS-CoV-2 diagnosis were utilised in this analysis. These samples were from people living in Lagos, Nigeria, who were seeking a diagnosis for SARS-CoV-2 during the peak of the pandemic, from the last week of March 2020 to the second week of April 2020. People coming to the Drive-through/Walk-in testing Centre for diagnosis were mostly asymptomatic, though very few symptomatic elderly persons were attended to. The population varied with the pandemic timeline, first being mostly travellers and their contacts, then expanding to health workers and people with fever or cough as community transmission began. As samples were deidentified prior to use, we do not have access to specific patient data. These samples included those negative and positive for SARS-CoV-2, especially the presumed low positives samples often on the borderlines of the cut-off CT values of the various assays. The negative and positive samples were determined using the Tib MolBiol assay, while the borderline samples were beyond the Tib MolBiol E-gene assay cut-off but determined as positive on one or more other assays.The Centre had the unique privilege of having five different diagnostic methods for SARS-CoV-2. It utilised these assays to determine their comparative performance on samples taken from people in Nigeria who were being assessed for possible infection with the virus.The QuantStudio 3 (Thermofisher), the CFX Connect (Bio-Rad) and the CFX96 Deep Well (Bio-Rad) were the real-time PCR platforms utilised for the laboratory analysis. The study was started with the QuantStudio 3 and expanded to utilising the CFX platforms as the pressure for testing increased..2 mL PCR tubes in volumes suitable for single-use during laboratory analysis to eliminate contamination and degradation of the control materials through frequent freeze-thaw cycles. Similarly, on opening each assay box, where necessary depending on the run size, an aliquot was made and were stored at -20°C. A template specifying the qPCR run conditions for each assay was developed for the three qPCR platforms. Following the manufacturer’s instruction and wherever acceptable, the baseline threshold setting was automated and verified during the review of each run. The automation of threshold setting minimised variation in assay parameters that could affect the Ct/Cq values. The blank/negative and positive controls of each run must meet the assay requirements before a run is accepted as valid.For quality control and quality assurance, negative/blank and positive controls for each assay were aliquoted into 0RNA from the samples used for testing were extracted using the Qiagen Viral RNA Kit . According to the manufacturer’s instruction, 140 μL of the viral transport medium containing the nasopharyngeal swabs were used for RNA extraction and eluted in 60 μL of AVE buffer. These were stored at -20°C in between same-day use and at -80°C for long period storage at the laboratory. Test determinations were first performed for the BGI, Da An and Tib MolBiol qPCR assays in April 2020 using freshly extracted and characterised samples. Testing with the Genesig and Liferiver qPCR assays were done in May 2020 using the well-preserved RNAs samples. The real-time PCR analysis were all done according to the manufacturer’s instruction.Data analysis to obtain sensitivity, specificity and accuracy were performed using MEDCALC® easy-to-use statistical software free online Diagnostic test version .Ethical approval was obtained from the Institutional Review Board of the Nigerian Institute of Medical Research for the Drive-through/Walk-in COVID-19 testing Centre, to cover collecting samples for diagnostic and research purposes (IRB/20/024). In addition, the Nigerian Institute of Medical Research set-up a website for persons seeking diagnosis to forestall the gathering of large crowds. They fill the form online before being invited for sampling and testing, at a specified date and time. All were informed that as a research institution, their de-identified samples may be used for research purposes and they gave their consent willingly. Routinely, samples are coded, and these codes replace any personal identifiers within the testing workflow. We utilised the leftover samples of people who gave consent for the use of their deidentified samples for research..910 and 37.230 on the ORF1ab and N-gene, respectively. However, repeat testing by Da An Gene assay for this one sample gave a negative result. Similarly, the Liferiver assay repeatedly detected another sample as positive for N-gene (Cts = 36.707/38.406) and E-gene (Cts = 36.766/37.991). Thus, one sample here had an inconclusive test result from the Liferiver assay.A total of 63 samples were tested using the five qPCR assays. Of the fifteen negative samples, the BGI and the Genesig assays determined all as negatives. The Da An Gene assay determined 14/15 samples were negative while one was positive with CT values of 39.3–31.4, all five qPCR assays were concordant on these samples being positive for SARS-CoV-2. The sensitivity, specificity, and accuracy of the five assays were determined using the negative and high positive samples, as shown in Of the fifteen high positive samples with Ct values of 23.0 for positive samples, revised down from the initial 39.0 (10). Thus, most samples herein presumed to be low positives would have been declared negative for the SARS-CoV-2.For the samples presumed to be low positive samples (n = 33), there were variable results on the different assays . The MDx.0–36.9 on Tib MolBiol’s E-gene assay as positive for the SARS-CoV-2, including full positive status on assays detecting multiple genes. One sample was exhausted and could not be tested by Genesig and Liferiver assays  were all positive on the other four assays. In addition, the two multiplex assays often detected one (Da An Gene assay) or two (Liferiver assay) genes consistently . This kind of error could be prevented by empirically determining an in-house cut-off, often more stringent than the manufacturer recommended cut-off. Liferiver assay picked only the N-gene and E-gene for another negative sample, without the critical ORF1ab gene detected, thus the test was inconclusive. In addition, the study sample size is small, and our estimation of sensitivity, specificity and accuracy could be impacted as shown by the wide 95% confidence intervals. Though not statistically significant, it appears the ORF1 target was detected more than the N-gene or E-gene targets (All assays were sensitive to the high positive samples, correctly identifying them, but performance was varied for samples of high Ct values and probably low viral copies. Three of the assays correctly identified all the SARS-CoV-2 negative samples. However, two of the assays identified one different negative sample as positive or inconclusive. One negative sample had Ct values close to the Da An assay cut-off (39 targets ..0, where samples with Ct < 36.0 are considered positive and eligible to further test with the RdRP assay. This study shows that 52% of these samples with Ct > 36.0 and considered negative, were determined as concordant positive for SARS-CoV-2 by the other four assays. If these are genuinely positive, a lot of false negatives would have been left in the population, which may fuel the spread of the virus.Given that this virus is novel now, the varied performance of the assays, especially on supposedly low-positive samples, is expected as the dynamics of the pathogenesis of SARS-CoV-2 in different population had not been fully elucidated. The Tib MolBiol assay used as the comparator has a revised cut-off of 36.0. In the evaluation by FIND [.43 for the lowest sample dilution was the least Ct recorded for that concentration by all the assays evaluated. Our study further demonstrated that samples with Ct above 33.0 on BGI tend to have a variable performance on the other assays. Thus, in-house optimization of the assays and where possible RNA sample volumes could guide in its use for detecting all categories of samples. This is imperative since different real-time PCR assays are in use globally.The Da An, Liferiver and Tib MolBiol assays utilise 5 μL of sample RNA, while Genesig and BGI use 8 μL and 10 μL of sample RNA, respectively. As viral copies may be low in these borderline positive samples, the higher volume of the sample taken may be instrumental in the ability of the Genesig, and BGI assays to detect more low positive samples. Thus, in-house optimization of assay RNA sample volumes could guide in its use for detecting all categories of samples. This is important given the high turnover in diagnostic assays for SARS-CoV-2 at the time of this study. Besides, the BGI assay seems well optimised as the Ct values are often much lower than their cut-off of 38A lot of issues can affect PCR assay performance and thus, this study has some limitations . As the The SARS-CoV-2 is novel; hence, diagnostic assays will have variable performance, especially on viral strains from different regions and more so those detecting multiple targets. One key factor to hold onto is consistency. It is imperative therefore that facilities empirically define a cut-off to accept samples as positive, and a range to repeat testing to confirm a positive signal is consistent.


Immune attacks are key issues for cell transplantation. To assess the safety and the immune reactions after iPS cells-derived retinal pigment epithelium (iPS-RPE) transplantation, we transplanted HLA homozygote iPS-RPE cells established at an iPS bank in HLA-matched patients with exudative age-related macular degeneration. In addition, local steroids without immunosuppressive medications were administered. We monitored immune rejections by routine ocular examinations as well as by lymphocytes-graft cells immune reaction (LGIR) tests using graft RPE and the patient’s blood cells. In all five of the cases that underwent iPS-RPE transplantation, the presence of graft cells was indicated by clumps or an area of increased pigmentation at 6 months, which became stable with no further abnormal growth in the graft during the 1-year observation period. Adverse events observed included corneal erosion, epiretinal membrane, retinal edema due to epiretinal membrane, elevated intraocular pressure, endophthalmitis, and mild immune rejection in the eye. In the one case exhibiting positive LGIR tests along with a slight fluid recurrence, we administrated local steroid therapy that subsequently resolved the suspected immune attacks. Although the cell delivery strategy must be further optimized, the present results suggest that it is possible to achieve stable survival and safety of iPS-RPE cell transplantation for a year. Immune cells are capable of discriminating between self and non-self cells by recognizing major histocompatibility complex (MHC) antigens that are expressed on the cell surface. In humans, these cell surface antigens indicate human MHC (=human leukocyte antigens (HLA)). When immune cells encounter MHC molecules they do not recognize, an immune response is triggered in order to get to rid the body of the foreign entity. Although this mechanism is important for protecting against antigens from pathogens, it can be a hindrance when performing cell transplantations from one individual to another. Recently, we experimentally demonstrated that immune responses could be avoided when retinal pigment epithelial (RPE) cells generated from iPS cells derived from an MHC homozygous donor were transplanted to a recipient with matched MHC ,2,3. UsiGlobally, age-related macular degeneration (AMD) is the leading cause of blindness in elderly populations ,5,6,7. SClinical trials in AMD patients or animal experiments using allogeneic RPE cell grafts have previously failed, which indicates that the grafts were not able to survive because of immune attacks ,11,12,13To be eligible for the study (inclusion criteria), subjects had to be HLA-matched patients with wet AMD who had previously exhibited a limited effect after anti-VEGF injections . Table SIn 2017, we announced the start of a new clinical research study (UMIN-CTR number: UMIN000026003), which included plans to use the iPS bank established at the CiRA at Kyoto University to derive RPE cells for allogeneic transplantations. In the present study, we have started to use allografts of iPS-RPE cells from an HLA homozygous donor for AMD patients who have the same HLA haplotype identity (=HLA matched).After reviews by the Institutional Review Boards and Ethics Committees at the collaborating sites, our protocol was approved by the Certified Special Committee for Regenerative Medicine and by the Ministry of Health, Labour, and Welfare (Japan). After informed consent was obtained from all of the AMD patients, the study was conducted in accordance with the tenets of the Declaration of Helsinki. All experiments that involved the use of samples obtained from humans (and which included patients) and all animal studies were approved by the Institutional Review Board at the Kobe City Medical Center General Hospital and RIKEN BDR. The authors in this study vouch for the completeness and accuracy of the data and analyses and for the fidelity of the study to the approved protocol.The primary objective of this study was the safety together with the feasibility of the transplantation surgery with iPS-RPE cells. Therefore, in order to achieve this objective, the primary endpoint was classified as the incidence and severity of the following: adverse events (AEs) associated with iPS-RPE cells a graft source and AEs associated with the transplantation procedure of the iPS-RPE cell suspensions. The AEs associated with iPS-RPE cells a graft source focus on: (1) Transplanted cell graft failure or immune rejection  fundoscopy does not show healthy transplanted cells; b) optical coherence tomography (OCT) shows increased retinal edema; and c) OCT shows increased subretinal fluid; and (2) excess cell proliferation or tumor formation . The AEs associated with the transplantation procedure of the iPS-RPE cell suspensions focus on (1) retinal/choroidal/vitreous hemorrhage, and (2) retinal detachment.The secondary objectives were to evaluate the safety of the transplantation surgery with iPS-RPE cell except for the aforementioned primary endpoint, and to evaluate the efficacy for visual function. Therefore, the secondary safety endpoints included the type, incidence, and severity of AEs associated with each of the iPS-RPE cells and the transplantation procedure except for the primary endpoint, and all other AEs, which were assessed by CTCAEv4.0 JCOG.The secondary efficacy endpoints included the following contents that evaluated at 12, 24, and 52 weeks after transplantation: OCT-based foveal retinal thickness, subretinal fluid, and retinal edema , retinal sensitivity as determined by multifocal electroretinogram (ERG) and microperimetry , visual acuity , dye leakage from CNV by fluorescence fundus angiography , time to need for anti-VEGF treatment because of CNV recurrence and number of anti-VEGF treatment doses given during the 1-year period after transplantation , and change in QOL (assess this according to changes in VFQ-25 scores).5 iPS-RPE cells into50 μL HLCR011 . Transplantation surgery was performed under local sub-Tenon’s anesthesia or general anesthesia. The surgical protocol included pars plana vitrectomy and the creation of a small bleb by injecting balanced saline solution into the subretinal space around the lesion followed by the subretinal transplantation of allogeneic iPS-RPE cells. The RPE cell suspension was loaded on a PolyTip® cannula 25g/38g  with a 0.2-mL syringe installed in a custom-made fluid injector WRRK-02 , which allowed for a gentle discharge of a precise amount of cell suspension into the subretinal space  software package operated in the R language.The methods for the HLA genotyping in this clinical study have been previously published . A perip+ helper T cells, CD8+ cytotoxic T cells, CD11b+ monocytes/macrophages, CD19+ B cells, and CD56+ NK cells using Ki-67 FACS analysis. In addition, we also measured IFN-γ in the supernatants of the co-cultures. We evaluated results of these six contents  in PBMC co-cultured with RPE or B cells (PC) and then compared the results with the data for PBMC only – LGIR positive = > 3 positive, LGIR suspected positive = 2 positive, LGIR negative = 1 or 0 positive. The specific details are presented in the methods [Results of the LGIR tests were used to diagnose the rejection after transplantation. We first collected blood cells from the AMD patient, and then separated the peripheral blood mononuclear cells (PBMC). Subsequently we added iPS-RPE cells or allogeneic B cells as a positive control (PC), and then evaluated the immune reactions by Ki-67 FACS and IFN-γ ELISA. Both target cells were irradiated 20 Gy) before the assay. As compared to the control data (PBMC only), the patient’s lymphocytes exhibited a significant reaction against transplanted iPS-RPE cells in vitro . This was considered to be “positive” for the immune rejection. We used the LGIR tests, as this made it possible to find which of the cells actually proliferate in PBMC after co-cultures. We monitored CD40 Gy befoIn order to determine the immune attacks after transplantation, we also performed an assay for detecting RPE-specific antibody (RSA) using transplanted iPS-RPE cells and the patient’s serum before or after surgery. After immunohistochemistry (IHC), we evaluated the RPE staining by serum or antibody using confocal microscopy or by measurements of mean fluorescence intensity. We previously reported the details of these measurements in our methods .The study was designed to primarily evaluate the safety of the transplantation surgery with iPS-RPE cells in a small number of patients. Accordingly, the sample size was not determined to allow the efficacy to be evaluated with statistically sufficient precision or power, and instead a sample size of five patients was targeted. The analysis population included all the participants who had received at least part of the transplantation surgery with iPS-RPE cell. In principle, the data are shown as observed for each of these participants or summarized with the use of descriptive statistics, with no imputation of missing data. Further details are provided in the protocol and statistical analysis plan.To protect patient privacy and comply with relevant regulations, identified data are unavailable. Requests from qualified researchers with appropriate ethics board approvals and relevant data use agreements for de-identified data will be processed by the Kobe City Medical Center General Hospital. To request access please contact the office by email: shigeki_yamasaki@kcho.jpHLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, and HLA-DPB1). We initially established iPSCs using non-integrating episomal vectors [In this study, we used iPS-RPE cells from an HLA homozygous donor . As seen in A*24:02, B*52:01, C*12:02, DRB1*15:02, DQB1*06:01, and DPB1*09:01  from a HLA homozygous donor that contained the following six HLA gene loci: B1*09:01 E. The esB1*09:01 . iPS-RPEn = 5, age 64–80 years), and all patients were HLA haplotype identity that matched the above HLA homozygote iPS-RPE cells  in our hospital [A*24:02-B*52:01-C*12:02-DRB1*15:02-DQB1*06:01-DPB1*09:01) in the AMD patients. After informed consent was obtained for the present patients, we enrolled the wet AMD patients who had the same HLA haplotype identity in this study.A typical subject in the present clinical study was a wet AMD patient with an atrophic RPE lesion over the CNV with periodic recurrences while undergoing anti-VEGF treatment, and whose neural retina was at risk of degeneration G. As seePE cells . In facthospital , there w5 cells, single cell suspensions) in the subretinal space, this was followed by a one-shot intravitreous injection of anti-VEGF drug (intravitreous injection of aflibercept: IVA) and local steroid (intravitreous injection of triamcinolone acetonide: IVTA)  H. All thThe primary endpoint of this study was the safety of the investigational treatment, i.e., the presence of any adverse events (AEs) attributable to iPS-RPE transplantation therapy as listed in our protocol. All the registered patients completed a 52-week follow-up after transplantation, and during this period, we observed no case with AEs associated with either 1) iPS-RPE cells such as poor engraftment of transplanted cells, immune rejection, excessive cell proliferation, and tumorigenicity (0/5 cases: 0.0%), or 2) surgical procedure such as retinal/choroidal/vitreous hemorrhage or retinal detachment (0/5 cases: 0.0%).other AEs because these events were not conclusively attributed to the presence of graft cells. We had one case with retinal edema associated with epiretinal membrane (ERM) , visual acuity (BCVA), dye leakage from CNV by FA, time to need for anti-VEGF treatment, and change in QOL (VFQ-25 scores). ERM developed in all cases. However, even though the eye in Case 2 developed diffuse edema that did not respond to either IVA or IVTA at 6 months, there was no worsening of the BCVA in this patient. There was rapid resolution of the edema after the ERM removal surgery at 7 months after the transplantation . AlthougIn the same eye of Case 2, the subretinal iPS-RPE cells were also observed to be stable, which was quantitatively assessed as a decrease in the size of the window defect (WD) that was evaluated on the binary images constructed from the early phase fluorescein angiography (FA) . First, As to the immune responses, which was a major concern for this clinical trial, the mild clinical sign of rejection that was observed in only one case consisted of a subtle change in the subretinal fluid over the graft cells at 5 weeks after the transplantation. However, FA indicated that there was no evidence of leakage at 1 month Case 1, .Since the recurrence of AMD and graft rejection often share very similar clinical features, laboratory monitoring provided important clues in the detection of immune rejections in this clinical study. The laboratory tests that were performed included LGIR tests using graft RPE cells and blood cells, peripheral blood mononuclear cells (PBMC), and RPE-specific antibody (RSA) tests that were used to detect alloreactive antibody in the serum. The specific details for the methods for both examinations have been previously reported ,3. The r+/Ki-67+ (proliferative helper T cells) and CD11b+/Ki-67+ (proliferative monocytes) were evaluated using flow cytometry. As seen in + T cells and CD11b+ monocytes in PBMC at 4 weeks after being exposed to graft-equivalent iPS-RPE cells (PBMC plus RPE). Although color fundus   J. In add1 weeks) .+/Ki-67+ double-positive cells at 12 weeks after surgery, we did not find any ocular inflammatory signs including at the graft site. After the STTA, these double-positive cells gradually decreased. In Cases 3 and 4, we observed no inflammatory signs in the eye, and the LGIR tests were also negative during the 1-year evaluation   , the patRecently, several clinical studies using RPE cell suspensions or sheets prepared from embryonic stem (ES) cells that have been conducted in AMD patients have proven the safety of these treatments ,28,29,30n = 105, mean age, 76.0 ± 7.8 years) in our hospital, there was an 18.8% HLA haplotype identity that matched the six HLA gene loci (A*24:02-B*52:01-C*12:02-DRB1*15:02-DQB1*06:01-DPB1*09:01) in the AMD patients [In Japan, we can cover around 80% of Japanese patients if we can find 70 donors of the HLA-3 locus homozygote  . In factpatients . After iBased on these preclinical studies, the major aim of the present study was to see if we could achieve successful iPS-RPE transplantation without having to use a systemic immune suppressant for the wet AMD patients whose MHC (HLA) matched with the donor iPS cells (HLA-class I and class II matched). Moreover, the eye is also known to be a unique organ with a unique environment that has both anti-inflammatory and immunosuppressive mechanisms ,17. ThusIn the present study, we performed two immunological examinations using the patient’s blood: (1) LGIR tests in PBMC, and (2) detection of RPE-specific antibody (RSA tests) in the serum. The recurrence of AMD and immune rejections often share the same ocular findings, such as subretinal fluids and retinal edema. In this clinical trial, we determined that the LGIR test was a key for diagnosing immune rejections, and thus, when we subsequently found the LGIR tests to be positive in Case 1, treatment of this patient by local steroid injection proved to be immediately effective. More importantly, the LGIR tests at 4 weeks were positive prior to the ocular immune rejection signs in which OCT showed there was a slight accumulation of subretinal fluid over the grafted RPE cells at 5 weeks see . AlthougThe present clinical trial also uncovered a couple of other issues that will require further elucidation in the future. One of these issues involves the dispersion and backflow of graft cells into the vitreous space, which was considered to be a cause of epiretinal membrane (ERM) in all cases. Although ERM is generally not a serious complication and is indeed removable when it causes complications such as persistent edema, it is still better that patients not to have to undergo any extra surgeries. Therefore, we need to optimize the surgical method that is used to deliver graft cells in the targeted area with minimal dispersion. Another issue involves the elevation of the intraocular pressure (IOP), especially in a steroid responder. Cases 2, 3, and 4 all exhibited an elevated IOP that was eventually controlled with glaucoma medication and/or by changing the use of the steroid eye drops.We confirmed the safety of the HLA-matched allogeneic iPS cells-derived retinal cell transplantation for a period of at least one year. We were able to control the immune attacks using HLA homozygote iPS-RPE cells and local steroids in transplanted wet AMD patients. In all cases, RPE grafts survived during a 1-year observation period. LGIR tests using patient’s blood cells and transplanted RPE cells proved to be useful for the rapid diagnosis of immune attacks. The recurrences of the polyp lesions during the 1-year follow-up period were observed after transplantation in many of these patients. In this study, the grafted cell location was not sufficiently controlled to access the efficacy of this therapeutic approach. We need to further optimize the surgical procedure for controlled cell delivery to evaluate the suitable cases for the human iPS-RPE cells transplantation in the future studies.


The presence of co-morbidities, including underlying respiratory problems, has been identified as a risk factor for severe COVID-19 disease. Information on the clinical course of SARS-CoV-2 infection in children with cystic fibrosis (CF) is limited, yet vital to provide accurate advice for children with CF, their families, caregivers and clinical teams.Cases of SARS-CoV-2 infection in children with CF aged less than 18 years were collated by the CF Registry Global Harmonization Group across 13 countries between 1 February and 7 August 2020.Data on 105 children were collated and analysed. Median age of cases was ten years (interquartile range 6–15), 54% were male and median percentage predicted forced expiratory volume in one second was 94% (interquartile range 79–104). The majority (71%) of children were managed in the community during their COVID-19 illness. Out of 24 children admitted to hospital, six required supplementary oxygen and two non-invasive ventilation. Around half were prescribed antibiotics, five children received antiviral treatments, four azithromycin and one additional corticosteroids. Children that were hospitalised had lower lung function and reduced body mass index Z-scores. One child died six weeks after testing positive for SARS-CoV-2 following a deterioration that was not attributed to COVID-19 disease.SARS-CoV-2 infection in children with CF is usually associated with a mild illness in those who do not have pre-existing severe lung disease. There are two individual case reports in children with varying levels of pre-existing lung disease Increased understanding about SARS-CoV-2 infection in the paediatric age group is crucial to provide accurate and balanced advice that will keep children with CF safe, and recognise the relative risks, but also enable them to lead their lives in the most fulfilling way possible during the ongoing pandemic. This is particularly important given the associated disadvantages and substantial negative effects on quality of life, mental health, schooling and delivery of healthcare of strict isolation measures 22.1The ‘Cystic Fibrosis Registry Global Harmonization Group’ is a collaborative international group of patient registries. At the start of the COVID-19 pandemic the group set up fortnightly videoconferences to share experience. Nineteen countries participated in the study that ran between 1 February and 7 August 2020.Children were defined as individuals aged less than 18 years. Cases were included if they had a confirmed diagnosis of CF and were either diagnosed with SARS-CoV-2 infection via a polymerase chain reaction (PCR) test on a respiratory sample or a clinical diagnosis of COVID-19 was made in a hospital setting. Cases reported via antibody testing alone or self-reporting were excluded.cystic fibrosis transmembrane conductance regulator (CFTR) genotype, body mass index (BMI), best percentage predicted forced expiratory volume in one second (ppFEV1) in the 12 months prior to infection, usual medications, pre-existing CF-related complications, respiratory microbiology and transplant status.CF centres that had reported SARS-CoV-2 cases to their registry were asked to complete an anonymised data collection form and a designated country representative then collated all national cases for the study. Representatives provided the most comprehensive data available on each individual case. Data collected included details of the clinical presentation, management and outcome along with relevant patient demographics, including 2.2P values of <0•05 were considered statistically significant.The United Kingdom CF Registry team and the Children's Respiratory Group from Newcastle University performed analyses of pooled cases submitted by participating countries. Data were regarded as missing where specific information was not available at the time of data collection. Results were summarised in tables as numbers and proportions for categorical data and median values with interquartile ranges for continuous data. For categories where some data were missing, the number of cases where data were available was cited in the table. BMI Z-scores adjusted for age and sex were calculated using the World Health Organisation Child Growth Standards 2.3Each participating country was responsible for ensuring research ethics or institutional approvals were in place for this work as per their individual patient registries.2.4The funding source had no involvement in the collection, analysis or interpretation of data, writing or decision to submit for publication.3Data on 105 cases of SARS-CoV-2 infection in children with CF were analysed. From the 19 countries that make up the CF Registry Global Harmonization Group, 13 reported cases . Case numbers for individual countries have not been specified to maintain confidentiality.Details of the method of diagnosis of COVID-19 were available in 96 children. This was via a positive PCR test for SARS-CoV-2 on a respiratory sample in 95 children and by clinical diagnosis of COVID-19 in a hospital setting in one.3.1CFTR mutation. Median best ppFEV1 within 12 months prior to infection for those over the age of five years was 94%. Thirty-one had Pseudomonas aeruginosa respiratory infection and 31 were on long-term azithromycin treatment. Fifty children were on a CFTR modulator. None were on an active list awaiting transplantation and two children were post-transplant (one lung and one liver).Case demographics and features of pre-existing CF disease in the cohort are summarised in 3.2A summary of the symptomatology, management and treatment of cases is provided in Varied levels of care were required with 71% of children being managed in the community and the remainder admitted to hospital. One child was admitted to intensive care six weeks after acute SARS-CoV-2 infection. A range of treatments were used, these included oral (37%) or intravenous (46%) antibiotics with the majority of intravenous antibiotics being administered in hospital. Four children received azithromycin acutely, five antiviral medications and one additional corticosteroids. Of those who were hospitalised, six required new supplementary oxygen therapy, two required non-invasive ventilation and one invasive ventilation. None were supported with extracorporeal membrane oxygenation. No children received experimental treatments for COVID-19 other than azithromycin, interferon or corticosteroids.At the time of writing, only two cases were described as unresolved, however no formal follow up information is available at this stage. Both solid organ transplant recipients recovered from COVID-19. There were no deaths directly attributed to COVID-19. One child died approximately six weeks after testing positive for SARS-CoV-2. This child had been unwell with deteriorating health during the preceding year including several severe pulmonary exacerbations requiring intensive care admission. During the COVID-19 illness the child was treated with intravenous antibiotics, lopinavir/ritonavir and managed in a ward setting. The child recovered from that acute illness and became negative on repeat SARS-CoV-2 testing. However, six weeks later the child experienced a further severe pulmonary exacerbation requiring admission to intensive care, intubation and mechanical ventilation, and sadly died from respiratory failure.3.3CFTR genotype in each group. Although the median age was higher in children admitted to hospital, this was not statistically significant. Median BMI Z-score was lower in children admitted to hospital. No pancreatic sufficient children were admitted to hospital with COVID-19. Around half of children with CF-related diabetes were admitted to hospital. Children admitted had a lower median ppFEV1 (73%) than those managed in the community (97%). Significantly fewer children were hospitalised who were receiving CFTR modulator therapy, however, it should be noted that access to modulator drugs varied between countries.The characteristics of children admitted to hospital with COVID-19 were compared with those managed in the community in an exploratory analysis using hospitalisation as the best available surrogate of disease severity in our dataset . There w4We describe to our knowledge the first case series of SARS-CoV-2 infection in children with CF. Information was captured on 105 children across 13 countries with a spread of ages ranging from infants through to adolescents. Some of these cases were included within a predominantly adult series, where, crucially, no analysis of the paediatric age group was performed 1 in the preceding year was in the normal range at 94%. This level of lung function is comparable with the median ppFEV1 for those aged <18 years in 2019 registry reports from the United Kingdom (89.5%) and United States (95%) P. aeruginosa infection. This compares to around 25% of children aged <18 years isolating P. aeruginosa in the last 12 months in the 2019 United States CF and United Kingdom registries This is a diverse cohort from multiple countries and in-depth comparisons as to how representative the cohort is of children with CF worldwide in general are not possible. The median best ppFEVThe distribution of symptoms associated with COVID-19 was similar to those reported in other paediatric, non-CF, cohort studies Most children were treated with oral or intravenous antibiotics as is standard practice for any CF respiratory exacerbation Some studies have found that those with solid organ transplants are at an increased risk of severe COVID-19 We compared the characteristics of children admitted to hospital with those managed in the community as the best available surrogate of COVID-19 disease severity in our dataset. Children admitted to hospital had lower lung function, reduced BMI Z-scores and were less likely to be maintained on a CFTR modulator. No differences in gender were found. Importantly, these comparisons were performed with caution and are exploratory due to the relatively small overall numbers, prevalence of missing data, variable access to CFTR modulators internationally and the important caveat that some children were likely to have been tested for SARS-CoV-2 in association with a hospital admission for other reasons.There was one clear exception in the cases reported. A child, who had experienced a worrying clinical trajectory over the last year with respect to their underlying CF disease, developed a severe pulmonary exacerbation six weeks after testing positive for SARS-CoV-2. The clinical team involved did not consider this final illness to be related to the SARS-CoV-2 infection or the paediatric multisystem inflammatory syndrome that has been described in temporal association with COVID-19 This dataset was collated during a global pandemic involving multiple national patient registries and there are inevitable limitations. There are missing values within the dataset partly due to variation in data collection methods between individual registries. This highlights the importance of ongoing work towards registry harmonisation to facilitate and optimise collaborative reporting A major strength of this work is that it is the only large detailed case series of COVID-19 specifically focussing on children with CF. This study provides valuable information that will guide children with CF and their families and clinical teams. In summary, our findings suggest that SARS-CoV-2 infection in children with CF is usually associated with a mild illness in those who do not have pre-existing severe lung disease. It remains important, however in our opinion, that children with CF are considered a priority for access to vaccination programmes for SARS-CoV-2 once regulatory approvals are in place. Follow up of children with CF who have experienced infection with SARS-CoV-2 will be important to assess for the development of any long term complications or impact on lung function.None related to this work.P-RB: Not related to this work: reports personal fees from Astra-Zeneca, Boehringer Ingelheim, Chiesi, GSK, Insmed, Novartis, Pfizer, grants and personal fees from Vertex, personal fees from Zambon SBC: Not related to this work: personal fees from Chiesi Pharmaceuticals, Zambon and Insmed and personal fees and non-financial support from Vertex Pharmaceuticals.CG: Not related to this work: reports grants from CF Foundation, European Commission, NIH, FDA; personal fees from Gilead Sciences (Chair of a Grant Review Committee), Novartis ; other fees Boehringer Ingelheim  and Vertex Pharmaceuticals .LN: Not related to this work: reports that he received institutional fees for site participation in clinical trials from Vertex Pharmaceuticals and Boehringer IngelheimLVRFSF Not related to this work: reports grants and personal fees from Vertex Pharmaceuticals and Boehringer Ingelheim, grants from Timpel and Diagnostics of America (DASA), personal fees from Roche do Brasil, AbbVie, Sanofi and Glenmark.MB Not related to this work: investigator-led research grants from Pfizer and Roche Diagnostics; speaker fees paid to Newcastle University from Novartis, Roche Diagnostics and TEVA. Travel expenses to educational meetings Boehringer Ingelheim and Vertex Pharmaceuticals.RB, RC, MZ, CC, HC, AF, AJ, NK, BM, MDP-V, PM-L, KGB, IJH: none


The HIT-6 was weakly correlated with PASS  and PCS . PASS-Total (p = 0.001), headache frequency (p = 0.003), and HADS-Anxiety (p = 0.028) were independent variables associated with HIT-6. Headache frequency (p < 0.001) was an independent variable associated with MIDAS. The structural equation model indicated that headache severity has direct loading on emotion and subsequently influenced migraine-related disability. Disability has a significant effect on the frequency of abortive medication use. Migraine patients have altered emotional responses to pain perception. Pain-related anxiety made an important contribution to headache-related disability. The present results suggest that the management of disability by considering various pain-related emotional factors may be necessary for the therapeutic aspects of migraine.The response to pain is highly individual and can be influenced by complex emotional perception. This study aims to investigate the status of the pain-related emotional response, and the influence on headache characteristics and disability in migraine. We studied the pain-related emotional response in 145 consecutive migraine patients using the Pain Anxiety Symptoms Scale (PASS), the Pain Catastrophizing Scale (PCS), and the Pain Sensitivity Questionnaire (PSQ) and compared them with 106 healthy controls. We investigated the relationship between emotional factors and migraine characteristics. The effect of pain-related emotion on migraine-related disability assessed with the Headache Impact Test-6 (HIT-6) and the Migraine Disability Assessment (MIDAS). Migraine patients showed significantly higher scores on total PASS ( Despite proper medical treatment, some migraine patients continue to experience disabling headaches, contributing to considerable social and economic loss2. The perception of pain and functional impairment related to migraine attacks appears to have an individual diversity, which surpasses the contributory role of objective factors associated with headache characteristics, including the frequency and severity of headache attacks3.Migraine is one of the most common headache disorders, with a worldwide prevalence ranging from 5 to 12%. The World Health Organization (WHO) has recognized migraine as an urgent public health priority and listed it as a major leading cause of disability4. As a result of inappropriate cognitive processes, patients could suffer from pain beyond the basis of physical conditions6. Enhanced pain perception or sensitivity might be a significant risk factor in the chronicity of pain4. Fear or anxiety about pain related to the excessive prediction of future pain in some patients contributes significantly to their somatic symptoms and disability8. The belief that current pain will persist or blind faith appears to have a negative impact on coping and compliance, regarding pain as mysterious and inexplicable are associated with poor outcome, psychological distress, and somatization9.The interpretation and response to pain are highly subjective and vary widely depending on the individual perception of pain10. In terms of experiencing recurrent headaches, it is theoretically plausible that the emotional properties of pain might play a role in influencing the functional outcome, including disability in migraine patients.Headache patients with a lack of self-efficacy awareness and inadequate response strategies were more prone to poor outcomes in overall disability12. The Pain Catastrophizing Scale (PCS), which mainly evaluated individuals’ tendencies to focus on the pain, and the Pain Sensitivity Questionnaire (PSQ), which assesses pain sensitivity of various pain stimulation, are also being used as clinical evaluation tools14. The present study investigates the impact of the pain-related emotional response on pain perception and disability in migraine, by using PASS, PCS, and PSQ.Several measurement tools have been proposed and widely used to evaluate pain-related emotional aspects. The Pain Anxiety Symptoms Scale (PASS) is a questionnaire to measure pain-related anxiety and physical symptoms3. The pain related-emotional factors aggravate the headache disability15. Base on these findings, we hypothesized that headache severity might aggravate the pain-related emotional factor, and pain-related emotional factors might influence headache-related disability and behavior. We set the structural equation model 18. Inclusion criteria of migraine patients were as follows: (1) age between 19 and 75 years and with a diagnosis of migraine, (2) at least two headache-days per month, and (3) stable headache characteristics for at least one year before study entry. We excluded patients if they presented any of the following: (1) secondary headache disorders, (2) any systemic medical disease associated with chronic pain , (3) any recent painful situation except headache , and (4) any psychological disease-associated anxiety or depression.Participants who met the inclusion criteria were enrolled from the neurology outpatient clinics of two hospitals. We use the smartphone headache diary system (SHD) for enrollment of migraine patients. The SHD system collects headache frequency, duration, intensity, disability, and other clinical data from migraine participantsWe recruit a healthy control group aged between 19 and 75 without a history of migraine and another headache disorder. Exclusion criteria of healthy control follows: (1) any headache or painful situation for the recent three months, (2) history of other systemic medical diseases, chronic pain disorder, and trauma (3) any psychological disease-associated anxiety or depression.Various factors, such as demographics, headache characteristics , and abortive medications were recorded. The psychological status was recorded using the Hospital Anxiety and Depression Scale (HADS). The Korean version of the Migraine Disability Assessment Scale (MIDAS) and the Korean version of Headache Impact Test-6 (HIT-6) were recorded to evaluate the baseline impact or disability related to migraine.20. The previous study reported the PASS of the clinical and nonclinical group21. Between migraine and healthy control, the calculated sample size  for the t-test was 52. For multiple regression analysis, the calculated sample size of migraine group was 142  using the previous result22.We determined the sample size by using G*power software (version 3.1.9.6)The protocol of this study was reviewed and approved by the Institutional Review Board/Ethics Committee of the Catholic University of Korea College of Medicine . The participants received a detailed explanation of the aim and procedure of the study and provided written informed consent. This study was conducted in ethical principles, as described in the Declaration of Helsinki.24. The PASS is used to measure pain-related anxiety. It consists of 20 items measured on a six-point scale from 0 (never) to 5 . There are four sub-scales with five items. These are fear of pain (FP), cognitive anxiety (CA), somatic anxiety (SA), and escape and avoidance (EA)7. We used the total score and the sum of each sub-scale as clinical outcomes. The PCS is a 13 items questionnaire to measure levels of pain catastrophizing. Each item is rated on a five-point scale, from 0  to 4 . There are three sub-scales includes Rumination (RM: 4 items), Magnification (MG: 3 items), and Helplessness (HP: 6 items)25. We used the total score of all 13 items and the sum of the scores of each sub-item as clinical outcomes. The PSQ assumes various daily pain situations and is based on a self-rating of pain severity on different body parts. It is composed of 17 items, and each item is rated on an 11-point scale, from 0  to 10 (seems like the most excruciating pain imaginable). There are two sub-scales with seven items that include Minor Pain (min) and Moderate Pain (mod)26. The PSQ-Total score was calculated as the average of the 14 items. PSQ-minor and moderate score were calculated as the average rating of seven items26.Included patients and healthy controls were requested to complete the Korean version of the PASS, PCS, and PSQ. The migraine patient group completed the questionnaire on the interictal stage without headache pain. All of the Korean versions have had their validity and reliability levels formerly verifiedp values of less than 0.05 were deemed to be statistically significant. We use a structural equation model to evaluate the relationship between headache disability, severity, emotional factors, and abortive medication use. To evaluate the fit of our model, the absolute fit index (χ2), the comparative fit index (CFI), the Tucker–Lewis index (TLI), normed fit index (NFI), and the root mean square error approximation (RMSEA) were used. All of the statistical analyses were performed using R (version 3.6.2).The total and sub-domain scores of PASS, PCS, and PSQ were compared between migraine patients and healthy control using an independent-sample t-test. In the migraine group, the Pearson correlation analysis was used to evaluate the association between PASS, PCS, PSQ, and migraine characteristics. The multiple regression analysis was performed in the migraine group to determine independent factors associated with each migraine-related disability (HIT and MIDAS). The migraine characteristics  and total score of pain-related emotional factors  are used as independent variables and stepwise selection methods used in multiple regression analysis. These analyses were correlated in order to ensure that multicollinearity did not exist. For this study, we enrolled 163 migraine participants. 18 individuals withdrew from the study due to the low frequency of headache attack, another painful situation, and an incomplete questionnaire. Finally, 145 migraine participants and 106 healthy control completed the study Fig. .Figure 2The mean age of the migraine group was 38.9 ± 10.1 years, and 125 participants (86.2%) were women. The mean age of healthy control 39.5 ± 12.2 years, and 82 participants (74.5%) were women. There were no statistically significant differences between the groups for all demographic variables (Table p < 0.001). In sub-domain analysis, PASS-FP , PASS-CA , PASS-SA , and PASS-EA  were all significantly higher than the control group.The score of the PASS-Total in migraine patients was significantly higher than the control group . In sub-domain analysis, PCS-RM , PCS-MG  and PCS-HP  were all significantly higher than the control group.The score of PCS-Total in migraine patients was significantly higher than the control group . In sub-domain analysis, both PSQ-min  and PSQ-mod  were significantly higher than the control group , PASS-FP , PASS-CA , and PCS-HP . The frequency of abortive medication usage was associated with PASS-SA , PCS-HP , and PSQ-min . The duration of migraine illness was associated with PCS-HP .Headache pain intensity measured by the visual analogue scale (VAS). Headache pain intensity showed a weak correlation to PASS-Total score , PASS-FP , PASS-CA , PASS-SA , PASS-EA , PCS-Total , PCS-RM , PCS-MG , and PCS-HP . The MIDAS showed a weak correlation with PASS-Total , PASS-FP , PASS-CA , PASS-SA , and PCS-HP . The headache characteristics of migraine patients and Pearson correlation results are summarized in Tables Migraine-related disability measured by HIT-6 and MIDAS. The HIT-6 showed a weak correlation with the PASS-Total score , HADS-A (p = 0.028), and headache frequency (p = 0.003) were independently associated with HIT-6. In MIDAS, headache frequency (p < 0.001) was identified as independent variables . Headache severity has significant direct loading  on pain-related emotion. Pain-related emotion significantly predicts severe headache disability . Headache disability was associated with abortive medication use . Pain-related emotion does not have a significant effect on abortive medication use .Results of the structural equation model are showed with standardized path estimates Fig. . Model fThe major outcomes of the present study were as follows. PASS, PCS, and PSQ scores of migraine patients were significantly higher than those of the control group. PASS and PCS were significantly associated with headache pain intensity, abortive medication use, and headache disability. PASS is one of the important factors contributing to the development of headache-related disability measured by HIT-6 and MIDAS. The structural equation model shows that headache severity leads to a change in the pain-related emotional state. The Pain-related emotion has significant direct loading on headache disability, which is consequently related to rescue usage.28. The emotional and psychological problems could exacerbate a headache in migraine30. The neuronal network for pain and emotion are overlapped and have a mutual influence on each other32. The periaqueductal gray (PAG) and rostral ventromedial medullar play an important role in migraine pathogenesis and are closely related to various parts of the brain, including the amygdala, hippocampus, orbitofrontal cortex, and insula, which are responsible for pain-related cognition and emotion34. These structural connections indicate that psychological factors may affect pain modulation in migraine patients.Anxiety or fear for predicting pain can cause different coping strategies, resulting in various perceived pain behaviors. A negative expectation can reverse the analgesic effect, whereas the expectation of pain relief is an important factor for the analgesic effect of a placebo35. The PCS score related to fear of pain, frequency, chronicity, and headache severity in pediatric headache and obese women with migraine36. In the headache patients, anxiety sensitivity associated with fear of pain and escape or avoidance3.The emotional contributions for pain were reported in various chronic pain disorders, including back pain, cancer pain, rheumatoid arthritis, and tissue injury by using PASS, PCS, and PSQ21. Other studies set the cut-off value of total PASS score: mild (0–33), moderate (34–67), and severe pain anxiety (68–100)37. The present study showed that migraine sufferers have a range of moderate to severe pain-related anxiety, according to the PASS score (47.4 ± 17.2). It is known that anxiety is significantly associated with an increased risk of migraine38. The PCS is a tool for evaluating catastrophizing chronic pain disorders. A total PCS score over 30 is associated with clinically significant catastrophizing14. Previous studies reported that the catastrophized group showed more emotional distress and severe pain intensities than the groups of the normal and chronic pain group without catastrophizing40. In our study, the migraine patients had a higher PCS score (24.4 ± 14.1) than the control group, even though it did not reach a clinically significant catastrophizing level. PSQ evaluates the pain perception based on the imagined painful daily situation. The PSQ-min and total scores were significantly higher than the control in chronic pain diseases such as chronic tension-type headache, chronic low back pain, chronic mandibular pain, and chronic mixed pain disorder13. Moreover, PSQ related to headache pain intensity, catastrophizing, and central sensitization41. In the present study, the migraine patient group had a higher PSQ score than the control, similar to previous studies41.The PASS score used to evaluate pain-related anxiety, and the score over 30 suggests the problematic pain-related anxiety in young adults without current and chronic painWe consider the possibility that migraine patients exhibited an altered emotional and response to a pain similar to other chronic pain disorders.44. Pain severity was associated with PASS-FP, CA, and SA in chronic back pain43. Other studies reported that all PASS subdomains were associated with pain severity in heterogeneous chronic pain patients42. In headache patients, headache severity had direct loading on all PASS subdomains3. Interestingly, the present study suggested that PASS-SA was related to the behavioral consequence of taking abortive medication for headache attacks. PASS-SA assessed symptoms reflecting physiological arousal associated with the experience of pain. At the start of the study, we assumed that PASS-EA was probably associated with abortive medication use because PASS-EA contains an item to take medication to avoid pain. However, many other items of PASS-EA also include directions to reduce or stabilize activity to avoid pain, which may dilute the possibility of medication use. From the present study, we can assume that abortive medication could have priority when a physical discomfort is noticeable in migraine patients.The correlation analysis showed that pain-related anxiety, especially FP and CA, weakly correlated to headache pain intensity in migraine patients. PASS-FP was intended to measure fearful thoughts related to the experience of pain or the anticipated negative consequences of pain. PASS-CA was to assess cognitive symptoms associated with the experience of pain, such as racing thoughts or impaired concentration. Although the results vary depending on the study, subdomains of PASS related to the severity of pain are reported36. As migraine becomes severe and more frequent, it could be considered that the feeling of helplessness may increase when in pain. The present study showed significant but weak correlations between pain-related emotion and migraine characteristics. Therefore, a careful approach is needed to interpret our correlation analysis results.The PCS-HP weakly correlates with headache duration, headache pain intensity, and abortive medication use, which were similar to in a previous study suggesting the relationship between headache pain intensity, duration, and frequency in obese women with migraine43. McCracken et al. reported that the PASS score showed a significant correlation and is predictive of disability in various chronic pain disorders7. Another study reported that pain-related anxiety is a unique predictor of headache disability using PASS and MIDAS in migraine and tension-type headache22. In patients with chronic migraine, depression, anxiety, and catastrophizing were associated with severe migraine-related disability45. These studies suggest that the headache-related emotion, especially anxiety, is highly related to migraine-related disability. From the structural equation model, we could better understand the causal link between headache-related anxiety and disability in migraine patients.The present study showed that PASS, headache frequency, and HADS-A has a significant contribution to HIT-6 in multiple regression analysis. Considering that the main factor determining the MIDAS score is the frequency of the headache itself, the relative contribution of PASS can be meaningful enough. The structural equation model showed that headache severity enhances the headache-related emotion and has significant loading to migraine-related disability. The PASS score significantly correlated to the pain-related disability in chronic back pain and musculoskeletal disorder3. The correlation between avoidance and pain-related disability is reported in various chronic pain disorders47. It seems appropriate to understand that abortive medication usage in migraine is an action that reflects active treatment-seeking behavior rather than an avoidance of pain.Our result showed that the use of abortive medication was directly related to disability more than the emotional factor. The fear of pain influences avoidance behavior in headache patientsIn conclusion, the present study showed that migraine patients had altered emotional and cognitive responses to pain perception. The contributions of PASS to HIT-6 and MIDAS highlight the effect of distorted pain perception on headache, especially anxiety, and may be related to disability in migraine. In the therapeutic aspect of migraines, the strategy for disability considering pain-related emotional factors could be important. Further study is also needed to analyze a clear temporal relationship between pain-related emotional factor and disability in migraine.This study is limited in several ways. The present research was based on a one-time point evaluation. Thus, we were unable to explain the causal relationship and examine test–retest reliability clearly. By analyzing a single model, the proposed structural equation model may not be perfect. Many prophylactic medications can directly influence the emotional status. In this study design, we did not properly clarify this issue. Further study would be necessary to conduct with the factors of prophylactic medication as an important variable. We lacked information and knowledge on behavioral factors that can affect individual pain perception, that is, the degree of social activity engagement, interpersonal relationship, and work-related activities.