Francesco Fontana

BACKGROUND: Histopathological assessment of transplant biopsies is currently the standard method to diagnose allograft rejection and can help guide patient management, but it is one of the most challenging areas of pathology, requiring considerable expertise, time, and effort. We aimed to analyse the utility of deep learning to preclassify histology of kidney allograft biopsies into three main broad categories (ie, normal, rejection, and other diseases) as a potential biopsy triage system focusing on transplant rejection. METHODS: We performed a retrospective, multicentre, proof-of-concept study using 5844 digital whole slide images of kidney allograft biopsies from 1948 patients. Kidney allograft biopsy samples were identified by a database search in the Departments of Pathology of the Amsterdam UMC, Amsterdam, Netherlands (1130 patients) and the University Medical Center Utrecht, Utrecht, Netherlands (717 patients). 101 consecutive kidney transplant biopsies were identified in the archive of the Institute of Pathology, RWTH Aachen University Hospital, Aachen, Germany. Convolutional neural networks (CNNs) were trained to classify allograft biopsies as normal, rejection, or other diseases. Three times cross-validation (1847 patients) and deployment on an external real-world cohort (101 patients) were used for validation. Area under the receiver operating characteristic curve (AUROC) was used as the main performance metric (the primary endpoint to assess CNN performance). FINDINGS: Serial CNNs, first classifying kidney allograft biopsies as normal (AUROC 0·87 [ten times bootstrapped CI 0·85-0·88]) and disease (0·87 [0·86-0·88]), followed by a second CNN classifying biopsies classified as disease into rejection (0·75 [0·73-0·76]) and other diseases (0·75 [0·72-0·77]), showed similar AUROC in cross-validation and deployment on independent real-world data (first CNN normal AUROC 0·83 [0·80-0·85], disease 0·83 [0·73-0·91]; second CNN rejection 0·61 [0·51-0·70], other diseases 0·61 [0·50-0·74]). A single CNN classifying biopsies as normal, rejection, or other diseases showed similar performance in cross-validation (normal AUROC 0·80 [0·73-0·84], rejection 0·76 [0·66-0·80], other diseases 0·50 [0·36-0·57]) and generalised well for normal and rejection classes in the real-world data. Visualisation techniques highlighted rejection-relevant areas of biopsies in the tubulointerstitium. INTERPRETATION: This study showed that deep learning-based classification of transplant biopsies could support pathological diagnostics of kidney allograft rejection. FUNDING: European Research Council; German Research Foundation; German Federal Ministries of Education and Research, Health, and Economic Affairs and Energy; Dutch Kidney Foundation; Human(e) AI Research Priority Area of the University of Amsterdam; and Max-Eder Programme of German Cancer Aid.

PURPOSE: Previous studies have shown that early intracavernous prostaglandin E1 injection may reduce significantly the incidence of veno-occlusive dysfunction before spontaneous erections recover after nerve sparing radical prostatectomy. We identify the more convenient postoperative timing for successful intracavernous injection rehabilitation in a series of patients who underwent nonnerve sparing radical prostatectomy. MATERIALS AND METHODS: A total of 73 patients with a normal preoperative International Index of Erectile Function score were randomly allocated to undergo dynamic color Doppler ultrasound study 20 mg. prostaglandin E1 at 1, 2 to 3, 4 to 6 and 7 to 12 months postoperatively, respectively. In all cases the peak systolic velocity, end diastolic velocity and resistance index were evaluated at 5, 10 and 20 minutes after injection. RESULTS: Of the patients 36 received the intracavernous injection within the first 3 months (group 1) and 37 received it at 4 to 12 months (group 2). A significantly higher proportion of group 1 patients had grade 3 erection compared with group 2. Peak systolic velocity less than 30 cm. per second in at least 1 cavernosal artery was recorded in 22.2% of group 1 patients and 51.3% of group 2 (p >0.05). CONCLUSIONS: Intracavernous injections after nonnerve sparing radical prostatectomy produce valid erectile responses in a significantly higher proportion of patients when started within month 3 after the operation. Injection given in postoperative month 1 gives the best response rate but with significant complications and poor patient compliance. Arteriogenic and venogenic factors seem to be involved with failure.

Coronavirus disease 2019 (COVID-19) pneumonia has been poorly reported in solid organ transplanted patients; prognosis is uncertain and best management unclear. We describe the case of a 61-year-old kidney transplant recipient with several comorbidities who was hospitalized and later received a diagnosis of COVID-19 pneumonia; the infection was successfully managed with the use of hydroxychloroquine and a single administration of tocilizumab, after immunosuppression reduction; the patient did not require mechanical ventilation. During the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, transplant clinicians should be readily informed about new cases of COVID-19 pneumonia in solid organ transplant recipients, with focus on therapeutic strategies employed and their outcome. Coronavirus disease 2019 (COVID-19) pneumonia has been poorly reported in solid organ transplanted patients; prognosis is uncertain and best management unclear. We describe the case of a 61-year-old kidney transplant recipient with several comorbidities who was hospitalized and later received a diagnosis of COVID-19 pneumonia; the infection was successfully managed with the use of hydroxychloroquine and a single administration of tocilizumab, after immunosuppression reduction; the patient did not require mechanical ventilation. During the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, transplant clinicians should be readily informed about new cases of COVID-19 pneumonia in solid organ transplant recipients, with focus on therapeutic strategies employed and their outcome. In late December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as a novel pathogen causing severe pneumonia cases, lately named coronavirus disease 2019 (COVID-19), in Wuhan, China.1Guan W, Ni Z, Hu Y, et al. Clinical characteristics of coronavirus disease 2019 in China. N Engl J Med. 2020:NEJMoa2002032. https://doi.org/10.1056/NEJMoa2002032.Google Scholar Since then, the infection has been demonstrating a rapid global spread, with a devastating evolution in northern Italy; there, several simultaneous clusters developed with a substantial number of critically ill patients and a very high case fatality rate, especially among the elderly and those with comorbidities.2Onder G, Rezza G, Brusaferro S. Case-fatality rate and characteristics of patients dying in relation to COVID-19 in Italy [published online ahead of print 2020]. JAMA. 2020. https://doi.org/10.1001/jama.2020.4683Google Scholar COVID-19 is considered as potentially having a more severe course in solid organ transplant recipients, due to the chronic immunosuppression these patients are exposed to for preventing rejection. Only a few reports of COVID-19 in kidney transplanted patients are currently available in the literature,3Zhu L, Xu X, Ma K, et al. Successful recovery of COVID-19 pneumonia in a renal transplant recipient with long-term immunosuppression [published online ahead of print 2020]. Am J Transplant. 2020. https://doi.org/10.1111/ajt.15869.Google Scholar, 4Guillen E, Pineiro GJ, Revuelta I, et al. Case report of COVID-19 in a kidney transplant recipient: does immunosuppression alter the clinical presentation? [published online ahead of print 2020]. Am J Transplant. 2020. https://doi.org/10.1111/ajt.15874.Google Scholar, 5Seminari E, Colaneri M, Sambo M, et al. SARS Cov2 infection in a renal transplanted patients. A case report [published online ahead of print 2020]. Am J Transplant. 2020. https://doi.org/10.1111/ajt.15902.Google Scholar, 6Gandolfini I, Delsante M, Fiaccadori E, et al. COVID-19 in kidney transplant recipients [published online ahead of print 2020]. Am J Transplant. 2020. https://doi.org/10.1111/ajt.15891.Google Scholar, 7Huang J, Lin H, Wu Y, et al. COVID-19 in post-transplantation patients- report of two cases [published online ahead of print 2020]. Am J Transplant. 2020. https://doi.org/10.1111/ajt.15896.Google Scholar and prognosis and recommended management for these patients are unclear. Moreover, the impact of treatments other than best supportive care is unknown. A 61-year-old man, who underwent kidney transplantation from a deceased donor in 2005 for end-stage renal disease due to chronic interstitial nephritis, was admitted to the nephrology unit for persistent fever and shivering over the last 48 hours. He reported no cough or dyspnea, he had not traveled outside town in the past 15 days, and had no history of contact with people positive or suspected for SARS-Cov-2 infection. The patient had chronic kidney disease stage IIIa (serum creatinine 1.5 mg/dL, estimated glomerular filtration rate of 50 mL/min); maintenance immunosuppression consisted of cyclosporine A (CyA) plus steroid. Past medical history included nodal marginal zone lymphoma in active hematological surveillance; previous unprovoked pulmonary embolism treated with warfarin in secondary prevention; and idiopathic Parkinson disease with motor complications treated with subthalamic neurostimulation, with neurogenic bladder managed with intermittent bladder catheterization and complicated by frequent urinary tract infections. At first evaluation, physical examination was unremarkable (apart from tremor related to chronic neurological condition); blood pressure was 136/72 mm Hg, and body temperature was 38°C; peripheral capillary oxygen saturation was 97% breathing ambient air. Laboratory blood tests were normal with blood cell count (5460 cells/mm3 with 79% neutrophils), mild acute kidney injury (serum creatinine 1.9 mg/L), and minimally elevated C-reactive protein (4.1 mg/dL); CyA levels were 90 ng/mL (basal) and 136 ng/mL (after 2 hours). Chest radiography showed minimal left pleural effusion. Specimens for urinary and blood cultures were collected; urinary tract infection was suspected and antibiotic treatment with meropenem was initiated, based on a previous isolate. On day 3 after admission, considering persistence of fever, negativity of urinary cultures and serum procalcitonin, SARS-CoV-2 infection was suspected and the patient isolated in a single room. Antibiotic treatment was stopped, oropharyngeal/nasal swab for SARS-CoV-2 research in reverse transcription polymerase chain reaction (RT-PCR) was performed; a repeated chest radiograph showed bilateral basal interstitial pneumonia; arterial blood gases were unremarkable (pO2 91 mm Hg breathing ambient air). In the following days, the patient remained stable with undulating fever and no dyspnea. Search for viral and bacterial pathogens in PCR from upper respiratory tract material resulted negative, as were cytomegalovirus DNA on blood and blood cultures collected at admission. Diagnostic oropharyngeal/nasal swabs for SARS-CoV-2 were repeated and, only at the third attempt on day 9 after admission, the test was positive. In the same week 3 other hospitalized patients and, the week after, 2 healthcare workers resulted positive for SARS-CoV-2 infection in our service; nevertheless, even if cases were probably related, it was not possible to track a clear chronological order. On the day of diagnosis, arterial pO2 dropped to 57 mm Hg, and low-flow oxygen through nasal cannula was initiated; the patient was hemodynamically stable. Hydroxycloroquine was started at the dose of 200 mg bid; CyA dose was reduced by a half; intravenous fluids were initiated. Laboratory exams showed leukopenia with lymphopenia (see Figure 1); serum lactate dehydrogenase, hemoglobin, platelets, and D-dimer levels were normal. Two days after, considering the lack of improvement in clinical conditions, CyA was withdrawn and oral steroid dose increased (methylprednisolone 16 mg per day); after discussion with infectious disease specialist and signature of informed consent by the patient, tocilizumab was administered off label at the dose of 324 mg via subcutaneous route. Interleukin-6 (IL-6) levels in blood proved to be high (280.86 pg/mL). The patient developed progressive leukopenia (with leukocyte nadir at 660/µL, neutrophils 400/µL). Suspecting that leukopenia with neutropenia could be an immune-mediated process related to tocilizumab, and in order to enhance anti-inflammatory and immunomodulatory response, we administered intravenous immunoglobulins (IVIG) at the dose of 0.3 g/kg. Leukocyte count rose to 2090/µL (neutrophils 1180/µL, lymphocytes 670/µL), with CD4 count 518/µL. On day 14 after admission, urinary culture was positive for multiresistant Pseudomonas aeruginosa, and, considering the risk for bacterial disseminated infection after tocilizumab, antibiotic treatment with meropenem was reinitiated. On day 14, chest radiograph showed multiple nonhomogeneous bilateral consolidations, and azithromycin was administered orally for 3 days to prevent bacterial superinfection. The radiographic picture was interpreted as imaging evolution of COVID-19 pneumonia. Since tocilizumab administration, the patient remained apyretic. Arterial pO2 showed progressive improvement with values always above 60 mm Hg, and oxygen treatment was stopped. IL-6 levels increased, as expected, 6 days after administration of tocilizumab (619.11 pg/mL). Kidney function remained substantially stable over the course of hospitalization (Figure 1). The patient was discharged home on day 22 after admission without fever and with peripheral oxygen saturation of 95% breathing ambient air; respiratory frequency was 14 acts per minute, blood pressure was 140/80 mm Hg; white blood cell count was 2970/µL. Oropharyngeal/nasal swab for SARS-Cov-2 was still positive 13 days after diagnosis, and appropriate isolation measures were recommended at home. Hydroxychloroquine was stopped, and CyA is presently still withheld. The world is now facing a pandemic of SARS-CoV-2 infection causing COVID-19, which is unique in terms of rapidity of growth and global involvement; no proven specific therapies are available, other than supportive care. Emerging infectious diseases represent an enormous threat of contagion to transplant recipients; these patients, in turn, are subjected to frequent ambulatory checks and hospitalizations and they can substantially contribute to the spreading of the infection if not promptly identified. Clinical presentation of COVID-19 is nonspecific, most of the time manifesting as a febrile illness with often mild upper respiratory manifestations. Symptoms and signs can be very subtle especially in the early phases of the infection, requiring a high degree of suspicion by clinicians and imposing logistic challenges in order to isolate suspected cases. Our report shows potential limitations in the diagnosis of SARS-CoV-2 infection with a single oropharyngeal/nasal swab. Although RT-PCR diagnostic for SARS-CoV-2 infection has been described to be extremely sensitive,8Chan JF-W, Yip CC-Y, To KK-W, et al. Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp/Hel real-time reverse transcription-polymerase chain reaction assay validated in vitro and with clinical specimens. J Clin Microbiol. 2020:JCM.00310-20. https://doi.org/10.1128/JCM.00310-20.Google Scholar false negative results on oropharyngeal/nasal swab have been reported.9Winichakoon P, Chaiwarith R, Liwsrisakun C, et al. Negative nasopharyngeal and oropharyngeal swab does not rule out COVID-19 [published online ahead of print 2020]. J Clin Microbiol. 2020. https://doi.org/10.1128/JCM.00297-20.Google Scholar It is postulated that several mechanisms can be responsible for this phenomenon, among them sampling techniques, timing of viral positivity after initial infection, and lack of expression of angiotensin-converting enzyme 2 (functional receptor of SARS-CoV-2) in nasal and pharyngeal mucosa.10Wan Y Shang J Graham R Baric RS Li F. Receptor recognition by novel coronavirus from Wuhan: an analysis based on decade-long structural studies of SARS.J Virol. 2020; 94 (https://doi.org/10.1128/jvi.00127-20.)Crossref Scopus (2878) Google Scholar,11Hamming I Timens W Bulthuis MLC Lely AT Navis GJ van Goor H. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis.J Pathol. 2004; 203: 631-637Crossref PubMed Scopus (3780) Google Scholar It appears then important to perform repeated oropharyngeal/nasal swabs, or consider obtaining bronchoalveolar lavage material if initial testing is negative, in patients with a consistent clinical suspicion for SARS-CoV-2 infection. Moreover, even in the absence of positive RT-PCR positivity, clinical indicators (especially suggestive radiological imaging) should guide clinical diagnosis indicating need for isolation of the patient.12Li Y, Yao L, Li J, et al. Stability issues of RT-PCR testing of SARS-CoV-2 for hospitalized patients clinically diagnosed with COVID-19 [published online ahead of print 2020]. J Med Virol. 2020. https://doi.org/10.1002/jmv.25786.Google Scholar The optimal management of a solid organ transplant recipient with SARS-CoV-2 infection is not clearly determined. It seems rational to reduce the immunosuppressive load, as it is common practice in most severe infections in transplanted patients; in general, our protocol is to withdraw mycophenolate mophetil and, as a second step, reduce and consider stopping calcineurin inhibitor. Nevertheless, because a great part of the pulmonary damage in COVID-19 pneumonia appears to be related to excessive inflammatory response of the host,13Wu C, Chen X, Cai Y, et al. Risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in Wuhan, China [published online ahead of print 2020]. JAMA Intern Med. 2020. https://doi.org/10.1001/jamainternmed.2020.0994.Google Scholar it could be argued that antirejection drugs could contribute to reducing this process. In our patient, we decided to reduce cyclosporine dose by half and later stop it, also considering the high risk for bacterial infection, maintaining low-dose steroid. Several drugs are being administered off label and are currently under investigation in clinical trials for COVID-19. There is mounting enthusiasm regarding the use of chloroquine and its analog hydroxychloroquine against SARS-CoV-2. Rationale for its use derived from in vitro studies from China demonstrating that chloroquine was able to block virus infection at low-micromolar concentration in vitro and possessed high selectivity index.14Wang M Cao R Zhang L et al.Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro.Cell Res. 2020; 30: 269-271Crossref PubMed Scopus (4567) Google Scholar Recently, a small nonrandomized clinical trial from France showed that infected patients treated with hydroxychloroquine were more likely to achieve virologic clearance at day 6;15Gautret P, Lagier J-C, Parola P, et al. Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial [published online ahead of print 2020]. Int J Antimicrob Agents. 2020. https://doi.org/10.1016/j.ijantimicag.2020.105949.Google Scholar nevertheless, this study has received extensive criticism about its methodology and its results have been described as overstating.16Dahly D, Gates S, Morris T. Statistical review of hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial [published online ahead of print 2020]. Zenodo. 2020 https://doi.org/10.5281/ZENODO.3725560.Google Scholar Despite poor quality evidence, given the lack of alternative effective treatments, the relatively safe toxicity profile and its wide availability, many centers in Italy have adopted hydroxychloroquine as a first-line strategy in patients with confirmed SARS-Cov-2 infection. In our patient, hydroxychloroquine was continued for a total of 13 days. It is believed that the severity of pulmonary involvement in SARS-CoV-2 infection is mainly driven by an excessive inflammatory response mounted by the host immune system in response to pathogen. Indeed, inflammation-related indices have been reported to be higher in patients with COVID-19 pneumonia who develop acute respiratory distress syndrome compared to those who do not; interestingly, IL-6 was significantly more elevated in these patients.13Wu C, Chen X, Cai Y, et al. Risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in Wuhan, China [published online ahead of print 2020]. JAMA Intern Med. 2020. https://doi.org/10.1001/jamainternmed.2020.0994.Google Scholar IL-6 is a multifunctional proinflammatory cytokine which stimulates T cell proliferation/differentiation to T helper cells and differentiation of B-lymphocytes to antibody-secreting plasma cells.17Nishimoto N Terao K Mima T Nakahara H Takagi N Kakehi T. Mechanisms and pathologic significances in increase in serum interleukin-6 (IL-6) and soluble IL-6 receptor after administration of an anti-IL-6 receptor antibody, tocilizumab, in patients with rheumatoid arthritis and Castleman disease.Blood. 2008; 112: 3959-3964Crossref PubMed Scopus (565) Google Scholar Tocilizumab is a humanized monoclonal antibody that competitively inhibits IL-6 by binding to both its soluble and membrane receptors;17Nishimoto N Terao K Mima T Nakahara H Takagi N Kakehi T. Mechanisms and pathologic significances in increase in serum interleukin-6 (IL-6) and soluble IL-6 receptor after administration of an anti-IL-6 receptor antibody, tocilizumab, in patients with rheumatoid arthritis and Castleman disease.Blood. 2008; 112: 3959-3964Crossref PubMed Scopus (565) Google Scholar its use in COVID-19 pneumonia is currently being investigated in a multicenter, single-arm, open-label, phase 2 study in Italy.18Tocilizumab in COVID-19 Pneumonia (TOCIVID-19) - ClinicalTrials.gov. https://clinicaltrials.gov/ct2/show/study/NCT04317092?term=tocilizumab&cond=covid&draw=2&rank=1. Accessed March 24, 2020.Google Scholar In order to reduce pulmonary inflammatory response, we decided to administer tocilizumab in our patient in an early phase, before overt lung damage ensued; our strategy was also supported by demonstrating high levels of IL-6 in blood. The subcutaneous route of administration was chosen according to local stock availability, because intravenous formulation was rapidly exhausted. Administered dose was double that recommended for rheumatoid arthritis and was chosen in the attempt to mimic pharmacodynamics of the intravenous administration. There is no clear consensus regarding a definite schedule of tocilizumab administration in COVID-19; main contraindications to tocilizumab are untreated active infection, aspartate transaminase/alanine transaminase elevation, neutrophil count <500/µL, and platelets <50 000/µL. Considering the good clinical response in our patient, we decided not to proceed with additional doses. It must be clarified that an increase in serum IL-6 levels was expected after tocilizumab administration; because IL-6 receptor is fully inhibited, the produced ligand will remain in the circulation until degradation. This phenomenon does not correspond to a lack in tocilizumab efficacy, because the IL-6 signaling pathway is expected to be completely blocked; IL-6 levels are expected to gradually decrease over time. In addition to that, the patient was also treated with IVIG for leukopenia with neutropenia possibly related to tocilizumab and to counteract aberrant inflammation related to COVID-19. Indeed, tocilizumab have been reported to induce neutropenia, considered to be immune mediated.19Shovman O Y neutropenia in rheumatoid arthritis patients with previous history of case and review of Res. Scopus Google Scholar Moreover, IVIG anti-inflammatory and immunomodulatory several are the postulated among them the decrease in the of proinflammatory immune in and inflammatory Engl J Med. PubMed Scopus Google Scholar which could have been of in our considering the of pulmonary damage in COVID-19. oropharyngeal/nasal swab was still positive for SARS-CoV-2 at CyA treatment was not the patient is currently and CyA will be after demonstrating absence of viral In we report a case of COVID-19 pneumonia in a kidney transplant recipient with several which was successfully managed with the use of multiple treatments and a single dose of and of The patient had no need for mechanical ventilation. We that results in a single case are to considering the multiple drugs and the course of COVID-19 in transplanted patients. Nevertheless, we consider that transplant clinicians should be of potentially treatments for COVID-19 pneumonia in solid organ transplant the extremely growth of the of SARS-CoV-2 we it is of to report management of cases in before results of clinical trials The of this have no of to as described by the of are available to the