Sample-to-answer, extraction-free, real-time RT-LAMP test for SARS-CoV-2 in nasopharyngeal, nasal, and saliva samples: Implications and use for surveillance testing.

The global COVID-19 pandemic has highlighted the need for rapid, accurate and accessible nucleic acid tests to enable timely identification of infected individuals. We optimized a sample-to-answer nucleic acid test for SARS-CoV-2 that provides results in <1 hour using inexpensive and readily available reagents. The test workflow includes a simple lysis and viral inactivation protocol followed by direct isothermal amplification of viral RNA using RT-LAMP. The assay was validated using two different instruments, a portable isothermal fluorimeter and a standard thermocycler. Results of the RT-LAMP assay were compared to traditional RT-qPCR for nasopharyngeal swabs, nasal swabs, and saliva collected from a cohort of patients hospitalized due to COVID-19. For all three sample types, positive agreement with RT-LAMP performed using the isothermal fluorimeter was 100% for samples with Ct <30 and 69-91% for samples with Ct <40. Following validation, the test was successfully scaled to test the saliva of up to 400 asymptomatic individuals per day as part of the campus surveillance program at Rice University. Successful development, validation, and scaling of this sample-to-answer, extraction-free real-time RT-LAMP test for SARS-CoV-2 adds a highly adaptable tool to efforts to control the COVID-19 pandemic, and can inform test development strategies for future infectious disease threats.

Human Herpesvirus 6 Infection in Pediatric Liver Transplantation: Single-Center Study of Incidence, Outcomes, and Management.

BACKGROUND: Distinctions between HHV-6 primary infection in seronegative patients and HHV-6 reactivation in seropositive patients remains largely undescribed in pediatric liver transplant (LT) recipients. METHODS: We implemented pretransplant serology testing of HHV-6 in a large pediatric hospital and retrospectively assessed the incidence, manifestations and outcomes of HHV-6 infections over a 3-year period. RESULTS: Among 101 pediatric LT recipients, 96 had pretransplant HHV-6 serologies; 34 (35.4%) were seronegative and 62 (64.6%) seropositive. Posttransplantation, 8/25 (32%) seronegative patients had HHV-6 DNAemia (primary infection) compared to 2/48 (4%) seropositive patients (p=0.002). Compared to seropositive patients, seronegative patients with HHV-6 DNAemia were younger, and had symptoms of fever and/or elevated aminotransferases in association with higher viral loads, in the first month post-transplant. More than 90% of seronegative patients and 77.8% of seropositive patients had HHV-6 detected by PCR in liver biopsy obtained for concerns of allograft rejection, but most had no detectable concomitant DNAemia. Active replication of virus in the liver was confirmed by in situ hybridization in select cases. While HHV-6 infection occurred among patients on prophylaxis doses of antivirals for CMV, HHV-6 DNAemia and presenting symptoms resolved on treatment doses. CONCLUSIONS: HHV-6 DNA-emia occurred more frequently in seronegative pediatric LT recipients, usually in the early posttransplant period, and was subsequently detected in allograft biopsies. HHV-6 cannot be ruled out as a cause of hepatitis in the absence of allograft tissue testing and specialized virological assays, as HHV-6 may disrupt local allograft immune homeostasis while evading traditional screening methods using blood or plasma. The assessment of pre-transplant HHV-6 serological status may be important for risk stratification and post-transplant management of pediatric LT recipients.

Evaluation of liver failure in a pediatric transplant recipient of a liver allograft with inherited chromosomally integrated HHV-6B.

BACKGROUND: Active infections of human herpesvirus 6B (HHV-6B) are frequent in immunocompromised recipients after transplantation. Nevertheless, they need to be distinguished from latent inherited chromosomally integrated genomes (iciHHV-6) present in about 1% of the population to avoid unnecessary administration of toxic antivirals. METHODS: A 5-year-old child presented with acute liver allograft rejection associated with HHV-6 DNA in plasma, which led to an unfavorable outcome. We investigated the possibility of HHV-6 infection derived from an iciHHV-6 present in the donor's liver using molecular and histopathology studies in various tissues, including quantification of HHV-6 DNA, genotyping, sequencing for antiviral resistance genes, relative quantification of viral transcripts, and detection of gB and gH viral proteins. RESULTS: The presence of iciHHV-6B was evidenced in the donor with signs of reactivation in the gallbladder and transplanted liver (detection of HHV-6B mRNA and late proteins). This localized expression could have played a role in liver rejection. Low viral loads in the recipient's plasma, with identical partial U39 sequences, were in favor of viral DNA released from the transplanted liver rather than a systemic infection. CONCLUSIONS: Determination of iciHHV-6 status before transplantation should be considered to guide clinical decisions, such as antiviral prophylaxis, viral load monitoring, and antiviral therapy.

Clinico-radiologic features of pleuroparenchymal fibroelastosis in children.

BACKGROUND: Pleuroparenchymal fibroelastosis (PPFE) may be underdiagnosed clinically and radiographically in children with a remote history of cancer, leading to a delay in care and unnecessary lung biopsies. OBJECTIVE: To describe the characteristic clinical and radiologic findings of PPFE in a cohort of children to facilitate recognition and noninvasive diagnosis. MATERIALS AND METHODS: Clinical presentation, history of chemotherapy or radiation therapy, lung or bone marrow transplantation, and lung function testing and outcome were retrospectively extracted from the electronic medical records of eight children treated at our institution's pulmonary medicine clinic with histopathology confirmation of PPFE from 2008 to 2018. Two pediatric radiologists evaluated the chest imaging studies for the presence or absence of published radiologic findings of PPFE in adults, including platythorax, pneumothorax, upper lobe predominant pleural and septal thickening, and bronchiectasis. Platythorax indices were calculated from the normal chest CT exams of eight age- and gender-matched individuals obtained via the radiology search engine. RESULTS: The mean presentation age was 12.9 years (range: 7-16 years). Seven of the eight had a history of chemotherapy and radiation therapy for cancer. Three of the eight had undergone bone marrow transplantation and none had undergone lung transplantation. The mean time between chemotherapy, radiation therapy, and/or bone marrow transplantation and the presentation of PPFE was 8.4 years (range: 5.6-12.1 years). Most of the patients presented with dyspnea (63%), cough (50%) and/or pneumothorax (38%). The mean percentage of predicted FEV1 (forced expiratory volume in one second) was 14.1 (range: 7.7-27.5). All eight patients demonstrated platythorax, bronchiectasis, pleural and septal thickening (upper lobes in four, upper and lower lobes in four) and six had pneumothorax. Five underwent lung biopsies, four of whom developed pneumothoraces. CONCLUSION: Clinical and radiologic findings of pediatric PPFE are similar to those in adults, although a majority of the former have a history of treated cancer. Clinical presentation of restrictive lung disease, dyspnea, cough or spontaneous pneumothorax years after treatment for childhood cancer combined with platythorax, upper lobe pleural and septal thickening and traction bronchiectasis on chest CT establishes a presumptive diagnosis of PPFE.

Lupus Nephritis.

Childhood-onset systemic lupus erythematosus (SLE) is a subset of SLE with an onset before 18 years of age. Patients with early onset SLE tend to have a greater genetic component to their disease cause, more multisystemic involvement, and a more severe disease course, which includes greater risks for developing nephritis and end-stage kidney disease. Five- and 10-year mortality is lower than in adult-onset SLE. Although patient and renal survival have improved with advances in induction and maintenance immunosuppression, accumulation of irreversible damage is common. Cardiovascular and infectious complications are frequent, as are relapses during adolescence and the transition to adulthood.

Twelve tips for the introduction of emotional intelligence in medical education.

Emotional intelligence (EI) is the ability to recognize, understand, and manage emotions in yourself and in others. EI has long been recognized as a critical component for individual and organizational success within the business realm, and there is emerging evidence that enhancing EI is equally important in the medical setting. EI can improve interpersonal communications, enable constructive conflict resolution, and promote a culture of professionalism. As healthcare becomes increasingly team-based, proficiency in EI will be required to build consensus among multidisciplinary stakeholders, and effect change in attitudes and behaviors that result in improved patient safety and clinical outcomes. Based on the existing literature and the authors' experiences, these 12 tips provide practical suggestions on how to introduce EI into a medical curriculum. These tips have broad applicability, and can be implemented in courses on topics such as professionalism, leadership development, empathy, patient safety, or wellness.

Idiopathic membranous nephropathy in children treated with rituximab: report of two cases.

BACKGROUND: Idiopathic membranous nephropathy is an uncommon cause of nephrotic syndrome in children and can present treatment challenges. The current treatment options of steroids and cyclophosphamide, cyclosporine, or mycophenolate require prolonged treatment durations and the associated side effects may result in nonadherence in children, especially in adolescents. CASE-DIAGNOSIS: We report two adolescent patients with idiopathic membranous nephropathy with nephrotic range proteinuria and elevated anti-phospholipase A2 receptor levels who did not achieve remission with steroids and were later treated with rituximab. Both patients received two doses of rituximab and responded with remission. In addition, anti-PLA2R antibody levels normalized and/or significantly improved. CONCLUSIONS: Rituximab seems to be a safe and effective treatment option in children with idiopathic membranous nephropathy due to anti-PLA2R. Further studies are needed to evaluate this effectiveness.

Pediatric Pathology Fellowship Recruitment-Report of a Survey Conducted by the Fellowship Committee of the Society for Pediatric Pathology.

Pediatric pathology (PP) is a subspecialty of pathology encompassing disease states during human development from the fetus to the young adult. Despite the existence of ACGME-accredited fellowship programs and opportunity for pediatric pathology subspecialty board certification, many pediatric pathology fellowship positions remain unfilled in North America. We sought to understand the difficulties in recruitment to the PP training programs by conducting a survey. A 3-pronged survey targeting pathology residents (PR), PP fellows and recent fellowship graduates (F&G), and PP training programs was conducted. Three separate questionnaires were prepared, one for each group; and administered online via SurveyMonkey. There were 175 responses to PR survey, 29 to F&G and 19 to programs survey. The results of the PR and F&G survey revealed that trainees select a subspecialty early in their residency training, primarily based on their interest, followed by prospects of employment. Nearly half of resident respondents had discounted pediatric pathology subspecialty training without prior exposure to the specialty. Senior residents and faculty members were reported as the main source for fellowship information for residents choosing subspecialty training and the choice of the training program was mostly dictated by geographic location. Most fellow recruits are racially diverse, female, and American medical graduates. Pathology residents decide on subspecialty training based on their interest; however, many are not exposed to pediatric pathology early on in training. The survey results suggest that existing PP fellowship positions likely will continue to exceed demand for subspecialty training. The results of the study could aid in developing strategies to boost recruitment to PP.

Post-transplant Recurrent Bile Salt Export Pump Disease: A Form of Antibody-mediated Graft Dysfunction and Utilization of C4d.

Recurrent bile salt export pump (rBSEP) disease has been reported in progressive familial intrahepatic cholestasis type 2 (PFIC2) patients following liver transplantation (LT) and is often refractory to standard anti-cellular rejection immunosuppressants. The mechanism of rBSEP disease is proposed to be a form of type II hypersensitivity reaction with de novo anti-BSEP antibodies blocking the function of allograft BSEP. Utilization of C4d has not been evaluated in rBSEP. We describe a girl with 3 episodes of rBSEP with severe pruritus at 8.9, 10.3, and 11.0 years post-LT, respectively. Patient's serum reacted with normal liver canaliculi by indirect immunofluorescence (IF), whereas patient's liver showed canalicular immunoglobulin G deposition. The histologic features of all 3 liver biopsies recapitulate PFIC2 with cholestatic giant cell hepatitis. Canalicular BSEP expression was not detected in areas of feathery degeneration by immunohistochemistry, but was retained in morphologically normal liver. By direct IF, C4d showed diffuse sinusoidal staining in the third biopsy. Patient responded well to rituximab with or without intravenous immunoglobulin with subsiding symptoms and normalization of serum bile acid levels. In conclusion, rBSEP disease should be considered in the differential diagnosis when evaluating for rejection in a PFIC2 patient post-LT presenting with pruritus. A portion of liver core may be snap frozen in OCT medium for possible direct IF for C4d, that can serve as a surrogate marker for complement activation and antibody-mediated graft dysfunction.

Clinical presentation and outcomes of childhood-onset membranous lupus nephritis.

BACKGROUND: Best practices for managing childhood-onset membranous lupus nephritis (MLN) are not yet established. Most studies involve primarily or exclusively adult cohorts or pediatric cohorts with combinations of pure or mixed membranous and proliferative nephritis. METHODS: We performed a single-center cohort study of consecutively diagnosed children with pure MLN from 1990 and 2016. Patients received care in Houston, Texas, one of the most diverse metropolitan areas in North America. Renal outcomes were obtained using consensus definitions from the Childhood Arthritis and Rheumatology Research Alliance (CARRA). Logistic regression was used to detect predictors of complete renal response. RESULTS: A total of 56 children with MLN were identified (82% females, 44% black, 35% Hispanic) with a median follow-up time of 4.1 years. The mean age of MLN onset was 13.7 ± 3.4 years. On initial presentation 69% had nephrotic syndrome and 11% had acute kidney injury. Glucocorticoids were prescribed in 96% of patients and anti-malarials in 88%. Mycophenolate mofetil was the most common non-steroid immunosuppressive agent (69%), followed by rituximab (25%), cyclophosphamide (18%), and azathioprine (9%). Renin-angiotensin aldosterone system blocking agents were prescribed in 78% of patients. Of 37 patients with ≥2 years of follow-up, 74% achieved complete renal response at 24 months. No predictor variable of complete renal response was identified in this small cohort. Renal flares occurred in 48% of patients (86% proteinuric, 14% nephritic). On subsequent renal biopsy, 13% patients had developed proliferative nephritis. CONCLUSIONS: This single-center cohort of childhood-onset MLN showed favorable outcomes. Utilizing pediatric renal outcomes definitions, we found that response rates were high, as were rates of renal flare.

Juvenile Granulosa Cell Tumor of the Ovary: A Clinicopathologic Study.

STUDY OBJECTIVE: To report on the clinical characteristics and outcome of pediatric patients with juvenile granulosa cell tumor (JGCT) of the ovary. DESIGN, SETTING, PARTICIPANTS, INTERVENTIONS, AND MAIN OUTCOME MEASURES: Patients with histopathologically confirmed ovarian JGCT diagnosed between 1990 and 2016 were identified. Data on the clinical presentation, surgical management, oncologic management, laboratory investigation, follow-up, and outcome were collected. Tumors were staged according to the International Federation of Gynecology and Obstetrics criteria. RESULTS: Eight patients were diagnosed with ovarian JGCT during the study period. The median age at presentation was 3 years (range, 0.7-14 years). Precocious puberty was the presenting symptom in all five prepubertal children; abdominal distension due to mass effect was the presenting symptom in three children older than 9 years of age. In patients who had preoperative serologic testing, estradiol (n = 3) and inhibin (n = 3) levels were elevated. Five patients had stage I disease, and three had stage III. All stage I patients underwent salpingo-oophorectomy as the only treatment. Stage III patients received adjuvant chemotherapy. After a median follow-up of 6.2 years, six patients (75%) were alive without evidence of disease. One stage I patient with germline p53 mutation and phosphatase and tensin homolog mutation, died because of subsequent liposarcoma. One patient with stage IIIB disease developed recurrence detected according to an elevated inhibin serum level, and died due to progressive disease despite receiving multiple chemotherapy regimens. CONCLUSION: Juvenile granulosa cell tumor has a favorable prognosis in patients with stage I disease after surgical resection alone. Adjuvant chemotherapy might be indicated in patients with higher-stage tumors.

Radiopathologic correlation of a tricuspid valve papillary fibroelastoma detected in an infant.

Papillary fibroelastomas are benign primary cardiac tumors that usually arise from the valve apparatus and are rare in the pediatric population. Involvement of the tricuspid valve is even less common with only a few cases reported in the literature. Cardiac magnetic resonance imaging is a valuable examination that aids in differentiating a tumor from a thrombus. We present the case of an 11-month-old girl referred by her pediatrician to investigate a murmur noted since birth. To our knowledge, this is the first report of a pathologically proven papillary fibroelastoma arising from the tricuspid valve characterized by magnetic resonance imaging in an infant.

Copa Syndrome: a Novel Autosomal Dominant Immune Dysregulatory Disease.

Inherently defective immunity typically results in either ineffective host defense, immune regulation, or both. As a category of primary immunodeficiency diseases, those that impair immune regulation can lead to autoimmunity and/or autoinflammation. In this review we focus on one of the most recently discovered primary immunodeficiencies that leads to immune dysregulation: "Copa syndrome". Copa syndrome is named for the gene mutated in the disease, which encodes the alpha subunit of the coatomer complex-I that, in aggregate, is devoted to transiting molecular cargo from the Golgi complex to the endoplasmic reticulum (ER). Copa syndrome is autosomal dominant with variable expressivity and results from mutations affecting a narrow amino acid stretch in the COPA gene-encoding COPα protein. Patients with these mutations typically develop arthritis and interstitial lung disease with pulmonary hemorrhage representing a striking feature. Immunologically Copa syndrome is associated with autoantibody development, increased Th17 cells and pro-inflammatory cytokine expression including IL-1ß and IL-6. Insights have also been gained into the underlying mechanism of Copa syndrome, which include excessive ER stress owing to the impaired return of proteins from the Golgi, and presumably resulting aberrant cellular autophagy. As such it represents a novel cellular disorder of intracellular trafficking associated with a specific clinical presentation and phenotype.

Diagnostic Yield of Clinical Tumor and Germline Whole-Exome Sequencing for Children With Solid Tumors.

IMPORTANCE: Whole-exome sequencing (WES) has the potential to reveal tumor and germline mutations of clinical relevance, but the diagnostic yield for pediatric patients with solid tumors is unknown. OBJECTIVE: To characterize the diagnostic yield of combined tumor and germline WES for children with solid tumors. DESIGN: Unselected children with newly diagnosed and previously untreated central nervous system (CNS) and non-CNS solid tumors were prospectively enrolled in the BASIC3 study at a large academic children's hospital during a 23-month period from August 2012 through June 2014. Blood and tumor samples underwent WES in a certified clinical laboratory with genetic results categorized on the basis of perceived clinical relevance and entered in the electronic health record. MAIN OUTCOMES AND MEASURES: Clinical categorization of somatic mutations; frequencies of deleterious germline mutations related to patient phenotype and incidental medically-actionable mutations. RESULTS: Of the first 150 participants (80 boys and 70 girls, mean age, 7.4 years), tumor samples adequate for WES were available from 121 patients (81%). Somatic mutations of established clinical utility (category I) were reported in 4 (3%) of 121 patients, with mutations of potential utility (category II) detected in an additional 29 (24%) of 121 patients. CTNNB1 was the gene most frequently mutated, with recurrent mutations in KIT, TSC2, and MAPK pathway genes (BRAF, KRAS, and NRAS) also identified. Mutations in consensus cancer genes (category III) were found in an additional 24 (20%) of 121 tumors. Fewer than half of somatic mutations identified were in genes known to be recurrently mutated in the tumor type tested. Diagnostic germline findings related to patient phenotype were discovered in 15 (10%) of 150 cases: 13 pathogenic or likely pathogenic dominant mutations in adult and pediatric cancer susceptibility genes (including 2 each in TP53, VHL, and BRCA1), 1 recessive liver disorder with hepatocellular carcinoma (TJP2), and 1 renal diagnosis (CLCN5). Incidental findings were reported in 8 (5%) of 150 patients. Most patients harbored germline uncertain variants in cancer genes (98%), pharmacogenetic variants (89%), and recessive carrier mutations (85%). CONCLUSIONS AND RELEVANCE: Tumor and germline WES revealed mutations in a broad spectrum of genes previously implicated in both adult and pediatric cancers. Combined reporting of tumor and germline WES identified diagnostic and/or potentially actionable findings in nearly 40% of newly diagnosed pediatric patients with solid tumors.

Recurrent internal tandem duplications of BCOR in clear cell sarcoma of the kidney.

The X-linked BCL-6 co-repressor (BCOR) gene encodes a key constituent of a variant polycomb repressive complex (PRC) that is mutated or translocated in human cancers. Here we report on the identification of somatic internal tandem duplications (ITDs) clustering in the C terminus of BCOR in 23 of 27 (85%) pediatric clear cell sarcomas of the kidney (CCSK) from two independent cohorts. We profile CCSK tumours using a combination of whole-exome, transcriptome and targeted sequencing. Identical ITD mutations are found in primary and relapsed tumour pairs but not in adjacent normal kidney or blood. Mutant BCOR transcripts and proteins are markedly upregulated in ITD-positive tumours. Transcriptome analysis of ITD-positive CCSKs reveals enrichment for PRC2-regulated genes and similarity to undifferentiated sarcomas harbouring BCOR-CCNB3 fusions. The discovery of recurrent BCOR ITDs defines a major oncogenic event in this childhood sarcoma with significant implications for diagnostic and therapeutic approaches to this tumour.

Persistent cat scratch disease requiring surgical excision in a patient with MPGN.

We present the case of a 13-year-old immunosuppressed patient with unrelenting cat scratch disease despite 9 months of antibiotic therapy. The patient was being treated with mycophenolate and prednisone for membranoproliferative glomerulonephritis (type 1) diagnosed 13 months before the onset of cat scratch disease. Cat scratch disease was suspected due to epitrochlear lymphadenitis and an inoculation papule on the ipsilateral thumb, and the diagnosis was confirmed by the use of acute and convalescent titers positive for Bartonella henselae. The patient experienced prolonged lymphadenitis despite azithromycin and rifampin therapy, and she developed a draining sinus tract ∼4 months after initial inoculation while receiving antibiotics. Acute exacerbation of the primary supratrochlear node prompted incision and drainage of the area, with no improvement in the disease course. Ultimately, excision of all affected nodes and the sinus tract 9 months after the initial diagnosis was required to achieve resolution. Bartonella was detected at a high level according to a polymerase chain reaction assay in the excised nodes. Persistent treatment with oral antibiotics may have prevented disseminated infection in this immunosuppressed patient. Surgical excision of affected nodes should be considered in patients with cat scratch disease that persists beyond 16 weeks.

Correlating surgical and pathological diagnoses in pediatric appendicitis.

BACKGROUND: The stratification of appendicitis into simple and complex variants has far-reaching implications. While the operative diagnosis made by the surgeon dictates clinical management, the pathologic diagnosis often differs and is frequently used for coding and reimbursement. The purpose of this study was to examine discrepancies between the operative and pathologic diagnoses with subsequent correlation to clinical outcomes. METHODS: Patients with acute appendicitis from July 2011 to July 2012 were identified. Diagnoses included simple (normal, acute, and suppurative) and complex (gangrenous and perforated). We evaluated the inter-rater reliability between pathologic and operative diagnoses in the five appendicitis categories. Clinical outcomes of deep and superficial surgical site infections were evaluated according to the pathologic and surgical diagnosis. RESULTS: During the study period, we identified 1166 patients with acute appendicitis. The surgeon and pathologist agreed on the specific diagnosis (acute, suppurative, gangrenous, perforated, normal) in 48% of patients (kappa 0.289, 95% CI 0.259-0.324, p=0.001). Agreement on disease severity (simple vs. complex) improved to 82%. The operative diagnosis more accurately predicted infectious complications than the pathologic diagnoses. CONCLUSION: Significant discordance exists between surgical and pathologic diagnoses. While the relevance of this discordance to clinical outcomes is still not clear, a potential for incorrect hospital coding and subsequent reimbursement exists. Future quality improvement projects should focus on standardizing the surgical and pathologic diagnoses.