Unmasking viral sequences by metagenomic next-generation sequencing in adult human blood samples during steroid-refractory/dependent graft-versus-host disease.

BACKGROUND: Viral infections are common complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Allo-HSCT recipients with steroid-refractory/dependent graft-versus-host disease (GvHD) are highly immunosuppressed and are more vulnerable to infections with weakly pathogenic or commensal viruses. Here, twenty-five adult allo-HSCT recipients from 2016 to 2019 with acute or chronic steroid-refractory/dependent GvHD were enrolled in a prospective cohort at Geneva University Hospitals. We performed metagenomics next-generation sequencing (mNGS) analysis using a validated pipeline and de novo analysis on pooled routine plasma samples collected throughout the period of intensive steroid treatment or second-line GvHD therapy to identify weakly pathogenic, commensal, and unexpected viruses. RESULTS: Median duration of intensive immunosuppression was 5.1 months (IQR 5.5). GvHD-related mortality rate was 36%. mNGS analysis detected viral nucleotide sequences in 24/25 patients. Sequences of ≥ 3 distinct viruses were detected in 16/25 patients; Anelloviridae (24/25) and human pegivirus-1 (9/25) were the most prevalent. In 7 patients with fatal outcomes, viral sequences not assessed by routine investigations were identified with mNGS and confirmed by RT-PCR. These cases included Usutu virus (1), rubella virus (1 vaccine strain and 1 wild-type), novel human astrovirus (HAstV) MLB2 (1), classic HAstV (1), human polyomavirus 6 and 7 (2), cutavirus (1), and bufavirus (1). CONCLUSIONS: Clinically unrecognized viral infections were identified in 28% of highly immunocompromised allo-HSCT recipients with steroid-refractory/dependent GvHD in consecutive samples. These identified viruses have all been previously described in humans, but have poorly understood clinical significance. Rubella virus identification raises the possibility of re-emergence from past infections or vaccinations, or re-infection. Video abstract.

Different degrees of ischaemic injury in the right and left ventricle in cases of severe, nonfatal, pulmonary embolism.

Pulmonary fat embolism (PFE) is a common complication of blunt force traumas with bone fractures. Severe forms cause impedance to right ventricular (RV) ejection, with eventual right heart ischaemia and failure. In a prospective study, we have investigated 220 consecutive autopsy cases (73 females, 147 males, mean age 52.1 years, min 14 years, max 91 years). PFE was detected in 52 cases that were divided into three groups according to the degree of PFE (1-3). A fourth group of cases of violent death without PFE was used for comparison. In each case, histology (H&E, Masson) and immunohistochemistry (fibronectin and C5b-9) were performed on six cardiac samples (anterior, lateral and posterior wall of both ventricles). The degree of cardiac damage was registered in each sample and the mean degree of damage was calculated in each case at the RV and left ventricle (LV). Moreover, a parameter ∆ that is the difference between the mean damage at the RV and the LV was calculated in each case. The results were compared within each group and between the groups. In the present study, we could not detect prevalent RV damage in cases of high degree PFE as we did in our previous investigation. In the group PFE3 the difference of the degree of damage between the RV and LV was higher than the one observed in the groups PFE0-2 with the antibody anti-fibronectin. Prevalent right ventricular stress in cases of severe PFE may explain this observation.

An extraordinary case of an intracranial foreign body after a gunshot to the head.

Intermediate targets (IT) can modify the morphology of an entrance wound, the trajectory of the bullet, and contaminate the path with fragments or material from the target. The penetration into the body of big fragments or even of an entire IT is exceptional and only rarely reported in the literature. The interpretation of a gunshot wound after contact of the bullet with IT can sometimes be very tricky as the classical morphology can be missing. The presented case is a rare example of atypical entrance wound and path due to a surprising intermediate target of a gunshot fired against the head.

Transplanted human pancreatic islets after long-term insulin independence.

Long-term insulin independence after islets of Langerhans transplantation is rarely achieved. The aims of this study were to identify the histological and immunological features of islets transplanted in a type 1 diabetic patient who died of a cerebral hemorrhage after >13 years insulin independence. Islets were pooled from two donors with respectively one and five HLA mismatches. Insulin-positive islets were found throughout the right and left liver, and absent in the pancreas. Two- and three-dimensional analysis showed that islets lost their initial rounded and compact morphology, had a mean diameter of 136 µm and were constituted of an unfolded epithelial band of 39.1 µm. Leukocyte phenotyping showed no evidence of a tolerogenic environment in the islet-containing portal spaces. Finally, HLA typing of microdissected islets showed HLA from the best matched donor in all 23 microdissection samples, compared to 1/23 for the least matched donor. This case report demonstrates that allogeneic islets can survive over 13 years while maintaining insulin independence. Allogeneic islets had unique morphologic features and implanted in the liver regardless of their size. Finally, our results suggest that, in this case, rejection had been prevalent over autoimmunity, although this hypothesis warrants further investigation.

Creutzfeldt-Jakob disease revealed by a logopenic variant of primary progressive aphasia.

Logopenic aphasia, mainly characterized by word anomia, sentence and phrase comprehension difficulties secondary to phonological loop deficits but relatively preserved single word comprehension and no agrammatism, is one of the 3 main variants of primary progressive aphasia (PPA). We describe the first case of PPA that fulfilled clinical criteria of logopenic aphasia but showed abnormal DWI hyperintensities that were predominant on the left hemisphere and compatible with Creutzfeldt-Jakob disease (CJD). After abnormally long isolated language deficits, the patient rapidly worsened and died. Autopsy performed 18 months after onset of language difficulties confirmed the diagnosis. We therefore advocate performing DWI sequences in any suspicion of PPA in order to rule out CJD.

Autosomal dominant nemaline myopathy: a new phenotype unlinked to previously known genetic loci.

We report a large family with a mild form of autosomal dominant nemaline myopathy and a new phenotype. Onset of symptoms was in infancy with hypotonia and motor delay. Weakness involved neck flexors, abdominal and proximal limb muscles. There was no bulbar, respiratory or foot dorsiflexion weakness and no slowness in movement. Patients had remarkably good physical endurance and no limitation in daily activities, but were slow runners since childhood. Nemaline rods were seen in less than 5% of muscle fibres. No linkage to the five known nemaline myopathy genes (alpha-tropomyosin-3, nebulin, alpha-actin, troponin T1 and beta-tropomyosin), to the ryanodine receptor gene (associated with core-rod myopathy) or to the 15q21-23 locus was found.

Morphological, clinical and genetic aspects in a family with a novel LAMP-2 gene mutation (Danon disease).

A family with several cases of severe cardiomyopathy and moderate myopathy is described, affecting two brothers and their cousin as well as their mothers. One boy died of sudden cardiac arrest at 17 years of age. The two brothers were treated with an implantable defibrillator and their mother died suddenly at 40 years of age. Muscle biopsy in males showed vacuolar myopathy in two cases, and no abnormality on standard staining in the third case. Cardiac biopsies showed hypertrophic and vacuolated fibres. Complete absence of LAMP-2 was demonstrated by immunohistochemistry on the vacuolated skeletal and cardiac muscle, but also on the morphologically normal skeletal muscle. Sequencing of LAMP-2 gene showed a novel S157X mutation in exon 4. Danon disease is a rare and potentially lethal cause of hypertrophic cardiomyopathy. Diagnosis can be made by immunohistochemistry performed on cardiac or muscle biopsy, and confirmed by genetic analysis, which also allows for easy family screening and counselling.

[Periarteritis nodosa and hepatitis C: a fortuitous association?]. / Périartérite noueuse et hépatite C: association fortuite?

Polyarteritis nodosa is a vasculitis of unknown origin which can be rarely associated with hepatitis B. A exceptional clinical situation of a polyarteritis nodosa associated with hepatitis C is described. This case is also the occasion to review the clinical manifestations, the diagnostic strategy und the therapeutic options of this rare vasculitis.

Massive haemorrhagic transformation in cardioembolic stroke: the role of arterial wall trauma and dissection.

The pathogenesis of massive haemorrhagic transformation is not well established. Fatal haemorrhagic transformation associated with in situ dissection after acute middle cerebral artery (MCA) occlusion in a patient with atrial fibrillation is reported. An 80 year old woman with atrial fibrillation developed mild hemiparesis and aphasia. Brain CT and MRI at 4 and 5 hours, respectively, of symptom onset showed proximal MCA trunk occlusion with developing striatocapsular infarct and hypoperfusion in the superficial MCA territory. A few hours later, she developed massive bleeding into the ischaemic area and died. Pathological examination showed MCA trunk dissection, surrounded by a subarachnoid clot which communicated with the cerebral haematoma. It is suggested that direct arterial wall trauma as a result of cardioembolic MCA occlusion caused bleeding into the infarct. Secondary in situ dissection may be an overlooked mechanism of haemorrhagic transformation.

Clinicopathological and molecular biological studies in a patient with neurolymphomatosis.

We describe a patient with a clinical disorder that resembled vasculitic neuropathy in which peripheral nerves were successively affected over several months, but without systemic involvement. An initial muscle biopsy near the involved nerves showed signs of nonspecific inflammation around the muscle and nerve fibers. Immunosuppressive treatment resulted in a dramatic reduction in pain, but relapses of the disease eventually occurred, and the patient died 22 months after onset of the first symptoms. Pathologically, a malignant non-Hodgkin's B-cell lymphoma, restricted to the intra- and extradural peripheral nervous system, was found. The demonstration by Southern blotting of immunoglobulin heavy chain gene rearrangement confirmed the monoclonal nature of the lymphomatous cells. In situ hybridization tests for Epstein-Barr and herpes virus subtypes were negative. Our case underlines i) how difficult diagnosis can be despite extensive investigations, ii) the usefulness of immunosuppressive treatment in the early stage of the disease, iii) the importance of immunostaining and genome analysis for distinguishing between different types of human neurolymphomatosis, and iv) the fact that the initial inflammatory process in the muscle biopsy may be interpreted either as a paraneoplastic effect of the lymphoma or as a viral inflammatory neuromyopathy that triggers the development of the malignant lymphoma.

Bilateral Krukenberg tumor of the ovary during pregnancy.

This case report concerns a 35-year-old woman suffering from gravidic cholestasis, thrombocytosis and iterative vomiting episodes who underwent an elective cesarean section at week 35 because of recent herpetic vulvitis. Large bilateral ovarian tumors were observed which were interpreted as pregnancy luteomas. Nevertheless a biopsy of the right ovary was performed. Histologic examination revealed massive luteinization of the ovarian stroma. In addition, large tumor cells were found dispersed throughout the ovary as well as in vascular spaces as either isolated or clustered signet-ring cells. In search of the primary tumor, gastroscopy revealed a gastric ulcer in the antrum. The biopsies of the ulcer margins as well as those taken at distance demonstrated signet-ring cells in the lamina propria. Bilateral salpingo-oophorectomy and total gastrectomy were performed. In spite of postoperative chemotherapy, the patient died of disease 5 months after diagnosis.

Familial cardiomyopathy and distal myopathy with abnormal desmin accumulation and migration.

Desminopathies form a heterogeneous group of myopathies characterised by pathological aggregations of desmin. We report a family, where mother and daughter presented with an atrioventricular block and a slowly progressive distal muscular weakness, with non-homogeneous focal atrophy on computed tomography scans. The mother developed a severe global heart insufficiency necessitating a heart transplantation at 56 years of age. Skeletal muscle biopsies were characterised by inclusion bodies strongly expressing desmin and alpha B-crystallin, with a predominantly subsarcolemmal localisation. Ultrastructurally most inclusions corresponded to non-membrane bound granulo-filamentous material with disruption of myofibrils. An immunoblot showed a hyperintense desmin band at 53 kDa and a second band at 49 kDa, the latter being absent in controls. The cardiac muscle of the explanted heart showed very similar inclusions. These cases illustrate that in this distinct subtype of desminopathies the cardiac muscle alterations are comparable with those observed in skeletal muscle, and suggest the possibility of a primary desmin pathology.

Astrocytes secrete a factor inducing the expression of HT7-protein and neurothelin in endothelial cells of chorioallantoic vessels.

Among the most specific markers of the blood-brain barrier phenotype of endothelial cells are the well-characterized immunoglobulin-like surface glycoprotein HT7 and the probably related or identical glycoprotein neurothelin. Both can be induced in chorioallantoic vessels by transplants of embryonic mouse brain. Other blood-brain barrier markers have been shown to be inducible by type-1 astrocytes in endothelial cells of non-neural origin. In the present work we tested the hypothesis that this cellular interaction between astrocytes and endothelial cells is mediated by a soluble factor(s). Chorioallantoic vessels of embryonic day 9 chick embryos were exposed for 4 or 10 days to a constant and localized delivery of astrocyte-conditioned medium by using a piece of gelfoam posed onto the chorioallantoic membrane, as a localized reservoir, which was connected to a miniosmotic pump system delivering astrocyte-conditioned medium at a steady rate. We found that in a significant number of chorioallantoic vessels located near the gelfoam, endothelial cells exposed to astrocyte-conditioned medium for a period of 4 or 10 days, but not to glioma-, fibroblast- or endothelial cell-derived conditioned medium, expressed the HT7 antigen and neurothelin. These results provide evidence that type-1-astrocytes are capable of inducing blood-brain barrier related properties in endothelial cells of non-neural origin through a soluble factor(s).

Induction of the blood-brain barrier specific HT7 and neurothelin epitopes in endothelial cells of the chick chorioallantoic vessels by a soluble factor derived from astrocytes.

Some blood-brain barrier properties of microvascular endothelial cells have been shown to be inducible by astrocytes. We tested the hypothesis that this cellular interaction is mediated by a soluble factor(s). Chick chorioallantoic vessels in ovo were constantly exposed to astrocyte-conditioned medium. We found that endothelial cells exposed to astrocyte-derived factors, but not to glioma- or endothelial cell-derived factors, expressed the HT7 antigen and neurothelin, two specific markers of the blood-brain barrier phenotype. These results indicate that a soluble factor(s) secreted by astrocytes is capable to induce specific blood-brain barrier properties in endothelial cells of non-neural origin.