SARS-CoV-2 Viral Replication Persists in the Human Lung for Several Weeks after Symptom Onset.

Rationale: In the upper respiratory tract, replicating (culturable) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is recoverable for ∼4-8 days after symptom onset, but there is a paucity of data about the frequency and duration of replicating virus in the lower respiratory tract (i.e., the human lung).Objectives: We undertook lung tissue sampling (needle biopsy) shortly after death in 42 mechanically ventilated decedents during the Beta and Delta waves. An independent group of 18 ambulatory patients served as a control group.Methods: Lung biopsy cores from decedents underwent viral culture, histopathological analysis, electron microscopy, transcriptomic profiling, and immunohistochemistry.Measurements and Main Results: Thirty-eight percent (16 of 42) of mechanically ventilated decedents had culturable virus in the lung for a median of 15 days (persisting for up to 4 wk) after symptom onset. Lung viral culture positivity was not associated with comorbidities or steroid use. Delta but not Beta variant lung culture positivity was associated with accelerated death and secondary bacterial infection (P < 0.05). Nasopharyngeal culture was negative in 23.1% (6 of 26) of decedents despite lung culture positivity. This hitherto undescribed biophenotype of lung-specific persisting viral replication was associated with an enhanced transcriptomic pulmonary proinflammatory response but with concurrent viral culture positivity.Conclusions: Concurrent rather than sequential active viral replication continues to drive a heightened proinflammatory response in the human lung beyond the second week of illness and was associated with variant-specific increased mortality and morbidity. These findings have potential implications for the design of interventional strategies and clinical management of patients with severe coronavirus disease (COVID-19).

T-Cell Homeostatic Imbalance in Placentas From Women With Human Immunodeficiency Virus in the Absence of Vertical Transmission.

BACKGROUND: Implementation of universal antiretroviral therapy (ART) has significantly lowered vertical transmission rates but has also increased numbers of human immunodeficiency virus (HIV)-exposed uninfected children, who remain vulnerable to morbid effects. In the current study, we investigated whether T-cell alterations in the placenta contribute to altered immune status in HIV-exposed uninfected. METHODS: We analyzed T cells from term placenta decidua and villous tissue and paired cord blood from pregnant women living with HIV (PWH) who initiated ART late in pregnancy (n = 21) with pregnant women not living with HIV (PWNH) (n = 9). RESULTS: Placentas from PWH showed inverted CD4/CD8 ratios and higher proportions of tissue resident CD8+ T cells in villous tissue relative to control placentas. CD8+ T cells in the fetal capillaries, which were of fetal origin, were positively correlated with maternal plasma viremia before ART initiation, implying that imbalanced T cells persisted throughout pregnancy. In addition, the expanded memory differentiation of CD8+ T cells was confined to the fetal placental compartment and cord blood but was not observed in the maternal decidua. CONCLUSIONS: T-cell homeostatic imbalance in the blood circulation of PWH is reflected in the placenta. The placenta may be a causal link between HIV-induced maternal immune changes during gestation and altered immunity in newborn infants in the absence of vertical transmission.

Renal dysfunction, rod-cone dystrophy, and sensorineural hearing loss caused by a mutation in RRM2B.

More than two decades ago, a recessive syndromic phenotype affecting kidneys, eyes, and ears, was first described in the endogamous Afrikaner population of South Africa. Using whole-exome sequencing of DNA from two affected siblings (and their carrier parents), we identified the novel RRM2B c.786G>T variant as a plausible disease-causing mutation. The RRM2B gene is involved in mitochondrial integrity, and the observed change was not previously reported in any genomic database. The subsequent screening revealed the variant in two newly presenting unrelated patients, as well as two patients in our registry with rod-cone dystrophy, hearing loss, and Fanconi-type renal disease. All patients with the c.786G>T variant share an identical 1.5 Mb haplotype around this gene, suggesting a founder effect in the Afrikaner population. We present ultrastructural evidence of mitochondrial impairment in one patient, to support our thesis that this RRM2B variant is associated with the renal, ophthalmological, and auditory phenotype.

Clinicopathologic Characterization of Children With B-Cell Non-Hodgkin Lymphoma Over 10 Years at a Tertiary Center in Cape Town, South Africa.

BACKGROUND: We characterized B-cell non-Hodgkin lymphoma (NHL) cases over 10 years at a tertiary children's hospital to contribute to the body of knowledge on pediatric lymphoma in developing countries with a high human immunodeficiency virus (HIV) burden. METHODS: A retrospective cohort study was carried out using clinical and laboratory records of children newly diagnosed with B-cell NHL from January 2005 to December 2014. RESULTS: Seventy-five children ≤15 years of age were included. The majority had Burkitt lymphoma (n=61). Overall, (n=19) were HIV positive and 16% (n=12) had concurrent active tuberculosis. Bulky disease was present in 65.7% (n=46) and 30.1% (n=22) were classified as Lymphomes Malins B risk group C. The 5-year survival estimates for HIV-negative and HIV-positive children were similar in our cohort: 81% versus 79% for event-free survival and 85% versus 83.9% for overall survival. Of 3 children with Burkitt lymphoma, HIV, and Lymphomes Malins B group C, 2 died within 1 year. CONCLUSIONS: Irrespective of HIV status, the survival of children in our B-cell NHL cohort compares favorably with cure rates in developed nations, although advanced disease remains associated with a poor prognosis. Characterization of childhood NHL cases contributes to accurate risk stratification and tailored treatment.

Characteristics and Outcomes of Autoimmune Hepatitis from a Tertiary Paediatric Centre, Cape Town, South Africa.

OBJECTIVES: To describe the clinical characteristics, biochemical and histological features, outcomes and predictors of prognosis of children with autoimmune hepatitis (AIH) from a paediatric centre in South Africa. METHODS: Thirty-nine children diagnosed with AIH at Red Cross War Memorial Children's Hospital between 2005 and 2015 were included. Relevant patient's data were retrieved from the hospital's medical records and database. Liver biopsy slides were reviewed. Ethical approval was obtained. Data were analysed using SPSS. RESULTS: Females were 29 (74%). Mean age at presentation was 7.27 ± 3.35 years and the mean follow-up was 4.5 ± 2.4 years. Jaundice was present in 97% of patients at presentation. An acute presentation was observed in 26 (67%) even though cirrhosis was detected in 22 (56%). Autoantibody screening was completed in 35 patients, 20 (57%) were AIH-1, 1 (3%) was AIH-2 and 14 (40%) were seronegative AIH. Of the 25 patients who underwent magnetic resonance cholangiography 17 (68%) had associated autoimmune sclerosing cholangitis. The remission rate was 79%. However, 11 children relapsed later. One child required liver transplantation and one demised. Seronegative and seropositive patients have comparable characteristics and outcomes. While a higher alanine transaminase (ALT) level at presentation is a significant predictor of remission, a lower ALT level and cirrhosis are significant risk factors for unfavourable outcome. Overall survival rate was 97%. CONCLUSION: AIH responds well to therapy with excellent survival. Hence, it should be considered in any child presenting with viral screen negative hepatitis and start therapy timeously to prevent disease progression.

Collagenous gastritis: An unusual cause of generalized oedema in a child.

Collagenous gastritis is an uncommon gastrointestinal disease in children. Its cause remains uncertain. It may present as severe hypoproteinaemia manifesting as generalized oedema. We report a 15 months old female who presented with pica, generalized body oedema and diarrhoea. Diagnostic workup revealed gastric replacement of the lamina propria by hyalinized collagen on histology. This case seeks to highlight the need for early paediatric gastroenterology referral including oesophagogastroduodenoscopy with multiple tissue biopsies as part of a broad diagnostic workup in children with non-specific gastrointestinal symptoms to improve diagnostic yield and enable accurate histologic diagnosis, so that appropriate therapy can be timeously applied.

Epigenetic changes as a common trigger of muscle weakness in congenital myopathies.

Congenital myopathies are genetically and clinically heterogeneous conditions causing severe muscle weakness, and mutations in the ryanodine receptor gene (RYR1) represent the most frequent cause of these conditions. A common feature of diseases caused by recessive RYR1 mutations is a decrease of ryanodine receptor 1 protein content in muscle. The aim of the present investigation was to gain mechanistic insight into the causes of this reduced ryanodine receptor 1. We found that muscle biopsies of patients with recessive RYR1 mutations exhibit decreased expression of muscle-specific microRNAs, increased DNA methylation and increased expression of class II histone deacetylases. Transgenic mouse muscle fibres over-expressing HDAC-4/HDAC-5 exhibited decreased expression of RYR1 and of muscle-specific miRNAs, whereas acute knock-down of RYR1 in mouse muscle fibres by siRNA caused up-regulation of HDAC-4/HDAC-5. Intriguingly, increased class II HDAC expression and decreased ryanodine receptor protein and miRNAs expression were also observed in muscles of patients with nemaline myopathy, another congenital neuromuscular disorder. Our results indicate that a common pathophysiological pathway caused by epigenetic changes is activated in some forms of congenital neuromuscular disorders.

Outcomes of children with rhabdomyosarcoma treated with intensive chemotherapy, surgery, and radiotherapy through a period of protocol revision at a South African center, 1990-2010.

A total of 75 children with biopsy-proven rhabdomyosarcoma were treated at our institution between 1990 and 2010. Five-year overall survival (OS) for the entire cohort was 58.7%. OS by stage was as follows: Stage 1 (80%), Stage 2 (80%), Stage 3 (54.1%), and Stage 4 (38.5%). There was a trend to suggest that revision of treatment approaches improved crude survival over time: pre-2003 (OS 42.1%); 2003-2005 (OS 50.0%); 2005-2010 (OS 60.8%).

Extraocular Muscle Findings in Myasthenia Gravis Associated Treatment-Resistant Ophthalmoplegia.

We report the histopathological and ultrastructural tissue analysis of extraocular muscle (EOM) obtained from a patient with seronegative myasthenia gravis (MG) with treatment-resistant ophthalmoplegia for 3.5 years. The EOM demonstrated predominantly myopathic features and ultrastructural evidence of mitochondrial dysfunction, but the most striking features were increased endomysial collagen and adipocyte replacement of muscle fibers. By contrast, control EOM from a patient undergoing strabismus surgery for a sensory exotropia in a nonseeing eye and a similar duration of deviation, showed normal muscle histology. Although the histopathological and ultrastructural findings largely resemble those of limb muscle in MG, the abundant endomysial collagen may be nonspecific and secondary to poor force generation as a result of chronic ophthalmoplegia.

Mutations in FAM111B cause hereditary fibrosing poikiloderma with tendon contracture, myopathy, and pulmonary fibrosis.

Congenital poikiloderma is characterized by a combination of mottled pigmentation, telangiectasia, and epidermal atrophy in the first few months of life. We have previously described a South African European-descent family affected by a rare autosomal-dominant form of hereditary fibrosing poikiloderma accompanied by tendon contracture, myopathy, and pulmonary fibrosis. Here, we report the identification of causative mutations in FAM111B by whole-exome sequencing. In total, three FAM111B missense mutations were identified in five kindreds of different ethnic backgrounds. The mutation segregated with the disease in one large pedigree, and mutations were de novo in two other pedigrees. All three mutations were absent from public databases and were not observed on Sanger sequencing of 388 ethnically matched control subjects. The three single-nucleotide mutations code for amino acid changes that are clustered within a putative trypsin-like cysteine/serine peptidase domain of FAM111B. These findings provide evidence of the involvement of FAM111B in congenital poikiloderma and multisystem fibrosis.

Orbital lymphomatoid granulomatosis - a rare cause of proptosis.

A 1-year-old girl with unilateral proptosis was found to have primary orbital lymphomatoid granulomatosis - a condition rarely occurring in children. This multisystem angiocentric, angiodestructive, lymphoproliferative disease typically involves the lungs, with ocular involvement being extremely uncommon. Our case serves to illustrate the imaging findings of this unusual condition and highlight a rare cause of proptosis.

Mitochondrial impairment observed in fibroblasts from South African Parkinson's disease patients with parkin mutations.

Parkinson's disease (PD), defined as a neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra in the midbrain. Loss-of-function mutations in the parkin gene are a major cause of autosomal recessive, early-onset PD. Parkin has been implicated in the maintenance of healthy mitochondria, although previous studies show conflicting findings regarding mitochondrial abnormalities in fibroblasts from patients harboring parkin-null mutations. The aim of the present study was to determine whether South African PD patients with parkin mutations exhibit evidence for mitochondrial dysfunction. Fibroblasts were cultured from skin biopsies obtained from three patients with homozygous parkin-null mutations, two heterozygous mutation carriers and two wild-type controls. Muscle biopsies were obtained from two of the patients. The muscle fibers showed subtle abnormalities such as slightly swollen mitochondria in focal areas of the fibers and some folding of the sarcolemma. Although no differences in the degree of mitochondrial network branching were found in the fibroblasts, ultrastructural abnormalities were observed including the presence of electron-dense vacuoles. Moreover, decreased ATP levels which are consistent with mitochondrial dysfunction were observed in the patients' fibroblasts compared to controls. Remarkably, these defects did not manifest in one patient, which may be due to possible compensatory mechanisms. These results suggest that parkin-null patients exhibit features of mitochondrial dysfunction. Involvement of mitochondria as a key role player in PD pathogenesis will have important implications for the design of new and more effective therapies.

Microsporidiosis in pediatric renal transplant patients in Cape Town, South Africa: two case reports.

Microsporidia are an emerging group of pathogens associated with life-threatening opportunistic infections in immunocompromised hosts, particularly human immunodeficiency virus (HIV)-infected individuals. There have, however, been recent reports of infection in adult solid organ transplant recipients. We report two cases in children, to our knowledge the first in the paediatric literature. Two 13-yr-old, HIV-seronegative females received deceased donor renal transplants from the same donor. Both patients suffered acute cell-mediated rejection and CMV infection reactivation, managed with intensified immunosuppression and ganciclovir. Pyrexia of unknown origin and intermittent diarrhea in both prompted extensive investigations. In both patients, numerous spores of a microsporidial species were demonstrated in renal tissue on biopsy and in the urine, using modified trichrome and quick-hot Gram-chromotrope staining. Electron microscopy and PCR confirmed Encephalitozoon cuniculi infections. Both patients were successfully treated with 400 mg twice daily of albendazole, with sustained clinical improvement. We recommend that microsporidiosis be considered in the differential diagnosis of pyrexia of unknown origin in severely immunocompromised pediatric solid organ transplant recipients, particularly when associated with diarrhea.

Magnetic resonance imaging versus histopathology in Wilms tumor and nephroblastomatosis: 3 examples of noncorrelation.

Magnetic resonance imaging (MRI) has become the principal tool for Wilms tumor (WT) assessment and follow-up. MRI and histopathologic findings were not congruent in 2 of the q30 scanned patients with renal masses (2008 to 2011). Three lesions thought to be WT on MRI were found to be a sclerotic nephrogenic rest (1), cystic renal dysplasia (1), and focal chronic pyelonephritis (1). The "typical" features suggesting nephroblastomatosis and WT on MRI are unreliable and such lesions require biopsy for histopathologic diagnosis, especially when nephron-sparing surgery is necessary to preserve renal function.

Investigating somatic variants and pathways in mismatch repair-deficient (dMMR) colorectal carcinoma in South Africa.

AIMS: Colorectal carcinoma (CRC) is a common cause of morbidity and mortality worldwide, and an emerging public health problem in sub-Saharan Africa. Several authors have described an increased frequency of mismatch repair-deficient (dMMR) CRC in sub-Saharan Africa, but these tumours remain poorly characterised molecularly. We sought to interrogate the somatic molecular genetic landscape of dMMR CRC in a cohort of young patients to better inform Lynch syndrome (LS) screening strategies and personalised medicine approaches in our setting. METHODS: 32 patients (aged <60 years) were identified with dMMR CRC. DNA was extracted from selected formalin-fixed paraffin-embedded (FFPE) tissue resection samples and subjected to amplicon-based next-generation sequencing (NGS). RESULTS: Pathogenic or likely pathogenic variants were detected in the corresponding MMR gene in 14 of 18 (78%) MLH1/PMS2-deficient tumours, 5 of 8 (63%) MSH2/MSH6-deficient tumours, 1 of 4 (25%) tumours with isolated MSH6 loss and 0 of 2 tumours with isolated PMS2 loss. Previously unreported variants were identified in MLH1 (three) and MSH2 (one). Cases with a variant allele frequency suggesting a germline mutation were identified in MLH1 (eight), MSH2 (two) and MSH6 (one). Only one MMR gene variant was detected in more than one patient (MLH1 p.Q510*). Four POLE/POLD1 exonuclease domain variants were identified, one of which was previously unreported. CONCLUSION: The spectrum of disease-causing MMR gene variants in our population necessitates NGS testing for LS screening. This study also highlights the role of somatic testing on readily available FFPE samples to generate data on the epidemiology of CRC in different settings.

RyR1 deficiency in congenital myopathies disrupts excitation-contraction coupling.

In skeletal muscle, excitation-contraction (EC) coupling is the process whereby the voltage-gated dihydropyridine receptor (DHPR) located on the transverse tubules activates calcium release from the sarcoplasmic reticulum by activating ryanodine receptor (RyR1) Ca(2+) channels located on the terminal cisternae. This subcellular membrane specialization is necessary for proper intracellular signaling and any alterations in its architecture may lead to neuromuscular disorders. In this study, we present evidence that patients with recessive RYR1-related congenital myopathies due to primary RyR1 deficiency also exhibit downregulation of the alfa 1 subunit of the DHPR and show disruption of the spatial organization of the EC coupling machinery. We created a cellular RyR1 knockdown model using immortalized human myoblasts transfected with RyR1 siRNA and confirm that knocking down RyR1 concomitantly downregulates not only the DHPR but also the expression of other proteins involved in EC coupling. Unexpectedly, this was paralleled by the upregulation of inositol-1,4,5-triphosphate receptors; functionally however, upregulation of the latter Ca(2+) channels did not compensate for the lack of RyR1-mediated Ca(2+) release. These results indicate that in some patients, RyR1 deficiency concomitantly alters the expression pattern of several proteins involved in calcium homeostasis and that this may influence the manifestation of these diseases.

HHV-8 large T-cell lymphoma: an unusual malignancy amidst more common B-cell lymphomas in HIV-positive children treated at a single institution.

Non-Hodgkin lymphomas of B-cell origin are the most commonly encountered HIV-related lymphomas in adults and children. These lymphomas are often extranodal, involve the central nervous system or other sites, and have been found to behave in a more aggressive manner. We report an unusual case of a child with HHV-8-positive large T-cell lymphoma.

Genomic landscape of colorectal carcinoma in sub-Saharan Africa.

Our understanding of the molecular classification of colorectal carcinoma (CRC) has evolved significantly over the past two decades. Tumours can be broadly categorised as microsatellite stable (MSS), microsatellite instability (MSI) or CpG island-methylator phenotype. Prognostic and predictive information is provided by these categories. The overwhelming majority of the data on which these categories are based have originated from Europe and North America. There is a dearth of information represented from Africa and indigenous African patients. However, some small studies and preliminary data have shown significant differences in all of these groups. The prevalence of MSI in Africa is consistently reported as almost double that of European and North American data. Interestingly, BRAF V600E mutations and MLH1 promotor hypermethylation seem to be uncommon in Africa. The high proportion of MSI tumours is only partly accounted for by germline mutations in mismatch repair genes (Lynch syndrome), suggesting that there are likely to be other mechanisms at play. Within the MSS group, preliminary data suggest that the typical molecular pathways (Wingless/Integrated pathway activation) may not be as dominant in Africa. The purpose of this review is to summarise the current state of the molecular genetic landscape of CRC in Africa and provide insights into areas for further study.

Mucoepidermoid carcinoma of the bronchus in two children: Case reports.

Childhood mucoepidermoid carcinomas (MEC) of the bronchus are rare. They present with non-specific symptoms and signs making diagnosis delayed. We present two children with bronchial MEC managed in a tertiary children's hospital in Cape Town, South Africa. The first was a 11-year male with recurrent haemoptysis and the second child was a 6-year female with recurrent unifocal pneumonia. Chest CT scan and bronchoscopy with biopsy confirmed the diagnosis. Both patients underwent treatment, including surgery and are doing well. It is important to exclude endobronchial lesions when children present with recurrent respiratory symptoms, since early diagnosis will enable lung-sparing treatment.

Carboplatin-based chemotherapy and surgery: a cost effective treatment strategy for malignant extracranial germ cell tumours in the developing world.

Nineteen children with malignant extracranial germ cell tumours (MEGCT), were treated at our institution with a combination of carboplatin-based chemotherapy and surgery from 1992 to 2008. Seventeen are long term survivors. Chemotherapy was administered on an outpatient basis and required no monitoring of GFR or auditory function. There were no deaths from toxicity or infection. Blood product use was low. Carboplatin-based therapy achieved excellent results in our patient population and constitutes more than a US $1,800 saving per patient compared to regimens containing cisplatin. This has application in resource limited settings.