Proteomic studies in VWA1-related neuromyopathy allowed new pathophysiological insights and the definition of blood biomarkers.

Bi-allelic variants in VWA1, encoding Von Willebrand Factor A domain containing 1 protein localized to the extracellular matrix (ECM), were linked to a neuromuscular disorder with manifestation in child- or adulthood. Clinical findings indicate a neuromyopathy presenting with muscle weakness. Given that pathophysiological processes are still incompletely understood, and biomarkers are still missing, we aimed to identify blood biomarkers of pathophysiological relevance: white blood cells (WBC) and plasma derived from six VWA1-patients were investigated by proteomics. Four proteins, BET1, HNRNPDL, NEFM and PHGDH, known to be involved in neurological diseases and dysregulated in WBC were further validated by muscle-immunostainings unravelling HNRNPDL as a protein showing differences between VWA1-patients, healthy controls and patients suffering from neurogenic muscular atrophy and BICD2-related neuromyopathy. Immunostaining studies of PHGDH indicate its involvement in apoptotic processes via co-localisation with caspase-3. NEFM showed an increase in cells within the ECM in biopsies of all patients studied. Plasma proteomics unravelled dysregulation of 15 proteins serving as biomarker candidates among which a profound proportion of increased ones (6/11) are mostly related to antioxidative processes and have even partially been described as blood biomarkers for other entities of neuromuscular disorders before. CRP elevated in plasma also showed an increase in the extracellular space of VWA1-mutant muscle. Results of our combined studies for the first time describe pathophysiologically relevant biomarkers for VWA1-related neuromyopathy and suggest that VWA1-patient derived blood might hold the potential to study disease processes of clinical relevance, an important aspect for further preclinical studies.

European Federation of Neurological Societies cutoff values significantly reduce creatine kinase sensitivity for diagnosing neuromuscular disorders.

INTRODUCTION: Elevated creatine kinase (CK) level was redefined by the European Federation of Neurological Societies)EFNS(as 1.5 times the upper limit of normal. In the current study we sought to determine the sensitivity and specificity of CK testing for the diagnosis of neuromuscular disorders. METHODS: Demographics and CK levels were retrospectively extracted from an electronic database for 234 patients with neuromuscular disorders. Sensitivity, specificity, and likelihood ratios and the area under curve were determined for each diagnosis and different cutoff CK values. RESULTS: Using the EFNS cutoff values significantly reduced CK test sensitivity. Creatine kinase values >1000 IU/L showed a high likelihood (11.04) for myopathies and a low likelihood for polyneuropathies (0). DISCUSSION: European Federation of Neurological Societies cutoff values significantly reduce CK sensitivity for diagnosing neuromuscular disorders. While low CK values cannot exclude a neuromuscular disease, values >1000 IU/L are associated with a high likelihood of myopathy.

Neuromuscular Involvement in Glycogen Storage Disease Type III in Fifty Tunisian Patients: Phenotype and Natural History in Young Patients.

BACKGROUND: Our aim was to describe the natural history of neuromuscular involvement (NMI) in glycogen storage disease type III (GSDIII). METHODS: We conducted a longitudinal study of 50 Tunisian patients, 9.87 years old in average. RESULTS: NMI was diagnosed at an average age of 2.66 years and was clinically overt in 85% of patients. Patients with clinical features were older (p = 0.001). Complaints were dominated by exercise intolerance (80%), noticed at 5.33 years in average. Physical signs, observed at 6.75 years in average, were dominated by muscle weakness (62%). Functional impairment was observed in 64% of patients, without any link with age (p = 0.255). Among 33 patients, 7 improved. Creatine kinase (CK) and aspartate aminotransferase (AST) levels were higher with age.Electrophysiological abnormalities, diagnosed in average at 6.5 years, were more frequent after the first decade (p = 0.0005). Myogenic pattern was predominant (42%). Nerve conduction velocities were slow in two patients. Lower caloric intake was associated with more frequent clinical and electrophysiological features. Higher protein intake was related to fewer complaints and physical anomalies. CONCLUSION: Neuromuscular investigation is warranted even in asymptomatic patients, as early as the diagnosis of GSDIII is suspected. Muscle involvement can be disabling even in children. Favorable evolution is possible in case of optimal diet.

Proteomic serum biomarkers for neuromuscular diseases.

INTRODUCTION: The clinical evaluation of neuromuscular symptoms often includes the assessment of altered blood proteins or changed enzyme activities. However, the blood concentration of many muscle-derived serum markers is not specific for different neuromuscular disorders and also shows alterations in the course of these diseases. Thus, the establishment of more reliable biomarker signatures for improved muscle diagnostics is required. Areas covered: To address the lack of muscle disease-specific marker molecules, mass spectrometry-based proteomics was applied to the systematic identification and biochemical characterization of new serum biomarker candidates. This article outlines serum proteomics in relation to neuromuscular disorders and reviews the bioanalytical results from recent proteomic profiling studies of representative neuromuscular disorders, including motor neuron disease, muscular dystrophies and sarcopenia of old age. Pathophysiological changes in the skeletal muscle proteome are reflected by serum alterations in a variety of sarcomeric proteins, metabolic enzymes and signaling proteins. Expert commentary: Based on the proteomic identification of actively secreted or passively released skeletal muscle proteins following pathophysiological insults, new biomarker candidates can now be used to develop liquid biopsy procedures for superior diagnostic approaches, design novel prognostic tools and establish more reliable methods for the systematic evaluation of experimental therapies to treat neuromuscular disease.

Age-dependent neuromuscular impairment in prion protein knockout mice.

INTRODUCTION: The cellular prion protein (PrP(C) ) is commonly recognized as the precursor of prions, the infectious agents of the fatal transmissible spongiform encephalopathies, or prion diseases. Despite extensive effort, the physiological role of PrP(C) is still ambiguous. Evidence has suggested that PrP(C) is involved in different cellular functions, including peripheral nerve integrity and skeletal muscle physiology. METHODS: We analyzed the age-dependent influence of PrP(C) on treadmill test-based aerobic exercise capacity and on a series of morphological and metabolic parameters using wild-type and genetically modified mice of different ages expressing, or knockout (KO) for, PrP(C) . RESULTS: We found that aged PrP-KO mice displayed a reduction in treadmill performance compared with PrP-expressing animals, which was associated with peripheral nerve demyelination and alterations of skeletal muscle fiber type. CONCLUSION: PrP-KO mice have an age-dependent impairment of aerobic performance as a consequence of specific peripheral nerve and muscle alterations.

Carbon dioxide levels during polygraphy in children with sleep-disordered breathing.

PURPOSE: The recent scoring rules of the American Academy of Sleep Medicine (AASM) define hypoventilation in children as a carbon dioxide (CO2) level of >50 mmHg for >25 % of total sleep time (partial pressure of CO2 (PCO2) > 50[>25 %]). As there is no validated level of nocturnal hypoventilation with regard to end-organ damage in children, we evaluated the prevalence of hypoventilation with the AASM definition but also with a lesser degree of elevated CO2 in children with sleep-disordered breathing (SDB). METHODS: Transcutaneous CO2 (PtcCO2) was recorded during overnight polygraphy (PG). Hypoventilation was defined according to four definitions: the AASM score (PCO2 > 50[>25 %]), the peak value of PtcCO2 > 50 mmHg (PtcCO2 > 50[peak]), a percentage of PtcCO2 > 50 mmHg > 2 % of nighttime recording (PtcCO2 > 50[>2 %]) or a nocturnal PtcCO2 > 10 mmHg above waking baseline level (PtcCO2[>10 mmHg]). PtcCO2 indices were correlated to the apnoea-hypopnoea index (AHI) and oxygenation indices. RESULTS: PGs from 221 children with suspicion of obstructive sleep apnoea (72 %), neuromuscular diseases (21 %), and lung diseases (7 %) were analysed. The prevalence of hypoventilation according to PCO2 > 50[>25 %], PtcCO2 > 50[peak], PtcCO2 > 50[>2 %] and PtcCO2[>10 mmHg] were 16, 27, 31 and 52 %, respectively, and did not differ between the three diagnostic groups. Significant but weak correlations were observed between hypoventilation and AHI and oxygenation indices. CONCLUSIONS: Nocturnal hypoventilation occurs in a large number of children referred for SDB, independent of the underlying disease, when more stringent criteria than those of the AASM are used. The poor correlation between hypoventilation and AHI or oxygenation indices is in favour of CO2 being a supplemental index of SDB.

Recognition and management of motor delay and muscle weakness in children.

Diagnosis of neuromuscular disorders in young children is often delayed for years after symptoms emerge, resulting in missed opportunities for therapy and genetic counseling. Identification of the weak child begins with careful attention to caregiver concerns and developmental surveillance at well-child visits. Family and medical histories can differentiate inherited from acquired causes of weakness. Physical examination should include observation of ageappropriate motor skills such as pull-to-sit, sitting, rising to stand, and walking/running. Serum creatine kinase levels should always be measured in children exhibiting neuromuscular weakness. Referrals to early intervention programs should not be postponed pending definitive diagnosis. If motor delay does not improve with early intervention, referral to a pediatric neurologist for diagnostic assessment is recommended. Tongue fasciculations, loss of motor milestones, or creatine kinase level greater than three times the normal limit should prompt immediate neurology referral. Once a neuromuscular disorder is diagnosed, the primary care clinician can help the family navigate subspecialty visits and consultations, advocate for services in the school and home, and help them cope with the emotional stresses of caring for a child with special needs.

Cramp-fasciculation syndrome in patients with and without neural autoantibodies.

INTRODUCTION: We investigated the clinical, electrophysiological and neural autoantibody characteristics in cramp-fasciculation syndrome (CFS) patients. METHODS: We reviewed Mayo Clinic records from 2000 to 2011 to identify clinically defined CFS patients who underwent neural autoantibody testing. Stored sera of patients who tested positive for antibodies to voltage-gated potassium channel complex (VGKC complex) were analyzed further for leucine-rich glioma-inactivated 1 (LGI1) or contactin-associated protein-2 immunoglobulin G (CASPR2-IgG) antibodies. RESULTS: Thirty-seven patients were identified. Twelve were seropositive for neural autoantibodies. Clinical manifestations were similar in seropositive and seronegative patients, although central and autonomic neuronal hyperexcitability symptoms were more common in seropositive cases. No patients had a malignancy. Repetitive tibial nerve stimulation at 10 Hz revealed longer afterdischarges in seropositive patients. Two of 7 patients with VGKC-complex autoimmunity demonstrated LGI1 or CASPR2-IgG antibodies. Only 2 of 12 seropositive patients required immunotherapy. CONCLUSIONS: VGKC-complex autoimmunity occurs in a minority of CFS patients. Antibody positivity was associated with extramuscular manifestations, typically without malignancy. Target antigens within the VGKC complex remain unknown in most patients.

Single-fiber electromyography in hyperCKemia: the value of fiber density.

Although persistently raised serum creatine kinase (sCK), or hyperCKemia, is considered the biological hallmark of neuromuscular diseases, pauci- or asymptomatic- or isolated-hyperCKemia can often be found. Single-fiber electromyography (SFEMG) is an electrophysiological technique of great value in the assessment of neuromuscular, neuropathic and myopathic disorders. We hypothesize that SFEMG fiber density (FD) evaluation is able to detect subclinical electrophysiological abnormalities indicating a myopathic process in subjects with hyperCKemia. Nineteen subjects with hyperCKemia without evident clinical signs of muscle involvement and 15 healthy controls were studied. Electrophysiological investigations including nerve conduction studies (NCS), quantitative EMG (QEMG), SFEMG with focus on FD measurements, and muscle biopsy were performed. NCS, QEMG, SFEMG were normal in all controls. In subjects with hyperCKemia, NCS were normal; QEMG was abnormal in 5, while both SFEMG and muscle biopsy disclosed abnormalities in 12 subjects. The mean FD value was 2.6 ± 0.5 in the control and 4 ± 1.4 (p = 0.003) in the hyperCKemia group. SFEMG revealed subclinical changes in the majority of subjects with hyperCKemia. To the best of our knowledge, this is the first study demonstrating that SFEMG FD evaluation is able to detect the presence of muscle diseases, which are in a subclinical phase and would remain unidentified otherwise. SFEMG may be used to distinguish hyperCKemia associated to asymptomatic muscle disorders from idiopathic hyperCKemia. We believe that SFEMG FD evaluation should be added to the routine examinations in the screening of idiopathic hyperCKemia.

Efficacy and comfort of volume-guaranteed pressure support in patients with chronic ventilatory failure of neuromuscular origin.

BACKGROUND AND OBJECTIVE: Although non-invasive ventilation is widely used for the management of patients with neuromuscular disease, it remains unclear which ventilatory mode is associated with optimal patient-ventilator interaction and provides more effective gas exchange. The effects of volume-guaranteed pressure support (PSV-VTG) as compared with pressure support ventilation (PSV) and assisted pressure controlled ventilation (APCV) on breathing pattern and patient-ventilator synchrony during sleep, blood gases and subjective comfort were evaluated in a group of stable patients with neuromuscular disease. METHODS: PSV-VTG, PSV and APCV were applied in random order on three consecutive nights, during which cardio-respiratory monitoring was performed. Arterial blood gases were measured at the end of each ventilatory session. Evaluation of subjective sleep quality, comfort and perception of patient-ventilator synchrony was performed using a 10-item visual analogue questionnaire. RESULTS: Twenty-eight patients were included in the study. The different ventilatory modes showed similar effects on breathing pattern during sleep, arterial blood gases and degree of subjective comfort. Ineffective efforts were only observed during PSV-VTG and were inversely correlated with the variability of leaks (r=-0.61, P=0.015). A greater number of prolonged inspirations was observed with PSV-VTG and PSV compared with APCV (P=0.048 and 0.029, respectively). There were no correlations between patient comfort and physiological variables or ventilator setting. CONCLUSIONS: PSV-VTG did not outperform older modes of ventilation in terms of efficacy on breathing pattern and blood gases. In addition, application of PSV-VTG was associated with a higher rate of patient-ventilator dyssynchrony.

Transcutaneous Carbon Dioxide Measurement in Adult Patients with Neuromuscular Disorders: A quality Level Assessment.

BACKGROUND: Carbon dioxide tension (PCO2) monitoring during sleep, is crucial to identify respiratory failure in patients with neuromuscular disorders (NMD). Transcutaneous PCO2 monitoring is an available technique to measure PCO2. OBJECTIVES: To assess the quality level of transcutaneous blood gas measurements via SenTec monitor. METHODS: A 12-month analysis of SenTec measurements was conducted in a Belgian Centre for Home Mechanical Ventilation (HMV). Over two consecutive nights; SpO2 and PCO2 measurements, the presence of PCO2 drift and drift correction with SenTec, were reviewed and scores (0, 1, 2 for poor, medium and high level) were assigned to estimate the quality of measurements. RESULTS: Sixty-nine NMD patients met the inclusion criteria, of which 48/69 used HMV. PCO2 drift and drift correction were present in 15% and 68% of the 138 recordings, respectively. The quality level of measurements throughout night 1, scored 1.55 (0-2). The relevance of our clinical findings from SenTec scoring 1.94 (1-2); was considered highly satisfactory. HMV was ineffective in 24/48 patients. Among 12 patients with hypercapnia, 8 patients improved PCO2 between night 1 and 2. Among 12 patients with hypocapnia, PCO2 improved in 4/12 patients, who reached the range of normal PCO2 (35-47 mmHg). CONCLUSIONS: The quality of SenTec measurements was acceptable in the majority of recordings and clinical findings were deemed satisfactory in all cases. A single SenTec measurement was sufficient to determine the need for NIV. However, two SenTec registrations were insufficient to both improve NIV effectiveness in 50% of cases, and, to ensure follow-up of our interventions.

Absence of significant clinical benefit for a systematic routine creatine phosphokinase measurement in asymptomatic patients treated with anti-programmed death protein (ligand) 1 immune checkpoint inhibitor to screen cardiac or neuromuscular immune-related toxicities.

AIM: Despite unprecedented results of anti-programmed death protein (ligand) 1 (PD-(L)1) immune checkpoint inhibitor in the oncology's armamentarium, immune-related adverse events (irAEs) represent a therapeutic hurdle. Currently, there is no consensual recommendation on a routinely monitored biomarker to early detect irAE. Biological markers such as serum creatine phosphokinase (CPK) are commonly used to measure muscular tissue injury. The potential of routine serum CPK monitoring to predict cardiac or neuromuscular irAE in patients treated with immunotherapy remains unknown. METHODS: In this retrospective study between January 2016 and December 2018 at Gustave Roussy Cancer Campus, 1151 cancer patients treated with anti-PD-(L)1 immunotherapy were systematically monitored with serum CPK measurements before each immunotherapy cycle. We considered significant CPK increases according to Common Terminology Criteria for Adverse Events v5.0 (CTCAEV5) of grade ≥2 severity. Comparisons were performed in patients with immune-related CPK (ir-CPK) elevations symptomatic versus asymptomatic. RESULTS: Overall, 53 of 1151 (4.6%) patients showed a CPK increase. Elevations of CPK were deemed to be immunotherapy-related in 31 of 1151 (2.7%) patients. Among them, 12 of 31 (38.7%) patients experienced symptomatic cardiac or neuromuscular irAE, whereas the other 19 of 31 (61.3%) patients remained asymptomatic. In patients with symptomatic irAE, the mean ir-CPK level was higher compared with asymptomatic patients (1271 versus 771 UI/L, P value = 0.02). In the asymptomatic group, all patients experienced a spontaneous resolution of the ir-CPK increase, and none required medical intervention. CONCLUSION: Most patients with immune-related CPK increase remained asymptomatic. The CPK serum increase did not alter the clinical management of asymptomatic patients. The results of this study did not support a significant clinical interest for a systematic routine CPK monitoring in patients amenable to anti-PD-(L)1 immunotherapy.

Nocturnal Transcutaneous Blood Gas Measurements in a Pediatric Neurologic Population: A Quality Assessment.

Objective: To assess the quality of SpO2 and PCO2 recordings via transcutaneous monitoring in children with neurological conditions.Methods: Overnight transcutaneous SpO2 and PCO2 were analyzed. The presence of drift and drift correction was noted, and the rate of disrupted recordings scored (0: absence, 1; presence). The quality of recordings was also scored (0, 1, 2 for poor, medium, and high).Results: A total of 228 recordings from 64 children aged 9.7 ± 6 years were analyzed of which 42 used positive pressure respiratory support. The mean quality of the recordings was scored as 1.27 (0-2). PCO2 drift, drift correction, and disrupted recordings were present in 25%, 58%, and 26% of recordings, respectively. Satisfactory clinical decisions were taken in 91% of cases.Conclusion: The quality of transcutaneous sensor recordings was acceptable and clinical findings were deemed as satisfactory in the large majority of cases. Correction of PCO2 drift was challenging.

Coenzyme Q10: Clinical Applications beyond Cardiovascular Diseases.

Coenzyme Q10 (CoQ10) is an essential cofactor in oxidative phosphorylation (OXPHOS), present in mitochondria and cell membranes in reduced and oxidized forms. Acting as an energy transfer molecule, it occurs in particularly high levels in the liver, heart, and kidneys. CoQ10 is also an anti-inflammatory and antioxidant agent able to prevent the damage induced by free radicals and the activation of inflammatory signaling pathways. In this context, several studies have shown the possible inverse correlation between the blood levels of CoQ10 and some disease conditions. Interestingly, beyond cardiovascular diseases, CoQ10 is involved also in neuronal and muscular degenerative diseases, in migraine and in cancer; therefore, the supplementation with CoQ10 could represent a viable option to prevent these and in some cases might be used as an adjuvant to conventional treatments. This review is aimed to summarize the clinical applications regarding the use of CoQ10 in migraine, neurodegenerative diseases (including Parkinson and Alzheimer diseases), cancer, or degenerative muscle disorders (such as multiple sclerosis and chronic fatigue syndrome), analyzing its effect on patients' health and quality of life.

Measures of nocturnal oxyhemoglobin desaturation in children with neuromuscular disease or Prader-Willi syndrome.

OBJECTIVES: Evidence for nocturnal oximetry interpretation in patients with abnormal neuromuscular function is limited. We aimed to compare children with neuromuscular disease (NMD) or Prader-Willi syndrome (PWS) to otherwise healthy subjects with obstructive sleep-disordered breathing (SDB) or without respiratory disorder (controls) regarding nocturnal oximetry parameters. METHODS: We analyzed recordings from children with: (a) NMD; (b) PWS; (c) snoring and adenotonsillar hypertrophy and/or obesity (SDB); and (d) controls. Outcomes included: (a) basal SpO2 ; (b) proportions of subjects with McGill oximetry score (MOS) >1 (clusters of desaturations); and (c) desaturation index (SpO2 drops ≥3%/h-ODI3). RESULTS: Data of 12 subjects with NMD (median age, 5.2 years; IQR, 2.7, 8.2), 14 children with PWS (5 years; 2.3, 6.9), 21 children with SDB (5.8 years; 4.6, 9.6), and 20 controls (6.2 years; 5.4, 11.2) were analyzed. Children with NMD, PWS, and SDB had lower basal SpO2 than controls (95.6% [94.5%, 96.9%], 96.2% [95.1%, 97.4%], 96.1% [95.8%, 97.5%] vs 97.8% [97.2%, 97.9%], respectively; (P < .01). NMD and PWS showed the greatest negative effect on basal SpO2 (P < .05). Children with SDB or PWS had a higher risk of MOS >1 than patients with NMD (OR, 25.9 [95% CI, 3.4-200.4] and 9.5 [1.5-62.6]). NMD, PWS, and SDB were similar regarding ODI3, which was elevated compared to ODI3 in controls (P < .05). Frequent desaturations predominated in NMD, while periods of sustained desaturation were noted in NMD and PWS. CONCLUSION: PWS and NMD have a negative effect on basal SpO2 , while clusters of desaturations are prevalent in patients with PWS or obstructive SDB.

A Randomized Trial of Initiation of Chronic Noninvasive Mechanical Ventilation at Home vs In-Hospital in Patients With Neuromuscular Disease and Thoracic Cage Disorder: The Dutch Homerun Trial.

BACKGROUND: There is an increasing demand for home mechanical ventilation (HMV) in patients with chronic respiratory insufficiency. At present, noninvasive ventilation is exclusively initiated in a clinical setting at all four centers for HMV in the Netherlands. In addition to its high societal costs and patient discomfort, commencing HMV is often delayed because of a lack of hospital bed capacity. RESEARCH QUESTION: Is HMV initiation at home, using a telemonitoring approach, noninferior to in-hospital initiation in a nationwide study? STUDY DESIGN AND METHODS: We conducted a nationwide, randomized controlled noninferiority trial, in which every HMV center recruited 24 patients (home [n = 12] vs hospital [n = 12]) with a neuromuscular disease or thoracic cage disorder, all with an indication to start HMV. Change in arterial CO2 (Paco2) over a 6-month period was considered the primary outcome, and quality of life and costs were assessed as secondary outcomes. RESULTS: A total of 96 patients were randomized, most of them diagnosed with neuromuscular disease. We found a significant improvement in Paco2 within both groups (home: from 6.1 to 5.6 kPa [P < .01]; hospital: from 6.3 to 5.6 kPa [P < .01]), with no significant differences between groups. Health-related quality of life showed significant improvement on various subscales; however, no significant differences were observed between the home and hospital groups. From a societal perspective, a cost reduction of more than €3,200 ($3,793) per patient was evident in the home group. INTERPRETATION: This nationwide, multicenter study shows that HMV initiation at home is noninferior to hospital initiation, as it shows the same improvement in gas exchange and health-related quality of life. In fact, from a patient's perspective, it might even be a more attractive approach. In addition, starting at home saves over €3,200 ($3,793) per patient over a 6-month period. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT03203577; URL: www.clinicaltrials.gov.

Cytokine biomarkers associated with clinical cases of acute flaccid myelitis.

Acute flaccid myelitis (AFM) is a serious neurological illness first recognized in the United States in 2014, with subsequent outbreaks every two years. Following extensive etiologic testing by multiple laboratories of hundreds of specimens collected from patients diagnosed with AFM, no consistent cause of AFM has been identified. However, viruses, including enteroviruses, have been implicated through detection in non-sterile site specimens and antibody studies. Cytokines and chemokines play important roles in the modulation of the innate and adaptive immune response to pathogens. In the current study, we measured levels of cytokines and chemokines in serum and CSF collected from confirmed AFM patients and non-AFM control patients, to identify unique biomarkers as potential hallmarks of AFM pathogenesis. Analysis of ratios of cytokines and chemokines in the CSF compared to the serum indicate that the pro-inflammatory cytokines/chemokines IP-10 and IL-6 were significantly elevated in AFM patients compared to non-AFM patients. These results may provide additional insight into potential etiologies, pathogenic mechanisms, and treatments for AFM.

Contribution of Enteroviruses to Acute Central Nervous System or Systemic Infections in Northern Italy (2015-2017): Is It Time to Establish a National Laboratory-Based Surveillance System?

BACKGROUND: Enteroviruses (EVs) can cause infections and outbreaks of mild to severe diseases, such as central nervous system (CNS) and systemic infections. The contribution of EVs to acute CNS/systemic infections requiring hospitalization was assessed by analysing data extracted from virology laboratory database. METHODS: Real-life data obtained from two molecular virology laboratories located in Northern Italy were retrieved from databases and analysed retrospectively. The queries used to extract the data were (i) requests for EV-RNA detection in clear cerebrospinal fluid (CSF) specimens collected from hospitalized patients with suspected acute CNS (including aseptic meningitis, encephalitis, and acute flaccid myelitis/paralysis) or systemic infections (sepsis-like illness or fever (≥ 38°C) of unknown origin), (ii) CSF samples collected from January 1st, 2015, to December 31st, 2017. RESULTS: 582 requests of EV-RNA detection in CSF samples collected from as many patients of any age were recorded. EV-RNA was detected in 4.5% of the CSF samples; 92.3% of EV-positive cases were patients < 15 years, 58.3% of whom were < 3 months. EVs circulated all-year-round, and the highest EV-positive rates were observed from May to August. The risk of EV infection and the relative illness ratio value among children < 1 - year - old were significantly higher than those observed for older patients. CONCLUSIONS: EV surveillance should be carried out for all pediatric patients < 15 years and especially children less than 1 year of age with clinically suspected CNS infection/systemic infections. The implementation of a laboratory-based surveillance established for analysing the virological data provided by laboratories that routinely perform EV molecular testing may enable us to determine the impact of EVs that can cause infections requiring hospitalization.

Patterns of neutrophil serine protease-dependent cleavage of surfactant protein D in inflammatory lung disease.

The manuscript presents definitive studies of surfactant protein D (SP-D) in the context of inflammatory lung fluids. The extent of SP-D depletion in bronchoalveolar lavage fluid (BALF) of children affected with cystic fibrosis (CF) is demonstrated to correlate best with the presence of the active neutrophil serine protease (NSP) elastase. Novel C-terminal SP-D fragments of 27 kDa and 11 kDa were identified in patient lavage fluid in addition to the previously described N-terminal, 35-kDa fragment by the use of isoelectrofocusing, modified blotting conditions, and region-specific antibodies. SP-D cleavage sites were identified. In vitro treatment of recombinant human SP-D dodecamers with NSPs replicated the fragmentation, but unexpectedly, the pattern of SP-D fragments generated by NSPs was dependent on calcium concentration. Whereas the 35- and 11-kDa fragments were generated when incubations were performed in low calcium (200 microM CaCl(2)), incubations in physiological calcium (2 mM) with higher amounts of elastase or proteinase-3 generated C-terminal 27, 21, and 14 kDa fragments, representing cleavage within the collagen and neck regions. Studies in which recombinant SP-D cleavage by individual NSPs was quantitatively evaluated under low and high calcium conditions showed that the most potent NSP for cleaving SP-D is elastase, followed by proteinase-3, followed by cathepsin G. These relative potency findings were considered in the context of other studies that showed that active NSPs in CF BALF are in the order: elastase, followed by cathepsin G, followed by proteinase-3. The findings support a pre-eminent role for neutrophil elastase as the critical protease responsible for SP-D depletion in inflammatory lung disease.

Antibodies to Enteroviruses in Cerebrospinal Fluid of Patients with Acute Flaccid Myelitis.

Acute flaccid myelitis (AFM) has caused motor paralysis in >560 children in the United States since 2014. The temporal association of enterovirus (EV) outbreaks with increases in AFM cases and reports of fever, respiratory, or gastrointestinal illness prior to AFM in >90% of cases suggest a role for infectious agents. Cerebrospinal fluid (CSF) from 14 AFM and 5 non-AFM patients with central nervous system (CNS) diseases in 2018 were investigated by viral-capture high-throughput sequencing (VirCapSeq-VERT system). These CSF and serum samples, as well as multiple controls, were tested for antibodies to human EVs using peptide microarrays. EV RNA was confirmed in CSF from only 1 adult AFM case and 1 non-AFM case. In contrast, antibodies to EV peptides were present in CSF of 11 of 14 AFM patients (79%), significantly higher than controls, including non-AFM patients (1/5 [20%]), children with Kawasaki disease (0/10), and adults with non-AFM CNS diseases (2/11 [18%]) (P = 0.023, 0.0001, and 0.0028, respectively). Six of 14 CSF samples (43%) and 8 of 11 sera (73%) from AFM patients were immunoreactive to an EV-D68-specific peptide, whereas the three control groups were not immunoreactive in either CSF (0/5, 0/10, and 0/11; P = 0.008, 0.0003, and 0.035, respectively) or sera (0/2, 0/8, and 0/5; P = 0.139, 0.002, and 0.009, respectively).IMPORTANCE The presence in cerebrospinal fluid of antibodies to EV peptides at higher levels than non-AFM controls supports the plausibility of a link between EV infection and AFM that warrants further investigation and has the potential to lead to strategies for diagnosis and prevention of disease.