RSV-CLASS -Clinical Assessment Severity Score: An easy-to-use clinical disease severity score for respiratory syncytial virus infection in hospitalized children.

Respiratory syncytial virus (RSV) is the most common cause of acute respiratory tract infection in infants and young children often leading to severe disease requiring hospitalization. However, validated tools for systematic assessment of disease severity are lacking. This study aimed at creating and validating a standardized, simple-to-use disease severity score for RSV infection in children-the RSV-CLASS (Clinical Assessment Severity Score). Therefore, data from over 700 RSV-infected children over six winter seasons (2014-2020) was analyzed using univariate and multiple regression analyses for the prediction of lower respiratory tract infection (LRTI) as a proxy for a severe course of the disease. Testing a broad range of respiratory symptoms, they eventually yielded seven items. Performing stepwise selection, these were reduced to the final four items: cough, tachypnea, rales, and wheezing, each receiving one point in the proposed score named RSV-CLASS. The score was calculated for children in two cohorts A and B, one for development and one for validation, with an area under the curve of 0.90 and 0.87, respectively. With a score value of 3 or 4, 97.8% and 100% of the children, respectively, were admitted with LRTI and classified correctly. The RSV-CLASS is a disease severity score based on a neutral, analytical approach using prospective data from a large study cohort. It will contribute to systematically assessing the disease severity of RSV infection and can be used for evidence-based clinical decision-making as well as for research settings.

Methods for L-ribooligonucleotide sequence determination using LCMS.

The ability to verify the sequence of a nucleic acid-based therapeutic is an essential step in the drug development process. The challenge associated with sequence identification increases with the length and nuclease resistance of the nucleic acid molecule, the latter being an important attribute of therapeutic oligonucleotides. We describe methods for the sequence determination of Spiegelmers, which are enantiomers of naturally occurring RNA with high resistance to enzymatic degradation. Spiegelmer sequencing is effected by affixing a label or hapten to the 5'-end of the oligonucleotide and chemically degrading the molecule in a controlled fashion to generate fragments that are then resolved and identified using liquid chromatography-mass spectrometry. The Spiegelmer sequence is then derived from these fragments. Examples are shown for two different Spiegelmers (NOX-E36 and NOX-A12), and the specificity of the method is shown using a NOX-E36 mismatch control.

Viral Etiology and Clinical Characteristics of Acute Respiratory Tract Infections in Hospitalized Children in Southern Germany (2014-2018).

Background: Viral acute respiratory tract infections (ARTIs) are a leading cause of hospitalization in infants and young children. Methods: During the winter seasons of 2014-2018, hospitalized children (<18 years) with symptoms of ARTI were prospectively included at the University Hospital Heidelberg, Germany. Nasopharyngeal swabs were obtained for multiplex molecular analysis of 10 groups of respiratory viruses, and clinical data were obtained using a standardized questionnaire. Results: Of 1353 children included in this study, 1142 (84.4%) were positive for ≥1 viral pathogen. Virus monoinfection was detected in 797 (69.8%) children, whereas 345 (30.2%) children had coinfections with 2-4 viral pathogens. Respiratory syncytial virus (RSV), rhinovirus, and influenza virus were the main pathogens detected. RSV-positive children had significantly more often lower ARTIs, including symptoms of severe cough, wheezing, chest indrawing, tachypnea, and pulmonary rales. Hospitalized children aged <6 months represented the largest age group with detection of ≥1 viral pathogen (455/528 [86.2%] children). Coinfection was more frequent in younger children and, particularly for RSV with rhinovirus, significantly associated with more severe respiratory symptoms (P = .01). Conclusions: A better understanding of the etiology of viral ARTIs among hospitalized children plays a key role for future strategies in prevention, control, and treatment of respiratory viral infections.

Topical glucocorticoid application causing iatrogenic Cushing's syndrome followed by secondary adrenal insufficiency in infants: two case reports.

BACKGROUND: Iatrogenic Cushing's syndrome induced by oral and parenteral glucocorticoid administration is a well-known complication. Immediate withdrawal from exogenous steroids can lead to life-threatening adrenal insufficiency. However, Cushing's syndrome caused by topical treatment with glucocorticoids, such as dexamethasone eye drops or dermal application, is rarely recognized. Young infants in particular are at high risk of suffering from iatrogenic Cushing's syndrome when treated with highly potent topical glucocorticoids. CASE PRESENTATION: We present a 6-month-old Syrian boy with cushingoid face after dermal clobetasol cream treatment and a 2-year-old Iranian girl with severe growth retardation after application of dexamethasone eye drops. Both families have a migration background and language barriers. In both cases no endogenous cortisol secretion was initially detected in serum and in 24-hour collected urine. After dose reduction of glucocorticoids, severity of symptoms was reversible and serum cortisol was detectable. DISCUSSION AND CONCLUSION: Young infants are at high risk of developing Cushing's syndrome from topically applied highly potent glucocorticoids. Precise recommendations of treatment dosage, duration, and frequency must be given to the parents, and if necessary, with the help of an interpreter. Monitoring of height and weight as well as regular pediatric follow-ups should be scheduled. Physicians should be aware of potential adrenal insufficiency following withdrawal from long-term topical glucocorticoid treatment, and hydrocortisone treatment should be considered.

RSVpredict: An Online Tool to Calculate the Likelihood of Respiratory Syncytial Virus Infection in Children Hospitalized With Acute Respiratory Disease.

BACKGROUND: Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection in young children. Early detection of RSV infection can avoid unnecessary diagnostic and therapeutic intervention and is required to prevent the nosocomial spread of RSV infection in pediatric hospitals. We developed a web tool to calculate the probability of RSV infection in children hospitalized with acute respiratory tract infection (ARTI) (RSVpredict). METHODS: During winter seasons 2014/2015 to 2017/2018, 1545 children hospitalized with clinical symptoms of ARTI at the University Hospital Heidelberg/Germany were prospectively included. Medical information was reported on a standardized data sheet, and nasopharyngeal swabs were obtained for multiplex real-time polymerase chain reaction analyses. We applied logistic regression to develop a prediction model and developed a web-based application to predict the individual probability of RSV infection. RESULTS: Duration of clinical symptoms ≥2 days on admission, calendar month of admission, admission for lower respiratory tract infection, the presence of cough and rale and younger age were associated with RSV infection (P < 0.05). Those data were included in the prediction model (RSVpredict, https://web.imbi.uni-heidelberg.de/rsv/). RSVpredict is a web-based application to calculate the risk of RSV infection in children hospitalized with ARTI. The prediction model is based on easily accessible clinical symptoms and predicts the individual probability of RSV infection risk immediately. CONCLUSIONS: Pediatricians might use the RSVpredict to take informed decisions on further diagnostic and therapeutic intervention, including targeted RSV testing in children with relevant RSV infection risk.

Automated detection of covalent adducts to human serum albumin by immunoaffinity chromatography, on-line solution phase digestion and liquid chromatography-mass spectrometry.

A generic method for the detection of covalent adducts to the cysteine-34 residue of human serum albumin (HSA) has been developed, based on an on-line combination of immunoaffinity chromatography for selective sample pre-treatment, solution phase digestion, liquid chromatography and tandem mass spectrometry. Selective anti-HSA antibodies immobilized on agarose were used for sample pre-concentration and purification of albumin from the chemically produced alkylated HSA. After elution, HSA and HSA adducts are mixed with pronase and directed to a reaction capillary kept at a digestion temperature of 70 degrees C. The digestion products were trapped on-line on a C18 SPE cartridge. The peptides were separated on a reversed-phase column using a gradient of organic modifier and subsequently detected using tandem mass spectrometry. Modified albumin samples consisted of synthetically alkylated HSA by the reactive metabolite of acetaminophen, N-acetyl-p-benzoquinoneimine (NAPQI), and using the alkylating agent 1-chloro-2,4-dinitrobenzene (CDNB) as reference. The resulting mixture of alkylated versus non-modified albumin has been applied to the on-line system, and alkylation of HSA is revealed by the detection of the modified marker tetra-peptide glutamine-cysteine-proline-phenylalanine (QCPF) adducts NAPQI-QCPF and CDNB-QCPF. Detection of alkylated species was enabled by the use of data comparison algorithms to distinguish between unmodified and modified HSA samples. The in-solution digestion proved to be a useful tool for enabling fast (less than 2 min) and reproducible on-line digestion of HSA. A detection limit of 1.5 micromol/L of modified HSA could be obtained by applying 10 microL of NAPQI-HSA sample.

Reverse-transcription loop-mediated isothermal amplification for rapid detection of respiratory syncytial virus directly from nasopharyngeal swabs.

Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infection in young infants and a major cause of nosocomial infection in pediatric care. Currently available RSV point-of-care tests are of limited sensitivity and relatively expensive. We developed and evaluated a novel RSV rapid test for use at point-of-care, based on reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for direct testing of nasopharyngeal swab specimens. RT-LAMP can detect RSV within 30min, without the need for RNA extraction. The sensitivity of our RT-LAMP assay was 70-80% in comparison to RT-PCR. The RT-LAMP test sensitivity is at least equivalent to currently available rapid antigen detection tests (RADT), and the cost of RT-LAMP test reagents is only approximately 10% of that of commercially available RADT tests. RT-LAMP appears to be an attractive alternative to RADT, particularly in settings with limited financial resources. Future improvements could include lyophilization of test reagents and automated read-out of RT-LAMP results.

Selective quantitative bioanalysis of proteins in biological fluids by on-line immunoaffinity chromatography-protein digestion-liquid chromatography-mass spectrometry.

A quantitative method for the determination of proteins in complex biological matrices has been developed based on the selectivity of antibodies for sample purification followed by proteolytic digestion and quantitative mass spectrometry. An immunosorbent of polyclonal anti-bovine serum albumin (BSA) antibodies immobilized on CNBR agarose is used in the on-line mode for selective sample pretreatment. Next, the purified sample is trypsin digested to obtain protein specific peptide markers. Subsequent analysis of the peptide mixture using a desalination procedure and a separation step coupled, on-line to an ion-trap mass spectrometer, reveals that this method enables selective determination of proteins in biological matrices like diluted human plasma. This approach enhances substantially the selectivity compared to common quantitative analysis executed with immunoassays and colorimetry, fluorimetry or luminescence detection. Hyphenation of the immunoaffinity chromatography with on-line digestion and chromatography-mass spectrometry is performed and a completely on-line quantification of the model protein BSA in bovine and human urine was established. A detection limit of 170 nmol/l and a quantification limit of 280 nmol/l is obtained using 50 microl of either standard or spiked biological matrix. The model system allows fully automated absolute quantitative mass spectrometric analysis of intact proteins in biological matrices without time-consuming labeling procedures.

RNA aptamers and spiegelmers: synthesis, purification, and post-synthetic PEG conjugation.

This unit describes the solid-phase synthesis and downstream processing for RNA oligonucleotides with a length of up to 40 to 50 nucleotides on a 1- to 4-mmol scale with subsequent conjugation to PEG using the L-RNA spiegelmer NOX-E36 as an example. Following synthesis and two-step deprotection, the crude oligonucleotide is purified by preparative reversed-phase HPLC and desalted by tangential flow ultrafiltration. The resulting intermediate amino-modified oligonucleotide is reacted with NHS-ester-activated PEG, and the oligonucleotide-PEG conjugate is obtained after preparative AX-HPLC purification, followed by ultrafiltration and lyophilization. Critical process parameters are described, as well as time considerations and examples for analytical methods used as in-process and quality controls.