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Respiratory syncytial virus (RSV) and influenza viruses are important global causes of morbidity and mortality. We evaluated the diagnostic accuracy of the Luminex NxTAG respiratory pathogen panels (RPPs)™ (index) against other RPPs (comparator) for detection of RSV and influenza viruses. Studies comparing human clinical respiratory samples tested with the index and at least one comparator test were included. A random-effect latent class meta-analysis was performed to assess the specificity and sensitivity of the index test for RSV and influenza. Risk of bias was assessed using the QUADAS-2 tool and certainty of evidence using GRADE. Ten studies were included. For RSV, predicted sensitivity was 99% (95% credible interval [CrI] 96–100%) and specificity 100% (95% CrI 98–100%). For influenza A and B, predicted sensitivity was 97% (95% CrI 89–100) and 98% (95% CrI 88–100) respectively; specificity 100% (95% CrI 99–100) and 100% (95% CrI 99–100), respectively. Evidence was low certainty. Although index sensitivity and specificity were excellent, comparators’ performance varied. Further research with clear patient recruitment strategies could ascertain performance across different populations.Protocol Registration: Prospero CRD42021272062.

Since the first reports of hepatitis of unknown aetiology occurring in UK children, over 1000 cases have been reported worldwide, including 268 cases in the UK, with the majority younger than 6 years old. Using genomic, proteomic and immunohistochemical methods, we undertook extensive investigation of 28 cases and 136 control subjects. In five cases who underwent liver transplantation, we detected high levels of adeno-associated virus 2 (AAV2) in the explanted livers. AAV2 was also detected at high levels in blood from 10/11 non-transplanted cases. Low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), both of which enable AAV2 lytic replication, were also found in the five explanted livers and blood from 15/17 and 6/9 respectively, of the 23 non-transplant cases tested. In contrast, AAV2 was detected at low titre in 6/100 whole bloods from child controls from cohorts with presence or absence of hepatitis and/or adenovirus infection. Our data show an association of AAV2 at high titre in blood or liver tissue, with unexplained hepatitis in children infected in the recent HAdV-F41 outbreak. We were unable to find evidence by electron microscopy, immunohistochemistry or proteomics of HAdV or AAV2 viral particles or proteins in explanted livers, suggesting that hepatic pathology is not due to direct lytic infection by either virus. The potential that AAV2, although not previously associated with disease, may, together with HAdV-F41 and/or HHV-6, be causally implicated in the outbreak of unexplained hepatitis, requires further investigation.

In late 2019, SARS-CoV-2 emerged in the Wuhan province of China. Rapid global spread led to the Covid-19 pandemic. Rapid and accurate detection of SARS-CoV-2 has become a vitally important tool in controlling the spread of the virus. Lateral flow devices (LFDs) offer the potential advantage of speed and on-site testing. The sensitivity of these devices compared to the gold standard RT-PCR has been questioned. We compared the performance of the Innova lateral flow kit, recommended by the UK government, with our rapid in-house RT-PCR protocol using stored positive patient samples. The LFD device was found to be 6,000-10,000 times less sensitive than RT-PCR for the detection of SARS-CoV-2. Overall, the LFD detected 46.2% of the positives detected by RT-PCR. 50% of the LFD results were observed to be weak positives, only visible after careful examination by experienced laboratory staff. At lower viral loads, such as 10,000-100,000 RNA copies/ml, the LFD detected 22.2% of positives. In addition, two strong positives (3 and 1.5 million RNA copies/ml) were not detected by the LFD. The argument for use of LFD kits, despite their lack of sensitivity, is that they detect infectious virus and hence contagious individuals. At present, there is a lack of scientific evidence supporting this claim. The LFD used in the UK fails to identify individuals with considerable viral loads and has been subject to a class I recall by the US FDA but is still approved and recommended for use by the UK government. We believe that using LFD testing for assessing SARS-CoV-2 infection risk is a strategy which has risks that outweigh any benefits.

Background A critical window in infancy has been proposed, during which the microbiota may affect subsequent health. The longitudinal development of the oropharyngeal microbiota is under-studied and may be associated with early-life wheeze. We aimed to investigate the temporal association of the development of the oropharyngeal microbiota with early-life wheeze. Methods A population-based birth cohort based in London, UK was followed for 24 months. We collected oropharyngeal swabs at six time-points. Microbiota was determined using sequencing of the V3-V5 region of the 16S rRNA-encoding gene. Medical records were reviewed for the outcome of doctor diagnosed wheeze. We used a time-varying model to investigate the temporal association between the development of microbiota and doctor-diagnosed wheeze. Findings 159 participants completed the study to 24 months and for 98 there was complete sequencing data at all timepoints and outcome data. Of these, 26 had doctor-diagnosed wheeze. We observed significant increase in the abundance of Neisseria between 9 and 24 months in children who developed wheeze (p = 0∙003), while in those without wheezing there was a significant increment in the abundance of Granulicatella (p = 0∙012) between 9 and 12 months, and of Prevotella (p = 0∙018) after 18 months. Interpretation A temporal association between the respiratory commensal Granulicatella and also Prevotella with wheeze (negative), and between Neisseria and wheeze (positive) was identified in infants prior to one year of age. This adds to evidence for the proposed role of the microbiota in the development of wheeze. Fund Research funding from the Winnicott Foundation, Meningitis Now and Micropathology Ltd.

Neisseria meningitidis, the causative agent of meningococcal disease (MD), evades complement-mediated clearance upon infection by ‘hijacking’ the human complement regulator factor H (FH). The FH protein family also comprises the homologous FH-related (FHR) proteins, hypothesized to act as antagonists of FH, and FHR-3 has recently been implicated to play a major role in MD susceptibility. Here, we show that, next to FH and FHR-3, the circulating levels of all FH family proteins are equally decreased during pediatric MD. We did not observe a specific consumption of FH or FHR-3 by N. meningitidis during the first days of infection and the levels recovered over time. MD severity associated predominantly with a loss of FH. Strikingly, loss of FH and FHRs associated strongly with renal failure, suggesting insufficient protection of host tissue by the FH protein family. Retaining higher levels of FH family proteins may thus limit tissue injury during MD.

Shortage of reagents and consumables required for the extraction and molecular detection of SARS-CoV-2 RNA in respiratory samples has led many laboratories to investigate alternative approaches for sample preparation. Fomsgaard et al 20205 recently presented results using heat-processing of respiratory samples prior to RT-qPCR as an economical method enabling an extremely fast streamlining of the processes at virtually no cost.Here, we present our results using this method and highlight some major pitfalls that diagnostics laboratories should be aware of before proceeding with this technique. We first investigated various treatments using different temperatures, incubation times and sample volumes based on the above study to optimise the heat-treatment conditions. Although the initial data confirmed the published results, further investigations revealed unexpected inhibitory properties of some commonly used virus transport media (VTMs) on some commercially available RT-qPCR mixes, emphasising the critical importance of a thorough validation process to determine the most adapted reagents to be used depending on the sample types to be tested.In conclusion, although the method works, with very consistent Ct values and an excellent sensitivity when compared to a conventional RNA extraction method, it is critical to include an internal control to check each sample for potential inhibition.

Background: Kawasaki disease (KD) is a vasculitis of early childhood mimicking several infectious diseases. Differentiation between KD and infectious diseases is essential as KD's most important complication—the development of coronary artery aneurysms (CAA)—can be largely avoided by timely treatment with intravenous immunoglobulins (IVIG). Currently, KD diagnosis is only based on clinical criteria. The aim of this study was to evaluate whether routine C-reactive protein (CRP) and additional inflammatory parameters myeloid-related protein 8/14 (MRP8/14 or S100A8/9) and human neutrophil-derived elastase (HNE) could distinguish KD from infectious diseases. Methods and Results: The cross-sectional study included KD patients and children with proven infections as well as febrile controls. Patients were recruited between July 2006 and December 2018 in Europe and USA. MRP8/14, CRP, and HNE were assessed for their discriminatory ability by multiple logistic regression analysis with backward selection and receiver operator characteristic (ROC) curves. In the discovery cohort, the combination of MRP8/14+CRP discriminated KD patients (n = 48) from patients with infection (n = 105), with area under the ROC curve (AUC) of 0.88. The HNE values did not improve discrimination. The first validation cohort confirmed the predictive value of MRP8/14+CRP to discriminate acute KD patients (n = 26) from those with infections (n = 150), with an AUC of 0.78. The second validation cohort of acute KD patients (n = 25) and febrile controls (n = 50) showed an AUC of 0.72, which improved to 0.84 when HNE was included. Conclusion: When used in combination, the plasma markers MRP8/14, CRP, and HNE may assist in the discrimination of KD from both proven and suspected infection.

Background: Sepsis is one of the main reasons for non-elective admission to pediatric intensive care units (PICUs), but little is known about determinants influencing outcome. We characterized children admitted with community-acquired sepsis to European PICUs and studied risk factors for mortality and disability.Methods: Data were collected within the collaborative Seventh Framework Programme (FP7)-funded EUCLIDS study, which is a prospective multicenter cohort study aiming to evaluate genetic determinants of susceptibility and/or severity in sepsis. This report includes 795 children admitted with community-acquired sepsis to 52 PICUs from seven European countries between July 2012 and January 2016. The primary outcome measure was in-hospital death. Secondary outcome measures were PICU-free days censured at day 28, hospital length of stay, and disability. Independent predictors were identified by multivariate regression analysis.Results: Patients most commonly presented clinically with sepsis without a source (n = 278, 35%), meningitis/encephalitis (n = 182, 23%), or pneumonia (n = 149, 19%). Of 428 (54%) patients with confirmed bacterial infection, Neisseria meningitidis (n = 131, 31%) and Streptococcus pneumoniae (n = 78, 18%) were the main pathogens. Mortality was 6% (51/795), increasing to 10% in the presence of septic shock (45/466). Of the survivors, 31% were discharged with disability, including 24% of previously healthy children who survived with disability. Mortality and disability were independently associated with S. pneumoniae infections (mortality OR 4.1, 95% CI 1.1–16.0, P = 0.04; disability OR 5.4, 95% CI 1.8–15.8, P P P Conclusions: Despite widespread immunization campaigns, invasive bacterial disease remains responsible for substantial morbidity and mortality in critically ill children in high-income countries. Almost one third of sepsis survivors admitted to the PICU were discharged with some disability. More research is required to delineate the long-term outcome of pediatric sepsis and to identify interventional targets. Our findings emphasize the importance of improved early sepsis-recognition programs to address the high burden of disease.

Comparison of the genome of the Gram negative human pathogen Haemophilus quentini MP1 with other species of Haemophilus revealed that, although it is more closely related to Haemophilus haemolyticus than Haemophilus influenzae, the pathogen is in fact genetically distinct, a finding confirmed by phylogenetic analysis using the H. influenzae multilocus sequence typing genes. Further comparison with two other H. quentini strains recently identified in Canada revealed that these three genomes are more closely related than any other species of Haemophilus; however, there is still some sequence variation. There was no evidence of acquired antimicrobial resistance within the H. quentini MP1 genome nor any mutations within the DNA gyrase or topoisomerase IV genes known to confer resistance to fluoroquinolones, which has been previously identified in other H. quentini isolates. We hope by presenting the annotation and genetic comparison of the H. quentini MP1 genome it will aid the future molecular detection of this potentially emerging pathogen via the identification of unique genes that differentiate it from other species of Haemophilus.

We compared the blood RNA transcriptome of children hospitalized with influenza A H1N1/09, respiratory syncytial virus (RSV) or bacterial infection, and healthy controls. Compared to controls, H1N1/09 patients showed increased expression of inflammatory pathway genes and reduced expression of adaptive immune pathway genes. This was validated on an independent cohort. The most significant function distinguishing H1N1/09 patients from controls was protein synthesis, with reduced gene expression. Reduced expression of protein synthesis genes also characterized the H1N1/09 expression profile compared to children with RSV and bacterial infection, suggesting that this is a key component of the pathophysiological response in children hospitalized with H1N1/09 infection.

Haemophilus quentini is a rare and distinct genospecies of Haemophilus that has been suggested as a cause of neonatal bacteremia and urinary tract infections in men. We present the draft whole-genome sequence of H. quentini MP1 isolated from an infant in the United Kingdom, aiding future identification and detection of this pathogen.

Remote Sensing applications of the marine environment, such as benthic mapping or in-water parameter retrieval, can be highly sensitive to in-scene glint. Glint can negatively impact retrieval algorithms rendering the image data unusable in extreme cases. This work seeks to identify the best sensor tasking for minimizing glint when collecting data from an aircraft or space-based imaging platform while maximizing image quality. This paper describes the development of a glint-avoidance tool through the use of statistical surface models, in-water radiative transfer models, and simple atmospheric models to optimize collection planning to reduce the probability of glint contamination in remotely sensed data.

A conventional multiplex PCR assay that detects herpes simplex virus type 1 (HSV-1), HSV-2, varicella-zoster virus, and enteroviruses for the diagnosis of central nervous system infections was modified to be performed using the LightCycler system. The sensitivity of detection of each of the viruses using the LightCycler assay was compared to that of the conventional assay using external quality assessment material. The assays had equivalent sensitivities, but the LightCycler assay was more rapid, reduced the risk of contamination, and used an amplicon detection format that demonstrated greater discrimination than a gel electrophoresis method.

We report an HIV-positive patient who developed a unilateral retinitis and subsequent intracranial lesions. The finding of Epstein Barr virus (EBV) DNA at a > 1-log greater concentration in the vitreous compared to blood raised the possibility of a primary CNS non-Hodgkin's lymphoma, which was subsequently confirmed on brain biopsy. EBV DNA quantification acted as a diagnostic marker that led to a change in the management of our patient.

The Department of Defense (DoD) Space Test Program (STP) was created in 1965 to provide access to space for DoD research and development activities. STP provides spaceflight opportunities for payloads vetted through the DoD Space Experiments Review Board (SERB). Small spacecraft acquisition is one of STP's key areas of excellence; it is a key aspect to the program that enables the program to fly multi-payload missions for low costs and provides flexibility to fly on a variety of launch vehicles. To facilitate the space flight of these experiments, the Evolved Expendable Launch Vehicle (EELV) Secondary Payload Adaptor (ESPA) was developed and flown under the direction of STP. The same payload interface requirements and environments defined for ESPA are now being applied to other multi-payload adaptors (MPAs) for other launch vehicles. To fully utilize the capabilities and opportunities presented by ESPA opportunities, STP is looking to take advantage of the ESPA form factor and success of the Standard Interface Vehicle (SIV) Program through lessons learned and innovative acquisition approaches. STP's goal is to access capable, low cost spacecraft buses through partnering and utilizing the pre-existing industry base while encouraging standardized mechanical and data interfaces where appropriate to maximize responsive spaceflight opportunities. These principles are at the foundation of STP and are influencing the future of small spacecraft acquisition and access-to-space across the space community.

We have developed a novel multilocus sequence typing (MLST) scheme and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and random amplification of polymorphic DNA typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (STs). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB1), representing 64 % of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants, which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore support an association between acne and P. acnes strains from the type IA cluster and highlight the role of a widely disseminated clonal genotype in this condition. Characterization of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.

Background. Molecular techniques can often reveal a broader range of pathogens in respiratory infections. We aim to investigate the prevalence and age pattern of viral co-infection in children hospitalized with lower tract acute respiratory infection (LT-ARI), using molecular techniques. Methods. A nested polymerase chain reaction approach was used to detect Influenza (A, B), metapneumovirus, respiratory syncytial virus (RSV), parainfluenza (1-4), rhinovirus, adenovirus (A-F), bocavirus and coronaviruses (NL63, 229E, OC43) in respiratory samples of children with acute respiratory infection prospectively admitted to any of the GENDRES network hospitals between 2011-2013. The results were corroborated in an independent cohort collected in the UK. Results. A total of 204 and 97 nasopharyngeal samples were collected in the GENDRES and UK cohorts, respectively. In both cohorts, RSV was the most frequent pathogen (52.9% and 36.1% of the cohorts, respectively). Co-infection with multiple viruses was found in 92 samples (45.1%) and 29 samples (29.9%), respectively; this was most frequent in the 12-24 months age group. The most frequently observed co-infection patterns were RSV-Rhinovirus (23 patients, 11.3%, GENDRES cohort) and RSV-bocavirus / bocavirus-influenza (5 patients, 5.2%, UK cohort). Conclusion. The presence of more than one virus in pediatric patients admitted to hospital with LT-ARI is very frequent and seems to peak at 12-24 months of age. The clinical significance of these findings is unclear but should warrant further analysis.

A decade after the first studies were performed it is justifiable to claim that molecular techniques have revolutionised the work of the clinical virology laboratory. Hitherto, the role of the virology laboratory was often a retrospective diagnosis based on virus isolation and serology. Nevertheless, the epidemiological data collected in this way justified the continued activity of clinical virology. The recent molecular revolution in laboratory methods has been timely because it has been in parallel with the emergence of new pathogens that have presented the clinical virologist with fresh diagnostic challenges. A concurrent development of specific antiviral compounds has increased the potential of rapid laboratory investigation to contribute to the management of acutely ill or immunosuppressed patients. Molecular assays for the detection of microorganisms can be designed even when only partial nucleic acid sequence information is available. This is valuable when identifying and diagnosing new diseases and emerging pathogens because there is the possibility of a rapid development of assays in house. However, the standardisation of assay design is a continuing problem because of the large number of nucleic acid sequences that can be used as a target for the detection of any one virus. Many of the methodologies associated with molecular techniques have now been incorporated into commercial kits, but in house molecular assays continue to be used. In house assays are often well evaluated and perform to a high standard, but differing assay sensitivities and specificities have been highlighted by quality assessment programmes. Some of the variations in performance can be accounted for by assay design, although it is probable that some in house assays do not travel well and perform badly because laboratory staff are inexperienced with molecular techniques. Molecular techniques in their present format, even in commercial kits, require a considerable degree of operator skill. If a …