Background: In low-resource settings, limited laboratory capacity adds to the burden of central nervous system (CNS) infections in children and spurs overuse of antibiotics. The commercially available BioFire® FilmArray® Meningitis/Encephalitis Panel (FA-ME) with its capability to simultaneously detect 14 pathogens in cerebrospinal fluid (CSF), could potentially narrow such a diagnostic gap. Methods: In Mbarara, Uganda, we compared clinical utility (clinical turnaround time [cTAT], microbial yield, and influence on patient outcome and antibiotic exposure) of FA-ME with bacterial culture, in children 0–12 years with suspected CNS infection. Results: Of 212 enrolled children, CSF was sampled from 194. All samples underwent bacterial culture, of which 193 also underwent FA-ME analyses. FA-ME analyses prospectively influenced care for 169 of the 193 patients, and they constituted an ‘Index group’. The remaining 43/212 patients constituted a ‘Reference group’. Of all 194 CSF-sampled patients, 87% (168) had received antibiotics before lumbar puncture. Median cTAT for FA-ME was 4.2 h, vs. two days for culture. Bacterial yield was 12% (24/193) and 1.5% (3/194) for FA-ME and culture, respectively. FA-ME viral yield was 12% (23/193). Fatality rate was 14% in the Index group vs. 19% in the Reference group (P = 0.20). From clinician receival of FA-ME results, median antibiotic exposure was 6 days for bacteria-negative vs. 13 days for bacteria-positive patients (P = 0.03). Median hospitalization duration was 7 vs. 12 days for FA-ME negative and positive patients, respectively (P < 0.01). Conclusions: In this setting, clinical FA-ME utility was found in a higher and faster microbial yield and shortened hospitalization and antibiotic exposure of patients without CSF pathology. More epidemiologically customized pathogen panels may increase FA-ME utility locally, although its use in similar settings would require major cost reductions. Trial registration: The trial was registered with clinicaltrials.gov (NCT03900091) in March 2019, and its protocol was published in November 2020.

Laboratory automation effectively increases the throughput in sample analysis, reduces human errors in sample processing, as well as simplifies and accelerates the overall logistics. Automating diagnostic testing workflows in peripheral laboratories and also in near-patient settings -like hospitals, clinics and epidemic control checkpoints- is advantageous for the simultaneous processing of multiple samples to provide rapid results to patients, minimize the possibility of contamination or error during sample handling or transport, and increase efficiency. However, most automation platforms are expensive and are not easily adaptable to new protocols. Here, we address the need for a versatile, easy-to-use, rapid and reliable diagnostic testing workflow by combining open-source modular automation (Opentrons) and automation-compatible molecular biology protocols, easily adaptable to a workflow for infectious diseases diagnosis by detection on paper-based diagnostics. We demonstrated the feasibility of automation of the method with a low-cost Neisseria meningitidis diagnostic test that utilizes magnetic beads for pathogen DNA isolation, isothermal amplification, and detection on a paper-based microarray. In summary, we integrated open-source modular automation with adaptable molecular biology protocols, which was also faster and cheaper to perform in an automated than in a manual way. This enables a versatile diagnostic workflow for infectious diseases and we demonstrated this through a low-cost N. meningitidis test on paper-based microarrays.

Objectives: Meningitis is a medical emergency, and it is crucial to diagnose it accurately and promptly in order to manage patients effectively. It would, therefore, be essential to introduce and have fast, accurate, and user-friendly methods to determine the cause of these infections. This study aimed to demonstrate a potentially cost-effective new approach for detecting meningitis using a paper-based vertical flow microarray, which could be useful in settings with limited resources. Methods: We describe a multiplex paper microarray for detecting Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, and Salmonella spp. by the passive vertical flow of PCR-amplified clinical samples. A multibiotinylated amplicon was obtained as a product of PCR in the presence of both a biotinylated primer and biotin-11-dUTP. An enhancement step based on an enzyme-free gold enhancement protocol was also used to facilitate visual detection. Results: This study showed that the vertical flow microarray (previously evaluated for one pathogen) can discriminately detect the amplification results down to the 102 copies of DNA limit for four meningitis pathogens in a multiplexed set-up. The study further demonstrated the ability of this device and setup to detect three of the four pathogens from clinical biosamples. Discussion: This study demonstrated the capacity of a vertical flow microarray device to detect amplification products for four prevalent meningitis pathogens in a multiplex format. The vertical flow microarray demonstrated consistent visualization of the expected gene amplification results; however, indicating limitations in the pre- and amplification steps. This study highlights the potential of this multiplexing method for diagnosing meningitis and other syndromic diseases caused by various pathogens, especially in resource-limited areas.

Objective: Discriminating between viral and bacterial lower respiratory tract infection (LRTI) in children is challenging, leading to an excessive use of antibiotics. Myxovirus resistance protein A (MxA) is a promising biomarker for viral infections. The primary aim of the study was to assess differences in blood MxA levels between children with viral and bacterial LRTI. Secondary aims were to assess differences in blood MxA levels between children with viral LRTI and asymptomatic controls and to assess MxA levels in relation to different respiratory viruses. Methods: Children with LRTI were enrolled as cases at Sachs' Children and Youth Hospital, Stockholm, Sweden. Nasopharyngeal aspirates and blood samples for analysis of viral PCR, MxA, and C-reactive protein were systematically collected from all study subjects in addition to standard laboratory/radiology assessment. Aetiology was defined according to an algorithm based on laboratory and radiological findings. Asymptomatic children with minor surgical disease were enrolled as controls. Results: MxA levels were higher in children with viral LRTI (n = 242) as compared to both bacterial (n = 5) LRTI (p < 0.01, area under the curve (AUC) 0.90, 95% CI: 0.81 to 0.99), and controls (AUC 0.92, 95% CI: 0.88 to 0.95). In the subgroup of children with pneumonia diagnosis, a cutoff of MxA 430 mg/l discriminated between viral (n = 29) and bacterial (n = 4) aetiology with 93% (95% CI: 78-99%) sensi-tivity and 100% (95% CI: 51-100%) specificity (AUC 0.98, 95% CI: 0.94 to 1.00). The highest MxA levels were seen in cases PCR positive for influenza (median MxA 1699 mg/l, interquartile range: 732 to 2996) and respiratory syncytial virus (median MxA 1115 mg/l, interquartile range: 679 to 2489). Discussion: MxA accurately discriminated between viral and bacterial aetiology in children with LRTI, particularly in the group of children with pneumonia diagnosis, but the number of children with bacterial LRTI was low.

(1) Immunization with pneumococcal conjugate vaccines has decreased the burden of community-acquired pneumonia (CAP) in children and likely led to a shift in CAP etiology. (2) The Trial of Respiratory infections in children for ENhanced Diagnostics (TREND) enrolled children 1-59 months with clinical CAP according to the World Health Organization (WHO) criteria at Sachs' Children and Youth Hospital, Stockholm, Sweden. Children with rhonchi and indrawing underwent "bronchodilator challenge". C-reactive protein and nasopharyngeal PCR detecting 20 respiratory pathogens, were collected from all children. Etiology was defined according to an a priori defined algorithm based on microbiological, biochemical, and radiological findings. (3) Of 327 enrolled children, 107 (32%) required hospitalization; 91 (28%) received antibiotic treatment; 77 (24%) had a chest X-ray performed; and 60 (18%) responded to bronchodilator challenge. 243 (74%) episodes were classified as viral, 11 (3%) as mixed viral-bacterial, five (2%) as bacterial, two (0.6%) as atypical bacterial and 66 (20%) as undetermined etiology. After exclusion of children responding to bronchodilator challenge, the proportion of bacterial and mixed viral-bacterial etiology was 1% and 4%, respectively. (4) The novel TREND etiology algorithm classified the majority of clinical CAP episodes as of viral etiology, whereas bacterial etiology was uncommon. Defining CAP in children <5 years is challenging, and the WHO definition of clinical CAP is not suitable for use in children immunized with pneumococcal conjugate vaccines.

Objectives In many resource-limited health systems, point-of-care tests (POCTs) are the only means for clinical patient sample analyses. However, the speed and simplicity of POCTs also makes their use appealing to clinicians in high-income countries (HICs), despite greater laboratory accessibility. Although also part of the clinical routine in HICs, clinician perceptions of the utility of POCTs are relatively unknown in such settings as compared with others. In a Swedish paediatric emergency department (PED) where POCT use is routine, we aimed to characterise healthcare providers' perspectives on the clinical utility of POCTs and explore their implementation in the local setting; to discuss and compare such perspectives, to those reported in other settings; and finally, to gather requests for ideal novel POCTs. Design Qualitative focus group discussions study. A data-driven content analysis approach was used for analysis. Setting The PED of a secondary paediatric hospital in Stockholm, Sweden. Participants Twenty-four healthcare providers clinically active at the PED were enrolled in six focus groups. Results A range of POCTs was routinely used. The emerging theme Utility of our POCT use is double-edged illustrated the perceived utility of POCTs. While POCT services were considered to have clinical and social value, the local routine for their use was named to distract clinicians from the care for patients. Requests were made for ideal POCTs and their implementation. Conclusion Despite their clinical integration, deficient implementation routines limit the benefits of POCT services to this well-resourced paediatric clinic. As such deficiencies are shared with other settings, it is suggested that some characteristics of POCTs and of their utility are less related to resource level and more to policy deficiency. To address this, we propose the appointment of skilled laboratory personnel as ambassadors to hospital clinics offering POCT services, to ensure higher utility of such services.

Background: A timely differential diagnostic is essential to identify the etiology of central nervous system (CNS) infections in children, in order to facilitate targeted treatment, manage patients, and improve clinical outcome. Objective: The Pediatric Infection-Point-of-Care (PI-POC) trial is investigating novel methods to improve and strengthen the differential diagnostics of suspected childhood CNS infections in low-income health systems such as those in Southwestern Uganda. This will be achieved by evaluating (1) a novel DNA-based diagnostic assay for CNS infections, (2) a commercially available multiplex PCR-based meningitis/encephalitis (ME) panel for clinical use in a facility-limited laboratory setting, (3) proteomics profiling of blood from children with severe CNS infection as compared to outpatient controls with fever yet not severely ill, and (4) Myxovirus resistance protein A (MxA) as a biomarker in blood for viral CNS infection. Further changes in the etiology of childhood CNS infections after the introduction of the pneumococcal conjugate vaccine against Streptococcus pneumoniae will be investigated. In addition, the carriage and invasive rate of Neisseria meningitidis will be recorded and serotyped, and the expression of its major virulence factor (polysaccharide capsule) will be investigated. Methods: The PI-POC trial is a prospective observational study of children including newborns up to 12 years of age with clinical features of CNS infection, and age-/sex-matched outpatient controls with fever yet not severely ill. Participants are recruited at 2 Pediatric clinics in Mbarara, Uganda. Cerebrospinal fluid (for cases only), blood, and nasopharyngeal (NP) swabs (for both cases and controls) sampled at both clinics are analyzed at the Epicentre Research Laboratory through gold-standard methods for CNS infection diagnosis (microscopy, biochemistry, and culture) and a commercially available ME panel for multiplex PCR analyses of the cerebrospinal fluid. An additional blood sample from cases is collected on day 3 after admission. After initial clinical analyses in Mbarara, samples will be transported to Stockholm, Sweden for (1) validation analyses of a novel nucleic acid-based POC test, (2) biomarker research, and (3) serotyping and molecular characterization of S. pneumoniae and N. meningitidis. Results: A pilot study was performed from January to April 2019. The PI-POC trial enrollment of patients begun in April 2019 and will continue until September 2020, to include up to 300 cases and controls. Preliminary results from the PI-POC study are expected by the end of 2020. Conclusions: The findings from the PI-POC study can potentially facilitate rapid etiological diagnosis of CNS infections in low-resource settings and allow for novel methods for determination of the severity of CNS infection in such environment.

Cell culture is a ubiquitous and flexible research method. However, it heavily relies on plastic consumables generating millions of tonnes of plastic waste yearly. Plastic waste is a major and growing global concern. Here we describe a new cell culture dish that offers a culture area equivalent to three petri dishes but that is on average 61% lighter and occupies 67% less volume. Our dish is composed of a lid and three thin containers surrounded by a light outer shell. Cell culture can be performed in each of the containers sequentially. The outer shell provides the appropriate structure for the manipulation of the dish as a whole. The prototype was tested by sequentially growing cells in each of its containers. As a control, sequential cultures in groups of 3 petri dishes were performed. No statistical differences were found between the prototype and the control in terms of cell number, cell viability or cell distribution.

Respiratory viral infections often mimic the symptoms of infections caused by bacteria; however, restricted and targeted administration of antibiotics is needed to combat growing antimicrobial resistance. This is particularly relevant in low-income settings. In this work, we describe the use of isothermal amplification of viral DNA at 37 °C coupled to a paper-based vertical flow microarray (VFM) setup that utilizes a colorimetric detection of amplicons using functionalized gold nanoparticles. Two oligonucleotide probes, one in-house designed and one known adenoviral probe were tested and validated for microarray detection down to 50 nM using a synthetic target template. Furthermore, primers were shown to function in a recombinase polymerase amplification reaction using both synthetic template and viral DNA. As a proof-of-concept, we demonstrate adenoviral detection with four different adenoviral species associated with respiratory infections using the paper-based VFM format. The presented assay was validated with selected adenoviral species using the in-house probe, enabling detection at 1 ng of starting material with intra- and inter-assay %CV of ≤ 9% and ≤ 13%. Finally, we validate our overall method using clinical samples. Based on the results, the combination of recombinase polymerase amplification, paper microarray analysis, and nanoparticle-based colorimetric detection could thus be a useful strategy towards rapid and affordable multiplexed viral diagnostics.

Background: There is a need to better distinguish viral infections from antibiotic-requiring bacterial infections in children presenting with clinical community-acquired pneumonia (CAP) to assist health care workers in decision making and to improve the rational use of antibiotics. Objective: The overall aim of the Trial of Respiratory infections in children for ENhanced Diagnostics (TREND) study is to improve the differential diagnosis of bacterial and viral etiologies in children aged below 5 years with clinical CAP, by evaluating myxovirus resistance protein A (MxA) as a biomarker for viral CAP and by evaluating an existing (multianalyte point-of-care antigen detection test system [mariPOC respi] ArcDia International Oy Ltd.) and a potential future point-of-care test for respiratory pathogens. Methods: Children aged 1 to 59 months with clinical CAP as well as healthy, hospital-based, asymptomatic controls will be included at a pediatric emergency hospital in Stockholm, Sweden. Blood (analyzed for MxA and C-reactive protein) and nasopharyngeal samples (analyzed with real-time polymerase chain reaction as the gold standard and antigen-based mariPOC respi test as well as saved for future analyses of a novel recombinase polymerase amplification-based point-of-care test for respiratory pathogens) will be collected. A newly developed algorithm for the classification of CAP etiology will be used as the reference standard. Results: A pilot study was performed from June to August 2017. The enrollment of study subjects started in November 2017. Results are expected by the end of 2019.Conclusions: The findings from the TREND study can be an important step to improve the management of children with clinical.