A fast extraction-free isothermal LAMP assay for detection of SARS-CoV-2 with potential use in resource-limited settings.

BACKGROUND: To retain the spread of SARS-CoV-2, fast, sensitive and cost-effective testing is essential, particularly in resource limited settings (RLS). Current standard nucleic acid-based RT-PCR assays, although highly sensitive and specific, require transportation of samples to specialised laboratories, trained staff and expensive reagents. The latter are often not readily available in low- and middle-income countries and this may significantly impact on the successful disease management in these settings. Various studies have suggested a SARS-CoV-2 loop mediated isothermal amplification (LAMP) assay as an alternative method to RT-PCR. METHODS: Four previously published primer pairs were used for detection of SARS-CoV-2 in the LAMP assay. To determine optimal conditions, different temperatures, sample input and incubation times were tested. Ninety-three extracted RNA samples from St. George's Hospital, London, 10 non-extracted nasopharyngeal swab samples from Great Ormond Street Hospital for Children, London, and 92 non-extracted samples from Queen Elisabeth Central Hospital (QECH), Malawi, which have previously been tested for SARS-Cov-2 by quantitative reverse-transcription RealTime PCR (qRT-PCR), were analysed in the LAMP assay. RESULTS: In this study we report the optimisation of an extraction-free colourimetric SARS-CoV-2 LAMP assay and demonstrated that a lower limit of detection (LOD) between 10 and 100 copies/µL of SARS-CoV-2 could be readily detected by a colour change of the reaction within as little as 30 min. We further show that this assay could be quickly established in Malawi, as no expensive equipment is necessary. We tested 92 clinical samples from QECH and showed the sensitivity and specificity of the assay to be 86.7% and 98.4%, respectively. Some viral transport media, used routinely to stabilise RNA in clinical samples during transportation, caused a non-specific colour-change in the LAMP reaction and therefore we suggest collecting samples in phosphate buffered saline (which did not affect the colour) as the assay allows immediate sample analysis on-site. CONCLUSION: SARS-CoV-2 LAMP is a cheap and reliable assay that can be readily employed in RLS to improve disease monitoring and management.

Inflammatory Phenotypes Predict Changes in Arterial Stiffness Following Antiretroviral Therapy Initiation.

BACKGROUND: Inflammation drives vascular dysfunction in HIV, but in low-income settings causes of inflammation are multiple, and include infectious and environmental factors. We hypothesized that patients with advanced immunosuppression could be stratified into inflammatory phenotypes that predicted changes in vascular dysfunction on ART. METHODS: We recruited Malawian adults with CD4 <100 cells/µL 2 weeks after starting ART in the REALITY trial (NCT01825031). Carotid femoral pulse-wave velocity (cfPWV) measured arterial stiffness 2, 12, 24, and 42 weeks post-ART initiation. Plasma inflammation markers were measured by electrochemiluminescence at weeks 2 and 42. Hierarchical clustering on principal components identified inflammatory clusters. RESULTS: 211 participants with HIV grouped into 3 inflammatory clusters representing 51 (24%; cluster-1), 153 (73%; cluster-2), and 7 (3%; cluster-3) individuals. Cluster-1 showed markedly higher CD4 and CD8 T-cell expression of HLADR and PD-1 versus cluster-2 and cluster-3 (all P < .0001). Although small, cluster-3 had significantly higher levels of cytokines reflecting inflammation (IL-6, IFN-γ, IP-10, IL-1RA, IL-10), chemotaxis (IL-8), systemic and vascular inflammation (CRP, ICAM-1, VCAM-1), and SAA (all P < .001). In mixed-effects models, cfPWV changes over time were similar for cluster-2 versus cluster-1 (relative fold-change, 0.99; 95% CI, .86-1.14; P = .91), but greater in cluster-3 versus cluster-1 (relative fold-change, 1.45; 95% CI, 1.01-2.09; P = .045). CONCLUSIONS: Two inflammatory clusters were identified: one defined by high T-cell PD-1 expression and another by a hyperinflamed profile and increases in cfPWV on ART. Further clinical characterization of inflammatory phenotypes could help target vascular dysfunction interventions to those at highest risk. CLINICAL TRIALS NETWORK: NCT01825031.

Dissimilarity of the gut-lung axis and dysbiosis of the lower airways in ventilated preterm infants.

BACKGROUND: Chronic lung disease of prematurity (CLD), also called bronchopulmonary dysplasia, is a major consequence of preterm birth, but the role of the microbiome in its development remains unclear. Therefore, we assessed the progression of the bacterial community in ventilated preterm infants over time in the upper and lower airways, and assessed the gut-lung axis by comparing bacterial communities in the upper and lower airways with stool findings. Finally, we assessed whether the bacterial communities were associated with lung inflammation to suggest dysbiosis. METHODS: We serially sampled multiple anatomical sites including the upper airway (nasopharyngeal aspirates), lower airways (tracheal aspirate fluid and bronchoalveolar lavage fluid) and the gut (stool) of ventilated preterm-born infants. Bacterial DNA load was measured in all samples and sequenced using the V3-V4 region of the 16S rRNA gene. RESULTS: From 1102 (539 nasopharyngeal aspirates, 276 tracheal aspirate fluid, 89 bronchoalveolar lavage, 198 stool) samples from 55 preterm infants, 352 (32%) amplified suitably for 16S RNA gene sequencing. Bacterial load was low at birth and quickly increased with time, but was associated with predominant operational taxonomic units (OTUs) in all sample types. There was dissimilarity in bacterial communities between the upper and lower airways and the gut, with a separate dysbiotic inflammatory process occurring in the lower airways of infants. Individual OTUs were associated with increased inflammatory markers. CONCLUSIONS: Taken together, these findings suggest that targeted treatment of the predominant organisms, including those not routinely treated, such as Ureaplasma spp., may decrease the development of CLD in preterm-born infants.

Novel Campylobacter concisus lipooligosaccharide is a determinant of inflammatory potential and virulence.

The pathogenicity of Campylobacter concisus, increasingly found in the human gastrointestinal (GI) tract, is unclear. Some studies indicate that its role in GI conditions has been underestimated, whereas others suggest that the organism has a commensal-like phenotype. For the enteropathogen C. jejuni, the lipooligosaccharide (LOS) is a main driver of virulence. We investigated the LOS structure of four C. concisus clinical isolates and correlated the inflammatory potential of each isolate with bacterial virulence. Mass spectrometric analyses of lipid A revealed a novel hexa-acylated diglucosamine moiety with two or three phosphoryl substituents. Molecular and fragment ion analysis indicated that the oligosaccharide portion of the LOS had only a single phosphate and lacked phosphoethanolamine and sialic acid substitution, which are hallmarks of the C. jejuni LOS. Consistent with our structural findings, C. concisus LOS and live bacteria induced less TNF-α secretion in human monocytes than did C. jejuni Furthermore, the C. concisus bacteria were less virulent than C. jejuni in a Galleria mellonella infection model. The correlation of the novel lipid A structure, decreased phosphorylation, and lack of sialylation along with reduced inflammatory potential and virulence support the significance of the LOS as a determinant in the relative pathogenicity of C. concisus.

Identification of bacterial pathogens in sudden unexpected death in infancy and childhood using 16S rRNA gene sequencing.

Background: Sudden unexpected death in infancy (SUDI) is the most common cause of post-neonatal death in the developed world. Following an extensive investigation, the cause of ~40% of deaths remains unknown. It is hypothesized that a proportion of deaths are due to an infection that remains undetected due to limitations in routine techniques. This study aimed to apply 16S rRNA gene sequencing to post-mortem (PM) tissues collected from cases of SUDI, as well as those from the childhood equivalent (collectively known as sudden unexpected death in infancy and childhood or SUDIC), to investigate whether this molecular approach could help identify potential infection-causing bacteria to enhance the diagnosis of infection. Methods: In this study, 16S rRNA gene sequencing was applied to de-identified frozen post-mortem (PM) tissues from the diagnostic archive of Great Ormond Street Hospital. The cases were grouped depending on the cause of death: (i) explained non-infectious, (ii) infectious, and (iii) unknown. Results and conclusions: In the cases of known bacterial infection, the likely causative pathogen was identified in 3/5 cases using bacterial culture at PM compared to 5/5 cases using 16S rRNA gene sequencing. Where a bacterial infection was identified at routine investigation, the same organism was identified by 16S rRNA gene sequencing. Using these findings, we defined criteria based on sequencing reads and alpha diversity to identify PM tissues with likely infection. Using these criteria, 4/20 (20%) cases of unexplained SUDIC were identified which may be due to bacterial infection that was previously undetected. This study demonstrates the potential feasibility and effectiveness of 16S rRNA gene sequencing in PM tissue investigation to improve the diagnosis of infection, potentially reducing the number of unexplained deaths and improving the understanding of the mechanisms involved.

'If I am on ART, my new-born baby should be put on treatment immediately': Exploring the acceptability, and appropriateness of Cepheid Xpert HIV-1 Qual assay for early infant diagnosis of HIV in Malawi.

Early infant diagnosis of HIV (EID-HIV) is key to reducing paediatric HIV mortality. Traditional approaches for diagnosing HIV in exposed infants are usually unable to optimally contribute to EID. Point-of-care testing such as Cepheid Xpert HIV-1 Qual assay-1 (XPertHIV) are available and could improve EID-HIV in resource constrained and high HIV burden contexts. We investigated the acceptability and perceived appropriateness of XpertHIV for EID-HIV in Mulanje Hospital, Malawi. Qualitative cross-sectional study using semi-structured interviews (SSI) among caregivers and health care workers at Mulanje District Hospital. The qualitative study was nested within a larger diagnostic study that evaluated the performance of XpertHIV using whole-blood-sample in a resource limited and high burden setting. A total of 65 SSIs were conducted among caregivers (n = 60) and health care providers (n = 5). Data were coded using deductive and inductive approaches while thematic approach was used to analyse data. Point-of-care XPertHIV was perceived to be acceptable among caregivers and health care providers. Caregivers' motivations for accepting XPertHIV HIV-testing for their infants included perceived risk of HIV emanating from child's exposure and validation of caregiver's own HIV sero-status. Although concerns about pain of testing and blood sample volumes taken from an infant remained amplified, overall, both caregivers and health care providers felt XpertHIV was appropriate because of its quick result turn-around-time which decreased anxiety and stress, the prospect of early treatment initiation and reduction in hospital visits and related costs. Implementation of XpertHIV has a great potential to improve EID-HIV in Malawi because of its quick turn-around-time and associated benefits including overcoming access-related barriers. Scaled implementation of this diagnostic technology require a robust community engagement strategy for managing caregivers and community myths and misconceptions towards the amount of blood sample collected from infants.

Impact of sub-optimal HIV viral control on activated T cells.

OBJECTIVE: HIV viral load (VL) monitoring is generally conducted 6-12 monthly in low- and middle-income countries, risking relatively prolonged periods of poor viral control. We explored the effects of different levels of loss of viral control on immune reconstitution and activation. DESIGN: Two hundred and eight participants starting protease inhibitor (PI)-based second-line therapy in the EARNEST trial (ISRCTN37737787) in Uganda and Zimbabwe were enrolled and CD38 + /HLA-DR + immunophenotyping performed (CD8-FITC/CD38-PE/CD3-PerCP/HLA-DR-APC; centrally gated) in real-time at 0, 12, 48, 96 and 144 weeks from randomization. METHODS: VL was assayed retrospectively on samples collected every 12-16 weeks and classified as continuous suppression (<40 copies/ml throughout); suppression with transient blips; low-level rebound (two or more consecutive VL >40, <5000 copies/ml); high-level rebound/nonresponse (two or more consecutive VL >5000 copies/ml). RESULTS: Immunophenotype reconstitution varied between that defined by numbers of cells and that defined by cell percentages. Furthermore, VL dynamics were associated with substantial differences in expression of CD4 + and CD8 + cell activation markers, with only individuals with high-level rebound/nonresponse (>5000 copies/ml) experiencing significantly greater activation and impaired reconstitution. There was little difference between participants who suppressed consistently and who exhibited transient blips or even low-level rebound by 144 weeks ( P  > 0.2 vs. suppressed consistently). CONCLUSION: Detectable viral load below the threshold at which WHO guidelines recommend that treatment can be maintained without switching (1000 copies/ml) appear to have at most, small effects on reconstitution and activation, for patients taking a PI-based second-line regimen.

Testing the effects of mass drug administration of azithromycin on mortality and other outcomes among 1-11-month-old infants in Mali (LAKANA): study protocol for a cluster-randomized, placebo-controlled, double-blinded, parallel-group, three-arm clinical trial.

BACKGROUND: Mass drug administration (MDA) of azithromycin (AZI) has been shown to reduce under-5 mortality in some but not all sub-Saharan African settings. A large-scale cluster-randomized trial conducted in Malawi, Niger, and Tanzania suggested that the effect differs by country, may be stronger in infants, and may be concentrated within the first 3 months after treatment. Another study found no effect when azithromycin was given concomitantly with seasonal malaria chemoprevention (SMC). Given the observed heterogeneity and possible effect modification by other co-interventions, further trials are needed to determine the efficacy in additional settings and to determine the most effective treatment regimen. METHODS: LAKANA stands for Large-scale Assessment of the Key health-promoting Activities of two New mass drug administration regimens with Azithromycin. The LAKANA trial is designed to address the mortality and health impacts of 4 or 2 annual rounds of azithromycin MDA delivered to 1-11-month-old (29-364 days) infants, in a high-mortality and malaria holoendemic Malian setting where there is a national SMC program. Participating villages (clusters) are randomly allocated in a ratio of 3:2:4 to three groups: placebo (control):4-dose AZI:2-dose AZI. The primary outcome measured is mortality. Antimicrobial resistance (AMR) will be monitored closely before, during, and after the intervention and both among those receiving and those not receiving MDA with the study drugs. Other outcomes, from a subset of villages, comprise efficacy outcomes related to morbidity, growth and nutritional status, outcomes related to the mechanism of azithromycin activity through measures of malaria parasitemia and inflammation, safety outcomes (AMR, adverse and serious adverse events), and outcomes related to the implementation of the intervention documenting feasibility, acceptability, and economic aspects. The enrolment commenced in October 2020 and is planned to be completed by the end of 2022. The expected date of study completion is December 2024. DISCUSSION: If LAKANA provides evidence in support of a positive mortality benefit resulting from azithromycin MDA, it will significantly contribute to the options for successfully promoting child survival in Mali, and elsewhere in sub-Saharan Africa. TRIAL REGISTRATION: ClinicalTrials.gov NCT04424511. Registered on 11 June 2020.

Statistical analysis plan for the LAKANA trial: a cluster-randomized, placebo-controlled, double-blinded, parallel group, three-arm clinical trial testing the effects of mass drug administration of azithromycin on mortality and other outcomes among 1-11-month-old infants in Mali.

BACKGROUND: The Large-scale Assessment of the Key health-promoting Activities of two New mass drug administration regimens with Azithromycin (LAKANA) trial in Mali aims to evaluate the efficacy and safety of azithromycin (AZI) mass drug administration (MDA) to 1-11-month-old infants as well as the impact of the intervention on antimicrobial resistance (AMR) and mechanisms of action of azithromycin. To improve the transparency and quality of this clinical trial, we prepared this statistical analysis plan (SAP). METHODS/DESIGN: LAKANA is a cluster randomized trial that aims to address the mortality and health impacts of biannual and quarterly AZI MDA. AZI is given to 1-11-month-old infants in a high-mortality setting where a seasonal malaria chemoprevention (SMC) program is in place. The participating villages are randomly assigned to placebo (control), two-dose AZI (biannual azithromycin-MDA), and four-dose AZI (quarterly azithromycin-MDA) in a 3:4:2 ratio. The primary outcome of the study is mortality among the intention-to-treat population of 1-11-month-old infants. We will evaluate relative risk reduction between the study arms using a mixed-effects Poisson model with random intercepts for villages, using log link function with person-years as an offset variable. We will model outcomes related to secondary objectives of the study using generalized linear models with considerations on clustering. CONCLUSION: The SAP written prior to data collection completion will help avoid reporting bias and data-driven analysis for the primary and secondary aims of the trial. If there are deviations from the analysis methods described here, they will be described and justified in the publications of the trial results. TRIAL REGISTRATION: ClinicalTrials.gov ID NCT04424511 . Registered on 11 June 2020.

Linking gastrointestinal microbiota and metabolome dynamics to clinical outcomes in paediatric haematopoietic stem cell transplantation.

BACKGROUND: Haematopoietic stem cell transplantation is a curative procedure for a variety of conditions. Despite major advances, a plethora of adverse clinical outcomes can develop post-transplantation including graft-versus-host disease and infections, which remain the major causes of morbidity and mortality. There is increasing evidence that the gastrointestinal microbiota is associated with clinical outcomes post-haematopoietic stem cell transplantation. Herein, we investigated the longitudinal dynamics of the gut microbiota and metabolome and potential associations to clinical outcomes in paediatric haematopoietic stem cell transplantation at a single centre. RESULTS: On admission (baseline), the majority of patients presented with a different gut microbial composition in comparison with healthy control children with a significantly lower alpha diversity. A further, marked decrease in alpha diversity was observed immediately post-transplantation and in most microbial diversity, and composition did not return to baseline status whilst hospitalised. Longitudinal trajectories identified continuous fluctuations in microbial composition, with the dominance of a single taxon in a significant proportion of patients. Using pam clustering, three clusters were observed in the dataset. Cluster 1 was common pre-transplantation, characterised by a higher abundance of Clostridium XIVa, Bacteroides and Lachnospiraceae; cluster 2 and cluster 3 were more common post-transplantation with a higher abundance of Streptococcus and Staphylococcus in the former whilst Enterococcus, Enterobacteriaceae and Escherichia predominated in the latter. Cluster 3 was also associated with a higher risk of viraemia. Likewise, further multivariate analysis reveals Enterobacteriaceae, viraemia, use of total parenteral nutrition and various antimicrobials contributing towards cluster 3, Streptococcaceae, Staphylococcaceae, Neisseriaceae, vancomycin and metronidazole contributing towards cluster 2. Lachnospiraceae, Ruminococcaceae, Bifidobacteriaceae and not being on total parenteral nutrition contributed to cluster 1. Untargeted metabolomic analyses revealed changes that paralleled fluctuations in microbiota composition; importantly, low faecal butyrate was associated with a higher risk of viraemia. CONCLUSIONS: These findings highlight the frequent shifts and dominations in the gut microbiota of paediatric patients undergoing haematopoietic stem cell transplantation. The study reveals associations between the faecal microbiota, metabolome and viraemia. To identify and explore the potential of microbial biomarkers that may predict the risk of complications post-HSCT, larger multi-centre studies investigating the longitudinal microbial profiling in paediatric haematopoietic stem cell transplantation are warranted. Video abstract.

SARS-CoV-2 infection and antibody seroprevalence in routine surveillance patients, healthcare workers and general population in Kita region, Mali: an observational study 2020-2021.

OBJECTIVE: To estimate the degree of SARS-CoV-2 transmission among healthcare workers (HCWs) and general population in Kita region of Mali. DESIGN: Routine surveillance in 12 health facilities, HCWs serosurvey in five health facilities and community serosurvey in 16 villages in or near Kita town, Mali. SETTING: Kita region, western Mali; local health centres around the central (regional) referral health centre. PARTICIPANTS: Patients in routine surveillance, HCWs in local health centres and community members of all ages in populations associated with study health centres. MAIN OUTCOME MEASURES: Seropositivity of ELISA test detecting SARS-CoV-2-specific total antibodies and real-time RT-PCR confirmed SARS-CoV-2 infection. RESULTS: From 2392 routine surveillance samples, 68 (2.8%, 95% CI: 2.2% to 3.6%) tested positive for SARS-CoV-2 by RT-PCR. The monthly positivity rate was 0% in June-August 2020 and gradually increased to 6% by December 2020 and 6.2% by January 2021, then declined to 5.5%, 3.3%, 3.6% and 0.8% in February, March, April and May 2021, respectively. From 397 serum samples collected from 113 HCWs, 175 (44.1%, 95% CI: 39.1% to 49.1%) were positive for SARS-CoV-2 antibodies. The monthly seroprevalence was around 10% from September to November 2020 and increased to over 40% from December 2020 to May 2021. For community serosurvey in December 2020, overall seroprevalence of SARS-CoV-2 antibodies was 27.7%. The highest age-stratified seroprevalence was observed in participants aged 60-69 years (45.5%, 95% CI: 32.3% to 58.6%). The lowest was in children aged 0-9 years (14.0%, 95% CI: 7.4% to 20.6%). CONCLUSIONS: SARS-CoV-2 in rural Mali is much more widespread than assumed by national testing data and particularly in the older population and frontline HCWs. The observation is contrary to the widely expressed view, based on limited data, that COVID-19 infection rates were lower in 2020-2021 in West Africa than in other settings.

FcMBL magnetic bead-based MALDI-TOF MS rapidly identifies paediatric blood stream infections from positive blood cultures.

Rapid identification of potentially life-threatening blood stream infections (BSI) improves clinical outcomes, yet conventional blood culture (BC) identification methods require ~24-72 hours of liquid culture, plus 24-48 hours to generate single colonies on solid media suitable for identification by mass spectrometry (MS). Newer rapid centrifugation techniques, such as the Bruker MBT-Sepsityper® IVD, replace culturing on solid media and expedite the diagnosis of BCs but frequently demonstrate reduced sensitivity for identifying clinically significant Gram-positive bacterial or fungal infections. This study introduces a protocol that utilises the broad-range binding properties of an engineered version of mannose-binding lectin linked to the Fc portion of immunoglobulin (FcMBL) to capture and enrich pathogens combined with matrix-assisted laser desorption-ionisation time-of-flight (MALDI-TOF) MS for enhanced infection identification in BCs. The FcMBL method identified 94.1% (64 of 68) of clinical BCs processed, with a high sensitivity for both Gram-negative and Gram-positive bacteria (94.7 and 93.2%, respectively). The FcMBL method identified more patient positive BCs than the Sepsityper® (25 of 25 vs 17 of 25), notably with 100% (3/3) sensitivity for clinical candidemia, compared to only 33% (1/3) for the Sepsityper®. Additionally, during inoculation experiments, the FcMBL method demonstrated a greater sensitivity, identifying 100% (24/24) of candida to genus level and 9/24 (37.5%) top species level compared to 70.8% (17/24) to genus and 6/24 to species (25%) using the Sepsityper®. This study demonstrates that capture and enrichment of samples using magnetic FcMBL-conjugated beads is superior to rapid centrifugation methods for identification of BCs by MALDI-TOF MS. Deploying the FcMBL method therefore offers potential clinical benefits in sensitivity and reduced turnaround times for BC diagnosis compared to the standard Sepsityper® kit, especially for fungal diagnosis.

Costing and cost-effectiveness of Cepheid Xpert HIV -1 Qual Assay using whole blood protocol versus PCR by Abbott Systems in Malawi.

Background: Timely diagnosis of HIV in infants and children is an urgent priority. In Malawi, 40,000 infants annually are HIV exposed. However, gold standard polymerase-chain-reaction (PCR) based testing requires centralised laboratories, causing turn-around times (TAT) of 2 to 3 months and significant loss to follow-up. If feasible and acceptable, minimising diagnostic delays through HIV Point-of-care-testing (POCT) may be cost-effective. We assessed whether POCT Cepheid Xpert HIV-1 Qual assay whole blood (XpertHIV) was more cost-effective than PCR. Methods: From July-August 2018, 700 PCR Abbott tests using dried blood spots (DBS) were performed on 680 participants who enrolled on the feasibility, acceptability and performance of the XpertHIV study. Newly identified HIV-positive We conducted a cost-minimisation and cost-effectiveness analysis of XpertHIV against PCR, as the standard of care. A random sample of 200 caregivers from the 680 participants had semi-structured interviews to explore costs from a societal perspective of XpertHIV at Mulanje District Hospital, Malawi. Analysis used TAT as the primary outcome measure. Results were extrapolated from the study period (29 days) to a year (240 working days). Sensitivity analyses characterised individual and joint parameter uncertainty and estimated patient cost per test. Results: During the study period, XpertHIV was cost-minimising at $42.34 per test compared to $66.66 for PCR. Over a year, XpertHIV remained cost-minimising at $16.12 compared to PCR at $27.06. From the patient perspective (travel, food, lost productivity), the cost per test of XpertHIV was $2.45. XpertHIV had a mean TAT of 7.10 hours compared to 153.15 hours for PCR. Extrapolates accounting for equipment costs, lab consumables and losses to follow up estimated annual savings of $2,193,538.88 if XpertHIV is used nationally, as opposed to PCR. Conclusions: This preliminary evidence suggests that adopting POCT XpertHIV will save time, allowing HIV-exposed infants to receive prompt care and may improve outcomes. The Malawi government will pay less due to XpertHIV's cost savings and associated benefits.

Characterising Post-mortem Bacterial Translocation Under Clinical Conditions Using 16S rRNA Gene Sequencing in Two Animal Models.

Sudden unexpected death in infancy (SUDI) is the sudden and unexpected death of an apparently healthy infant occurring within the first year of life where the cause is not immediately obvious. It is believed that a proportion of unexplained infant deaths are due to an infection that remains undiagnosed. The interpretation of post-mortem microbiology results is difficult due to the potential false-positives, a source of which is post-mortem bacterial translocation. Post-mortem bacterial translocation is the spread of viable bacteria from highly colonised sites to extra-intestinal tissues. We hypothesise that although post-mortem bacterial translocation occurs, when carcasses are kept under controlled routine clinical conditions it is not extensive and can be defined using 16S rRNA gene sequencing. With this knowledge, implementation of the 16S rRNA gene sequencing technique into routine clinical diagnostics would allow a more reliable retrospective diagnosis of ante-mortem infection. Therefore, the aim of this study was to establish the extent of post-mortem bacterial translocation in two animal models to establish a baseline sequencing signal for the post-mortem process. To do this we used 16S rRNA gene sequencing in two animal models over a 2 week period to investigate (1) the bacterial community succession in regions of high bacterial colonisation, and (2) the bacterial presence in visceral tissues routinely sampled during autopsy for microbiological investigation. We found no evidence for significant and consistent post-mortem bacterial translocation in the mouse model. Although bacteria were detected in tissues in the piglet model, we did not find significant and consistent evidence for post-mortem bacterial translocation from the gastrointestinal tract or nasal cavity. These data do not support the concept of significant post-mortem translocation as part of the normal post-mortem process.

Intestinal barrier dysfunction plays an integral role in arthritis pathology and can be targeted to ameliorate disease.

BACKGROUND: Evidence suggests an important role for gut-microbiota dysbiosis in the development of rheumatoid arthritis (RA). The link between changes in gut bacteria and the development of joint inflammation is missing. Here, we address whether there are changes to the gut environment and how they contribute to arthritis pathogenesis. METHODS: We analyzed changes in markers of gut permeability, damage, and inflammation in peripheral blood and serum of RA patients. Serum, intestines, and lymphoid organs isolated from K/BxN mice with spontaneous arthritis or from wild-type, genetically modified interleukin (IL)-10R-/-or claudin-8-/-mice with induced arthritis were analyzed by immunofluorescence/histology, ELISA, and flow cytometry. FINDINGS: RA patients display increased levels of serum markers of gut permeability and damage and cellular gut-homing markers, both parameters positively correlating with disease severity. Arthritic mice display increased gut permeability from early stages of disease, as well as bacterial translocation, inflammatory gut damage, increases in interferon γ (IFNγ)+and decreases in IL-10+intestinal-infiltrating leukocyte frequency, and reduced intestinal epithelial IL-10R expression. Mechanistically, both arthritogenic bacteria and leukocytes are required to disrupt gut-barrier integrity. We show that exposing intestinal organoids to IFNγ reduces IL-10R expression by epithelial cells and that mice lacking epithelial IL-10R display increased intestinal permeability and exacerbated arthritis. Claudin-8-/-mice with constitutively increased gut permeability also develop worse joint disease. Treatment of mice with AT-1001, a molecule that prevents development of gut permeability, ameliorates arthritis. CONCLUSIONS: We suggest that breakdown of gut-barrier integrity contributes to arthritis development and propose restoration of gut-barrier homeostasis as a new therapeutic approach for RA. FUNDING: Funded by Versus Arthritis (21140 and 21257) and UKRI/MRC (MR/T000910/1).

Inflammatory pathways amongst people living with HIV in Malawi differ according to socioeconomic status.

BACKGROUND: Non-communicable diseases (NCDs) are increased amongst people living with HIV (PLWH) and are driven by persistent immune activation. The role of socioeconomic status (SES) in immune activation amongst PLWH is unknown, especially in low-income sub-Saharan Africa (SSA), where such impacts may be particularly severe. METHODS: We recruited Malawian adults with CD4<100 cells/ul two weeks after starting ART in the REALITY trial (NCT01825031), as well as volunteers without HIV infection. Clinical assessment, socioeconomic evaluation, blood draw for immune activation markers and carotid femoral pulse wave velocity (cfPWV) were carried out at 2- and 42-weeks post-ART initiation. Socioeconomic risk factors for immune activation and arterial stiffness were assessed using linear regression models. RESULTS: Of 279 PLWH, the median (IQR) age was 36 (31-43) years and 122 (44%) were female. Activated CD8 T-cells increased from 70% amongst those with no education to 88% amongst those with a tertiary education (p = 0.002); and from 71% amongst those earning less than 10 USD/month to 87% amongst those earning between 100-150 USD/month (p = 0.0001). Arterial stiffness was also associated with higher SES (car ownership p = 0.003, television ownership p = 0.012 and electricity access p = 0.029). Conversely, intermediate monocytes were higher amongst those with no education compared to a tertiary education (12.6% versus 7.3%; p = 0.01) and trended towards being higher amongst those earning less than 10 USD/month compared to 100-150 USD/month (10.5% versus 8.0%; p = 0.08). Water kiosk use showed a protective association against T cell activation (p = 0.007), as well as endothelial damage (MIP1ß, sICAM1 and sVCAM1 p = 0.047, 0.026 and 0.031 respectively). CONCLUSIONS: Socioeconomic risk factors for persistent inflammation amongst PLWH in SSA differ depending on the type of inflammatory pathway. Understanding these pathways and their socioeconomic drivers will help identify those at risk and target interventions for NCDs. Future studies assessing drivers of inflammation in HIV should include an SES assessment.

Cervical Gene Delivery of the Antimicrobial Peptide, Human ß-Defensin (HBD)-3, in a Mouse Model of Ascending Infection-Related Preterm Birth.

Approximately 40% of preterm births are preceded by microbial invasion of the intrauterine space; ascent from the vagina being the most common pathway. Within the cervical canal, antimicrobial peptides and proteins (AMPs) are important components of the cervical barrier which help to prevent ascending vaginal infection. We investigated whether expression of the AMP, human ß-defensin-3 (HBD3), in the cervical mucosa of pregnant mice could prevent bacterial ascent from the vagina into the uterine cavity. An adeno-associated virus vector containing both the HBD3 gene and GFP transgene (AAV8 HBD3.GFP) or control AAV8 GFP, was administered intravaginally into E13.5 pregnant mice. Ascending infection was induced at E16.5 using bioluminescent Escherichia coli (E. coli K1 A192PP-lux2). Bioluminescence imaging showed bacterial ascent into the uterine cavity, inflammatory events that led to premature delivery and a reduction in pups born alive, compared with uninfected controls. Interestingly, a significant reduction in uterine bioluminescence in the AAV8 HBD3.GFP-treated mice was observed 24 h post-E. coli infection, compared to AAV8 GFP treated mice, signifying reduced bacterial ascent in AAV8 HBD3.GFP-treated mice. Furthermore, there was a significant increase in the number of living pups in AAV HBD3.GFP-treated mice. We propose that HBD3 may be a potential candidate for augmenting cervical innate immunity to prevent ascending infection-related preterm birth and its associated neonatal consequences.

Microbiota-Derived Metabolites Suppress Arthritis by Amplifying Aryl-Hydrocarbon Receptor Activation in Regulatory B Cells.

The differentiation of IL-10-producing regulatory B cells (Bregs) in response to gut-microbiota-derived signals supports the maintenance of tolerance. However, whether microbiota-derived metabolites can modulate Breg suppressive function remains unknown. Here, we demonstrate that rheumatoid arthritis (RA) patients and arthritic mice have a reduction in microbial-derived short-chain fatty acids (SCFAs) compared to healthy controls and that in mice, supplementation with the SCFA butyrate reduces arthritis severity. Butyrate supplementation suppresses arthritis in a Breg-dependent manner by increasing the level of the serotonin-derived metabolite 5-Hydroxyindole-3-acetic acid (5-HIAA), which activates the aryl-hydrocarbon receptor (AhR), a newly discovered transcriptional marker for Breg function. Thus, butyrate supplementation via AhR activation controls a molecular program that supports Breg function while inhibiting germinal center (GC) B cell and plasmablast differentiation. Our study demonstrates that butyrate supplementation may serve as a viable therapy for the amelioration of systemic autoimmune disorders.

1,4-benzodiazepines as inhibitors of respiratory syncytial virus. The identification of a clinical candidate.

Respiratory syncytial virus (RSV) is the cause of one-fifth of all lower respiratory tract infections worldwide and is increasingly being recognized as representing a serious threat to patient groups with poorly functioning or immature immune systems. Racemic 1,4-benzodiazepines show potent anti-RSV activity in vitro. Anti-RSV evaluation of 3-position R- and S-benzodiazepine enantiomers and subsequent optimization of this series resulted in selection of a clinical candidate. Antiviral activity was found to reside mainly in the S-enantiomer, and the R-enantiomers were consistently less active against RSV. Analogues of 1,4-(S)-benzodiazepine were synthesized as part of the lead optimization program at Arrow and tested in the XTT assay. From this exercise, (S)-1-(2-fluorophenyl)-3-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]-diazepin-3-yl)-urea, 17b (RSV-604) was identified as a clinical candidate, exhibiting potent anti-RSV activity in the XTT assay, which was confirmed in secondary assays. Compound 17b also possessed a good pharmacokinetic profile and has now progressed into the clinic.

1,4-Benzodiazepines as inhibitors of respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the cause of one-fifth of all lower respiratory tract infections worldwide and is increasingly being recognized as a serious threat to patient groups with poorly functioning immune systems. Our approach to finding a novel inhibitor of this virus was to screen a 20 000-member diverse library in a whole cell XTT assay. Parallel assays were carried out in the absence of virus in order to quantify any associated cell toxicity. This identified 100 compounds with IC(50)'s less than 50 muM. A-33903 (18), a 1,4-benzodiazepine analogue, was chosen as the starting point for lead optimization. This molecule was moderately active and demonstrated good pharmacokinetic properties. The most potent compounds identified from this work were A-58568 (47), A-58569 (44), and A-62066 (46), where modifications to the aromatic substitution enhanced potency, and A-58175 (42), where the amide linker was modified.