Detection of a historic reservoir of bedaquiline/clofazimine resistance-associated variants in Mycobacterium tuberculosis.

BACKGROUND: Drug resistance in tuberculosis (TB) poses a major ongoing challenge to public health. The recent inclusion of bedaquiline into TB drug regimens has improved treatment outcomes, but this advance is threatened by the emergence of strains of Mycobacterium tuberculosis (Mtb) resistant to bedaquiline. Clinical bedaquiline resistance is most frequently conferred by off-target resistance-associated variants (RAVs) in the mmpR5 gene (Rv0678), the regulator of an efflux pump, which can also confer cross-resistance to clofazimine, another TB drug. METHODS: We compiled a dataset of 3682 Mtb genomes, including 180 carrying variants in mmpR5, and its immediate background (i.e. mmpR5 promoter and adjacent mmpL5 gene), that have been associated to borderline (henceforth intermediate) or confirmed resistance to bedaquiline. We characterised the occurrence of all nonsynonymous mutations in mmpR5 in this dataset and estimated, using time-resolved phylogenetic methods, the age of their emergence. RESULTS: We identified eight cases where RAVs were present in the genomes of strains collected prior to the use of bedaquiline in TB treatment regimes. Phylogenetic reconstruction points to multiple emergence events and circulation of RAVs in mmpR5, some estimated to predate the introduction of bedaquiline. However, epistatic interactions can complicate bedaquiline drug-susceptibility prediction from genetic sequence data. Indeed, in one clade, Ile67fs (a RAV when considered in isolation) was estimated to have emerged prior to the antibiotic era, together with a resistance reverting mmpL5 mutation. CONCLUSIONS: The presence of a pre-existing reservoir of Mtb strains carrying bedaquiline RAVs prior to its clinical use augments the need for rapid drug susceptibility testing and individualised regimen selection to safeguard the use of bedaquiline in TB care and control.

Socioeconomic and Demographic Risk Factors for SARS-CoV-2 Seropositivity Among Healthcare Workers in a UK Hospital: A Prospective Cohort Study.

BACKGROUND: To protect healthcare workers (HCWs) from the consequences of disease due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to understand the risk factors that drive exposure and infection within hospitals. Insufficient consideration of key socioeconomic variables is a limitation of existing studies that can lead to bias and residual confounding of proposed risk factors for infection. METHODS: The Co-STARs study prospectively enrolled 3679 HCWs between April 2020 and September 2020. We used multivariate logistic regression to comprehensively characterize the demographic, occupational, socioeconomic, and environmental risk factors for SARS-CoV-2 seropositivity. RESULTS: After adjusting for key confounders, relative household overcrowding (odds ratio [OR], 1.4 [95% confidence interval {CI}, 1.1-1.9]; P = .006), Black, Black British, Caribbean, or African ethnicity (OR, 1.7 [95% CI, 1.2-2.3]; P = .003), increasing age (ages 50-60 years: OR, 1.8 [95% CI, 1.3-2.4]; P < .001), lack of access to sick pay (OR, 1.8 [95% CI, 1.3-2.4]; P < .001). CONCLUSIONS: Socioeconomic and demographic factors outside the hospital were the main drivers of infection and exposure to SARS-CoV-2 during the first wave of the pandemic in an urban pediatric referral hospital. Overcrowding and out-of-hospital SARS-CoV-2 contact are less amenable to intervention. However, lack of access to sick pay among externally contracted staff is more easily rectifiable. Our findings suggest that providing easier access to sick pay would lead to a decrease in SARS-CoV-2 transmission and potentially that of other infectious diseases in hospital settings. CLINICAL TRIALS REGISTRATION: NCT04380896.

Salivary IgA and vimentin differentiate in vitro SARS-CoV-2 infection: A study of 290 convalescent COVID-19 patients.

SARS-CoV-2 initially infects cells in the nasopharynx and oral cavity. The immune system at these mucosal sites plays a crucial role in minimizing viral transmission and infection. To develop new strategies for preventing SARS-CoV-2 infection, this study aimed to identify proteins that protect against viral infection in saliva. We collected 551 saliva samples from 290 healthcare workers who had tested positive for COVID-19, before vaccination, between June and December 2020. The samples were categorized based on their ability to block or enhance infection using in vitro assays. Mass spectrometry and enzyme-linked immunosorbent assay experiments were used to identify and measure the abundance of proteins that specifically bind to SARS-CoV-2 antigens. Immunoglobulin (Ig)A specific to SARS-CoV-2 antigens was detectable in over 83% of the convalescent saliva samples. We found that concentrations of anti-receptor-binding domain IgA >500 pg/µg total protein in saliva correlate with reduced viral infectivity in vitro. However, there is a dissociation between the salivary IgA response to SARS-CoV-2, and systemic IgG titers in convalescent COVID-19 patients. Then, using an innovative technique known as spike-baited mass spectrometry, we identified novel spike-binding proteins in saliva, most notably vimentin, which correlated with increased viral infectivity in vitro and could serve as a therapeutic target against COVID-19.

The effect of M. tuberculosis lineage on clinical phenotype.

Six lineages of Mycobacterium tuberculosis sensu stricto (which excludes M. africanum) are described. Single-country or small observational data suggest differences in clinical phenotype between lineages. We present strain lineage and clinical phenotype data from 12,246 patients from 3 low-incidence and 5 high-incidence countries. We used multivariable logistic regression to explore the effect of lineage on site of disease and on cavities on chest radiography, given pulmonary TB; multivariable multinomial logistic regression to investigate types of extra-pulmonary TB, given lineage; and accelerated failure time and Cox proportional-hazards models to explore the effect of lineage on time to smear and culture-conversion. Mediation analyses quantified the direct effects of lineage on outcomes. Pulmonary disease was more likely among patients with lineage(L) 2, L3 or L4, than L1 (adjusted odds ratio (aOR) 1.79, (95% confidence interval 1.49-2.15), p<0.001; aOR = 1.40(1.09-1.79), p = 0.007; aOR = 2.04(1.65-2.53), p<0.001, respectively). Among patients with pulmonary TB, those with L1 had greater risk of cavities on chest radiography versus those with L2 (aOR = 0.69(0.57-0.83), p<0.001) and L4 strains (aOR = 0.73(0.59-0.90), p = 0.002). L1 strains were more likely to cause osteomyelitis among patients with extra-pulmonary TB, versus L2-4 (p = 0.033, p = 0.008 and p = 0.049 respectively). Patients with L1 strains showed shorter time-to-sputum smear conversion than for L2. Causal mediation analysis showed the effect of lineage in each case was largely direct. The pattern of clinical phenotypes seen with L1 strains differed from modern lineages (L2-4). This has implications for clinical management and could influence clinical trial selection strategies.

Prioritizing persons deprived of liberty in global guidelines for tuberculosis preventive treatment.

In this Policy Forum piece, Aditya Narayan and colleagues discuss the challenges and opportunities for tuberculosis preventive treatment in carceral settings.

Within-host diversity improves phylogenetic and transmission reconstruction of SARS-CoV-2 outbreaks.

Accurate inference of who infected whom in an infectious disease outbreak is critical for the delivery of effective infection prevention and control. The increased resolution of pathogen whole-genome sequencing has significantly improved our ability to infer transmission events. Despite this, transmission inference often remains limited by the lack of genomic variation between the source case and infected contacts. Although within-host genetic diversity is common among a wide variety of pathogens, conventional whole-genome sequencing phylogenetic approaches exclusively use consensus sequences, which consider only the most prevalent nucleotide at each position and therefore fail to capture low-frequency variation within samples. We hypothesized that including within-sample variation in a phylogenetic model would help to identify who infected whom in instances in which this was previously impossible. Using whole-genome sequences from SARS-CoV-2 multi-institutional outbreaks as an example, we show how within-sample diversity is partially maintained among repeated serial samples from the same host, it can transmitted between those cases with known epidemiological links, and how this improves phylogenetic inference and our understanding of who infected whom. Our technique is applicable to other infectious diseases and has immediate clinical utility in infection prevention and control.

During an infectious disease outbreak, tracing who infected whom allows public health scientists to see how a pathogen is spreading and to establish effective control measures. Traditionally, this involves identifying the individuals an infected person comes into contact with and monitoring whether they also become unwell. However, this information is not always available and can be inaccurate. One alternative is to track the genetic data of pathogens as they spread. Over time, pathogens accumulate mutations in their genes that can be used to distinguish them from one another. Genetically similar pathogens are more likely to have spread during the same outbreak, while genetically dissimilar pathogens may have come from different outbreaks. However, there are limitations to this approach. For example, some pathogens accumulate genetic mutations very slowly and may not change enough during an outbreak to be distinguishable from one another. Additionally, some pathogens can spread rapidly, leaving less time for mutations to occur between transmission events. To overcome these challenges, Torres Ortiz et al. developed a more sensitive approach to pathogen genetic testing that took advantage of the multiple pathogen populations that often coexist in an infected patient. Rather than tracking only the most dominant genetic version of the pathogen, this method also looked at the less dominant ones. Torres Ortiz et al. performed genome sequencing of SARS-CoV-2 (the virus that causes COVID-19) samples from 451 healthcare workers, patients, and patient contacts at participating London hospitals. Analysis showed that it was possible to detect multiple genetic populations of the virus within individual patients. These subpopulations were often more similar in patients that had been in contact with one another than in those that had not. Tracking the genetic data of all viral populations enabled Torres Ortiz et al. to trace transmission more accurately than if only the dominant population was used. More accurate genetic tracing could help public health scientists better track pathogen transmission and control outbreaks. This may be especially beneficial in hospital settings where outbreaks can be smaller, and it is important to understand if transmission is occurring within the hospital or if the pathogen is imported from the community. Further research will help scientists understand how pathogen population genetics evolve during outbreaks and may improve the detection of subpopulations present at very low frequencies.

Quantifying patient- and hospital-level antimicrobial resistance dynamics in Staphylococcus aureus from routinely collected data.

Introduction. Antimicrobial resistance (AMR) to all antibiotic classes has been found in the pathogen Staphylococcus aureus. The reported prevalence of these resistances varies, driven by within-host AMR evolution at the patient level, and between-host transmission at the hospital level. Without dense longitudinal sampling, pragmatic analysis of AMR dynamics at multiple levels using routine surveillance data is essential to inform control measures.Gap Statement. The value and limitations of routinely collected hospital data to gain insight into AMR dynamics at the hospital and individual levels simultaneously are unclear.Methodology. We explored S. aureus AMR diversity in 70 000 isolates from a UK paediatric hospital between 2000-2021, using electronic datasets containing multiple routinely collected isolates per patient with phenotypic antibiograms and information on hospitalization and antibiotic consumption.Results. At the hospital level, the proportion of isolates that were meticillin-resistant (MRSA) increased between 2014-2020 from 25-50 %, before sharply decreasing to 30%, likely due to a change in inpatient demographics. Temporal trends in the proportion of isolates resistant to different antibiotics were often correlated in MRSA, but independent in meticillin-susceptible S. aureus. Ciprofloxacin resistance in MRSA decreased from 70-40 % of tested isolates between 2007-2020, likely linked to a national policy to reduce fluoroquinolone usage in 2007. At the patient level, we identified frequent AMR diversity, with 4 % of patients ever positive for S. aureus simultaneously carrying, at some point, multiple isolates with different resistances. We detected changes over time in AMR diversity in 3 % of patients ever positive for S. aureus. These changes equally represented gain and loss of resistance.Conclusion. Within this routinely collected dataset, we found that 65 % of changes in resistance within a patient's S. aureus population could not be explained by antibiotic exposure or between-patient transmission of bacteria, suggesting that within-host evolution via frequent gain and loss of AMR genes may be responsible for these changing AMR profiles. Our study highlights the value of exploring existing routine surveillance data to determine underlying mechanisms of AMR. These insights may substantially improve our understanding of the importance of antibiotic exposure variation, and the success of single S. aureus clones.

The effect of M. tuberculosis lineage on clinical phenotype.

Eight lineages of Mycobacterium tuberculosis sensu stricto are described. Single-country or small observational data suggest differences in clinical phenotype between lineages. We present strain lineage and clinical phenotype data from 12,246 patients from 3 low-incidence and 5 high-incidence countries. We used multivariable logistic regression to explore the effect of lineage on site of disease and on cavities on chest radiography, given pulmonary TB; multivariable multinomial logistic regression to investigate types of extra-pulmonary TB, given lineage; and accelerated failure time and Cox proportional-hazards models to explore the effect of lineage on time to smear and culture-conversion. Mediation analyses quantified the direct effects of lineage on outcomes. Pulmonary disease was more likely among patients with lineage(L) 2, L3 or L4, than L1 (adjusted odds ratio (aOR) 1.79, (95% confidence interval 1.49-2.15), p<0.001; aOR=1.40(1.09-1.79), p=0.007; aOR=2.04(1.65-2.53), p<0.001, respectively). Among patients with pulmonary TB, those with L1 had greater risk of cavities on chest radiography versus those with L2 (aOR=0.69(0.57-0.83), p<0.001) and L4 strains (aOR=0.73(0.59-0.90), p=0.002). L1 strains were more likely to cause osteomyelitis among patients with extra-pulmonary TB, versus L2-4 (p=0.033, p=0.008 and p=0.049 respectively). Patients with L1 strains showed shorter time-to-sputum smear conversion than for L2. Causal mediation analysis showed the effect of lineage in each case was largely direct. The pattern of clinical phenotypes seen with L1 strains differed from modern lineages (L2-4). This has implications for clinical management and could influence clinical trial selection strategies.

Aberrant olfactory network functional connectivity in people with olfactory dysfunction following COVID-19 infection: an exploratory, observational study.

Background: Olfactory impairments and anosmia from COVID-19 infection typically resolve within 2-4 weeks, although in some cases, symptoms persist longer. COVID-19-related anosmia is associated with olfactory bulb atrophy, however, the impact on cortical structures is relatively unknown, particularly in those with long-term symptoms. Methods: In this exploratory, observational study, we studied individuals who experienced COVID-19-related anosmia, with or without recovered sense of smell, and compared against individuals with no prior COVID-19 infection (confirmed by antibody testing, all vaccine naïve). MRI Imaging was carried out between the 15th July and 17th November 2020 at the Queen Square House Clinical Scanning Facility, UCL, United Kingdom. Using functional magnetic resonance imaging (fMRI) and structural imaging, we assessed differences in functional connectivity (FC) between olfactory regions, whole brain grey matter (GM) cerebral blood flow (CBF) and GM density. Findings: Individuals with anosmia showed increased FC between the left orbitofrontal cortex (OFC), visual association cortex and cerebellum and FC reductions between the right OFC and dorsal anterior cingulate cortex compared to those with no prior COVID-19 infection (p < 0.05, from whole brain statistical parametric map analysis). Individuals with anosmia also showed greater CBF in the left insula, hippocampus and ventral posterior cingulate when compared to those with resolved anosmia (p < 0.05, from whole brain statistical parametric map analysis). Interpretation: This work describes, for the first time to our knowledge, functional differences within olfactory areas and regions involved in sensory processing and cognitive functioning. This work identifies key areas for further research and potential target sites for therapeutic strategies. Funding: This study was funded by the National Institute for Health and Care Research and supported by the Queen Square Scanner business case.

Scaling up evidence-based approaches to tuberculosis screening in prisons.

People deprived of liberty have among the highest rates of tuberculosis globally. The incidence of tuberculosis is ten times greater than the incidence of tuberculosis in the general population. In 2021, WHO updated its guidance to strongly recommend systematic screening for tuberculosis in prisons and penitentiary systems. Which case-finding strategies should be adopted, and how to effectively implement these strategies in these settings, will be crucial questions facing ministries of health and justice. In this Viewpoint, we review the evidence base for tuberculosis screening and diagnostic strategies in prisons, highlighting promising approaches and knowledge gaps. Drawing upon past experiences of implementing active case-finding and care programmes in settings with a high tuberculosis burden, we discuss challenges and opportunities for improving the tuberculosis diagnosis and treatment cascade in these settings. We argue that improved transparency in reporting of tuberculosis notifications and outcomes in prisons and renewed focus and resourcing from WHO and other stakeholders will be crucial for building the commitment and investments needed from countries to address the continued crisis of tuberculosis in prisons.

Quantifying patient- and hospital-level antimicrobial resistance dynamics in Staphylococcus aureus from routinely collected data.

Antimicrobial resistance (AMR) to all antibiotic classes has been found in the pathogen Staphylococcus aureus . The reported prevalence of these resistances vary, driven by within-host AMR evolution at the patient level, and between-host transmission at the hospital level. Without dense longitudinal sampling, pragmatic analysis of AMR dynamics at multiple levels using routine surveillance data is essential to inform control measures. We explored S. aureus AMR diversity in 70,000 isolates from a UK paediatric hospital between 2000-2020, using electronic datasets containing multiple routinely collected isolates per patient with phenotypic antibiograms, hospitalisation information, and antibiotic consumption. At the hospital-level, the proportion of isolates that were meticillin-resistant (MRSA) increased between 2014-2020 from 25 to 50%, before sharply decreasing to 30%, likely due to a change in inpatient demographics. Temporal trends in the proportion of isolates resistant to different antibiotics were often correlated in MRSA, but independent in meticillin-susceptible S. aureus . Ciprofloxacin resistance in MRSA decreased from 70% to 40% of tested isolates between 2007-2020, likely linked to a national policy to reduce fluoroquinolone usage in 2007. At the patient level, we identified frequent AMR diversity, with 4% of patients ever positive for S. aureus simultaneously carrying, at some point, multiple isolates with different resistances. We detected changes over time in AMR diversity in 3% of patients ever positive for S. aureus . These changes equally represented gain and loss of resistance. Within this routinely collected dataset, we found that 65% of changes in resistance within a patient’s S. aureus population could not be explained by antibiotic exposure or between-patient transmission of bacteria, suggesting that within-host evolution via frequent gain and loss of AMR genes may be responsible for these changing AMR profiles. Our study highlights the value of exploring existing routine surveillance data to determine underlying mechanisms of AMR. These insights may substantially improve our understanding of the importance of antibiotic exposure variation, and the success of single S. aureus clones.

Genome-Wide Association with Uncertainty in the Genetic Similarity Matrix.

Genome-wide association studies (GWASs) are often confounded by population stratification and structure. Linear mixed models (LMMs) are a powerful class of methods for uncovering genetic effects, while controlling for such confounding. LMMs include random effects for a genetic similarity matrix, and they assume that a true genetic similarity matrix is known. However, uncertainty about the phylogenetic structure of a study population may degrade the quality of LMM results. This may happen in bacterial studies in which the number of samples or loci is small, or in studies with low-quality genotyping. In this study, we develop methods for linear mixed models in which the genetic similarity matrix is unknown and is derived from Markov chain Monte Carlo estimates of the phylogeny. We apply our model to a GWAS of multidrug resistance in tuberculosis, and illustrate our methods on simulated data.

Evaluation of three alternatives cost-effective culture media for Mycobacterium tuberculosis detection and drug susceptibility determination using the microscopic observation drug susceptibility (MODS) assay.

Tuberculosis phenotypic detection assays are commonly used in low-resource countries. Therefore, reliable detection methods are crucial for early diagnosis and treatment. The microscopic observation drug susceptibility (MODS) assay is a culture-based test to detect Mycobacterium tuberculosis and characterize drug resistance in 7-10 days directly from sputum. The use of MODS is limited by the availability of supplies necessary for preparing the enriched culture. In this study, we evaluated three dry culture media that are easier to produce and cheaper than the standard one used in MODS [1]: an unsterilized powder-based mixed (Boldú et al., 2007) [2], a sterile-lyophilized medium, and (Sengstake et al., 2017) [3] an irradiated powder-based mixed. Mycobacterial growth and drug susceptibility were evaluated for rifampin, isoniazid, and pyrazinamide (PZA). The alternative cultures were evaluated using 282 sputum samples with positive acid-fast smears. No significant differences were observed in the positivity test rates. The positivity time showed high correlations (Rho) of 0.925, 0.889, and 0.866 between each of the three alternative media and the standard. Susceptibility testing for MDR and PZA showed an excellent concordance of 1 compared to the reference test. These results demonstrate that dry culture media are appropriate and advantageous for use in MODS in low-resource settings.

Distorted TCR repertoires define multisystem inflammatory syndrome in children.

While the majority of children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) display mild or no symptoms, rare individuals develop severe disease presenting with multisystem inflammatory syndrome (MIS-C). The reason for variable clinical manifestations is not understood. Here, we carried out TCR sequencing and conducted comparative analyses of TCR repertoires between children with MIS-C (n = 12) and mild (n = 8) COVID-19. We compared these repertoires with unexposed individuals (samples collected pre-COVID-19 pandemic: n = 8) and with the Adaptive Biotechnologies MIRA dataset, which includes over 135,000 high-confidence SARS-CoV-2-specific TCRs. We show that the repertoires of children with MIS-C are characterised by the expansion of TRBV11-2 chains with high junctional and CDR3 diversity. Moreover, the CDR3 sequences of TRBV11-2 clones shift away from SARS-CoV-2 specific T cell clones, resulting in distorted TCR repertoires. In conclusion, our study reports that CDR3-independent expansion of TRBV11-2+ cells, lacking SARS-CoV-2 specificity, defines MIS-C in children.

Infant BCG vaccination and risk of pulmonary and extrapulmonary tuberculosis throughout the life course: a systematic review and individual participant data meta-analysis.

BACKGROUND: BCG vaccines are given to more than 100 million children every year, but there is considerable debate regarding the effectiveness of BCG vaccination in preventing tuberculosis and death, particularly among older children and adults. We therefore aimed to investigate the age-specific impact of infant BCG vaccination on tuberculosis (pulmonary and extrapulmonary) development and mortality. METHODS: In this systematic review and individual participant data meta-analysis, we searched MEDLINE, Web of Science, BIOSIS, and Embase without language restrictions for case-contact cohort studies of tuberculosis contacts published between Jan 1, 1998, and April 7, 2018. Search terms included "mycobacterium tuberculosis", "TB", "tuberculosis", and "contact". We excluded cohort studies that did not provide information on BCG vaccination or were done in countries that did not recommend BCG vaccination at birth. Individual-level participant data for a prespecified list of variables, including the characteristics of the exposed participant (contact), the index case, and the environment, were requested from authors of all eligible studies. Our primary outcome was a composite of prevalent (diagnosed at or within 90 days of baseline) and incident (diagnosed more than 90 days after baseline) tuberculosis in contacts exposed to tuberculosis. Secondary outcomes were pulmonary tuberculosis, extrapulmonary tuberculosis, and mortality. We derived adjusted odds ratios (aORs) using mixed-effects, binary, multivariable logistic regression analyses with study-level random effects, adjusting for the variable of interest, baseline age, sex, previous tuberculosis, and whether data were collected prospectively or retrospectively. We stratified our results by contact age and Mycobacterium tuberculosis infection status. This study is registered with PROSPERO, CRD42020180512. FINDINGS: We identified 14 927 original records from our database searches. We included participant-level data from 26 cohort studies done in 17 countries in our meta-analysis. Among 68 552 participants, 1782 (2·6%) developed tuberculosis (1309 [2·6%] of 49 686 BCG-vaccinated participants vs 473 [2·5%] of 18 866 unvaccinated participants). The overall effectiveness of BCG vaccination against all tuberculosis was 18% (aOR 0·82, 95% CI 0·74-0·91). When stratified by age, BCG vaccination only significantly protected against all tuberculosis in children younger than 5 years (aOR 0·63, 95% CI 0·49-0·81). Among contacts with a positive tuberculin skin test or IFNγ release assay, BCG vaccination significantly protected against tuberculosis among all participants (aOR 0·81, 95% CI 0·69-0·96), participants younger than 5 years (0·68, 0·47-0·97), and participants aged 5-9 years (0·62, 0·38-0·99). There was no protective effect among those with negative tests, unless they were younger than 5 years (0·54, 0·32-0·90). 14 cohorts reported on whether tuberculosis was pulmonary or extrapulmonary (n=57 421). BCG vaccination significantly protected against pulmonary tuberculosis among all participants (916 [2·2%] in 41 119 vaccinated participants vs 334 [2·1%] in 16 161 unvaccinated participants; aOR 0·81, 0·70-0·94) but not against extrapulmonary tuberculosis (106 [0·3%] in 40 318 vaccinated participants vs 38 [0·2%] in 15 865 unvaccinated participants; 0·96, 0·65-1·41). In the four studies with mortality data, BCG vaccination was significantly protective against death (0·25, 0·13-0·49). INTERPRETATION: Our results suggest that BCG vaccination at birth is effective at preventing tuberculosis in young children but is ineffective in adolescents and adults. Immunoprotection therefore needs to be boosted in older populations. FUNDING: National Institutes of Health.

Alternative cost-effective media to facilitate MODS culture for diagnostics of tuberculosis.

Most culture-based methods for tuberculosis diagnosis remain low-cost options for low- and mid-income countries. The MODS culture is a rapid and low-cost assay to diagnose tuberculosis and determine drug susceptibility. However, its implementation is limited due to the low accessibility to supplies required for the enriched medium. In this study, we evaluate two alternative culture media: A powder-based mixed (PM) and a lyophilized media (LM). Catalase, PANTA, and gamma irradiation were evaluated as additions to PM and LM. The culture performance of the alternative media was compared with the standard MODS medium (MM) using Mycobacterium tuberculosis isolates and positive acid-fast smear sputum samples. Overall, no significant difference was observed in the bacterial growth between PM and LM with MM. However, PANTA and gamma irradiation combined reduced bacterial growth significantly in all media variants. A median positivity day of 6 ± 5 days was observed for sputum samples, regardless of the culture medium. The preliminary results show that the two variants culture media have a similar performance to the standard MODS medium. The powder-based media with PANTA (PM_P) showed a time-to-positivity and sensitivity similar to the standard MODS medium. It is the simplest to prepare and does not require any sterilization process.

The influence of time on the sensitivity of SARS-CoV-2 serological testing.

Sensitive serological testing is essential to estimate the proportion of the population exposed or infected with SARS-CoV-2, to guide booster vaccination and to select patients for treatment with anti-SARS-CoV-2 antibodies. The performance of serological tests is usually evaluated at 14-21 days post infection. This approach fails to take account of the important effect of time on test performance after infection or exposure has occurred. We performed parallel serological testing using 4 widely used assays (a multiplexed SARS-CoV-2 Nucleoprotein (N), Spike (S) and Receptor Binding Domain assay from Meso Scale Discovery (MSD), the Roche Elecsys-Nucleoprotein (Roche-N) and Spike (Roche-S) assays and the Abbott Nucleoprotein assay (Abbott-N) on serial positive monthly samples collected as part of the Co-STARs study ( www.clinicaltrials.gov , NCT04380896) up to 200 days following infection. Our findings demonstrate the considerable effect of time since symptom onset on the diagnostic sensitivity of different assays. Using a time-to-event analysis, we demonstrated that 50% of the Abbott nucleoprotein assays will give a negative result after 175 days (median survival time 95% CI 168-185 days), compared to the better performance over time of the Roche Elecsys nucleoprotein assay (93% survival probability at 200 days, 95% CI 88-97%). Assays targeting the spike protein showed a lower decline over the follow-up period, both for the MSD spike assay (97% survival probability at 200 days, 95% CI 95-99%) and the Roche Elecsys spike assay (95% survival probability at 200 days, 95% CI 93-97%). The best performing quantitative Roche Elecsys Spike assay showed no evidence of waning Spike antibody titers over the 200-day time course of the study. We have shown that compared to other assays evaluated, the Abbott-N assay fails to detect SARS-CoV-2 antibodies as time passes since infection. In contrast the Roche Elecsys Spike Assay and the MSD assay maintained a high sensitivity for the 200-day duration of the study. These limitations of the Abbott assay should be considered when quantifying the immune correlates of protection or the need for SARS-CoV-2 antibody therapy. The high levels of maintained detectable neutralizing spike antibody titers identified by the quantitative Roche Elecsys assay is encouraging and provides further evidence in support of long-lasting SARS-CoV-2 protection following natural infection.

Within-host diversity improves phylogenetic and transmission reconstruction of SARS-CoV-2 outbreaks.

Accurate inference of who infected whom in an infectious disease outbreak is critical for the delivery of effective infection prevention and control. The increased resolution of pathogen whole-genome sequencing has significantly improved our ability to infer transmission events. Despite this, transmission inference often remains limited by the lack of genomic variation between the source case and infected contacts. Although within-host genetic diversity is common among a wide variety of pathogens, conventional whole-genome sequencing phylogenetic approaches to reconstruct outbreaks exclusively use consensus sequences, which consider only the most prevalent nucleotide at each position and therefore fail to capture low frequency variation within samples. We hypothesized that including within-sample variation in a phylogenetic model would help to identify who infected whom in instances in which this was previously impossible. Using whole-genome sequences from SARS-CoV-2 multi-institutional outbreaks as an example, we show how within-sample diversity is stable among repeated serial samples from the same host, is transmitted between those cases with known epidemiological links, and how this improves phylogenetic inference and our understanding of who infected whom. Our technique is applicable to other infectious diseases and has immediate clinical utility in infection prevention and control.

The Influence of Time on the Sensitivity of SARS-CoV-2 Serological Testing.

Background: Serological testing is used to quantify SARS-CoV-2 seroprevalence, guide booster vaccination and select patients for anti-SARS-CoV-2 antibodies therapy. However, our understanding of how serological tests perform as time passes after infection is limited. Methods: Four assays were compared in parallel: 1) the multiplexed spike, nucleoprotein and receptor binding domain Meso Scale Discovery (MSD) assay 2) the Roche Elecsys-Nucleoprotein assay (Roche-N) 3) the Roche Spike assay (Roche-S) and 4) the Abbott Nucleoprotein assay (Abbott-N) on serial positive monthly samples from hospital staff up to 200 days following infection as part of the Co-Stars study. Results: We demonstrate that 50% of the Abbott-N assays give a negative result after 175 days (median survival time 95% CI 168-185 days) while the Roche-N assay (93% survival probability at 200 days, 95% CI 88-97%) maintained seropositivity. The MSD spike (97% survival probability at 200 days, 95% CI 95-99%) and the Roche-S assay (95% survival probability at 200 days, 95% CI 93-97%) also remained seropositive. The best performing quantitative Roche-S assay showed no evidence of waning Spike antibody titres over 200-days. Conclusions: The Abbott-N assay fails to detect SARS-CoV-2 antibodies as time passes since infection. In contrast the Roche and the MSD assays maintained high sensitivity. The limitations of the Abbott assay must be considered in clinical decision making. The long duration of detectable neutralizing spike antibody titres by the quantitative Roche-S assay provides further evidence in support of long-lasting SARS-CoV-2 protection to pre-existing strains of SARS-CoV-2 following natural infection. Trial registration : Co-STARs study was registered with ClinicalTrials.gov on May 8th, 2020, with trial number NCT04380896 (www.clinicaltrials.gov, NCT04380896).

T cell responses to SARS-CoV-2 in healthy controls and primary immunodeficiency patients.

Understanding the T cell response to SARS-CoV-2 is key in patients who lack antibody production. We demonstrate the applicability of a functional assay to measure the T cell response in a cohort of patients with immunodeficiency.