Development of a Two-Component Nanoparticle Vaccine Displaying an HIV-1 Envelope Glycoprotein that Elicits Tier 2 Neutralising Antibodies.

Despite treatment and other interventions, an effective prophylactic HIV vaccine is still an essential goal in the control of HIV. Inducing robust and long-lasting antibody responses is one of the main targets of an HIV vaccine. The delivery of HIV envelope glycoproteins (Env) using nanoparticle (NP) platforms has been shown to elicit better immunogenicity than soluble HIV Env. In this paper, we describe the development of a nanoparticle-based vaccine decorated with HIV Env using the SpyCatcher/SpyTag system. The Env utilised in this study, CAP255, was derived from a transmitted founder virus isolated from a patient who developed broadly neutralising antibodies. Negative stain and cryo-electron microscopy analyses confirmed the assembly and stability of the mi3 into uniform icosahedral NPs surrounded by regularly spaced CAP255 gp140 Env trimers. A three-dimensional reconstruction of CAP255 gp140 SpyTag-SpyCatcher mi3 clearly showed Env trimers projecting from the centre of each of the pentagonal dodecahedral faces of the NP. To our knowledge, this is the first study to report the formation of SpyCatcher pentamers on the dodecahedral faces of mi3 NPs. To investigate the immunogenicity, rabbits were primed with two doses of DNA vaccines expressing the CAP255 gp150 and a mosaic subtype C Gag and boosted with three doses of the NP-developed autologous Tier 2 CAP255 neutralising antibodies (Nabs) and low levels of heterologous CAP256SU NAbs.

A metagenomic prospective cohort study on gut microbiome composition and clinical infection in small bowel transplantation.

Two-thirds of small-bowel transplantation (SBT) recipients develop bacteremia, with the majority of infections occurring within 3 months post-transplant. Sepsis-related mortality occurs in 31% of patients and is commonly caused by bacteria of gut origin, which are thought to translocate across the implanted organ. Serial post-transplant surveillance endoscopies provide an opportunity to study whether the composition of the ileal and colonic microbiota can predict the emergence as well as the pathogen of subsequent clinical infections in the SBT patient population. Five participants serially underwent aspiration of ileal and colonic bowel effluents at transplantation and during follow-up endoscopy either until death or for up to 3 months post-SBT. We performed whole-metagenome sequencing (WMS) of 40 bowel effluent samples and compared the results with clinical infection episodes. Microbiome composition was concordant between participants and timepoint-matched ileal and colonic samples. Four out of five (4/5) participants had clinically significant infections thought to be of gut origin. Bacterial translocation from the gut was observed in 3/5 patients with bacterial infectious etiologies. In all three cases, the pathogens had demonstrably colonized the gut between 1-10 days prior to invasive clinical infection. Recipients with better outcomes received donor grafts with higher alpha diversity. There was an increase in the number of antimicrobial resistance genes associated with longer hospital stay for all participants. This metagenomic study provides preliminary evidence to support the pathogen translocation hypothesis of gut-origin sepsis in the SBT cohort. Ileal and colonic microbiome compositions were concordant; therefore, fecal metagenomic analysis could be a useful surveillance tool for impeding infection with specific gut-residing pathogens.

A plant-produced SARS-CoV-2 spike protein elicits heterologous immunity in hamsters.

Molecular farming of vaccines has been heralded as a cheap, safe and scalable production platform. In reality, however, differences in the plant biosynthetic machinery, compared to mammalian cells, can complicate the production of viral glycoproteins. Remodelling the secretory pathway presents an opportunity to support key post-translational modifications, and to tailor aspects of glycosylation and glycosylation-directed folding. In this study, we applied an integrated host and glyco-engineering approach, NXS/T Generation™, to produce a SARS-CoV-2 prefusion spike trimer in Nicotiana benthamiana as a model antigen from an emerging virus. The size exclusion-purified protein exhibited a characteristic prefusion structure when viewed by transmission electron microscopy, and this was indistinguishable from the equivalent mammalian cell-produced antigen. The plant-produced protein was decorated with under-processed oligomannose N-glycans and exhibited a site occupancy that was comparable to the equivalent protein produced in mammalian cell culture. Complex-type glycans were almost entirely absent from the plant-derived material, which contrasted against the predominantly mature, complex glycans that were observed on the mammalian cell culture-derived protein. The plant-derived antigen elicited neutralizing antibodies against both the matched Wuhan and heterologous Delta SARS-CoV-2 variants in immunized hamsters, although titres were lower than those induced by the comparator mammalian antigen. Animals vaccinated with the plant-derived antigen exhibited reduced viral loads following challenge, as well as significant protection from SARS-CoV-2 disease as evidenced by reduced lung pathology, lower viral loads and protection from weight loss. Nonetheless, animals immunized with the mammalian cell-culture-derived protein were better protected in this challenge model suggesting that more faithfully reproducing the native glycoprotein structure and associated glycosylation of the antigen may be desirable.

Augmenting glycosylation-directed folding pathways enhances the fidelity of HIV Env immunogen production in plants.

Heterologous glycoprotein production relies on host glycosylation-dependent folding. When the biosynthetic machinery differs from the usual expression host, there is scope to remodel the assembly pathway to enhance glycoprotein production. Here we explore the integration of chaperone coexpression with glyco-engineering to improve the production of a model HIV-1 envelope antigen. Calreticulin was coexpressed to support protein folding together with Leishmania major STT3D oligosaccharyltransferase, to improve glycan occupancy, RNA interference to suppress the formation of truncated glycans, and Nicotiana benthamiana plants lacking α1,3-fucosyltransferase and ß1,2-xylosyltransferase was used as an expression host to prevent plant-specific complex N-glycans forming. This approach reduced the formation of undesired aggregates, which predominated in the absence of glyco-engineering. The resulting antigen also exhibited increased glycan occupancy, albeit to a slightly lower level than the equivalent mammalian cell-produced protein. The antigen was decorated almost exclusively with oligomannose glycans, which were less processed compared with the mammalian protein. Immunized rabbits developed comparable immune responses to the plant-produced and mammalian cell-derived antigens, including the induction of autologous neutralizing antibodies when the proteins were used to boost DNA and modified vaccinia Ankara virus-vectored vaccines. This study demonstrates that engineering glycosylation-directed folding offers a promising route to enhance the production of complex viral glycoproteins in plants.

Development of a synthetic nanoparticle vaccine presenting the HIV-1 envelope glycoprotein.

Two-component self-assembling virus-like particles (VLPs) are promising scaffolds for achieving high-density display of HIV-1 envelope (gp140) trimers, which can improve the induction of neutralising antibodies (NAbs). In this study gp140 was displayed on the surface of VLPs formed by the AP205 phage coat protein. The CAP256 SU gp140 antigen was selected as the patient who this virus was isolated from developed broadly neutralising antibodies (bNAbs) shortly after superinfection with this virus. The CAP256 SU envelope is also sensitive to several bNAbs and has shown enhanced reactivity for certain bNAb precursors. A fusion protein comprising the HIV-1 CAP256 SU gp140 and the SpyTag (ST) (gp140-ST) was produced in HEK293 cells, and trimers were purified to homogeneity using gel filtration. SpyCatcher (SC)-AP205 VLPs were produced inEscherichia coliand purified by ultracentrifugation. The gp140-ST trimers and the SC-AP205 VLPs were mixed in varying molar ratios to generate VLPs displaying the glycoprotein (AP205-gp140-ST particles). Dynamic light scattering, negative stain electron microscopy and 2D classification indicated that gp140-ST was successfully bound to the VLPs, although not all potential binding sites were occupied. The immunogenicity of the coupled VLPs was evaluated in a pilot study in rabbits. One group was injected four times with coupled VLPs, and the second group was primed with DNA vaccines expressing Env and a mosaic Gag, followed by modified vaccinia Ankara expressing the same antigens. The animals were then boosted twice with coupled VLPs. Encouragingly, gp140-ST displayed on SC-AP205 VLPs was an effective boost to heterologously primed rabbits, leading to induction of autologous Tier 2 neutralising antibodies in 2/5 rabbits. However, four inoculations of coupled VLPs alone failed to elicit any Tier 2 antibodies. These results demonstrate that the native-like structure of HIV-1 envelope trimers and selection of a geometrically-suitable nanoparticle scaffold to achieve a high-density display of the trimers are important considerations that could improve the effect of nanoparticle-displayed gp140.

Cryo-EM reveals mechanisms of angiotensin I-converting enzyme allostery and dimerization.

Hypertension (high blood pressure) is a major risk factor for cardiovascular disease, which is the leading cause of death worldwide. The somatic isoform of angiotensin I-converting enzyme (sACE) plays a critical role in blood pressure regulation, and ACE inhibitors are thus widely used to treat hypertension and cardiovascular disease. Our current understanding of sACE structure, dynamics, function, and inhibition has been limited because truncated, minimally glycosylated forms of sACE are typically used for X-ray crystallography and molecular dynamics simulations. Here, we report the first cryo-EM structures of full-length, glycosylated, soluble sACE (sACES1211 ). Both monomeric and dimeric forms of the highly flexible apo enzyme were reconstructed from a single dataset. The N- and C-terminal domains of monomeric sACES1211 were resolved at 3.7 and 4.1 Å, respectively, while the interacting N-terminal domains responsible for dimer formation were resolved at 3.8 Å. Mechanisms are proposed for intradomain hinging, cooperativity, and homodimerization. Furthermore, the observation that both domains were in the open conformation has implications for the design of sACE modulators.

The structures of the C146A variant of the amidase from Pyrococcus horikoshii bound to glutaramide and acetamide suggest the basis of amide recognition.

The nitrilase superfamily enzymes from Pyrococcus abyssi and Pyrococcus horikoshii hydrolyze several different amides. No nitriles that we tested were hydrolyzed by either enzyme. Propionamide and acetamide were the most rapidly hydrolyzed of all the substrates tested. Amide substrate docking studies on the wild-type and C146A variant P. horikoshii enzymes suggest a sequence in which the incoming amide substrate initially hydrogen bonds to the amino group of Lys-113 and the backbone carbonyl of Asn-171. When steric hindrance is relieved by replacing the cysteine with alanine, the amide then docks such that the amino group of Lys-113 and the backbone amide of Phe-147 are hydrogen-bonded to the substrate carbonyl oxygen, while the backbone carbonyl oxygen of Asn-171 and the carboxyl oxygen of Glu-42 are hydrogen-bonded to the amino group of the substrate. Here, we confirm the location of the acetamide and glutaramide ligands experimentally in well-resolved crystal structures of the C146A mutant of the enzyme from P. horikoshii. This ligand location suggests that there is no direct interaction between the substrate amide and the other active site glutamate, Glu-120, and supports an active-site geometry leading to the formation of the thioester intermediate via an attack on the si-face of the amide by the sulfhydryl of the active site cysteine.

Diagnosis of coeliac disease by flow cytometry of intraepithelial lymphocytes: a new 'gold' standard?

Objective: The analysis of intraepithelial lymphocytes (IELs) by flow cytometry of duodenal biopsies-the 'IEL' lymphogram-has been proposed as a diagnostic test for coeliac disease. However, its clinical applicability has been limited due to variability in methods and definitions. This study set out to define useful parameters for the application of the IEL lymphogram to the diagnosis of coeliac disease. Design: Flow cytometry was performed on 117 sets of duodenal biopsies in 107 adult patients with active coeliac disease, long-term coeliac disease on a gluten free diet and a control group. The initial 95 samples were used for hypothesis generation for the subsequent samples comprising 12 patients with coeliac disease and 10 controls. Results: Rather than using single linear cut-offs for CD3 and T-cell receptor γδ (TCRγδ)+ve IELs, a discriminant function was identified as %CD3+ve IELs+2x(%TCRγδ+IELs)>100. This differentiated coeliac disease from control biopsies in the hypothesis generating group. These results were replicated in the validation group and found to be independent of histology in patients on long-term gluten free diet up to 12 years (combined sensitivity, 98.5%; specificity, 97.7%). Conclusions: Flow cytometric analysis of IELs is a highly sensitive and specific adjunct to serology and histological examination for the diagnosis of coeliac disease, even in individuals with coeliac disease following a gluten free diet who exhibit normal duodenal histology.

Transplant-associated thrombotic microangiopathy and immune haematological complications following intestine-containing organ transplantation: experience from over 100 consecutive cases.

Descriptions of passenger lymphocyte syndrome (PLS), immune cytopenias and transplant-associated thrombotic microangiopathy (TA-TMA) after intestine-containing transplants remain scarce. We describe our centre's experience of these complications from 2007 to 2019. Ninety-six patients received 103 transplants. PLS occurred in 9 (9%) patients (median 12 days post-transplant); all due to ABO antibodies. There were 31 minor ABO mismatch transplants. No patient required change in immunosuppression. Immune cytopenias (excluding PLS) occurred in six patients at an incidence of 1·7/100 patient years; three immune haemolysis, one immune thrombocytopenia, one acquired Glanzmann's and one immune neutropenia; 50% occurred with other cytopenias. All cases eventually responded to treatment, with a median of four treatments (range 1-8) and 5/6 were treated with rituximab. One patient with immune haemolysis required bortezomib. Complications were common in patients with immune cytopenias; 4/6 with infection needing intravenous antibiotics and 3/6 with venous thromboembolism. In 3/6 cases, a secondary cause for the immune cytopenia was evident. Switching from tacrolimus to ciclosporin was not necessary. There were five cases of transplant-associated thrombotic microangiopathy (TA-TMA; 1·5/100 patient years) requiring calcineurin inhibitor withdrawal; two cases associated with acute rejection. Two cases were managed with plasma exchange, one with plasma infusions and one with eculizumab. Further research in this patient group is required.

Multicenter cohort study of patients with buried bumper syndrome treated endoscopically with a novel, dedicated device.

BACKGROUND AND AIMS: Buried bumper syndrome (BBS) is a rare adverse event of percutaneous endoscopic gastrostomy (PEG) placement in which the internal bumper migrates through the stomal tract to become embedded within the gastric wall. Excessive tension between the internal and external bumpers, causing ischemic necrosis of the gastric wall, is believed to be the main etiologic factor. Several techniques for endoscopic management of BBS have been described using off-label devices. The Flamingo set is a novel, sphincterotome-like device specifically designed for BBS management. We aimed to evaluate the effectiveness of the Flamingo device in a large, homogeneous cohort of patients with BBS. METHODS: A guidewire was inserted through the external access of the PEG tube into the gastric lumen. The Flamingo device was then introduced into the stomach over the guidewire. This dedicated tool can be flexed by 180 degrees, exposing a sphincterotome-like cutting wire, which is used to incise the overgrown tissue until the PEG bumper is exposed. A retrospective, international, multicenter cohort study was conducted on 54 patients between December 2016 and February 2019. RESULTS: The buried bumper was successfully removed in 53 of 55 procedures (96.4%). The median time for the endoscopic removal of the buried bumper was 22 minutes (range, 5-60). Periprocedural endoscopic adverse events occurred in 7 procedures (12.7%) and were successfully managed endoscopically. A median follow-up of 150 days (range, 33-593) was performed in 29 patients (52.7%), during which no significant adverse events occurred. CONCLUSIONS: Through our experience, we found this dedicated novel device to be safe, quick, and effective for minimally invasive, endoscopic management of BBS.

How to manage adult coeliac disease: perspective from the NHS England Rare Diseases Collaborative Network for Non-Responsive and Refractory Coeliac Disease.

Adult coeliac disease (CD) affects approximately 1% of the population. Most patients diagnosed will respond to a gluten-free diet; however, up to 30% may have persisting symptoms. Such patients may have ongoing issues associated with adherence, non-responsive CD or refractory CD. This article provides a clinical overview of how to manage this group of patients with persisting symptoms, including an investigational algorithm and details of how to contact the National Health Service England Rare Diseases Collaborative Network for Non-Responsive and Refractory Coeliac Disease. We hope this will be a valuable source of contemporary information for all UK gastroenterologists and internationally.

Shotgun EM of mycobacterial protein complexes during stationary phase stress.

There is little structural information about the protein complexes conferring resistance in Mycobacterium tuberculosis (Mtb) to anti-microbial oxygen and nitrogen radicals in the phagolysosome. Here, we expose the model Mycobacterium, Mycobacterium smegmatis, to simulated oxidative-stress conditions and apply a shotgun EM method for the structural detection of the resulting protein assemblies. We identified: glutamine synthetase I, essential for Mtb virulence; bacterioferritin A, critical for Mtb iron regulation; aspartyl aminopeptidase M18, a protease; and encapsulin, which produces a cage-like structure to enclose cargo proteins. After further investigation, we found that encapsulin carries dye-decolourising peroxidase, a protein antioxidant, as its primary cargo under the conditions tested.

The Long and Short of IT: intestinal failure-associated liver disease (IFALD) in adults-recommendations for early diagnosis and intestinal transplantation.

Intestinal failure-associated liver disease (IFALD) often presents in adults unexpectedly with advanced disease. Non-invasive tests can be falsely reassuring. Patients with 'ultrashort' intestine (<20 cm) ending in a stoma are at particular risk of developing IFALD, which may occur rapidly. Recent experience and studies suggest that IFALD can be reversed by isolated intestine transplant occurring before the development of high grade fibrosis or cirrhosis. Post-transplant survival is superior for isolated intestinal grafts compared with liver containing intestinal grafts; waiting time and waiting list mortality is higher for a combined graft, and donor liver supply is limited. Therefore, the aim of clinicians treating patients with intestinal failure should be to identify IFALD early and refer to an intestinal transplant centre while isolated intestine transplantation can be contemplated and before the liver disease has progressed to a stage requiring consideration of combined liver and intestinal transplantation.

Cryo-EM and directed evolution reveal how Arabidopsis nitrilase specificity is influenced by its quaternary structure.

Nitrilases are helical enzymes that convert nitriles to acids and/or amides. All plants have a nitrilase 4 homolog specific for ß-cyanoalanine, while in some plants neofunctionalization has produced nitrilases with altered specificity. Plant nitrilase substrate size and specificity correlate with helical twist, but molecular details of this relationship are lacking. Here we determine, to our knowledge, the first close-to-atomic resolution (3.4 Å) cryo-EM structure of an active helical nitrilase, the nitrilase 4 from Arabidopsis thaliana. We apply site-saturation mutagenesis directed evolution to three residues (R95, S224, and L169) and generate a mutant with an altered helical twist that accepts substrates not catalyzed by known plant nitrilases. We reveal that a loop between α2 and α3 limits the length of the binding pocket and propose that it shifts position as a function of helical twist. These insights will allow us to start designing nitrilases for chemoenzymatic synthesis.

Substrate specificity of plant nitrilase complexes is affected by their helical twist.

Nitrilases are oligomeric, helix-forming enzymes from plants, fungi and bacteria that are involved in the metabolism of various natural and artificial nitriles. These biotechnologically important enzymes are often specific for certain substrates, but directed attempts at modifying their substrate specificities by exchanging binding pocket residues have been largely unsuccessful. Thus, the basis for their selectivity is still unknown. Here we show, based on work with two highly similar nitrilases from the plant Capsella rubella, that modifying nitrilase helical twist, either by exchanging an interface residue or by imposing a different twist, without altering any binding pocket residues, changes substrate preference. We reveal that helical twist and substrate size correlate and when binding pocket residues are exchanged between two nitrilases that show the same twist but different specificities, their specificities change. Based on these findings we propose that helical twist influences the overall size of the binding pocket.

Fatal breakthrough mucormycosis in a multivisceral transplant patient receiving micafungin: Case report and literature review.

Introduction: Antifungal agents are routinely used in the post-transplant setting for both prophylaxis and treatment of presumed and proven fungal infections. Micafungin is an echinocandin-class antifungal with broad antifungal cover and favorable side effect profile but, notably, it has no activity against molds of the order Mucorales. Presentation of case: A 47-year-old woman underwent multivisceral transplantation for intestinal failure-associated liver disease. She had a prolonged post-operative recovery complicated by invasive candidiasis and developed an intolerance to liposomal amphotericin B. In view of her immunosuppression, she was commenced on micafungin as prophylaxis to prevent invasive fungal infection. However, she developed acute graft versus host disease with bone marrow failure complicated by disseminated mucormycosis which was only diagnosed post mortem. Discussion: Non-Aspergillus breakthrough mold infections with micafungin therapy are rare with only eight other cases having been described in the literature. Breakthrough infections have occurred within one week of starting micafungin. Diagnosis is problematic and requires a high degree of clinical suspicion and microscopic/histological examination of an involved site. The management of these aggressive infections involves extensive debridement and appropriate antifungal cover. Conclusion: A high level of suspicion of invasive fungal infection is required at all times in immunosuppressed patients, even those receiving antifungal prophylaxis. Early biopsy is required. Even with early recognition and aggressive treatment of these infections, prognosis is poor.

Nasal unsedated seated percutaneous endoscopic gastrostomy (nuPEG): a safe and effective technique for percutaneous endoscopic gastrostomy placement in high-risk candidates.

OBJECTIVE: Percutaneous endoscopic gastrostomy (PEG) tube placement is associated with a high risk of cardiorespiratory complications in patients with significant respiratory compromise. This study reports a case series of high-risk patients undergoing PEG placement using a modified technique-nasal unsedated seated PEG (nuPEG) placement. DESIGN: Retrospective review of 67 patients at high risk of complications undergoing PEG placement between September 2012 and December 2016. SETTING: UK specialist tertiary centre for clinical nutrition support. INTERVENTIONS: Patients underwent 'push' PEG placement using nasal endoscopy without sedation in a seated position. MAIN OUTCOME MEASURES: Procedural success and tolerability, short term (within 24 hours), medium term (24 hours to 30 days) complications and survival were recorded. RESULTS: 67 patients underwent 68 nuPEG placements. The majority had motor neuron disease (46/67). One patient developed a lower respiratory tract infection the following day. Two patients experienced accidental displacement of their PEG within 2 weeks. One patient died within 30 days of nuPEG insertion due to reasons unrelated to the procedure. Endoscopic comfort scores of 1 or 2 (98.0%) indicated good tolerance. A failure rate of 10.5% was attributed to intrathoracic displacement of the stomach, almost certainly due to the advanced stage of the neurological disease and associated diaphragmatic weakness. CONCLUSIONS: Our experience with the nuPEG technique suggests that it is safe and well tolerated in high-risk patients. As a result, it has now entirely supplanted radiologically inserted gastrostomy insertion in our institution and we recommend it as the method of choice for gastrostomy tube insertion in such patients.

Addition of Insulin to Parenteral Nutrition for Control of Hyperglycemia.

Administration of parenteral nutrition (PN) may result in hyperglycemia in patients with preexisting diabetes or disease-related insulin resistance, and it can be associated with increased rates of complications. Treatment requires insulin therapy. Insulin can be administered subcutaneously, intravenously via a variable rate sliding scale, or by adding it directly to the PN. The last method is a potentially attractive technique for a number of reasons-it could deliver the insulin intravenously at a steady rate alongside carbohydrates, and in malnourished patients with little subcutaneous tissue, it may prevent the need for frequent insulin injections. Despite such potential advantages, the addition of insulin to PN remains controversial, largely with respect to the bioavailability of insulin in PN and resultant concerns of the risk of hypoglycemia. There is a paucity of long-term quality controlled studies to address this question. The available literature suggests that, at least in the short term, insulin addition to PN can achieve reasonable glycemic control with low rates of hypoglycemia, and the technique compares favorably with the use of long-acting insulin preparations. This literature review finds a wide range of values reported for insulin availability via PN, ranging from 44% to 95% depending on the type of PN container material used and the presence of added vitamins and trace elements. Few studies looking at glycemic control among patients receiving home PN were found, and larger prospective trials are needed to assess the efficacy and safety of this technique in this patient group.

Detection of Mycobacterium tuberculosis bacilli in bio-aerosols from untreated TB patients.

Background: Tuberculosis (TB) is predominantly an airborne disease. However, quantitative and qualitative analysis of bio-aerosols containing the aetiological agent, Mycobacterium tuberculosis (Mtb), has proven very challenging. Our objective is to sample bio-aerosols from newly diagnosed TB patients for detection and enumeration of Mtb bacilli. Methods: We monitored each of 35 newly diagnosed, GeneXpert sputum-positive, TB patients during 1 hour confinement in a custom-built Respiratory Aerosol Sampling Chamber (RASC). The RASC (a small clean-room of 1.4m ) incorporates aerodynamic particle size detection, viable and non-viable sampling devices, real-time CO 2 monitoring, and cough sound-recording. Microbiological culture and droplet digital polymerase chain reaction (ddPCR) were used to detect Mtb in each of the bio-aerosol collection devices. Results:  Mtb was detected in 27/35 (77.1%) of aerosol samples; 15/35 (42.8%) samples were positive by mycobacterial culture and 25/27 (92.96%) were positive by ddPCR. Culturability of collected bacilli was not predicted by radiographic evidence of pulmonary cavitation, sputum smear positivity. A correlation was found between cough rate and culturable bioaerosol.  Mtb was detected on all viable cascade impactor stages with a peak at aerosol sizes 2.0-3.5µm. This suggests a median of 0.09 CFU/litre of exhaled air (IQR: 0.07 to 0.3 CFU/l) for the aerosol culture positives and an estimated median concentration of 4.5x10 CFU/ml (IQR: 2.9x10 -5.6x10 ) of exhaled particulate bio-aerosol. Conclusions:  Mtb was identified in bio-aerosols exhaled by the majority of untreated TB patients using the RASC. Molecular detection was more sensitive than mycobacterial culture on solid media, suggesting that further studies are required to determine whether this reflects a significant proportion of differentially detectable bacilli in these samples.