Genetic Diversity in Mycobacterium tuberculosis Clinical Isolates and Resulting Outcomes of Tuberculosis Infection and Disease.

Tuberculosis claims more human lives than any other bacterial infectious disease and represents a clear and present danger to global health as new tools for vaccination, treatment, and interruption of transmission have been slow to emerge. Additionally, tuberculosis presents with notable clinical heterogeneity, which complicates diagnosis, treatment, and the establishment of nonrelapsing cure. How this heterogeneity is driven by the diversity ofclinical isolates of the causative agent, Mycobacterium tuberculosis, has recently garnered attention. Herein, we review advances in the understanding of how naturally occurring variation in clinical isolates affects transmissibility, pathogenesis, immune modulation, and drug resistance. We also summarize how specific changes in transcriptional responses can modulate infection or disease outcome, together with strain-specific effects on gene essentiality. Further understanding of how this diversity of M. tuberculosis isolates affects disease and treatment outcomes will enable the development of more effective therapeutic options and vaccines for this dreaded disease.

Peptidoglycan biosynthesis and remodeling revisited.

The bacterial peptidoglycan layer forms a complex mesh-like structure that surrounds the cell, imparting rigidity to withstand cytoplasmic turgor and the ability to tolerate stress. As peptidoglycan has been the target of numerous clinically successful antimicrobials such as penicillin, the biosynthesis, remodeling and recycling of this polymer has been the subject of much interest. Herein, we review recent advances in the understanding of peptidoglycan biosynthesis and remodeling in a variety of different organisms. In order for bacterial cells to grow and divide, remodeling of cross-linked peptidoglycan is essential hence, we also summarize the activity of important peptidoglycan hydrolases and how their functions differ in various species. There is a growing body of evidence highlighting complex regulatory mechanisms for peptidoglycan metabolism including protein interactions, phosphorylation and protein degradation and we summarize key recent findings in this regard. Finally, we provide an overview of peptidoglycan recycling and how components of this pathway mediate resistance to drugs. In the face of growing antimicrobial resistance, these recent advances are expected to uncover new drug targets in peptidoglycan metabolism, which can be used to develop novel therapies.

ß-lactam resistance: The role of low molecular weight penicillin binding proteins, ß-lactamases and ld-transpeptidases in bacteria associated with respiratory tract infections.

Disruption of peptidoglycan (PG) biosynthesis in the bacterial cell wall by ß-lactam antibiotics has transformed therapeutic options for bacterial infections. These antibiotics target the transpeptidase domains in penicillin binding proteins (PBPs), which can be classified into high and low molecular weight (LMW) counterparts. While the essentiality of the former has been extensively demonstrated, the physiological roles of LMW PBPs remain poorly understood. Herein, we review the function of LMW PBPs, ß-lactamases and ld-transpeptidases (Ldts) in pathogens associated with respiratory tract infections. More specifically, we explore their roles in mediating ß-lactam resistance. Using a comparative genomics approach, we identified a high degree of genetic redundancy for LMW PBPs which retain the motifs, SxxN, SxN and KTG required for catalytic activity. Differences in domain architecture suggest distinct physiological roles, possibly related to bacterial cell cycle and/or adaptation to various environmental conditions. Many of the LMW PBPs play an important role in ß-lactam resistance either through mutation or variation in abundance. In all of the bacterial genomes assessed, at least one ß-lactamase homologue is present, suggesting that enzymatic degradation of ß-lactams is a highly conserved resistance mechanism. Furthermore, the presence of Ldt homologues in the majority of species surveyed suggests that alternative PG crosslinking may further mediate ß-lactam drug resistance. A deeper understanding of the interplay between these different mechanisms of ß-lactam resistance will provide a framework for new therapeutics, which are urgently required given the rapid emergence of antimicrobial resistance. © 2018 IUBMB Life, 70(9):855-868, 2018.

Detection and Quantification of Differentially Culturable Tubercle Bacteria in Sputum from Patients with Tuberculosis.

RATIONALE: Recent studies suggest that baseline tuberculous sputum comprises a mixture of routinely culturable and differentially culturable tubercle bacteria (DCTB). The latter seems to be drug tolerant and dependent on resuscitation-promoting factors (Rpfs). OBJECTIVES: To further explore this, we assessed sputum from patients with tuberculosis for DCTB and studied the impact of exogenous culture filtrate (CF) supplementation ex vivo. METHODS: Sputum samples from adults with tuberculosis and HIV-1 and adults with no HIV-1 were used for most probable number (MPN) assays supplemented with CF and Rpf-deficient CF, to detect CF-dependent and Rpf-independent DCTB, respectively. MEASUREMENTS AND MAIN RESULTS: In 110 individuals, 19.1% harbored CF-dependent DCTB and no Rpf-independent DCTB. Furthermore, 11.8% yielded Rpf-independent DCTB with no CF-dependent DCTB. In addition, 53.6% displayed both CF-dependent and Rpf-independent DCTB, 1.8% carried CF-independent DCTB, and 13.6% had no DCTB. Sputum from individuals without HIV-1 yielded higher CF-supplemented MPN counts compared with counterparts with HIV-1. Furthermore, individuals with HIV-1 with CD4 counts greater than 200 cells/mm3 displayed higher CF-supplemented MPN counts compared with participants with HIV-1 with CD4 counts less than 200 cells/mm3. CF supplementation allowed for detection of mycobacteria in 34 patients with no culturable bacteria on solid media. Additionally, the use of CF enhanced detection of sputum smear-negative individuals. CONCLUSIONS: These observations demonstrate a novel Rpf-independent DCTB population in sputum and reveal that reduced host immunity is associated with lower prevalence of CF-responsive bacteria. Quantification of DCTB in standard TB diagnosis would be beneficial because these organisms provide a putative biomarker to monitor treatment response and risk of disease recurrence.

Activity of phosphino palladium(II) and platinum(II) complexes against HIV-1 and Mycobacterium tuberculosis.

Treatment of human immunodeficiency virus (HIV) is currently complicated by increased prevalence of co-infection with Mycobacterium tuberculosis. The development of drug candidates that offer the simultaneous management of HIV and tuberculosis (TB) would be of great benefit in the holistic treatment of HIV/AIDS, especially in sub-Saharan Africa which has the highest global prevalence of HIV-TB coinfection. Bis(diphenylphosphino)-2-pyridylpalladium(II) chloride (1), bis(diphenylphosphino)-2-pyridylplatinum(II) chloride (2), bis(diphenylphosphino)-2-ethylpyridylpalladium(II) chloride (3) and bis(diphenylphosphino)-2-ethylpyridylplatinum(II) (4) were investigated for the inhibition of HIV-1 through interactions with the viral protease. The complexes were subsequently assessed for biological potency against Mycobacterium tuberculosis H37Rv by determining the minimal inhibitory concentration (MIC) using broth microdilution. Complex (3) showed the most significant and competitive inhibition of HIV-1 protease (p = 0.014 at 100 µM). Further studies on its in vitro effects on whole virus showed reduced viral infectivity by over 80 % at 63 µM (p < 0.05). In addition, the complex inhibited the growth of Mycobacterium tuberculosis at an MIC of 5 µM and was non-toxic to host cells at all active concentrations (assessed by tetrazolium dye and real time cell electronic sensing). In vitro evidence is provided here for the possibility of utilizing a single metal-based compound for the treatment of HIV/AIDS and TB.

bis-Molybdopterin guanine dinucleotide is required for persistence of Mycobacterium tuberculosis in guinea pigs.

Mycobacterium tuberculosis is able to synthesize molybdopterin cofactor (MoCo), which is utilized by numerous enzymes that catalyze redox reactions in carbon, nitrogen, and sulfur metabolism. In bacteria, MoCo is further modified through the activity of a guanylyltransferase, MobA, which converts MoCo to bis-molybdopterin guanine dinucleotide (bis-MGD), a form of the cofactor that is required by the dimethylsulfoxide (DMSO) reductase family of enzymes, which includes the nitrate reductase NarGHI. In this study, the functionality of the mobA homolog in M. tuberculosis was confirmed by demonstrating the loss of assimilatory and respiratory nitrate reductase activity in a mobA deletion mutant. This mutant displayed no survival defects in human monocytes or mouse lungs but failed to persist in the lungs of guinea pigs. These results implicate one or more bis-MGD-dependent enzymes in the persistence of M. tuberculosis in guinea pig lungs and underscore the applicability of this animal model for assessing the role of molybdoenzymes in this pathogen.

Cleavage of the moaX-encoded fused molybdopterin synthase from Mycobacterium tuberculosis is necessary for activity.

BACKGROUND: Molybdopterin cofactor (MoCo) biosynthesis in Mycobacterium tuberculosis is associated with a multiplicity of genes encoding several enzymes in the pathway, including the molybdopterin (MPT) synthase, a hetero tetramer comprising two MoaD and two MoaE subunits. In addition to moaD1, moaD2, moaE1, moaE2, the M. tuberculosis genome also contains a moaX gene which encodes an MPT-synthase in which the MoaD and MoaE domains are located on a single polypeptide. In this study, we assessed the requirement for post-translational cleavage of MoaX for functionality of this novel, fused MPT synthase and attempted to establish a functional hierarchy for the various MPT-synthase encoding genes in M. tuberculosis. RESULTS: Using a heterologous Mycobacterium smegmatis host and the activity of the MoCo-dependent nitrate reductase, we confirmed that moaD2 and moaE2 from M. tuberculosis together encode a functional MPT synthase. In contrast, moaD1 displayed no functionality in this system, even in the presence of the MoeBR sulphurtransferase, which contains the rhodansese-like domain, predicted to activate MoaD subunits. We demonstrated that cleavage of MoaX into its constituent MoaD and MoaE subunits was required for MPT synthase activity and confirmed that cleavage occurs between the Gly82 and Ser83 residues in MoaX. Further analysis of the Gly81-Gly82 motif confirmed that both of these residues are necessary for catalysis and that the Gly81 was required for recognition/cleavage of MoaX by an as yet unidentified protease. In addition, the MoaE component of MoaX was able to function in conjunction with M. smegmatis MoaD2 suggesting that cleavage of MoaX renders functionally interchangeable subunits. Expression of MoaX in E. coli revealed that incorrect post-translational processing is responsible for the lack of activity of MoaX in this heterologous host. CONCLUSIONS: There is a degree of functional interchangeability between the MPT synthase subunits of M. tuberculosis. In the case of MoaX, post-translational cleavage at the Gly82 residue is required for function.

Energy metabolism and drug efflux in Mycobacterium tuberculosis.

The inherent drug susceptibility of microorganisms is determined by multiple factors, including growth state, the rate of drug diffusion into and out of the cell, and the intrinsic vulnerability of drug targets with regard to the corresponding antimicrobial agent. Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant source of global morbidity and mortality, further exacerbated by its ability to readily evolve drug resistance. It is well accepted that drug resistance in M. tuberculosis is driven by the acquisition of chromosomal mutations in genes encoding drug targets/promoter regions; however, a comprehensive description of the molecular mechanisms that fuel drug resistance in the clinical setting is currently lacking. In this context, there is a growing body of evidence suggesting that active extrusion of drugs from the cell is critical for drug tolerance. M. tuberculosis encodes representatives of a diverse range of multidrug transporters, many of which are dependent on the proton motive force (PMF) or the availability of ATP. This suggests that energy metabolism and ATP production through the PMF, which is established by the electron transport chain (ETC), are critical in determining the drug susceptibility of M. tuberculosis. In this review, we detail advances in the study of the mycobacterial ETC and highlight drugs that target various components of the ETC. We provide an overview of some of the efflux pumps present in M. tuberculosis and their association, if any, with drug transport and concomitant effects on drug resistance. The implications of inhibiting drug extrusion, through the use of efflux pump inhibitors, are also discussed.

A modified BCG with depletion of enzymes associated with peptidoglycan amidation induces enhanced protection against tuberculosis in mice.

Mechanisms by which Mycobacterium tuberculosis (Mtb) evades pathogen recognition receptor activation during infection may offer insights for the development of improved tuberculosis (TB) vaccines. Whilst Mtb elicits NOD-2 activation through host recognition of its peptidoglycan-derived muramyl dipeptide (MDP), it masks the endogenous NOD-1 ligand through amidation of glutamate at the second position in peptidoglycan side-chains. As the current BCG vaccine is derived from pathogenic mycobacteria, a similar situation prevails. To alleviate this masking ability and to potentially improve efficacy of the BCG vaccine, we used CRISPRi to inhibit expression of the essential enzyme pair, MurT-GatD, implicated in amidation of peptidoglycan side-chains. We demonstrate that depletion of these enzymes results in reduced growth, cell wall defects, increased susceptibility to antibiotics, altered spatial localization of new peptidoglycan and increased NOD-1 expression in macrophages. In cell culture experiments, training of a human monocyte cell line with this recombinant BCG yielded improved control of Mtb growth. In the murine model of TB infection, we demonstrate that depletion of MurT-GatD in BCG, which is expected to unmask the D-glutamate diaminopimelate (iE-DAP) NOD-1 ligand, yields superior prevention of TB disease compared to the standard BCG vaccine. In vitro and in vivo experiments in this study demonstrate the feasibility of gene regulation platforms such as CRISPRi to alter antigen presentation in BCG in a bespoke manner that tunes immunity towards more effective protection against TB disease.

Tuberculosis is the leading cause of death from an infectious disease worldwide, partially due to a lack of access to drug treatments in certain countries where the disease is common. The only available tuberculosis vaccine ­ known as the BCG vaccine ­ is useful for preventing cases in young children, but is ineffective in teenagers and adults. So, there is a need to develop new vaccines that offer better, and longer lasting, durable protection in people of all ages. During an infection, our immune system recognizes markers known as PAMPs on the surface of bacteria, viruses or other disease-causing pathogens. The recognition of PAMPs by the immune system enables the body to distinguish foreign invading organisms from its own cells and tissues, thus triggering a response that fights the infection. If the body encounters the infectious agent again in the future, the immune system is able to quickly recognize and eliminate it before it can cause disease. Vaccines protect us by mimicking the appearance of the pathogen to trigger the first immune response without causing the illness. The BCG vaccine contains live bacteria that are closely related to the bacterium responsible for tuberculosis called Mycobacterium tuberculosis. Both M. tuberculosis and the live bacteria used in the BCG vaccine are able to hide an important PAMP, known as the NOD-1 ligand, from the immune system, making it harder for the body to detect them. The NOD-1 ligand forms part of the bacterial cell wall and modifying the BCG bacterium so it cannot disguise this PAMP may lead to a new, more effective vaccine. To investigate this possibility, Shaku et al. used a gene editing approach to develop a modified version of the BCG bacterium which is unable to hide its NOD-1 ligand when treated with a specific drug. Immune cells trained with the modified BCG vaccine were more effective at controlling the growth of M. tuberculosis than macrophages trained using the original vaccine. Furthermore, mice vaccinated with the modified BCG vaccine were better able to limit M. tuberculosis growth in their lungs than mice that had received the original vaccine. These findings offer a new candidate vaccine in the fight against tuberculosis. Further studies will be needed to modify the vaccine for use in humans. More broadly, this work demonstrates that gene editing can be used to expose a specific PAMP present in a live vaccine. This may help develop more effective vaccines for other diseases in the future.

Essential roles for imuA'- and imuB-encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis.

In Mycobacterium tuberculosis (Mtb), damage-induced mutagenesis is dependent on the C-family DNA polymerase, DnaE2. Included with dnaE2 in the Mtb SOS regulon is a putative operon comprising Rv3395c, which encodes a protein of unknown function restricted primarily to actinomycetes, and Rv3394c, which is predicted to encode a Y-family DNA polymerase. These genes were previously identified as components of an imuA-imuB-dnaE2-type mutagenic cassette widespread among bacterial genomes. Here, we confirm that Rv3395c (designated imuA') and Rv3394c (imuB) are individually essential for induced mutagenesis and damage tolerance. Yeast two-hybrid analyses indicate that ImuB interacts with both ImuA' and DnaE2, as well as with the beta-clamp. Moreover, disruption of the ImuB-beta clamp interaction significantly reduces induced mutagenesis and damage tolerance, phenocopying imuA', imuB, and dnaE2 gene deletion mutants. Despite retaining structural features characteristic of Y-family members, ImuB homologs lack conserved active-site amino acids required for polymerase activity. In contrast, replacement of DnaE2 catalytic residues reproduces the dnaE2 gene deletion phenotype, strongly implying a direct role for the alpha-subunit in mutagenic lesion bypass. These data implicate differential protein interactions in specialist polymerase function and identify the split imuA'-imuB/dnaE2 cassette as a compelling target for compounds designed to limit mutagenesis in a pathogen increasingly associated with drug resistance.

Production and Performance Assessment of a Severe Acute Respiratory Syndrome Coronavirus 2 Biomimetic in a Verification Program for Pandemic Readiness.

During the early stages of the 2019 coronavirus disease (COVID-19) pandemic in South Africa, one of many challenges included availability of control material for laboratory proficiency testing programs. Proficiency testing control material using live severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or RNA extracted from cell culture was either biohazardous or costly, particularly in resource-limited settings. This study reports the development and application of a noninfectious SARS-CoV-2 biomimetic Mycobacterium smegmatis strain that mimics a positive result in the GeneXpert SARS-CoV-2 Xpert Xpress cartridge. Nucleotide sequences located in genes encoding the RNA-dependent RNA polymerase, the nucleocapsid, and the envelope proteins were used. The resulting biomimetic strain was prepared as a positive proficiency testing control and distributed in South Africa for verification of laboratories before their testing of clinical specimens. Between April and December 2020, a total of 151 GeneXpert instruments with 2532 modules were verified to bring COVID-19 mass testing in South Africa online. An average concordance of 98.6% was noted in the entire laboratory network, allowing identification of false-positive/false-negative results and instrument errors. This noninfectious, easily scalable proficiency testing control material became available within 2 months after the start of the pandemic in South Africa and represents a useful approach to consider for other diseases and future pandemics.

Development of primer-probe sets to rapidly distinguish single nucleotide polymorphisms in SARS-CoV-2 lineages.

Ongoing SARS-CoV-2 infections are driven by the emergence of various variants, with differential propensities to escape immune containment. Single nucleotide polymorphisms (SNPs) in the RNA genome result in altered protein structures and when these changes occur in the S-gene, encoding the spike protein, the ability of the virus to penetrate host cells to initiate an infection can be significantly altered. As a result, vaccine efficacy and prior immunity may be diminished, potentially leading to new waves of infection. Early detection of SARS-CoV-2 variants using a rapid and scalable approach will be paramount for continued monitoring of new infections. In this study, we developed minor groove-binding (MGB) probe-based qPCR assays targeted to specific SNPs in the S-gene, which are present in variants of concern (VOC), namely the E484K, N501Y, G446S and D405N mutations. A total of 95 archived SARS-CoV-2 positive clinical specimens collected in Johannesburg, South Africa between February 2021 and March 2022 were assessed using these qPCR assays. To independently confirm SNP detection, Sanger sequencing of the relevant region in the S-gene were performed. Where a PCR product could be generated and sequenced, qPCR assays were 100% concordant highlighting the robustness of the approach. These assays, and the approach described, offer the opportunity for easy detection and scaling of targeted detection of variant-defining SNPs in the clinical setting.

A modified BCG with depletion of enzymes associated with peptidoglycan amidation induces enhanced protection against tuberculosis in mice.

Mechanisms by which Mycobacterium tuberculosis (Mtb) evades pathogen recognition receptor activation during infection may offer insights for the development of improved tuberculosis (TB) vaccines. Whilst Mtb elicits NOD-2 activation through host recognition of its peptidoglycan-derived muramyl dipeptide (MDP), it masks the endogenous NOD-1 ligand through amidation of glutamate at the second position in peptidoglycan sidechains. As the current BCG vaccine is derived from pathogenic mycobacteria, a similar situation prevails. To alleviate this masking ability and to potentially improve efficacy of the BCG vaccine, we used CRISPRi to inhibit expression of the essential enzyme pair, MurT-GatD, implicated in amidation of peptidoglycan sidechains. We demonstrate that depletion of these enzymes results in reduced growth, cell wall defects, increased susceptibility to antibiotics and altered spatial localization of new peptidoglycan. In cell culture experiments, training of monocytes with this recombinant BCG yielded improved control of Mtb growth. In the murine model of TB infection, we demonstrate that depletion of MurT-GatD in BCG, resulting in unmasking of the D-glutamate diaminopimelate (iE-DAP) NOD-1 ligand, yields superior prevention of TB disease compared to the standard BCG vaccine. This work demonstrates the feasibility of gene regulation platforms such as CRISPRi to alter antigen presentation in BCG in a bespoke manner that tunes immunity towards more effective protection against TB disease.

Amidation of glutamate residues in mycobacterial peptidoglycan is essential for cell wall cross-linking.

Introduction: Mycobacteria assemble a complex cell wall with cross-linked peptidoglycan (PG) which plays an essential role in maintenance of cell wall integrity and tolerance to osmotic pressure. We previously demonstrated that various hydrolytic enzymes are required to remodel PG during essential processes such as cell elongation and septal hydrolysis. Here, we explore the chemistry associated with PG cross-linking, specifically the requirement for amidation of the D-glutamate residue found in PG precursors. Methods: Synthetic fluorescent probes were used to assess PG remodelling dynamics in live bacteria. Fluorescence microscopy was used to assess protein localization in live bacteria and CRISPR-interference was used to construct targeted gene knockdown strains. Time-lapse microscopy was used to assess bacterial growth. Western blotting was used to assess protein phosphorylation. Results and discussion: In Mycobacterium smegmatis, we confirmed the essentiality for D-glutamate amidation in PG biosynthesis by labelling cells with synthetic fluorescent PG probes carrying amidation modifications. We also used CRISPRi targeted knockdown of genes encoding the MurT-GatD complex, previously implicated in D-glutamate amidation, and demonstrated that these genes are essential for mycobacterial growth. We show that MurT-rseGFP co-localizes with mRFP-GatD at the cell poles and septum, which are the sites of cell wall synthesis in mycobacteria. Furthermore, time-lapse microscopic analysis of MurT-rseGFP localization, in fluorescent D-amino acid (FDAA)-labelled mycobacterial cells during growth, demonstrated co-localization with maturing PG, suggestive of a role for PG amidation during PG remodelling and repair. Depletion of MurT and GatD caused reduced PG cross-linking and increased sensitivity to lysozyme and ß-lactam antibiotics. Cell growth inhibition was found to be the result of a shutdown of PG biosynthesis mediated by the serine/threonine protein kinase B (PknB) which senses uncross-linked PG. Collectively, these data demonstrate the essentiality of D-glutamate amidation in mycobacterial PG precursors and highlight the MurT-GatD complex as a novel drug target.

The performance of tongue swabs for detection of pulmonary tuberculosis.

Introduction: Oral and/or tongue swabs have demonstrated ability to detect Mycobacterium tuberculosis (Mtb) in adults with pulmonary tuberculosis (TB). Swabs provide useful alternative specimens for diagnosis of TB using molecular assays however, the diagnostic pickup by culture requires further improvement and development. Several studies identified the presence of differentially culturable tubercle bacilli (DCTB) populations in a variety of clinical specimens. These organisms do not grow in routine laboratory media and require growth factors in the form of culture filtrate (CF) from logarithmic phase cultures of Mtb H37Rv. Methods: Herein, we compared the diagnostic performance of sputum and tongue swabs using Mycobacterial Growth Indicator Tube (MGIT) assays, Auramine smear, GeneXpert and DCTB assays supplemented with or without CF. Results: From 89 eligible participants, 83 (93%), 66 (74%) and 79 (89%) were sputum positive by MGIT, smear and GeneXpert, respectively. The corresponding tongue swabs displayed a lower sensitivity with 39 (44%), 2 (2.0%) and 18 (20%) participants respectively for the same tests. We aimed to improve the diagnostic yield by utilizing DCTB assays. Sputum samples were associated with a higher positivity rate for CF-augmented DCTB at 82/89 (92%) relative to tongue swabs at 36/89 (40%). Similarly, sputum samples had a higher positivity rate for DCTB populations that were CF-independent at 64/89 (72%) relative to tongue swabs at 26/89 (29%). DCTB positivity increased significantly, relative to MGIT culture, for tongue swabs taken from HIV-positive participants. We next tested whether the use of an alternative smear stain, DMN-Trehalose, would improve diagnostic yield but noted no substantial increase. Discussion: Collectively, our data show that while tongue swabs yield lower bacterial numbers for diagnostic testing, the use of growth supplementation may improve detection of TB particularly in HIV-positive people but this requires further interrogation in larger studies.

Opportunities and challenges of leveraging COVID-19 vaccine innovation and technologies for developing sustainable vaccine manufacturing capabilities in Africa.

The COVID-19 pandemic heralded unprecedented resource mobilisation and global scientific collaboration to rapidly develop effective vaccines. Regrettably, vaccine distribution has been inequitable, particularly in Africa where manufacturing capacity remains nominal. To address this, several initiatives are underway to develop and manufacture COVID-19 vaccines in Africa. Nevertheless, diminishing demand for COVID-19 vaccines, the cost competitiveness of producing goods locally, intellectual property rights issues, and complex regulatory environments among other challenges can undermine these ventures. We outline how extending COVID-19 vaccine manufacturing in Africa to include diverse products, multiple vaccine platforms, and advanced delivery systems will ensure sustainability. Possible models, including leveraging public-academic-private partnerships to enhance success of vaccine manufacturing capacity in Africa are also discussed. Intensifying research in vaccine discovery on the continent could yield vaccines that further bolster sustainability of local production, ensuring greater pandemic preparedness in resource-constrained environments, and long-term health systems security.

Survival and detection of SARS-CoV-2 variants on dry swabs post storage.

COVID-19 has resulted in nearly 598 million infections and over 6.46 million deaths since the start of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in 2019. The rapid onset of the pandemic, combined with the emergence of viral variants, crippled many health systems particularly from the perspective of coping with massive diagnostic loads. Shortages of diagnostic kits and capacity forced laboratories to store clinical samples resulting in huge backlogs, the effects of this on diagnostic pickup have not been fully understood. Herein, we investigated the impact of storing SARS-CoV-2 inoculated dry swabs on the detection and viability of four viral strains over a period of 7 days. Viral load, as detected by qRT-PCR, displayed no significant degradation during this time for all viral loads tested. In contrast, there was a ca. 2 log reduction in viral viability as measured by the tissue culture infectious dose (TCID) assay, with 1-3 log viable virus detected on dry swabs after 7 days. When swabs were coated with 102 viral copies of the Omicron variant, no viable virus was detected after 24 hours following storage at 4°C or room temperature. However there was no loss of PCR signal over 7 days. All four strains showed comparable growth kinetics and survival when cultured in Vero E6 cells. Our data provide information on the viability of SARS-CoV-2 on stored swabs in a clinical setting with important implications for diagnostic pickup and laboratory processing protocols. Survival after 7 days of SARS-CoV-2 strains on swabs with high viral loads may impact public health and biosafety practices in diagnostic laboratories.

Detection of differentially culturable tubercle bacteria in sputum from drug-resistant tuberculosis patients.

Several studies described the presence of non-replicating, drug-tolerant differentially culturable tubercle bacteria (DCTB) in sputum from patients with active tuberculosis (TB). These organisms are unable to form colonies on agar but can be recovered in liquid media supplemented with culture filtrate as a source of growth factors. Herein, we undertook to investigate the response of DCTB during the treatment of individuals with drug-resistant TB. A cohort of 100 participants diagnosed with rifampicin-resistant TB were enrolled and prospectively followed to monitor response to therapy using routine culture and limiting dilution assays, supplemented with culture filtrate (CF) to quantify DCTB. Fifteen participants were excluded due to contamination, and of the remaining 85 participants, 29, 49, and 7 were infected with rifampicin mono-resistant (RMR), multidrug-resistant (MDR), or extremely drug-resistant (XDR) TB, respectively. Analysis of baseline sputum demonstrated that CF supplementation of limiting dilution assays detected notable amounts of DCTB. Prevalence of DCTB was not influenced by smear status or mycobacterial growth indicator tube time to positivity. CF devoid of resuscitation promoting factors (Rpfs) yielded a greater amount of DCTB in sputum from participants with MDR-TB compared with those with RMR-TB. A similar effect was noted in DCTB assays without CF supplementation, suggesting that CF is dispensable for the detection of DCTB from drug-resistant strains. The HIV status of participants, and CD4 count, did not affect the amount of DCTB recovered. During treatment with second-line drug regimens, the probability of detecting DCTB from sputum specimens in liquid media with or without CF was higher compared with colony forming units, with DCTB detected up to 16 weeks post treatment. Collectively, these data point to differences in the ability of drug-resistant strains to respond to CF and Rpfs. Our findings demonstrate the possible utility of DCTB assays to diagnose and monitor treatment response for drug-resistant TB, particularly in immune compromised individuals with low CD4 counts.

Culture filtrate supplementation can be used to improve Mycobacterium tuberculosis culture positivity for spinal tuberculosis diagnosis.

Culture remains the gold standard to diagnose spinal tuberculosis (STB) despite the paucibacillary nature of the disease. Current methods can take up to 42 days to yield a result, delaying the ability to rapidly detect drug resistance. Studies have demonstrated the use of supplementation with culture filtrate (CF) from an axenic culture of Mycobacterium tuberculosis (Mtb) as a source of growth factors to improve culture rates. Our objective was to test a modified culture assay, utilizing CF supplemented media (CFSM), to improve culture positivity rates for suspected STB. Twelve patients with suspected STB were assessed by conventional culture (BACTEC™ MGIT 960), GeneXpert™ and standard histopathological examination. Spinal biopsies were taken from areas of diseased vertebral tissue or abscess, predetermined from MRI. Additional biopsies were obtained to assess CFSM for improved detection and faster culture of Mtb. All cases were diagnosed as STB and treated empirically for tuberculosis based on either bacteriological evidence (GeneXpert™, MGIT and/or CFSM positive), or based on clinical presentation. 5 specimens (45.45%) were positive for Mtb DNA as detected by GeneXpert™ and 1 specimen (8.33%) was cultured using MGIT (time to detection; 18 days). CFSM was able to culture 7 specimens (58.3%), with all CFSM positive specimens yielding a culture within 14 days. Two samples were positive only using the CFSM assay pointing to additional yield for diagnostic workup. Modification of standard culture can improve detection of Mtb and reduce time to positivity in individuals with STB where culture material is a requirement.

Differentially culturable tubercle bacteria as a measure of tuberculosis treatment response.

Introduction: Routine efficacy assessments of new tuberculosis (TB) treatments include quantitative solid culture or routine liquid culture, which likely miss quantification of drug tolerant bacteria. To improve these assessments, comparative analyses using additional measures such as quantification of differentially culturable tubercle bacteria (DCTB) are required. Essential for enabling this is a comparative measure of TB treatment responses using routine solid and liquid culture with liquid limiting dilutions (LLDs) that detect DCTB in sputum. Methods: We recruited treatment-naïve TB patients, with and without HIV-infection, and serially quantified their sputum for DCTB over the course of treatment. Results: Serial sputum sampling in 73 individuals during their first 14 days of treatment demonstrated that clearance of DCTB was slower compared to routine solid culture. Treatment response appeared to be characterized by four patterns: (1) Classic bi-phasic bacterial clearance; (2) early non-responders with slower clearance; (3) paradoxical worsening with an increase in bacterial count upon treatment initiation; and (4) non-responders with no change in bacterial load. During treatment, LLDs displayed greater bacterial yield when compared with quantitative solid culture. Upon treatment completion, 74% [46/62] of specimens displayed residual DCTB and within this group, two recurrences were diagnosed. Residual DCTB upon treatment completion was associated with a higher proportion of MGIT culture, GeneXpert, and smear positivity at two months post treatment. No recurrences occurred in the group without residual DCTB. Discussion: These data indicate that DCTB assays detect distinct subpopulations of organisms in sputum that are missed by routine solid and liquid culture, and offer important alternatives for efficacy assessments of new TB treatments. The residual DCTB observed upon treatment completion suggests that TB treatment does not always eliminate all bacterial populations, a finding that should be investigated in larger cohorts.