BACKGROUND: Multiplex PCR amplifies numerous targets in a single tube reaction and is essential in molecular biology and clinical diagnostics. One of its most important applications is in the targeted sequencing of pathogens. Despite this importance, few tools are available for designing multiplex primers. RESULTS: We developed primerJinn, a tool that designs a set of multiplex primers and allows for the in silico PCR evaluation of primer sets against numerous input genomes. We used primerJinn to create a multiplex PCR for the sequencing of drug resistance-conferring gene regions from Mycobacterium tuberculosis, which were then successfully sequenced. CONCLUSIONS: primerJinn provides a user-friendly, efficient, and accurate method for designing multiplex PCR primers for targeted sequencing and performing in silico PCR. It can be used for various applications in molecular biology and bioinformatics research, including the design of assays for amplifying and sequencing drug-resistance-conferring regions in important pathogens.

turboLysis is a novel mechanical cell lysis device that utilizes small beads to efficiently lyse tough cells like Mycobacterium, Saccharomyces, and Arabidopsis. We compared turboLysis to bead beating using the BeadBug 6 for several concentrations of Mycobacterium tuberculosis roughly correlated to the bacterial load commonly seen in patient samples. turboLysis performed similarly to the BeadBug at low bacterial concentrations and outperformed it at high concentrations above 2x105 CFU/ml (p < 0.005). Thus, turboLysis offers good cell lytic performance in a small form factor at a low cost.

Targeted amplicon sequencing to identify pathogens, resistance-conferring mutations, and strain types is an important tool in diagnosing and treating infections. However, due to the short read limitations of Illumina sequencing, many applications require the splitting of limited clinical samples between two reactions. Here, we outline hairpin Illumina single-tube sequencing PCR (hissPCR) which allows for the generation of overlapping amplicons containing Illumina indexes and adapters in a single tube, effectively extending the Illumina read length while maintaining reagent and sample input requirements.

Non-polyadenylated RNA includes a large subset of crucial regulators of RNA expression and constitutes a substantial portion of the transcriptome, playing essential roles in gene regulation. For example, enhancer RNAs are long non-coding RNAs that perform enhancer-like functions, are bi-directionally transcribed, and usually lack polyA tails. This paper presents a novel method, selSeq, that selectively removes mRNA and pre-mRNA from samples enabling the selective sequencing of crucial regulatory elements, including non-polyadenylated RNAs such as long non-coding RNA, enhancer RNA, and non-canonical mRNA.

Circular DNA offers benefits over linear DNA in diagnostic and field assays, but currently, circular DNA generation is lengthy, inefficient, highly dependent on the length and sequence of DNA, and can result in unwanted chimeras. We present streamlined methods for generating PCR-targeted circular DNA from a 700 bp amplicon of rv0678, the high GC content (65%) gene implicated in Mycobacterium tuberculosis bedaquiline resistance, and demonstrate that these methods work as desired. We employ self-circularization with and without splints, a Gibson cloning-based approach, and novel 2 novel methods for generating pseudocircular DNA. The circular DNA can be used as a template for rolling circle PCR followed by long-read sequencing, allowing for the error correction of sequence data, and improving the confidence in the drug resistance determination and strain identification; and, ultimately, improving patient treatment. IMPORTANCE Antimicrobial resistance is a global health threat, and drug resistant tuberculosis is a principal cause of antimicrobial resistance-related fatality. The long turnaround time and the need for high containment biological laboratories of phenotypic growth-based Mycobacterium tuberculosis drug susceptibility testing often commit patients to months of ineffective treatment, and there is a groundswell of effort in shifting from phenotypic to sequencing-based genotypic assays. Bedaquiline is a key component to newer, all oral, drug resistant, tuberculosis regimens. Thus, we focus our study on demonstrating the circularization of rv0678, the gene that underlies most M. tuberculosis bedaquiline resistance. We present 2 novel methods for generating pseudocircular DNA. These methods greatly reduce the complexity and time needed to generate circular DNA templates for rolling circle amplification and long-read sequencing, allowing for error correction of sequence data, and improving confidence in the drug resistance determination and strain identification.

BACKGROUND: Bedaquiline is a crucial drug for control of rifampicin-resistant tuberculosis. Molecular drug resistance assays could facilitate effective use of bedaquiline and surveillance of drug resistance emergence. To facilitate molecular assay development, we aimed to identify genomic markers of bedaquiline resistance. METHODS: In this systematic review and individual isolate analysis, we searched Europe PubMed Central and Scopus for studies published from the inception of each database until Oct 19, 2020, that assessed genotypic and phenotypic bedaquiline resistance in clinical or non-clinical Mycobacterium tuberculosis isolates. All studies reporting on the assessment of variants in the four genes of interest (Rv0678, atpE, pepQ, and Rv1979c) and phenotypic bedaquiline data in both clinical and non-clinical samples were included. We collated individual isolate data from eligible studies to assess the association between genomic variants with phenotypic bedaquiline resistance, using a standardised method endorsed by WHO. Risk of bias of the extracted data was independently assessed by two authors using the Quality Assessment of Diagnostic Accuracy Studies tool for clinical studies and Systematic Review Center for Laboratory Animal Experimentation tool for animal studies. The primary outcome was to identify mutations associated with resistance in four genes of interest (Rv0678, atpE, pepQ, and Rv1979c); for each genomic variant, the odds ratio (OR), 95% CI, and p value were calculated to identify resistance markers associated with bedaquiline resistance. This study is registered with PROSPERO, CRD42020221498. FINDINGS: Of 1367 studies identified, 41 published between 2007 and 2020 were eligible for inclusion. We extracted data on 1708 isolates: 1569 (91·9%) clinical isolates and 139 (8·1%) non-clinical isolates. We identified 237 unique variants in Rv0678, 14 in atpE, 28 in pepQ, and 11 in Rv1979c. Most clinical isolates with a single variant reported in Rv0678 (229 [79%] of 287 variants), atpE (14 [88%] of 16 variants), pepQ (32 [100%] of 32 variants), or Rv1979c (115 [98%] of 119 variants) were phenotypically susceptible to bedaquiline. Except for the atpE 187G→C (OR ∞, [95% CI 13·28-∞]; p<0·0001) and Rv0678 138_139insG (OR 6·91 [95% CI 1·16-47·38]; p=0·016) variants, phenotypic-genotypic associations were not significant (p≥0·05) for any single variant in Rv0678, atpE, pepQ, and Rv1979c. INTERPRETATION: Absence of clear genotypic-phenotypic associations for bedaquiline complicates the development of molecular drug susceptibility tests. A concerted global effort is urgently needed to assess the genotypic and phenotypic drug susceptibility of M tuberculosis isolates, especially in patients who have received unsuccessful bedaquiline-containing regimens. Treatment regimens should be designed to prevent emergence of bedaquiline resistance and phenotypic drug susceptibility tests should be used to guide and monitor treatment. FUNDING: Research Foundation Flanders, South African Medical Research Council, Department of Science and Innovation - National Research Foundation, National Institute of Health Institute of Allergy and Infectious Diseases, and Doris Duke Charitable Foundation.

Pyrazinamide is an important component of both drug-susceptible and drug-resistant tuberculosis treatment regimens. Although approximately 50% of rifampin-resistant isolates are also resistant to pyrazinamide, pyrazinamide susceptibility testing is not routinely performed due to the challenging nature of the assay. We investigated the diagnostic accuracy of genotypic and phenotypic methods and explored the occurrence of pyrazinamide heteroresistance. We assessed pyrazinamide susceptibility among 358 individuals enrolled in the South African EXIT-RIF cohort using Sanger and targeted deep sequencing (TDS) of the pncA gene, whole-genome sequencing (WGS), and phenotypic drug susceptibility testing. We calculated the diagnostic accuracy of the different methods and investigated the prevalence and clinical impact of pncA heteroresistance. True pyrazinamide susceptibility status was assigned to each isolate using the Köser classification and expert rules. We observed 100% agreement across genotypic methods for detection of pncA fixed mutations; only TDS confidently identified three isolates (0.8%) with minor variants. For the 355 (99.2%) isolates that could be assigned true pyrazinamide status with confidence, phenotypic DST had a sensitivity of 96.5% (95% confidence interval [CI], 93.8 to 99.3%) and specificity of 100% (95% CI, 100 to 100%), both Sanger sequencing and WGS had a sensitivity of 97.1% (95% CI, 94.6 to 99.6%) and specificity of 97.8% (95% CI, 95.7 to 99.9%), and TDS had sensitivity of 98.8% (95% CI, 97.2 to 100%) and specificity of 97.8% (95% CI, 95.7 to 99.9%). We demonstrate high sensitivity and specificity for pyrazinamide susceptibility testing among all assessed genotypic methods. The prevalence of pyrazinamide heteroresistance in Mycobacterium tuberculosis isolates was lower than that identified for other first-line drugs.

Mycobacterium tuberculosis (Mtb) infects lung myeloid cells, but the specific Mtb-permissive cells and host mechanisms supporting Mtb persistence during chronic infection are incompletely characterized. We report that after the development of T cell responses, CD11clo monocyte-derived cells harbor more live Mtb than alveolar macrophages (AM), neutrophils, and CD11chi monocyte-derived cells. Transcriptomic and functional studies revealed that the lysosome pathway is underexpressed in this highly permissive subset, characterized by less lysosome content, acidification, and proteolytic activity than AM, along with less nuclear TFEB, a regulator of lysosome biogenesis. Mtb infection does not drive lysosome deficiency in CD11clo monocyte-derived cells but promotes recruitment of monocytes that develop into permissive lung cells, mediated by the Mtb ESX-1 secretion system. The c-Abl tyrosine kinase inhibitor nilotinib activates TFEB and enhances lysosome functions of macrophages in vitro and in vivo, improving control of Mtb infection. Our results suggest that Mtb exploits lysosome-poor lung cells for persistence and targeting lysosome biogenesis is a potential host-directed therapy for tuberculosis.

BACKGROUND: The relationship between tuberculosis (TB), one of the leading infectious causes of death worldwide, and the microbiome, which is critical for health, is poorly understood. METHODS: To identify potential microbiome-host interactions, profiling of the oral, sputum and stool microbiota [n = 58 cases, n = 47 culture-negative symptomatic controls (SCs)] and whole blood transcriptome were done in pre-treatment presumptive pulmonary TB patients. This was a cross-sectional study. Microbiota were also characterised in close contacts of cases (CCCs, n = 73) and close contacts of SCs (CCSCs, n = 82) without active TB. FINDINGS: Cases and SCs each had similar α- and β-diversities in oral washes and sputum, however, β-diversity differed in stool (PERMANOVA p = 0•035). Cases were enriched with anaerobes in oral washes, sputum (Paludibacter, Lautropia in both) and stool (Erysipelotrichaceae, Blautia, Anaerostipes) and their stools enriched in microbial genes annotated as amino acid and carbohydrate metabolic pathways. In pairwise comparisons with their CCCs, cases had Megasphaera-enriched oral and sputum microbiota and Bifidobacterium-, Roseburia-, and Dorea-depleted stools. Compared to their CCSCs, SCs had reduced α-diversities and many differential taxa per specimen type. Cases differed transcriptionally from SCs in peripheral blood (PERMANOVA p = 0•001). A co-occurrence network analysis showed stool taxa, Erysipelotrichaceae and Blautia, to negatively co-correlate with enriched death receptor and EIF2 signalling pathways whereas Anaerostipes positively correlated with enriched interferon signalling, Nur77 signalling and inflammasome pathways; all of which are host pathways associated with disease severity. In contrast, none of the taxa enriched in SCs correlated with host pathways. INTERPRETATION: TB-specific microbial relationships were identified in oral washes, induced sputum, and stool from cases before the confounding effects of antibiotics. Specific anaerobes in cases stool predict upregulation of pro-inflammatory immunological pathways, supporting the gut microbiotas role in TB. FUNDING: European & Developing Countries Clinical Trials Partnership, South African-Medical Research Council, National Institute of Allergy and Infectious Diseases.

BACKGROUND: Atypical Beijing genotype Mycobacterium tuberculosis strains are widespread in South Africa and have acquired resistance to up to 13 drugs on multiple occasions. It is puzzling that these strains have retained fitness and transmissibility despite the potential fitness cost associated with drug resistance mutations. METHODS: We conducted Illumina sequencing of 211 Beijing genotype M. tuberculosis isolates to facilitate the detection of genomic features that may promote acquisition of drug resistance and restore fitness in highly resistant atypical Beijing forms. Phylogenetic and comparative genomic analysis was done to determine changes that are unique to the resistant strains that also transmit well. Minimum inhibitory concentration (MIC) determination for streptomycin and bedaquiline was done for a limited number of isolates to demonstrate a difference in MIC between isolates with and without certain variants. RESULTS: Phylogenetic analysis confirmed that two clades of atypical Beijing strains have independently developed resistance to virtually all the potent drugs included in standard (pre-bedaquiline) drug-resistant TB treatment regimens. We show that undetected drug resistance in a progenitor strain was likely instrumental in this resistance acquisition. In this cohort, ethionamide (ethA A381P) resistance would be missed in first-line drug-susceptible isolates, and streptomycin (gidB L79S) resistance may be missed due to an MIC close to the critical concentration. Subsequent inadequate treatment historically led to amplification of resistance and facilitated spread of the strains. Bedaquiline resistance was found in a small number of isolates, despite lack of exposure to the drug. The highly resistant clades also carry inhA promoter mutations, which arose after ethA and katG mutations. In these isolates, inhA promoter mutations do not alter drug resistance, suggesting a possible alternative role. CONCLUSION: The presence of the ethA mutation in otherwise susceptible isolates from ethionamide-naïve patients demonstrates that known exposure is not an adequate indicator of drug susceptibility. Similarly, it is demonstrated that bedaquiline resistance can occur without exposure to the drug. Inappropriate treatment regimens, due to missed resistance, leads to amplification of resistance, and transmission. We put these results into the context of current WHO treatment regimens, underscoring the risks of treatment without knowledge of the full drug resistance profile.