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Potential Mycobacterium tuberculosis (Mtb) transmission during different pulmonary tuberculosis (TB) disease states is poorly understood. We quantified viable aerosolized Mtb from TB clinic attendees following diagnosis and through six months' follow-up thereafter. Presumptive TB patients (n=102) were classified by laboratory, radiological, and clinical features into Group A: Sputum-Xpert Ultra-positive TB (n=52), Group B: Sputum-Xpert Ultra-negative TB (n=20), or Group C: TB undiagnosed (n=30). All groups were assessed for Mtb bioaerosol release at baseline, and subsequently at 2 wk, 2 mo, and 6 mo. Groups A and B were notified to the national TB program and received standard anti-TB chemotherapy; Mtb was isolated from 92% and 90% at presentation, 87% and 74% at 2 wk, 54% and 44% at 2 mo and 32% and 20% at 6 mo, respectively. Surprisingly, similar numbers were detected in Group C not initiating TB treatment: 93%, 70%, 48% and 22% at the same timepoints. A temporal association was observed between Mtb bioaerosol release and TB symptoms in all three groups. Persistence of Mtb bioaerosol positivity was observed in ~30% of participants irrespective of TB chemotherapy. Captured Mtb bacilli were predominantly acid-fast stain-negative and poorly culturable; however, three bioaerosol samples yielded sufficient biomass following culture for whole-genome sequencing, revealing two different Mtb lineages. Detection of viable aerosolized Mtb in clinic attendees, independent of TB diagnosis, suggests that unidentified Mtb transmitters might contribute a significant attributable proportion of community exposure. Additional longitudinal studies with sputum culture-positive and -negative control participants are required to investigate this possibility.
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Bacillus , Mycobacterium tuberculosis , Tuberculosis Pulmonar , Tuberculosis , Humanos , Esputo/microbiología , Tuberculosis Pulmonar/diagnóstico , Tuberculosis/microbiología , Firmicutes , Sensibilidad y EspecificidadRESUMEN
Interrupting transmission is an attractive anti-tuberculosis (TB) strategy but it remains underexplored owing to our poor understanding of the events surrounding transfer of Mycobacterium tuberculosis (Mtb) between hosts. Determining when live, infectious Mtb bacilli are released and by whom has proven especially challenging. Consequently, transmission chains are inferred only retrospectively, when new cases are diagnosed. This process, which relies on molecular analyses of Mtb isolates for epidemiological fingerprinting, is confounded by the prolonged infectious period of TB and the potential for transmission from transient exposures. We developed a Respiratory Aerosol Sampling Chamber (RASC) equipped with high-efficiency filtration and sampling technologies for liquid-capture of all particulate matter (including Mtb) released during respiration and non-induced cough. Combining the mycobacterial cell wall probe, DMN-trehalose, with fluorescence microscopy of RASC-captured bioaerosols, we detected and quantified putative live Mtb bacilli in bioaerosol samples arrayed in nanowell devices. The RASC enabled non-invasive capture and isolation of viable Mtb from bioaerosol within 24 hours of collection. A median 14 live Mtb bacilli (range 0-36) were isolated in single-cell format from 90% of confirmed TB patients following 60 minutes bioaerosol sampling. This represented a significant increase over previous estimates of transmission potential, implying that many more organisms might be released daily than commonly assumed. Moreover, variations in DMN-trehalose incorporation profiles suggested metabolic heterogeneity in aerosolized Mtb. Finally, preliminary analyses indicated the capacity for serial image capture and analysis of nanowell-arrayed bacilli for periods extending into weeks. These observations support the application of this technology to longstanding questions in TB transmission including the propensity for asymptomatic transmission, the impact of TB treatment on Mtb bioaerosol release, and the physiological state of aerosolized bacilli.
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Pruebas Respiratorias , Tos/microbiología , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Pulmonar/microbiología , Adulto , Estudios de Cohortes , Humanos , Microscopía Fluorescente , Nanotecnología/instrumentaciónRESUMEN
Rationale: South African adolescents carry a high tuberculosis disease burden. It is not known if schools are high-risk settings for Mycobacterium tuberculosis (MTB) transmission. Objectives: To detect airborne MTB genomic DNA in classrooms. Methods: We studied 72 classrooms occupied by 2,262 students in two South African schools. High-volume air filtration was performed for median 40 (interquartile range [IQR], 35-54) minutes and assayed by droplet digital PCR (ddPCR)-targeting MTB region of difference 9 (RD9), with concurrent CO2 concentration measurement. Classroom data were benchmarked against public health clinics. Students who consented to individual tuberculosis screening completed a questionnaire and sputum collection (Xpert MTB/RIF Ultra) if symptom positive. Poisson statistics were used for MTB RD9 copy quantification. Measurements and Main Results: ddPCR assays were positive in 13/72 (18.1%) classrooms and 4/39 (10.3%) clinic measurements (P = 0.276). Median ambient CO2 concentration was 886 (IQR, 747-1223) ppm in classrooms versus 490 (IQR, 405-587) ppm in clinics (P < 0.001). Average airborne concentration of MTB RD9 was 3.61 copies per 180,000 liters in classrooms versus 1.74 copies per 180,000 liters in clinics (P = 0.280). Across all classrooms, the average risk of an occupant inhaling one MTB RD9 copy was estimated as 0.71% during one standard lesson of 35 minutes. Among 1,836/2,262 (81.2%) students who consented to screening, 21/90 (23.3%) symptomatic students produced a sputum sample, of which one was Xpert MTB/RIF Ultra positive. Conclusions: Airborne MTB genomic DNA was detected frequently in high school classrooms. Instantaneous risk of classroom exposure was similar to the risk in public health clinics.
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Microbiología del Aire , ADN Bacteriano/análisis , Exposición por Inhalación/análisis , Mycobacterium tuberculosis/aislamiento & purificación , Instituciones Académicas , Tuberculosis/transmisión , Adolescente , Estudios Transversales , Femenino , Humanos , Exposición por Inhalación/efectos adversos , Exposición por Inhalación/estadística & datos numéricos , Masculino , Mycobacterium tuberculosis/genética , Riesgo , Sudáfrica , Tuberculosis/diagnósticoRESUMEN
BACKGROUND: Congregate settings, such as healthcare clinics, may play an essential role in Mycobacterium tuberculosis (Mtb) transmission. Using patient and environmental data, we studied transmission at a primary care clinic in South Africa. METHODS: We collected patient movements, cough frequency, and clinical data, and measured indoor carbon dioxide (CO2) levels, relative humidity, and Mtb genomes in the air. We used negative binomial regression model to investigate associations. RESULTS: We analyzed 978 unique patients who contributed 14 795 data points. The median patient age was 33 (interquartile range [IQR], 26-41) years, and 757 (77.4%) were female. Overall, median CO2 levels were 564 (IQR 495-646) parts per million and were highest in the morning. Median number of coughs per day was 466 (IQR, 368-503), and overall median Mtb DNA copies/µL/day was 4.2 (IQR, 1.2-9.5). We found an increased presence of Mtb DNA in the air of 32% (95% credible interval, 7%-63%) per 100 additional young adults (aged 15-29 years) and 1% (0-2%) more Mtb DNA per 10% increase of relative humidity. Estimated cumulative transmission risks for patients attending the clinic monthly for at least 1 hour range between 9% and 29%. CONCLUSIONS: We identified young adults and relative humidity as potentially important factors for transmission risks in healthcare clinics. Our approach should be used to detect transmission and evaluate infection control interventions.
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Mycobacterium tuberculosis , Tuberculosis , Dióxido de Carbono/análisis , Femenino , Humanos , Masculino , Mycobacterium tuberculosis/genética , Atención Primaria de Salud , Sudáfrica/epidemiología , Tuberculosis/diagnóstico , Adulto JovenRESUMEN
Stigma and mistrust generate significant barriers to the uptake of biomedical, clinical and public health measures to combat infectious diseases. Many pandemics such as HIV, TB and COVID-19 disproportionately affect poorer communities, and the social and public health impact is connected through socio-political histories and contexts. This chapter describes activities and reflections of a South African public engagement programme, Eh!woza, that aims to bring together the biomedicine of disease with its social context and impact. We describe experiences working on tuberculosis-related public engagement programmes in South Africa, and how these approaches were refocused to address the COVID-19 pandemic. We reflect on the lesson learned and considerations around visualising the social impact of disease and making the visualisation of accurate information relatable to younger audiences. Much of the discussion is situated within description and reflection, touching on both the historical and contemporary cultural and political conditions in which infectious diseases have flourished. Finally, the challenges we faced when effectively disseminating media on large-scale digital platforms are highlighted, raising important questions around representation, mass targets, and impactful dissemination of public engagement outputs.
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COVID-19 , Tuberculosis , COVID-19/epidemiología , Humanos , Pandemias , Salud Pública , Estigma Social , Tuberculosis/epidemiologíaRESUMEN
Cobalamin is an essential cofactor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. Mycobacterium smegmatis, an environmental saprophyte frequently used as surrogate for the obligate human pathogen M. tuberculosis, carries approximately 30 genes predicted to be involved in de novo cobalamin biosynthesis. M. smegmatis also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase that catalyzes the final reaction in methionine biosynthesis. In addition to metH, M. smegmatis possesses a cobalamin-independent methionine synthase, metE, suggesting that enzyme use-MetH versus MetE-is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling metE expression in M. tuberculosis Here, we apply a targeted mass spectrometry-based approach to confirm de novo cobalamin biosynthesis in M. smegmatis during aerobic growth in vitro We also demonstrate that M. smegmatis can transport and assimilate exogenous cyanocobalamin (CNCbl; also known as vitamin B12) and its precursor, dicyanocobinamide ([CN]2Cbi). However, the uptake of CNCbl and (CN)2Cbi in this organism is restricted and seems dependent on the conditional essentiality of the cobalamin-dependent methionine synthase. Using gene and protein expression analyses combined with single-cell growth kinetics and live-cell time-lapse microscopy, we show that transcription and translation of metE are strongly attenuated by endogenous cobalamin. These results support the inference that metH essentiality in M. smegmatis results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between M. smegmatis and M. tuberculosis provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity.IMPORTANCE Alterations in cobalamin-dependent metabolism have marked the evolution of Mycobacterium tuberculosis into a human pathogen. However, the role(s) of cobalamin in mycobacterial physiology remains poorly understood. Using the nonpathogenic saprophyte M. smegmatis, we investigated the production of cobalamin, transport and assimilation of cobalamin precursors, and the role of cobalamin in regulating methionine biosynthesis. We confirm constitutive de novo cobalamin biosynthesis in M. smegmatis, in contrast with M. tuberculosis, which appears to lack de novo cobalamin biosynthetic capacity. We also show that uptake of cyanocobalamin (vitamin B12) and its precursors is restricted in M. smegmatis, apparently depending on the cofactor requirements of the cobalamin-dependent methionine synthase. These observations establish M. smegmatis as an informative foil to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.
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Regulación Bacteriana de la Expresión Génica , Metionina/biosíntesis , Mycobacterium smegmatis/metabolismo , Vitamina B 12/biosíntesis , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/genética , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Metiltransferasas/genética , Metiltransferasas/metabolismo , Mycobacterium smegmatis/genética , RiboswitchRESUMEN
Tuberculosis (TB) is the leading cause of death among HIV-1-infected individuals and Mycobacterium tuberculosis (Mtb) co-infection is an early precipitate to AIDS. We aimed to determine whether Mtb strains differentially modulate cellular susceptibility to HIV-1 infection (cis- and trans-infection), via surface receptor interaction by their cell envelope lipids. Total lipids from pathogenic (lineage 4 Mtb H37Rv, CDC1551 and lineage 2 Mtb HN878, EU127) and non-pathogenic (Mycobacterium bovis BCG and Mycobacterium smegmatis) Mycobacterium strains were integrated into liposomes mimicking the lipid distribution and antigen accessibility of the mycobacterial cell wall. The resulting liposomes were tested for modulating in vitro HIV-1 cis- and trans-infection of TZM-bl cells using single-cycle infectious virus particles. Mtb glycolipids did not affect HIV-1 direct infection however, trans-infection of both R5 and X4 tropic HIV-1 strains were impaired in the presence of glycolipids from M. bovis, Mtb H37Rv and Mtb EU127 strains when using Raji-DC-SIGN cells or immature and mature dendritic cells (DCs) to capture virus. SL1, PDIM and TDM lipids were identified to be involved in DC-SIGN recognition and impairment of HIV-1 trans-infection. These findings indicate that variant strains of Mtb have differential effect on HIV-1 trans-infection with the potential to influence HIV-1 disease course in co-infected individuals.
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Infecciones Oportunistas Relacionadas con el SIDA/metabolismo , Coinfección/metabolismo , Glucolípidos/metabolismo , VIH-1/fisiología , Liposomas/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculosis/metabolismo , Infecciones Oportunistas Relacionadas con el SIDA/virología , Moléculas de Adhesión Celular/metabolismo , Pared Celular/metabolismo , Células HEK293 , Humanos , Lectinas Tipo C/metabolismo , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium bovis/metabolismo , Mycobacterium smegmatis/metabolismo , Receptores de Superficie Celular/metabolismo , Tuberculosis/microbiología , Internalización del VirusRESUMEN
An amendment to this paper has been published and can be accessed via the original article.
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BACKGROUND: Tuberculosis (TB) is transmitted in bioaerosols containing Mycobacterium tuberculosis (Mtb). Despite being central to ongoing TB transmission, no routine diagnostic assay exists to measure Mtb in bioaerosols. Furthermore, published studies of Mtb in bioaerosol samples have been limited to individuals with sputum-positive pulmonary TB. Notably, TB diagnosis is based on clinical symptoms and sputum laboratory findings. This is despite the fact that approximately half of all patients commencing TB treatment are sputum-negative, resulting in a high proportion of presumptive treatments. Here, we propose to use a sensitive air sampling protocol to investigate the prevalence of Mtb-containing bioaerosols in both sputum-positive and sputum-negative TB suspects, at the same time evaluating the potential to identify unrecognized transmitters of TB. METHODS: Our parallel-group design will identify viable Mtb in bioaerosols produced by individuals attending a TB clinic in South Africa. Sampling will be performed on eligible individuals presenting with symptoms indicative of TB and repeated at 14 days if initially positive. Participants will be prospectively classified into three distinct groups based on National TB Control Program (NTBCP) criteria: Group A, TB notification with sputum-based laboratory confirmation; Group B, TB notification with empiric diagnosis; and Group C, individuals not notified. Group C individuals with detectable Mtb bioaerosol will be monitored until resolution of clinical and laboratory status. Collection of bioaerosol specimens will be via two consecutive sampling modalities: (1) direct sampling following a specific respiratory manoeuvre; and (2) indirect sampling during passive respiratory activity. Bioaerosol specimens will be analyzed for viable Mtb using DMN-trehalose staining and live-cell fluorescence microscopy. Mtb genomes and mycobacterial and host lipids will be detected using droplet digital PCR and mass spectrometry analyses, respectively. The primary objective is to determine the prevalence of Mtb bioaerosols in all TB clinic attendees and in each of the groups. Secondary objectives are to investigate differences in prevalence of Mtb bioaerosol by HIV status and current isoniazid preventive therapy (IPT) use; we will also determine the impact of anti-TB chemotherapy on Mtb-containing bioaerosol production. DISCUSSION: Respiratory bioaerosol has a potential role in non-invasive TB diagnosis, infectivity measurement and treatment monitoring. TRIAL REGISTRATION: ClinicalTrials.gov: NCT04241809 . Date of Registration: 27/1/2020.
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Aerosoles/análisis , Manejo de Especímenes/métodos , Tuberculosis Pulmonar/diagnóstico , Adulto , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Femenino , Humanos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Sudáfrica , Esputo/microbiologíaRESUMEN
Much remains unknown about Mycobacterium tuberculosis transmission. Seminal experimental studies from the 1950s demonstrated that airborne expulsion of droplet nuclei from an infectious tuberculosis (TB) patient is the primary route of transmission. However, these findings did not rule out other routes of M. tuberculosis transmission. We reviewed historical scientific evidence from the late 19th/early 20th century and contemporary studies investigating the presence, persistence and infectiousness of environmental M. tuberculosis We found both experimental and epidemiological evidence supporting the presence and viability of M. tuberculosis in multiple natural and built environments for months to years, presumably following contamination by a human source. Furthermore, several studies confirm M. tuberculosis viability and virulence in the environment using guinea pig and mouse models. Most of this evidence was historical; however, several recent studies have reported consistent findings of M. tuberculosis detection and viability in the environment using modern methods. Whether M. tuberculosis in environments represents an infectious threat to humans requires further investigation; this may represent an untapped source of data with which to further understand M. tuberculosis transmission. We discuss potential opportunities for harnessing these data to generate new insights into TB transmission in congregate settings.
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Tos/microbiología , Mycobacterium tuberculosis/aislamiento & purificación , Investigación/tendencias , Tuberculosis Pulmonar/historia , Tuberculosis Pulmonar/transmisión , Aerosoles , Animales , Modelos Animales de Enfermedad , Polvo , Cobayas , Historia del Siglo XIX , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Ratones , Esputo/microbiologíaRESUMEN
HIV significantly affects the immunological environment during tuberculosis coinfection, and therefore may influence the selective landscape upon which M. tuberculosis evolves. To test this hypothesis whole genome sequences were determined for 169 South African M. tuberculosis strains from HIV-1 coinfected and uninfected individuals and analyzed using two Bayesian codon-model based selection analysis approaches: FUBAR which was used to detect persistent positive and negative selection (selection respectively favoring and disfavoring nonsynonymous substitutions); and MEDS which was used to detect episodic directional selection specifically favoring nonsynonymous substitutions within HIV-1 infected individuals. Among the 25,251 polymorphic codon sites analyzed, FUBAR revealed that 189-fold more were detectably evolving under persistent negative selection than were evolving under persistent positive selection. Three specific codon sites within the genes celA2b, katG, and cyp138 were identified by MEDS as displaying significant evidence of evolving under directional selection influenced by HIV-1 coinfection. All three genes encode proteins that may indirectly interact with human proteins that, in turn, interact functionally with HIV proteins. Unexpectedly, epitope encoding regions were enriched for sites displaying weak evidence of directional selection influenced by HIV-1. Although the low degree of genetic diversity observed in our M. tuberculosis data set means that these results should be interpreted carefully, the effects of HIV-1 on epitope evolution in M. tuberculosis may have implications for the design of M. tuberculosis vaccines that are intended for use in populations with high HIV-1 infection rates.
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Mycobacterium tuberculosis/metabolismo , Tuberculosis Pulmonar/metabolismo , Teorema de Bayes , Codón , Coinfección/metabolismo , Evolución Molecular , Variación Genética , Infecciones por VIH/complicaciones , Infecciones por VIH/genética , VIH-1/genética , Humanos , Modelos Genéticos , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Selección Genética , Tuberculosis Pulmonar/complicacionesRESUMEN
In 2015, there were an estimated 10.4 million new cases of tuberculosis (TB) globally, making it one of the leading causes of death due to an infectious disease. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC), with human disease resulting from infection by M. tuberculosis sensu stricto and M. africanum. Recent progress in genotyping techniques, in particular the increasing availability of whole genome sequence data, has revealed previously under appreciated levels of genetic diversity within the MTBC. Several studies have shown that this genetic diversity may translate into differences in TB transmission, clinical manifestations of disease, and host immune responses. This suggests the existence of MTBC genotype-dependent host-pathogen interactions which may influence the outcome of infection and progression of disease. In this review, we highlight the studies demonstrating differences in innate and adaptive immunological outcomes consequent on MTBC genetic diversity, and discuss how these differences in immune response might influence the development of TB vaccines, diagnostics and new therapies.
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Inmunidad Adaptativa , Variación Antigénica , Variación Genética , Inmunidad Innata , Mycobacterium tuberculosis/genética , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/inmunología , Animales , Genotipo , Interacciones Huésped-Patógeno , Humanos , Mycobacterium tuberculosis/inmunología , Filogenia , Especificidad de la Especie , Tuberculosis/microbiologíaRESUMEN
Eh!woza is a public engagement initiative that explores the biomedical and social aspects of tuberculosis (TB) in South Africa. The project is a collaboration between scientists based in an infectious disease research institute, a local conceptual/visual artist, a youth-based educational non-governmental organization (NGO) and young learners from a high-burden TB community. The learners participate in a series of interactive science and media production workshops: initially presented with biomedical knowledge about TB and, in later sessions, are trained in creating documentary films and engage with ideas around visual representation. The participants are encouraged to make use of this newly acquired knowledge to tell stories from their chosen communities in Khayelitsha, a township in Cape Town. Through its engagement with the complex manner in which TB is experienced, framed and understood by biomedical scientists, young people, and those who have been affected by the disease, Eh!woza presents alternative ways of exploring the complexities of human illness. The integration and interrogation of biomedical understandings, lay narratives and the young participants' framing of the disease poses questions about 'knowing', and the meanings people attribute to ways of 'knowing' and the actions they impel. The project also presents contrasting reflections on cure-from a biomedical perspective, and care-from the perspective of TB-affected young people and community members. In this article, we describe the Eh!woza project, present thoughts from the participating students on the science and media workshops, and detail the narratives of ill-health and disease from people within their neighbourhoods. We conclude with a critical analysis of the complexities of knowledge communication, notions of cure versus care, and a consideration of the potential contribution of this project to the growth of medical humanities in Africa.
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Comprensión , Humanidades , Conocimiento , Narración , Investigadores , Estudiantes , Tuberculosis , Adolescente , Arte , Actitud , Investigación Biomédica , Comunicación , Atención a la Salud , Humanos , Películas Cinematográficas , Organizaciones , Características de la Residencia , SudáfricaRESUMEN
Pioneering studies linking symptomatic disease and cough-mediated release of Mycobacterium tuberculosis (Mtb) established the infectious origin of tuberculosis (TB), simultaneously informing the pervasive notion that pathology is a prerequisite for Mtb transmission. Our prior work has challenged this assumption: by sampling TB clinic attendees, we detected equivalent release of Mtb-containing bioaerosols by confirmed TB patients and individuals not receiving a TB diagnosis, and we demonstrated a time-dependent reduction in Mtb bioaerosol positivity during six-months' follow-up, irrespective of anti-TB chemotherapy. Now, by extending bioaerosol sampling to a randomly selected community cohort, we show that Mtb release is common in a TB-endemic setting: of 89 participants, 79.8% (71/89) produced Mtb bioaerosols independently of QuantiFERON-TB Gold status, a standard test for Mtb infection; moreover, during two-months' longitudinal sampling, only 2% (1/50) were serially Mtb bioaerosol negative. These results necessitate a reframing of the prevailing paradigm of Mtb transmission and infection, and may explain the current inability to elucidate Mtb transmission networks in TB-endemic regions.
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OBJECTIVE: Novel approaches are needed to understand and disrupt Mycobacterium tuberculosis transmission. In this proof-of-concept study, we investigated the use of environmental air samplings to detect and quantify M. tuberculosis in different clinic settings in a high-burden area. DESIGN: Cross-sectional, environmental sampling. SETTING: Primary-care clinic. METHODS: A portable, high-flow dry filter unit (DFU) was used to draw air through polyester felt filters for 2 hours. Samples were collected in the waiting area and TB room of a primary care clinic. Controls included sterile filters placed directly into collection tubes at the DFU sampling site, and filter samplings performed outdoors. DNA was extracted from the filters, and droplet digital polymerase chain reaction (ddPCR) was used to quantify M. tuberculosis DNA copies. Carbon dioxide (CO2) data loggers captured CO2 concentrations in the sampled areas. RESULTS: The median sampling time was 123 minutes (interquartile range [IQR], 121-126). A median of 121 (IQR, 35-243) M. tuberculosis DNA copies were obtained from 74 clinic samplings, compared to a median of 3 (IQR, 1-33; P < .001) obtained from 47 controls. At a threshold of 320 DNA copies, specificity was 100%, and 18% of clinic samples would be classified as positive. CONCLUSIONS: This proof-of-concept study suggests that the potential for airborne M. tuberculosis detection based on M. tuberculosis DNA copy yield to enable the identification of high-risk transmission locations. Further optimization of the M. tuberculosis extraction technique and ddPCR data analysis would improve detection and enable robust interpretation of these data.
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Mycobacterium tuberculosis , Tuberculosis , Humanos , Mycobacterium tuberculosis/genética , Tuberculosis/diagnóstico , Dióxido de Carbono , Estudios Transversales , Reacción en Cadena de la Polimerasa/métodosRESUMEN
BACKGROUND: Mycobacterium tuberculosis whole-genome sequencing (WGS) has been widely used for genotypic drug susceptibility testing (DST) and outbreak investigation. For both applications, Illumina technology is used by most public health laboratories; however, Nanopore technology developed by Oxford Nanopore Technologies has not been thoroughly evaluated. The aim of this study was to determine whether Nanopore sequencing data can provide equivalent information to Illumina for transmission clustering and genotypic DST for M tuberculosis. METHODS: In this genomic analysis, we analysed 151 M tuberculosis isolates from Madagascar, South Africa, and England, which were collected between 2011 and 2018, using phenotypic DST and matched Illumina and Nanopore data. Illumina sequencing was done with the MiSeq, HiSeq 2500, or NextSeq500 platforms and Nanopore sequencing was done on the MinION or GridION platforms. Using highly reliable PacBio sequencing assemblies and pairwise distance correlation between Nanopore and Illumina data, we optimise Nanopore variant filters for detecting single-nucleotide polymorphisms (SNPs; using BCFtools software). We then used those SNPs to compare transmission clusters identified by Nanopore with the currently used UK Health Security Agency Illumina pipeline (COMPASS). We compared Illumina and Nanopore WGS-based DST predictions using the Mykrobe software and mutation catalogue. FINDINGS: The Nanopore BCFtools pipeline identified SNPs with a median precision of 99·3% (IQR 99·1-99·6) and recall of 90·2% (88·1-94·2) compared with a precision of 99·6% (99·4-99·7) and recall of 91·9% (87·6-98·6) using the Illumina COMPASS pipeline. Using a threshold of 12 SNPs for putative transmission clusters, Illumina identified 98 isolates as unrelated and 53 as belonging to 19 distinct clusters (size range 2-7). Nanopore reproduced 15 out of 19 clusters perfectly; two clusters were merged into one cluster, one cluster had a single sample missing, and one cluster had an additional sample adjoined. Illumina-based clusters were also closely replicated using a five SNP threshold and clustering accuracy was maintained using mixed Illumina and Nanopore datasets. Genotyping resistance variants with Nanopore was highly concordant with Illumina, having zero discordant SNPs across more than 3000 SNPs and four insertions or deletions (indels), across 60 000 indels. INTERPRETATION: Illumina and Nanopore technologies can be used independently or together by public health laboratories performing M tuberculosis genotypic DST and outbreak investigations. As a result, clinical and public health institutions making decisions on which sequencing technology to adopt for tuberculosis can base the choice on cost (which varies by country), batching, and turnaround time. FUNDING: Academy for Medical Sciences, Oxford Wellcome Institutional Strategic Support Fund, and the Swiss South Africa Joint Research Award (Swiss National Science Foundation and South African National Research Foundation).
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Mycobacterium tuberculosis , Secuenciación de Nanoporos , Tuberculosis , Humanos , Mycobacterium tuberculosis/genética , Pruebas de Sensibilidad Microbiana , Análisis de Secuencia de ADN , Genómica , Tuberculosis/diagnóstico , Tuberculosis/tratamiento farmacológico , Tuberculosis/epidemiología , Brotes de EnfermedadesRESUMEN
Despite the availability of effective drug treatment, Mycobacterium tuberculosis (Mtb), the causative agent of TB disease, kills ~1. 5 million people annually, and the rising prevalence of drug resistance increasingly threatens to worsen this plight. We previously showed that sublethal exposure to the frontline anti-TB drug, rifampicin, resulted in substantial adaptive remodeling of the proteome of the model organism, Mycobacterium smegmatis, in the drug-sensitive mc2155 strain [wild type (WT)]. In this study, we investigate whether these responses are conserved in an engineered, isogenic mutant harboring the clinically relevant S531L rifampicin resistance-conferring mutation (SL) and distinguish the responses that are specific to RNA polymerase ß subunit- (RpoB-) binding activity of rifampicin from those that are dependent on the presence of rifampicin alone. We verified the drug resistance status of this strain and observed no phenotypic indications of rifampicin-induced stress upon treatment with the same concentration as used in WT (2.5 µg/ml). Thereafter, we used a cell wall-enrichment strategy to focus attention on the cell wall proteome and observed 253 proteins to be dysregulated in SL bacteria in comparison with 716 proteins in WT. We observed that decreased abundance of ATP-binding cassette (ABC) transporters and increased abundance of ribosomal machinery were conserved in the SL strain, whereas the upregulation of transcriptional machinery and the downregulation of numerous two-component systems were not. We conclude that the drug-resistant M. smegmatis strain displays some of the same proteomic responses observed in WT and suggest that this evidence supports the hypothesis that rifampicin exercises effects beyond RpoB-interaction alone and that mycobacteria recognise rifampicin as a signaling molecule in an RpoB-independent manner at sublethal doses. Taken together, our data indicates mixed RpoB-independent and RpoB-dependent proteomic remodeling in WT mycobacteria, with evidence for RpoB-independent ABC transporter downregulation, but drug activity-based transcriptional upregulation and two-component system downregulation.
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Detection and accurate quantitation of viable Mycobacterium tuberculosis is fundamental to understanding mycobacterial pathogenicity, tuberculosis (TB) disease progression and outcomes; TB transmission; drug action, efficacy and drug resistance. Despite this importance, methods for determining numbers of viable bacilli are limited in accuracy and precision owing to inherent characteristics of mycobacterial cell biology-including the tendency to clump, and "differential" culturability-and technical challenges consequent on handling an infectious pathogen under biosafe conditions. We developed an absolute counting method for mycobacteria in liquid cultures using a bench-top flow cytometer, and the low-cost fluorescent dyes Calcein-AM (CA) and SYBR-gold (SG). During exponential growth CA + cell counts are highly correlated with CFU counts and can be used as a real-time alternative to simplify the accurate standardisation of inocula for experiments. In contrast to CFU counting, this method can detect and enumerate cell aggregates in samples, which we show are a potential source of variance and bias when using established methods. We show that CFUs comprise a sub-population of intact, metabolically active mycobacterial cells in liquid cultures, with CFU-proportion varying by growth conditions. A pharmacodynamic application of the flow cytometry method, exploring kinetics of fluorescent probe defined subpopulations compared to CFU is demonstrated. Flow cytometry derived Mycobacterium bovis bacillus Calmette-Guérin (BCG) time-kill curves differ for rifampicin and kanamycin versus isoniazid and ethambutol, as do the relative dynamics of discrete morphologically-distinct subpopulations of bacilli revealed by this high-throughput single-cell technique.