Detection of Minority Variants and Mixed Infections in Mycobacterium tuberculosis by Direct Whole-Genome Sequencing on Noncultured Specimens Using a Specific-DNA Capture Strategy

Lozano, Nuria; Lanza, Val F.; Suárez‐González, Julia; Herránz, Marta; Sola‐Campoy, Pedro J.; Rodríguez‐Grande, Cristina; Buenestado‐Serrano, Sergio; Ruiz‐Serrano, María Jesús; Tudó, Griselda; Alcaide, Fernando; Muñóz, Patricia; Viedma, Darı́o Garcı́a de; Pérez‐Lago, Laura

doi:10.1128/msphere.00744-21

Cited by 10 publications

(5 citation statements)

References 33 publications

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“…These data indicate that while WGS is feasible on bioaerosols, sufficient DNA is required to generate data of high enough quality to identify specific Mtb lineages. Future work to facilitate WGS of a broader number of samples could include optimization of DNA extraction techniques, culture, and possibly Mtb DNA enrichment prior to WGS ( 35 – 37 ).…”

Section: Discussionmentioning

confidence: 99%

Aerosolization of viable Mycobacterium tuberculosis bacilli by tuberculosis clinic attendees independent of sputum-Xpert Ultra status

Patterson,

Dinkele,

Gessner

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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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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Section: Discussionmentioning

confidence: 99%

Aerosolization of viable Mycobacterium tuberculosis bacilli by tuberculosis clinic attendees independent of sputum-Xpert Ultra status

Patterson,

Dinkele,

Gessner

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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“…The objective was the direct detection in patient sputum so that minority populations of resistant strains could be identified, and the most appropriate treatment could be utilized as soon as possible. This approach used a platform for capturing M. tuberculosis -specific DNA that was designed to enrich the clinical specimen and obtain quality sequences ( 23 ). It is expected that continued progress for direct specimen WGS will continue to be refined and will play an important role in clinical diagnosis and public health in the near future.…”

Section: Discussionmentioning

confidence: 99%

A novel hybridization capture method for direct whole genome sequencing of clinical specimens to inform Legionnaires’ disease investigations

Weeber,

Singh,

Lapierre

et al. 2024

J Clin Microbiol

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The unprecedented precision and resolution of whole genome sequencing (WGS) can provide definitive identification of infectious agents for epidemiological outbreak tracking. WGS approaches, however, are frequently impeded by low pathogen DNA recovery from available primary specimens or unculturable samples. A cost-effective hybrid capture assay for Legionella pneumophila WGS analysis directly on primary specimens was developed. DNA from a diverse range of sputum and autopsy specimens PCR-positive for L. pneumophila serogroup 1 (LPSG1) was enriched with this method, and WGS was performed. All tested specimens were determined to be enriched for Legionella reads (up to 209,000-fold), significantly improving the discriminatory power to compare relatedness when no clinical isolate was available. We found the WGS data from some enriched specimens to differ by less than five single-nucleotide polymorphisms (SNPs) when compared to the WGS data of a matched culture isolate. This testing and analysis retrospectively provided previously unconfirmed links to environmental sources for clinical specimens of sputum and autopsy lung tissue. The latter provided the additional information needed to identify the source of these culture-negative cases associated with the South Bronx 2015 Legionnaires’ disease (LD) investigation in New York City. This new method provides a proof of concept for future direct clinical specimen hybrid capture enrichment combined with WGS and bioinformatic analysis during outbreak investigations. IMPORTANCE Legionnaires’ disease (LD) is a severe and potentially fatal type of pneumonia primarily caused by inhalation of Legionella -contaminated aerosols from man-made water or cooling systems. LD remains extremely underdiagnosed as it is an uncommon form of pneumonia and relies on clinicians including it in the differential and requesting specialized testing. Additionally, it is challenging to obtain clinical lower respiratory specimens from cases with LD, and when available, culture requires specialized media and growth conditions, which are not available in all microbiology laboratories. In the current study, a method for Legionella pneumophila using hybrid capture by RNA baiting was developed, which allowed us to generate sufficient genome resolution from L. pneumophila serogroup 1 PCR-positive clinical specimens. This new approach offers an additional tool for surveillance of future LD outbreaks where isolation of Legionella is not possible and may help solve previously unanswered questions from past LD investigations.

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“…Capture methods may be promising for enrichment in clinical sputum samples, which could facilitate culture-free MTB WGS. Other researchers propose to find low abundance sequences by hybridization (FLASH) to enrich targeted sequences [ 42 ], or propose a pipeline that can help to clean contaminant reads from sputum samples and/or detect mixed infections [ 42 , 43 , 44 , 45 ].…”

Section: Discussionmentioning

confidence: 99%

The Role of Next-Generation Sequencing (NGS) in the Management of Tuberculosis: Practical Review for Implementation in Routine

Beviere,

Reissier,

Penven

et al. 2023

Pathogens

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Next-generation sequencing (NGS) has modernized the field of tuberculosis (TB) research by enabling high-throughput sequencing of the entire genome of Mycobacterium tuberculosis (MTB), which is the causative agent of TB. NGS has provided insights into the genetic diversity of MTB, which are crucial for understanding the evolution and transmission of the disease, and it has facilitated the identification of drug-resistant strains, enabling rapid and accurate tailoring of treatment. However, the high cost and the technical complexities of NGS currently limit its widespread use in clinical settings. International recommendations are thus necessary to facilitate the interpretation of polymorphisms, and an experimental approach is still necessary to correlate them to phenotypic data. This review aims to present a comparative, step-by-step, and up-to-date review of the techniques available for the implementation of this approach in routine laboratory workflow. Ongoing research on NGS for TB holds promise for improving our understanding of the disease and for developing more efficacious treatments.

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Detection of Minority Variants and Mixed Infections in Mycobacterium tuberculosis by Direct Whole-Genome Sequencing on Noncultured Specimens Using a Specific-DNA Capture Strategy

Cited by 10 publications

References 33 publications

Aerosolization of viable Mycobacterium tuberculosis bacilli by tuberculosis clinic attendees independent of sputum-Xpert Ultra status

Aerosolization of viable Mycobacterium tuberculosis bacilli by tuberculosis clinic attendees independent of sputum-Xpert Ultra status

A novel hybridization capture method for direct whole genome sequencing of clinical specimens to inform Legionnaires’ disease investigations

The Role of Next-Generation Sequencing (NGS) in the Management of Tuberculosis: Practical Review for Implementation in Routine

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