IS6110 the Double-Edged Passenger

Menéndez, María del Carmen; Samper, Sofía; Otal, Isabel; García, María Jesús García

doi:10.5772/32046

Cited by 7 publications

(4 citation statements)

References 101 publications

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“…We anticipate that our assay can be used to continue to study TB urine cfDNA trends across subgroups and answer important unresolved questions regarding optimal sample collection techniques, processing methods, and storage conditions that have been the focus of recent work ( 38 – 40 ). As a caveat, the cfDNA concentrations measured by our assay may be confounded by the variable copy number of IS 6110 (0 to 25 copies) ( 25 ) and by differences in participants’ hydration status. In the future, strain typing and normalizing cfDNA concentration to urine creatinine may help better elucidate trends in cfDNA concentration.…”

Section: Discussionmentioning

confidence: 99%

“…We designed PCR primers targeting the insertion sequence IS 6110 , which is an established target for TB diagnosis present at multiple, variable copy number across ∼99% of Mycobacterium tuberculosis complex strains ( 25 ). Our PCR primers ( Table 1 ) amplify a short 40-bp target within a subregion of IS 6110 that is conserved and specific to the Mycobacterium tuberculosis (MTB) complex ( 26 ).…”

Section: Methodsmentioning

confidence: 99%

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Diagnosing Pulmonary Tuberculosis by Using Sequence-Specific Purification of Urine Cell-Free DNA

et al. 2021

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Transrenal urine cell-free DNA (cfDNA) is a promising tuberculosis (TB) biomarker, but is challenging to detect because of the short length (<100 bp) and low concentration of TB-specific fragments. We aimed to improve the diagnostic sensitivity of TB urine cfDNA by increasing recovery of short fragments during sample preparation. We developed a highly sensitive sequence-specific purification method that uses hybridization probes immobilized on magnetic beads to capture short TB cfDNA (50 bp) with 91.8% average efficiency. Combined with short-target PCR, the assay limit of detection was ≤5 copies of cfDNA in 10 mL urine. In a clinical cohort study in South Africa, our urine cfDNA assay had 83.7% sensitivity (95% CI: 71.0–91.5%) and 100% specificity (95% CI: 86.2–100%) for diagnosis of active pulmonary TB when using sputum Xpert MTB/RIF as the reference standard. The detected cfDNA concentration was 0.14–2804 copies/mL (median 14.6 copies/mL) and was inversely correlated with CD4 count and days to culture positivity. Sensitivity was non-significantly higher in HIV-positive (88.2%) compared to HIV-negative patients (73.3%), and was not dependent on CD4 count. Sensitivity remained high in sputum smear-negative (76.0%) and urine LAM-negative (76.5%) patients. With improved sample preparation, urine cfDNA is a viable biomarker for TB diagnosis. Our assay has the highest reported accuracy of any TB urine cfDNA test to date and has the potential to enable rapid non-sputum-based TB diagnosis across key underserved patient populations.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Diagnosing Pulmonary Tuberculosis by Using Sequence-Specific Purification of Urine Cell-Free DNA

et al. 2021

View full text Add to dashboard Cite

show abstract

“…We anticipate that our assay can be used to continue to study TB urine cfDNA trends across sub-groups and answer important unresolved questions regarding optimal sample collection techniques, processing methods, and storage conditions that have been the focus of recent work (40–42). As a caveat, the cfDNA concentrations measured by our assay may be confounded by the variable copy number of IS 6110 (0–25 copies) (25) and by differences in participants’ hydration statuses. In the future, strain typing and normalizing cfDNA concentration to urine creatinine may help better elucidate trends in cfDNA concentration.…”

Section: Discussionmentioning

confidence: 99%

“…We designed PCR primers targeting the insertion sequence IS 6110 , which is an established target for TB diagnosis present at multiple, variable copy number across ∼99% of Mycobacterium tuberculosis complex strains (25). Our PCR primers (Table 1) amplify a short 40 bp target within a subregion of IS 6110 that is conserved and specific to the Mycobacterium tuberculosis (MTB) complex (26).…”

Section: Methodsmentioning

confidence: 99%

Diagnosing pulmonary tuberculosis using sequence-specific purification of urine cell-free DNA

Oreskovic¹,

Panpradist²,

Marangu³

et al. 2021

Preprint

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Transrenal urine cell-free DNA (cfDNA) is a promising tuberculosis (TB) biomarker, but is challenging to detect because of the short length (<100 bp) and low concentration of TB-specific fragments. We aimed to improve the diagnostic sensitivity of TB urine cfDNA by increasing recovery of short fragments during sample preparation. We developed a highly sensitive sequence-specific purification method that uses hybridization probes immobilized on magnetic beads to capture short TB cfDNA (50 bp) with 91.8% average efficiency. Combined with short-target PCR, the assay limit of detection was ≤5 copies of cfDNA in 10 mL urine. In a clinical cohort study in South Africa, our urine cfDNA assay had 83.7% sensitivity (95% CI: 71.0– 91.5%) and 100% specificity (95% CI: 86.2–100%) for diagnosis of active pulmonary TB when using sputum Xpert MTB/RIF as the reference standard. The detected cfDNA concentration was 0.14–2804 copies/mL (median 14.6 copies/mL) and was inversely correlated with CD4 count and days to culture positivity. Sensitivity was non-significantly higher in HIV-positive (88.2%) compared to HIV-negative patients (73.3%), and was not dependent on CD4 count. Sensitivity remained high in sputum smear-negative (76.0%) and urine LAM-negative (76.5%) patients. With improved sample preparation, urine cfDNA is a viable biomarker for TB diagnosis. Our assay has the highest reported accuracy of any TB urine cfDNA test to date and has the potential to enable rapid non-sputum-based TB diagnosis across key underserved patient populations.

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Preparation and Preclinical Evaluation of Inhalable Particles Containing Rapamycin and Anti-Tuberculosis Agents for Induction of Autophagy

et al. 2016

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IS6110 the Double-Edged Passenger

Cited by 7 publications

References 101 publications

Diagnosing Pulmonary Tuberculosis by Using Sequence-Specific Purification of Urine Cell-Free DNA

Diagnosing Pulmonary Tuberculosis by Using Sequence-Specific Purification of Urine Cell-Free DNA

Diagnosing pulmonary tuberculosis using sequence-specific purification of urine cell-free DNA

Preparation and Preclinical Evaluation of Inhalable Particles Containing Rapamycin and Anti-Tuberculosis Agents for Induction of Autophagy

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