Development of a Multiplex Real-Time PCR Assay for Mycobacterium bovis BCG and Validation in a Clinical Laboratory

Duffy, Shannon C.; Venkatesan, M.; Chothe, Shubhada K.; Poojary, Indira; Verghese, Valsan Philip; Kapur, Vivek; Behr, Marcel A.; Michael, Joy Sarojini

doi:10.1128/spectrum.01098-21

Cited by 7 publications

(3 citation statements)

References 21 publications

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“…As a result, samples D and E were correctly called as XDR whereas sample F was called ‘MDR’ due to the identification of the RpoB S450L, as well as a BCG-associated mutation in MmaA3 that has been linked to low-level INH resistance. 19 …”

Section: Resultsmentioning

confidence: 99%

Challenging the gold standard: the limitations of molecular assays for detection ofMycobacterium tuberculosisheteroresistance

Danchuk,

Solomon,

Kohl

et al. 2024

Thorax

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ObjectivesHeteroresistant infections are defined as infections in which a mixture of drug-resistant and drug-susceptible populations are present. InMycobacterium tuberculosis(M. tb), heteroresistance poses a challenge in diagnosis and has been linked with poor treatment outcomes. We compared the analytical sensitivity of molecular methods, such as GeneXpert and whole genome sequencing (WGS) in detecting heteroresistance when compared with the ‘gold standard’ phenotypic assay: the agar proportion method (APM).MethodsUsing two rounds of proficiency surveys with defined monoresistant BCG strains and mixtures of susceptible/resistantM. tb, we determined the limit of detection (LOD) of known resistance associated mutations.ResultsThe LOD for rifampin-R (RIF-R) detection was 1% using APM, 60% using GeneXpert MTB/RIF, 10% using GeneXpert MTB/RIF Ultra and 10% using WGS. While WGS could detect mutations beyond those associated with RIF resistance, the LOD for these other mutations was also 10%. Additionally, we observed instances where laboratories did not report resistance in the majority population, yet the mutations were present in the raw sequence data.ConclusionThe gold standard APM detects minority resistant populations at a lower proportion than molecular tests.Mycobacterium bovisBCG strains with defined resistance and extracted DNA fromM. tbprovided concordant results and can serve in quality control of laboratories offering molecular testing for resistance. Further research is required to determine whether the higher LOD of molecular tests is associated with negative treatment outcomes.

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Section: Resultsmentioning

confidence: 99%

Challenging the gold standard: the limitations of molecular assays for detection ofMycobacterium tuberculosisheteroresistance

Danchuk,

Solomon,

Kohl

et al. 2024

Thorax

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“…Quantification of miRNA-137 in the hippocampus was determined using the relative gene expression technique of qRT-PCR, which used actin, a housekeeping gene, as an internal reference and the 2Ct method to compute fold changes in the target gene from normal rats as a baseline. 74 , 75 Following the manufacturer’s instructions, the total RNA purification kit extracted the pure RNA from the separated hippocampus after homogenization with a lysis solution. It was done by loading the RNA onto the purification column, which was then eluted with pure RNA under low-ionic strength conditions.…”

Section: Methodsmentioning

confidence: 99%

Brain Targeting of Citicoline Sodium via Hyaluronic Acid-Decorated Novel Nano-Transbilosomes for Mitigation of Alzheimer’s Disease in a Rat Model: Formulation, Optimization, in vitro and in vivo Assessment

AbouElhassan¹,

Sarhan²,

Hussein³

et al. 2022

IJN

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Background Alzheimer’s disease (AD) is one of the furthermost advanced neurodegenerative disorders resulting in cognitive and behavioral impairment. Citicoline sodium (CIT) boosts the brain’s secretion of acetylcholine, which aids in membrane regeneration and repair. However, it suffers from poor blood–brain barrier (BBB) permeation, which results in lower levels of CIT in the brain. Purpose This study targeted to encapsulate CIT into novel nano-platform transbilosomes decorated with hyaluronic acid CIT-HA*TBLs to achieve enhanced drug delivery from the nose to the brain. Methods A method of thin-film hydration was utilized to prepare different formulae of CIT-TBLs using the Box–Behnken design. The optimized formula was then hyuloranated via integration of HA to form the CIT-HA*TBLs formula. Furthermore, AD induction was performed by aluminum chloride (Alcl 3 ), animals were allocated, and brain hippocampus tissue was isolated for ELISA and qRT-PCR analysis of malondialdehyde (MDA), nuclear factor kappa B (NF-kB), and microRNA-137 (miR-137) coupled with immunohistochemical amyloid-beta (Aβ 1– 42) expression and histopathological finding. Results The hyuloranated CIT-HA*TBLs formula, which contained the following ingredients: PL (300 mg), Sp 60 (43.97 mg), and SDC (20 mg). They produced spherical droplets at the nanoscale (178.94 ±12.4 nm), had a high entrapment efficiency with 74.92± 5.54%, had a sustained release profile of CIT with 81.27 ±3.8% release, and had ex vivo permeation of CIT with 512.43±19.58 μg/cm 2 . In vivo tests showed that CIT-HA*TBL thermogel dramatically reduces the hippocampus expression of miR-137 and (Aβ 1– 42) expression, boosting cholinergic neurotransmission and decreasing MDA and NF-kB production. Furthermore, CIT-HA*TBLs thermogel mitigate histopathological damage in compared to the other groups. Conclusion Succinctly, the innovative loading of CIT-HA*TBLs thermogel is a prospectively invaluable intranasal drug delivery system that can raise the efficacy of CIT in Alzheimer’s management.

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“…Mycobacterial culture from the skin papule and gastric aspirate detected rifampicin-susceptible Mycobacterium tuberculosis complex (MTBC), which was identified as M. bovis BCG strain using real-time polymerase chain reaction (PCR). [ 1 ]…”

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confidence: 99%

Disseminated Bacillus Calmette-Guerin Infection: The Harbinger of Severe Combined Immunodeficiency Disease

Shajil,

Sathishkumar,

Jyothirmayi

et al. 2024

Indian Journal of Dermatology

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Development of a Multiplex Real-Time PCR Assay for Mycobacterium bovis BCG and Validation in a Clinical Laboratory

Cited by 7 publications

References 21 publications

Challenging the gold standard: the limitations of molecular assays for detection ofMycobacterium tuberculosisheteroresistance

Challenging the gold standard: the limitations of molecular assays for detection ofMycobacterium tuberculosisheteroresistance

Brain Targeting of Citicoline Sodium via Hyaluronic Acid-Decorated Novel Nano-Transbilosomes for Mitigation of Alzheimer’s Disease in a Rat Model: Formulation, Optimization, in vitro and in vivo Assessment

Disseminated Bacillus Calmette-Guerin Infection: The Harbinger of Severe Combined Immunodeficiency Disease

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