Next-generation sequencing to characterise pyrazinamide resistance in Mycobacterium tuberculosis isolates from two Balkan countries

Sodja, Eva; Koren, Simon; Toplak, Nataša; Truden, Sara; Žolnir-Dovč, Manca

doi:10.1016/j.jgar.2021.09.019

Cited by 10 publications

(4 citation statements)

References 30 publications

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“…The investigators deduced that the effect of the drug resistance pattern on conversion of sputum culture is most evident at eight weeks, and less evident at four and 12 weeks of treatment. 18 A related study conducted in 2016 by Zheng et al also further indicated that the presence of a pncA gene mutation, which confers drug resistance to pyrazinamide, [19][20][21] remained and significantly decreased both the rate of culture conversion at eight weeks and treatment success. Additional drug resistance to fluoroquinolones was also found to significantly reduce the likelihood of treatment success among MDR-TB patients in China.…”

Section: Discussionmentioning

confidence: 94%

Second-line drug resistance markers as proxy indicators of time to sputum culture conversion among second-line drug resistant tuberculosis patients tested in Uganda: A cross-sectional study

et al. 2023

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Background Increased tuberculosis disease burden arises as a result of low treatment success rates stemming from the emergence of second-line drug resistance. We aimed at determining the usefulness of second-line drug (SLD) resistance markers as proxy indicators of time to sputum culture conversion; a renowned predictor of Tuberculosis treatment outcome, among SLD-resistant tuberculosis (TB) patients tested at the Uganda National TB Reference Laboratory (NTRL). Methods A cross-sectional study was conducted on 72 bacteriologically confirmed SLD resistant TB patients with datasets including culture conversion time and second line probe assay mutation profiles between 01/06/2017 and 31/12/2019. The data were then imported into STATA v15 for descriptive statistical analysis, Univariate cox proportional hazard model analysis and Kaplan-Meier survival curves at a 5% level of significance; p-value ≤0.05. Results Results indicate the median time was achieved at 3 (0–12) months across the studied patients. The rrs G1484T mutation associated with conferring drug resistance to injectable agents was observed to have the shortest median conversion time of 1.5 months, longest by the gryB E540D at 5 months. A single mutation in the gryA gene locus showed higher converted proportions 70.8% (58.9–81.0) than those that had two 8.3% (3.1–17.3) or three 2.7% (0.3–10.0) mutations. Conclusions The studied second-line drug resistance markers had no statistically significant association with the time to sputum culture conversion, although increased drug resistance levels reduced the converted proportions and stressed the need to utilize molecular diagnostics data and other crucial variables to better comprehend proxy indicators of SLD resistant tuberculosis management.

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Section: Discussionmentioning

confidence: 94%

Second-line drug resistance markers as proxy indicators of time to sputum culture conversion among second-line drug resistant tuberculosis patients tested in Uganda: A cross-sectional study

et al. 2023

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“…The number of those is slowly rising. In 2015, the number of NTM isolates have outnumbered for the rst time the Mycobacterium tuberculosis isolates among Slovenian patients [28,29]. To our knowledge, this is the rst study reporting cervicofacial NTM lymphadenitis in children in Balkan Peninsula.…”

Section: Discussionmentioning

confidence: 99%

Paediatric cervicofacial lymphadenitis caused by non-tuberculous mycobacteria: nation- wide overview in the period 2000-2020

Sodja,

Šivic,

Truden

et al. 2024

Preprint

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Purpose Cervicofacial lymphadenitis caused by non-tuberculous mycobacteria (NTM) is a rare but increasing infection in children worldwide. The purpose of this study was to analyse and evaluate all microbiologically proven cases of NTM lymphadenitis in children under the age 14 years and to discuss possible reasons why there is no Mycobacterium haemophilum isolates among cases of paediatric NTM lymphadenitis in Slovenia. Methods Between 2000 and 2020, we retrospectively reviewed laboratory and medical records for some basic demographic and microbiological data. Different clinical samples were collected in medical centres and regional hospitals from all over Slovenia. Results In the period before mandatory BCG vaccination was discontinued (2000–2005), we did not observe any case of paediatric NTM lymphadenitis. After discontinuation of non-selective BCG vaccination of new-borns (2006–2020), we identified 55 cases of microbiologically confirmed NTM lymphadenitis in BCG-unvaccinated children with median age 26.0 months (range: 15.0–75.0 months). Mean annual incidence of paediatric NTM lymphadenitis accounted for 1.26 (range: 0.35–2.38) per 100,000 children. The main causative agents were Mycobacterium avium (38/55; 69.1%) and M. intracellulare (9/55; 16.4%). We did not find any M. haemophilum isolate. Since 2006, each year we microbiologically confirm sporadic cases of paediatric NTM lymphadenitis, a condition not diagnosed before. Conclusions Strong evidence suggests that the main reason for that is discontinuation of universal BCG vaccination in March 2005. Nevertheless, we could not neglect the effect of improved diagnostic procedures used in recognizing NTM as causative agents of paediatric lymphadenitis.

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“…Genotypic drug susceptibility testing (gDST) methods, such as Sanger sequencing and whole-genome sequencing, are employed to identify specific genetic mutations in the pncA gene linked to PZA resistance (PZAr) [14], [15]. These methods can also detect mutations in other genes, including rpsA , panD , and clpC1 [16]–[19].…”

Section: Introductionmentioning

confidence: 99%

Quantitative MODS-Wayne Assay for Rapid Detection of Pyrazinamide Resistance inMycobacterium tuberculosisfrom Sputum Samples

Toscano-Guerra,

Alcántara,

Gilman

et al. 2024

Preprint

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BackgroundTuberculosis (TB) remains a significant global health challenge, particularly with the rise of drug-resistant strains, such as Pyrazinamide (PZA)-resistant TB. This resistance hampers treatment effectiveness. Currently, there’s a lack of affordable and precise quantitative tests for detecting PZA resistance, highlighting the need for accessible diagnostic tools. Our study introduces a direct, accurate, and accessible susceptibility test for PZA resistance by quantifying pirazinoic acid (POA).MethodsWe analyzed 264 TB-positive samples (MP and TBN) to assess PZA susceptibility by measuring POA production. This was done using the MODS-Wayne qualitative assay and our developed quantitative approach (MODS-WQ). For MODS-WQ, TBN samples were processed in 7H9 media (MODS-WQ 7H9) and MP samples in citrate buffer (MODS-WQ CB). We measured POA levels using spectrophotometry against a calibration curve, with PZA susceptibility determined by a composite standard. We also investigated the correlation between POA levels and pyrazinamidase mutations. Multidrug-resistant samples were assessed using the MODS test, and statistical analyses were conducted.ResultsApproximately 23.48% of the samples showed PZA resistance, which included 62.8% of the multidrug-resistant (MDR) samples. The MODS-Wayne assay demonstrated an 87.8% sensitivity and 95.9% specificity for MP samples, and 62.5% sensitivity with 93.7% specificity for TBN samples. The MODS-WQ identified specific cut-off points—123.25 μM for MODS-WQ 7H9 and 664.7 μM for MODS-WQ CB—with corresponding sensitivities of 81.25% and 92.31%, and specificities of 77.22% and 95.93%. Significantly lower POA levels were observed in samples with mutations in the metal binding site compared to mutations on the enzyme’s periphery.ConclusionsThe citrate-buffered MODS-WQ demonstrates high sensitivity and specificity in quantifying POA. It offers a significant improvement over qualitative methods, effectively overcoming their subjectivity limitations in PZA resistance detection.

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Next-generation sequencing to characterise pyrazinamide resistance in Mycobacterium tuberculosis isolates from two Balkan countries

Cited by 10 publications

References 30 publications

Second-line drug resistance markers as proxy indicators of time to sputum culture conversion among second-line drug resistant tuberculosis patients tested in Uganda: A cross-sectional study

Second-line drug resistance markers as proxy indicators of time to sputum culture conversion among second-line drug resistant tuberculosis patients tested in Uganda: A cross-sectional study

Paediatric cervicofacial lymphadenitis caused by non-tuberculous mycobacteria: nation- wide overview in the period 2000-2020

Quantitative MODS-Wayne Assay for Rapid Detection of Pyrazinamide Resistance inMycobacterium tuberculosisfrom Sputum Samples

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