Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04

    • gamma secretase inhibitor The second part of this study eval

    2018-10-23

    The second part of this study evaluated the detection of NTM in clinical specimens by PCR in comparison to culture (Fig. 2). Here we used the Roche COBAS™ MTB assay supplemented with an additional pan Mycobacterium genus probe. The Mycobacterium genus probe is highly sensitive but compromised by limited specificity requiring subsequent confirmation testing, i.e., sequence analysis of the PCR amplicon. Due to its high sensitivity, the Mycobacterium genus probe readily detects contaminating DNA from environmental NTM present in laboratory chemicals and/or non-sterile specimens (especially sputum). The large number of NTM PCR positive specimens which were negative by culture (n=393, of which n=139 represent pathogenic NTM) is in part explained by the optimization of established cultural procedures towards recovery of MTB rather than NTM (Manual of Clinical Microbiology, 2015). As is true for any environmental or opportunistic pathogen the mere finding of an NTM does not establish disease. For NTM, pathogenicity is largely a species-specific trait (Tortoli, 2014). Many of the environmental contaminants are readily identified by gamma secretase inhibitor assignment as unlike pathogens, e.g. M. gordonae and most of the rapid growers (Table 1). Guidelines issued by ATS/IDSA have summarized the criteria which establish the clinical significance of an NTM (Griffith et al., 2007). These criteria include: (i) species identified, (ii) positive smear microscopy, (iii) detection in multiple patients\' specimens, and (iv) clinical history. Discordant results between culture and PCR results in our study were resolved by evaluation of the patients\' clinical history and the availability of additional patients\' specimens analyzed (Tables S6, S7 and S8 in Supplemental materials). The results show that diagnosis of NTM by culture would have missed 5 patients and that PCR would have missed 5 patients (Fig. 2), which was shown as a non-significant difference by statistical analysis. These results testify that PCR and culture exhibit comparable sensitivity in detecting clinically relevant NTM disease. The significant workload associated with the interpretation of positive genus PCR results – not the least DNA sequence analysis of the amplicon – is compensated by the faster time-to-result (approximately 2days including decontamination of the sample) in comparison with culture (1–3weeks, especially for slow growing NTM) and by the prospects to possibly replace resource-costly cultural detection methods in diagnostic mycobacteriology by molecular screening. In the third part of this study we evaluated the performance of the AID TB resistance line probe assay (LPA) to rapidly identify drug susceptible MTB in clinical samples that were MTB PCR positive. Our results show an excellent performance of the AID TB resistance line probe assay for smear positive samples (respiratory and non-respiratory specimens, Tables 2 and 3). Inherently, the line probe assay as a multiplex PCR is less sensitive than the COBAS™ TaqMan™ MTB PCR. Consequently, of the 438 specimens positive in the MTB PCR assay, only 311 (71.0%) gave an interpretable LPA result. Increasing the number of MTB PCR positive samples subjected to LPA analysis increases the likelihood of an interpretable result. This is possible as the specificity of the LPA is exceptionally high (>99%). The results of molecular DST of clinical specimens and phenotypic DST on isolates recovered by culture showed an excellent concordance of 98.5%. Overall the data indicate that treatment of drug-susceptible TB can be initiated based on genotypic resistance results. We observed a number of minor discrepancies when comparing molecular with phenotypic DST (Table 4; Table S10 in Supplemental materials). 2 isolates showed an isolated low-level INH resistance and no mutation in inhA or katG – neither by LPA nor by gene sequencing. One isolate had a wild-type rpoB LPA result but was resistant to rifampicin at 1.0mg/l, the corresponding deletion of 9bps in rpoB was not detected by the LPA. Given the current limitations of molecular DST and the in part controversial discussions on genotype – phenotype relationships, resistant LPA results should be confirmed phenotypically to identify possible discrepancies, e.g. some mutations are missed in MGIT critical concentration testing, nonsynonymous mutation in LPA targets may result in a false resistance result (Van Deun et al., 2009; Plinke et al., 2011; Sirgel et al., 2012a; Sirgel et al., 2012b; Sirgel et al., 2013; Böttger, 2011; Kaswa et al., 2014). We deliberately decided to use screening for mutations in inhA, katG, and rpoB by LPA as a surrogate marker of any type of drug resistance. Any isolate with a mutation found in inhA, katG, or rpoB is categorized as drug resistant and is suspect of MDR/XDR until ruled out. Based on this interpretation of LPA results, LPA readily separated drug-susceptible wild-type MTB from drug-resistant MTB isolates.