How well do routine molecular diagnostics detect rifampicin heteroresistance in<i>Mycobacterium tuberculosis</i>?

Ng, Kamela Charmaine S.; Supply, Philip; Cobelens, Frank; Gaudin, Cyril; González-Martín, Juliàn; Jong, Bouke C. de; Rigouts, Leen

doi:10.1101/632851

Cited by 2 publications

(1 citation statement)

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“…In addition, the heteroresistance is another important cause of the discordant LFX susceptibility results (21). On one hand, the coexistence of wide-type and mutant tubercle bacilli may result in a shift toward lower MIC value compared with that of mutant bacilli (16).…”

Section: Discussionmentioning

confidence: 99%

SpecificgyrAgene mutations correlate with high prevalence of discordant levofloxacin resistance inMycobacterium tuberculosisisolates from Beijing, China

Huo

et al. 2019

Preprint

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17Although molecular diagnostics are highly sensitive in the diagnosis of fluroquinolone 18 (FQ)-resistant tuberculosis (TB), the discordant results with the sequential phenotypic 19 drug susceptibility testing (DST) invariably occur. To investigate the prevalence and 20 the cause of discordant results between molecular and phenotypic DST for 21 levofloxacin (LFX), the cases who were determined to be LFX-susceptible using the 22 2 phenotypic DST but LFX-resistant using the MeltPro assay were retrospectively 23 reviewed in Beijing, China. We measured LFX minimal inhibitory concentrations 24 (MICs) using Middlebrook 7H9 broth. Sanger sequencing was used to determine 25 genotypic characteristics of discordant isolates. Between January and December 2018, 26 126 (22.1%) out of 571 smear-positive TB patients were identified as LFX-resistant 27 TB by MeltPro assay. Among the 126 LFX-resistant TB, there were 34 isolates 28 identified as LFX-susceptible TB by phenotypical DST. This result demonstrated a 29 discordance prevalence of 27.0%. LFX MICs were majorly centered around the 30 critical concentration, and 7 (21.2%) and 13 (39.4%) had MICs of 2.0 mg/l and 4.0 31 mg/l, respectively. The most prevalent mutations conferring discordant LFX 32 resistance were the amino acid substitutions of Ser to Leu in 90 codon (13, 39.4%) 33 and Asp to Ala in 94 codon of gyrA (11, 33.3mg/l belonged to Ala90Val and 34 Asp94Ala group. In conclusion, our data demonstrate that more than one quarter of 35 LFX-resistant isolates by molecular method are susceptible to LFX using the 36 phenotypic DST. The high prevalence of discordant LFX resistance is majorly due to 37 the isolates with specific mutations within gyrA gene conferring proximity of MICs to 38 the critical concentration. 39 gyrA 41 42 126 The PCR mixture were prepared as follows: 25 μl 2×PCR Mixture, 2 μl of DNA 127 template and 0.2 μM of each primer set. After completion of the thermal cycler 128 program, the amplicons were sent to Tsingke Company (Beijing, China) for DNA 129 sequencing service. DNA sequences were compared with the homologous sequences 130 of MTB H37Rv strain to identify whether the sequences had genetic mutations within 131 QRDR. 132 7 133 RESULTS 134Between January and December 2018, 571 smear-positive TB patients were screened 135 by MeltPro assay (Fig. 2). Of these, 126 (22.1%) were identified as LFX-resistant TB. 136 Phenotypical DST showed that 92 isolates were LFX-resistant, while the remaining 137 34 were LFX-susceptible, demonstrating a discordance prevalence of 27.0%. After 138

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Section: Discussionmentioning

confidence: 99%