| Objectives Our research conducted on-site surveys and employed Whole Genome Sequencing(WGS)technology,which revealed mutation sites of the target genes(gyr A and gyr B)and the allelic gene mutation frequency of Fluoroquinolones(FQs)resistant Mycobacterium tuberculosis(MTB)clinical isolates.An analysis was performed to assess the efficacy of resistance mutation sites and the cumulative allele mutation frequency burden in diagnosing FQs-resistant MTB isolates.This study ultimately seeks to provide additional data support and a theoretical basis for the diagnosis of FQs-resistant MTB.Methods 1 In the Meta-analysis study,literature from January 1,2010 to December 31,2021,pertaining to the target gene mutation diagnosis of FQs-resistant MTB was identified on CNKI,Wanfang Medical,VIP Medical,China Bio Med,Pub Med,and Ovid databases using a combined search approach of subject words and free words.Relevant data from these sources were extracted and subsequently analyzed using statistical software,including Stata 12.0,Meta Di Sc 1.4,and Revman 5.3.2 To evaluate the efficacy of target gene mutation in diagnosing FQs-resistant MTB,a case-control study was carried out,involving an epidemiological survey on patients with pulmonary tuberculosis receiving inpatient treatment at the Fifth Hospital of Shijiazhuang City from June 1,2020,to August31,2021.Following minimum inhibitory concentration(MIC)testing,56 and 83 strains were included in the FQs-resistant and control groups,respectively.WGS analysis on 139clinical isolates was performed,and mutation sites linked with resistance were analyzed using the World Health Organization(WHO)drug resistance database.In addition,we executed an in vitro drug resistance test for a newly identified locus in the Quinolone Resistance Determining Regions(QRDR).An analysis followed this,evaluating the efficacy of diagnosing FQs resistance using target gene mutation(mutation sites and allele gene mutation frequency),and the stability of the diagnostic threshold of cumulative allele gene mutation frequency burden.All gathered data were saved in Excel for an established database.SPSS 23.0 was used for analyzing FQs-resistant MTB-related factors while R software plotted the ROC curve and enabled efficiency comparisons.Arbitrary criteria were evaluated using the area under the ROC curve(AUC),compared using De Long’s test analysis,and the calibration accuracy of diagnostic indicators was assessed using the Net Reclassification Index(NRI).All tests were bilateral and had a test level ofα=0.05.Results The findings of this study are divided into four parts:To start,the meta-analysis study included data from 32 articles,constituting 38 research datasets.The combined sensitivity of the WGS and the currently recommended Genotype MTBDRsl VER 2.0method by the WHO for detecting target gene mutations during diagnosis of FQs-resistant MTB both exceeded 90%.Nevertheless,a comparative analysis using the Meta regression showed no statistically significant difference,with the combined Relative Diagnostic Odds Ratio(RDOR)and its 95%CI established at 4.45(0.60～33.09).Secondly,among the 139clinical isolates,ten FQs-resistant related amino acid mutations are identified.These include G88C,G88A,A90V,S91P,D94H,D94Y,D94A,and D94G amino acid mutations in the gyr A gene and S447F and N499D amino acid mutations in the gyr B gene.After the exclusion of 46 isolates carrying resistance mutation sites,there remained a distribution difference in the QRDR mutation frequency burden between the FQs resistance group and sensitive group within the 93 clinical isolates(P=0.008).In the third part of the study,we validated the relationship between the newly discovered amino acid mutations and fluoroquinolones(FQs)resistance in M.smegmatis.In comparison to the wild type p MV361-gyr B recombinant strain,the MIC value of Lfx for the p MV361-gyr B-G520D and p MV361-gyr B-G520T recombinant strain increased from 0.13μg/m L to 0.25μg/m L.Similarly,the MIC value of Mfx increased from 0.06μg/m L to 0.13μg/m L.These findings confirm that the G520D and G520T amino acid mutations in the gyr B gene are associated with resistance to both Lfx and Mfx.Fourthly,the inclusion of G520D and G520T amino acid mutations into the current FQs resistance-related mutations revealed a high diagnostic efficiency for FQs,Lfx,and Mfx resistance in clinical isolates,particularly in terms of resistance mutation sites and the QRDR mutation frequency burden.Additionally,further analysis showed that the AUC for diagnosing Lfx resistance using the QRDR mutation frequency burden(0.97)was significantly higher than that of the resistance mutation sites(0.93)with a statistically significant difference(P=0.03).Moreover,when compared to the resistance mutation sites,the correct classification ability of Lfx-resistant MTB clinical isolates improved by 3.60%(NRI=3.60%,P<0.001)when the QRDR mutation frequency burden threshold was set at 1.31.However,in diagnosing Mfx resistance,there was no statistical difference in the AUC between the resistance mutation sites(0.91)and the QRDR mutation frequency burden(0.94)(P=0.25).The stability of the QRDR mutation frequency burden threshold was validated using different MDR and CD4~+/CD8~+groupings for internal validation,as well as 22 strains of MTB clinical isolates from outpatients with pulmonary tuberculosis for external validation.The results showed that regardless of whether the QRDR mutation frequency burden was treated as a continuous variable or a binary variable with a threshold of 1.31,no statistical differences were found in the AUC for diagnosing resistance to FQs,Lfx,or Mfx in either the internal or external validation tests(all P>0.05).Conclusions Amino acid mutations G520D and G520T in the gyr B gene are associated with FQs resistance.The diagnostic efficiency of QRDR mutation frequency burden for FQs resistance may be higher than the current drug resistance mutation sites,especially for diagnosing Lfx-resistant MTB strains.Figure 28;Table 21;Reference 219... |
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