| Objectives1.To investigate the resistance to second-line anti-TB drugs among multidrug-resistant tuberculosis(MDR-TB)patients in Chongqing,so as to provide a reference and basis for the development of rational MDR-TB chemotherapy regimen and the suitable MDR-TB control strategy in Chongqing.2.To analyze the characteristics of genes’ mutations including gyr A,gyr B,rrs and eis genes among MDR-TB isolates from Chongqing and their associations with the resistance to the second-line anti-tuberculosis drugs.Therefore,to provide the scientific evidences for exploring the molecular mechanism of second-line drugs resistance and establishment of rapid molecular diagnosis methods for detection second-line drugs resistance in this area.Methods1.698 smear-positive tuberculosis patients were registered,whichwere collected in local TB dispensaries from 39 districts of Chongqing from January 2015 to August 2016.The specimens were cultured in P-nitrobenzoic acid(PNB)/2-Thiophene carborylic acid hydrazide(TCH)medium.Drug susceptibility testing was performed to select the multidrug-resistant tuberculosis isolates and obtain the drug susceptibility results against the second-line anti-tuberculosis drugs.2.Gene sequencing after DNA extraction and amplification from the strains to detect the gene mutation.The gyr A,gyr B,rrs and eis genes from168 MDR Mycobacterium tuberculosis clinical isolates were analyzed respectively to acquaint with mutation characteristics of Ofx/Km resistance genes from Chongqing.Results1.There were 168 MDR-TB patients,among which 83 cases(49.4%,83/168)were sensitive to all 6 second-line anti-TB drugs while 85 cases(50.6%,85/168)were resistant to at least one of the 6 anti-TB drugs.Among 168 MDR-TB patients,the proportion of resistance to Ofx was highest,accounting for 41.7%(70/168),and the proportions of resistance to other drugs were as follows: Km(16.7%,28/168),PAS(14.3%,24/168),Am(13.1%,22/168),Cm(8.3%,14/168)and Pto(7.7%,13/168).The overall proportion of extensively drug-resistant tuberculosis(XDR-TB)and pre-extensively drug-resistant tuberculosis(pre-XDR-TB)cases was 45.8%(77/168),and the proportion of XDR-TB cases and pre-XDR-TB cases was13.1%(22/168)and 32.7%(55/168)respectively.Among the different registration category MDR-TB cases,the overall proportion of XDR-TB and pre-XDR-TB was higher(60.9%,14/23)in retreatment failure patients.2.Among 168 MDR-TB isolates,70 cases were resistant to Ofx.These cases contained 57 gyr A mutation isolates(81.4%,57/70),and the all mutations distributed at codon 74,88,89,90,91,and 94 respectively.The highest mutation frequency was at codon 94,the rate was 52.9%(37/70).Besides,5 of the 70 ofloxacin-resistant isolates(7.1%)carried double point mutations.The mutation type of gyr A gene were mainly Asp94Gly(n=17),Ala90Val(n=14)and Asp94Ala(n=10).Mutations within gyr B were observed in 5 ofloxacin-resistant isolates(5/70,6.1%),with Asp461Asn(n=1),and Gly512Arg(n=3).3.Among all 168 MDR-TB isolates,28 isolates were resistant to KAN,and 19(67.9%,19/28)of these contained mutations within the rrs or and 4(14.3%,4/28)within eis region of interest respectively.The most frequent rrs region mutation was A1401 G,which was observed in 18isolates(64.3%).The other mutation within rrs region was G1339A(n=1).Mutations within the promoter region of eis included the G(-10)A(n=3)and C(-14)T(n=1).4.When setting the traditional proportion method as a gold standard,the method by using gyr A as the Ofx-resistant marker showed a high sensitivity(81.4%),specificity(92.9%),positive predictive value(PPV)(89.1%)and negative predictive value(NPV)(87.5%).The sensitivity,specificity,PPV and NPV of gyr B for Ofx resistance were 7.1%,94.9%,50.0% and 58.9% respectively.By using rrs as the Km-resistant marker,the sensitivity,specificity,PPV and NPV of rrs for Km resistance were 67.9%,99.3%,95.0% and 93.9% respectively.The specificity,PPV and NPV of eis for detecting drug-resistance of Km were 99.3%,80.0% and 85.3%respectively,but the sensitivity was only 14.3%.Conclusions1.Among MDR-TB patients in Chongqing,the situation of second-line anti-TB drugs resistance especially to Ofx is serious.The proportions of XDR-TB and pre-XDR-TB among MDR-TB patients are high in Chongqing.Drug-resistance monitoring among MDR-TB patients should be strengthened and MDR-TB detection and treatment should be improved,so as to prevent the development of pre-XDR-TB or even XDR-TB from MDR-TB.2.The gyr A gene mutation is the major mechanism of Ofx resistance,and the mainly mutations distribute at codon 90,91,94.The gyr A gene could be a reliable molecular marker for Ofx resistance detection,such method shows a fair clinic application value.While there was no significant correlation between gyr B gene mutations and Ofx resistance,and gyr B is not a good molecular marker for Ofx resistance diagnosis.3.An A1401 G mutation in the rrs gene is the main mechanism of Kmresistance,besides,mutations in the eis promoter region also associate with resistance to Km.It is feasible that rrs gene is used for a rapid screening test to Km resistance in Chongqing.The eis promoter appears to be a very promising marker,and the combination testing of rrs gene and eis promoter will be in favor of improving both sensitivity and accuracy of detection of Km resistance. |
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