Pathogen Identification And Drug Sensitivity Analysis Of HIV-Associated NTM Disease In Western Yunnan Province

Objective: Clinical isolates of suspected HIV-associated nontuberculous mycobacteria(NTM)in western Yunnan Province were identified.We genotyped the obtained dominant bacterium mycobacterium avium(MAV)and the MAV collected and identified by our research group using MATR-VNTR method,and discussed the genotype characteristics and epidemic trend of the dominant bacteria.The drug sensitivity of MAV was tested by proportional method to explore the status of HIV-associated mycobacterium avium drug resistance and the correlation between gene polymorphism and drug resistance.Methods: 1.Identification of strains: Acid-fast staining was performed on NTM,and PNB and TCH identification medium were used for preliminary identification.DNA of the initially identified strains was extracted,and PCR amplification,electrophoresis and sequencing were performed with the gene primers 16 S r RNA,rpo B and hsp65,respectively.The sequenced gene sequences were input into the Gen Bank of NCBI for BLAST comparison,and their homology was analyzed to obtain the results of strain identification.2.Genotyping: MATR-VNTR genotyping was performed on the identified dominant strain MAV.12 MATR gene loci were selected and these loci were used as primers for PCR amplification and agarose gel electrophoresis.DNA amplification bands of MAV strain were obtained.According to the formula,the number of repetitions and the value of HGDI are calculated.The combined number of repeats was imported into Bio Numerics(6.6)software,and UPGM and MST functions were used to make cluster analysis tree and minimum spanning tree respectively.3.Drug sensitivity experiment:The drug sensitivity test of dominant strain MAV was carried out by proportional method.Ten drugs,amikacin(AMI),kanamycin(K),streptomycin(SM),capreomycin(CPM),linezolid(LINE),clarithromycin(CLAR),levofloxacin(LFX),rifambudin(RBU),isoniazid(INH)and ethambutol(EMB),were selected for drug sensitivity test.4.Statistical analysis: Statistical software SPSS 26.0 was used to analyze the correlation between MAV genotype and ethambutol sensitivity.Results: 1.Strain identification: Among the 50 strains of HIV-associated NTM in western Yunnan Province,44 strains of mycobacterium avium(88%),2 strains of mycobacterium gordon(4%),2 strains of mycobacterium tuberculosis(4%),1 strain of mycobacterium abscess(1%),and 1 strain of intracellular mycobacterium(2%)were identified.Mycobacterium avium was the most dominant strain in this study.2.Genotyping: After genotyping,10 MATR loci(MATR 1,2,3,4,5,6,7,9,13,15)had HGDI values greater than 0.5,indicating high diversity index.By UPGM and MST analysis,the 67 MAV were divided into 5 major gene groups(Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ)and61 independent strain patterns.There were clusters of strains,and there were 3 clusters of strains(in group Ⅰ and group Ⅳ).Each cluster contained 2 strains,among which 2clusters were from the same isolated place.The clustering rate was 8.96%(6/67),which was low,indicating that the MATR-VNTR genotyping method had a high resolution for MAV in western Yunnan Province.3.Drug sensitivity test: All 44 MAV were sensitive to rifambudin(resistance rate 0%,0/44),and all except 6 strains were resistant to ethambutol(resistance rate 86%,38/44).All strains were resistant to the remaining eight drugs.4.Statistical analysis: There was no significant correlation between MAV genotype and ethambutol sensitivity.Conclusions: 1.Mycobacterium avium was the dominant strain of NTM in western Yunnan Province.2.MATR-VNTR genotyping method is suitable for MAV genotyping in western Yunnan Province.The resolution of MATR gene loci is high,which indicates that MAV has gene polymorphism and regionalism.3.All the 44 MAV were sensitive to rifambudin,which could provide reference for clinical treatment.4.There was no significant correlation between MAV genotype and ethambutol sensitivity.