Study On The Etiology And Biological Characteristics Of HIV-related NTM Diseases In Some Areas Of Yunnan Province

Objective: A total of 64 samples of suspected nontuberculous mycobacteria(NTM)clinical dual infection(HIV combined with NTM)were collected.They were from Baoshan City,Dehong Dai and Jingpo Autonomous Prefecture,and Dali Bai Autonomous Prefecture.Through a series of experiments on clinical dual-sensitivity strains,including physiological,biochemical and genetic sequencing identification,MIRU-VNTR genotyping and polymorphism analysis,drug sensitivity experiments,etc.,to understand the distribution of non-tuberculous mycobacteria in some areas of Yunnan Province,Identification of dominant strains and drug resistance,provide reference and help for the research and clinical prevention,diagnosis and treatment of traceable pathogenic bacteria.Methods:1.First,the samples were inoculated on two differentiating media of p-nitrobenzoic acid(PNB)and thiophene 2-carboxylic acid hydrazine(TCH),and then the colony results were observed,and the clinical isolates were initially identified.At the same time,acid-fast staining was performed to observe the stain-positive strains.Then,three primers,16 S r RNA,hsp65,and rpo B,were selected to perform polymerase chain reaction(PCR)amplification on the sample after the initial screening,and then subjected to agarose gel electrophoresis,laying a good foundation for further strain identification.After cutting the glue under the ultraviolet light,the pure purposeized strips are stored for later use.Finally,the sequenced base sequence is placed in NCBI BLAST for sequence comparison,and the result is checked.2.Through the above experimental results,it is found that the slow-growing bacteria Mycobacterium avium(MAV)in this batch of non-tuberculous mycobacteria is absolutely dominant.Therefore,the MIRU-VNTR method was used for genotyping research on Mycobacterium avium,15 effective sites were selected,PCR and agarose gel electrophoresis were used to obtain bands,and gradient PCR was required to explore the annealing temperature of some sites.Use Quantity One software to calculate the tape size of the entry,and use the formula to calculate the number of repetitions to save the data for later use.Import the above data and strains separately into the Bionumerics 6.6 software,use the UPGM function to make a cluster analysis graph and the MAT function to generate a minimal dendrogram.Finally,by calculating the resolution of each site,the site with the highest resolution can be obtained,and the site with the lowest resolution can be eliminated.3.Use streptomycin(SM),isoniazid(INH),ethambutol(EMB),kanamycin(K),rifabutin(RBU)for drug sensitivity analysis of Mycobacterium avium.After using the proportional method to gradually dilute the bacterial solution,select the appropriate high dilution and low dilution bacterial suspension to inoculate the above 5 kinds of drug sensitive medium and the control medium respectively.Place the incubator at 36°C±1°C for 4 weeks and read the results.Result:1.The samples collected in Baoshan City,Dehong Dai Jingpo Autonomous Prefecture,and Dali Bai Autonomous Prefecture were identified and found that 16 strains of Mycobacterium avium,11 strains of Mycobacterium.intracellulare,7 strains of Gordonia,and Mycobacterium maraseillense 4 strains,4 strains of Mycobacterium triplex,3 strains of Nocardia farcinica,2 strains of Mycobacterium tuberculosis,1 strain of Mycobacterium chelonae,1 strain of Mycobacterium abscessus,1 strain of Mycobacterium paraintracellulare.Mycobacterium avium has the largest number.2.The Mycobacterium avium identified in this experiment was combined with the 2019 data of this research group.UPGM and MAT analysis showed that 23 Mycobacterium avium were divided into 3 gene groups,and group A accounted for 60.9%(14/23).Group B accounted for 26.1(6/23),group C accounted for 13%(3/23).Baoshan and Dehong regions are divided into group A genes,Dali City,Eryuan County,Jianchuan County,and Yongping County in Dali Prefecture are divided into group B genes,and 3 strains in Binchuan County,Dali Prefecture are divided into group C genes.As a result of the minimal dendrogram,there are significant differences between Mycobacterium avium and Japanese strains in parts of Yunnan,and there are also differences between strains in parts of Yunnan and Portuguese strains,but not as significant as those in Japan.Among the 15 sites,the highest resolution is0.714 at MATR-3 site,and the lowest resolution is 0.143 at MATR-6 site.3.The proportional drug susceptibility method showed that the 16 strains of Mycobacterium avium had a resistance rate of 0 to rifabutin,and the drug resistance rate to the remaining 4 drugs reached more than 80%,and the resistance rate of isoniazid was 93.75%.The rate of resistance to butamine was 81.25%,the rate of resistance to kanamycin was 87.5%,and the rate of streptomycin resistance was 87.5%.Conclusions:1.The main isolates of HIV-related nontuberculous mycobacteria in Baoshan City of Yunnan Province,Dehong Dai and Jingpo Autonomous Prefecture,and Dali Bai Autonomous Prefecture are Mycobacterium avium,which can be used as the dominant flora.2.Using MATR-VNTR method to calculate the genetic polymorphism of M.avium,MATR-3 bit resolution is the highest,in addition to MATR-6 bit resolution is low,the other14 bits can be used as the identification of mycobacteria in the region.Some regions of Yunnan differ greatly from the mycobacteria gene of birds in Japan and Portugal.3.This batch of Mycobacterium avium is sensitive to rifabutin,but has a high resistance to isoniazid,ethambutol,kanamycin,and streptomycin.The drug susceptibility results show that rifabutin has more advantages in the treatment of Mycobacterium avium disease.