Immunogenicity of Whole / 生物医学与环境科学（英文）

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Immunological Evaluation of a Novel Mycobacterium tuberculosis Antigen Rv0674 / 生物医学与环境科学（英文）

OBJECTIVE@#This study aimed to characterize the diagnostic and vaccine potential of a novel Mycobacterium tuberculosis antigen Rv0674.@*METHODS@#To evaluate the diagnostic potential and antigenicity of Rv0674, IgG was evaluated using ELISA and interferon (IFN)-γ was done by using ELISpot assay among TB patients and healthy donors. For immunogenicity evaluation, BALB/c mice were immunized with Rv0674. Cytokine production was determined by cytokine release assay using an ELISA kit, and the antibodies were tested using ELISA.@*RESULTS@#The results of serum Elisa tests showed that Rv0674 specific immunoglobulin G (IgG) response was higher in TB patients than negative controls. And Rv0674 had good performance in serological test with sensitivity and specificity of 77.1% and 81.1%, respectively. While it shows poor sensitivity and specificity of 26.23% and 79.69% for IFN-γ tests. In BALB/c mice, Rv0674 adjuvant by DDA/Poly I:C could also induce a high level of IFN-γ, interleukin-2 and interleukin-6 as well as a high IgG titer in both high- and low-dose groups indicating that Rv0674 is essential in humoral and cellular immunity. Moreover, the cytokine profile and IgG isotype characterized Rv0674 as a Th1/Th2-mixed-type protective immunity with the predominance of Th1 cytokines.@*CONCLUSION@#Rv0674 may be a good potential candidate for the development of TB serological diagnosis and a new TB vaccine.

Application value of detecting Mycobacterium tuberculosis specific IgG/IgM antibodies for tuberculosis diagnosis with colloidal gold immunochromatography assay / 中国人兽共患病学报

In this study,we estimated the application value of detecting Mycobacterium tuberculosis (MTB) specific IgG/IgM antibodies for tuberculosis diagnosis with colloidal gold immunochromatography assay (GICA).We collected 332 effective serum samples and their background information,including 260 patients with tuberculosis and 72 healthy individuals.The means of GICA was used to detect MTB specific IgG/IgM antibodies.Results were compared with the clinical diagnosis and the results of bacteriological tests.The SPSS 22.0 software was used to analyze the results,and when P＜0.05 the difference was statistically significant.The sensitivity and specificity of GICA were 41.15％ and 91.67％,and the sensitivity of the bacterial positive and negative patients were 51.38％ and 33.77％,respectively.The positive rate of IgG/IgM antibodies detection with GICA (41.15％) was much higher than that of bacteria with acid-fast stain of sputum smear (18.84％) and sputum bacteria cultivation (36.15 ％) (P ＜ 0.05) respectively.The positive rate of the combination of tuberculosis antibody detection,sputum bacterial culture and sputum smear was 61.54％,higher than the result of single method or combination of two methods.The detection of specific antibodies against MTB in serum with GICA is sensitive,specific,rapid and convenient,which can be used in clinical screening.Meanwhile,there are still certain limitations of this method,and the sensitivity and specificity need to be improved.Therefore,the GICA can be used as an auxiliary diagnosis combined with sputum bacteriology,imaging test and clinical features rather than diagnose tuberculosis alone.

Preliminary Study on Drug Susceptibility Profile and Resistance Mechanisms to Macrolides of Clinical Isolates of Non-tuberculous Mycobacteria from China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Macrolide susceptibility and drug resistance mechanisms of clinical non-tuberculous mycobacteria (NTM) isolates were preliminarily investigated for more accurate diagnosis and treatment of the infection in China.</p><p><b>METHODS</b>Four macrolides, including clarithromycin (CLAR), azithromycin (AZM), roxithromycin (ROX), and erythromycin (ERY), were used to test the drug susceptibility of 310 clinical NTM isolates from six provinces of China with the broth microdilution method. Two resistance mechanisms, 23S rRNA and erm, were analyzed with nucleotide sequence analysis.</p><p><b>RESULTS</b>Varied effectiveness of macrolides and species-specific resistance patterns were observed. Most Mycobacterium abscessus subsp. massiliense were susceptible and all M. fortuitum were highly resistant to macrolides. All the drugs, except for erythromycin, exhibited excellent activities against slow-growing mycobacteria, and drug resistance rates were below 22.2%. Only four highly resistant strains harbored 2,058/2,059 substitutions on rrl and none of other mutations were related to macrolide resistance. G2191A and T2221C on rrl were specific for the M. abscessus complex (MABC). Seven sites, G2140A, G2210C, C2217G, T2238C, T2322C, T2404C, and A2406G, were specifically carried by M. avium and M. intracellulare. Three sites, A2192G, T2358G, and A2636G, were observed only in M. fortuitum and one site G2152A was specific for M. gordonae. The genes erm(39) and erm(41) were detected in M. fortuitum and M. abscessus and inducible resistance was observed in relevant sequevar.</p><p><b>CONCLUSION</b>The susceptibility profile of macrolides against NTM was demonstrated. The well-known macrolide resistance mechanisms, 23S rRNA and erm, failed to account for all resistant NTM isolates, and further studies are warranted to investigate macrolide resistance mechanisms in various NTM species.</p>

First Report in China on the Identification and Drug Sensitivity of Mycobacterium elephantis Isolated from the Milk of a Cow with Mastitis / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>In this study, milk from a cow with mastitis was analyzed to determine the presence of mycobacterial infection. Milk quality and security problems pertaining to the safe consumption of dairy products were also discussed in this study.</p><p><b>METHODS</b>Milk was preprocessed with 4% NaOH. Then, mycobacteria were isolated from the milk sample on L-J medium. The isolate was identified using multiple loci Polymerase Chain Reaction (PCR) and multi-locus sequence analysis with 16S rRNA, sodA, hsp65, and ITS genes. The drug sensitivity of the isolate to 27 antibiotics was tested through alamar blue assay.</p><p><b>RESULTS</b>Smooth, moist, pale yellow colonies appeared on the L-J medium within a week after inoculation. Based on the results of multiple loci PCR analysis, the isolate was preliminarily identified as non-tuberculous mycobacteria. The 16S rRNA, SodA, hsp65, and ITS gene sequences of the isolate exhibited 99%, 99%, 99%, and 100% similarities, respectively, with those of the published reference strains of Mycobacterium elephantis (M. elephantis). The drug sensitivity results showed that the strain is resistant to isoniazid, p-aminosalicylic acid, and trimesulf but is sensitive to ofloxacin, rifampicin, amikacin, capreomycin, moxifloxacin, kanamycin, levofloxacin, cycloserine, ethambutol, streptomycin, tobramycin, rifabutin, ciprofloxacin, linezolid, cefoxitin, clarithromycin, and minocycline.</p><p><b>CONCLUSION</b>To the best of our knowledge, this study is initially to report the isolation of M. elephantis from the milk of a cow with mastitis in China.</p>

Molecular Characteristics and Drug Susceptibility of Mycobacterium tuberculosis Isolates from Patients Co-infected with Human Immunodeficiency Virus in Beijing, China / 生物医学与环境科学（英文）

70 clinical Mycobacterium tuberculosis strains isolated from AIDS patients in two HIV/AIDS referral hospitals in Beijing were used in this study. M. tuberculosis and non-tuberculosis mycobacterium (NTM) were identified by using multi-locus PCR. M. tuberculosis was genotyped by using 15-locus MIRU-VNTR technique and spoligotyping afterwards. Meanwhile, the drug susceptibilities of the strains to the four first-line anti TB drugs (rifampin, isoniazid, streptomycin, and ethambutol) and the four second-line anti-TB drugs (capreomycin, kanamycin, ofloxacin, and ethionanide) were tested with proportional method. In this study, M. tuberculosis and NTM strains isolated from AIDS patients with TB-like symptoms were identified and genotyping analysis indicated that Beijing genotype was the predominant genotype. In addition, the prevalence of drug-resistant TB, especially the prevalence of XDR-TB, was higher than that in TB patients without HIV infection.

Molecular characterization and drug-resistance of Mycobacterium tuberculosis strains in Xuzhou, China / 生物医学与环境科学（英文）

To understand the genetic diversity and drug resistance status of Mycobacterium tuberculosis (M. tuberculosis) circulating in Xuzhou of China, the spacer-oligonucleotide typing (Spoligotyping) and multi-loci VNTRs (variable number tandem repeats) analysis (MLVA) were utilized for the genotyping of the isolates. Drug susceptibility test (DST) was performed by the proportion method on the Lowenstein-Jensen (L-J) medium using isoniazid, rifampicin, ethambutol, and streptomycin. By Spoligotyping, 287 M. tuberculosis isolates were differentiated into 14 clusters. Then with 15-loci MLVA, these strains could be divided into 32 clusters, 228 genotypes. Of 15 VNTRs, 6 loci had the highly discriminatory powers, 6 loci presented moderate discrimination and 3 loci demonstrated less polymorphism. The DST results showed that 46 strains were resistant to at least one first-line anti-tuberculosis agent. There was a difference in the isoniazid resistance between Beijing and non-Beijing genotype strains. We concluded that the combination of Spoligotyping and 15 VNTR loci as the genotyping in our study was applicable for this region, the drug resistant isolates were identified, and the Beijing family was the most prevalent genotype in the rural counties of Xuzhou.

Evaluation of four candidate VNTR Loci for genotyping 225 Chinese clinical Mycobacterium tuberculosis complex strains / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To evaluate four candidate variable number tandem repeat (VNTR) loci for genotyping Mycobacterium tuberculosis complex strains.</p><p><b>METHODS</b>Genomic sequences for two M. tuberculosis strains (CCDC5079 and CCDC5180) were generated, and using published sequence data, four candidate VNTR loci were identified. The VNTRs were used to genotype 225 Chinese clinical M. tuberculosis complex strains. The discriminatory power of the VNTRs was evaluated using BioNumerics 5.0 software.</p><p><b>RESULTS</b>The Hunter-Gaston Index (HGI) for BJ1, BJ2, BJ3, and BJ4 loci was 0.634, 0.917, 0.697, and 0.910, respectively. Combining all four loci gave an HGI value of 0.995, thus confirming that the genotyping had good discriminatory power. The HGI values for BJ1, BJ2, BJ3, and BJ4, obtained from Beijing family strain genotyping, were 0.447, 0.878, 0.315, and 0.850, respectively. Combining all four loci produced an HGI value of 0.988 for genotyping the Beijing family strains. We observed unique patterns for M. bovis and M. africanum strains from the four loci.</p><p><b>CONCLUSION</b>We have shown that the four VNTR loci can be successfully used for genotyping M. tuberculosis complex strains. Notably, these new loci may provide additional information about Chinese M. tuberculosis isolates than that currently afforded by established VNTR loci typing.</p>

A new multilocus sequence analysis scheme for Mycobacterium tuberculosis / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Tuberculosis remains one of the most serious infectious diseases in the world. In this study, a scheme of Mycobacterium tuberculosis (M. tuberculosis) multilocus sequence analysis (MLSA) was established for the phylogenetic and epidemiology analysis.</p><p><b>METHODS</b>To establish the scheme of M. tuberculosis MLSA, the genome of H37Rv, CCDC5079 and CCDC5180 were compared, and some variable genes were chosen to be the MLSA typing scheme. 44 M. tuberculosis clinical isolates were typed by MLSA, IS6110-RFLP, and soligotyping, to evaluate the MLSA methods.</p><p><b>RESULTS</b>After comparison of the genome, seven high discrimination gene loci (recX, rpsL, rmlC, rpmG1, mprA, gcvH, ideR) were chosen to be the MLSA typing scheme finally. 11 variable SNP sites of those seven genes were found among the 44 M. tuberculosis isolate strains and 11 sequence types (STs) were identified. Based on the Hunter-Gaston Index (HGI), MLSA typing was not as good for discrimination at the strain level as IS6110-RFLP, but the HGI was much better than that of spoligotyping. In addition, the MEGA analysis result of MLSA data was similar to spoligotyping/PGG lineage, showing a strong phylogenetic signal in the modern strains of M. tuberculosis. The MLSA data analysis by eBURST revealed that 4 sequence types (ST) came into a main cluster, showing the major clonal complexes in those 44 strains.</p><p><b>CONCLUSION</b>MLSA genotyping not only can be used for molecular typing, but also is an ideal method for the phylogenetic analysis for M. tuberculosis.</p>