The author tested 34 urine samples with 5s-23srRNA gene spacer RFLP analysis and compared PCR results with serodiagnosis results.8 pafients of urine are positive by PCR. 7 patients were infected by B. garinii, 1 patient by B afzelii. These genospecies infecting patients are identical with those infecting local vectors. The results showed that the accuracy of laboratory diagnosis oould be improved by simultaneously testing samples with PCR and serodiagnosis. The method of 5s-23srRNA gene spacer RFLP analysis will probably be used widely in studying the relationship of clinical manifestation and genospecies and in epidemiology investigations.

ABSTRACT:To understand the species distribution of nontuberculous mycobacteria (NTM ) in Fujian Province of China , we collected clinical Mycobacterium isolates in the Fuzhou Pulmonary Hospital from 2009 to 2012 .A total of 6 362 clinical My‐cobacteria isolates were identified as 5 713 (89 .8% ) M .tuberculosis complex and 649 (10 .2% ) NTM strains by conventional identification method .Then ,by means of hsp65‐and rpoB‐PCR‐RFLP methods ,649 NTM strains were identified as 24 spe‐cies or complex of NTM ,in which the top three species or complex with the highest occurrence frequency were M .intracellular , M .avium and M .abscessus ,accounting for 48 .5% ,21 .3% and 12 .5% respectively .The prevalence rate of NTM was 10 .2%among Mycobacterium culture‐positive patients .There are lots of NTM species infecting human being ,and the most prevalence NTM species was M .avium complex accounting for 67 .8% in Fujian Province .

Slowly growing mycobacteria (SGM ) are distributed in the environment ,for example in soil and dirty water . SGM can cause human infections ,especially lung diseases .In this article ,first and second line antituberculous agents were ex‐amined in order to identify the optimum drugs for the treatment of SGM disorders .The fewest SGM in our study (4/34) were susceptible to isoniazid .Rifampicin (13/34) and ethambutol (14/34) were effective against similar numbers of strains .Ofloxa‐cin (23/34) ,kanamycin (26/34 ) , tobramycin (26/34 ) and streptomycin (27/34 ) were active against most of the tested strains .Ciprofloxacin (31/34) ,levofloxacin (31/34) ,amikacin (33/34) and capreomycin (33/34) showed an excellent range of activity .Moxifloxacin (34/34) showed the widest range of activity against the SGM species .Among the tested SGM spe‐cies ,M .simiae and M .af ricanum were resistant to the highest number of drugs .M .szulgai and M .duvalii were susceptible to all the first and second line antituberculous agents tested .Overall ,the second‐line antituberculous agents were good candi‐dates for the treatment of infection by SGM species and can be widely used in the therapy of SGM diseases .

To identify the species of Mycobacterium clinical isolates by molecular biology techniques,six clinical isolates which were preliminarily recognized as Mycobacterium tuberculosis by the TCH and PNB culture methods were selected in this study.PCR was applied to amplify the oxyR-ahpC interval resistant-zone(intergenic region) and the length of PCR product in four strains was the same as that of H37Rv,while the length in the other two strains was different from that of H37Rv but same as Mycobacterium intracellulare 95002.With sequencing and on-line homology comparison with H37Rv,U18263 and U71061,the DNA sequence of oxyR-ahpC intergenic region displayed a 99% homology with Mycobacterium intracellulare U71061 and an 84% homology with Mycobacterium tuberculosis H37Rv.In addition,the results of hsp65 PCR-restriction fragment length polymorphism analysis and multi-locus PCR amplification in the two strains were identical with those of Mycobacterium intracellulare 95002.These two clinical isolates which were preliminarily recognized as Mycobacterium tuberculosis by PNB and TCH culture methods were finally identified as Mycobacterium intracellulare.Results predicted that the application associated with various techniques of molecular biology would provide a faster,easier and more correct way for the species identification of Mycobacterium.

Objective: To examine the association between the cross-resistance to ethionamide (Eto) and isoniazid (INH) and mutations of drug resistant genes in Mycobacterium tuberculosis (MTB), so as to provide the evidence for clinical diagnosis and treatment for multidrug-resistant (MDR) tuberculosis. Methods: Totally 126 MTB clinical isolates were selected, including 88 MDR-MTB clinical isolates and 38 INH- and rifampicin (RFP)-sensitive isolates. The resistance to INH and Eto was tested in MTB clinical isolates using the drug susceptibility test, and the mutations in the spacer region of INH and Eto resistance-related katG, inhA, ethA, mshA, ndh, spacer region of oxyR-ahpC and inhA promoter were detected using PCR assay. The phenotypic resistance served as a gold standard, and the sensitivity, specificity and accuracy of gene mutation tests were calculated for detection of MTB clinical isolates cross-resistant to INH and Eto. Results: Of the 126 MTB clinical isolates, there were 37 isolates cross-resistant to INH and Eto (29.37%), 51 isolates with resistance to INH and susceptibility to Eto (40.48%), 4 isolates with susceptibility to INH and resistance to Eto (3.17%) and 34 isolates with susceptibility to INH and Eto (26.98%). Among the 41 Eto-resistant MTB clinical isolates, there were 37 isolates with resistance to INH (90.24%). There were 64 MTB clinical isolates detected with katG mutations (50.79%), 4 isolates with mutation in the spacer region of oxyR-ahpC (3.17%), 2 isolates with inhA mutations (1.59%), and these isolates were all resistant to INH. There were 11 MTB clinical isolates detected with mutation in the inhA promoter (8.73%) and one isolate with ndh mutation, and all these isolates were cross-resistant to INH and Eto. There were 23 MTB clinical isolates detected with ethA mutations (18.25%) and 40 isolates with mshA mutations (31.75%), in which Eto-susceptible and -resistant isolates were detected. The diagnostic sensitivity, specificity and accuracy of inhA promoter tests for detection of cross-resistance to INH and Eto were 29.73% (95%CI: 16.44%-47.17%), 100.00% (95%CI: 87.36%-100.00%) and 63.38% (95%CI: 51.76%-73.63%) in MTB clinical isolates. Conclusions The prevalence of INH resistance is high in Eto-resistant MTB clinical isolates. Mutation in the inhA promoter region correlates with the cross-resistance to INH and Eto in MTB clinical isolates, and detection of mutation in the inhA promoter may be feasible to detect the cross-resistance to INH and Eto in MTB clinical isolates.

Objective To investigate and evaluate the PCR-RFLP method for identification of Mycobacterium abscessus (M.abscessus) group.Methods 46 clinical acid-fast bacilli (AFB) isolates from Pulmonary Hospital of Fujian Province,Center for Disease Control and Prevention of Zhaoyang District in Beijing and Beijing People Liberation Army General Hospital were collected in 2009 -2010 and identified by traditional bacteriological characteristics according to clinical laboratory handbook of mycobacteria (2004).The PCR-RFLP method was used for species identification of M.abscessus group using hsp65 (441 bp) or rpoB (380 bp) gene fragment as specific target,while the direct DNA sequencing was performed as a control method.Results Of 46 AFB isolates,30 strains were identified as Mycobacterium tuberculosis by its traditional bacteriological characteristics and 16 strains were identified as non-tuberculosis mycobacterium (NTM).10 strains of the NTM strains had identical bacteriological characteristics with the reference strain M.abscessus ATCC 19977.Identified by hsp65 PCR-RFLP,9 of these 10 strains got the same pattern of 235bp and 200 bp(BstE Ⅱ )/145 bp,70 bp,60 bp,55 bp,50 bp and 40 bp(Hae Ⅲ ),and 1 got pattern of 235 bp and 200 bp(BstE Ⅱ )/200 bp,70 bp,60 bp and 50 bp(Hae Ⅲ ).While identified by rpoB PCRRFLP,all 10 strains got the same pattern of 105 bp,95 bp and 80 bp( Msp Ⅰ )/130 bp,100 bp and 90 bp (Hae Ⅲ ).By analysis of the DNA sequence,hsp65 and rpoB sequence of these 9 strains showed 100％similarity with those of M.abscessus,while the hsp65 and rpoB sequence of the other one strain showed 100％similarity with those of M.massiliense.Conclusion PCR-RFLP is a rapid and effective method for species identification of M.abscessus group,and hsp65 PCR-RFLP is more effective than rpoB PCR-PFLP in the species identification of M.abscessus group.

Objective To examine the foasibility of a new method for Mycobacterium tuberculosis (M. tuberculosis)"Beijing family"strain identifjcation——RD105 deletion test. Methods Two methods,Spoligotyping and RD105 deletion test,were used for M. tuberculosis"Beijing family"strain identification,respectively. The difference of the two identification methods was compared. Results Three hundred and forty-two clinical isolates from four areas(Beijing,Fujian,Xinjiang and Jilin)were assayed in this study.Among the total samples,261 isolates belonged to"Beijing family"accounting for 76.32%,while the other 81 isolates belonged to"non-Beijing family"accounting for 23.68%. The coincidence rate for these two methods was 100%. Conclusion The simple and rapid new method——RD105 deletion test can be used to identify"Beijing family"instead of the traditional method——Spoligotyping.

Objective To clone and express the outer surface protein C ( OspC) from a Chinese Borrelia afzelli FP1 strain and to evaluate the immune protectivity of the recombinant OspC protein ( rOspC) . Methods The gene encoding OspC protein of Borrelia afzelli FP1 strain was amplified by polymerase chain reaction (PCR) and then inserted into pET-30a plasmid to construct the recombinant expression plasmid pET-30a-OspC. The transformed E. coli BL21 strains carrying pET-30a-OspC plasmid were induced by IPTG to express OspC protein. The expressed proteins were purified by Ni-IDA resin chromatography and analyzed by SDS-PAGE and Western blot assay. Indirect immunofluorescence assay ( IFA) was performed to detect anti-rOspC protein antibodies in serum samples from rabbits immunized with rOspC protein. In vitro neutral-ization test was performed for evaluation the immune protectivity of rOspC protein. Results The recombi-nant expression plasmid pET-30a-OspC was successfully constructed and highly expressed in E. coli BL21. A strong antigen-antibody reaction between the rOspC protein and polyclonal antibody against Borrelia afzelli FP1 strain was detected by Western blot assay. The titers of IgG in serum samples from rabbits immunized with rOspC protein were significantly elevated. The in vitro neutralization test indicated that 106/ml of Borre-lia afzelli FP1 strains were neutralized by every anti-OspC protein serum sample from the experiment group. Conclusion The rOspC protein showed a strong immune protectivity against Borrelia afzelli, which could be used in the development of polyvalent subunit vaccine against lyme disease.

The Mycobacterium chelonae/abscessus (M.chelonae/abscessus) complex belongs to the rapidly growing genus Mycobacterium (RGM).It is one of the most important pathogenic members of Mycobacterium leading to nosocomial infections and outbreaks.It includes members of M.chelonae,M.immnunogenum,M.abscessus,M.massiliense,and M.bolletii.In order to investigate the epidemiological characteristics of the M.chelonae/abscessus complex in China and to conduct the molecular methods for species identification of M.chelonae/abscessus,we collected clinical M.chelonae/abscessus complex strains identified by phenotypic tests.Members were verified by sequencing of 16S rRNA,Species and subspecies were identified by hsp65 and rpoB PCR RFLP methods.In total,27 clinical specimens were identified as Mycobacterium chelonae/abscessus complex by phenotypic tests.16s rRNA gene sequence analysis of all 27 clinical samples shared over 99.7％ similarity with M.chelonae and M.abscessus.Species identification with hsp65 PCR-RFLP and rpoB PCR-RFLP revealed that 18 specimens were M.abscessus and 4 were M.absecces.The remaining 5 samples displayed a pattern that failed to match any previously reported pattern.Thus,this might represent a novel species that is part of the Mycobacterium chelonae/abscessus complex.We identified that a majority of the chronic lung infection in China is caused by the M.chelonae/abscessus complex.Specifically,the M.abscessus species might be the most infectious,while other species in the complex can still cause infection.Interestingly,there may be a novel or previously unidentified species that is a part of the complex.Finally,we show that species identification can be carried out more accurately by combined use of hsp65 and rpoB PCR-RFLP.

Objective To evaluate the antigenicity of two proteins of Mycobacteium tuberculosis (M. tuberculosis), Dnak(Rv0350) and MPT83(Rv2873), in order to provide a scientific basis for immuno-logical diagnosis of tuberculosis and research on vaccines. Methods The two antigen proteins, Dnak (Rv0350) and MPT83(Rv2873), were cloned, expressed and purified using the methods of genetic recom-bination and protein purification technology. Blood samples were collected from subjects including tuberculo-sis patients ( TB) , non-tuberculosis patients with other pulmonary diseases ( non-TB) and healthy volunteers (HV). To analyze the immunological properties of the recombinant Dnak (Rv0350) and MPT83 (Rv2873) proteins, they were used as antigens to detect humoral and cellular immunity in the subjects with enzyme linked immunosorbent assay ( ELISA ) and effector T cell enzyme-linked immunospot assay ( ELISPOT ) . Results The recombinant and purified Dnak (Rv0350) and MPT83 (Rv2873) proteins of M. tuberculosis were successfully obtained and used as antigens in the detection of humoral and cellular immunity in the sub-jects. Specific antibodies ( IgG) in the serum samples of 135 TB, 56 non-TB and 94 HV were tested with ELISA. The results showed that the sensitivity, specificity and accuracy of Dnak ( Rv0350 ) protein were 77. 80％ (105/135), 56. 70％ (85/150) and 66. 67％ (190/285). Similarly, the sensitivity, specificity and accuracy of MPT83 (Rv2873) protein were 76. 30％ (103/135), 43. 30％ (65/150) and 58. 95％(168/285). Cellular immunity was tested with the levels of IFN-γproduced by effector T lymphocytes after stimulating peripheral blood monouclear cells ( PBMC) collected form subjects of 59 TB, 65 non-TB and 64 HV with Dnak (Rv0350) and MPT83 (Rv2873) protein antigens. The results showed that the sensitivity, specificity and accuracy of Dnak (Rv0350) and MPT83 (Rv2873) proteins were 66. 10％ (39/59), 62. 79％ (81/129) and 63. 83％ (120/188), and 47. 46％ (28/59), 79. 84％ (103/129) and 69. 68％(131/188), respectively. Conclusions M. tuberculosis Dnak (Rv0350) and MPT83 (Rv2873) proteins have good antigenicity and could stimulate T cells to produce stronger immune responses. The two proteins used in combination might have promising potential in the research of immunodiagnosis of tuberculosis and the development of new anti-tuberculosis vaccines.