| Research backgroundThe prevention and cure of pulmonary tuberculosis remain severe extremely in21century in our country. The epidemic situation of tuberculosis shows5characters: high infection rate, high prevalence, high incidence, high resistance rate, high death rate and slow tuberculosis control process. The epidemic situation of tuberculosis in countryside is higher than that in city. The prevalence and death rate of young adults are higher, which harm the life safety of the people severely. Mycobacterium tuberculosis (MTB) can form cell wall-deficient mutants (L forms) easily, and M. tuberculosis L forms (MTB-L) are able to survive under harsh environmental conditions. MTB-L can survive over long periods in vivo and can be isolated and cultivated from blood samples of patients with active sarcoidosis. The biological characteristics, pathogenicity, and drug susceptibility of MTB-L differ from those of MTB, and these differences might be important factors underlying tardy progression, aggravation, recurrence, and drug resistance in tuberculosis. Approximately70%of active pulmonary tuberculosis cases are reported to be smear and culture negative. However, the MTB-L positive rate exceeds30%for sputum specimens from smear- and culture-negative pulmonary tuberculosis cases, and the MTB-L positive rate exceeds50%for sputum specimens from multidrug-resistant tuberculosis cases. Thus, a fast and convenient method to detect MTB and MTB-L would be useful in determining the nature of a tuberculosis infection, selecting a treatment strategy, and identifying preventive measures.Research purposeTo aim directly at severe situation of the prevention and cure of pulmonary tuberculosis, and the bionomics and pathogenic characters of Mycobacterium tuberculosis L forms; we planed:①to establish a novel, high sensibility, high specificity method for smear dyeing and drug sensitivity test of Mycobacterium tuberculosis L forms;②to analyze the infection and drug fast of tuberculosis in Luohu district of Shenzhen city systematically and to evaluate the diagnosis and treatment value of the modified method.Research contents①to establish a novel, high sensibility, high specificity method for smear dyeing and drug sensitivity test of Mycobacterium tuberculosis L forms, and to evaluate the diagnosis and treatment value of the modified method.②the sputum specimens of dubious tuberculosis patients and directly observed therapy, short-course tuberculosis patients from Luohu Chronic Disease Prevention and Cure Hospital during January,2010to December,2011were detected for Mycobacterium tuberculosis and Mycobacterium tuberculosis L forms by the modified acid-fast staining.③the drug sensitivity test of MTB-L of the above samples were carried out by the modified MTB-L culture medium. ④the drug sensitivity test of MTB of the above samples were carried out by the modified Roche medium.⑤the epidemic situation of tuberculosis in Luohu district was analyzed, the affection of the drug fast to Mycobacterium tuberculosis and Mycobacterium tuberculosis L forms on the clinical diagnosis and treatment prognosis of tuberculosis was evaluated.Research methods1The improvement and methodological evaluation of MTB-L acid-fast staining method1.1Procedures of our modified acid-fast staining method (modified MTB-L acid-fast staining method), modified IK acid-fast staining method and traditional acid-fast staining methodModified IK acid-fast staining method and traditional Ziehl-Neelsen acid-fast staining method were executed as previously reported. Colorants should be added onto the slide repeatedly and heating was needed in the process of traditional acid-fast staining method. Heating was not needed in the process of modified IK acid-fast staining method; but the process should be sustained for24hours. However, heating was not needed in the process of our modified acid-fast staining method either and the process should be sustained for about5minutes only. The procedures of the three methods were presented in the table below. 1.2Fluorescent quantitation PCR methodMTB DNA was extracted from50sputum samples from patients with definite tuberculosis by using the commercially available Mycobacterium tuberculosis (TB) fluorescent polymerase chain reaction diagnostic kit (Da’an Gene) according to the manufacturer’s instructions. Extracted DNA was stored at-20℃in extract buffer until analysis.MTB DNA was analyzed by using a previously reported real-time PCR method. Briefly, real-time PCR was carried out by using the commercially available Mycobacterium tuberculosis (TB) fluorescent polymerase chain reaction diagnostic kit (Da’an Gene) in a40-μl reaction mixture containing PCR buffer, deoxynucleoside triphosphates (i.e., dATP, dTTP, dCTP, and dGTP), forward and reverse primers (5’-TCGCCCGTCTACTTGGTGTT-3’;5’-TGATGTGGTCGTAGTAGGTC-3’), and the fluorescence probe (5’-ACAACGCCGAATTGCGAAGGGC-3’), along with3μl Taq DNA polymerase and2μl of the extracted DNA sample. The thermal cycler was programmed as follows:one cycle of93℃×2min,10cycles of93℃×45s and55℃×1min, and30cycles of93℃×30s and55℃×45s. The fluorescence signals of FAM were acquired at the end of the third program (i.e., after55℃×45s). Fluorescence data analysis was conducted using SDS software (Version1.4; Applied Biosystems). The threshold fluorescence level, used to derive threshold cycle (Ct) values, was automatically determined by the SDS software.1.3Evaluation of our modified acid-fast staining methodThe sensitivity, specificity, positive predictive value, negative predictive value, positive rate, and diagnostic efficiency of the modified MTB-L acid-fast staining method were evaluated by using the PCR method as the comparison’s gold standard. Briefly, fifty sputum samples from patients with definite tuberculosis were analyzed by comparing results from our modified acid-fast staining method and the PCR method.2Clinical application of the modified MTB-L acid-fast staining methodNine hundred and sixty sputum samples from patients with definite tuberculosis were smeared onto individual glass slides to form egg-shaped sputum membranes of size10×20mm (6smears per sample). Two of the six smears were analyzed using the traditional acid-fast staining method; two, using the modified IK acid-fast staining method; and two, using our modified acid-fast staining method. The positive detection rates for MTB and MTB-L in the smears of the960samples treated with each of the three methods were compared. The epidemic situation of tuberculosis in Luohu district, the infection status of MTB and MTB-L, and the affection of MTB and MTB-L on clinical diagnosis and treatment prognosis to tuberculosis were analyzed.3The improvement and methodological evaluation of MTB-L medium3.1The routine cultivation using Roche mediumThe sputum samples were collected from clinically diagnosed patients with tuberculosis. Then the samples were liquefied through pre-treatment by4%sodium hydroxide. About0.1ml liquefied samples were inoculated onto the Roche medium at37℃. After3～7days, the colonies were observed once a week and defined using acid-fast staining method. Negative results were reported when no colonies were found till8th week.3.2The cultivation using modified tryptone soybean tryptone ager for MTB-L (TSA-L medium)The sputum samples were collected from clinically diagnosed patients with tuberculosis. Then the samples were liquefied through pre-treatment by4%sodium hydroxide. About0.1ml liquefied samples were inoculated onto the TSA-L at37℃with5%～10%CO2. The colonies were observed once a week. The doubtful colonies were defined using microscope.3.3The cultivation using our modified medium for MTB-L (MTB-L medium)The prescription of TSA-L were improved, that is to say, cholesterol and lecithin in favor of mycobacteria growth were added into TSA-L. Then the sputum samples were collected from clinically diagnosed patients with tuberculosis. The samples were liquefied through pre-treatment by4%sodium hydroxide. About0.1ml liquefied samples were inoculated onto the MTB-L at37℃with5%～10%CO2. The colonies were observed once a week. The doubtful colonies were defined using microscope.3.4Evaluation of our modified acid-fast staining methodFifty MTB-L positive samples defined by Fluorescent quantitation PCR method were cultivated using Roche medium, TSA-L medium and MTB-L medium respectively. The positive rates and required time of colonies growth were analyzed.4Clinical application of the modified MTB-L mediumDrug sensitivity test for MTB-L positive samples were carried out using the modified MTB-L medium. The resistant states of MTB-L to first line drug were analyzed. Drug sensitivity test for MTB positive samples were carried out using Roche medium. The resistant states of MTB to first line drug were analyzed.Statistical treatment①Differences between values of MTB-L acid-fast staining method, Z-N acid-fast staining method, modified IK acid-fast staining method and PCR method were assessed by using McNemar’s chi square test.②Differences among values of MTB-L acid-fast staining method, modified IK acid-fast staining method, and Z-N acid-fast staining method, and differences among values of negative conversion rate of MTB、MTB-L were assessed by using group design chi-square test.③Differences among values of culture positive rate of MTB-L culture method and TAS-L culture method were assessed by using group design chi-square test. Days of MTB-L culture positive by the two methods were recorded as mean±SD, and the differences of mean±SD were assessed by using Student’s t-test.④Differences among smear-positive and culture-negative rate of MTB-L culture method and TAS-L culture method, and differences among drug resistance rate of MTB、MTB-L were assessed by using group design chi-square test.⑤SPSS13.0was used to define the differences.Research results⑥Sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic efficiency of our modified acid-fast staining method were similar to the modified IK acid-fast staining method. As in the modified IK acid-fast staining method, no heating is needed in our method. In addition, our method only needs about5min to complete, which is markedly shorter than the24h required to complete the modified IK acid-fast staining method. The results obtained using our methods have satisfactory concordance with those obtained by PCR. Moreover, the MTB and MTB-L positive detection rates of our method were significantly higher than those of the routine acid-fast staining method.②Except1case of MTB-L positive patient, the detection rate of MTB and MTB-L in tuberculosis initial treated patients in Shenzhen Luohu district changed into negative after5month normalization treatment. The sputum negative conversion rates of MTB positive patients at the end of2,3months were higher than those of MTB and MTB-L positive patients and MTB-L positive patients sharply. Except2cases of MTB and MTB-L positive patients, and1case of MTB-L positive patient, the detection rate of MTB and MTB-L in tuberculosis retreated patients in Shenzhen Luohu district changed into negative after4month normalization treatment.③MTB-L could grow more quickly on our modified mycobacterium tuberculosis solid medium than on modified TSA-L solid medium; the positive rates and required time of colonies growth of MTB-L using our modified solid medium were higher than those on modified TSA-L solid medium④Initial treatment resistance rate of tuberculosis in Luohu district was lower than that of the fifth national tuberculosis epidemiological survey in2010; meanwhile, Retreated resistance rate of the former was than that of the latter.Research conclusion⑤A new, modified acid-fast staining method, which is convenient, specific, and sufficiently sensitive to examine sputum for the presence of MTB-L and MTB quickly and routinely, had been established successfully. Our method can be easily utilized in a general tuberculosis laboratory.②A new, modified cultural method, which is convenient, specific, and sufficiently sensitive to drug sensitivity test of MTB-L routinely, had been established successfully. Our method can be easily utilized in a general tuberculosis laboratory.③The sputum negative conversion rates of MTB positive patients in tuberculosis initial treated and retreated patients were higher than those of MTB+MTB-L or MTB-L positive patients. The sputum negative conversion rates between MTB+MTB-L positive patients and MTB-L positive patients changed little. So, MTB-L is an important reason for the lower sputum negative conversion rates of tuberculosis patients.④The rates of initial resistance and retreated resistance of MTB-L were both46.7%, which indicated that the status of MTB-L drug resistance is still quite serious.⑤The rates of initial resistance and retreated resistance of MTB in Luohu district were lower than those of national epidemiological sampling survey of tuberculosis in2010(22.5%vs.42.7%,33.3%vs.38.5%), which indicated that satisfactory progress is made in the control and prevention of MTB in Luohu district.⑥The rates of initial resistance and retreated resistance of MTB-L were higher than those of MTB respectively, which indicated that MTB-L and the severity of drug fast is closely related. |
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