The Effect Of Yangyinqingfei Method In The Treatment Of Pulmonary Tuberculosis And The Study Of Mycobacteria Identification

Background and ObjectivesTuberculosis is one of China's major infectious diseases, and has been included in the "National Science and Technology major projects-the prevention and control of such as AIDS and viral hepatitis of major infectious diseases-the study of Traditional Chinese Medicine prevention of tuberculosis" project. But so far, it has not completed TCM symptoms of tuberculosis and Integrative Medicine therapy studies programs. Has not been seen the large systems report of the TCM treatment of pulmonary tuberculosis, the identification of Mycobacterium tuberculosis takes a long (48 days), complicated, and inconvenient for clinical treatment. Therefore, this article aims to study the law TCM symptoms of pulmonary tuberculosis and integrative medicine treatment options to improve the symptoms of tuberculosis of the Chinese Classification and treatment programs and to explore the new technology (PCR-RLB technology) of Mycobacterium identification, to speed up pathogen Identification of learning and enhance their sensitivity and accuracy. Thus for the prevention and treatment of tuberculosis provides a theoretical basis and experimental evidence.This paper is divided into three parts to study.The first part the study of syndromes of Traditional Chinese Medicine of initial treatment secondary pulmonary tuberculosis (This is the "National Science and Technology major projects-the comprehensive study of TB medicine" project, topic ID: 2008ZX10005-010).The second part the clinical observation of the Yangyinqingfei joint western medicine treatment smear positive initial treatment secondary pulmonary tuberculosis.The third part the clinical value and study on method of PCR-reverse line blot hybridization assay of identification of mycobacterium.The first part The study of syndromes of Traditional Chinese Medicine of initial treatment secondary pulmonary tuberculosis Research Content1. Collected 2009.3-2009.11 the patient of Complied with a standard in Shenzhen Third People's Hospital Pulmonary inpatient wards and out, a total of 100 cases. First in accordance with the look, smell, questioning, pulse feeling, and then collection tongue and pulse with the tongue apparatus, Sphygmography. Collected by the four diagnostic medicine specialist, a full-time observation and fill out the question naire. Chief Physician of a Chinese guide. According to established diagnostic criteria for TCM syndrome differentiation.2. Statistical Methods:SPSS statistical software for statistical analysis, count data with chi-square test. Research Results1.100 cases initial treatment of secondary pulmonary symptoms and signs were recorded in all:5 cases with weak cough,2 patients with a low voice cough, shortness of breath cough in 3 cases,67 cases of cough shortly, 1 case of shortness of breath and low voice; 80 cases of cough with least sputum,15 cases of thick yellow sputum viscosity,5 cases of sputum thin white; 14 cases of hemoptysis color red; 21 cases of afternoon fever,2 cases of the afternoon hot flushes,67 cases of hand, foot and heart heat; 94 cases of sweating,3 cases of spontaneous,3 cases of spontaneous and sweating; 3 cases of afternoon zygomatic Red; 15 cases of chest faint,3 cazes of pain of chest and hypochondrium,3 cases of chest distress; 87 cases of yellow urine; 81 cases of pulse rate; 13 cases of body weight loss; 11 cases of insomnia,7 cases of poor appetite; 6 cases of tired; 6 cases of weakness; 4 cases of dry mouth throat; 4 cases of dizziness; 3cases of fear the wind;3 cases of shortness of breath and disinclination to talk; 2 cases of diarrhea; 2 cases of lassitude weakness; 1 case of irritability; 1 case of mouth and throat pain; 1 cases of man nocturnal emission; The woman was 1 case of small or closed; 1 case of hand-foot-not warm; 1 case of abdominal distention; 1 case of pale; 1 case of pale complexion.2.100 cases of untreated secondary tuberculosis of the tongue, the occurrence of pulse number of patients:Tongue:60 cases of red tongue,12 cases of red tongue and less-chun,3 cases of light pale tongue with scalloped edges; 20 cases of red tongue and dry; 5 cases of pale tongue。Tongue coating:2 cases of a small fur,91 cases of thin yellow fur,3 cases of yellow and greasy fur; 4 cases of coating of whitePulse:67 cases of Pulse breakdown,22 cases of fine pulse,3 cases of pulse thin and the number,3 cases of slippery pulse,1 case of pulse a few strings,3 cases of pulse weak,1 case of thready and weak pulse。3.100 cases of TCM Syndrome Types in initially treated secondary pulmonary tuberculosis:TCM syndrome differentiation according to the standard of 100 patients were classified syndrome, in which 67 cases of Yin deficiency syndrome,20 cases of Yin deficiency and fire excess, 3 cases of deficiency of both Qi and Yin,1 case of deficiency of Qi in spleen and lung,2 cases of deficiency of Yin in lung and Kidney, 3 cases of Deficiency of lung Qi,1 case of liver fire attacking lung,3 cases of phlegm-heat accumulated in the lung card. The incidence of Yin deficiency Syndrome (67%) was significantly higher than other syndromes (P<0.01), followed by Yin deficiency and fire excess (20%) higher than the incidence of other than except of Yin deficiency. (P<0.01).The second part The clinical observation of the Yangyinqingfei joint western medicine treatment smear positive initial treatment secondary pulmonary tuberculosis.Research Content1. Collected 2009.3-2009.11 the patient of Complied with a standard in Shenzhen Third People's Hospital Pulmonary inpatient wards, a total of 76 cases. Division by the number of sequential principle (odd as the control group, even for the treatment group) was divided into treatment group and control group,38 cases in each group. The treatment of the control group is 2HRZE/4HR. The treatment of the treatment group is Western medicine treatment group (treatment with the control group)+Yangyinqingfei decoction, a total of 2 weeks. Before treatment and after treatment were observed indices and clinical symptoms scores (cough, night sweats, hot hand-foot center, chest dull pain, blood in sputum, dry mouth and throat, less phlegm); sputum smear amount of acid-fast staining bacteria; DR lesion area was observed by anteroposterior chest, empty number; effect observation; the incidence of adverse reactions. 2. Statistical Methods:SPSS statistical software for statistical analysis, count data with chi-square test, measurement data with t test.Research results1. The comparison of the basic conditions of the treatment group and control group in before treatment:According to the principles of sequential the 76 cases of cases divided into treatment group and control group, treatment group 38 cases,38 cases of the control group. Two groups in age, sex, weight, symptom score, duration, rate of sputum acid-fast staining, DR chest, with or without disease, identification of Mycobacterium is not significant difference comparable2. The comparison.of symptom scores of treatment group and control group in before and after treatment:The treatment and control groups after treatment than before treatment symptom score improved (P<0.05); the score of treatment group slightly lower than the control groups, but both were no significant differences between (P> 0.05).3. The comparition of sputum acid-fast staining score of treatment group and control group in before and after treatment: The acid-fast staining score of treatment group and control group after treatment decreased slightly than before treatment, but no significant difference (P>0.05); the acid-fast staining scores between the two groups showed no significant difference after treatment (P>0.05).4. The comparison of chest of treatment group and control group in before and after treatment:The lesions chest area and several empty of treatment group and control group did not change in after treatment and before treatment.5. The effect observation of treatment group and control group therapy:After 2 weeks of treatment, the total improvement rate of the treatment group and control group is 100%, there are mainly significant improvement in clinical performance, and sputum smear and chest was no significant change. There are not significant difference in treatment group and control group (P>0.05).6. The comparition of the incidence of adverse reactions of the treatment group and control group after treatment:1 case of the reatment groupand 2 cases of the control group suffered from liver damage, are present only as ALT, AST increased, the maximum value is less than 2 times the upper limit of normal (80U/L), bilirubin normal, no clinical symptoms, for 1 week after treatment were normal liver protection; 1 case of the treatment group and 2 cases of the control group suffered from nausea, vomiting, gastrointestinal reactions, the symptoms of gastrointestinal reactions disappeared when the RFP is changed to after meals; 1 case of the control group had skin rash, itching, performance, disable the symptoms gradually disappeared after isoniazid treatment was no occurrence of allergic dermatitis; there were not renal abnormalities, optic neuritis, peripheral neuritis, gouty arthritis adverse reactions in treatment group and control group. The treatment group and control group in the incidence of adverse reactions was no significant difference (P>0.05).The third part The clinical value and study on method of PCR-reverse line blot hybridization assay of identification of mycobacterium.Research Content1. The first part of the experimentThere are 42 strains of (40 species) standard strains and 546 mycobacterium clinical isolates strains by the membrane chip, DNA extraction, PCR amplification, reverse hybridization line point, gas chromatography, high performance liquid chromatography, PCR product direct sequencing. The standard strains of Mycobacterium is hybrid with the PCR-RLB hybridization conditions to compare the PCR-RLB probe specificity; diluted with ultra pure water the bacteria Mycobacterium tuberculosis H37Rv is 0.5 Maxwell unit, extracting DNA, and then 500pg/μl,50 pg/μl,5 pg/μl,500 fg/μl,50 fg/μl were serially diluted, PCR amplified products of PCR-RLB hybridization was observed imaging spots in the nylon membrane to determine the sensitivity of Mycobacterium probe; by comparing the PCR-RLB hybridization, GC identification, HPLC identification, DNA sequencing, the PCR-RLB test in the identification of Mycobacterium Bacillus species, subtypes, and the specificity of mixed infections. 2. The second part of the experimentCollection of 771 clinical samples, erery clinical samples divided into 4 portions, respectively, according to the steps to do the AFB smear, the mycobacterial culture and identification, the fluorescence quantitative polymerase chain reaction, the DNA extraction, the reverse line dot blot, the PCR product direct sequencing. By comparing the quantitative results of FQ-PCR and PCR-RLB hybridization, the PCR amplification products of M. Tuberculosis is related with the PCR-RLB; the comparing the results of conventional culture and identification, DNA sequencing to PCR-RLB hybridization is the sign of PCR-RLB experimental clinical specificity; the PCR-RLB positive rates were compared with the results for AFB smear, culture and identification results, FQ-PCR quantitative results. Mycobacterial culture and identification were compared with the blot, the required time for analysis.Research ResultsThe first part of the experiment1. PCR-RLB primers and probe specificity:All mycobacterium species are corresponding reaction with Mycobacterium, but some fragments of very similar strains interval, as to M. marinum and M. ulcerans,there is cross-reactive between M. senegalance and FOR1(M. fortuitum a/c and M. senegalance), but M. fortuitumwith and SEN (M. senegalance) does not react, the PCR-RLB hybridization can identify M. chelonae, M. abscessus, M. kansasii and M. gastri, M. avium, M. intracellulare, but the group of sub-species of M. tuberculo sis complex can not identify.2. PCR-RLB probe Sensitivity: The concentration of standard strains of Mycobacterium tuberculosis bacteria is 500pg/μl,50pg/μl,5pg/μl every time, PCR-RLB's nylon membrane were visible imaging, when the concentration of is 500fg/μl, the nylon membrane imaging point looming, when the concentration is 50fg/μl, the nylon membrane is no imaging to determine. the sensitivity of Mycobacterium tuberculosis probe 500fg/L.3. Clinical isolates of detection:Strains of mycobacterium tuberculosis complex in a total of 312, which detected 245 GC, HPLC detection of 67 are the M. tuberculosis complex group, the PCR-RLB results suggest that except for 1 strain of M. Scrofulaceum, inconsistent with GC results, the other group are M. tuberculosis complex, the strains by DNA sequencing confirmed to M. scrofulaceum; M. avium complex in a total are five, five by GC identification were M. avium complex group. The PCR-RLB identified results suggest that 5 is M. intracellular, consistent with the DNA sequencing; a total of 39 strains is M. avium,39 strains were identified M. avium by HPLC, the PCR-RLB identification of 18 strains is M. avium, consistent with the DNA sequencing,17 strains of M. intracel bronchiectasis lular are consistent with the sequencing,4 strains of M. avium and M. kansasii mixed a/b, DNA sequence can not read; M. abscessus complex is 28 strains by GC, by PCR-RLB 26 strains are M. abscessus, the other one was M. chelonae a/b, the other one was M. chelonae c, there were two M. chelonae by DNA sequencing; by HPLC there were 4 M. chelonae, but there were 3 M. chelonae a/b by PCR-RLB identification, consistent with DNA sequencing and 1 M. chelonae mixed M. fortuitum that DNA sequencing can't be read. There were 29 strains, which were 22 M. fortuitum by GC and 7 M. fortuitum by HPLC as the result of PCR-RLB identification. There were 12 M. phlei by GC as according to the PCR-RLB identification results of.4 strains were identified by HPLC the M. intracellular; PCR-RLB identified 2 strains as M. avium,2 strains as M. intracellular, consistent with the DNA sequencing. There were 30 M. scrofulaceum; including GC identification of 16 M. scrofulaceum, but PCR-RLB identified 14 strains as M. scrofulaceum,2 M. scrofulaceum mixed M. Intracellular strains can't read by DNA sequencing, sequence. The other strains identified as M. scrofullaceum by the HPLC consistent with the PCR-RLB. There were 2 M. smegmatis identified by HPLC consistent with PCR-RLB results. There were 6 M. kansasii, including 2 M. kansasii by GC, which was M. kansasii a/b by the PCR-RLB,14 M. kansasii were by HPLC identified. The PCR-RLB identified 2 as m. kansasii a/b,2 as M. kansasii c; the results of DNA sequencing were M. Kansasii. The 2 strains were identified by HPLC as M. ulcerans, PCR-RLB identified as M. marinum/M. ulcerans, the results of DNA sequencing is M. ulcerans. There were 51 M. gordon, of which 36 strains identified by the GC, while the 31 strains is M. gordon, the PCR-RLB identificate 31 as M. gordon,3 mixed Mycobacterium abscessus,2 Mycobacterium mixed M. fortuitum, can not be read by DNA sequencing. The 15 strains by the HPLC and PCR-RLB identification are M. gordon; the 4 strains by HPLC, PCR-RLB and DNA sequencing were M. triple. The 2 strains by HPLC, PCR-RLB and DNA sequencing were M. malmoense. The 2 strains by HPLC, PCR-RLB and DNA sequencing were M. xenopi. The 2 strains by HPLC, PCR-RLB and DNA sequencing were M. szulgai. The 4 strains by HPLC, PCR-RLB and DNA sequencing were M.lentiflavum. The 8 strains by PCR-RLB and DNA sequencing were M. lepraemurium.The second part of the experiment1. PCR-RLB probe sensitivity:Will be identified as Mycobacterium tuberculosis complex in specimens of FQ-PCR and PCR-RLB results were compared, DNA quantification in 5×102-1×104copies/ml group specimen PCR-RLB hybridization showed no positive reaction, DNA Quantitative in 1×104-1×105copies/ml group of samples,49.8% samples PCR-RLB hybridization shows positive results, significantly higher than DNA quantification in 5×102-1×104 copies/ml group (x2 64.9, P<0.01), and DNA quantification in the 1×105-2.3×108 copies/ml of specimen is available on the PCR-RLB hybridization in see positive results, significantly higher than the DNA quantification in the 1×104-1×105 copies/ml group (x2=68.46, P <0.01).2. PCR-RLB specific comparison:Traditional culture and identification results, PCR-RLB hybridization, DNA sequencing results were compared, PCR-RLB identified as 35 strains of NTM, and the results are consistent with the traditional culture and identification; 35 specimens of birds blot, PCR-RLB identified as 7 strains of M. avium, and DNA sequencing results are consistent; 8 strains of M. intracellular, as the result of DNA sequencing; 12 strains of M. absessus, and DNA sequencing results are consistent; 3 strains of M.kansasii,2 strains of M. Kansasii a/b,1 strain of M. Kansasii c, and the sequencing results are 3 strains of M. Kansasii; 2 strains of M. gordon,1 strain of M. chelonae,2 strains of M. fortuitum are consistent with the sequencing. 3. Clinical specimens positive rate comparison:The PCR-RLB hybridization results with the smear for AFB, bacterial culture results and the results were compared with FQ-PCR positive rate. PCR-RLB blot positive rate 39.8%, significantly higher than for AFB smear positive rate of 21.4%(x=61.57, P<0.01), with culture results positive rate of 35.7% was no significant difference (x2=2.65, P>0.05), but significantly lower than the FQ-PCR positive rate of 65.9%(x2=13.67, P<0.01).4. The traditional culture and identification methods and PCR-RLB hybridization and time-consuming comparison:The traditional culture and identification methods spends 28-56 days, while the PCR-RLB hybridization identification of Mycobacterium is cost 1 day, significantly shorter than traditional methods.ConclusionBy studying our secondary pulmonary tuberculosis patients newly diagnosed multiple TCM as lung Yin deficiency syndrome (67%) and Yin deficiency and fire excess (20%) was significantly higher with other syndromes. This understanding of traditional Chinese medicine is consistent also with our clinical symptoms of tuberculosis incidence, tongue, and pulse results consistent.According to the first part of the study, we chose the most common TCM stasis to early Yangyinqingfei treatment of secondary pulmonary smear positive patients with Yin deficiency syndrome in 38 cases; found that short-term efficacy was better than western medicine and no specific adverse reactions.The PCR-RLB test is based on 16S-23S rRNA gene spacer sequence for the target sequences of primers and probe, through the establishment of PCR-RLB, the optimum conditions, and with the standard strains of mycobacteria and clinical isolates of hybridization confirmed the specificity and sensitivity of direct hybridization with the clinical specimens, and with the smear, culture, TB-DNA quantitative comparison, confirmed that the experimental method is sensitive, highly specific, rapid, and is worthy of promotion.