肺结核患儿胃液样本结核分枝杆菌培养阳性率提高方法的探讨

目的:建立提高肺结核患儿胃液样本结核分枝杆菌(Mycobacterium tuberculosis,MTB)培养阳性率的方法，同时，探讨培养液的pH值对MTB抗原-胶体金检测试剂正确判读结果的影响和磷酸盐缓冲液(phosphate buffer solution, PBS)对结核样本前处理液pH值的调节作用。方法:将70份疑似肺结核患儿胃液样本同时用两步培养法、传统培养法和Xpert Ultra进行检测。两步培养法为先将胃液样本直接接种到罗氏培养基上，约20～30 d后收集可疑MTB菌落再行常规的碱处理-中和离心法处理进行分离培养(传统培养法)。比较传统培养法和两步法阳性检出率的差异；以样本的Xpert Ultra检测结果作为菌载量标准，分析两步法在不同菌载量样本中的阳性检出率差异。同时，配制系列pH值梯度的溶液，确定MTB抗原-胶体金检测试剂获得最佳显示结果的最适pH值范围；用系列体积的PBS缓冲液(pH=6.8)中和3%或4%NaOH与等体积样本的混合液，确定pH值至7～9时所需的体积稀释倍数。结果:传统培养法阳性检出率为26.5%(18/68),两步法阳性检出率为23.1%(15...

Exploration for improving the culture-positive rate of Mycobacterium tuberculosis in gastric fluid specimens from pediatric patients with pulmonary tuberculosis

Objective: To establish a method to improve the positive rate of culture for Mycobacterium tuberculosis (MTB) in gastric fluid specimens from pediatric patients with pulmonary tuberculosis, and to investigate the effect of culture medium pH on the correct interpretation of a tuberculosis antigen-colloidal gold assay and the effect of phosphate buffer solution (PBS) buffer on the pH of the NaOH-sample mix-solution. Methods: Seventy gastric juice samples received from suspected tuberculosis children were detected by self-established two-step culture method, traditional culture method and Xpert Ultra at the same time. The two-step culture method was to inoculate the gastric fluid specimens directly on the Lowenstein-Jensen (L-J) media first, and then, collect the suspected MTB colonies after about 20 to 30 days and treat them with sodium hydroxide-neutralization-centrifugation (traditional method) for MTB isolation. The difference of positive detection rate between traditional culture method and two-step method was compared. Take the Xpert Ultra test results of the sample as the bacterial load standard, the difference of positive detection rate of the two-step method in different bacterial load samples was analyzed. Meanwhile, a series of solutions with gradient pH were prepared to determine the optimal pH value for MTB antigen-colloidal gold diagnosis kit; the mixture of 3% or 4% NaOH and equal volume sample were neutralized with PBS buffer (pH=6.8) of a series of volumes, and the volume dilution ratio required when the pH value reaches 7-9 were determined. Results: The MTB positive rates were 26.5% (18/68) by the traditional method and 23.1% (15/65) by the two-step culture method, there was no statistically significant difference (χ²=0.125, P>0.05). The two-step method raised the positive detection rate from 26.5% (18/68) to 32.4% (22/68) based on the traditional method. The positive isolation rate of the two-step method in samples with bacterial load ≥low grade (44.0%, 11/25) was significantly higher than that in samples with load ≤very low grade (11.8%, 4/34) (χ²=7.896, P=0.005). The optimal pH range for the chosen tuberculosis antigen-colloidal gold diagnosis kit was 6-9. After being treated with 3% or 4% NaOH, adjustment of pH to 7-9 required at least 10- or 14-fold dilution with PBS buffer (pH=6.8) for gastric fluid simulative samples, and at least 12- or 16-fold dilution for sputum simulative samples. Conclusion: The positive detection rate of the two-step isolation culture method was not found better than the traditional method, however, it could be used as a supplement to the conventional method to improve the overall positive isolation rate of gastric fluid samples, and was especially recommended for the MTB detection in children with highly suspected but difficultly diagnosed pulmonary tuberculosis. In addition, this study investigated the optimal pH range for the use of MTB antigen-colloidal gold diagnosis kit and the optimal dilution ratio of neutralizing the NaOH-sample solution with PBS (pH=6.8), which could provide scientific basis for standardizing the isolation of MTB in China.