我国卡介苗生产用菌种工作种子批及成品过度皮肤反应试验

Test for excessive dermal reactivity of seeds from working seed lot and final product of BCG in China

目的进行我国皮内注射用卡介苗生产用菌种工作种子批及成品的过度皮肤反应试验,探讨在菌种及成品检定项目下是否需要增加过度皮肤反应试验。方法对种子批及种子批制备的样品进行复溶和连续10倍稀释,分别经体重不低于250 g的健康豚鼠皮内接种0.1 m L复溶疫苗、1∶10稀释菌液、1∶100稀释菌液,连续4周观察注射部位的反应情况,测量皮肤反应直径,记录皮肤坏死与结节形成情况,并与相同稀释度的有关参考品及国外卡介苗制品进行比较。分析皮肤反应直径大小与卡介苗制品浓度、沉降率及活菌数的相关性。结果国内皮内注射用卡介苗生产用菌种工作种子批复溶疫苗、1∶10稀释菌液注射4周后的皮肤反应直径分别为(3.28±0.41)和(2.76±0.36)mm,1∶100稀释菌液已完全愈合,未测出。47批国内皮内注射用卡介苗制品的复溶疫苗、1∶10稀释菌液的皮肤反应直径分别为(3.92±0.45)和(2.61±0.69)mm,1∶100稀释菌液有31批未测出,16批的反应直径为(0.72±1.02)mm,与卡介苗活菌数国家参考品及生产用菌种工作种子批反应直径无差异。国外卡介苗制品3个浓度皮肤反应直径分别为(4.66±0.60)、(3.70±0.68)和(1.99±1.73)mm,反应程度高于国产卡介苗制品。皮肤反应直径大小与沉降率呈正相关(P=0.000),与卡介苗制品浓度及活菌数无显著相关性。结论过度皮肤反应可能主要与菌种有关;在确定的菌种情况下,可能与疫苗制造过程中颗粒大小的均匀度、浓度及活菌数的相关性更大;我国卡介苗规程通过对浓度、沉降率、活菌数的控制,可以从生产过程中控制成品引起皮肤反应的程度。目前在我国卡介苗规程中设置过度皮肤反应试验的意义并不大。

Objective To discuss the necessity of adding the item of the test for excessive dermal reactivity of seeds from working seed lot and final product of BCG in China. Methods The seeds from seed lot as well as the samples prepared with the seeds were reconstituted and 10-fold serially diluted. Healthy guinea-pigs with body weights of not less than 250 g were injected i. d. with 0. 1 m L of the reconstituted BCG vaccine and the vaccines at dilutions of 1 ∶ 10 and 1 ∶ 100 respectively,and observed for the reaction in injection site for 4 weeks,while the diameter of the skin reaction was measured,and the skin necrosis and formation of duration were recorded. The results were compared with those of references and imported BCG products, at the same dilutions. The relationships of skin reaction diameter to the concentration,sedimentation rate and viable count of BCG product were analyzed. Results The skin reaction diameters 4 weeks after injection with reconstituted BCG vaccine prepared with working seeds and the vaccine at a dilution of 1 ∶ 10 were（3. 28 ± 0. 41）and（2. 76 ± 0. 36）mm respectively,while that with vaccine at a dilution of 1 ∶ 100 was undetected.The reaction skin diameters of 47 batches of reconstituted BCG vaccine for intradermal injection and the vaccine at a dilution of 1 ∶ 10 were（3. 92 ± 0. 45） and（2. 61 ± 0. 69） mm respectively,while were undetected in 37 batches of vaccine at a dilution of 1 ∶ 100,and（0. 72 ± 1. 02）mm in the other 16 batches,which were basically consistent with those of national reference for BCG viable count and the seeds from seed lot. However,the skin reaction diameters of imported BCG vaccines at the three dilutions were（4. 66 ± 0. 60）,（3. 70 ± 0. 68）and（1. 99 ± 1. 73）mm respectively. The skin reaction diameter was positively related to the sedimentation rate（P = 0. 000）,while showed no relationship to the concentration and viable count of BCG product. Conclusion Excessive skin reaction may be mainly related to the BCG strain. In the case of given strain,it may be more relevant to the uniformity,concentration and viable count of the particles in the vaccine manufacturing process. In the requirements for BCG vaccine,the degree of skin reaction caused by the final product may be controlled through the control of concentration,sedimentation rate and the viable count during the production. Therefore,it is not significant to set the test for excessive dermal reactivity in the requirements for BCG vaccine in China at present.