NHP MGIA methods optimisation experiments

In order to minimise the variability associated with low-titre mycobacterial inocula, two stock parameters were assessed: a) time from thawing to inoculation and b) de-clumping methods. Mycobacteria were thawed and added to duplicate human PBMC co-cultures every hour for 5 hours after resting on the bench at room temperature. Mycobacterial viability showed a progressive, albeit modest, decrease for the first 3 hours, before beginning to recover at 4 hours (Figure 5A). Six methods of de-clumping were compared using 6 replicate in-tube co-cultures containing cells from the same human sample for each method: 1) vortexing for 5 minutes on the highest speed, 2) standing on the bench for 5 minutes to allow clumps to settle and then removing only the top fraction, 3) centrifuging at a low speed to bring clumps down and then removing only the top fraction, 4) sonicating for 2 minutes, 5) vortexing with 1mm borosilicate solid-glass beads for 2 minutes, and 6) syringing through a 5µM cellulose acetate filter.<br>Mycobacterial recovery was highest using the glass beads method, while other methods (particularly centrifuging and filtering) resulted in some loss of mycobacteria. BCG growth was significantly higher (lower TTP) following vortexing with glass beads compared with centrifuging or filtering (p=0.0002, delta mean TTP = 90 hours; and p=0.008, delta mean TTP = 83 hours respectively; Kruskal Wallis with Dunn's multiple comparisons test, p=0.0002, Figure 5B). Reproducibility between replicates was greatest for glass beads and filtering (coefficient of variation, CV = 2.2% and 1.2% respectively), and poorest for vortexing (CV = 13%). Based on these findings, we recommend that mycobacterial stocks suffering from clumping should be vortexed with sterile 1mm borosilicate glass beads (Sigma Aldrich, UK) for 2 minutes prior to inoculation, and that inoculation should be conducted as soon after thawing as possible.<br>Due to limitations regarding the maximum blood volume permitted for collection from macaques, plasma may be a more feasible alternative to serum. As specific antibodies are likely the main component of serum contributing to control of mycobacterial growth in the MGIA, we compared levels of PPD-specific IgM, IgG and IgA between serum and plasma from matched animals at baseline. In all cases there was a strong correlation, although serum contained modestly but significantly higher levels of specific antibodies at most time-points measured. We therefore compared the use of autologous serum vs. autologous plasma in the direct NHP MGIA co-culture (n=7 animals), in which other components such as complement factors may also contribute to functional control of mycobacterial growth, and observed an intraclass correlation coefficient (ICC) of 0.58 (moderate agreement). As shown by Bland-Altman analysis relating the difference between paired measurements to the mean of the pair, there was minimal bias between the two methods (mean bias = 0.025). Furthermore, all samples were within the 95% limits of agreement (the interval of 1.96 standard deviations of the measurement differences either side of the mean difference), which extended from -0.20 (95% CI, -0.50 to -0.13) to 0.25 (95% CI, 0.18 to 0.55) log10 CFU (Figure 6). Although the sample size was small and there is some in inherent intra-assay variability, this suggests that plasma may be substituted where serum is unavailable or limited in volume, but we do not recommend using the two samples interchangeably within a single experiment or direct comparison.<br>The effect of heat inactivating serum was assessed by measuring mycobacterial growth at the end of in-tube n=6 human PBMC co-cultures. Mycobacterial growth was lower when co-cultures contained serum that had been heat-inactivated compared with serum that had not been heat inactivated, but this was not statistically significant by Wilcoxon (p=0.06, delta mean TTP = 24 hours; Wilcoxon, Figure 8A). It has been reported that heat inactivation of serum decreases uptake of mycobacteria into monocytes due to the destruction of complement. As monocytes provide the target host cell for mycobacterial survival and replication, a decrease in monocyte invasion may lead to decreased mycobacterial growth. Finally, we compared serum/plasma separated from blood collected in either serum clot-activator or Ethylenediaminetetraacetic acid (EDTA) vacutainers. Adding plasma separated from an EDTA vacutainer to the MGIA co-culture resulted in significant inhibition of mycobacterial growth (p=0.03, delta mean TTP = 68 hours; Wilcoxon, Figure 8B). EDTA has been shown to have anti-tubercular activity and has even been suggested for potential use in treatment of drug-resistant TB. Based on these findings, we recommend that autologous serum/plasma should be added to a final concentration of 20%, should not be heat-inactivated and should not be collected in vacutainers containing EDTA.

为降低与低滴度分枝杆菌接种物相关的实验变异，本研究评估了两项分枝杆菌储备液参数：a）从解冻至接种的时间间隔，b）去团块方法。将分枝杆菌解冻后，于室温静置在实验台面上，每小时将其接种至重复的人外周血单个核细胞（Peripheral Blood Mononuclear Cell, PBMC）共培养体系中，持续5小时。结果显示，分枝杆菌活力在最初3小时呈渐进式小幅下降，直至第4小时才开始恢复（图5A）。
本研究共比较了6种去团块方法，每种方法均使用来自同一人体样本的细胞开展6次重复管内共培养：1）最高转速涡旋振荡5分钟；2）静置实验台5分钟使团块沉降，随后仅取上清组分；3）低速离心使团块沉降，随后仅取上清组分；4）超声处理2分钟；5）加入1mm硼硅酸盐实心玻璃珠后涡旋振荡2分钟；6）通过5μm醋酸纤维素滤器过滤接种物。
采用玻璃珠法的分枝杆菌回收率最高，其余方法（尤其是低速离心与过滤法）均会造成一定程度的分枝杆菌损失。与低速离心或过滤法相比，经玻璃珠涡旋处理后的卡介苗（Bacillus Calmette-Guérin, BCG）生长显著更优（时间到阳性（Time to Positivity, TTP）更短）：前者组间比较的P值为0.0002，平均TTP差值为90小时；后者P值为0.008，平均TTP差值为83小时（采用克鲁斯卡尔-沃利斯（Kruskal Wallis）检验结合邓恩（Dunn）多重比较检验，总P=0.0002，图5B）。重复样本间的重现性以玻璃珠法与过滤法最佳，变异系数（Coefficient of Variation, CV）分别为2.2%与1.2%；涡旋振荡法的重现性最差，CV为13%。基于上述结果，我们建议：存在团块问题的分枝杆菌储备液，应在接种前加入无菌1mm硼硅酸盐玻璃珠（西格玛奥德里奇（Sigma Aldrich），英国）涡旋振荡2分钟；且接种应尽可能在解冻后尽早开展。

鉴于从非人灵长类动物（non-human primate, NHP）体内采集血液的最大体积存在限制，血浆或许是血清更为可行的替代样本。由于特异性抗体可能是血清中控制分枝杆菌生长的核心组分，我们在基线时比较了配对非人灵长类动物血清与血浆中结核菌素纯蛋白衍生物（Purified Protein Derivative, PPD）特异性IgM、IgG与IgA水平。结果显示，二者间存在强相关性，但多数检测时间点的血清特异性抗体水平均显著小幅高于血浆。因此，我们在直接非人灵长类分枝杆菌生长抑制实验（Mycobacterial Growth Inhibition Assay, MGIA）共培养体系（n=7只动物）中比较了自体血清与自体血浆的应用效果——该体系中补体等其他成分也可能参与分枝杆菌生长的功能调控，结果得到组内相关系数（intraclass correlation coefficient, ICC）为0.58，提示二者具有中等一致性。如Bland-Altman分析所示（将配对测量值的差值与配对均值进行关联分析），两种方法间的偏倚极小（平均偏倚为0.025）。此外，所有样本均落在95%一致性限值范围内（即均值差值两侧各1.96倍测量差值标准差的区间），该区间范围为-0.20（95%置信区间（Confidence Interval, CI）：-0.50~-0.13）至0.25（95% CI：0.18~0.55）log10 CFU（菌落形成单位（Colony-Forming Unit, CFU））（图6）。尽管样本量较小且存在固有的批内变异，但上述结果提示：当血清不可获取或体积有限时，可采用血浆作为替代样本；但我们不建议在单次实验中或直接比较时互换使用这两种样本。

本研究通过测量管内n=6例人PBMC共培养体系终点的分枝杆菌生长情况，评估了血清热灭活的影响。相较于未经过热灭活的血清，添加热灭活血清的共培养体系中分枝杆菌生长水平更低，但经威尔科克森（Wilcoxon）检验分析，该差异未达到统计学显著性（P=0.06，平均TTP差值为24小时；图8A）。已有研究表明，血清热灭活会破坏补体，从而降低单核细胞对分枝杆菌的摄取能力。而单核细胞是分枝杆菌存活与复制的宿主靶细胞，单核细胞侵袭能力的下降可能会导致分枝杆菌生长水平降低。最后，我们比较了分别使用血清促凝管与乙二胺四乙酸（Ethylenediaminetetraacetic acid, EDTA）真空采血管采集血液后分离得到的血清/血浆的效果。将EDTA真空采血管分离得到的血浆添加至MGIA共培养体系，会显著抑制分枝杆菌生长（P=0.03，平均TTP差值为68小时；威尔科克森检验，图8B）。已有研究证实EDTA具有抗结核活性，甚至被提议用于耐药结核病的潜在治疗。基于上述结果，我们建议：自体血清/血浆的最终添加浓度应为20%，且不应进行热灭活，同时不应使用含EDTA的真空采血管采集血液。