Post-mortem Nasopharyngeal Microbiome Analysis of Zambian Infants with and without Respiratory Syncytial Virus Disease: A Nested Case Control Study

Our study compares the nasopharyngeal microbiome of the deceased RSV+ and deceased RSV- infants from the Zambia Pertussis and RSV Infant Mortality Estimation (ZPRIME) study. Infants were between four days and six months old and enrolled in the ZPRIME study within 48 hours of death. We selected a subset of community deaths for our analysis who were RSV+ with a PCR Ct<35. We selected 50 RSV+ infants via random sampling from and matched those to 50 RSV- infants from the same study. Due to age- and seasonality-related variations in RSV impact, we matched infants by age at death (4-28 days (0+ months), 29-61 days (1+ month), 62-152 days (2+ months) and 152-182 days (4+ months)) and date of death (+/- 1.5 months). All decedents underwent one-time, post-mortem NP sampling. NP samples were tested for RSV by reverse transcriptase quantitative PCR following the testing protocol developed by the Centers for Disease Control and Surveillance. 16S ribosomal DNA was amplified using PCR with primers specific to the V3-V4 region.

本研究对比了赞比亚百日咳与呼吸道合胞病毒（Respiratory Syncytial Virus, RSV）婴儿死亡率估算（ZPRIME）研究中，RSV阳性已故婴儿与RSV阴性已故婴儿的鼻咽微生物组（nasopharyngeal microbiome）。纳入研究的婴儿月龄介于4天至6个月之间，且于死亡后48小时内入组ZPRIME研究。本研究选取该研究中社区死亡的亚组样本进行分析，这些样本的RSV检测结果为阳性且聚合酶链式反应（Polymerase Chain Reaction, PCR）循环阈值（Ct）<35。我们通过随机抽样选取50名RSV阳性婴儿，并将其与同研究中的50名RSV阴性婴儿进行匹配。鉴于RSV感染的影响存在年龄与季节相关的差异，我们按照死亡年龄（4~28天（0+月龄）、29~61天（1+月龄）、62~152天（2+月龄）及152~182天（4+月龄））与死亡日期（±1.5个月）对婴儿进行匹配。所有死者均接受了一次性死后鼻咽拭子采样。参照美国疾病控制与监测中心制定的检测方案，采用逆转录定量聚合酶链式反应（reverse transcriptase quantitative PCR, RT-qPCR）对鼻咽样本进行RSV检测。随后采用针对V3-V4区的特异性引物，通过PCR扩增16S核糖体DNA（16S ribosomal DNA）。