Fitzgerald Felicity, Harris Kathryn, Doyle Ronan, Alber Dagmar, Klein Nigel
1 Paediatrics Department, North Middlesex University Hospital , London, United Kingdom .
AIDS Res Hum Retroviruses. 2013 Dec;29(12):1589-93. doi: 10.1089/AID.2013.0097. Epub 2013 Oct 2.
Microbial translocation (MT) from the gut is implicated in driving immune activation, increasing morbidity and mortality in HIV. We used bacterial 16S rDNA PCR, Sanger sequencing, and high-throughput sequencing to identify microbial DNA in the bloodstream of HIV-infected children in London, United Kingdom. Blood samples were collected from sequential children attending the HIV clinic at Great Ormond Street Hospital, London. DNA extraction, broad range 16S rDNA PCR, and standard Sanger sequencing were carried out. A subset of positive samples was analyzed by high-throughput sequencing (Roche 454 platform). Of 105 samples collected from sequential children, nine were positive using broad range 16S rDNA PCR (8.6%; 95% CI 4.4-16%). From three amplicons, 16S rDNA sequences were identified as Streptococcus, Propionibacterium acnes, and coagulase-negative Staphylococcus. Four positive samples were analyzed by high-throughput sequencing. In the three samples in which organisms were identified by Sanger sequencing, the same species were identified. Further species, in differing proportions, were identified in all four samples. The identified organisms included known gut orders Bifidobacteriaceae, Lactobacillaceae, Bacteroidales, and Clostridiales. In immunocompetent children of equivalent age, no bacterial DNA was detected in blood using this approach. This is the first study to our knowledge using molecular techniques to identify MT in children in the developed world. Our data indicate that 16S rDNA is detectable in 8.6% of HIV-infected children. Levels of DNA were low and from multiple bacterial species. Further studies are needed to ascertain the importance of MT in HIV-infected children.
肠道微生物易位（MT）被认为与免疫激活、增加HIV患者的发病率和死亡率有关。我们使用细菌16S rDNA聚合酶链反应（PCR）、桑格测序和高通量测序来鉴定英国伦敦HIV感染儿童血液中的微生物DNA。从伦敦大奥蒙德街医院HIV门诊的连续就诊儿童中采集血样。进行DNA提取、广谱16S rDNA PCR和标准桑格测序。对一部分阳性样本进行高通量测序（罗氏454平台）。在从连续儿童中采集的105份样本中，9份使用广谱16S rDNA PCR呈阳性（8.6%；95%置信区间4.4 - 16%）。从三个扩增子中，16S rDNA序列被鉴定为链球菌、痤疮丙酸杆菌和凝固酶阴性葡萄球菌。对4份阳性样本进行高通量测序。在通过桑格测序鉴定出微生物的3份样本中，鉴定出了相同的物种。在所有4份样本中还鉴定出了比例不同的其他物种。鉴定出的微生物包括已知的肠道菌目双歧杆菌科、乳杆菌科、拟杆菌目和梭菌目。在年龄相当的免疫功能正常儿童中，使用这种方法在血液中未检测到细菌DNA。据我们所知，这是第一项在发达国家使用分子技术鉴定儿童MT的研究。我们的数据表明，在8.6%的HIV感染儿童中可检测到16S rDNA。DNA水平较低且来自多种细菌物种。需要进一步研究以确定MT在HIV感染儿童中的重要性。
