Using RNA Sequencing of High-Risk Placentas to Diagnose Congenital Infection

Background:
Diagnosis of congenital infection and early onset neonatal sepsis is inadequate [1]. Neonatal serology, blood cultures and polymerase chain reaction (PCR) diagnostics are limited by false positive immuno-assays, passive maternal immunoglobulin transfer, small sample volumes and low pathogen load [2]. The current "gold standard" diagnosis of congenital infection requires pathogen specific assessment of culture, serology and PCR, often at the level of the National Reference Laboratory. Great Ormond Street Hospital and the Institute of Child Health are leaders in the field of central nervous system (CNS) pathogen diagnosis using next generation RNA (Ribonucleic Acid) sequencing [3]. Despite the abundance of placental material and the critical importance of the placenta in vertically transmitted infection, no published study has yet performed next generation RNA sequencing on placental samples in which congenital infection is clinically suspected. It is well established that placental histopathology, although of low sensitivity, is a proxy for congenital infection [4]. RNA sequencing has the advantage of unbiased sensitive detection of pathogen RNA which negates the need to send multiple individual diagnostic samples. The prevalence of vertically transmitted infection in the UK is too low to power a single centre study [5]. The proposed PhD project benefits from the ability to recruit patients and collect samples from three existing studies already underway in Peru, Bolivia and Uganda where the prevalence of infection is significantly higher.

Aims/Objectives:
1)  Collect high risk placentas from field sites (either Uganda or Peru) as part of existing studies;
2)  Learn RNA extraction and analytical techniques and perform this on the high-risk placentas;
3)  Write PhD, peer-reviewed publications and feed information back to health professionals.

Methods:
Ethical and institutional approval has already been approved by Universidad Peruana Cayetano Heredia, Harvard School of Medicine and Johns Hopkins School of Public Health as well as the Peruvian Ministry of Health. Additional ethical approval will also be obtained from UCL before the study commences. High risk placentas will be selected using the risk factors proposed by Ford-Jones et al as well as control samples with no risk factors. Metadata including gestational age, co-morbidities (e.g. pre-eclampsia), gender, parity and maternal age will also be collected as these have been shown to influence the placental host RNA expression. Two cores will be stored immediately in RNAlater to preserve sample RNA, the third sample will be stored in formalin for histological analysis in future studies. Total RNA will be purified from placental samples at the Institute of Child Health, London using the Qiagen RNeasy kit and 100bp paired-end sequencing will be performed on the Illumina NextSeq 500 platform. Taxonomer, a recently published web-based platform for rapid and sensitive metagenomic analysis will also be used for pathogen sorting and diagnosis.

Timeline:

References:

1.  Ng S, Strunk T, Jiang P, Muk T, Sangild PT, Currie A. Precision Medicine for Neonatal Sepsis. Front Mol Biosci. 2018;5: 70. doi:10.3389/fmolb.2018.00070.
2.  Klingenberg C, Kornelisse RF, Buonocore G, Maier RF, Stocker M. Culture-Negative Early-Onset Neonatal Sepsis - At the Crossroad Between Efficient Sepsis Care and Antimicrobial Stewardship. Front Pediatr. 2018;6: 285. doi:10.3389/fped.2018.00285.
3.  Morfopoulou S, Brown JR, Davies EG, Anderson G, Virasami A, Qasim W, et al. Human Coronavirus OC43 Associated with Fatal Encephalitis [Internet]. 2016. doi:10.1056/NEJMc1509458.
4.  Heerema-McKenney A. Defense and infection of the human placenta. APMIS Acta Pathol Microbiol Immunol Scand. 2018;126: 570–588. doi:10.1111/apm.12847.
5.  Abdel-Fattah SA, Bhat A, Illanes S, Bartha JL, Carrington D. TORCH test for fetal medicine indications: only CMV is necessary in the United Kingdom. Prenat Diagn. 2005;25: 1028–1031. doi:10.1002/pd.1242.