Detection of pathogens associated with early-onset neonatal sepsis in cord blood at birth using quantitative PCR

Abstract

Background: Early onset neonatal sepsis (EONS) typically begins prior to, during or soon after birth and may be rapidly fatal. There is paucity of data on the aetiology of EONS in sub-Saharan Africa due to limited diagnostic capacity in this region, despite the associated significant mortality and long-term neurological impairment. Methods: We compared pathogens detected in cord blood samples between neonates admitted to hospital with possible serious bacterial infection (pSBI) in the first 48 hours of life (cases) and neonates remaining well (controls). Cord blood was systematically collected at Kilifi County Hospital (KCH) from 2011-2016, and later tested for 21 bacterial, viral and protozoal targets using multiplex PCR via TaqMan Array Cards (TAC). Results: Among 603 cases (101 [17%] of whom died), 179 (30%) tested positive for ≥1 target and 37 (6.1%) tested positive for multiple targets. Klebsiella oxytoca, Escherichia coli/Shigella spp., Pseudomonas aeruginosa, and Streptococcus pyogenes were commonest. Among 300 controls, 79 (26%) tested positive for ≥1 target, 11 (3.7%) were positive for multiple targets, and K. oxytoca and P. aeruginosa were most common. Cumulative odds ratios across controls: cases (survived): cases (died) were E. coli/Shigella spp. 2.6 (95%CI 1.6-4.4); E. faecalis 4.0 (95%CI 1.1-15); S. agalactiae 4.5 (95%CI 1.6-13); Ureaplasma spp. 2.9 (95%CI 1.3-6.4); Enterovirus 9.1 (95%CI 2.3-37); and Plasmodium spp. 2.9 (95%CI 1.4-6.2). Excluding K. oxytoca and P. aeruginosa as likely contaminants, aetiology was attributed in 9.4% (95%CI 5.1-13) cases using TAC. Leading pathogen attributions by TAC were E. coli/Shigella spp. (3.5% (95%CI 1.7-5.3)) and Ureaplasma spp. (1.7% (95%CI 0.5-3.0)). Conclusions: Cord blood sample may be useful in describing EONS pathogens at birth, but more specific tests are needed for individual diagnosis. Careful sampling of cord blood using aseptic techniques is crucial to minimize contamination. In addition to culturable bacteria, Ureaplasma and Enterovirus were causes of EONS.

Keywords: Neonate; PCR; aetiology; molecular; sepsis.

Figure 1.. Organisms included in the whole blood TaqMann Array Card panel.

Pathogens interrogated with TaqMan Array Card (TAC) in whole blood. The TAC is a 384-well real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based platform consisting of 8 (shown as no. 1 to 8 above) individual microfluidic channels that can be loaded with PCR reactions containing nucleic acid extract from a clinical specimen or control material. The TAC was customised to include 21 targets (16 bacterial, 4 viral and 1 protozoal organism, tested in duplicate) and two controls (MS2 bacteriophage and phocine Herpesvirus (PhHV)). The 21 targets are shown in alphabetical order as follows: Acinetobacter baumanii, Cytomegalovirus, Enterococcus faecalis, Enterovirus, Escherichia coli/ Shigella spp., Haemophilus influenzae, Herpes Simplex Virus 1, Herpes Simplex Virus 2, Klebsiella oxytoca, Klebsiella pneumoniae, Listeria monocytogenes, Mycobacterium tuberculosis, Neisseria meningitidis, Plasmodium spp., Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, and Ureaplasma spp.

Figure 2.. Study Participant Flow.

Selection of cases and controls from a cohort of 15,409 deliveries at Kilifi County Hospital (KCH) between March 2011 and March 2016. Cases were hospitalised within the first 48 hours of life, resident of the Kilifi Health Demographic Surveillance System (KHDSS) and presented with one or more of the WHO-defined criteria for possible serious bacterial infection (pSBI). Controls were resident of the KHDSS and not hospitalised within the first 60 days of life.

Figure 3.. Patterns of detection of Taqmann PCR Targets.

Organisms included in the TAC were detected in 4 distinct groups: Group 1 (Herpes Simplex Virus 1, Herpes Simplex Virus 2, Staphylococcus aureus, Salmonella enterica, Streptococcus pneumoniae, and Acinetobacter baumanii); Group 2 ( Hemophilus influenzae, Neisseria meningitidis, Enterococcus faecalis, Enterovirus, Plasmodium spp., and Streptococcus agalactiae); Group 3 (Cytomegalovirus, Streptococcus pyogenes, Pseudomonas aeruginosa, and Klebsiella oxytoca); and Group 4 ( Klebsiella pneumoniae, Ureaplasma spp. and Escherichia coli/Shigella spp.). Weighting of cases (represented as cases/2) was done to allow for improved accuracy in assessing the distribution of organisms tested, comparing cases to controls, since cases (n=603) were ~twice more than controls (n=300). Weighting was done by calculating the number of eligible cases/controls divided by the number of enrolled cases/controls i.e. the inverse of the sampling fraction for cases/controls.