Bacterial DNA detection in very preterm infants assessed for risk of early onset sepsis

Subjects

This prospective study was conducted in a single tertiary neonatal intensive care unit from October 2017 until September 2018. Inclusion criteria were: preterm delivery ≤32 weeks of gestation, inborn neonates and collection of blood samples completed within the first 2 h after delivery. Exclusion criteria were: incomplete neonatal records, insufficient sample volume and congenital malformations. The study protocol has complied with all the relevant national guidelines, institutional policies and was in accordance with the tenets of the Helsinki Declaration, and has been approved by local Ethics Committee (2015/06-02-4) and local Committee on Human Research. Written informed consent was obtained from parents of each enrolled infant.

Clinical and histological chorioamnionitis

Clinical chorioamnionitis was defined as: maternal body temperature ≥37.8 °C and a minimum of two other criteria from: maternal or fetal tachycardia, uterine tenderness, malodorous vaginal discharge and leukocytosis ≥15,000 mm⁻³. Acute inflammatory lesions of the placenta were histologically characterized by the infiltration of neutrophils in the chorion and amnion (acute chorioamnionitis) or in the placenta (intervillositis, deciduitis, or villositis). Funisitis was referred to when the inflammatory process involved the umbilical cord (umbilical vein, umbilical artery, and the Wharton’s jelly) [14].

Blood sampling and tests

Whole blood sample was collected aseptically from the neonate within the first 2 h after delivery through a newly inserted catheter (umbilical vein, umbilical artery or peripheral vein). Blood culture (1 mL) and bacterial DNA analysis were performed, whole blood count, C-reactive protein (CRP), procalcitonin (PCT) and interleukin 6 (IL-6) were determined. The real-time PCR sample was collected into a special tube (volume 1.6 mL – BD Vacutainer Plus Plastic with sodium citrate 0.129 M). The volume of blood collected was 500 μL.

Blood culture

Blood cultures were investigated using the BacT/Alert automated blood culture monitoring system (Bact/Alert, BioMerieux, US). Immediately after sample collection, the blood samples were transported to the microbiology laboratory. Here they were loaded into the Bact/Alert culture instrument and were incubated for a standard period of 5 days (120 h) before being flagged as negative. Bottles flagged up as positive were subcultured on Columbia agar +5% sheep blood (BioMerieux), UriSelect™ 4 Medium (BioRad) and Schaedler agar +5% sheep blood (BioMerieux) plates and a microscopic slide for Gram stain was prepared. After 4–6 h of incubation, the streaked agar plates were inspected and in case of a detectable growth, the visible colonies were identified using the Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) on a MicroFlex LT/SH smart platform (Bruker Daltonik GmbH).

Conventional microbiological methods

High Vaginal Swabs were collected from all women with imminent preterm delivery. Neonatal swabs (ear, stomach and axilla) were taken immediately after admission to NICU. Cotton-tipped swabs were placed in AMIES agar (Micro-Trans Swab, Med-Lab Trade, Czech Republic), then inoculated to sheep blood agar, chocolate agar, and MacConkey agar and incubated aerobically. Organisms were identified by standard microbiological methods.

Bacterial DNA extraction

Bacterial DNA was extracted from citrated blood samples by using a QIAamp DSP DNA Mini Kit, according to the manufacturer’s protocol (QIAGEN, Hilden, Germany). Briefly, DNA was extracted from 500 µL of citrated blood and eluated into 50 µL of elution buffer. The eluates were stored at −20 °C until further analysis.

Real time PCR analysis

Two multiplex real time PCR assays were used during the study period to detect bacterial nucleic acid for the evaluation of infections in neonates. BactoPlexx Real-Time PCR Kit (KITGEN, Třinec, Czech Republic) and FTD Neonatal sepsis (Fast Track Diagnostics, Luxembourg) were used for the amplification and detection of the pathogens. Nucleic acid input was 2 µL for BactoPlex and 10 µL for FTD Neonatal sepsis. Initial denaturation and thermocycling (°C/seconds) were 94/300 and 94/30, 58/30, 72/30 for BactoPlex assay, for FTD Neonatal sepsis assay they were 94/180 and 94/8, 60/34 respectively. Cycles setting was 50 for Bactoplex and 40 for FTD Neonatal sepsis. Cycles positivity assessment was ≤35 for both assays. The quantitative real-time PCR assays were processed on Rotor-Gene Q (QIANGEN, Hilden, Germany). BactoPlex Real-Time PCR Kit was used until an assay validated for neonatal samples became available. It enables to detect a wide scale of bacterial pathogens: Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa/putida, Serratia marcescens, Stenotrophomonas maltophilia, Enterococcus faecalis, Enterococcus faecium, coagulase-negative staphylococci, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus spp. FTD Neonatal sepsis assay was used for the detection of Group B Streptococcus/Streptococcus agalactiae, Listeria monocytogenes, E. coli, S. aureus, Chlamydia trachomatis and Ureaplasma urealyticum/parvum. Each PCR test was performed in conjunction with three controls; positive control, negative control and an internal control. The assay uses Streptococcus equi as an internal control which is introduced into each sample and it also shows the correctness of the extraction process. The individual pathogen specific PCRs had been evaluated previously. Primer and probes used for each reaction are available from the manufacturer.

The presence of specific pathogen sequences in the reaction was detected by an increase in observed fluorescence from the relevant dual-labeled probe, and was reported as a cycle threshold value (Ct) by the Real-Time thermocycler. Subsequently, Ct analysis was performed. The number of cycles was used to quantify bacterial DNA load (BDL).

Neonatal blood count and biomarkers

Blood counts were measured with a Coulter Micro Dif II (Coulter Electronics Ltd., Fullerton, US). CRP was measured by immunoturbidimetry (Cobas 6000, c501 module, Roche Diagnostics, Mannheim, Germany). Immunoluminometric assay (Lumitest PCT, Brahms, Germany) was used for PCT analysis. Luminescence was measured automatically in a Berilux Analyser (Behring Diagnostics, Germany). Serum IL-6 level was assessed by electrochemiluminescence immunoassay (Cobas 6000, e601 module, Roche Diagnostics, Mannheim, Germany).

Early onset sepsis and neonatal outcome

Data were collected from neonatal and maternal medical records. The definition of EOS was based on positive blood culture and clinical signs of infection or negative blood culture with manifested clinical symptoms and abnormal levels of two or more biomarkers in the first 72 h of life. The range of 5,000–20,000 white blood cells (WBC) in a microliter of blood was considered as normal. An I:T ratio of ≥0.2, CRP >10 mg/L, PCT >2 μg/L and IL-6 >200 ng/L were considered as abnormal [3, 15, 16]. Clinical signs of an early onset infection included: hypothermia, respiratory instability (apnea, desaturations, respiratory distress syndrome with ongoing mechanical ventilation), cardiac (cyanosis, bradycardia, poor peripheral perfusion, hypotension) and neurological (lethargy, suspected seizures) symptoms [17]. Higher risk of EOS was considered in the presence of clinical and/or laboratory chorioamnionitis, Group B Streptococcal (GBS) and/or E. coli (E. coli) colonization of the mother, preterm premature rupture of membranes (PPROM) and preterm onset of labor [18]. Antibiotics were started by the attending physician according to the presence of risk factors, clinical and/or laboratory signs of infection. After 48 h, a decision was made whether to continue with antibiotic treatment according to blood culture findings and the dynamics of clinical signs, blood count and levels of serum biomarkers. Attending physicians were made aware of the PCR result once available, but the obtained result was not considered crucial for the treatment algorithm.

Other neonatal outcomes (respiratory distress syndrome, patent ductus arteriosus, intraventricular hemorrhage, necrotizing enterocolitis, periventricular leukomalacia, retinopathy of prematurity and bronchopulmonary dysplasia) were followed up according to the Vermont-Oxford definition [19].

Statistical analysis

Data were reported using descriptive statistical methods. Univariate analyses were performed using Chi-square, Fisher’s exact and Mann-Whitney U tests. All reported p-values are two-sided and not adjusted for multiplicity. A value of p<0.05 was considered statistically significant. Data analysis was performed using the IBM SPSS Statistics 25.0.0.0 software (IBM Corp., Armonk, NY).

PCR positive group

Twenty-nine infants were assessed by BactoPlex Real-Time PCR Kit, 53 by FTD Neonatal sepsis. Nineteen neonates were PCR positive. Seven positive results were assessed by BactoPlex Real-Time PCR Kit, 12 by FTD Neonatal sepsis. No difference was found in the capability to detect bacterial DNA between both tests (24% vs. 23% respectively, p>0.99). E. coli was the most frequently detected strain (12 episodes, 63%). The remaining seven positive results included Streptococcus species (2 episodes), S. aureus (2), S. maltophilia (1), Ureaplasma parvum (1) and Coagulase-negative Staphylococci (1). The summary of microbiological and PCR findings of positive patients is expressed in Table 2. Only one patient had a positive blood culture with a corresponding PCR result (E. coli). Three patients regarded as infected had a negative blood culture and positive PCR result (S. aureus, U. parvum, E. coli). Eleven patients were treated as infected despite no pathogen detection. This shows a low PCR sensitivity of 27% for EOS. In two cases the clinical team decided to discontinue antibiotic treatment after 48 h despite a positive PCR result (S. maltophilia, E. coli). Forty-two patients received no antibiotic treatment for EOS and had no clinical and/or laboratory signs of early infection. Thirteen of them were positive for PCR. This shows a moderate specificity and a negative predictive value of 78% and 83%, respectively. The accuracy of the PCR test for EOS was 68%. We found no association of positive PCR results with clinical or histological chorioamnionitis and neonatal outcomes. WBC count was significantly lower in the PCR positive group, but it did not meet the EOS criteria. Comparison of clinical data and laboratory findings over the period of 2 h after delivery between PCR positive and negative patients are summarized in Table 3.

E. coli PCR positive sub-group

E. coli was the most frequent pathogen found in swabs of both mothers (16 cases, 20%) and neonates (13 cases, 16%), however there was no statistically significant link between them (p=0.71). No relationship was detected between neonatal E. coli swabs (ear, stomach, axilla) and bacterial PCR blood stream assessment results. Statistically significant association was revealed between vaginal E. coli colonization of mothers and E. coli PCR positivity in infants (44% PCR positive neonates of mothers with E. coli positive vaginal swabs vs. 8% PCR positive neonates of E. coli negative mothers, p=0.001), regardless of PPROM presence and mode of delivery. A positive E. coli vaginal swab result was not associated with higher frequency of chorioamnionitis and funisitis. No differences in neonatal morbidity, neonatal infection, mode of delivery and blood sampling procedure were found between E. coli PCR positive and negative subjects.

Use of antibiotics

Antibiotic treatment was commenced in 40 infants overall, for suspected EOS and the presence of risk factors. In 25 patients with negative blood culture results, the antibiotics were discontinued after 48 h. In 15 infants with persistent clinical and laboratory findings (including one case with positive blood culture), antibiotics were continued beyond 48 h.