Patients and case definitions
This prospective feasibility study was carried out from January 2016 to May 2018 at the PICU in Vietnam National Children’s Hospital, Hanoi, Vietnam. Children who met the following inclusion criteria within 72 hours of PICU admission were enrolled: (1) admission to the PICU with infectious pneumonia; (2) age of 1 month to 15 years; (3) DIC scores defined by the International Society of Thrombosis and Haemostasis (ISTH) ≥ 5; (4) PaO2/FiO2 ratio (P/F ratio) ≤ 100 mm Hg (based on the Berlin definition of 2012) ; (5) No severe left ventricular dysfunction; (6) Chest X-ray showing an abnormal shadow (consolidation, ground-glass opacity, or nodular shadow); and (7) Written informed consent prior to initiation of any study procedures, obtained from the patient’s parents. Patients with severe renal dysfunction (creatinine clearance <10 mL/min) or bleeding symptoms, or those whom the treating doctor deemed ineligible for any reason, were excluded from this study.
Similarly to the administration of rTM to adults, rTM (380 U/kg) was administered for a maximum of 6 consecutive days via intravenous drip infusion for 30 minutes. All necessary treatments performed by PICU doctors were acceptable, except administration or co-administration of anticoagulants, thrombolytic agents, and platelet aggregation inhibitors. Blood samples were taken on the day of admission to the PICU (day X), before the first administration of rTM, and on α days after day X (day X+α). Blood samples were used to evaluate plasma concentrations of TM, cytokines/chemokines, biomarkers, blood cell counts, blood chemistry and DIC scores. The rTM administration was discontinued in case that patients developed severe renal dysfunction or bleeding in vital organs. We observed the clinical time course including vital signs and physical examination findings, bleeding symptoms, P/F ratio, oxygenation index, ventilator settings, and radiographic findings.
Blood and tracheal lavage fluid (TLF) were collected on the day of admission to the PICU. Microbiological cultures were prepared according to standard microbiological procedures. To detect and differentiate up to 25 pathogenic microbial DNA types in blood samples, the LightCycler SeptiFast Test (Roche Diagnostics GmbH, Mannheim, Germany) was used . Total nucleic acids from the TLF samples were extracted with a MagNA Pure LC total nucleic acid isolation kit using a Roche MagNA Pure LC instrument according to the manufacturer's instructions (Roche Diagnostics). The extracts were tested by multiplex real-time reverse transcriptase (rRT) polymerase chain reaction (PCR) (rRT-PCR) using the FTD Respiratory Pathogens 33 Kit according to the manufacturer's instructions (Fast Track Diagnostics, Junglinster, Luxembourg) to screen 33 kinds of respiratory pathogens and by an in-house developed conventional single-target rRT-PCR and real-time PCR (rPCR) to detect genomes of cytomegalovirus (CMV), human immunodeficiency virus (HIV), and varicella zoster virus (VZV).
TM and biomarker assay
The plasma concentration of TM, soluble receptor of advanced glycation end products (sRAGE), pulmonary surfactant protein D (SP-D), angiopoietin-2 (Ang-2) and high-mobility group box 1 (HMGB-1) were measured using enzyme-linked immunosorbent assay (ELISA) kits and a microplate reader (Multiskan FC; Thermo Fisher Scientific K.K., Tokyo, Japan). ELISA kits for TM, sRAGE, SP-D and Ang-2 were purchased from R&D Systems (Tokyo, Japan) and for HMGB-1 from Shino-Test Corporation (Tokyo, Japan). All samples were run in duplicate and the average concentrations were used for statistical analysis.
Cytokine and chemokine assay
The levels of cytokines/chemokines in the plasma were measured using the Human Cytokine Magnetic 25-Plex Panel Kit (Invitrogen, Carlsbad, CA, USA) on a Magpix® system (Merck Vietnam Co., Ltd, Vietnam). All samples were run in duplicate and the average concentrations were used for statistical analysis.
DIC scores were shown as medians with the interquartile range (IQR), and the groups (day X and day X+6) were compared using the Wilcoxon signed rank test. P < 0.05 was considered statistically significant. Data were statistically analyzed using Prism 5.0 for Mac (GraphPad Software Inc., La Jolla, CA, USA) with a two-tailed hypothesis.