Next-generation sequencing-based detection of Ureaplasma in the gastric fluid of neonates with respiratory distress and chorioamnionitis

Abstract

Background

Respiratory distress is common in neonates admitted to neonatal intensive care units. Additionally, infectious diseases such as intrauterine infections or vertical transmission are important underlying causes of respiratory failure. However, pathogens often cannot be identified in neonates, and there are many cases in which antibacterial drugs are empirically administered. Next-generation sequencing (NGS) is advantageous in that it can detect trace amounts of bacteria that cannot be detected by culturing or bacteria that are difficult to cultivate. However, there are few reports on the diagnosis of infectious diseases using NGS in the neonatal field, especially those targeting respiratory distress.

Objective

The purpose of our study was to investigate the microorganisms associated with neonatal respiratory distress and to determine whether less invasive collection specimens such as plasma and gastric fluid are useful.

Methods

Neonates were prospectively recruited between January and August 2020 from Nagoya University Hospital. The inclusion criteria were as follows: 1) admission to the neonatal intensive care unit; 2) respiratory distress presentation within 48 h of birth; and 3) suspected infection, collection of blood culture, and administration of antibiotics. Plasma samples and blood cultures were simultaneously collected. Gastric fluid samples were also collected if the patient was not started on enteral nutrition. Information on the patients and their mothers were collected from the medical records. DNA was extracted from 140 µL of plasma and gastric fluid samples. DNA sequencing libraries were prepared, and their quality was analyzed. DNA libraries were sequenced using high-throughput NGS. The NGS data of plasma and gastric fluid samples were analyzed using the metagenomic pipeline PATHDET, which calculated the number of reads assigned to microorganisms and their relative abundance. Putative pathogens were listed.

Results

Overall, 30 plasma samples and 25 gastric fluid samples from 30 neonates were analyzed. Microorganism-derived reads of gastric fluid samples were significantly higher than those of plasma samples. Transient tachypnea of the newborn was the most common cause of respiratory distress with 13 cases (43%), followed by respiratory distress syndrome with 7 cases (23%). There were 8 cases (29%) of chorioamnionitis and 7 cases (25%) of funisitis pathologically diagnosed. All blood cultures were negative, and only two gastric fluid cultures were positive for group B Streptococcus (Patient 15) and Candida albicans (Patient 24). Putative pathogens that met the positive criteria for PATHET were detected in four gastric fluid samples, one of which was group B Streptococcus from Patient 15. In the gastric fluid sample of Patient 24, Candida albicans were detected by NGS but did not meet the positive criteria for PATHDET. Cluster analysis of the plasma samples divided them into two study groups, and the indicator genera of each cluster (Phormidium or Toxoplasma) are shown in Figure 1. Clinical findings did not show any significant differences between the two groups. Cluster analysis of the gastric fluid samples divided them into three study groups, and the indicator genera of each cluster (Ureaplasma, Nostoc, and Streptococcus) are shown in Figure 2. The incidence rate of chorioamnionitis was significantly higher in Ureaplasma group than in the other two groups.

Conclusion

Gastric fluid may be useful for assessing neonatal patients with respiratory distress. To the best of our knowledge, this was the first study to reveal that the presence of Ureaplasma in the gastric fluid of neonates with respiratory distress was associated with chorioamnionitis. The early diagnosis of intra-amniotic infections using gastric fluid and its treatment may change the treatment strategy for neonatal respiratory distress. Screening for Ureaplasma in neonates with respiratory distress may reduce the need for empirical antibiotic administration. Further research is required to confirm these findings.

Keywords:

• Chorioamnionitis
• gastric fluid
• neonate
• next-generation sequencing
• Ureaplasma

Acknowledgements

The authors acknowledge all the children and parents who participated in this study.

Ethics approval and consent to participate

The study was approved by the Institutional Review Board of Nagoya University Graduate School of Medicine (approval no. 2019-0373). This study was carried out in compliance with the Declaration of Helsinki guidelines. Written consent was obtained from the parents of the neonates who participated in this study.

Authors’ contributions

T.Ok., K.Ho. M.H., and Y.I. designed the study. Y.Sa., K.Ha., and M.Y. collected clinical samples. T.O., N.T. Y.Su., and J.K reviewed the medical records for the clinical assessment of patients. T.Ok., K.Ho., M.Y., T.S., and Y.T. conducted experiments. T.Ok., K.Ho., and T.Og. performed the bioinformatics analysis. T.O., K.H., N.T., Y.S. and Y.I. drafted the manuscript. All authors have read and approved the final manuscript.

Disclosure statement

The authors report there are no competing interests to declare.

Data availability statement

Full datasets supporting the findings of this study are available from the corresponding author upon reasonable request. Sequencing data generated and/or analyzed during the current study are available in the DDBJ Sequence Read Archive under experiment number DRR402161-DRR402215.

Additional information

Funding

This work was supported by a Grant-in-Aid for Young Scientists [grant number 20 K17464] and the Takeda Science Foundation; K.H. is the recipient of the grants. This work was also supported by Japan Society for the Promotion of Science [22K07818].