The rapid diagnosis of intraamniotic infection with nanopore sequencing
16S; 16S PCR; 16S rDNA genes; 16S rRNA genes; amniocentesis; amniotic cavity; amniotic fluid; bacteria; bacterial infection; cultivation; culture; DNA; DNA sequencing; Escherichia coli (E. coli); infection; intraamniotic infection; intraamniotic inflammation; long-read sequencing; microbial invasion of the amniotic cavity (MIAC); microorganism; molecular microbiologic technique; Mycoplasma species; nanopore; Oxford nanopore technology (ONT); polymerase chain reaction (PCR); pregnancy; preterm; preterm labor; preterm prelabor rupture of the membranes (PROM); preterm PROM; rapid sequencing; removal of false positive; RNA; Sanger sequencing; sequencing; short-read; sterile inflammation; third-generation sequencing; Ureaplasma species
Background: Intraamniotic infection (defined as intraamniotic inflammation with microorganisms) is an important cause of the preterm labor syndrome. Methods for the detection of microorganisms in amniotic fluid are culture and/or polymerase chain reaction assay. However, both methods take time, and the results are rarely available for clinical decision-making. Nanopore sequencing technology offers real-time, long-read sequencing that can produce rapid results. Objective: To determine 1) the diagnostic performance of the 16S rDNA nanopore sequencing method for the identification of microorganisms in patients with intraamniotic inflammation and 2) the relationship between microbial burden and the intensity of the amniotic fluid inflammatory response. Study Design: We performed a prospective cohort study that included singleton pregnancies presenting with symptoms of preterm labor with intact membranes or of preterm prelabor rupture of the membranes. Amniotic fluid samples were obtained for the evaluation of bacteria in the amniotic cavity using cultivation and polymerase chain reaction-based 16S Sanger sequencing methods. Participants were classified into 4 groups according to the results of an amniotic fluid culture, 16S Sanger sequencing, and an amniotic fluid interleukin 6 concentration: 1) no intraamniotic infection and intraamniotic inflammation (interleukin 6 <2.6 ng/mL, and no microorganisms in the amniotic cavity, as determined by culture or 16S Sanger sequencing); 2) microbial invasion of the amniotic cavity without intraamniotic inflammation, defined by the presence of bacteria detected by culture or 16S Sanger sequencing; 3) sterile intraamniotic inflammation (interleukin 6 ?2.6 ng/mL without microbial invasion of the amniotic cavity); and 4) intraamniotic infection (interkeukin 6 ?2.6 ng/mL with microbial invasion of the amniotic cavity). Patients who underwent a mid-trimester amniocentesis, had no intraamniotic infection or intraamniotic inflammation, and delivered at term represented the control group. 16S rDNA nanopore sequencing was performed and the diagnostic indices for the identification of intraamniotic infection were determined. Bioinformatic analysis was carried out to identify microorganisms, and a read count of at least 100 or a read count exceeding that of the background species from the control group, along with a relative abundance of no less than 1%, was used. Results: 1) The 16S nanopore sequencing had a sensitivity of 88.9% (8/9), specificity of 95.4% (41/43), positive predictive value of 80.0% (8/10), negative predictive value of 97.6% (41/42), positive likelihood ratio of 19.1 (95% confidence interval, 4.875.4), negative likelihood ratio of 0.1 (95% confidence interval, 0.020.7), and an accuracy of 94.2% (49/52) for the identification of intraamniotic infection (prevalence, 17% [9/52]); 2) the microbial load determined by the 16S nanopore sequencing had a strong positive correlation with the intensity of an intraamniotic inflammatory response (amniotic fluid interleukin 6 concentration; Spearman's correlation 0.9; P=.002); and 3) a subgroup of patients with intraamniotic inflammation did not have bacteria determined by culture, Sanger sequencing, or nanopore 16S, thus confirming the existence of sterile intraamniotic inflammation. Conclusion: The 16S nanopore sequencing has high diagnostic indices, predictive values, likelihood ratios, and accuracy in the diagnosis of intraamniotic infection. 2025 The Author(s)
Chaemsaithong, Piya; Romero, Roberto; Pongchaikul, Pisut; Warintaksa, Puntabut; Mongkolsuk, Paninee; Bhuwapathanapun, Maolee; Kotchompoo, Kanyaphat; Nimsamer, Pattaraporn; Kruasuwan, Worarat; Amnuaykiatlert, Orrakanya; Vivithanaporn, Pornpun; Meyyazhagan, Arun; Awonuga, Awoniyi; Settacomkul, Rapeewan; Singhsnaeh, Arunee; Laolerd, Warawut; Santanirand, Pitak; Thaipisuttikul, Iyarit; Wongsurawat, Thidathip; Jenjaroenpun, Piroon
American Journal of Obstetrics and Gynecology;Volume;233;Issue;3;pp.1930-1.93e+20
All Open Access; Hybrid Gold Open Access
https://doi.org/10.1016/j.ajog.2025.02.011 https://www.scopus.com/pages/publications/105000367380?origin=resultslist
Chaemsaithong P., Department of Obstetrics and Gynecology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, Program in Translational Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Romero R., Pregnancy Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, United States Department of Health and Human Services, Bethesda, MD, United States, Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, United States, Department of Epidemiology and Biostatistics, Michigan State University, East Lansing, MI, United States; Pongchaikul P., Chakri Naruebodindra Medical Institute, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Samut Prakarn, Thailand, Integrative Computational BioScience Center, Mahidol University, Nakhon Pathom, Thailand, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, United Kingdom; Warintaksa P., Department of Obstetrics and Gynecology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Mongkolsuk P., Chakri Naruebodindra Medical Institute, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Samut Prakarn, Thailand; Bhuwapathanapun M., Department of Obstetrics and Gynecology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Kotchompoo K., Department of Obstetrics and Gynecology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Nimsamer P., Division of Medical Bioinformatics, Research Department, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand, Siriraj Long-Read Lab (Si-LoL), Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; Kruasuwan W., Division of Medical Bioinformatics, Research Department, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand, Siriraj Long-Read Lab (Si-LoL), Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; Amnuaykiatlert O., Mahidol University International Demonstration School, Mahidol University, Nakhon Pathom, Thailand; Vivithanaporn P., Chakri Naruebodindra Medical Institute, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Samut Prakarn, Thailand; Meyyazhagan A., Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI, United States, Department of Life Sciences, Christ University, Bengaluru, India; Awonuga A., Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI, United States; Settacomkul R., Chakri Naruebodindra Medical Institute, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Samut Prakarn, Thailand; Singhsnaeh A., Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Laolerd W., Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Santanirand P., Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Thaipisuttikul I., Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; Wongsurawat T., Division of Medical Bioinformatics, Research Department, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand, Siriraj Long-Read Lab (Si-LoL), Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; Jenjaroenpun P., Division of Medical Bioinformatics, Research Department, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand, Siriraj Long-Read Lab (Si-LoL), Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand