The clinical utility of Nanopore 16S rRNA gene sequencing for direct bacterial identification in normally sterile body fluids

Hiu-Yin Lao; Lily Lok-Yee Wong; Yan Hui; Timothy Ting-Leung Ng; Chloe Toi-Mei Chan; Hazel Wing-Hei Lo; Miranda Chong-Yee Yau; Eddie Chi-Man Leung; River Chun-Wai Wong; Alex Yat-Man Ho; Kam-Tong Yip; Jimmy Yiu-Wing Lam; Viola Chi-Ying Chow; Kristine Shik Luk; Tak-Lun Que; Franklin Wang Ngai Chow; Gilman Kit-Hang Siu

doi:10.3389/fmicb.2023.1324494

The clinical utility of Nanopore 16S rRNA gene sequencing for direct bacterial identification in normally sterile body fluids

Front Microbiol. 2024 Jan 9:14:1324494. doi: 10.3389/fmicb.2023.1324494. eCollection 2023.

Authors

Hiu-Yin Lao¹, Lily Lok-Yee Wong¹, Yan Hui¹, Timothy Ting-Leung Ng¹, Chloe Toi-Mei Chan¹, Hazel Wing-Hei Lo¹, Miranda Chong-Yee Yau², Eddie Chi-Man Leung², River Chun-Wai Wong³, Alex Yat-Man Ho⁴, Kam-Tong Yip⁵, Jimmy Yiu-Wing Lam², Viola Chi-Ying Chow³, Kristine Shik Luk⁴, Tak-Lun Que⁵, Franklin Wang Ngai Chow¹, Gilman Kit-Hang Siu¹

Affiliations

¹ Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, China.
² Department of Clinical Pathology, Pamela Youde Nethersole Eastern Hospital, Chai Wan, Hong Kong SAR, China.
³ Department of Microbiology, Prince of Wales Hospital, Shatin, Hong Kong SAR, China.
⁴ Department of Pathology, Princess Margaret Hospital, Kowloon, Hong Kong SAR, China.
⁵ Department of Clinical Pathology, Tuen Mun Hospital, Tuen Mun, Hong Kong SAR, China.

Abstract

The prolonged incubation period of traditional culture methods leads to a delay in diagnosing invasive infections. Nanopore 16S rRNA gene sequencing (Nanopore 16S) offers a potential rapid diagnostic approach for directly identifying bacteria in infected body fluids. To evaluate the clinical utility of Nanopore 16S, we conducted a study involving the collection and sequencing of 128 monomicrobial samples, 65 polymicrobial samples, and 20 culture-negative body fluids. To minimize classification bias, taxonomic classification was performed using 3 analysis pipelines: Epi2me, Emu, and NanoCLUST. The result was compared to the culture references. The limit of detection of Nanopore 16S was also determined using simulated bacteremic blood samples. Among the three classifiers, Emu demonstrated the highest concordance with the culture results. It correctly identified the taxon of 125 (97.7%) of the 128 monomicrobial samples, compared to 109 (85.2%) for Epi2me and 102 (79.7%) for NanoCLUST. For the 230 cultured species in the 65 polymicrobial samples, Emu correctly identified 188 (81.7%) cultured species, compared to 174 (75.7%) for Epi2me and 125 (54.3%) for NanoCLUST. Through ROC analysis on the monomicrobial samples, we determined a threshold of relative abundance at 0.058 for distinguishing potential pathogens from background in Nanopore 16S. Applying this threshold resulted in the identification of 107 (83.6%), 117 (91.4%), and 114 (91.2%) correctly detected samples for Epi2me, Emu, and NanoCLUST, respectively, in the monomicrobial samples. Nanopore 16S coupled with Epi2me could provide preliminary results within 6 h. However, the ROC analysis of polymicrobial samples exhibited a random-like performance, making it difficult to establish a threshold. The overall limit of detection for Nanopore 16S was found to be about 90 CFU/ml.

Keywords: 16S rRNA gene sequencing; Nanopore sequencing; body fluids; direct bacterial identification; rapid diagnosis.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Health and Medical Research Fund (HMRF) (ref no. 21200092). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.