Mycobacterium marinum (M. marinum) is a slow-growing, non-tuberculous mycobacterium (NTM) and the leading cause of extra-respiratory NTM infections worldwide. It was first isolated from fish in 1926 (Streit et al., 2006; Ackleh et al., 2015). The first human case of M. marinum infection was reported in 1951 by Norden and Linell (Hashish et al., 2018), the infection presents as a nodular granulomatous disease (Johnson and Stout, 2015). M. marinum culture is time-consuming and has a low positivity rate (70%) (Yu et al., 2023) for pathogen detection. M. marinum is a photochromogen that produces yellow pigment after exposure to light. Ziehl-Neelsen staining of specimens is rarely positive (Akram and Aboobacker, 2022), and even positive smear microscopy analysis cannot distinguish M. marinum from other mycobacteria (Aubry et al., 2017). Traditionally, M. marinum infections have been diagnosed in the laboratory using culture-based detection which requires a significant amount of time to isolate the bacterium from the clinical specimens and identify the species. M. marinum grows best at 30 °C but is inhibited at 37 °C (Aubry et al., 2017). Therefore, microscopic and culture-based results are often negative in clinical microbiological studies. PCR and serological assays are rapid and culture-independent techniques; however, both require prior knowledge of the types of pathogenic microorganisms. Moreover, these methods focus on individual pathogens rather than entire populations (Rajapaksha et al., 2019). Fortunately, recent technological advances in nanopore sequencing by Oxford Nanopore Technologies (ONT) have the potential to address these challenges.

Pocket-sized MinION was the first commercially available third-generation nanopore sequencing device developed by ONT in 2014 (Reuter et al., 2015). which is powered directly by a USB port from a laptop computer. The basic working principle of nanopore sequencing is to monitor electrical current changes caused by nucleic acids passing through a nanopore protein. Subsequently, the resulting signals are decoded to provide a specific DNA or RNA sequence (Wang et al., 2015). This new sequencing technology has many advantages, including short sequencing time, long read length, and low cost (Walter et al., 2017; Taylor et al., 2019a). The maximum turnaround time from sample collection to the delivery of the results is 6 h (Greninger et al., 2015), with data acquisition completed within 10 min once the sample is loaded onto the MinION (Greninger et al., 2015). Because of its portability, a MinION nanopore sequencer can be taken directly to the patient’s bedside and used to detect pathogens in many different environments (Reuter et al., 2015). In addition, nanopore sequencing is becoming more clinically viable owing to continued improvements in its accuracy and throughput. This technology has made nanopore sequencing an attractive diagnostic technique that produces longer reads to improve the clinical diagnosis, research, and epidemiological tracking of infectious diseases. Many studies have reported on the analysis of Mycobacterium tuberculosis (MTB) using nanopore sequencing (Smith et al., 2020; Gliddon et al., 2021; Dippenaar et al., 2022); however, there are few reports on NTM, such as M. marinum (Xing et al., 2022). Here, we report the use of third-generation nanopore sequencing to diagnose a case of M. marinum.

Both NTM and MTB belong to the genus Mycobacterium and have similar pathogenic mechanisms and clinical manifestations, which often result in misdiagnosis. Culture-based detection is a conventional method for diagnosing NTM infections. However, M. marinum is a slow-growing microorganism that is difficult to culture. Typically, it takes over two weeks to grow, and optimal growth requires low temperatures (approx. 30 °C) (Aubry et al., 2017). The isolation culture-based detection is generally performed at 37°C; therefore, a risk of culture-negative infections misdiagnosis emerges. In addition, M. marinum and M. ulcerans share more than 98% of their genomes, and no current PCR techniques can distinguish between them (Aubry et al., 2017). Next generation sequencing (NGS) platforms can identify all microorganisms in a sample within 24 h (Naccache et al., 2014; van Dijk et al., 2014). However, short sequencing reads make it difficult to parse the complex genomic structures of microorganisms. In addition, the high cost, lengthy and tedious processes, and large equipment size remain major deterrents to its routine use in clinical settings. Therefore, there is an urgent need to develop rapid and accurate methods to detect pathogenic microorganisms. Nanopore sequencing is increasingly recognized for its advantageous characteristics, including short turnaround time (< 6 h), long read lengths, small equipment size, and affordability, making it a valuable asset in a clinical microbiology laboratory (Jain et al., 2016; Petersen et al., 2019). Recently nanopore sequencing has enabled the sequencing of bacterial, viral, and fungal genomes (Ashton et al., 2015; Loman et al., 2015; Istace et al., 2017).

In our case, the patient had abscesses with yellow pus and granulomas that were visible on pathological examination, suggesting a possible MTB or NTM infection. Ziehl-Neelsen and fluorescent staining for AFB were positive. However, microscopy alone cannot distinguish between cases of MTB and NTM, and it was difficult to determine which microorganism was responsible for the primary infections. MTB DNA and NTM DNA tests were negative in our study. No microorganisms were observed to grow in the culture medium. Nanopore sequencing was used to identify the infectious agent accurately. Fortunately, nanopore sequencing confirmed that the infection was caused by M. marinum. These findings indicate that Nanopore MinION sequencing can be used as a rapid diagnostic tool for infectious diseases.

Nanopore sequencing is a valuable sequencing technology, in which different nucleotide bases are distinguished by changes in the current when an ionic current is passes across the flow cell during sequencing (Laver et al., 2015). High-throughput sequencing is used in various clinical fields, such as identification, characterization, and surveillance of pathogenic microorganisms; detection of drug resistance genes and evaluation of the resistance phenotype; and description of disease-related microbial community (Zhang et al., 2022). This method offers many solutions to the current challenges in genome sequencing. However, a major limitation of this approach is its read accuracy, which can be compromised by a high error rate and insufficient sequencing depth when the technology is used repeatedly on the same sequence (Petersen et al., 2019; Taylor et al., 2019a). In addition, there is no genus-level identification of an organism, and the utility and diagnostic yield of metagenomic sequencing is considerably reduced in other clinical situations. Most software developed to interpret nanopore sequences requires relatively high bioinformatics skills, which are unavailable to most biologists. With continued improvements in the accuracy and throughput of nanopore sequencing platforms, the sequencing error rate has reached a clinically acceptable range (Taylor et al., 2019b). In conclusion, nanopore sequencing is a reliable method for the rapid detection of pathogens in suspected infections. We believe that nanopore sequencing will become a routine diagnostic tool in the field of clinical infectious disease diagnosis and healthcare surveillance in the near future.

The datasets presented in this study can be found in online repositories. The name of the repository and accession number can be found below: https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA967818.

This study was approved by the Ethics Committee of the Hangzhou Red Cross Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the individuals for the publication of any potentially identifiable images or data included in this article. Written informed consent was obtained from the participant/patient(s) for the publication of this case report.

Y-YH wrote the manuscript. S-PH and A-HS reviewed and edited the manuscript. Q-SL and Z-DL reviewed the clinical data. Y-YH and Q-SL supervised the microbiological investigations. Z-DL and S-PH reviewed the pathological data. All authors contributed to the article and approved the submitted version.