Kidney transplant recipients face substantially elevated risks of life-threatening fungal infections due to chronic immunosuppression. Although invasive fungal infections represent less than 5% of post-transplant complications (Anastasopoulos et al., 2015; Sommerer et al., 2022), they carry disproportionately high mortality rates (Pappas et al., 2010; Singh and Husain, 2013; Silva et al., 2018). Donor-derived infection (DDI), while uncommon, results in significant morbidity and mortality, with approximately one-third of recipients experiencing graft loss or death - rates that are notably elevated in cases involving fungal diseases (Kaul et al., 2021). The reported incidence of DDI is approximately 0.2% among all deceased donor organ transplantations (Ison and Grossi, 2013), with fungal infections accounting for approximately 15.5% of DDI (Shingde et al., 2018). Timely diagnosis remains a major challenge, as conventional culture methods are slow and insensitive, and non-culture-based assays have significant limitations (Anesi and Baddley, 2016).

Metagenomic next generation sequencing (mNGS) addresses the limitations of current diagnosis and has been increasingly applied to pathogen detection in clinical practice. Prior antibiotic exposure does not affect it, enabling faster and more accurate identification. Some studies have demonstrated that mNGS can improve the sensitivity of fungal infection (Zhao et al., 2022; Shi et al., 2023). In contrast, identification of filamentous molds, such as Aspergillus, by mNGS remains challenging due to difficulties in extracting DNA from thicker polysaccharide cell walls and the relatively low fungal load in bronchoalveolar lavage fluid (BALF) (Clarke et al., 2018; Han et al., 2019).

However, few studies have investigated the utility of mNGS for diagnosing fungal infections in renal transplant patients. Furthermore, its role in detecting and characterizing donor-derived fungal infections remains largely uninvestigated. In this study, we used mNGS to identify pathogens from multiple sample types in patients with suspected fungal infections, evaluated the types and prevalence of detected pathogens, compared the results with those obtained by conventional methods, and evaluated the clinical impact on antimicrobial therapy management. Crucially, this study provides novel insights into the potential of mNGS for uncovering donor-derived fungal infections, an area where current diagnostic approaches are insufficient.

Effective management of post-transplant infections depends on prevention, early diagnosis, and targeted therapy. Although less frequent than bacterial infections, invasive fungal infections (IFIs) in transplant recipients are associated with high mortality rates, ranging from 15% to 50% in kidney transplant patients (Seok et al., 2020; van Delden et al., 2020; Wu et al., 2023). While mNGS has advanced infectious disease diagnostics, its specific application in profiling fungal infections in kidney transplant recipients remains underexplored. In this study, we conducted a comprehensive analysis of fungal infections in these patients using mNGS and compared its performance with CMTs. Our aim was to characterize the fungal pathogen spectrum, including mixed and donor-derived infections, and to evaluate the clinical impact of mNGS on antifungal therapy.

Currently, fungal smear and culture, serum (1,3)-b-D-glucan (G) or galactomannan (GM) tests, and PCR are used for microbiological fungal analysis. Traditional culture methods offer valuable information on drug susceptibility but have limitations such as low positive rates, reduced sensitivity, high false-positive rates, and lengthy processing times. Kidney transplant recipients often receive multiple medications, which may affect the accuracy of these diagnostic methods. Conventional blood cultures may fail to diagnose candidiasis in up to 25–50% of cases (Bassetti et al., 2016). In contrast, mNGS, characterized by rapid detection, high sensitivity, and broad coverage, effectively compensates for these shortcomings.

The significantly higher fungal detection rate by mNGS (90.67% vs. 26.67%) is consistent with emerging evidence from other immunocompromised cohorts (Decker et al., 2019; Chen et al., 2020; Chien et al., 2022; Wang et al., 2022; Overbeek et al., 2024). This discrepancy is especially notable for fastidious organisms like Pneumocystis jirovecii, which cannot be routinely cultured, and fungi with fragile structures prone to damage during transport or processing. Although most reports emphasize its advantages in fungal diagnosis, some studies note limitations, particularly for filamentous molds such as Aspergillus (Miao et al., 2018). One study even suggested that conventional methods may outperform mNGS in diagnosing pulmonary fungal infections (Peng et al., 2021), indicating that mNGS’s diagnostic value in mycology is still somewhat contested. However, the heightened sensitivity of mNGS introduces the critical challenge of distinguishing true invasive infections from colonization or environmental contamination, especially for organisms like Candida and Aspergillus, which can be commensal. In our practice, a positive mNGS result was never interpreted in isolation; clinical decisions were always based on integrating mNGS findings with the patient’s clinical trajectory, radiological findings, and immune status. This multi-parameter approach is essential to mitigate the risk of overdiagnosis and unnecessary treatment, converting a potentially high false-positive rate into clinically actionable insights.

A particular strength of mNGS is its ability to identify fastidious, non-culturable fungi, which is crucial in immunocompromised hosts vulnerable to opportunistic pathogens. Consistent with other studies reporting frequent detection of Pneumocystis jirovecii, Candida, and Aspergillus (Zhao et al., 2021; Wang et al., 2022; Fishman, 2017), our findings confirm the utility of mNGS in profiling the fungal spectrum in kidney transplant recipients. Notably, mNGS demonstrates exceptional performance in diagnosing Pneumocystis jirovecii, a major pathogen in this population, with sensitivity and specificity surpassing conventional staining and biomarker assays (Jiang et al., 2021; Xu et al., 2021; Duan et al., 2022; Sun et al., 2022). Regarding specific pathogens, Candida species remain the most prevalent fungi among solid organ transplant recipients, a pattern also evident in our cohort. Invasive aspergillosis continues to carry a poor prognosis, especially in lung transplant recipients (Singh and Husain, 2013). Increasingly, studies - including ours - support mNGS for superior detection of Aspergillus compared to traditional methods (Hoenigl et al., 2023; Shi et al., 2023; Zhan et al., 2023; Niu et al., 2024), underscoring its emerging role in managing these high-risk infections.

mNGS demonstrates strong performance in identifying fungal species and co-infecting pathogens, highlighting its potential to guide antimicrobial therapy. Multiple studies have confirmed the clear advantages of mNGS in detecting mixed infections (Liang et al., 2022; Zhang et al., 2022), consistent with our findings. Additionally, mNGS identified polymicrobial fungal infections in 9 samples, whereas CMTs detected only one. The comprehensive fungal profile provided by mNGS offers valuable epidemiological insights into fungal infections among transplant recipients.

The impact of antibacterial drugs on mNGS is less pronounced than on CMTs, allowing tailored treatment strategies based on these findings. Accurate strain identification is crucial for guiding antifungal treatment. Several studies reported that mNGS prompted treatment modifications in 39.3%–45.1% of cases (Liang et al., 2022; Zhang et al., 2022; Shi et al., 2023). Consistent with these reports, our findings confirm that mNGS directly influences antifungal management by supporting informed initiation of targeted therapy and guided discontinuation of unnecessary medication. The rapid availability of mNGS results delivers timely microbiological evidence to support clinical decision-making, facilitating antimicrobial stewardship and potentially improving patient outcomes.

A key clinical finding of our study pertains to DDIs. Although less common than bacterial or viral counterparts, DDIs pose a severe threat in transplantation, with an overall DDI-associated mortality of 15% (Kaul et al., 2021). Current guidelines emphasize blood and urine cultures from deceased donors but do not routinely recommend culturing preservation fluid (Singh et al., 2012; Malinis and Boucher, 2019). A considerably higher DDI rate of 7.5% was identified in our cohort, where both mNGS and CMTs were systematically applied to preservation and drainage fluids. This contrasts with the 4% incidence reported in an earlier meta-analysis (Oriol et al., 2018). The elevated donor-derived fungal infection rate in our study may be attributed to the combined detection strategy employing both mNGS and CMTs for detection. A study further note that renal transplant recipients are at elevated risk for urinary tract infections and systemic complications from donor-derived pathogens, highlighting the importance of broad-spectrum screening strategies like those employed in our study (Fiorentino et al., 2019). Prior research indicates that donor-derived fungal infections are predominantly caused by Candida (24%), Cryptococcus (20%), and Aspergillus (13%), with associated mortality rates of 10.0%, 7.7%, and 33.3%, respectively (Kaul et al., 2021). In our study, all donor-derived Candida infections were successfully managed without recipient mortality, which may reflect our center’s protocol of routine postoperative caspofungin prophylaxis and subsequent therapy adjustments informed by etiological evidence. This outcome undermucorscores the importance of targeted antifungal strategies in recipients of grafts from donors with fungal colonization, consistent with current guideline recommendations (Singh et al., 2012). One fatal case of donor-derived mycosis was also identified, underscoring the indispensable role of sensitive detection tools like mNGS in identifying rare but high-risk pathogens that conventional screening might fail to detect.

The present study had several limitations. First, traditional detection methods such as the G test, GM test, cryptococcal capsular polysaccharide antigen test, and Grocott methenamine silver stain were not comprehensively employed. Fungi like Mucor and Pneumocystis jirovecii are difficult to culture, and these detection methods help diagnose these fungi. Therefore, a direct comparison of mNGS with these methods was not feasible. Second, mNGS cannot reliably distinguish between colonization and active infection, as fungi like Candida or Aspergillus commonly exist as commensals. Therefore, positive results require cautious interpretation within the full clinical context-including symptoms, radiological findings, and immune status-to avoid overdiagnosis and unnecessary treatment. Third, mNGS does not provide susceptibility results, although fungal resistance mechanisms tend to be less complex than bacterial resistance. Fourth, the thick cell wall of Aspergillus complicates nucleic acid extraction, leading to a false-negative result.

In summary, while mNGS enables rapid and sensitive detection of fungal infections-particularly those caused by unculturable pathogens-in kidney transplant recipients, its clinical application necessitates standardized yet adaptable protocols tailored to individual patient scenarios.