Commonly, cutaneous infectious granulomas (CIG) are localized and chronic skin infection caused by a variety of pathogens such as protozoans, bacteria, worms, viruses and fungi. The diagnosis of CIG depends mainly on the clinical examination of the skin, microbiological and histopathological examination. However, microbiological examination, including direct smear and culture, shows low sensitivity. Moreover, the histomorphological findings of CIG caused by different pathogens are commonly very similar so the exact identity of the infectious graulomas is hardly to work out [8]. Although pathogen detection is important, histopathology even with special staining can rarely identify pathogens to species level [3]. As a result, the CIG usually remains undiagnosed for several years and resulting in delayed or inadequate treatment, prolonged stays, readmissions. According to our findings, it took an average of 36 months from the start of symptoms until the collection of the samples. [Interquartile range (IQR) 3.25–10 months], so our study consistent with those conclusions. The epidemiological pathogens of CIG vary depending on geographical regions throughout the world [2]. In general, fungi and mycobacteria are the two most common types of CIG, our study consistent with those conclusions. In the present study, the most common pathogen was Non-tuberculous Mycobacteria (NTM), while positive result of fungi are six (C.parapsilosis, M.restricta, S. globosa, P. jirovecii, Talaromyces marneffei, Fonsecaea monophora). The main reason is that fungi generally bigger in diameter, so that fungi structure are more easily to be found through the examination of histomophology and direct smear. Although some of the studies stated that fungi cell wall is so thick that mNGS with lower ratio of positive results [9], mNGS is able to detect the fungi of Fonsecaea spp., which presented as muriform cells with thick cell wall in tissue.

Secondly, our study’s conclusion is that, in the following areas, mNGS produced higher-quality detection than traditional examination.1) mNGS results have a greater positive rate than culture (80.77% vs. 57.7%). The positive rate of mNGS ranged from 41.3 to 82.14% in earlier research, which was often greater than the culture rate [10,11,12]. 2) Multiple pathogens can be simultaneously detected by mNGS in a single specimen. Generally, it needs to collect multiple samples for different examination, including bacteria, fungi, mycobacteria, etc. Therefore, mNGS provides an effective alternative to optimize microbiological detection. 3) mNGS not only can be used to detect the sample of fresh tissue and pus, but also the sample of paraffin-embedded tissues. In many cases, sample of tissue is not sent for cultures since gurnulomatous inflammation is not found until microscopy is performed. Also, a sample may have been sent for culture but without positive results neither in bacterial culture nor fungal culture. Thus, mNGS provides an effective alternative to test with the samples of paraffin-embedded tissue, which are the only material available for testing. 4) The total time for detection by using mNGS is much shorter than by culture. As we know, the culture-based method for microbiological detection is time-concuming. mNGS could be done within two days, while median time for culture is 19.6 ± 12.4 days. Totally, 21 patients showed clinical improvement by the 30-day follow up. Eighteen of them were adjusted treatment and fifteen of them continuous treatment based on the results of mNGS. So, it showed that mNGS results may affect the clinical prognosis resulting from enabling the patients to initiate timely treatment.

In other hand, although we demonstratd some merits of mNGS, there are still some limitations as follows: it is needed to combine with comprehensive assessment of cases, including patients’ information, clinical manifestation, location of specimen collection and microbiological characteristics, for clinical interpretation of positive results of mNGS. In addition, there are still some samples got negative mNGS, but positive in special straining of histopathology (Patient 16) or T-SPOTⓇ. TB test (Patient 11). Except for the patient 11, there are totally nine patients, including one patient infected by M. tuberculosis and six patients infected by M. marinum, with positive T-SPOTⓇ. TB test (9/18, 50%). T-SPOTⓇ. TB test used to diagnose latent tuberculosis, pulmonary tuberculosis, or extrapulmonary tuberculosis is the interferon-γ (IFN-γ) release assay (IGRA), which has a high sensitivity for the specific detection of these conditions. But some NTMs, like M. marinum, could show false positive results in IGRA test [13]. Moreover, GeneXpert is frequently employed for tuberculosis detection. Findings from a limited number of current studies indicate that GeneXpert and mNGS have comparable specificity. However, in terms of sensitivity, mNGS appears to be marginally superior to GeneXpert [14, 15]. Nevertheless, we believe further research is necessary before drawing definitive conclusions. So, more detection methods should be used spontaneously to confirm the findings and make proper diagnosis.

In summary, our data show that mNGS detected NTM, M. tuberculosis, fungi and bacteria in cutaneous infectious granulomas. Compared to culture, mNGS showed a higher positive rate with high sensitivity rate and negative predictive value. In addition, mNGS can detect more pathogens in one sample and can be used to detect variable samples including the samples of paraffin-embedded tissue. However, our study had some limitations: Our limited study population and the risk of selection bias affect the representativeness and generalizability of the experimental results. Nonetheless, this study reveals preliminary trends and provides direction for subsequent exploration. Future studies should aim to expand the sample size and provide more scientific evidence for the application of mNGS in the prevention and treatment of CIG. In addition, mNGS has an extremely broad detection range of microorganisms, a feature that brings both advantages and challenges: It is difficult to accurately distinguish between colonization or contamination during the interpretation of results of mNGS. For the samples used in this study, the colonizing bacteria usually originated from the skin surface, such as Cutibacterium acnes, Staphylococcus epidermidis, and Malassezia globosa, and were relatively easy to distinguish. On the other hand, contamination is related to originate from the reagents themselves, the experimental environment, and cross-contamination between different samples. These sources of contamination may lead to misinterpretation of mNGS results. In recognition of this, researchers should fully understand and consider the microbial community characteristics in the study background when interpreting mNGS results, which is the key to improving the accuracy of mNGS results interpretation and guiding clinical decision-making, as well as a direction worthy of continuous efforts in our future research and practice.

In conclusion, although mNGS provides a potentially rapid and effective alternative test for the diagnosis of cutaneous infectious granulomas, a comprehensive judgment must be made in conjunction with clinical evaluation, microbial culture results, and histopathological examination to ensure the accuracy and reliability of the diagnosis in practical application.