The Application Value of Metagenomic Next-generation Sequencing in the Diagnosis of Pulmonary Mucormycosis

The Application Value of Metagenomic Next-generation Sequencing in the Diagnosis of Pulmonary Mucormycosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Application Value of Metagenomic Next-generation Sequencing in the Diagnosis of Pulmonary Mucormycosis Xiao Yao, Haiyang Sang, Xiaohang Hu, Jinyan Yan, Ting Liu, Shuguang Gao, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6308222/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Although pulmonary mucormycosis is rare, it is highly invasive and carries a significant mortality rate. Due to its nonspecific clinical manifestations, it is often misdiagnosed as other invasive fungal diseases. Metagenomic next-generation sequencing is a rapid, precise, and comprehensive method for pathogen detection, showing great potential in the early diagnosis of pulmonary mucormycosis. It provides clinicians with faster and more accurate etiological information, thereby improving patient outcomes and reducing mortality rates. Methods This study retrospectively analyzed the clinical data of 14 patients diagnosed with pulmonary mucormycosis between 1/6/2021 and 30/6/2024. Results Males accounted for 57.14% of the cases. Diabetes mellitus was present in 12 patients (85.71%, 12/14), and fever was observed in 12 patients (85.71%, 12/14). The 14 patients were categorized as confirmed cases (4 cases), probable cases (4 cases), and possible cases (6 cases). Two patients (14.29%, 2/14) were diagnosed with disseminated mucormycosis. Chest Computed Tomography scans revealed cavities in half of the patients (50.00%, 7/14). Fungal hyphae were identified in 3 out of 4 histopathological examinations. mNGS detected Mucorales pathogens in 100% of cases, significantly outperforming the positive rates of the 1,3-β-D-glucan test test (35.71%, 5/14), Galactomannan test test (42.86%, 6/14), histopathology (75.00%, 3/4), and fungal culture (28.57%). Additionally, mNGS identified bacterial and viral co-infections, with 11 patients diagnosed as having mixed infections. All patients received timely and targeted treatment adjustments, resulting in 9 patients improving and being discharged, 2 patients being transferred to higher-level hospitals, and 2 patients discontinuing treatment. The 90-day follow-up revealed a mortality rate of 28.51%. Conclusions Metagenomic next-generation sequencing can serve as an important complement to traditional diagnostic methods, enabling rapid and accurate differentiation of Mucorales from other fungi. This allows patients to receive timely and targeted antifungal therapy, playing a critical role in early intervention and improving prognosis. Pulmonary mucormycosis Clinical features Metagenomic next-generation sequencing Diagnosis Treatment strategy Figures Figure 1 Figure 2 Introduction Mucormycosis is an invasive fungal infection caused by Mucorales fungi. Although relatively rare, it progresses rapidly, shows no age predilection, is highly destructive, and carries a high mortality rate (approximately 40%-80%)( 1 ). Its incidence ranges from 1 to 12.3 cases per million people in Asia( 2 ). Mucorales can invade the lungs, nose, orbits, gastrointestinal tract, kidneys, skin, and other tissues, often leading to multi-organ involvement and disseminated infections( 1 , 3 , 4 ). In recent years, solid organ transplantation, malignancies, hypoxia, and inappropriate corticosteroid use have become significant risk factors for mucormycosis with advances in medicine( 5 ). However, diabetes remains the primary risk factor in developing countries( 6 , 7 ). Additionally, studies indicate that males are more susceptible to mucormycosis than females( 8 , 9 ). Pulmonary mucormycosis (PM), as the second most common type, has garnered significant attention due to its invasiveness and mortality rate exceeding 50%( 10 , 11 ). It is primarily caused by the inhalation of sporangiospores and, although commonly seen in immunocompromised patients, can also occur in individuals without any identifiable risk factors. Due to its nonspecific clinical manifestations, PM is often misdiagnosed as bacterial pneumonia, aspergillosis, or other infectious diseases, particularly in cases of mixed infections, making diagnosis even more challenging( 12 , 13 ). Therefore, early and rapid diagnosis poses a significant challenge for clinicians. The diagnosis of PM currently relies mainly on serological tests, such as 1,3-β-D-glucan test (G-test) and Galactomannan test (GM-test), microbiological cultures, imaging, and histopathological analysis. However, these methods have certain limitations. Serological tests, while sensitive to various fungi, cannot directly identify Mucorales, resulting in insufficient specificity. In microbiological diagnostics, direct microscopy has a low positivity rate and cannot differentiate fungal species, while fungal cultures are time-consuming and also exhibit low positivity rates( 14 ), making them inadequate for rapid diagnosis. Imaging studies, although helpful in diagnosing lung diseases, face challenges in distinguishing PM from other fungal infections. Histopathology is considered the gold standard for diagnosis, but its positivity rate is only about 50%, and obtaining representative tissue samples is difficult, especially in the early stages of the disease( 15 ). In recent years, advancements in molecular diagnostic technologies have provided new possibilities for the diagnosis of PM. Techniques such as qPCR, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and restriction fragment length polymorphism have shown diagnostic value but are limited by issues such as high false-positive rates or operational complexity( 16 ). In contrast, metagenomic next-generation sequencing (mNGS) has demonstrated significant advantages. It can detect the genomic information of all microorganisms in a sample without relying on traditional culture methods or pathogen prediction, enabling simultaneous detection of bacteria, viruses, fungi, and parasites. This is particularly useful for rare or difficult-to-culture pathogens, such as Mucorales( 17 , 18 , 19 ). Compared to traditional methods, mNGS offers higher sensitivity and significantly reduces detection time( 20 ), facilitating early diagnosis and timely treatment. mNGS holds significant potential in the diagnosis of PM, providing clinicians with faster and more accurate pathogen information, thereby improving patient outcomes and reducing mortality rates. Materials and Methods Study Design and Patients This retrospective study strictly adhered to inclusion and exclusion criteria, consecutively enrolling 14 hospitalized patients with PM at the Affiliated Hospital of Jining Medical University between 1/6/2021 and 30/6/2024. The inclusion criteria were as follows: age ≥ 18 years; complete clinical data; and simultaneous performance of serological tests, fungal cultures, and mNGS. The date of admission was defined as D0. All patients had received treatment at local hospitals prior to admission. To promptly identify the infectious pathogens, all enrolled patients underwent bronchoalveolar lavage fluid (BALF) fungal culture and mNGS within D4. Based on the definitions of invasive fungal diseases by the European Organization for Research and Treatment of Cancer/National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/NIAIDMSG)( 21 ), invasive fungal infections were classified into three categories: I) Proven cases: defined by the presence of fungi in lung histopathology or BALF fungal culture; II) Probable cases: meeting clinical criteria (i.e., imaging, bronchoscopy, or sinus analysis) and mycological criteria (specific fungal antigens and cell wall components); III) Possible cases: meeting clinical criteria but lacking mycological evidence. After obtaining written informed consent from the patients' families, clinicians collected BALF through standard bronchoalveolar lavage procedures and promptly sent the samples for fungal culture and mNGS. Clinical data collected for all enrolled patients included gender, age, comorbidities, imaging and laboratory test results, clinical manifestations, treatment modalities, hospital stay duration, and discharge outcomes. A three-month follow-up on survival status was conducted via telephone. Traditional Detection Methods After trypsin digestion of BALF, 10 µL was inoculated onto Sabouraud dextrose agar for fungal culture. The cultures were incubated at 56°C in a Thermo Scientific 371 CO 2 incubator (Thermo Fisher Scientific,USA). Following pure culture isolation, the samples were inoculated onto chromogenic media for fungal identification and subjected to antifungal susceptibility testing using the YEASTONE kit (Thermo Fisher Scientific, USA). Simultaneously, samples were smeared onto glass slides for Gram staining and morphological observation. Antifungal susceptibility results were interpreted according to the 2018 Clinical and Laboratory Standards Institute (CLSI) breakpoints. Peripheral blood samples were collected for complete blood count, reactive protein (CRP), G-test, and GM-test analyses. Lung tissue samples were sent to the pathology laboratory for histological sectioning. Tissue sections were stained with hematoxylin-eosin, periodic acid-Schiff, and Grocott's methenamine silver and examined under an optical microscope (Olympus BX53, Japan). All patients underwent Chest Computed Tomography scans using either a GE Optima CT660 scanner or a SIEMENS Definition Flash scanner after admission, and the reports were issued by two senior radiologists. mNGS Detection A 3 mL aliquot of BALF was inactivated at 65°C for 30 minutes, and DNA was extracted strictly following the instructions of the DNA purification kit (BGI Genomics, Wuhan, China). DNA libraries were constructed at appropriate concentrations, including DNA fragmentation, end repair, adapter ligation, and PCR amplification. The DNA library and sequencing library preparations were performed using the PMseq™ high-throughput DNA detection kit (BGI Genomics). Quality-qualified libraries were used to prepare DNA nanoballs using the MiSeq™ Dx reagent kit (BGI Genomics). Finally, sequencing was performed on the BGI200 platform( 19 ). Negative and positive controls were included throughout the entire process to ensure quality control and minimize contamination. Each sample required a minimum of 20 million high-quality sequencing reads. Low-quality reads, low-complexity reads, adapter contamination, and short sequences (< 35 bps) were removed. The filtered sequences were mapped to the human reference database using Burrows-Wheeler Alignment (Version 0.7.10) software to identify and exclude human host sequences. The remaining data were compared against the Pathogen Metagenomic Database. All parameters of the detected pathogens, including strict unique mapped reads number(SMRN), relative abundance, genome coverage, and depth, were classified and recorded. Each batch of experiments included negative controls (NC) and positive controls (PC), which were processed alongside the samples and included in the bioinformatics analysis. An infectious pathogen was considered positive by mNGS if it met one of the following criteria: I) When the SMRN of the NC was greater than 50, the SMRN of the sample had to be at least 3 times that of the NC; II) When SMRN of the NC was less than 50, the SMRN of the sample had to be at least 5 times that of the NC; III) When the NC was negative, the SMRN thresholds were set as follows: bacteria and fungi ≥ 3, viruses ≥ 1, and parasites ≥ 100. Among the qualifying genera, the one with the highest SMRN was selected as the final positive result. However, whether the reported pathogen was truly pathogenic required further clinical judgment by physicians based on the patient's clinical manifestations. Results Baseline Characteristics of Patients This study included 14 patients with PM, with a mean age of 63.4 years (range: 38–77). Males accounted for 57.14% (8/14), and the average hospital stay was 14 days (range: 4–34). Among the 14 patients, only 1 (7.15%, 1/14) had no underlying diseases, while the remaining 13 (92.85%, 13/14) had a history of comorbidities. Specifically, 12 patients (85.71%, 12/14) had diabetes, 2 (14.29%, 2/14) had a history of fungal infections, and others had conditions such as coronary heart disease and hypertension. Fever was observed in 12 patients (85.71%, 12/14), with the highest recorded temperature reaching 40°C. Chest pain was reported in 7 patients (50.00%, 7/14), and all patients exhibited symptoms of cough, sputum production, and dyspnea. However, none experienced abdominal pain or coma. Blood cell analysis on the day of admission revealed that 5 patients (35.71%, 5/14) had elevated white blood cell counts, with the highest reaching 31.31×10^9/L, and 8 patients (57.14%, 8/14) had elevated neutrophil percentages. All patients showed elevated C-reactive protein (CRP) levels, indicating significant inflammatory responses. After active treatment, 9 patients improved and were discharged, 2 were transferred to higher-level hospitals due to rapid disease progression, and 2 discontinued treatment for financial reasons. The 14 patients in this study were categorized as follows: 4 proven cases (numbered 1–4 in this study), 4 probable cases (numbered 5–8), and 6 possible cases (numbered 9–14). Among them, 12 patients (85.71%, 12/14) had infections confined to the lungs, while 2 (14.29%, 2/14) had disseminated mucormycosis involving the chest and brain, respectively. Chest CT findings primarily included cavities in 7 patients (50.00%, 7/14), consolidation in 3 (21.43%, 3/14), high-density shadows in 2 (14.29%, 2/14), nodules in 1 (7.14%, 2/14), and pleural effusion in 1 (7.14%, 1/14). The baseline clinical characteristics of the enrolled patients are summarized in Table 1 . Table 1 Clinical characteristics of 14 mucormycosis patients Characteristics Number (%) Gender, Male 8(57.14) Age, years, mean (Q1, Q3) 63.4(56,71) Comorbidities Diabetes 12(85.71) Cardiovascular disease 6(42.85) Pulmonary disease 4(28.57) Hypertension 3(21.43) Hematological disease 2(14.29) Cerebrovascular disease 1(7.14) Clinical manifestations Fever 12(85.71) Cough 14(100.00) Dyspnea 14(100.00) Sputum 14(100.00) Chest pain 7(50.00) Abdominal pain 0 Coma 0 Clinical forms Pulmonary 12(85.71) Dissemination 2(14.29) Classification Proven 4(28.57) Probable 4(28.57) Possible 6(42.85) Surgery 0 Length of stay, d, means (Q1, Q3) 14( 8 , 18 ) Outcomes Cure 9(64.29) Improvement 5(35.71) Survival rate of 90 days 11(78.57) Laboratory Examination WBC(> 10×10 9 /L) 5(35.71) LEU(> 70%) 8(57.14) CRP(> 8 mg/L) 14(100.00) Imaging features Cavities 7(50.00) Consolidation 3(21.43) High density images 2(14.29) Multiple solid nodules 1(7.14) Pleural effusion 1(7.14) Comparison of Serological Tests, Histopathology, and mNGS Among the 14 enrolled patients, 5 (35.71%, 5/14) tested positive for the G-test, and 6 (42.86%, 6/14) tested positive for the GM-test. Among the 4 proven cases, only 1 showed positivity for both tests, while 1 was negative for both. In the 4 probable cases, all tested positive for the GM-test, but only 2 were positive for the G-test. Lung histopathological examination was performed in 4 cases, with 3 (cases 1–3) showing acute and chronic inflammatory cell infiltration and the presence of fungal hyphae. In case 4, only acute and chronic inflammatory cell infiltration and alveolar histiocyte aggregation were observed, without fungal hyphae. mNGS results detected PM pathogens in all patients, identifying a total of 5 species and 16 strains. These included Rhizopus microsporus (43.75%, 7/16), Rhizopus delemar (18.75%, 3/16), Rhizomucor pusillus (18.75%, 3/16), Rhizopus oryzae (12.50%, 2/16), and Lichtheimia ramosa (6.25%, 1/16). In proven cases 1–3, both mNGS and histopathology were positive, but only case 1 was positive for both serological tests, while case 2 was positive only for the G-test. In proven case 4, mNGS was positive, but only the GM-test was positive. Among probable cases 5–8, only cases 5 and 6 were positive for both the G-test and GM-test, while cases 7 and 8 were positive only for the GM-test. In possible cases 9–14, mNGS was positive in all cases, with only case 10 showing positivity for the G-test and the remaining cases negative for serological tests. Notably, mNGS results were obtained prior to histopathological findings in all patients. These results demonstrate that the positivity rate of mNGS (100%, 14/14) was significantly higher than that of histopathology (75.00%, 3/4), the G-test (35.71%, 5/14), and the GM-test (42.86%, 6/14). The detection results for all enrolled patients are summarized in Table 2 . Table 2 The detection results and treatment strategy for 14 fungal mucormycosis patients Case G test GM test Histopathologic Culture mNGS sampling time Treatment Change Treatment 1 P P P Aspergillus fumigatus Rhizopus microsporus D2 D4 VOR + AMB 2 P N P Aspergillus fumigatus Rhizopus delsoni D4 D5 ISA 3 N N P N Rhizopus oryzae D2 D4 AMB 4 N P N Rhizopus Rhizopus oryzae;Rhizopus microsporus D1 D3 AMB + POS 5 P P NT N Rhizophorus minutus D3 D5 POS 6 P P NT Aspergillus fumigatus Rhizopus microsporus D1 D3 VOR + AMB 7 N P NT N Lichtheimia ramosa;Rhizopus microsporus D1 D3 ISA 8 N P NT N Rhizopus minutus D1 D4 AMB 9 N N NT N Rhizopus delsoni D2 D4 AMB 10 P N NT N Rhizopus oryzae D4 D6 VOR + POS 11 N N NT N Rhizopus microsporus D2 D4 AMB 12 N N NT N Rhizopus microsporus D1 D3 POS 13 N N NT N Rhizopus delsoni D3 D5 AMB + ISA 14 N N NT N Rhizopus microsporus D1 D3 POS Positive rate(%) 35.71 50.00 75.00 35.71 100 P: Positive; N: negative; NT: No tested; AMB: Amphotericin B; POS: Posaconazole; ISA: Isavuconazole; VOR: Voriconazole; G-test: 1,3-β-D-glucan test; GM-test: Galactomannan test Comparison of mNGS and Fungal Culture Results All patients underwent timely collection of bronchoalveolar lavage fluid (BALF) for both mNGS and fungal culture after admission. Samples were collected on D1 for 6 patients (42.85%, 6/14), D2 for 4 patients (28.57%, 4/14), D3 for 2 patients (14.29%, 2/14), and D4 for 2 patients (14.29%, 2/14). mNGS results were reported within 18–48 hours after sample submission, significantly faster than fungal culture, which required at least 5 days (Fig. 1 ). Among the 14 enrolled patients, mNGS detected a total of 42 pathogenic strains, including 12 bacteria (28.57%, 12/42), 25 fungi (59.52%, 25/42), and 5 viruses (11.91%, 5/42). Fungi accounted for the highest proportion, with Aspergillus fumigatus being the most common (14.29%, 6/42). The pathogens causing PM detected in this study included Rhizopus microsporus (11.90%, 5/42), Rhizomucor pusillus (7.14%, 3/42), Rhizopus delsoni (7.14%, 3/42), Rhizopus oryzae (7.14%, 3/42), and Lichtheimia ramosa (2.38%, 1/42). Additionally, Candida albicans and Candida glabrata were detected. Among the bacteria, besides common pathogens such as Staphylococcus aureus (7.14%, 3/42) and Haemophilus influenzae (4.76%, 2/42), mNGS also identified Pneumocystis jirovecii (4.76%, 2/42) and Ureaplasma urealyticum (2.38%, 1/42), which are difficult to detect using traditional methods. Detected viruses included Human gammaherpesvirus type 4 (4.76%, 2/42), Human betaherpesvirus type 5 (4.76%, 2/42), and Human alphaherpesvirus type 1 (2.38%, 1/42). Pathogens were detected in all patients. While 3 patients had only one fungal species detected, the remaining 11 had two or more pathogens, including cases with two fungi (case 4), bacteria and fungi (cases 2, 3, 5, 6, 8, 11), fungi and viruses (cases 1, 7), and bacteria, fungi, and viruses (cases 10, 14). Case 10 exhibited a complex mixed infection with S. aureus , A. fumigatus , C. albicans , R. oryzae , and H. gammaherpesvirus type 4 . Case 14 showed Acinetobacter pittii , P. jirovecii , Aspergillus , R. microsporus , and H. betaherpesvirus type 5 , representing the most complex mixed infection. Fungal culture identified fungi in 4 cases, with only 1 case (case 4) detecting Rhizopus, while the other 3 cases (cases 1, 2, 6) identified A. fumigatus . In cases 1, 2, 4, and 6, mNGS not only detected the corresponding Rhizopus and A. fumigatus but also identified additional bacteria, fungi, and viruses (Fig. 2 ). The overall mismatch rate between the two methods was 71.43% (10/14), with a partial match rate of 28.57% (4/14). These results demonstrate that mNGS significantly outperforms fungal culture in terms of positivity rate, detection speed, and breadth of pathogen identification, particularly for rare pathogens and mixed infections. The specific pathogens detected are illustrated in Fig. 2 . Impact of mNGS on Treatment and Outcomes Among the 14 enrolled patients, all had received treatment at local hospitals prior to admission, with 9 (64.29%, 9/14) prophylactically administered voriconazole for antifungal therapy upon admission. Based on the mNGS results, clinicians promptly adjusted the treatment regimens for all patients. Cases 3 and 8 received amphotericin B via nebulization, cases 9 and 11 received intravenous amphotericin B, cases 2 and 7 were treated with oral isavuconazole, cases 5, 12, and 14 received oral posaconazole, case 4 was treated with a combination of intravenous amphotericin B and posaconazole, and case 13 received intravenous amphotericin B and isavuconazole. Cases 1, 6, and 10, which involved mixed infections of Aspergillus and Mucorales, were treated with a combination of voriconazole and amphotericin B (Table 2 ). Additionally, all enrolled patients were administered broad-spectrum antibiotics. Among the 14 patients, 2 (cases 4 and 13) were diagnosed with disseminated mucormycosis. Case 4 involved the lungs and brain, while case 13 involved the lungs and pleura. In case 4, chest CT upon admission indicated severe pneumonia, and the patient was initially treated with meropenem for infection. mNGS results identified Rhizopus delemar and Rhizopus microsporus, and cranial CT revealed a brain abscess, likely due to Rhizopus invasion. The clinician immediately initiated intravenous amphotericin B and posaconazole for antifungal treatment. However, due to the severity of the condition and high treatment costs, the family decided to discontinue treatment, and the patient died 26 days after discharge. In case 13, piperacillin-tazobactam was initially administered for infection. mNGS results detected R. delsoni , prompting the clinician to add isavuconazole for antifungal therapy. Subsequently, the patient developed chest pain, suspected to be caused by Mucorales invading the pleura. The treatment was adjusted to include isavuconazole and amphotericin B cholesteryl sulfate complex infusion. Follow-up chest CT after one week showed improvement in lung infection and abscesses, and the patient was discharged after 10 days of treatment. After active symptomatic treatment, 9 patients improved and were discharged, 2 (cases 2 and 5) were transferred to higher-level hospitals due to lack of improvement, and 3 (cases 4, 6, and 8) discontinued treatment for financial reasons. A 90-day telephone follow-up revealed that all 9 patients who improved and were discharged survived. Among the 5 patients who were discharged due to severe illness, only case 2, who was transferred to a higher-level hospital, survived. The remaining 4 (cases 4, 5, 6, and 8) died due to the severity of their conditions, resulting in a mortality rate of 28.57%. All 4 deceased patients had at least two underlying conditions, including diabetes and myocardial infarction. Case 5 also had acute myeloid leukemia and did not undergo full-course treatment. Discussion Mucormycosis, caused by Mucorales, is characterized by rapid filamentous growth, widespread distribution, spore production, unique structural features, adaptability to diverse environments, and interactions with host immune defenses. These factors collectively contribute to the establishment and progression of infection, making mucormycosis the second most common angioinvasive fungal disease after invasive aspergillosis. PM, the second most prevalent form, occurs through the inhalation of airborne sporangiospores. These spores secrete lytic enzymes and toxins (such as mucorubin) and can spread through lung parenchyma to the chest wall, central nervous system, or gastrointestinal tract, inducing tissue thrombosis and necrosis. This can lead to complications such as encephalitis, brain abscesses, and gastrointestinal ulcers( 22 , 23 ), Due to its severe infection, rapid progression, and high mortality rate( 1 , 11 ), early etiological diagnosis is critical for timely treatment. PM is commonly observed in immunocompromised individuals with conditions such as diabetes, hematologic malignancies, solid organ transplantation, neutropenia, long-term corticosteroid use, and other underlying diseases( 24 ). Numerous studies have identified diabetes as the most frequent predisposing factor, present in 50–70% of patients diagnosed with mucormycosis( 8 , 25 ). Hyperglycemia impairs phagocyte function, intracellular killing, and chemotaxis, while hypoxia in lung diseases creates a favorable environment for fungal spore germination and proliferation, increasing the risk of mucormycosis. Notably, mucormycosis can also occur in individuals without any predisposing conditions( 10 , 26 ). In this study, 92.85% of patients had underlying diseases, with 85.71% having diabetes, consistent with previous studies( 8 , 9 , 25 ), This highlights diabetes as a significant risk factor for PM, while other comorbidities (e.g., coronary heart disease, hypertension) may further increase susceptibility. Previous studies have also reported a higher prevalence of mucormycosis in males( 8 , 9 ), which aligns with our findings (57.14% male vs. 42.86% female). Mucorales can invade not only the lungs but also the brain, nose, orbits, gastrointestinal tract, kidneys, skin, and other tissues. In this study, 12 cases involved only the lungs, while 2 cases involved the lungs and pleura or central nervous system, diagnosed as disseminated mucormycosis. The clinical symptoms of PM, including high fever (> 38°C), cough, dyspnea, and chest pain[1], are nonspecific. In this study, 85.71% of patients presented with fever, 50.00% with chest pain, and all patients exhibited cough, sputum production, and dyspnea. In the early stages, imaging may only show perivascular ground-glass opacities, progressing to pulmonary nodules, consolidation, cavities, and the reverse halo sign in later stages( 27 ),The primary imaging findings in our patients were cavities (50.00%) and consolidation (21.43%). Based solely on clinical manifestations and imaging, it is challenging for clinicians to distinguish PM from other pulmonary diseases. Although the G test and GM test used for fungal screening cannot be directly applied to the diagnosis of mucormycosis, they hold certain value in differentiating invasive aspergillosis( 14 , 28 ). In this study, the positive rates of these two tests were 35.71% and 42.86%, respectively. While the GM-test was positive in 3 culture-positive cases (cases 1, 2, and 6), it was also positive in culture-negative cases (cases 5, 7, and 8), possibly due to low fungal concentrations or prior antifungal prophylaxis. However, negative results cannot rule out mucormycosis. Although definitive diagnosis of PM can rely on BALF fungal culture and lung histopathology, invasive procedures may not be initially suitable for patients without risk factors, and culture results take considerable time. In this study, all cases underwent BALF fungal culture, with only 4 yielding positive results, including just 1 case of Rhizopus , resulting in a diagnostic positivity rate of only 7.14%. Among the 4 cases with histopathological examination, fungal hyphae were identified in only 3, yielding a positivity rate of 75%. mNGS directly sequences all microbial nucleic acids in a sample without prior knowledge of the pathogen, significantly increasing the likelihood of detecting Mucorales by identifying specific gene sequences. It serves as an important complement to histopathology and microbiological diagnostics. Recently, mNGS has been increasingly used to assist in the clinical diagnosis of invasive fungal infections, including mucormycosis( 29 , 30 , 31 ). Wang C. et al. studied 310 patients with suspected invasive pulmonary fungal infections and found that mNGS had a sensitivity and specificity of 86.76% and 86.98%, respectively, outperforming traditional microbiological methods in accuracy and speed (AUC = 0.967)( 30 ). Wei E. et al. analyzed 3 pediatric cases of rare invasive mold brain abscesses, detecting Rhizomucor miehei and Rhizomucor pusillus in cerebrospinal fluid via mNGS, enabling timely and precise treatment and discharge( 31 ). In this study, all enrolled patients underwent BALF mNGS, which detected 25 fungal strains, including 15 cases of Mucorales. The most common pathogen was R. microsporus , followed by Rhizomucor pusillus and R. delsoni , consistent with previous studies( 32 , 33 ). However, regional variations in pathogenic organisms may exist due to environmental factors and temperature-dependent growth characteristics. Additionally, metagenomic sequencing can detect other microorganisms coexisting with Mucorales. Studies have shown that mucormycosis can co-occur with other invasive mold infections, most commonly aspergillosis, as reported in hematologic malignancies( 34 ), COVID-19-associated mucormycosis (CAM)( 35 ), and other immunocompromised patients( 36 ). In this study, 50.00% of patients had co-detected Aspergillus. Besides fungi, mNGS also identified bacteria and viruses, including P. jirovecii and U. urealyticum , which are difficult to detect using traditional methods. Among the 11 cases with mixed infections, two or more pathogens were detected. These results demonstrate that mNGS outperforms traditional methods in terms of reporting speed, comprehensiveness, and positivity rate, providing more timely, comprehensive, and accurate diagnoses of PM, particularly in mixed infections, consistent with previous studies( 37 ). In this study, all 14 patients had their treatment regimens adjusted based on mNGS results. Targeted antifungal therapies, including isavuconazole, posaconazole, amphotericin B, or voriconazole, were administered individually or in combination as needed. Amphotericin B is the most active drug against Mucorales in vitro and has shown efficacy in treating mucormycosis when used alone( 38 , 39 ). In this study, 8 of the 14 patients received amphotericin B alone or in combination with other antifungals. After precise and active treatment, 9 patients improved and were discharged. A 90-day follow-up revealed that 4 patients died due to severe illness, delayed treatment, and underlying comorbidities, resulting in a mortality rate of 28.57%, lower than previously reported rates( 40 , 41 ). This may be attributed to differences in the study population or the timely and accurate diagnostic results provided by mNGS, enabling clinicians to develop more personalized treatment plans and improve patient outcomes. This study highlights the superior diagnostic and prognostic advantages of mNGS over traditional methods in invasive mucormycosis, allowing clinicians to rapidly adjust treatment strategies and enhance clinical efficiency. This study has certain limitations. First, as a single-center retrospective analysis with a small sample size, the conclusions may be subject to bias. Second, Mucorales are ubiquitous in the environment, making it difficult to distinguish colonization from infection. Additionally, the lack of standardized bioinformatics analysis protocols for mNGS necessitates comprehensive clinical judgment. In conclusion, mNGS significantly improves the early diagnosis of PM through its rapid and comprehensive pathogen detection capabilities, particularly in cases where traditional methods yield negative results or in mixed infections. Although challenges remain in the clinical application of mNGS, our study provides new insights into its use for the early diagnosis of PM. Conclusion As an emerging pathogen detection technology, mNGS demonstrates a significantly high positive rate in the detection of Mucorales pathogens. It is particularly suitable for infectious diseases with negative results from traditional detection methods or complex clinical conditions. mNGS assists clinicians in achieving early diagnosis and rapidly adjusting treatment plans, thereby improving patient survival rates. Declarations Ethics approval Based on the "Administrative Measures for Ethical Review of Biomedical Research Involving Human Beings", this study was approved by the Institutional Medical Ethics Committee of Affiliated Hospital of Jining Medical University (Ethics Approval Number: 2025-01-C024). Clinical trial not applicable Consent for publication not applicable Availability of data and material Data is provided within the manuscript Competing interests We declare no conflicts of interest. Funding This work was supported by the Research Fund for Shandong Provincial Natural Science Foundation (No. ZR2023MH325), Jining Medical University Affiliated Hospital Doctoral Research Fund (No. 2022-BS-01), Jining Medical University Affiliated Hospital “Zhi-Xing” Project (No. ZX-ZD-2023-01), Academician Lin He New Medicine (No. JYHL2022MS05) and the Jining City Key Research and Development Plan Project (No. 2023YXNS178). Authors' contributions X Y, MK and LQ J contributed to the design and execution of the study and the writing of the manuscript. XH H, HY S, SG G, H C, T L, HX D, JY Y GH P and XJ M contributed to the raw sequence data upload process and provided valuable assistance with the project. A cknowledgments We are grateful to all the participants. We also deeply appreciate the doctors' assistance with sample collection. References Cornely OA, Alastruey-Izquierdo A, Arenz D, Chen SCA, Dannaoui E, Hochhegger B, et al. Global guideline for the diagnosis and management of mucormycosis: an initiative of the European Confederation of Medical Mycology in cooperation with the Mycoses Study Group Education and Research Consortium. Lancet Infect Dis. 2019;19(12):e405–21. Hassan MIA, Voigt K. Pathogenicity patterns of mucormycosis: epidemiology, interaction with immune cells and virulence factors. Med Mycol. 2019;57(Suppl 2):S245–56. Singla K, Samra T, Bhatia N. Primary Cutaneous Mucormycosis in a Trauma Patient with Morel-Lavallée Lesion. Indian J Crit Care Med Peer-Rev Off Publ Indian Soc Crit Care Med. 2018;22(5):375–7. Rodriguez CJ, Tribble DR, Malone DL, Murray CK, Jessie EM, Khan M, et al. Treatment of Suspected Invasive Fungal Infection in War Wounds. Mil Med. 2018;183(suppl2):142–6. Muthu V, Agarwal R, Rudramurthy SM, Thangaraju D, Shevkani MR, Patel AK, et al. Multicenter Case–Control Study of COVID-19–Associated Mucormycosis Outbreak, India. Emerg Infect Dis. 2023;29(1):8–19. Prakash H, Chakrabarti A. Global Epidemiology of Mucormycosis. J Fungi. 2019;5(1):26. Sahu M, Shah M, Mallela VR, Kola VR, Boorugu HK, Punjani AAR, et al. COVID-19 associated multisystemic mucormycosis from India: a multicentric retrospective study on clinical profile, predisposing factors, cumulative mortality and factors affecting outcome. Infection. 2023;51(2):407–16. Manade VV, Kotecha MR. A clinical study of rhino-orbital-cerebral mucormycosis during the COVID-19 pandemic in western Maharashtra. J Fam Med Prim Care. 2024;13(9):3730–4. Kumari K, Rathod D, Meshram T, Mohammed S, Raju S, Sharma A, et al. Perioperative anesthesia challenges and outcomes of patients with Rhino-Orbito-Cerebral Mucormycosis during the second wave of COVID-19 pandemic: An observational study. J Anaesthesiol Clin Pharmacol. 2023;39(4):615–21. Jeong W, Keighley C, Wolfe R, Lee WL, Slavin MA, Kong DCM, et al. The epidemiology and clinical manifestations of mucormycosis: a systematic review and meta-analysis of case reports. Clin Microbiol Infect. 2019;25(1):26–34. Stone N, Gupta N, Schwartz I. Mucormycosis: time to address this deadly fungal infection. Lancet Microbe. 2021;2(8):e343–4. Danion F, Duval C, Séverac F, Bachellier P, Candolfi E, Castelain V, et al. Factors associated with coinfections in invasive aspergillosis: a retrospective cohort study. Clin Microbiol Infect. 2021;27(11):1644–51. Millon L, Caillot D, Berceanu A, Bretagne S, Lanternier F, Morio F, et al. Evaluation of serum Mucorales PCR for the diagnosis of Mucormycoses: The MODIMUCOR prospective trial. Clin Infect Dis. 2022;75(5):777–85. Lass-Flörl C. Zygomycosis: conventional laboratory diagnosis. Clin Microbiol Infect. 2009;15:60–5. Walsh TJ, Gamaletsou MN, McGinnis MR, Hayden RT, Kontoyiannis DP. Early Clinical and Laboratory Diagnosis of Invasive Pulmonary, Extrapulmonary, and Disseminated Mucormycosis (Zygomycosis). Clin Infect Dis. 2012;54(suppl1):S55–60. Danion F, Coste A, Le Hyaric C, Melenotte C, Lamoth F, Calandra T, et al. What Is New in Pulmonary Mucormycosis? J Fungi. 2023;9(3):307. Liu Y, Wu W, Xiao Y, Zou H, Hao S, Jiang Y. Application of metagenomic next-generation sequencing and targeted metagenomic next-generation sequencing in diagnosing pulmonary infections in immunocompetent and immunocompromised patients. Front Cell Infect Microbiol. 2024;14:1439472. Gu W, Miller S, Chiu CY. Clinical Metagenomic Next-Generation Sequencing for Pathogen Detection. Annu Rev Pathol. 2019;14:319–38. Liu H, Zhang Y, Chen G, Sun S, Wang J, Chen F, et al. Diagnostic Significance of Metagenomic Next-Generation Sequencing for Community-Acquired Pneumonia in Southern China. Front Med. 2022;9:807174. Zhang Y, Wei E, Niu J, Yan K, Zhang M, Yuan W, et al. Clinical features of pediatric mucormycosis: role of metagenomic next generation sequencing in diagnosis. Front Cell Infect Microbiol. 2024;14:1368165. Donnelly JP, Chen SC, Kauffman CA, Steinbach WJ, Baddley JW, Verweij PE, et al. Revision and Update of the Consensus Definitions of Invasive Fungal Disease From the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium. Clin Infect Dis Off Publ Infect Dis Soc Am. 2019;71(6):1367–76. Steinbrink JM, Miceli MH. Clinical Review of Mucormycosis. Infect Dis Clin North Am. 2021;35(2):435–52. Soliman SSM, Baldin C, Gu Y, Singh S, Gebremariam T, Swidergall M, et al. Mucoricin is a Ricin-Like Toxin that is Critical for the Pathogenesis of Mucormycosis. Nat Microbiol. 2021;6(3):313–26. Singh AK, Singh R, Joshi SR, Misra A. Mucormycosis in COVID-19: A systematic review of cases reported worldwide and in India. Diabetes Metab Syndr. 2021;15(4):102146. Prakash H, Ghosh AK, Rudramurthy SM, Singh P, Xess I, Savio J, et al. A prospective multicenter study on mucormycosis in India: Epidemiology, diagnosis, and treatment. Med Mycol. 2019;57(4):395–402. Zhang Y, Wei E, Niu J, Yan K, Zhang M, Yuan W, et al. Clinical features of pediatric mucormycosis: role of metagenomic next generation sequencing in diagnosis. Front Cell Infect Microbiol. 2024;14:1368165. Agrawal R, Yeldandi A, Savas H, Parekh ND, Lombardi PJ, Hart EM. Pulmonary Mucormycosis: Risk Factors, Radiologic Findings, and Pathologic Correlation. Radiographics. 2020;40(3):656–66. Smith LD, Ahmad M, Ashraf DC, Kersten RC, Winn BJ, Grob SR, et al. Cutaneous mucormycosis of the eyelid treated with subcutaneous liposomal amphotericin B injections. Ophthal Plast Reconstr Surg. 2024;40(2):e42–5. Boulware DR, Atukunda M, Kagimu E, Musubire AK, Akampurira A, Tugume L, et al. Oral Lipid Nanocrystal Amphotericin B for Cryptococcal Meningitis: A Randomized Clinical Trial. Clin Infect Dis Off Publ Infect Dis Soc Am. 2023;77(12):1659–67. Wang C, You Z, Fu J, Chen S, Bai D, Zhao H, et al. Application of metagenomic next-generation sequencing in the diagnosis of pulmonary invasive fungal disease. Front Cell Infect Microbiol. 2022;12:949505. Wei E, Niu J, Zhang M, Zhang Y, Yan K, Fang X, et al. Metagenomic next-generation sequencing could play a pivotal role in validating the diagnosis of invasive mold disease of the central nervous system. Front Cell Infect Microbiol. 2024;14:1393242. Pham D, Howard-Jones AR, Sparks R, Stefani M, Sivalingam V, Halliday CL, et al. Epidemiology, Modern Diagnostics, and the Management of Mucorales Infections. J Fungi. 2023;9(6):659. Özbek L, Topçu U, Manay M, Esen BH, Bektas SN, Aydın S, et al. COVID-19–associated mucormycosis: a systematic review and meta-analysis of 958 cases. Clin Microbiol Infect. 2023;29(6):722–31. Miller MA, Molina KC, Gutman JA, Scherger S, Lum JM, Mossad SB, et al. Mucormycosis in Hematopoietic Cell Transplant Recipients and in Patients With Hematological Malignancies in the Era of New Antifungal Agents. Open Forum Infect Dis. 2020;8(2):ofaa646. Sasani E, Pakdel F, Khodavaisy S, Salehi M, Salami A, Sohrabi M, et al. Mixed Aspergillosis and Mucormycosis Infections in Patients with COVID-19: Case Series and Literature Review. Mycopathologia. 2024;189(1):10. Loubet D, Sarton B, Lelièvre L, Grouteau G, Iriart X, Chauvin P, et al. Fatal mucormycosis and aspergillosis coinfection associated with haemophagocytic lymphohistiocytosis: A case report and literature review. J Med Mycol. 2023;33(1):101325. Hu X, Jiang L, Liu X, Chang H, Dong H, Yan J, et al. The diagnostic value of bronchoalveolar lavage fluid metagenomic next-generation sequencing in critically ill patients with respiratory tract infections. Microbiol Spectr. 2024;12(8):e00458–24. Pomorska A, Malecka A, Jaworski R, Radon-Proskura J, Hare RK, Nielsen HV, et al. Isavuconazole in a Successful Combination Treatment of Disseminated Mucormycosis in a Child with Acute Lymphoblastic Leukaemia and Generalized Haemochromatosis: A Case Report and Review of the Literature. Mycopathologia. 2019;184(1):81–8. Ashkenazi-Hoffnung L, Bilavsky E, Levy I, Grisaru G, Sadot E, Ben-Ami R, et al. Isavuconazole As Successful Salvage Therapy for Mucormycosis in Pediatric Patients. Pediatr Infect Dis J. 2020;39(8):718–24. Claustre J, Larcher R, Jouve T, Truche AS, Nseir S, Cadiet J, et al. Mucormycosis in intensive care unit: surgery is a major prognostic factor in patients with hematological malignancy. Ann Intensive Care. 2020;10:74. Lin CY, Wang IT, Chang CC, Lee WC, Liu WL, Huang YC, et al. Comparison of Clinical Manifestation, Diagnosis, and Outcomes of Invasive Pulmonary Aspergillosis and Pulmonary Mucormycosis. Microorganisms. 2019;7(11):531. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6308222","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":442545774,"identity":"40032c12-5d5b-4512-a156-f9cc4f2784ee","order_by":0,"name":"Xiao Yao","email":"","orcid":"","institution":"Affiliated Hospital of Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Yao","suffix":""},{"id":442545775,"identity":"6a86c2e4-ac96-4846-b2cc-b6a25d4adf54","order_by":1,"name":"Haiyang Sang","email":"","orcid":"","institution":"Affiliated Hospital of Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Haiyang","middleName":"","lastName":"Sang","suffix":""},{"id":442545777,"identity":"8785e2cd-4233-4249-85c5-6ed3991b9925","order_by":2,"name":"Xiaohang Hu","email":"","orcid":"","institution":"Affiliated Hospital of Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaohang","middleName":"","lastName":"Hu","suffix":""},{"id":442545778,"identity":"aac4d538-0d62-477a-8232-7918f12c64cb","order_by":3,"name":"Jinyan Yan","email":"","orcid":"","institution":"Affiliated Hospital of Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jinyan","middleName":"","lastName":"Yan","suffix":""},{"id":442545780,"identity":"e15fe218-67c1-42fc-8490-44d769070159","order_by":4,"name":"Ting Liu","email":"","orcid":"","institution":"Affiliated Hospital of Jining Medical 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University","correspondingAuthor":false,"prefix":"","firstName":"Liqing","middleName":"","lastName":"Jiang","suffix":""},{"id":442545789,"identity":"b5a971f9-ef8c-456a-bf50-f6ce4ebe3d7f","order_by":11,"name":"Min Kong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYJACZijVACRsePj5G4jWwghSmiYjOeMAaVoO2xg0JOBXLt9++PDnwja7PHkHxsbHPBXneQwYDjB++JiDWwtjT1qC8cy25GLDA4zNxjxnbvOYMzcwS87chs9ROQbJvG3MiRsbGNskZ7bd5rFsOMDGzItHCxv/G4PDvG31UC3/zvEYHEjAr4VHIsewmbftcOJ8BsY2iY8NBwhrkZB4lszMc+544gZmxmaDD8eSeSRnHGzG6xf5/uTDn3nKqhPntzcffJBQY2fPz9988MNHPFrgwOAwnAmOICKAPJHqRsEoGAWjYAQCAOCvTkjjUbuoAAAAAElFTkSuQmCC","orcid":"","institution":"Medical Laboratory of Jining Medical University, Lin He's Academician Workstation of New Medicine and Clinical Translation in Jining Medical University, Jining Medical University","correspondingAuthor":true,"prefix":"","firstName":"Min","middleName":"","lastName":"Kong","suffix":""}],"badges":[],"createdAt":"2025-03-26 03:38:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6308222/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6308222/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80814083,"identity":"9195e49b-d178-4eac-9776-bfa4e04076b8","added_by":"auto","created_at":"2025-04-17 10:46:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":26714,"visible":true,"origin":"","legend":"\u003cp\u003ethe time interval from sampling to report issuance for mNGS and fungal culture. The blue line represents the interval for mNGS, with all patients having a duration of 1–3 days. The yellow line represents the interval for fungal culture, with all patients having a duration of 2–5 days.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6308222/v1/4c1bed8749b878af50ffc9d6.png"},{"id":80814991,"identity":"50d19af1-66f9-4763-a366-5e266c261a42","added_by":"auto","created_at":"2025-04-17 10:54:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42659,"visible":true,"origin":"","legend":"\u003cp\u003ethe types of pathogens detected by mNGS and fungal culture in all enrolled patients. Numbers 1–14 represent the types of pathogens detected in each patient, including bacteria, fungi, and viruses. The labels 1C, 2C, 4C, and 6C indicate the types of pathogens detected in patients with positive fungal cultures, with only one type of fungus detected in each case.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6308222/v1/0d98e183f1ec0a4b82b46f96.png"},{"id":83234076,"identity":"d2a3ef8d-d81d-4fef-8f64-a5fd19625a6b","added_by":"auto","created_at":"2025-05-21 14:01:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":983331,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6308222/v1/ad9b7324-1e27-47cd-bc61-efddc75d4abe.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Application Value of Metagenomic Next-generation Sequencing in the Diagnosis of Pulmonary Mucormycosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMucormycosis is an invasive fungal infection caused by Mucorales fungi. Although relatively rare, it progresses rapidly, shows no age predilection, is highly destructive, and carries a high mortality rate (approximately 40%-80%)(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Its incidence ranges from 1 to 12.3 cases per million people in Asia(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Mucorales can invade the lungs, nose, orbits, gastrointestinal tract, kidneys, skin, and other tissues, often leading to multi-organ involvement and disseminated infections(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). In recent years, solid organ transplantation, malignancies, hypoxia, and inappropriate corticosteroid use have become significant risk factors for mucormycosis with advances in medicine(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). However, diabetes remains the primary risk factor in developing countries(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Additionally, studies indicate that males are more susceptible to mucormycosis than females(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Pulmonary mucormycosis (PM), as the second most common type, has garnered significant attention due to its invasiveness and mortality rate exceeding 50%(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). It is primarily caused by the inhalation of sporangiospores and, although commonly seen in immunocompromised patients, can also occur in individuals without any identifiable risk factors. Due to its nonspecific clinical manifestations, PM is often misdiagnosed as bacterial pneumonia, aspergillosis, or other infectious diseases, particularly in cases of mixed infections, making diagnosis even more challenging(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Therefore, early and rapid diagnosis poses a significant challenge for clinicians.\u003c/p\u003e \u003cp\u003eThe diagnosis of PM currently relies mainly on serological tests, such as 1,3-β-D-glucan test (G-test) and Galactomannan test (GM-test), microbiological cultures, imaging, and histopathological analysis. However, these methods have certain limitations. Serological tests, while sensitive to various fungi, cannot directly identify Mucorales, resulting in insufficient specificity. In microbiological diagnostics, direct microscopy has a low positivity rate and cannot differentiate fungal species, while fungal cultures are time-consuming and also exhibit low positivity rates(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), making them inadequate for rapid diagnosis. Imaging studies, although helpful in diagnosing lung diseases, face challenges in distinguishing PM from other fungal infections. Histopathology is considered the gold standard for diagnosis, but its positivity rate is only about 50%, and obtaining representative tissue samples is difficult, especially in the early stages of the disease(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In recent years, advancements in molecular diagnostic technologies have provided new possibilities for the diagnosis of PM. Techniques such as qPCR, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and restriction fragment length polymorphism have shown diagnostic value but are limited by issues such as high false-positive rates or operational complexity(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). In contrast, metagenomic next-generation sequencing (mNGS) has demonstrated significant advantages. It can detect the genomic information of all microorganisms in a sample without relying on traditional culture methods or pathogen prediction, enabling simultaneous detection of bacteria, viruses, fungi, and parasites. This is particularly useful for rare or difficult-to-culture pathogens, such as Mucorales(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Compared to traditional methods, mNGS offers higher sensitivity and significantly reduces detection time(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), facilitating early diagnosis and timely treatment. mNGS holds significant potential in the diagnosis of PM, providing clinicians with faster and more accurate pathogen information, thereby improving patient outcomes and reducing mortality rates.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design and Patients\u003c/h2\u003e \u003cp\u003eThis retrospective study strictly adhered to inclusion and exclusion criteria, consecutively\u003c/p\u003e \u003cp\u003eenrolling 14 hospitalized patients with PM at the Affiliated Hospital of Jining Medical University\u003c/p\u003e \u003cp\u003ebetween 1/6/2021 and 30/6/2024. The inclusion criteria were as follows: age\u0026thinsp;\u0026ge;\u0026thinsp;18 years;\u003c/p\u003e \u003cp\u003ecomplete clinical data; and simultaneous performance of serological tests, fungal cultures, and\u003c/p\u003e \u003cp\u003emNGS. The date of admission was defined as D0. All patients had received treatment at local\u003c/p\u003e \u003cp\u003ehospitals prior to admission. To promptly identify the infectious pathogens, all enrolled patients\u003c/p\u003e \u003cp\u003eunderwent bronchoalveolar lavage fluid (BALF) fungal culture and mNGS within D4. Based on\u003c/p\u003e \u003cp\u003ethe definitions of invasive fungal diseases by the European Organization for Research and\u003c/p\u003e \u003cp\u003eTreatment of Cancer/National Institute of Allergy and Infectious Diseases Mycoses Study Group\u003c/p\u003e \u003cp\u003e(EORTC/NIAIDMSG)(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), invasive fungal infections were classified into three categories:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eI) Proven cases: defined by the presence of fungi in lung histopathology or BALF fungal\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eculture; II) Probable cases: meeting clinical criteria (i.e., imaging, bronchoscopy, or sinus analysis) and mycological criteria (specific fungal antigens and cell wall components); III) Possible cases: meeting clinical criteria but lacking mycological evidence.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eAfter obtaining written informed consent from the patients' families, clinicians collected BALF through standard bronchoalveolar lavage procedures and promptly sent the samples for fungal culture and mNGS. Clinical data collected for all enrolled patients included gender, age, comorbidities, imaging and laboratory test results, clinical manifestations, treatment modalities, hospital stay duration, and discharge outcomes. A three-month follow-up on survival status was conducted via telephone.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTraditional Detection Methods\u003c/h3\u003e\n\u003cp\u003eAfter trypsin digestion of BALF, 10 \u0026micro;L was inoculated onto Sabouraud dextrose agar for\u003c/p\u003e \u003cp\u003efungal culture. The cultures were incubated at 56\u0026deg;C in a Thermo Scientific 371 CO\u003csub\u003e2\u003c/sub\u003e incubator\u003c/p\u003e \u003cp\u003e(Thermo Fisher Scientific,USA). Following pure culture isolation, the samples were inoculated\u003c/p\u003e \u003cp\u003eonto chromogenic media for fungal identification and subjected to antifungal susceptibility testing\u003c/p\u003e \u003cp\u003eusing the YEASTONE kit (Thermo Fisher Scientific, USA). Simultaneously, samples were\u003c/p\u003e \u003cp\u003esmeared onto glass slides for Gram staining and morphological observation. Antifungal\u003c/p\u003e \u003cp\u003esusceptibility results were interpreted according to the 2018 Clinical and Laboratory Standards\u003c/p\u003e \u003cp\u003eInstitute (CLSI) breakpoints. Peripheral blood samples were collected for complete blood count,\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003ereactive protein (CRP), G-test, and GM-test analyses. Lung tissue samples were sent to the\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003epathology laboratory for histological sectioning. Tissue sections were stained with hematoxylin-eosin, periodic acid-Schiff, and Grocott's methenamine silver and examined under an optical microscope (Olympus BX53, Japan). All patients underwent Chest Computed Tomography scans using either a GE Optima CT660 scanner or a SIEMENS Definition Flash scanner after admission, and the reports were issued by two senior radiologists.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e\n\u003ch3\u003emNGS Detection\u003c/h3\u003e\n\u003cp\u003eA 3 mL aliquot of BALF was inactivated at 65\u0026deg;C for 30 minutes, and DNA was extracted\u003c/p\u003e \u003cp\u003estrictly following the instructions of the DNA purification kit (BGI Genomics, Wuhan, China).\u003c/p\u003e \u003cp\u003eDNA libraries were constructed at appropriate concentrations, including DNA fragmentation, end\u003c/p\u003e \u003cp\u003erepair, adapter ligation, and PCR amplification. The DNA library and sequencing library\u003c/p\u003e \u003cp\u003epreparations were performed using the PMseq\u0026trade; high-throughput DNA detection kit (BGI\u003c/p\u003e \u003cp\u003eGenomics). Quality-qualified libraries were used to prepare DNA nanoballs using the MiSeq\u0026trade;\u003c/p\u003e \u003cp\u003eDx reagent kit (BGI Genomics). Finally, sequencing was performed on the BGI200 platform(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNegative and positive controls were included throughout the entire process to ensure quality\u003c/p\u003e \u003cp\u003econtrol and minimize contamination. Each sample required a minimum of 20\u0026nbsp;million high-quality\u003c/p\u003e \u003cp\u003esequencing reads. Low-quality reads, low-complexity reads, adapter contamination, and short\u003c/p\u003e \u003cp\u003esequences (\u0026lt;\u0026thinsp;35 bps) were removed. The filtered sequences were mapped to the human reference\u003c/p\u003e \u003cp\u003edatabase using Burrows-Wheeler Alignment (Version 0.7.10) software to identify and exclude\u003c/p\u003e \u003cp\u003ehuman host sequences. The remaining data were compared against the Pathogen Metagenomic\u003c/p\u003e \u003cp\u003eDatabase. All parameters of the detected pathogens, including strict unique mapped reads\u003c/p\u003e \u003cp\u003enumber(SMRN), relative abundance, genome coverage, and depth, were classified and recorded.\u003c/p\u003e \u003cp\u003eEach batch of experiments included negative controls (NC) and positive controls (PC), which\u003c/p\u003e \u003cp\u003ewere processed alongside the samples and included in the bioinformatics analysis. An infectious\u003c/p\u003e \u003cp\u003epathogen was considered positive by mNGS if it met one of the following criteria: I) When the\u003c/p\u003e \u003cp\u003eSMRN of the NC was greater than 50, the SMRN of the sample had to be at least 3 times that of\u003c/p\u003e \u003cp\u003ethe NC; II) When SMRN of the NC was less than 50, the SMRN of the sample had to be at least 5\u003c/p\u003e \u003cp\u003etimes that of the NC; III) When the NC was negative, the SMRN thresholds were set as follows:\u003c/p\u003e \u003cp\u003ebacteria and fungi\u0026thinsp;\u0026ge;\u0026thinsp;3, viruses\u0026thinsp;\u0026ge;\u0026thinsp;1, and parasites\u0026thinsp;\u0026ge;\u0026thinsp;100. Among the qualifying genera, the one with\u003c/p\u003e \u003cp\u003ethe highest SMRN was selected as the final positive result. However, whether the reported\u003c/p\u003e \u003cp\u003epathogen was truly pathogenic required further clinical judgment by physicians based on the\u003c/p\u003e \u003cp\u003epatient's clinical manifestations.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBaseline Characteristics of Patients\u003c/h2\u003e \u003cp\u003eThis study included 14 patients with PM, with a mean age of 63.4 years (range: 38\u0026ndash;77).\u003c/p\u003e \u003cp\u003eMales accounted for 57.14% (8/14), and the average hospital stay was 14 days (range: 4\u0026ndash;34).\u003c/p\u003e \u003cp\u003eAmong the 14 patients, only 1 (7.15%, 1/14) had no underlying diseases, while the remaining 13\u003c/p\u003e \u003cp\u003e(92.85%, 13/14) had a history of comorbidities. Specifically, 12 patients (85.71%, 12/14) had\u003c/p\u003e \u003cp\u003ediabetes, 2 (14.29%, 2/14) had a history of fungal infections, and others had conditions such as\u003c/p\u003e \u003cp\u003ecoronary heart disease and hypertension. Fever was observed in 12 patients (85.71%, 12/14), with\u003c/p\u003e \u003cp\u003ethe highest recorded temperature reaching 40\u0026deg;C. Chest pain was reported in 7 patients (50.00%,\u003c/p\u003e \u003cp\u003e7/14), and all patients exhibited symptoms of cough, sputum production, and dyspnea. However,\u003c/p\u003e \u003cp\u003enone experienced abdominal pain or coma. Blood cell analysis on the day of admission revealed\u003c/p\u003e \u003cp\u003ethat 5 patients (35.71%, 5/14) had elevated white blood cell counts, with the highest reaching\u003c/p\u003e \u003cp\u003e31.31\u0026times;10^9/L, and 8 patients (57.14%, 8/14) had elevated neutrophil percentages. All patients\u003c/p\u003e \u003cp\u003eshowed elevated C-reactive protein (CRP) levels, indicating significant inflammatory responses.\u003c/p\u003e \u003cp\u003eAfter active treatment, 9 patients improved and were discharged, 2 were transferred to higher-level\u003c/p\u003e \u003cp\u003ehospitals due to rapid disease progression, and 2 discontinued treatment for financial reasons.\u003c/p\u003e \u003cp\u003eThe 14 patients in this study were categorized as follows: 4 proven cases (numbered 1\u0026ndash;4 in this study), 4 probable cases (numbered 5\u0026ndash;8), and 6 possible cases (numbered 9\u0026ndash;14). Among them, 12 patients (85.71%, 12/14) had infections confined to the lungs, while 2 (14.29%, 2/14) had disseminated mucormycosis involving the chest and brain, respectively. Chest CT findings primarily included cavities in 7 patients (50.00%, 7/14), consolidation in 3 (21.43%, 3/14), high-density shadows in 2 (14.29%, 2/14), nodules in 1 (7.14%, 2/14), and pleural effusion in 1 (7.14%, 1/14). The baseline clinical characteristics of the enrolled patients are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical characteristics of 14 mucormycosis patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender, Male\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8(57.14)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge, years, mean (Q1, Q3)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63.4(56,71)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eComorbidities\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiabetes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12(85.71)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCardiovascular disease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6(42.85)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePulmonary disease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4(28.57)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypertension\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3(21.43)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHematological disease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2(14.29)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCerebrovascular disease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1(7.14)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eClinical manifestations\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFever\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12(85.71)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14(100.00)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDyspnea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14(100.00)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSputum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14(100.00)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChest pain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7(50.00)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbdominal pain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eComa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eClinical forms\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePulmonary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12(85.71)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDissemination\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2(14.29)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eClassification\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProven\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4(28.57)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProbable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4(28.57)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePossible\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6(42.85)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSurgery\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLength of stay, d, means (Q1, Q3)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOutcomes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9(64.29)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImprovement\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5(35.71)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSurvival rate of 90 days\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11(78.57)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLaboratory Examination\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWBC(\u0026gt;\u0026thinsp;10\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5(35.71)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLEU(\u0026gt;\u0026thinsp;70%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8(57.14)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCRP(\u0026gt;\u0026thinsp;8 mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14(100.00)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eImaging features\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCavities\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7(50.00)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConsolidation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3(21.43)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh density images\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2(14.29)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMultiple solid nodules\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1(7.14)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePleural effusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1(7.14)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eComparison of Serological Tests, Histopathology, and mNGS\u003c/h2\u003e \u003cp\u003eAmong the 14 enrolled patients, 5 (35.71%, 5/14) tested positive for the G-test, and 6\u003c/p\u003e \u003cp\u003e(42.86%, 6/14) tested positive for the GM-test. Among the 4 proven cases, only 1 showed\u003c/p\u003e \u003cp\u003epositivity for both tests, while 1 was negative for both. In the 4 probable cases, all tested positive\u003c/p\u003e \u003cp\u003efor the GM-test, but only 2 were positive for the G-test. Lung histopathological examination was\u003c/p\u003e \u003cp\u003eperformed in 4 cases, with 3 (cases 1\u0026ndash;3) showing acute and chronic inflammatory cell infiltration\u003c/p\u003e \u003cp\u003eand the presence of fungal hyphae. In case 4, only acute and chronic inflammatory cell infiltration\u003c/p\u003e \u003cp\u003eand alveolar histiocyte aggregation were observed, without fungal hyphae.\u003c/p\u003e \u003cp\u003emNGS results detected PM pathogens in all patients, identifying a total of 5 species and 16 strains. These included \u003cem\u003eRhizopus microsporus\u003c/em\u003e (43.75%, 7/16), \u003cem\u003eRhizopus delemar\u003c/em\u003e (18.75%, 3/16), \u003cem\u003eRhizomucor pusillus\u003c/em\u003e (18.75%, 3/16), \u003cem\u003eRhizopus oryzae\u003c/em\u003e (12.50%, 2/16), and \u003cem\u003eLichtheimia ramosa\u003c/em\u003e (6.25%, 1/16). In proven cases 1\u0026ndash;3, both mNGS and histopathology were positive, but only case 1 was positive for both serological tests, while case 2 was positive only for the G-test. In proven case 4, mNGS was positive, but only the GM-test was positive. Among probable cases 5\u0026ndash;8, only cases 5 and 6 were positive for both the G-test and GM-test, while cases 7 and 8 were positive only for the GM-test. In possible cases 9\u0026ndash;14, mNGS was positive in all cases, with only case 10 showing positivity for the G-test and the remaining cases negative for serological tests. Notably, mNGS results were obtained prior to histopathological findings in all patients. These results demonstrate that the positivity rate of mNGS (100%, 14/14) was significantly higher than that of histopathology (75.00%, 3/4), the G-test (35.71%, 5/14), and the GM-test (42.86%, 6/14). The detection results for all enrolled patients are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe detection results and treatment strategy for 14 fungal mucormycosis patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG test\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGM test\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHistopathologic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCulture\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003emNGS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003esampling time\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTreatment Change\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eAspergillus fumigatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eRhizopus microsporus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eVOR\u0026thinsp;+\u0026thinsp;AMB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eAspergillus fumigatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eRhizopus delsoni\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eISA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eRhizopus oryzae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAMB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eRhizopus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eRhizopus oryzae;Rhizopus microsporus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAMB\u0026thinsp;+\u0026thinsp;POS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eRhizophorus minutus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePOS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eAspergillus fumigatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eRhizopus microsporus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eVOR\u0026thinsp;+\u0026thinsp;AMB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eLichtheimia ramosa;Rhizopus microsporus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eISA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eRhizopus minutus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAMB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eRhizopus delsoni\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAMB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eRhizopus oryzae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eVOR\u0026thinsp;+\u0026thinsp;POS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eRhizopus microsporus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAMB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eRhizopus microsporus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePOS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eRhizopus delsoni\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAMB\u0026thinsp;+\u0026thinsp;ISA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eRhizopus microsporus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePOS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive rate(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eP: Positive; N: negative; NT: No tested; AMB: Amphotericin B; POS: Posaconazole; ISA: Isavuconazole; VOR: Voriconazole; G-test: 1,3-β-D-glucan test; GM-test: Galactomannan test\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eComparison of mNGS and Fungal Culture Results\u003c/h3\u003e\n\u003cp\u003eAll patients underwent timely collection of bronchoalveolar lavage fluid (BALF) for both\u003c/p\u003e \u003cp\u003emNGS and fungal culture after admission. Samples were collected on D1 for 6 patients (42.85%,\u003c/p\u003e \u003cp\u003e6/14), D2 for 4 patients (28.57%, 4/14), D3 for 2 patients (14.29%, 2/14), and D4 for 2 patients\u003c/p\u003e \u003cp\u003e(14.29%, 2/14). mNGS results were reported within 18\u0026ndash;48 hours after sample submission,\u003c/p\u003e \u003cp\u003esignificantly faster than fungal culture, which required at least 5 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the 14 enrolled patients, mNGS detected a total of 42 pathogenic strains, including 12\u003c/p\u003e \u003cp\u003ebacteria (28.57%, 12/42), 25 fungi (59.52%, 25/42), and 5 viruses (11.91%, 5/42). Fungi accounted for the highest proportion, with \u003cem\u003eAspergillus fumigatus\u003c/em\u003e being the most common (14.29%, 6/42). The pathogens causing PM detected in this study included \u003cem\u003eRhizopus microsporus\u003c/em\u003e (11.90%, 5/42), \u003cem\u003eRhizomucor pusillus\u003c/em\u003e (7.14%, 3/42), \u003cem\u003eRhizopus delsoni\u003c/em\u003e (7.14%, 3/42), \u003cem\u003eRhizopus oryzae\u003c/em\u003e (7.14%, 3/42), and \u003cem\u003eLichtheimia ramosa\u003c/em\u003e (2.38%, 1/42). Additionally, \u003cem\u003eCandida albicans\u003c/em\u003e and \u003cem\u003eCandida glabrata\u003c/em\u003e were detected. Among the bacteria, besides common pathogens such as \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (7.14%, 3/42) and \u003cem\u003eHaemophilus influenzae\u003c/em\u003e (4.76%, 2/42), mNGS also identified \u003cem\u003ePneumocystis jirovecii\u003c/em\u003e (4.76%, 2/42) and \u003cem\u003eUreaplasma urealyticum\u003c/em\u003e (2.38%, 1/42), which are difficult to detect using traditional methods. Detected viruses included \u003cem\u003eHuman gammaherpesvirus type 4\u003c/em\u003e (4.76%, 2/42), \u003cem\u003eHuman betaherpesvirus type 5\u003c/em\u003e (4.76%, 2/42), and \u003cem\u003eHuman alphaherpesvirus type 1\u003c/em\u003e (2.38%, 1/42).\u003c/p\u003e \u003cp\u003ePathogens were detected in all patients. While 3 patients had only one fungal species detected, the remaining 11 had two or more pathogens, including cases with two fungi (case 4), bacteria and fungi (cases 2, 3, 5, 6, 8, 11), fungi and viruses (cases 1, 7), and bacteria, fungi, and viruses (cases 10, 14). Case 10 exhibited a complex mixed infection with \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eA. fumigatus\u003c/em\u003e, \u003cem\u003eC. albicans\u003c/em\u003e, \u003cem\u003eR. oryzae\u003c/em\u003e, and \u003cem\u003eH. gammaherpesvirus type 4\u003c/em\u003e. Case 14 showed \u003cem\u003eAcinetobacter pittii\u003c/em\u003e, \u003cem\u003eP. jirovecii\u003c/em\u003e, \u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003eR. microsporus\u003c/em\u003e, and \u003cem\u003eH. betaherpesvirus type 5\u003c/em\u003e, representing the most complex mixed infection.\u003c/p\u003e \u003cp\u003eFungal culture identified fungi in 4 cases, with only 1 case (case 4) detecting Rhizopus, while the other 3 cases (cases 1, 2, 6) identified \u003cem\u003eA. fumigatus\u003c/em\u003e. In cases 1, 2, 4, and 6, mNGS not only detected the corresponding \u003cem\u003eRhizopus\u003c/em\u003e and \u003cem\u003eA. fumigatus\u003c/em\u003e but also identified additional bacteria, fungi, and viruses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The overall mismatch rate between the two methods was 71.43% (10/14), with a partial match rate of 28.57% (4/14). These results demonstrate that mNGS significantly outperforms fungal culture in terms of positivity rate, detection speed, and breadth of pathogen identification, particularly for rare pathogens and mixed infections. The specific pathogens detected are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eImpact of mNGS on Treatment and Outcomes\u003c/h3\u003e\n\u003cp\u003eAmong the 14 enrolled patients, all had received treatment at local hospitals prior to\u003c/p\u003e \u003cp\u003eadmission, with 9 (64.29%, 9/14) prophylactically administered voriconazole for antifungal\u003c/p\u003e \u003cp\u003etherapy upon admission. Based on the mNGS results, clinicians promptly adjusted the treatment\u003c/p\u003e \u003cp\u003eregimens for all patients. Cases 3 and 8 received amphotericin B via nebulization, cases 9 and 11\u003c/p\u003e \u003cp\u003ereceived intravenous amphotericin B, cases 2 and 7 were treated with oral isavuconazole, cases 5,\u003c/p\u003e \u003cp\u003e12, and 14 received oral posaconazole, case 4 was treated with a combination of intravenous\u003c/p\u003e \u003cp\u003eamphotericin B and posaconazole, and case 13 received intravenous amphotericin B and\u003c/p\u003e \u003cp\u003eisavuconazole. Cases 1, 6, and 10, which involved mixed infections of Aspergillus and Mucorales,\u003c/p\u003e \u003cp\u003ewere treated with a combination of voriconazole and amphotericin B (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, all\u003c/p\u003e \u003cp\u003eenrolled patients were administered broad-spectrum antibiotics.\u003c/p\u003e \u003cp\u003eAmong the 14 patients, 2 (cases 4 and 13) were diagnosed with disseminated mucormycosis. Case 4 involved the lungs and brain, while case 13 involved the lungs and pleura. In case 4, chest CT upon admission indicated severe pneumonia, and the patient was initially treated with meropenem for infection. mNGS results identified Rhizopus delemar and Rhizopus microsporus, and cranial CT revealed a brain abscess, likely due to Rhizopus invasion. The clinician immediately initiated intravenous amphotericin B and posaconazole for antifungal treatment. However, due to the severity of the condition and high treatment costs, the family decided to discontinue treatment, and the patient died 26 days after discharge. In case 13, piperacillin-tazobactam was initially administered for infection. mNGS results detected \u003cem\u003eR. delsoni\u003c/em\u003e, prompting the clinician to add isavuconazole for antifungal therapy. Subsequently, the patient developed chest pain, suspected to be caused by Mucorales invading the pleura. The treatment was adjusted to include isavuconazole and amphotericin B cholesteryl sulfate complex infusion. Follow-up chest CT after one week showed improvement in lung infection and abscesses, and the patient was discharged after 10 days of treatment.\u003c/p\u003e \u003cp\u003eAfter active symptomatic treatment, 9 patients improved and were discharged, 2 (cases 2 and 5) were transferred to higher-level hospitals due to lack of improvement, and 3 (cases 4, 6, and 8) discontinued treatment for financial reasons. A 90-day telephone follow-up revealed that all 9 patients who improved and were discharged survived. Among the 5 patients who were discharged due to severe illness, only case 2, who was transferred to a higher-level hospital, survived. The remaining 4 (cases 4, 5, 6, and 8) died due to the severity of their conditions, resulting in a mortality rate of 28.57%. All 4 deceased patients had at least two underlying conditions, including diabetes and myocardial infarction. Case 5 also had acute myeloid leukemia and did not undergo full-course treatment.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMucormycosis, caused by Mucorales, is characterized by rapid filamentous growth,\u003c/p\u003e \u003cp\u003ewidespread distribution, spore production, unique structural features, adaptability to diverse\u003c/p\u003e \u003cp\u003eenvironments, and interactions with host immune defenses. These factors collectively contribute\u003c/p\u003e \u003cp\u003eto the establishment and progression of infection, making mucormycosis the second most common\u003c/p\u003e \u003cp\u003eangioinvasive fungal disease after invasive aspergillosis. PM, the second most prevalent form,\u003c/p\u003e \u003cp\u003eoccurs through the inhalation of airborne sporangiospores. These spores secrete lytic enzymes and\u003c/p\u003e \u003cp\u003etoxins (such as mucorubin) and can spread through lung parenchyma to the chest wall, central\u003c/p\u003e \u003cp\u003enervous system, or gastrointestinal tract, inducing tissue thrombosis and necrosis. This can lead to\u003c/p\u003e \u003cp\u003ecomplications such as encephalitis, brain abscesses, and gastrointestinal ulcers(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), Due to its\u003c/p\u003e \u003cp\u003esevere infection, rapid progression, and high mortality rate(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), early etiological diagnosis is\u003c/p\u003e \u003cp\u003ecritical for timely treatment.\u003c/p\u003e \u003cp\u003ePM is commonly observed in immunocompromised individuals with conditions such as diabetes, hematologic malignancies, solid organ transplantation, neutropenia, long-term corticosteroid use, and other underlying diseases(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Numerous studies have identified diabetes as the most frequent predisposing factor, present in 50\u0026ndash;70% of patients diagnosed with mucormycosis(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Hyperglycemia impairs phagocyte function, intracellular killing, and chemotaxis, while hypoxia in lung diseases creates a favorable environment for fungal spore germination and proliferation, increasing the risk of mucormycosis. Notably, mucormycosis can also occur in individuals without any predisposing conditions(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In this study, 92.85% of patients had underlying diseases, with 85.71% having diabetes, consistent with previous studies(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), This highlights diabetes as a significant risk factor for PM, while other comorbidities (e.g., coronary heart disease, hypertension) may further increase susceptibility. Previous studies have also reported a higher prevalence of mucormycosis in males(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), which aligns with our findings (57.14% male vs. 42.86% female). Mucorales can invade not only the lungs but also the brain, nose, orbits, gastrointestinal tract, kidneys, skin, and other tissues. In this study, 12 cases involved only the lungs, while 2 cases involved the lungs and pleura or central nervous system, diagnosed as disseminated mucormycosis.\u003c/p\u003e \u003cp\u003eThe clinical symptoms of PM, including high fever (\u0026gt;\u0026thinsp;38\u0026deg;C), cough, dyspnea, and chest pain[1], are nonspecific. In this study, 85.71% of patients presented with fever, 50.00% with chest pain, and all patients exhibited cough, sputum production, and dyspnea. In the early stages, imaging may only show perivascular ground-glass opacities, progressing to pulmonary nodules, consolidation, cavities, and the reverse halo sign in later stages(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e),The primary imaging findings in our patients were cavities (50.00%) and consolidation (21.43%). Based solely on clinical manifestations and imaging, it is challenging for clinicians to distinguish PM from other pulmonary diseases.\u003c/p\u003e \u003cp\u003eAlthough the G test and GM test used for fungal screening cannot be directly applied to the diagnosis of mucormycosis, they hold certain value in differentiating invasive aspergillosis(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In this study, the positive rates of these two tests were 35.71% and 42.86%, respectively. While the GM-test was positive in 3 culture-positive cases (cases 1, 2, and 6), it was also positive in culture-negative cases (cases 5, 7, and 8), possibly due to low fungal concentrations or prior antifungal prophylaxis. However, negative results cannot rule out mucormycosis. Although definitive diagnosis of PM can rely on BALF fungal culture and lung histopathology, invasive procedures may not be initially suitable for patients without risk factors, and culture results take considerable time. In this study, all cases underwent BALF fungal culture, with only 4 yielding positive results, including just 1 case of \u003cem\u003eRhizopus\u003c/em\u003e, resulting in a diagnostic positivity rate of only 7.14%. Among the 4 cases with histopathological examination, fungal hyphae were identified in only 3, yielding a positivity rate of 75%.\u003c/p\u003e \u003cp\u003emNGS directly sequences all microbial nucleic acids in a sample without prior knowledge of the pathogen, significantly increasing the likelihood of detecting Mucorales by identifying specific gene sequences. It serves as an important complement to histopathology and microbiological diagnostics. Recently, mNGS has been increasingly used to assist in the clinical diagnosis of invasive fungal infections, including mucormycosis(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Wang C. \u003cem\u003eet al.\u003c/em\u003e studied 310 patients with suspected invasive pulmonary fungal infections and found that mNGS had a sensitivity and specificity of 86.76% and 86.98%, respectively, outperforming traditional microbiological methods in accuracy and speed (AUC\u0026thinsp;=\u0026thinsp;0.967)(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Wei E. \u003cem\u003eet al.\u003c/em\u003e analyzed 3 pediatric cases of rare invasive mold brain abscesses, detecting \u003cem\u003eRhizomucor miehei\u003c/em\u003e and \u003cem\u003eRhizomucor pusillus\u003c/em\u003e in cerebrospinal fluid via mNGS, enabling timely and precise treatment and discharge(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). In this study, all enrolled patients underwent BALF mNGS, which detected 25 fungal strains, including 15 cases of Mucorales. The most common pathogen was \u003cem\u003eR. microsporus\u003c/em\u003e, followed by \u003cem\u003eRhizomucor pusillus\u003c/em\u003e and \u003cem\u003eR. delsoni\u003c/em\u003e, consistent with previous studies(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). However, regional variations in pathogenic organisms may exist due to environmental factors and temperature-dependent growth characteristics. Additionally, metagenomic sequencing can detect other microorganisms coexisting with Mucorales. Studies have shown that mucormycosis can co-occur with other invasive mold infections, most commonly aspergillosis, as reported in hematologic malignancies(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), COVID-19-associated mucormycosis (CAM)(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), and other immunocompromised patients(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). In this study, 50.00% of patients had co-detected Aspergillus. Besides fungi, mNGS also identified bacteria and viruses, including \u003cem\u003eP. jirovecii\u003c/em\u003e and \u003cem\u003eU. urealyticum\u003c/em\u003e, which are difficult to detect using traditional methods. Among the 11 cases with mixed infections, two or more pathogens were detected. These results demonstrate that mNGS outperforms traditional methods in terms of reporting speed, comprehensiveness, and positivity rate, providing more timely, comprehensive, and accurate diagnoses of PM, particularly in mixed infections, consistent with previous studies(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In this study, all 14 patients had their treatment regimens adjusted based on mNGS results. Targeted antifungal therapies, including isavuconazole, posaconazole, amphotericin B, or voriconazole, were administered individually or in combination as needed. Amphotericin B is the most active drug against Mucorales in vitro and has shown efficacy in treating mucormycosis when used alone(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). In this study, 8 of the 14 patients received amphotericin B alone or in combination with other antifungals. After precise and active treatment, 9 patients improved and were discharged. A 90-day follow-up revealed that 4 patients died due to severe illness, delayed treatment, and underlying comorbidities, resulting in a mortality rate of 28.57%, lower than previously reported rates(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). This may be attributed to differences in the study population or the timely and accurate diagnostic results provided by mNGS, enabling clinicians to develop more personalized treatment plans and improve patient outcomes. This study highlights the superior diagnostic and prognostic advantages of mNGS over traditional methods in invasive mucormycosis, allowing clinicians to rapidly adjust treatment strategies and enhance clinical efficiency.\u003c/p\u003e \u003cp\u003eThis study has certain limitations. First, as a single-center retrospective analysis with a small sample size, the conclusions may be subject to bias. Second, Mucorales are ubiquitous in the environment, making it difficult to distinguish colonization from infection. Additionally, the lack of standardized bioinformatics analysis protocols for mNGS necessitates comprehensive clinical judgment. In conclusion, mNGS significantly improves the early diagnosis of PM through its rapid and comprehensive pathogen detection capabilities, particularly in cases where traditional methods yield negative results or in mixed infections. Although challenges remain in the clinical application of mNGS, our study provides new insights into its use for the early diagnosis of PM.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAs an emerging pathogen detection technology, mNGS demonstrates a significantly high positive rate in the detection of Mucorales pathogens. It is particularly suitable for infectious diseases with negative results from traditional detection methods or complex clinical conditions. mNGS assists clinicians in achieving early diagnosis and rapidly adjusting treatment plans, thereby improving patient survival rates.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the \u0026quot;Administrative Measures for Ethical Review of Biomedical Research Involving Human Beings\u0026quot;, this study was approved by the Institutional Medical Ethics Committee of Affiliated Hospital of Jining Medical University (Ethics Approval Number: 2025-01-C024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Research Fund for Shandong Provincial Natural Science Foundation (No. ZR2023MH325), Jining Medical University Affiliated Hospital Doctoral Research Fund (No. 2022-BS-01), Jining Medical University Affiliated Hospital \u0026ldquo;Zhi-Xing\u0026rdquo; Project (No. ZX-ZD-2023-01), Academician Lin He New Medicine (No. JYHL2022MS05) and the Jining City Key Research and Development Plan Project (No. 2023YXNS178).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX Y, MK and LQ J contributed to the design and execution of the study and the writing of the manuscript. XH H, HY S, \u0026nbsp; SG G, H C, T L, HX D, JY Y GH P and XJ M contributed to the raw sequence data upload process and provided valuable assistance with the project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003ecknowledgments\u003c/strong\u003e\u003cbr\u003e\u0026nbsp; \u0026nbsp;We are grateful to all the participants. We also deeply appreciate the doctors\u0026apos; assistance with sample collection.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCornely OA, Alastruey-Izquierdo A, Arenz D, Chen SCA, Dannaoui E, Hochhegger B, et al. Global guideline for the diagnosis and management of mucormycosis: an initiative of the European Confederation of Medical Mycology in cooperation with the Mycoses Study Group Education and Research Consortium. Lancet Infect Dis. 2019;19(12):e405\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHassan MIA, Voigt K. 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J Med Mycol. 2023;33(1):101325.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu X, Jiang L, Liu X, Chang H, Dong H, Yan J, et al. The diagnostic value of bronchoalveolar lavage fluid metagenomic next-generation sequencing in critically ill patients with respiratory tract infections. Microbiol Spectr. 2024;12(8):e00458\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePomorska A, Malecka A, Jaworski R, Radon-Proskura J, Hare RK, Nielsen HV, et al. Isavuconazole in a Successful Combination Treatment of Disseminated Mucormycosis in a Child with Acute Lymphoblastic Leukaemia and Generalized Haemochromatosis: A Case Report and Review of the Literature. Mycopathologia. 2019;184(1):81\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAshkenazi-Hoffnung L, Bilavsky E, Levy I, Grisaru G, Sadot E, Ben-Ami R, et al. Isavuconazole As Successful Salvage Therapy for Mucormycosis in Pediatric Patients. Pediatr Infect Dis J. 2020;39(8):718\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClaustre J, Larcher R, Jouve T, Truche AS, Nseir S, Cadiet J, et al. Mucormycosis in intensive care unit: surgery is a major prognostic factor in patients with hematological malignancy. Ann Intensive Care. 2020;10:74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin CY, Wang IT, Chang CC, Lee WC, Liu WL, Huang YC, et al. Comparison of Clinical Manifestation, Diagnosis, and Outcomes of Invasive Pulmonary Aspergillosis and Pulmonary Mucormycosis. Microorganisms. 2019;7(11):531.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pulmonary mucormycosis, Clinical features, Metagenomic next-generation sequencing, Diagnosis, Treatment strategy","lastPublishedDoi":"10.21203/rs.3.rs-6308222/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6308222/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAlthough pulmonary mucormycosis is rare, it is highly invasive and carries a significant mortality rate. Due to its nonspecific clinical manifestations, it is often misdiagnosed as other invasive fungal diseases. Metagenomic next-generation sequencing is a rapid, precise, and comprehensive method for pathogen detection, showing great potential in the early diagnosis of pulmonary mucormycosis. It provides clinicians with faster and more accurate etiological information, thereby improving patient outcomes and reducing mortality rates.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study retrospectively analyzed the clinical data of 14 patients diagnosed with pulmonary mucormycosis between 1/6/2021 and 30/6/2024.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMales accounted for 57.14% of the cases. Diabetes mellitus was present in 12 patients (85.71%, 12/14), and fever was observed in 12 patients (85.71%, 12/14). The 14 patients were categorized as confirmed cases (4 cases), probable cases (4 cases), and possible cases (6 cases). Two patients (14.29%, 2/14) were diagnosed with disseminated mucormycosis. Chest Computed Tomography scans revealed cavities in half of the patients (50.00%, 7/14). Fungal hyphae were identified in 3 out of 4 histopathological examinations. mNGS detected Mucorales pathogens in 100% of cases, significantly outperforming the positive rates of the 1,3-β-D-glucan test test (35.71%, 5/14), Galactomannan test test (42.86%, 6/14), histopathology (75.00%, 3/4), and fungal culture (28.57%). Additionally, mNGS identified bacterial and viral co-infections, with 11 patients diagnosed as having mixed infections. All patients received timely and targeted treatment adjustments, resulting in 9 patients improving and being discharged, 2 patients being transferred to higher-level hospitals, and 2 patients discontinuing treatment. The 90-day follow-up revealed a mortality rate of 28.51%.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eMetagenomic next-generation sequencing can serve as an important complement to traditional diagnostic methods, enabling rapid and accurate differentiation of Mucorales from other fungi. This allows patients to receive timely and targeted antifungal therapy, playing a critical role in early intervention and improving prognosis.\u003c/p\u003e","manuscriptTitle":"The Application Value of Metagenomic Next-generation Sequencing in the Diagnosis of Pulmonary Mucormycosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 10:46:24","doi":"10.21203/rs.3.rs-6308222/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"491e494c-1d54-4ead-a136-b7eb6f48ff90","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-21T13:53:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-17 10:46:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6308222","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6308222","identity":"rs-6308222","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}