Patient
A 40-year-old man was diagnosed with acute myeloid leukemia (AML) in high-risk group in April 18th, 2018. After diagnosis, the patient underwent twice chemotherapy treatments. Unfortunately, the patient later relapsed on July 25, 2018. Laboratory results indicated that he was converted to mixed phenotype leukemia (MPAL) no-remission (NR). On August 14, 2018, the patient received haplo-HSCT, and the transplant was successfully reconstructed on August 29, 2018. Then, the patient underwent post-transplant consolidation: 3 times of decitabine therapy until May 07, 2019. Meanwhile,he kept immunosuppressive therapy with Anti-rejection agent (Cyclosporine, 50mg BID) for three years,to prevent graft-versus-host disease(GVHD). The patient had never relapsed with any hematonosis until this admission. However, he developed “chest tightness and asthma in the past six months, and worsened half a month ago”. Therefore, he was admitted to a local hospital because of the aggravated pulmonary symptoms on February 13, 2021. And he was transferred to our hospital on March 3, 2021 in view of his pulmonary infection symptoms did not improve after anti-infection treatment at the local hospital. After admission, the doctor systematically performed relevant examinations for him. A chest computed tomography (CT) examination presented one infectious cavity in the upper lobe of the left lung (Fig.1). In addition, the scattered small dots and patches in both lungs were new foci. Many routine laboratory examinations also suggested infection. To identify the pathogen, fiberoptic bronchoscopy was performed twice to collect bronchoalveolar lavage fluid (BALF) for culture, 1,3-β-D-glucan (GM) and mNGS. Given these findings, posaconazole and caspofungin were used for antifungal therapy, meropenem and co-sulfamethoxazole for antibacterial, ganciclovir and ribavirin for antiviral. In a word, antifungal drugs had always been used for a long time, as clinical symptoms, CT examination, and laboratory examinations supported fungal infection in the lung. Finally, all these therapies failed to control this fatal pulmonary, and he died of respiratory failure on March 20, 2021. The patient's disease progression is shown in Fig. 2.
Results of laboratory tests for the patient
Blood cell analysis suggested mild anemia (hemoglobin 106g/L) with normal white blood cell count(WBC:4.82×10^9/L, NEUT#:4.42×10^9/L, LYM#:0.27×10^9/L↓). As for inflammatory markers, the level of procalcitonin (PCT) was 0.18 ng/mL, and hypersensitive C-reactive protein (hs-CRP, 59.33mg/L) were increased significantly. No significant abnormalities were found in blood coagulation function. Liver and kidney function were mild abnormality (UA:9.03mmol/L, ALT:82U/L). Sputum had been repeatedly for bacterial culture and no pathogenic microorganisms grew. BALF was collected on February 18, 2021. GM tests were positive (GM:5.65 positive) and mNGS presented Nocardia nova with 785 reads, cytomegalovirus (CMV) with 109 reads, Epstein-barr virus (EBV) with 26 reads in this BALF. As symptoms worsen, BALF from left upper bronchus on March 10, 2021 was secondly collected for mNGS. Nocardia nova (248 reads), Enterococcus (257reads), Aspergillus (60 reads), Parvovirus (71 reads) and Cytomegalovirus (20reads) were detected by mNGS in the BALF. Some white velvety colonies grew in the BALF after 3 days incubation (Fig. 3), with no evidence of mycobacterial, or additional fungal pathogens isolated. Because the accurate morphology of this fungus had not been established and no M. cirrosus were listed in MALDI-TOF-MS library, this fungus had never been correctly identified as a Microascus species until a month later. In a word, many results presented some fungal infection in the lung, including GM, mNGS, and pure fungal colonies.
Morphological and physiological characteristics of this fungus
This fungus was detected from the BALF of the patient by growing in both Columbia blood plate and chocolate agar plate for two days. The primary colonies were white and approximately 3-4 mm in diameter (Fig. 3a). Then it was incubated on many media to observe growing at different temperature. This fungus can grow on Columbia blood plate, chocolate agar plate, nutrient agar, Sabouraud's agar and LB broth (Fig. 3b) respectively, and grow well at 35℃ and 28℃. It could grow faster and bigger on chocolate agar plate than Sabouraud’s agar. As the prolonged cultivation, it presented different conies. Overall, white and approximately 3-4 mm colonies could be observed on culture medium for three days (Fig. 3c). Over 12 mm colonies with folds around the periphery and dark pigment in the center could be observed on culture medium for about a week (Fig. 3d). It could also grow at the bottom of a liquid medium. After about one week of cultivation on chocolate agar, the ball-shaped, smooth conidia and septate hyphae could be observed under the microscope by dyeing with Lactophenol cotton blue (Fig. 3e). For about four weeks, in the dark and brown part of the fungal colony, ascoma and ascospores could be formed as the forms of sexual reproduction of the fungus. Microscopically, ascoma were large and spherical-shaped, containing ascospores which were half-moon shaped (Fig. 3f-3h). Based on the presence of branched conidiophores bearing cylindrical annellides in brush-like groups and on the development of small black ascomata (cleistothecia) after four weeks, it was morphologically identified as Microascus sp. Compared with the morphological description of M. cirrosus from seven clinical cases (Table 1), the M. cirrosus presented typical colonies and microscopic morphology. And compared with other filamentous fungi such as Aspergillus, Microascus species usually has only short chains of conidia without apical cysts.
Fungal drug sensitivity
The minimum inhibitory concentrations of this fungus against amphotericin B, caspofungin, fluconazole and fluorocytosine were as following: (amphotericin B ≥16 μg/mL, caspofungin ≥16 μg/mL, fluconazole ≥64 μg/mL, fluorocytosine ≥64 μg/mL), according to the criteria [22], these antifungal drugs are all insensitive. This is generally consistent with the results of in vitro drug susceptibility studies on Microascus sp by Sandoval-Denis M et al and Gao L et al [11, 16].
Multi-site sequence analysis
The fungal nucleic acid was amplified by PCR with the primers described as Brasch Jochen et al [24]. and four products were obtained (LSU568bp, ITS618bp, EF-1α898bp and TUB523bp). The analysis revealed that this fungus represented a new genotype in the genus closely related to M. cirrosus (Fig.4). Based on its genetic characteristics and on its distinct morphological features, it is proposed below as a new genotype of M. cirrosus named by M. cirrosus SZ 2021.
The whole genome sequence of this fungus by second-generation sequencing
By performing De novo sequencing on the case fungus, we obtained its whole nucleic acid information. The total number of bases after quality control is approximately 2.693 billion and GC Bases Ratio is 53.98%. There is no whole M. cirrosus genome available for reference in the NCBI database currently. Thus, we have upload the assembled sequence of M. cirrosus SZ 2021 to the public database NCBI for research. This second-generation sequencing project was deposited in the NCBI under BioProject: PRJNA835605, BioSample:SAMN28105390 and GenBank: JAMBUN000000000. The version described in this paper is version JAMBUN010000000.
Re-analysis of sequences from BALF detected by mNGS
Because no the whole genomic information of Microascus species were available in public databases, no sequences of this fungus had been reported by mNGS. The results of mNGS from BALF had always recommended nocardia and aspergillus for dozens of reads. We reanalyzed the initial mNGS sequence from BALF by comparing the whole genome sequence of M. cirrosus SZ 2021. No Aspergillus gene sequence was reported in the former BALF on February 18, 2021 in the local hospital, whereas 4 reads from M. cirrosus could be detected by retrospective bioinformatics analysis. In the BALF sample where the M. cirrosus colonies were cultured, more than a thousand sequences (1000 reads) could be aligned, including 660 no-human-reads and unclassfiled-reads. We also reanalyzed the initial BALF mNGS sequence from the HSCT patients who had underwent pulmonary infection but failed to cure in our hospital. Different number of sequences of M.cirrosus could be detected in another two cases, 3 reads and 13 reads respectively, with no verification of cultural colonies, but favorited by GM tests. In conclusion, the sequences from BALF detected by mNGS demonstrated that M. cirrosus could be detected correctly and rapidly by mNGS, as there is correct genomic information in the database.
Distinguishing features of M. cirrosus detected by MALDI-TOF-MS
We obtained the protein fingerprint of M. cirrsus SZ 2021 by MALDI-TOF-MS (Fig 5), but did not identify the strain name, probably because the mass spectrometry identification database did not contain strain information. Then, we constructed main spectrum profile (MSP) dendrogram by combining M. cirrsus SZ 2021 with other Microascus spp, Aspergillus fumigatus and Nocardia nova. It was found that M. cirrosus SZ 2021 was relatively close to Microascus gracilis, classified as a different cluster from Aspergillus fumigatus and Nocardia nova (Fig 6).