We searched for English resources from PubMed, Google Scholar, and Embase. At the same time, we also searched for Chinese resources from China National Knowledge Internet (CNKI), WanFang and WeiPu. We used the strategy “(China OR Taiwan OR Hong Kong OR Macao) and (phaeohyphomycosis OR black OR dematiaceous OR melanized OR phaeoid)” and the genus names of known clinical major dematiaceous fungi. Cases reported from 1987 to 2021were enrolled.

Criteria for study inclusion:

Criteria for study exclusion:

Refer to the most authoritative reviews published by Sanjay G. Revankar and Flavio Queiroz Telles in 2002, 2004, 2010, and 2017, cases were classified as (Revankar et al., 2002; Revankar et al., 2004; Revankar and Sutton, 2010; Queiroz-Telles et al., 2017):

We calculated descriptive statistics (frequencies, means, medians) for demographic, clinical, and, laboratory variables. We applied chi-square analysis with Fisher’s exact test to compare groups if needed. Excel and SPSS were used in data collection and analysis. P-value ≤ 0.05 is statistically significant.

Since 2000, there had been a rapid growth in the number of reported phaeohyphomycosis in China. The number of cases in the last 20 years was about 6.6 times that of 20 years ago ( Figure 1 ). There were 118 (68%) patients from the mainland, 39 (22%) patients from Taiwan, and 17 (10%) patients from Hong Kong. Most cases were sporadic in tropical and subtropical regions, although some cases were in temperate regions, mainly between latitudes of 3°N and 53°N. Most patients came from southern China, including Taiwan, Zhejiang, Jiangsu, and Guangdong provinces ( Figure 2 ).

A total of 174 patients were enrolled in this study, including 107 males and 67 females. The ratio of male to female patients was 1·6:1. The mean age at diagnosis was 48 years (range, 2–89 years; median age, 53years). The most common risk factors for phaeohyphomycosis infections were traumas (37%), diabetes (11%), and corticosteroid use (11%). Inherited CARD9 deficiency (7%) was found in either severe refractory or mild PHM infections. Malnutrition (6%), tumors (5%), kidney transplantations (3%), and chemotherapy (2%) were frequent risk factors in patients with disseminated, CNS, deep local, and pulmonary infections. Another 22% of the patients did not find any obvious risk factors ( Table 1 ).

In 2017, Flavio Queiroz Telles et al. proposed the spectrum of fungal diseases caused by melanized fungi in order from superficial to deep (Queiroz-Telles et al., 2017). Based on their spectrum of diseases, we reclassified the types of phaeohyphomycosis. The mortality rate was prioritized. Cerebral infections were ranked first. Disseminated infections were ranked second and pulmonary infections were ranked third. Given the highest incidence of subcutaneous and corneal infections, they were separated from the deep-local infection types, and therefore seven applicable subclasses were divided. The spectrum of clinical syndromes was summarized as follows ( Table 2 ):

▲ Eumycetoma (not included)

▲ Chromoblastomycosis (not included)

Fast diagnosis is often critical for the survival of fatal invasive fungal infections, especially crucial for the drugs of systemic phaeohyphomycosis as the usually poor outcome of treatment. The European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and European Confederation of Medical Mycology (ECMM) joint clinical guidelines recommended the diagnosis of systemic PHM in 2014 including histopathology, culture, and sequencing for definitive species identification (Chowdhary et al., 2014).

Traditionally, the diagnosis of PHM requires a combination of the patient’s clinical manifestations and mycological confirmations ( Figure 5 ). The most specific and persuasive diagnostic elements are yellowish-brown hyphae with or without budding cells in skin scrapings, stained tissue slices, aspirated pus, or surgical drainage (Chowdhary et al., 2014). The evidence can be obtained directly by KOH microscopic examination, pathology, fungal culture, or animal models. When the infections are superficial or on the corneal, diagnosis can be made by direct microscopy, and biopsy is not needed. However, pathological biopsy remains the gold standard when diagnosing cerebral, pulmonary, and deep-local infections, as it is not uncommon that the early-stage CSF, BALF and drainage fluid examinations generally show negative fungal cultures or smears. In our study, pieces of evidence of fungal elements were found in 88% (153 of 174) cases that underwent pathological examinations. In subcutaneous infections, pathological diagnosis is based on the clinical manifestations of PHM lesions (Queiroz-Telles et al., 2017). Microscopically, the nodular cyst mainly has palisading epithelioid macrophages at the inner edge of the abscess. The lumen of the abscess often contains necrotic debris mixed with polymorphonuclear leukocytes and the mycelium is prominently seen in the wall of the abscess. The solid papules revealed a thickening epidermis near the hair follicles. Dermal edema, capillary dilation with inflammatory cell infiltration, and acanthosis with granuloma formation are common. Dark fungal elements can be seen within giant cells in the tissue. The old verrucous plaques show hyperkeratosis, parakeratosis, and acanthosis. Lymphocytes, histiocytes, plasma cells, and giant cells are always surrounded by a thick zone of fibroblasts and collagen. The fungal cells are present as yeastlike cells in short chains or toruloid hyphae. The detection rate of fungal elements can be improved in pathologic examination by special staining. Hematoxylin and eosin stain (H&E) stain are the most used, Fontana-Masson stain and Gomori methenamine silver (GMS) stain are sensitive for the detection of fungal cells when fungal elements are scarce (Yan et al., 2019).

However, different species of dark molds cannot be distinguished by pathological sections alone. That’s why we emphasize the necessity of simultaneous culture. Isolation on common sabouraud dextrose agar (SDA) is feasible. It is recommended to turn to plant-based media such as potato dextrose agar (PDA), corn, and oat dextrose agar due to the poor sporulation in part of dark fungi. Most pathogens can develop visible colonies during 1–2 weeks from the initial pale white to the dark colony. The specimens ought to be incubated at 25° to 30°C for 4 weeks before being discarded as negative. Nearly 99% (173 of 174) cases in China obtained dark fungi strains through cultures. Morphological identifications were done in 167 cases. However, confirmation of phenotypic characteristics highly depends on experienced specialists and needs a long cycle. And its limitation of identification to the genus level makes it impossible to provide an early definitive specific diagnosis.

Conservative culture methods have demerits and cannot offer reliable and rapid information to achieve optimal disease management. Sequence analysis of the internal transcribed spacer (ITS) and D1/D2 regions of rDNA are recommended for effective and accurate molecular identification of dark fungi, among which the ITS region has been widely regarded as the universal barcode for the classification of dark fungi. Nearly 54% (94 of 174) cases in China obtained definitive species based on sequence analysis combined with morphological characteristics except for one patient directly through ITS sequencing without fungal culture. It confirms the theory that molecular identification directly from clinical samples is another alternative approach for species identification. The molecular approach especially nuclear ITS sequencing was the most used, accounting for 85% (80 of 94). In addition, 5.8s rDNA, 18s rDNA, 26s rDNA, 28s rDNA, β-tubulin, β-actin, Chitin synthase, Plasma membrane ATPase, (glyceraldehyde-3-phosphate dehydrogenase) gpd, (translation elongation factor 1-alpha) TEF, (RNA polymerase II largest subunit) RPB1, and (the second largest subunit of RNA polymerase II) RPB2 were used in combination with ITS sequences to verify inter/intra specific variations and to identify new species.

Although the molecular methods are faster than controversial techniques, they have higher cost and are susceptible to contamination due to multi-step processes. Sometimes DNA fragments encoding melanin may inhibit PCR amplification and the limited existing GenBank database may bring difficulty in identifying dark fungi strains (Paul et al., 2017). Around 2010, a technology named MALDI-TOF MS was introduced into the clinical microbiology territory as a more effective and faster diagnostic technique than DNA sequencing (Lau, 2021). It would take 5-9 days for sequence identification of melanized fungi whereas it would take shorter times about 3-7 days for MALDI-TOF MS (Paul et al., 2019). There was also one strain identified as Phialemonium species by the use of matrix-assisted laser desorption ionization time–of–flight mass spectrometry (MALDI-TOF MS). With the continuous improvement of the database in the future, this technology will become mature and more widely used for dark mold identification.

In our study, there were a total of 55 dark fungi strains that caused PHM in China ( Supplementary file Table 2 ). The most common species of strains were Exophiala spp. (33%, 58 of 174) Twelve strains were first reported to cause PHM in the world ( Table 5 ). The species identification in China mainly depends on a combination of phenotypic and morphologic features. And also, we found plenty of points of differences in clinical pathogenic strains between China and foreign countries ( Table 6 ).

Clinically, clinical presentations of phaeohyphomycosis are particularly non-specific in the early stages. They can mimic a variety of infectious and other diseases ( Table 7 ). It is super important to distinguish PHM from a big variety of other confusable diseases as early as possible.

In this study, we have made a detailed framework to link the treatment with the prognosis of the phaeohyphomycosis patients in China ( Table 8 , Supplementary Table 3 ). The main antifungal agents used in cerebral PHM were triazoles plus amphotericin B deoxycholate (AmB-DOC)/liposomal amphotericin B (L-AmB), among triazoles voriconazole was the most used. Cerebral cysts were usually combined with surgeries. The disseminated infections mainly used triazoles plus AmB-DOC/L-AmB, terbinafine, or 5–fluorocytosine (5-FC). Voriconazole, itraconazole, fluconazole, and ketoconazole all had been tried. Most pulmonary infections were initially treated with combination therapy, including caspofungin plus AmB-DOC or voriconazole, and maintained with oral itraconazole or voriconazole in the stable phases. Deep-local infections were mainly used AmB-DOC plus voriconazole or itraconazole. Most mild subcutaneous infections and keratitis were initially treated with triazoles, including itraconazole, voriconazole, and posaconazole. Severe subcutaneous mainly chose triazoles combined with AmB-DOC and terbinafine. Terbinafine is lipophilic and widely distributed in organizations, especially in the stratum corneum, which has been mostly used for subcutaneous and superficial infections. But as we counted, triazoles are still the dominant antifungal agents in skin infections. Itraconazole was given in doses of 200mg to 400mg daily, voriconazole in doses of 100mg to 800mg daily, and terbinafine in doses of 250mg to 500mg daily. All corneal infections had resorted to keratoplasty when drug therapies were not effective. Among echinocandins, only caspofungin was used in pulmonary infections, mainly in combination with voriconazole and AmB-DOC. Posaconazole, a new triazole, was infrequently used and was tried as a single agent in one patient with cerebral PHM and another patient with severe subcutaneous PHM. The median treatment duration of all PHM in China was 60 days (range, 1–4745days).

Other treatments included surgeries and physical therapies. The operative rates were 60% (6 of 10), 36% (4 of 11), 35% (9 of 26), and 12% (15 of 84) respectively in the deep-local, CNS, corneal, and subcutaneous infections. The physical therapies contained thermotherapy, cryotherapy, laser, and 5-aminolevulinic acid-photodynamic therapy (ALA-PDT). Hyperthermia was used marginally in subcutaneous PHM (8%, 7 of 84) and disseminated PHM (9%, 1 of 11).

AmB-DOC/L-AmB and voriconazole were used most frequently in CNS, disseminated and pulmonary infections that improved or cured at the end of follow-up. The efficacy of L-AmB was better than AmB-DOC in some individual cases, whereas AmB-DOC was mainly used in China as we counted. The overall mortality rates of these three infections were 82% (9 of 11), 55% (6 of 11), and 75% (6 of 8) and their authentic mortality rates due to fungal infections were 55% (6 of 11), 36% (4 of 11) and 25% (2 of 8) respectively. The effective rates in the late stages were extremely low. Four patients with severe pulmonary infections all died despite attempts to use a variety of antifungal drugs including caspofungin. The cure rate of early surgical excision of localized brain cysts was 75% (3 of 4) and the effective rate was 100% (4 of 4). The subcutaneous PHM had an overall effective rate of 66% (56 of 85), and a cure rate of 45% (38 of 85). The subcutaneous nodules were resected in only 10 patients of whom the cure rate was 70% (7 of 10). In addition, among the 48 PHM patients who used a single antifungal agent–itraconazole at a certain stage, 11 cases had little effect and 24 cases were cured, so the effective rate was 73% (35 of 48) and the cure rate was 50% (24 of 48). The effective rates of keratitis and superficial PHM were 77% (20 of 26) and 70% (16 of 23). Their prognosis was favorable except for the permanent vision loss in a portion of keratitis.

In this study, 38 clinical fungal isolates from clinical specimens provided the minimum inhibitory concentration (MIC)/minimal effective concentration (MEC) values ( Supplementary Table 4 ). We further linked the 16 dematiaceous fungal isolates’ MIC values to their clinical efficacy when a single triazole agent–itraconazole was used ( Supplementary Table 5 ). At present, there is no clinical or epidemiological cut-off point for drug susceptibility of melanized fungi, so we reported the results based on the previous literature (Revankar and Sutton, 2010), MIC/MEC of ≤1 μg/ml was used as the index of potential sensitivity to most antifungal drugs to treat melanized molds, flucytosine (5-FC) (<50 μg/ml) excluded.

As a result, the drug resistance rate to fluconazole among the clinical dematiaceous fungi was the highest, accounting for 93% (25 of 27). The average MICs of echinocandins were higher than triazole drugs and had high variability in vitro activity. Most clinical strains were susceptible to terbinafine (89%, 16 of 18), voriconazole (86%, 18 of 21), and itraconazole (77%, 27 of 35). The average MICs of terbinafine were the lowest among all antifungal agents. AmB (56%, 18 of 32) was relatively less susceptible in vitro. Furthermore, the coincidence rate of itraconazole between MICs and clinical efficacy was 81% (13 of 16).