Fungi can cause serious infections worldwide, with the number of serious cases reaching 150 million (Bongomin et al., 2017). Immunocompromised hosts, such as those with organ transplantation, hematopoietic stem cell transplantation, malignant tumors receiving radiotherapy and chemotherapy, and autoimmune disease receiving immunosuppressive therapy, are at risk of invasive pulmonary mycosis, which may lead to poor prognosis. On the basis of discharge diagnoses, the incidence of invasive fungal infections in France was 5.9/100 000 cases/year, with a mortality of 27.6%, both of which increased during the observation period (2001–2010) (von Lilienfeld-Toal et al., 2019). While, the proportion of pulmonary fungal infection increased annually from 26.5 per 1000 inpatients in 2013 to 42.6 in 2019 and from 3.07 per 1000 outpatients in 2013 to 8.48 in 2019 in Guangzhou, China (Li et al., 2021). The most common pulmonary mycoses are Aspergillus, Candida, Cryptococcus species, and Pneumocystis jirovecii (Bongomin et al., 2017; Azoulay et al., 2019; Zhan et al., 2021). Mixed fungal infections have also been reported (Wang et al., 2018; Awari et al., 2020); however, their characteristics have not yet been described in detail, and most are in immunocompromised hosts.

Existing data show that pulmonary fungal disease is not limited to immunosuppressed hosts. Reports of pulmonary fungal diseases in immunocompetent hosts are gradually increasing, such as cryptococcosis, Talaromycosis marneffei infection, and aspergillosis, although the latter is one of the common infections in primary immunodeficiency diseases such as chronic granulomatous diseases (Mortaz et al., 2019; Abd Elaziz et al., 2020). In China, most patients with cryptococcosis do not have an immunocompromised status (Ye et al., 2012; Cheng et al., 2021). Talaromycosis marneffei infection, an endemic fungal disease in Asian populations, also occurs in HIV-negative immunocompetent populations (Lee et al., 2019; Pan et al., 2020). Although some patients have been confirmed to have nonclassical immunodeficiency, such as hyper-IgE syndrome and anti-IFN-γ autoantibody-associated immunodeficiency syndrome, most patients do not have clear immunodeficiency or hypofunction (Lee et al., 2019; Pan et al., 2020). Due to the lack of specificity in the clinical manifestations of pulmonary mycosis in immunocompetent hosts, mycosis is often not the first consideration during clinical diagnosis and treatment. Incorrect diagnosis often delays treatment, which may affect the prognosis.

Diagnosis and treatment are even more difficult in mixed fungal disease because of unsatisfied positivity in culture and pathology. The need for timely inoculation, destruction of hyphae by tissue grinding, and empiric use of antifungal drugs is one of the reasons for negative culture. The morphological similarity of mold makes pathological diagnosis dependent on experienced technicians. Awari et al. and Wang et al. reported some cases of mixed mycoses of Aspergillus and Cryptococcus in both immunocompromised and immunocompetent patients (Wang et al., 2018; Awari et al., 2020). Coinfection with Talaromyces marneffei and Cryptococcus neoformans has also been reported in a non-HIV patient (He et al., 2020). However, the characteristics of fungal coinfection are still undefined. Here, we retrospectively analyzed a group of successively managed mixed mycoses to explore the clinical characteristics and main points of diagnosis and treatment of mixed mycoses.

This was a retrospective study conducted at the First Affiliated Hospital of Guangzhou Medical University. Between Jan 1, 2011 and Dec 31, 2019, patients who were hospitalized in the Department of Pulmonary and Critical Care Medicine with a diagnosis of mixed mycosis that involved pulmonary were searched from the electronic medical record database and enrolled as the study group. This study was approved by the ethics committee of the First Affiliated Hospital of Guangzhou Medical University (2018-119). Because this is a retrospective study, informed consent was not required.

In the past, it was believed that pulmonary mycosis occurred only in immunosuppressed hosts, with Candida, Aspergillus and Cryptococcus as the most common opportunistic fungi (Pfaller and Diekema, 2004), but increasing evidence shows that pulmonary mycosis in patients without apparent immune deficiency diseases is not uncommon, especially pulmonary cryptococcosis, which mainly occurs in immunocompetent hosts in mainland China. In our study, patients without apparent immunocompromising disease or immunosuppressive treatment accounted for an extremely large proportion.

It should be noted that only 2 cases in this study were in classical immunocompromised hosts, and one of them developed a mixed infection with Talaromyces marneffei in the present study. Another two cases of Talaromyces marneffei mixed infection were confirmed to have atypical types of immunodeficiency after the occurrence of Talaromyces marneffei infection, one with STAT3 hyper-IgE syndrome and the other with anti-IFN-γ autoantibody-associated immunodeficiency syndrome. The former was originally characterized by recurrent cold staphylococcal abscesses, pneumonia, eczema, hyperextensibility, and extreme elevation of IgE levels (Holland et al., 2007). The latter is associated with severe disseminated nontuberculous mycobacterial infections and other disseminated opportunistic infections (Salmonella, Histoplasma, and Cryptococcus) in previously healthy adults (Hong et al., 2020). However, recent studies have shown that these populations are also susceptible to infection with Talaromyces marneffei, which was previously a significant infectious complication in HIV/AIDS patients but has now increased in incidence in patients with other immune defects (Cao et al., 2019; Lee et al., 2019). Therefore, it is necessary to identify the potential cause of immunodeficiency in cases of Talaromyces marneffei infection, and people with unapparent immunodeficiency need to pay attention to the possibility of mycoses, especially those caused by Talaromyces marneffei.

In the present study, all but one patient infected by Mucor had type 2 diabetes mellites. The incidence of diabetes mellites was more common in the mucormycosis group than in the non-mucormycosis group. This result was consistent with the literature indicating that diabetes mellites, with or without ketoacidosis, is one of the risk factors for Mucor infection (Cornely et al., 2019). Although the mechanism of immunodeficiency in diabetic patients is still unclear and needs in-depth research, if diabetic patients with pulmonary mycosis do not have a good response to the existing antifungal treatment that does not cover Mucor, attention should be given to the possibility of Mucor coinfection. Additionally, there were 3 non-immunocompromised hosts with mixed fungal diseases and no underlying diseases. It is valuable information for clinicians because not only single pulmonary fungal disease but also mixed fungal diseases can occur in patients without apparent immune deficiency diseases. According to our experience, during the follow-up, if the patients with proven pulmonary fungal disease showed a poor response to antifungal treatment, the existence of another fungal disease needs to be considered even in patients without apparent immune deficiency diseases.

Regardless of the immune status, among the single IFDs, Aspergillus and Cryptococcus are the most common pathogens, while Mucor and Talaromyces marneffei are relatively rare (Letourneau et al., 2014; Yan et al., 2016; Cao et al., 2019). Pneumocystis jiroveci is also one of the most common pathogens in immunosuppressed hosts (Azoulay et al., 2019). However, the mixed mycoses reported in the literature are mainly mixed infections of Aspergillus and Cryptococcus (Lin et al., 2006; Enoki et al., 2012; Wang et al., 2018; Awari et al., 2020). There have also been reports of Aspergillus and Mucor coinfection in the lung or outside the lungs (Bergantim et al., 2013; Mahadevaiah et al., 2013; Webb et al., 2013). Most Cryptococcus and Talaromyces marneffei coinfections have been reported in HIV patients (Le et al., 2010; Groll et al., 2021). However, in the present study, mixed fungal infections with Aspergillus and Mucor were the most common types, followed by Aspergillus and Cryptococcus coinfection, and coinfection with Talaromyces marneffei and other fungi was not uncommon in HIV-negative patients. This information is valuable for clinicians. When the initial antifungal treatment is not effective and mixed fungal diseases are considered, the possibility of Mucor should be considered, especially in patients who have diabetes mellites. Therefore, fungus coinfection is worthy of consideration.

The symptoms, signs and imaging findings of mixed fungal lung infection lack specificity. There is no halo sign or crescent sign, which is not consistent with the imaging features of previous invasive pulmonary aspergillosis with a “halo sign at the early stage and crescent sign at the later stage” (Park et al., 2010). Classical chest CT images of Mucor infection, such as reversed halo signs and vascular occlusion, were also lacking in the present Mucor patients. Although Mucor patients more commonly showed multiple nodules and/or masses, effusion or consolidation and enlarged mediastinal lymph nodes in chest CT scans than non-Mucor coinfection patients, it was still difficult to diagnose Mucor coinfection, for which the prescription of targeted treatment amphotericin B liposome is recommended (Cornely et al., 2019). Hence, the diagnosis of proven invasive pulmonary mycosis mostly depends on histopathologic, cytopathologic, or direct microscopic examination of a specimen obtained by needle aspiration or biopsy.

However, the reality is that the diagnosis of pulmonary mycosis based solely on pathology may lead to misdiagnosis and missed diagnosis. Especially when distinguishing various molds, such as Aspergillus and Mucor, confusion occurs easily because of the similarity in the morphology of these two molds. Discrimination highly depends on skill and experience. In this study, the pathological judgment of some cases also exhibited this problem. It is worth noting that for 3 patients, mixed mycosis was missed in the biopsy in the hospital to which they were first admitted. The diagnosis of mucormycosis was made after reviewing the original pathological slides from the hospitals to which they were first admitted. As stated in the EORTC/MSGERC consensus definitions of IFDs, amplification of fungal DNA by PCR combined with DNA sequencing when molds are seen in formalin-fixed paraffin-embedded tissue can be used as one of the criteria for the diagnosis of infection by molds in these situations (Donnelly et al., 2019).

Additionally, lesions in 7 patients re-enlarged after the original effective treatment and were diagnosed after a second biopsy and pathological examination. Hence, based on the comprehensive patient situation, clinicians must also have rigorous procedures in the management of patients and analyze the reasons for ineffectiveness of original treatment or re-enlargement of lesions, including the sufficient triazole concentration in blood (Di Paolo et al., 2021), drug resistance (Yousefian et al., 2021), and the possibility of mixed infection with multiple pathogens such as bacteria, tuberculosis, other fungi, etc. As shown in this study, mixed mycosis is also one of the factors that needs to be considered, even in immunocompetent patients. Hence, we recommend that the following procedures can be considered in patients with pulmonary mycosis when poor response to initial antifungal treatment happens or the initial lesions grow again, including whether treatment drug monitor shows adequate concentrations of azoles (Di Paolo et al., 2021), whether there is new occurrence of symptoms or characteristic imaging of other types fungal infections other than tuberculosis or non-tuberculosis mycobacteria, whether current in use antifungal agents could cover suspected pathogens such as fluconazole is invalid for aspergillus and voriconazole is invalid for mucor. At this time, targeted fungal etiology and pathological examination are required to confirm the diagnosis.

Except for early complete surgical treatment being strongly supported for mucormycosis whenever possible, systemic antifungal treatment is recommended as the first choice for other fungi by guidelines (Cornely et al., 2019; Garcia-Vidal et al., 2019; Groll et al., 2021). In the present study, except for one patient who died because of refusing further treatment, all mucormycosis patients recovered after receiving systemic amphotericin B treatment. One mucormycosis patient also received surgery after systemic antifungal treatment. Three cases with non-Mucor mixed infection received surgery after poor response to systemic antifungal treatment or for diagnosis purposes. Hence, surgery is an alternative to systemic antifungal therapy in some mixed pulmonary mycoses with a poor response. In contrast to the high mortality in immunocompromised hosts in the literature, most mixed mycosis cases in the present study had a good response.

The major limitations of our study were that it was a retrospective, single-center study. There may have a bias in patient enrollment. Another limitation was that not all patients were screened for gene mutations, such as the STAT 3 gene and the level of anti-interferon γ antibody. In the present study, only three cases with Talaromyces marneffei infection were tested for the STAT 3 gene and anti-interferon γ antibody, with one of them positive for each.

Mixed pulmonary mycosis is not uncommon in immunocompetent hosts. During the management of mycosis, we recommend keeping mixed mycosis in mind for patients with a poor response to initial antifungal treatment, even in immunocompetent populations. A rigorous procedure may help to differentiate mixed mycosis from single mycosis.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

This study was approved by the ethics committee of the First Affiliated Hospital of Guangzhou Medical University (2018-119). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

Conception and design: YZ, CL, and FY. Collection and assembly of data: YZ, CL, SL, JZ, ZL, and YG. Data analysis and interpretation: YZ. Manuscript writing: YZ, CL, and FY. Final approval of manuscript: YZ, CL, SL, JZ, ZL, YG, and FY. All authors contributed to the article and approved the submitted version.

This research was funded by the ZHONGNANSHAN MEDICAL FOUNDATION OF GUANGDONG PROVINCE (ZNSA-2020003); Independent Fund of the State Key Laboratory of Respiratory Diseases (SKLRD-Z-202019); the Guangzhou Institute of Respiratory Health Open Project (2019GIRHZ06).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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