Talaromyces (Penicillium) marneffei infection in non-HIV-infected patients

Talaromyces (Penicillium) marneffei is an important pathogenic thermally dimorphic fungus causing systemic mycosis in Southeast Asia. The clinical significance of T. marneffei became evident when the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome epidemic arrived in Southeast Asia in 1988. Subsequently, a decline in the incidence of T. marneffei infection among HIV-infected patients was seen in regions with access to highly active antiretroviral therapy and other control measures for HIV. Since the 1990s, an increasing number of T. marneffei infections have been reported among non-HIV-infected patients with impaired cell-mediated immunity. Their comorbidities included primary adult-onset immunodeficiency due to anti-interferon-gamma autoantibodies and secondary immunosuppressive conditions including other autoimmune diseases, solid organ and hematopoietic stem cell transplantations, T-lymphocyte-depleting immunsuppressive drugs and novel anti-cancer targeted therapies such as anti-CD20 monoclonal antibodies and kinase inhibitors. Moreover, improved immunological diagnostics identified more primary immunodeficiency syndromes associated with T. marneffei infection in children. The higher case-fatality rate of T. marneffei infection in non-HIV-infected than HIV-infected patients might be related to delayed diagnosis due to the lack of clinical suspicion. Correction of the underlying immune defects and early use of antifungals are important treatment strategies. Clinicians should be familiar with the changing epidemiology and clinical management of T. marneffei infection among non-HIV-infected patients.


INTRODUCTION
Talaromyces (Penicillium) marneffei is an important pathogenic thermally dimorphic fungus causing systemic mycosis in Southeast Asia. 1-3 T. marneffei is a member of the family Trichocomaceae, order Eurotiales, class Eurotiomycetes, division Ascomycota. It is the only member in the Talaromyces genus which is considered to be an important human pathogen. T. marneffei infection is endemic in tropical regions, especially Thailand, Vietnam, northeastern India, Southern China, Hong Kong, Taiwan, Laos, Malaysia, Myanmar, Cambodia and Laos. 1 The fungus was first isolated from the hepatic lesions of a bamboo rat (Rhizomys sinensis) which died spontaneously from the infection in 1956. 4 Subsequent studies showed that bamboo rats (Rhizomys sp. and Cannomys sp.) and soil from their burrows were important enzootic and environmental reservoirs of T. marneffei, respectively. [4][5][6][7] The prevalence of T. marneffei infection in these susceptible animal species varies widely across Southeast Asia. Historically, T. marneffei infection in human has been considered to be exclusively associated with acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV) infection. 1,8 In some regions such as Hong Kong and southern China, T. marneffei infection has long been considered as one of the top three AIDSdefining opportunistic infections, alongside tuberculosis and cryptococcosis. 2,9 In recent years, improved treatment of HIV infection with highly active antiretroviral therapy and control of the HIV/ AIDS epidemic with other measures have led to a change in the epidemiology of T. marneffei infection, with an increasing number and proportion of cases being reported in non-HIV-infected patients who had other immunocompromising conditions ( Figure 1). T. marneffei infection in non-HIV-infected children has been discussed elsewhere. 10 In this article, we thoroughly reviewed the epidemiological and clinical characteristics of T. marneffei infection among non-HIV-infected adult patients, and discussed on the specific management strategies for each at-risk group.

THE CHANGING EPIDEMIOLOGY OF T. MARNEFFEI INFECTION
The first human case of T. marneffei infection occurred as a laboratory-acquired infection in 1959 11 (Figure 2). A laboratory researcher accidentally inoculated the fungus into his own finger while performing experiments on mice and caused a localized small nodule at the inoculation site. 11 The first natural human case of infection was reported in 1973 and involved an American minister with Hodgkin's disease who resided in Southeast Asia. 12 Over the next 10 to 15 years, a few more sporadic cases were reported in Thailand, Hong Kong and southern China. [13][14][15][16][17][18][19][20][21][22] The HIV status of most of these patients was not known as the virus was not discovered until 1981 and laboratory diagnostics for HIV infection was not readily available in Southeast Asia in the early 1980s. The incidence rate of T. marneffei infection markedly increased after the HIV/AIDS epidemic arrived in Southeast Asia in 1988. 1 T. marneffei infection was reported not only among HIV-infected patients residing in endemic areas, but also in HIV-infected patients who had traveled to these endemic areas. 1 The economic boom in Southeast Asian countries since the 1990s was accompanied by an improvement in their healthcare infrastructures. These included better control of HIV infection and improved diagnosis of non-AIDS conditions associated with impaired cellmediated immunity. The availability of highly active antiretroviral therapy and other control measures for the HIV/AIDS epidemic led to a decrease in the incidence rate of HIV-associated T. marneffei infection. 1 On the other hand, T. marneffei infection was increasingly reported in different groups of patients with primary or secondary immunocompromising conditions (Figure 2). The use of potent immunosuppressive drugs in patients with transplantation and autoimmune diseases was associated with an increased incidence of T. marneffei infection among these non-HIV-infected patients since the mid 1990s. Improved genetic testing for various primary immunodeficiency syndromes led to the recognition of more cases of T. marneffei infection in non-HIV-infected children. 10,23 The recently identified association between T. marneffei infection and the adult-onset immunodeficiency syndrome caused by anti-interferon-gamma (anti-IFN-γ) autoantibodies helped to explain many previous cases of T. marneffei infection among non-HIV-infected adult Asian patients who had no other comorbidities. 24 Recently, T. marneffei infection was also observed in non-HIV-infected hematology patients who were treated with novel targeted therapies including anti-CD20 monoclonal antibodies and kinase inhibitors. 25   T. marneffei proliferates in macrophages and disseminates via the reticuloendotheial system. 1 Clinically, the infection is characterized by fungal invasion of multiple body organ systems, especially blood, bone marrow, skin, lungs and reticuloendothelial tissues. 1 Similar to other intracellular pathogens, the activation of macrophages by T-lymphocyte-derived cytokines, especially those of the Th1 response such as interleukin (IL)-12, IFN-γ and tumor necrosis factor (TNF)-α, is important for host defense against T. marneffei infection. 27 This is supported by the observation that T. marneffei infection in nude or T-lymphocyte-depleted mice was invariably fatal, whereas the fungus could be cleared within three weeks in healthy mice. 28,29 A polarized Th1 response prevented immunoevasion by T. marneffei in parasitized mononuclear phagocytes and stimulated macrophage killing of intracellular T. marneffei via the L-arginine-dependent nitric oxide pathway. 30,31 Furthermore, granuloma formation, which is important for containment of the fungus, was found in wild-type mice but not in IFN-γ-knocked out mice. 27 These evidences suggest that patients with defective cell-mediated immunity may be at risk of developing T. marneffei infection. The severity of the infection varies among patients with different degrees of immunosuppression. In HIV-infected patients, T. marneffei infection is often disseminated and involves multiple organs. 1,2 In non-HIV-infected patients, the infection may be focal or disseminated, depending on the underlying immunocompromising condition and the timing of diagnosis. In a retrospective cohort study involving 116 HIV-infected and 34 non-HIV-infected patients with T. marneffei infection in Thailand, it was found that the non-HIV-infected patients were significantly older, less likely to have fever, splenomegaly and umbilicated skin lesions, and more likely to have Sweet's syndrome and osteoarticular lesions. 32 The non-HIV-infected patients also had higher leukocyte, CD4 lymphocyte and platelet counts, and lower alanine transaminase level and blood culture-positive rate. 32 To further analyze the clinical characteristics of non-HIV-infected patients with T. marneffei infection, we reviewed the reports of non-HIV-related T. marneffei infection in the English-language literature found in a PubMed search using the key words 'Talaromyces', 'Penicillium', 'marneffei' and 'penicilliosis' on 1 October 2015. Reports involving patients with uncertain HIV status were excluded. A total of 119 patients with detailed clinical information were identified (Table 1). There were 65 males and 54 females. Their median age was 42.8 years (range, 22 to 79 years). Common clinical features of T. marneffei infection among these non-HIV-infected patients included fever, malaise, weight loss, skin and soft tissue lesions, hepatosplenomegaly, lymphadenopathy, cough and dyspnea ( Table 2). Some patients also had osteoarticular involvement and abdominal symptoms such as abdominal pain and diarrhea due to mesenteric lymphadenopathy or terminal ileitis mimicking Crohn's disease. Less common clinical features included tracheomediastinal fistula and neurological manifestations such as seizure and confusion due to the presence of intracranial lesions. Laboratory investigations often revealed leukocytosis or leukopenia, anemia, thrombocytosis, deranged liver function test results and elevated inflammatory marker levels including those of C-reactive protein and erythrocyte sedimentation rate. Patients with pulmonary involvement exhibited various chest X-ray abnormalities, including uni-or multi-lobar consolidations, cavities, interstitial infiltrates, pleural effusion, pericardial effusion and enlarged hilar shadow due to mediastinal and hilar lymphadenopathies.
Overall, 33/119 (27.7%) non-HIV-infected patients with T. marneffei infection died despite most of them having received antifungal treatment with anti-T. marneffei activity, such as amphotericin B, itraconazole and voriconazole (Table 2). This case-fatality rate was similar to that of non-HIV-infected patients with T. marneffei infection in another study, in which 10/34 (29.4%) died. 32 Both of these rates were higher than that of HIV-infected patients (24/116, 20.7%) in the same cohort, and might reflect delayed diagnosis of T. marneffei infection among non-HIV-infected patients due to the lack of clinical suspicion in the early stage. Notably, many of the non-HIV-infected patients with T. marneffei infection were initially misdiagnosed and empirically treated as tuberculosis because both infections are endemic in Southeast Asia, have similar predisposing factors and overlapping clinical manifestations ( Table 2). The diagnosis of T. marneffei infection in these patients was often established weeks to months later when the clinical condition failed to improve to empirical anti-tuberculosis treatment. Moreover, some of the patients residing in non-endemic regions only became symptomatic months to years after returning from endemic areas. 56,59 These factors likely led to delayed commencement of appropriate antifungal treatment. Familiarity with the non-AIDS conditions associated with T. marneffei infection would facilitate clinicians to improve the clinical management of the infection among these at-risk patients.

SPECIFIC CHARACTERISTICS OF NON-AIDS CONDITIONS ASSOCIATED WITH T. MARNEFFEI INFECTION
Anti-IFN-γ autoantibodies Immunodeficiency due to anti-IFN-γ autoantibodies is an emerging adult-onset immunodeficiency syndrome first described in 2004. 68,69 The affected patients have high-titer serum neutralizing anti-IFN-γ autoantibodies that inhibit STAT1 phosphorylation and IL-12 production, leading to a severely compromised Th1 response. 70 As a result, these patients develop recurrent, severe and/or disseminated opportunistic infections caused by various intracellular pathogens. 24,70 Over the past decade, this condition has been increasingly reported among adult Asian patients, including Filipino, Thai, Vietnamese, Japanese and Chinese residents in Hong Kong, Taiwan and mainland China. 24,57,[70][71][72][73][74][75] This ethnic predilection is likely related to genetic predispositions among Asians. Recently, the association between anti-IFN-γ autoantibodies and HLA class II alleles, including HLA-DR*15:02/16:02 and HLA-DQ*05:01/05:02, was reported. 72,76 As the initial reports mostly involved patients residing in areas nonendemic of T. marneffei, only non-tuberculous mycobacteriosis was recognized as an important opportunistic pathogen in these patients. 68,69,77 It was not until 2010 when the association between anti-IFN-γ autoantibodies and T. marneffei infection was described among eight Chinese patients living in Hong Kong. 24 The susceptibility of patients with anti-IFN-γ autoantibodies to other intracellular pathogens including non-typhoidal Salmonella sp., Burkholderia sp., varicella zoster virus and less commonly, Histoplasma capsulatum and Cryptococcus neoformans, was also recognized subsequently. 24,70 T. marneffei infections in patients with anti-IFN-γ autoantibodies usually manifest as fever of unknown origin, cervical lymphadenitis and/or mild symptomatic infection with positive serology. 24 Occasionally, T. marneffei infection or non-tuberculous mycobacteriosis might precipitate the development of reactive dermatoses such as Sweet's syndrome, erythema nodosum, exanthematous pustulosis and pustular psoriasis, or cause direct infective cutaneous lesions in these patients. 51 The treatment of T. marneffei infection in patients with anti-IFN-γ autoantibodies comprises of both effective antifungal therapy and immunomodulation to control the underlying immunological defect. These patients often responded poorly or developed recurrent infections when they were treated with antifungal therapy alone. The most effective immunomodulating treatment available currently is rituximab, an anti-CD20 antibody which targets B lymphocytes to reduce the production of serum-neutralizing anti-IFN-γ autoantibodies. 78,79 However, since rituximab has recently been identified as a potential risk factor for T. marneffei infection, a delicate balance to minimize the level of anti-IFN-γ autoantibodies, while not over-suppressing the immune system, needs to be established. 25 The dosing regimen and time intervals of administering rituximab in patients with anti-IFN-γ autoantibodies should be further evaluated in larger clinical trials.
In patients with organ-or tissue-specific conditions, such as Sjögren's syndrome, primary biliary cirrhosis, primary immune thrombocytopenia and autoimmune hemolytic anemia, the degree of systemic immunosuppression was usually not severe. Therefore, T. marneffei infection usually occurred when these patients received high-dose or prolonged treatment with T-lymphocyte-depleting drugs, including corticosteroids, cyclosporine, azathioprine, tacrolimus and mycophenolate mofetil. Treatment of T. marneffei in these patients usually required a reduction of immunosuppressive drugs together with  Abbreviation: HIV, human immunodeficiency virus. a All reports in Table 1 were included. Only data presented in the reports were included.
T. marneffei infection in non-HIV-infected adults JFW Chan et al antifungal therapy. In patients with SLE or mixed connective tissue disease who had more severely compromised cell-mediated immunity, T. marneffei infection might occur even with less immunosuppressive therapy. In patients with active SLE, marked lymphopenia may occur in the absence of immunosuppressive therapy and is an important risk factor for not only T. marneffei infection, but also opportunistic infections caused by other intracellular organisms such as Mycobacterium tuberculosis, non-tuberculous mycobacteria, Nocardia sp., Rhodococcus sp., Burkholderia pseudomallei, C. neoformans, Pneumocytis jirovecii, Toxoplasma gondii and herpesviruses. 81,82 The management of T. marneffei infection in these patients thus requires both effective antifungal agents and careful titration of immunosuppressive therapy to control the underlying lupus activity.
Organ transplantation T. marneffei infection has been occasionally reported in solid organ transplantation and hematopoietic stem cell transplantation (HSCT) recipients. Most of these patients developed T. marneffei infection when escalated doses of immunosuppressive drugs were used to treat graft rejection. T. marneffei infection was otherwise uncommon among transplantation recipients who had minimal maintenance anti-rejection therapy. The most common type of transplantation associated with T. marneffei infection was renal transplantation, with at least 12 cases having been reported. 22,36,38,41,[44][45][46]52,56,83 T. marneffei infection has also been uncommonly reported in liver transplantation, lung transplantation and HSCT recipients on multiple T-lymphocytedepleting immunosuppressive drugs. 47,66,80,84 Due to the small number of cases, it is difficult to determine the reasons for the apparently higher incidence of T. marneffei infection among renal than other solid organ transplantation and HSCT recipients. Possible reasons include the earlier adoption and higher annual number of renal transplantation than the other types of transplantation in T. marneffei-endemic areas, and the different antifungal prophylaxis regimens used in these transplantation recipients. For example, in Hong Kong, the first cadaveric and living-related donor renal transplantations were performed in 1969 and 1980, respectively. 85 The first HSCT was performed in 1990, and the first liver and lung transplantations were performed in 1991 and 1995, respectively. [86][87][88] Furthermore, renal transplantation has consistently remained as one of the most common types of solid organ transplantation performed each year in Hong Kong over the past few decades. While HSCT recipients are generally considered to have more severe immunosuppression and thus higher risk of developing invasive fungal infections than solid organ transplantation recipients, they tend to receive more potent and prolonged antifungal prophylaxis and/or empirical antifungal treatment with activities against T. marneffei, such as itraconazole, voriconazole, posaconazole and amphotericin B. [89][90][91][92][93] In contrast, fluconazole and nystatin are commonly used as antifungal prophylaxis in patients with solid organ transplantations including renal transplantation, and thus may not be effective against T. marneffei infection.

Hematological malignancies and novel anti-cancer targeted therapies
In addition to a HSCT recipient with IgA myeloma, T. marneffei infection has also been reported in a few adult patients with hematological malignancies or proliferative diseases including non-Hodgkin's lymphoma, Waldenström's macroglobulinemia and Langerhans cell histiocytosis. 32,42,43,47,48,67,80 The incidence of T. marneffei infection in this group of patients has remained low in the past decades. Recently, however, we were alerted by four unprecedented cases of disseminated T. marneffei infection among hematology patients who received novel targeted therapies including anti-CD20 monoclonal antibodies and kinase inhibitors. 25,26 Rituximab and obinutuzumab are types I and II anti-CD20 monoclonal antibodies, respectively, that predominantly target B lymphocytes. In contrast to Th1 response, the role of B-lymphocytemediated humoral response in T. marneffei infection is not welldefined. Patients with B-lymphocyte dysfunction may have impaired production of neutralizing antibodies against key virulence factors of T. marneffei identified in genome sequencing, proteome profiling and other downstream studies. [94][95][96][97][98][99][100][101][102][103][104][105][106][107][108] Treatment with rituximab induces long-lasting B-lymphocyte-depleting effects. B-lymphocyte reconstitution, characterized by the expansion of functionally immature B lymphocytes and decreased memory B lymphocytes, may take more than one year after treatment completion. 109 During this period, latent infections such as hepatitis B or even T. marneffei infection may become reactivated. 110 The newer obinutuzumab is even more potent than rituximab in depleting B lymphocytes. 111 Therefore, T. marneffei infection should be considered not only in patients who are receiving, but also those who have already completed treatment with anti-CD20 monoclonal antibodies, when they develop compatible clinical features.
Kinase inhibitors such as ruxolitinib and sorafenib have been increasingly used to treat hematological and solid organ malignancies and/or benign conditions such as psoriasis and alopecia acreata in recent years. Ruxolitinib is a selective Janus kinase (JAK) 1 and 2 inhibitor that interferes with the signal transduction for types I and II cytokines including IFN-γ and the JAK-STAT pathway. 112 Besides T. marneffei infection, opportunistic infections and reactivation due to other intracellular organisms, including M. tuberculosis, C. neoformans, herpes simplex virus and hepatitis B virus, have also been reported in patients who received treatment with ruxolitinib. [113][114][115][116] Sorafenib is a multi-kinase inhibitor that exhibits various immunomodulatory effects, including impairment of T-lymphocyte proliferation, IFN-γ production, natural killer cell activity, dendritic cell function and proinflammatory cytokine secretion. [117][118][119] The use of sorafenib has been associated with reactivation of latent tuberculosis. 120 With the expanding list of targeted therapies becoming available in the market and being used in endemic regions of T. marneffei, it would be important for clinicians to maintain a high index of suspicion and possibly perform serial serological surveillance for T. marneffei infection in patients who have received these agents to avoid a delay in diagnosis and treatment. 121 Other non-AIDS conditions Sporadic cases of T. marneffei infection have been reported in a few other non-HIV-infected patients. Their underlying conditions included idiopathic CD4+ thrombocytopenia, Job's syndrome, diabetes mellitus, splenectomy and colonic, breast and buccal cancers. 32,43,45,49,50,54,62,65,67 However, it is difficult to assess the exact role of these conditions in T. marneffei infection because of the limited number of cases. Interestingly, an increasing number of non-HIVinfected Asian patients with T. marneffei infection who were previously considered to have no underlying comorbidities were subsequently found to be positive for anti-IFN-γ autoantibodies. 32 Advances in immunological diagnostics may help to identify more novel immunodeficiency syndromes and their association with T. marneffei infection in the future.

CONCLUDING REMARKS
The epidemiology of T. marneffei infection has changed significantly in the past three decades. It is now widely recognized that the infection is not limited to HIV-infected patients. Looking ahead, more cases of T. marneffei infection are likely to be reported in the future because of several reasons. First, improvement in the healthcare systems of developing countries in Southeast Asia, such as mainland China, Thailand and Vietnam, will likely lead to an enlarging population of non-HIV-infected patients at risk of the infection, including transplantation recipients and cancer patients on targeted therapies. The discovery of novel immunodeficiency syndromes in children and adults will continue to identify more at-risk patient groups. The availability of new diagnostic and typing methods will facilitate the detection and molecular characterization of the T. marneffei strains infecting the patients in these areas. 9,[121][122][123][124][125][126] Finally, further studies to address key questions regarding the use of prospective surveillance and optimal treatment strategies will become feasible with this continuously expanding population of non-HIV-infected patients with T. marneffei infection.