Talaromyces marneffei Infection: Virulence Factors and Rapid Diagnostics

Abstract

Talaromyces (Penicillium) marneffei is a thermally dimorphic fungus that causes talaromycosis, and the pathogen is found throughout tropical and subtropical Asia. T. marneffei has specifically emerged as an opportunistic fungal pathogen in individuals with advanced HIV disease and, to a lesser extent, other immunocompromised conditions, but more recently talaromycosis is increasingly described in immunocompetent people. Due to the high mortality rate of up to 50%, understanding T. marneffei interactions with host immune responses and diagnostic modalities is vital to the development of strategies to reduce morbidity and mortality. In this chapter, we describe T. marneffei virulence factors that enhance the fungus’ capacity for survival and growth in the host to lead to disease. We also discuss approaches for early diagnosis, which are essential to reduce the mortality rate in talaromycosis. Talaromycosis remains a neglected disease, but advances in our understanding of host-pathogen dynamics as well as the ongoing development of new diagnostic approaches are poised to enhance our capacity to combat this disease.

1. Introduction

Talaromyces (Penicillium) marneffei is a thermally dimorphic fungus endemic in the tropical and subtropical regions of Asia (Figure 1) [1, 2]. It is by far the species that most commonly causes human illness in immunocompromised patients, especially those with AIDS over the last three decades, especially in endemic areas of Southeast Asia (Thailand, Vietnam, Myanmar), East Asia (southern mainland China, Hong Kong, and Taiwan area), and north-eastern India, resulting in a rapid increase in incidence [2, 3]. Talaromycosis is not only recognized in endemic areas, but it is also increasingly being recorded in travelers from non-endemic areas such as Australia, Belgium, France, Germany, Japan, the Netherlands, Oman, Sweden, Switzerland, Togo, the United Kingdom, and the United States [4, 5, 6, 7, 8, 9, 10]. Fever, weight loss, anemia, lymphadenopathy, hepatosplenomegaly, respiratory symptoms, and skin lesions were all common clinical manifestations of T. marneffeiinfection. Furthermore, amphotericin B, itraconazole, and voriconazole were the most often used first-line treatments, either alone or in combination with other drugs. The majority of patients die if they are not treated.

Figure 1.

Geographic distribution of talaromycosis.

Since 1994, talaromycosis is the fourth most general opportunistic infection, after tuberculosis, pneumocystis, and cryptococcosis in AIDS patients in Thailand [11, 12]. Currently, the number of T. marneffei infections has declined in the last few years because of a decreased incidence of HIV and widespread accessibility of antiretroviral therapy [13]. Talaromycosis is not limited to people living with HIV. It is becoming more common in non-HIV-infected people who have other immunosuppressive conditions, such as primary immunodeficiency, autoimmune diseases, cancer, and solid organ and bone marrow transplants [14]. Common clinical manifestations of infection caused by T. marneffei included fever, weight loss, anemia, lymphadenopathy, hepatosplenomegaly, respiratory signs, and skin lesions. Due to increased migration and global travel, talaromycosis is becoming more common outside of endemic areas [4]. Although most people with talaromycosis are immunocompromised, healthy people can be affected as well, although rarely [15]. Patients with advanced HIV disease (CD4 cell counts <100 cells/m³) are at high risk; they typically occur with disseminated disease affecting the lungs, liver, spleen, gastrointestinal system, bloodstream, skin, and bone marrow [15]. Individuals without HIV are less likely than individuals with HIV to have skin lesions and positive blood cultures. As a result, as compared to HIV-positive persons, diagnosis is delayed (180 days vs. 45 days) and death is greater (29% vs. 21%) [16]. Table 1 summarizes the laboratory findings and clinical prognosis of talaromycosis in patients with and without HIV infection.

Variable criteria	HIV-infected related	HIV-infected unrelated	Reference
Positive blood culture (%)	76.7	47.1	[17]
White blood cells (x 10^3cells/mm^3)	4.1	15.6
CD4 (%)	3	30
Lymphocytes (%)	11.8	16.4	[16]
Neutrophils (%)	81.2	75.2
Skin lesions (%)	53.4	31.6	[16]
Diagnosis delayed; (days)	45	180	[17]
Medium treatment duration (days)	84	180	[17]
Death (%)	21	29	[17]

Table 1.

The laboratory characteristics and clinical prognosis of talaromycosis in individuals with and without HIV infection.

Talaromycosis can affect both immunocompetent and immunocompromised patients, and the disease can be localized or systemic [18].

The ecology and route of transmission of T. marneffei infection are unknown. The organism has been isolated from the internal organs of four species of bamboo rats (Rhizomys sinensis, R. pruinosus, R. sumatranensis, and the reddish-brown subspecies of Cannomys badius) as well as the soil environment in which they dwell [19, 20]. A recent occupational history or other exposures to fine soil dust during the rainy season were determined to be the most important risk factors for illness. Contact with or consumption of bamboo rats do not appear to be significant risk factors for T. marneffei infection [21].

2. Culture and morphological characteristics

2.1 Macroscopic and microscopic appearances

T. marneffei is classified as a thermally dimorphic fungus which grows as a saprophytic mold. The fungus produces abundant conidia at 25°C and converts to yeast cells at 37°C. Mycelial colonies grow relatively quickly on Potato dextrose agar (PDA) at 25°C, as do other Penicillium species, and appear as flat, powdery white colonies. With continued culture, the periphery of the colony becomes more rugose, with radial folds. The color of the fungal colony changes from white to light brown and becomes light green after 10 days of culture. The colony produces diffusible red pigments into the agar and the underside of the colony (Figure 2A). This pigment production is one of the most characteristic features of T. marneffei. The microscopic examination of the mycelia phase reveals typical morphology of Penicillium or Talaromyces species. The microscopic examination of the mycelial form of T. marneffei is recognized as dense brush-like, spore-bearing structures (Figure 2B). The conidiophores can be simple or branched structures with clusters of flask-shaped phialides at the ends. The conidiophores are hyaline, smooth-walled and have terminal verticils of 3 to 5 metulae, each with 3 to 7 phialides [1].

Figure 2.

Thermal dimorphism of Talaromyces marneffei: (A) at 25°C, T. marneffei was grown on PDA as a mold, producing greenish-yellow to yellow conidia and secreting a distinctive diffusible red pigment; (B) conidiophores have phialides and conidia chains that resemble those of Penicillium species; (C) at 37°C, T. marneffei grows as a yeast with a dark-brown colony on BHI agar, producing brown pigment; (D) yeast cells are divided by fission rather than budding. Bars, 5 μm.

At 37°C on Brain heart infusion (BHI) agar, T. marneffei can convert to yeast phase growth. Macroscopically, yeast-like colonies appear cebriform, convoluted, or smooth. Colonies are glabrous and beige-colored and take up to 10–14 days to exhibit full growth. Pigment production is both decreased and altered; the pigment released from T. marneffei yeast cultures appears closer to brown in color in comparison with the red pigment released from T. marneffei mycelial cultures (Figure 2C). Microscopically, yeast cells of T. marneffei are spherical to ellipsoidal yeast like cells separating by single septum, measuring 2–3 to 2–6 μm (Figure 2D) [22].

3. Virulence attributes of T. marneffei

It is generally believed that inhalation of T. marneffei conidia is the likely route for infection, in line with the mode of infection for other molds such as Aspergillus fumigatus [23, 24] and Histoplasma capsulatum [25]. Indeed, T. marneffei conidia are presumably small enough (2 μm in diameter) to reach the alveoli of the lung and then are subsequently phagocytized by pulmonary histiocytes. T. marneffei, however, is able to live and develop inside this hostile intracellular environment rather than being killed by the action of these immune cells (Figure 3). Then, T. marneffei can be disseminated throughout the body once established within the phagocyte and cause systemic infection if the host’s immunological state is impaired. Indeed, a better knowledge of how the virulence pathways of T. marneffei interact with the immunological response of the host has helped us in redefining the pathogenesis of this fungus.

Figure 3.

A depiction of the acquisition process of mycelial T. marneffei from the environment to the lung with the transformation to fission yeast in macrophage after deposition in alveoli.

3.1 Adherence to host tissues

Adherence to host tissues by T. marneffei conidia may play an extremely important role in the establishment of talaromycosis. Although the infective propagule and route of entry have not been definitively confirmed, inhalation of fungal conidia is likely to be the proposed route of infection [1]. Indeed, T. marneffei conidia are tiny enough (2 μm in diameter) to reach the lung’s alveoli, and the identify of a potential conidial laminin/fibronectin receptor in T. marneffei suggests a plausible mode of conidia attachment to the pulmonary epithelium [26, 27]. T. marneffei also bind extracellular matrix (ECM)-associated glycosaminoglycans, chondroitin sulfate B, heparin, and highly sulfated chitosan CP-3, which are major constituents of many tissues particularly the basal lamina [28]. These ECM may become exposed in the lung as a result of tissue damage facilitating conidia adhesion to the bronchoalveolar epithelium. Nonetheless, this hypothesis has yet to be confirmed in animal models. Following that, T. marneffei glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified and acted as an adherence protein to facilitate conidia attachment to the host’s bronchoalveolar epithelium suggesting that this protein may play an important role in the establishment of disease [29]. Indeed, knowledge of the adherence mechanisms in T. marneffei is still limited. The development of proteomic tools and the availability of genomic data will be of great importance to elucidate the mechanisms of the host-fungus interplay, and particularly of its adherence to the host tissues.

3.2 Dimorphic switching

Fungal morphogenesis appears to be a critical factor in infection establishment. Indeed, dimorphic switching between mycelial and yeast phases is regarded to be a significant virulence component in dimorphic pathogenic fungi including H. capsulatum, Coccidioides immitis, Paracoccidioides brasiliensis, Blastomyces dermatitis, and T. marneffei [30]. Conversion to the yeast form may provide protection against killing by neutrophils, monocytes, and macrophages. Thermal dimorphism of T. marneffei is essential for survival in host cells that are responsible for the host temperature change. During the past decade, significant progress has been made in the understanding of the phase transition to yeast forms. T. marneffei has the ability to change morphology from hyphal mold in the environment to pathogenic yeast cells once conidia are inhaled into the lung of a mammalian host. Within the human host, T. marneffei conidia are engulfed and destroyed by the host’s phagocytes, particularly alveolar macrophages. After internalized conidia, T. marneffei can differentiate into yeast cells and proliferate within alveolar macrophages [1]. The conversion of conidia to the yeast phase is the first critical process that permits T. marneffei to establish an infection, which is supported by the deletion of genes involved in phase transition altering the host response. The dimorphic transition of T. marneffei is a complex process involved by a number of genetic factors [31, 32].

According to the study of Yang et al. [33], the transcription factor madsA gene, a member of the MADS-box gene family, functions as a global regulator involved in the conidiation and germination, especially in the dimorphic transition of T. marneffei. In addition, overexpression of madsA in T. marneffei induced mycelium growth at 37°C, indicating that madsA is involved in the control of the dimorphic transition from yeast to mold. The deletion mutant and a complemented mutant of madsA in T. marneffei were then constructed and to identify its involvement in morphogenesis, dimorphic transition, and stress response [34]. When compared to the wild type and complementary strains, the ΔmadsA demonstrated a faster transition from yeast (37°C) to mycelium (25°C) with abnormal morphogenesis. This study suggested that madsA functions as a regulator of yeast-to-mycelium transition and is closely related to conidiation and germination in T. marneffei although its roles in the survival, pathogenicity, and transmission require more investigation.

Despite the fact that temperature is only established stimulus controlling dimorphism in fungi, little study has been performed on the cellular changes or intracellular processes between the mycelial and yeast forms of T. marneffei. Based on yeast-phase specific proteins involved in virulence, the expression of yeast antigens of T. marneffei during phase transition was recently studied using a yeast-specific monoclonal antibody (MAb) 4D1 [35]. The MAb 4D1, yeast phase-specific MAb against T. marneffei, was produced using a modification of standard hybridoma technology with incorporating of cyclophosphamide without cross-reactivity to a panel of dimorphic and common fungal antigens [36, 37]. In addition, the MAb 4D1 was reactive against a 50–180 kDa broad high-molecular-weight smear of yeast phase mannoprotein antigen in T. marneffei. Recently, the MAb 4D1 was used to track cellular events in T. marneffei during phase transition and demonstrated that conidia were directly converted to fission yeast cells, with the expression of the yeast-specific antigen occurring 12 hours after phagocytosis by human THP-1 macrophage. These phenomena were clearly exhibited by overlapping signals between the green color of fluorescence isothiocyanate (FITC)-labeled conidia and the red color of MAb4D1 specific to yeast antigens, resulting in a yellow co-localized signal 12 hours after macrophage internalization (Figure 4). When compared to the results in artificial cultivation media, this experiment demonstrated that the phase transitional ability of T. marneffei conidia in culture medium was converted to yeast cells at a slower rate than in the host macrophage THP-1 environment. Thus, MAb 4D1 can be applied as a biomolecular tool for understanding the phase transition of T. marneffei and provides strong evidence for this fungal shift from an environmental saprophyte to a pathogenic fungus.

Figure 4.

The overlapping signals between the green of FITC-labeled conidia and the red of MAb4D1 which gives the co-localized signal as a yellow at 12 hours after internalization (white arrows). T. marneffei yeast cells were labeled with MAb 4D1 and Alexaflor 555-conjugated goat anti-mouse IgG antibody. A: Light microscopic image; B: Fluorescence image showing the green channel (FITC-labeled conidia); C: Fluorescence image of the red channel (MAb 4D1-positive yeast cells); D: THP-1 nuclei were stained with DAPI (blue) a merged channel showing the overlapping of images. Bars, 5 μm.

The readily reversible nature of the mycelial to yeast and yeast to mycelial transformation processes in T. marneffei indicates that they are genetically controlled. A number of molecular biology studies have focused on the genetic factor that influences dimorphic switching in T. marneffei. Indeed, abaA expression is significantly upregulated during hyphal to yeast transformation in fungi [38]. The abaA deletion mutant also displays aberrant yeast morphology as both the developing transitory-state arthroconidial filaments and the yeast cells fail to couple nuclear and cellular division, resulting in multiple nuclei in both the arthroconidial compartments and yeast cells. Furthermore, during the yeast to mycelia transition, transient upregulation of expression of cflA [39] and cflB genes [40] was observed. However, mutations in these genes resulting in altered function to do not block the dimorphic property of T. marneffei. Further study has employed two-dimensional difference gel electrophoresis to investigate proteins expressed differently in the yeast and mycelial phases, as well as peptide mass fingerprinting to identify these T. marneffei differentially expressed proteins [41]. These two enzymes are required for T. marneffei to survive as yeast inside phagocytes, where it is protected from the host defense system. Isocitratelyase, in particular, is the key rate-limiting enzyme in the glyoxylate bypass, a metabolic pathway that supplements the tricarboxylic acid cycle and is required for the survival of some intracellular pathogenic fungi such as P. brasiliensis and Cryptococcus neoformans [42, 43, 44]. This demonstrates the requirement of T. marneffei in sustaining the glyoxylate cycle under the host’s severe nutrient-depleted environment.

Transition to the yeast phase may provide protection from phagocyte destruction. Thermal dimorphism of this fungus plays an important role for survival in host phagocytes. However, the phenomenon that regulates this transit has remained an enigma. A number of molecular biology studies have concentrated upon the genetic element influenced in the dimorphic switching in T. marneffei. Many of those previously investigated including stuA, stlA, gasA, gasC, and cflB have no role in yeast cell development or the dimorphic switch. However, Borneman and his colleagues (2000) had found that abaA deletion mutant displayed aberrant mold to yeast conversion as both the developing transitory state arthroconidial filament and the yeast cells fail to couple nuclear and cell division, where multiple nuclei were observed within either arthroconidia or yeast cells. However, once conidia began to develop yeasts, a second series of genes appeared to take over the coupling of cell division events [38].

3.3 Oxidative stress response and heat-induced fungal adaptation proteins

Oxidative stress is one of the native defenses produced by the phagocytes to kill parasitic microorganisms. The phagocytes play a crucial role in eliminating fungal pathogens by producing reactive oxygen or nitrogen species, including superoxide radical anion (O²⁻), hydrogen peroxide (H₂O₂), hydroxyl radicals (OH⁻), and nitric oxide (NO) [45]. The reactive oxygen species (ROS) can damage pathogens by readily altering or inactivating proteins, membrane, nucleic acid, and they have potent immunoregulatory effects on the host immune system that affect the efficacy of the host response [46].

3.4 Catalase

Catalase peroxidase is capable of either reducing H₂O₂ with an external reductant or exchanging it to water and oxygen. The enzyme has been shown to be a virulence factor of Mycobacterium tuberculosis and A. fumigatus [47]. The catalase-peroxidase encoding gene (CpeA) in T. marneffei is associated with the upregulated expression of CpeA transcript both in yeast phase and under macrophage environment [48]. In recent years, Pongpom and her collaborators showed that CpeA controlled the fungus tolerance to H₂O₂ but not to heat stress response. H₂O₂ treatments induced high expression of this gene in both mold and yeast phase. It is therefore proposed that the CpeA of T. marneffei is utilized to protect the conidia and yeast cells from oxidative stress in the host macrophage environment [49].

3.5 Superoxide dismutase (SOD)

Superoxide dismutase (SOD) is an enzyme that alternately catalyzes the dismutation of the superoxide radical (O₂⁻) into either ordinary molecular oxygen (O₂) or hydrogen peroxide (H₂O₂). T. marneffei has been shown to survive and replicate as yeast inside the macrophage phagosome. Previously, Thirach and her colleagues investigated the fungal superoxide dismutase encoding gene (sodA) and found that the putative SodA peptide consisted of 154 amino acid residues and shared identity to fungal copper, zinc superoxide dismutase. The results suggested that sodA might play a role in stress response and in the adaptation of T. marneffei inside the macrophage [50].

3.6 High-temperature-induced fungal adaptation proteins

Since the pathogenic phase of T. marneffei is closely linked with the higher temperature for normal growth in the environment, the heat-shock proteins (HSPs) are proposed as potential virulence factors. HSP always serve as a molecular chaperone, control protein folding, and transport intracellular proteins, as well as repair or destroy proteins. HSPs are a group of proteins produced by eukaryotic cells in response to exposure to stressful conditions, as they could be upregulated upon infection to prevent misfolding of damaged proteins [51]. The Hsp 70 of T. marneffei was first isolated and identified by Kummasook and colleagues (2007) [52]. The results showed that the hsp70 transcription was upregulated during the mycelium to yeast transition. Upregulation was also observed when mycelial or yeast cells confronted to a heat stress environment at 39°C. It has been suggested that Hsp 70 may play an important role to prevent the yeast proteins from damage during temperature increase. Subsequently, Vanittanakom and her colleagues investigated the hsp30 of this fungus and showed high transcription degree in yeast phase grown at 37°C, but undetectable transcript level was observed in mycelium phase at 25°C. These researchers suggested that Hsp 30 may play an important role in heat-shock response and in cellular adaptation during infection [53]. Based on the role of HSPs in temperature adaptation, the Hsp70 and Hsp 30 have definite functions in the host intracellular response; therefore, further study in T. marneffei is necessary.

3.7 Fungal melanin

Melanin is a high-molecular-weight dark brown or black pigment produced by oxidative polymerization of phenolic or indolic compounds. Melanins are produced by a wide range of organisms, including bacteria, fungi, plants, and animals. Although different types of melanins can be produced by fungal organisms, the majority of fungal melanins are 1,8-dihydroxynaph thalene (DHN) melanins and L-3,4-dihydroxyphenylalanine (DOPA) melanins [54]. In the DHN pathway, 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN), which is derived from acetyl-CoA or propionyl-CoA with malonyl-CoA or methylmalonyl-CoA, is the first product of a polyketide synthases (PKS) pathway. This compound is then sequentially converted to scytalone, 1,3,8-trihydroxynaphthalene (1,3,8-THN), vermelone, and lastly 1,8-DHN as demonstrated in Figure 5 [55]. Finally, oxidative polymerization produces the end product, DHN-melanin [55]. The DHN melanin biosynthesis gene cluster of T. marneffei was studied [57]. A cluster of six-genes, alb1, arp2, arp1, abr1, abr2, and ayg1 are associated with conidial pigment synthesis in this organism. The genes alb1 (pks4), arp1, and arp2 encode for a polyketide synthase (PKS), a scytalone dehydratase, and a 1,3,6,8-tetrahydroxynaphthalene reductase, while abr1 and abr2 appear to encode two oxidases, respectively. Furthermore, all of these genes are phylogenetically linked to the A. fumigatus counterparts, and the production of DHN-melanin in T. marneffei is thought to be comparable to that of A. fumigatus [58]. Tricyclazole which inhibits two hydroxynaphthalene reductases in the DHN-melanin synthesis pathway was used to confirm the DHN melanin of T. marneffei [59].

Figure 5.

Biosynthesis pathway of DHN melanin in fungi. Scheme adapted from [54, 55, 56].

In T. marneffei, DOPA melanin is produced by yeast cells and composed of spherical granular particles in a beaded arrangement in the innermost cell wall [60, 61]. Bell and Wheeler [62] proposed a biosynthesis pathway for fungal DOPA melanin. There is experimental evidence for some of the proposed intermediates of that pathway [62, 63, 64]. In brief, laccase or tyrosinase catalyzes the hydroxylation of L-tyrosine to dopaquinone or the oxidation of L-DOPA to dopaquinone. Dopaquinone is a highly reactive intermediate which then forms leucodopachrome, which is oxidized to dopachrome. Hydroxylation (and decarboxylation) yields dihydroxyindoles that can polymerize to form DOPA melanin (Figure 6).

Figure 6.

The biosynthesis pathway of the dihydroxyphenylalanine (DOPA) melanin in fungi. Scheme adapted from [54, 56].

Both T. marneffei mycelial grown on culture medium at 25°C and yeast cells cultivated in a defined liquid minimal medium (MM) with L-DOPA were reactive with anti-melanin MAb 8D6, a melanin-binding MAb generated against A. fumigatus conidial melanin [60]. In mycelial phase, conidia, phialides, and hyphae were all positive with the anti-melanin MAb 8D6 (Figure 7). Thus, the melanization of T. marneffei was confirmed in both the mold and yeast forms.

Figure 7.

The melanin production of T. marneffei was detected in both mycelial and yeast forms using anti-melanin MAb 8D6. Corresponding immunofluorescence (A,C) and bright field (B,D) microscopy images demonstrating the labeling of mycelial phase (A,B) and yeast cells (C,D) of T. marneffei by anti-melanin MAb 8D6. Bar, 5 μm.

Melanins have been influenced in virulence in many pathogenic fungi including H. capsulatum, P. brasiliensis, C. neoformans, A. fumigatus and Sporothrix schenckii. Melanin synthesis can help fungi survive in a variety of environments [65] and increase their resistance to host immune responses, such as reducing macrophage oxidative burst capacity [66], inhibiting apoptosis in macrophages [67], and inhibiting cytokine production in the host [68]. Youngchim and colleagues (2005) were the first to describe melanin in T. marneffei. The study demonstrated that melanins were produced both in vitro and during infection by presenting the melanization of yeast cells inside skin tissue from talaromycosis. Furthermore, sera from T. marneffei inoculated mice produced a significant antibody response against melanin, suggesting that melanin can act as an immunologically active molecule recognized by the immune system [69]. Melanin extracted from T. marneffei was also effective in inhibiting the production of TNF-α by the human monocyte cell line THP-1 indicating that melanin could conceal the organism from initial recognition by the immune system. In this respect, melanin is thought to contribute to T. marneffei virulence by allowing the organism to survive and grow within host tissue. The study of Woo et al. [70] confirmed that knocking down the melanin-biosynthesis gene cluster, alb1, in T. marneffei resulted in a loss of virulence in mice when compared to the wild type. The mutant also had a 50% reduction in conidial survival when exposed to hydrogen peroxide compared to the wild type. In fact, melanin has been named “an antifungal resistance factor” due to its ability to make melanized cells less susceptible to antifungal drugs [71, 72]. It was confirmed from our previous study that melanin appears to protect T. marneffei by making it more resistant to antifungal drugs including amphotericin B, clotrimazole, fluconazole, itraconazole, and ketoconazole [73].

The melanin produced by T. marneffei may play some roles in the virulence factor of this fungus. Melanin acts as a power protector for T. marneffei in vitro by decreasing phagocytosis and increasing resistance to macrophage intracellular digestion [61]. Interestingly, heat-shock proteins (HSPs), particularly HSP90 in T. Marneffei, were found to be significantly more expressed in DOPA melanin yeast cells compared to non-melanized yeast cells using proteomic analysis [74]. By making further analysis in the proteomic pathway, heat-shock proteins were enriched in multiple important metabolic pathways, including stress response pathway and phagosome development indicating that HSP90 plays an important role in melanin synthesis pathway of T. marneffei.

3.8 Fungal laccase

P-diphenol dioxygen oxidoreductases or laccases are multi-copper containing oxidoreductase that catalyzed the oxidation of organic and inorganic substances including phenol containing amino acid, methoxy phenol, and aromatics amine, with the concomitant 4 electrons reduction of oxygen to water [75]. The essential properties of fungal laccases have been investigated and were shown to influence fungal development, to control phenotype and morphogenesis, to detoxify toxins, and to control pathogenesis in pathogenic fungi and stress response adaptation [76]. Laccases have been associated as contributors to virulence in many fungal pathogens such as A. fumigatus and C. neoformans. In C. neoformans, this enzyme can also promote the pathogenicity of C. neoformans by catalyzing the formation of melanin precursors. Melanized C. neoformans cell were more negatively charged on the cell wall, and this phenomenon could interfere with the phagocytosis mechanism [71]. The role of laccases in the virulence factor and pathogenesis in T. marneffei were characterized by Sapmak and colleagues [77]. It was found that quadruple deletions of laccase encoding genes (lac1, lac2, lac3, and arb2) in T. marneffei mutant were more sensitive to oxidative stressor, cell wall stressor, and antifungal agents including itraconazole, fluconazole, and clotrimazole. Subsequently, the results showed that the mutant strain of T. marneffei was more susceptible to killing by human macrophages, THP-1, than the wild-type T. marneffei. Moreover, the observation on the pro-inflammatory cytokine production in THP-1 human macrophages showed that the mutant T. marneffei stimulated a significantly higher production of TNF-α, IL-1β, and IL-6 compared to the wild type. Altogether, these results defined the role of laccases that influenced T. marneffei resistance to the host immune response [77].

3.9 Fungal cell wall, mannoproteins Mp1p

The fungal cell wall is a critical structure with high flexibility that is important for cellular integrity and vitality. Mannoproteins are one of the most important structural components of the fungal cell wall. In fact, substantial study with yeast has demonstrated that mannoproteins perform a variety of biological functions, including defining cell shape, stimulating cell growth and morphological change, functioning as a protective factor, aiding sex agglutination, and regulating cell wall porosity [78, 79, 80, 81]. Mp1p is an antigenic cell wall mannoprotein found in yeast, hyphae, and conidia of T. marneffei and has been effectively employed in serodiagnosis and infection prevention [57, 82, 83, 84, 85]. Mp1p is a 462-amino-acid protein having three domains: ligand-binding domain 1 (LBD1), ligand-binding domain 2 (LBD2), and a serine and threonine-rich domain near the C terminus [84]. Mp1p is a new virulence factor of T. marneffei through knockout and knockdown research employing an intracellular survival assay with murine macrophage cells and mice challenge models [86]. For a mouse model, the mice could live for up to 60 days without talaromycosis after being challenged with a Mp-knockout strain of T. marneffei, but the wild-type strain killed the mice within 21 days. In addition, the organ fungal burden and inflammatory response in mice infected with the MP1 knockout mutant were significantly reduced compared to the wild type.

Based on the structure of Mp1p, Mp1p-LBD2, a ligand-binding domain, is a strong arachidonic acid (AA) binder by forming a five-helix bundle monomeric structure with a long hydrophobic central cavity for high-affinity encapsulation of cellular AA [87]. AA is a key pro-inflammatory mediator because it is produced as a main eicosanoid precursor in response to microbial infection, which can generate many downstream prostaglandins and common markers of pro-inflammatory responses, including TNF-α and IL-6 [88]. Subsequently, Lam et al. [89] demonstrated that not only Mp1p-LBD2, but Mp1p-LBD1 is also a strong AA-binding domain in Mp1p. Thus, Mp1p is an effective AA-capturing protein that uses two AA-binding domains, Mp1p-LBD1 and Mp1p-LBD2, to capture released AA during the early stages of pro-inflammatory reactions. According to the crystal structure, Mp1p-LBD1-LBD2 are likely to function independently and equally important in terms of AA capturing, with each domain capable of accommodating two AA molecules. Taken together, Mp1p represents a novel class of fatty acid-binding proteins with the function of targeting key pro-inflammatory signaling lipid to suppress the host innate immune response.

3.10 Iron and calcium are essential cations required for growth and virulence

Ca²⁺ signaling plays an essential role in various processes, including cation homeostasis, pH adaptation, glucose metabolism, morphogenesis, and virulence in fungi [30, 90]. The Ca²⁺-binding protein calmodulin and the Ca²⁺/calmodulin-dependent phosphatase calcineurin are two major mediators of calcium signals in eukaryotic cells [91]. Calcineurin is a serine/threonine phosphatase that composed of two subunits of catalytic (CnaA) and regulatory (CnaB) that is activated through the binding of Ca²⁺-calmodulin (CaM) [92].

Calcineurin plays a crucial role in fungal virulence such as A. fumigatus [93, 94], C. neoformans [95], Candida spp. [96, 97, 98] and Pacoccidiodes brasiliensis [99]. Recent studies reveal a role of calcineurin in growth and virulence of T. marneffei [100]. In T. marneffei, deletion of the cnaA gene resulted in substantial defects in conidiation, germination, morphogenesis, cell wall integrity, and tolerance to several stresses. The importance of calcineurin functions in cell wall integrity of T. marneffei was supported by the study of MICs against caspofungin and micafungin, which revealed lower MICs in the cnaA mutant when compared to wild type. These two antifungal agents belong to the echinocandins that inhibit fungal cell wall biosynthesis by inhibiting cell wall β-(1,3)-D glucan synthesis [101]. In addition, the cnaA mutant conidia were not only more susceptible to salt, H₂O₂, and osmotic stress in vitro, but they also rarely germinated or processed yeast morphogenesis after being phagocytosed by macrophages. Calcineurin is also required for full virulence in a murine model of invasive T. marneffei infection. Thus, calcineurin homolog (cnaA) regulates fungal morphogenesis and the response of T. marneffei to external stressors, as well as the host immunological response and fungal pathogenicity.

Iron is an important trace element that is often limited for pathogens during infection; hence, adaptability to iron deficiency is critical for virulence [102, 103]. Indeed, iron has been demonstrated to be essential for T. marneffei development and pathogenicity. Iron overload also significantly decreased the antifungal activity of macrophages [104]. As T. marneffei lacks an iron excretion mechanism, controlling iron uptake, metabolism, and regulation can play an important role in iron homeostasis. Fungi have developed two methods to get iron in iron-limited environments: reductive iron assimilation (RIA) and siderophore-mediated iron acquisition [105, 106]. RIA begins with the reduction of ferric iron sources to more soluble ferrous iron by plasma membrane-localized ferrireductases [107]. Then, the ferrous iron is re-oxidized and imported by a protein complex composed of the ferroxidase, FetC, and the iron permease, FtrA. Both fetC and ftrA gene expression was higher in yeast cells (37°C) than in hyphal cells (25°C) with a clear upregulation in response to iron limitation [108]. Deletion of ftrA results in a defective RIA system, which reduces the growth of yeast cells but not hyphal cells under low iron conditions [109]. For siderophore biosynthesis pathway, sidD and sidF genes involved in the biosynthesis of extracellular siderophores of T. marneffei were upregulated early during yeast morphogenesis switching from 25 to 37°C and late during yeast cell growth [108].

Based on the functions of sidA and sidX, these two genes encoded the enzyme ornithine N5-oxygenase, which catalyzed ornithine to hydroxyornithine in an early step of the siderophore biosynthesis pathway. SidA is involved in extracellular siderophore formation in the mycelial phase, whereas SidX is involved in both intracellular and extracellular siderophore production in the yeast phase [109]. Mutant analysis revealed that T. marneffei yeast cells can utilize RIA for iron acquisition, providing another system in this cell type that varies extensively from hyphal cells. For example, the expression of fetC (involved in RIA) was significantly elevated in ΔsidA and ΔsidX yeast cells but not in hyphal cells.

Furthermore, T. marneffei has recently been studied for the expression of acuM and acuK, which have been found to be involved in gluconeogenesis and iron metabolism [110]. In fact, AcuM and AcuK are homologous Zn2Cys6 transcription factors previously identified as gluconeogenesis and iron metabolism regulators in other pathogenic fungi such as A. nidulans [111] and A. fumigatus [112]. T. marneffei transcript levels of acuM and acuK were sequentially downregulated when the fungus was grown in increasing iron concentrations [110]. As a result, the transcription factors AcuM and AcuK may play a role in iron metabolism by either reducing iron uptake or alleviating iron toxicity. In contrast, the acuM transcript was upregulated in the gluconeogenic condition, but the acuK transcript was only elevated in the acetate medium during the yeast phase. Taken together, the genes acuM and acuK have been linked to iron homeostasis and gluconeogenesis in T. marneffei. Deletion of acuK gene in T. marneffei resulted in a growth defect under iron-deficient conditions since the mutant produced fewer siderophores [113]. The fetC transcript in ΔacuK was significantly increased than in the wild type, indicating that acuK may be a negative regulator of fetC expression, the gene encoding an RIA enzyme in T. marneffei. In contrast, the sidA and sidX transcripts involved in the first step of siderophore biosynthesis in ΔacuK were relatively low. This finding implied that sidA and sidX may be controlled by Acuk, and the detailed mechanism has yet to be investigated. As iron assimilation is the complex system, more studies are required to completely understand its regulation mechanism.

3.11 Extracellular vesicles (EVs)

Extracellular vesicles (EVs), a type of nanoscale lipid bilayer membrane structure, play a function in transporting molecules to the extracellular space and are referred to as “virulence bags” [114, 115]. The characteristics and potential roles of these vesicles in virulence have been studied in a number of pathogenic fungi such as C. neoformans, C. albicans, H. capsulatum, and P. brasiliensis [116, 117, 118, 119].

In T. marneffei, EVs had a typical spherical shape with a diameter of 30 to 300 nm under the nanoparticle tracking analysis (NTA) and TEM [120]. The functions of EVs released by T. marneffei could promote the expression levels of reactive oxygen species (ROS), nitric oxide, and some inflammatory factors including interleukin-1β, interleukin-6, interleukin-10, and tumor necrosis factor in RAW 264.7 macrophage cells. It was also reported that T. marneffei could secrete EVs loaded with some active molecules including heat-shock protein, mannoprotein 1 (MP1p), and peroxidase. As an important carrier containing a variety of molecules, EVs play a crucial role in intercellular communication with host immune responses.

4. Laboratory diagnosis of talaromycosis

4.1 Staining and culture methods

Microbiological culture and histological staining are commonly used for diagnosis of T. marneffei infection. Clinical specimens including bone marrows aspirates, lymph node biopsies, blood, sputum, pleural fluid, cerebrospinal fluid (CSF), urine, and liver biopsies were used for diagnosis of T. marneffei infection [11, 121]. In addition, Wright’s staining of bone marrows aspirates and touch smear of skin biopsy or lymph node biopsies is a rapid diagnostic method [122] (Figure 8). The fungus can be seen in histological sections stained with Grocott methenamine silver (GMS) or periodic acid–Schiff (PAS). In contrast, T. marneffei yeast cells may result in the false impression that a capsule mimics to H. capsulatum when staining with hematoxylin and eosin (H&E) [123].

Figure 8.

Typical disfiguring central-umbilicated skin lesions on the face of a patient with advanced HIV and disseminated talaromycosis in Thailand (A), calcofluor white stained touch skin smear, showing fission yeast cells (B) and sausage-shaped yeasts with binary fission outside macrophage (C). The arrow heads highlight the midline septum in a dividing yeast cell characteristic of T. marneffei. Scale bar represents 5 μm.

Microbiological cultivation is a gold standard for diagnosis of talaromycosis. The bone marrows gave the highest yield for culture positive, approaching 100%, followed by culture of other specimens obtained from skin biopsy (90%) and hemoculture (76%) [11]. However, most of the fungal isolates in microbiological laboratory screening are usually obtained from hemoculture of HIV-infected patients and need to confirm the dimorphic transition of this fungus. T. marneffei was confirmed by morphology and thermal dimorphism. T. marneffei was confirmed by macroscopic and microscopic examination. However, a limitation of the culture method is time-consuming, taking about 1 to 2 weeks. Given that ineffective fungal therapy of T. marneffei is associated with poor prognosis and can be fatal, more rapid diagnosis of infection is preferable.

4.2 Serodiagnosis

Many serodiagnostic assays have been developed for the detection of T. marneffei antigen from various clinical specimens, as shown in Table 2. Based on a potent immunogenic protein known as Mp1p, the protein is made up of galactomannoprotein which located throughout the cell wall of T. marneffei yeast [85, 123] have developed monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs) against cell wall mannoprotein Mp1p expressed in Pichia pastoris. These antibodies were applied to detect antigens by using antigen capture ELISA. The method exhibited the sensitivities and specificities of 55% and 99.6% for the MAbs-MAbs based method and 75% and 99.4% for the MAbs-PAbs based method. There was no cross-reactivity found in 11 common pathogenic fungi, including Cryptococcus, Candida, Aspergillus, and Histoplasma. The Mp1p EIA was then applied to plasma samples of 372 patients who had culture-proven talaromycosis from blood and 517 individuals without talaromycosis (338 healthy volunteers and 179 with other infections) in Vietnam, demonstrating 98.1% specificity and 86.3% sensitivity [124]. In addition, paired plasma and urine testing in the same patients (n = 269) significantly improved sensitivity when compared to testing plasma or urine alone.

#	Methods	Diagnostic antigen	Diagnostic sensitivity*	Diagnostic specificity**
1	Immunodiffusion (microimmuno-diffusion)	Exoantigen	25 (2/8)	N/A
2	Immunodiffusion	Fission arthroconidia filtrate	11.7% (2/17)	100% (0/40)
3	Indirect immunofluorescent assay	Germinating conidia and yeast—hyphae cells	100%(8/8) IgG titer >160	N/A
4	Immunoblotting	Secreted yeast early stationary phase exoantigen profiles	200 kDa: 72.7% (24/33) 88 kDa: 94% (31/33) 54 kDa: 60.6% (20/33) 50 kDa: 57.6% (19/33)	200 kDa: 79.3% (23/29) 88 kDa: 93.1% (27/29) 54 kDa: 13.8% (4/29) 50 kDa: 10.8% (3/29) (for AIDS patients without Talaromycosis)
5	Immunoblotting	38 kDa of mycelial cell culture filtrate	45% (23/51)	28% (11/39) cross- reacted with Cryptococcosis 21% (6/28) cross- reacted with Candidiasis
6	Immunoblotting	Cytoplasmic yeast antigen (TM CYA) profiles	61 kDa: 48% (10/21) 54 kDa: 71% (15/21) 50 kDa: 48% (10/21)	100% (0/80)
			86% (18/21) Recognized at least one bandof TM CYA
7	Indirect ELISA	Recombinant fusion Mp1p (expressed in Escherichia coli)	82% (14/17)	100% (0/165)
8	Immunoblotting	Recombinant Mplp6	95% (19/20)	100% (0/35)
9	Immunoblotting	Recombinant Hsp30 fusion protein	20% (2/10)	100% (0/10)
10	Indirect Mp1p IgG ELISA	Recombinant Mp1p (expressed in Pichia pastoris)	30% (6/20)	98.5% (532/540)
Footnote

Table 2.

Summarized immunological methods for detection of T. marneffei specific antibodies.

^*

Compared with culture confirmed talaromycosis

^**

Compared with other microorganism infection, healthy individual living in endemic area and asymptomatic HIV infection living in endemic area

N/A: No data or inconclusive data

The most recent antigen detection assays developed for the diagnosis of T. marneffei infection have demonstrated the potential diagnostic application of MAb 4D1 [36]. In addition, MAb 4D1 (an IgG1) recognizes a 50–180 kDa manoproteins and the MAb shows specificity without cross-reactivity to a panel of dimorphic and common fungal antigens. Then, a new inhibitory ELISA using MAb 4D1 was designed to determine the antigenic concentration of T. marneffei in patient sera. The test identified antigenemia in all 45 (100%) talaromycosis, with a mean antigen concentration of 4.32 μg/ml. No cross-reactivity in this assay was found in patients with other fungal or bacterial infections, and healthy controls. This result showed that the detection of circulating antigens in talaromycosis was beneficially useful not only for diagnostic purposes but also as a tool to evaluate the clearance of fungal burden during treatment.

Currently, there is no commercially available diagnostic kit for talaromycosis. The available alternative serodiagnostic method for talaromycosis in routine laboratory is based on Platelia Aspergillus EIA which is designed for the detection of circulating A. fumigatus GM antigens. It has been reported that the GM antigens of T. marneffei and Aspergillus are very similar, and the EIA test could therefore give high degree of diagnostic sensitivity for talaromycosis [20, 125]. As a result, the intimate concordance rate between Mp1p antigen detection and the GM antigen assay in antigenemia of talaromycosis was demonstrated, which is extremely important. Several studies have revealed significant false-positive due to the cross-reaction of the MAb against GM (Rat MAb EB-A2) with GM antigen from non-Aspergillus spp., e.g. Geotrichum capitatum, H. capsulatum, P. brasiliensis, B. dermatitidis, Mycobacterium tuberculosis, galactoxylomannan from C. neoformans, and C. gattii and serum of patient treated with piperacillin-tazobactam or amoxicillin-clavulanic acid. It is not surprising that GM antigen is a “Pan-specific” marker for the fungal infection. Similarity between the (1–3)-β-D-glucan (BG) or “Fungitell” has been used for the diagnosis of filamentous fungal infections [126]. However, the lack of specificity means was unable to discriminate between Aspergillus spp. and other pathogenic fungi.

4.3 Rapid lateral flow immunochromatographic assay (ICA)

Recently, the rapid lateral flow ICAs have been developed for immunodiagnosis of the infection due to the clinically important fungi, e.g. polysaccharide antigen detection for C. neoformans [127], detection of specific IgG against Pythium insidiosum [128], hyphal-specific antigen detection of Candida species [129], and MAb against secreted glycoprotein of A. fumigatus for the diagnosis of invasive aspergillosis [130].

A “point-of-care” diagnosis of talaromycosis is urgently needed [2, 131]. Recently, we demonstrated a novel inhibition format of an ICT strip for rapid detection of the T. marneffei antigen from clinical urine samples. In this study, T. marneffei cytoplasmic yeast antigens (TM CYA) and the corresponding MAb 4D1 conjugated with nanoparticles of gold colloid were used. The inhibition (inh)-ICT strip was evaluated for its diagnostic performance in urine samples from both talaromycosis patients and a control group. The inh-ICT was highly specific against antigenuria from T. marneffei only, and it did not detect antigenuria of other clinically important microorganisms. The limit of the detection was 3.12 μg/mL of fungal antigen. The inh-ICT was used to test urine samples from 66 patients with confirmed T. marneffei infection, 40 patients with other microbial infections, and 72 healthy individuals from endemic area. The test exhibited diagnostic sensitivity, specificity, and accuracy of 87.87%, 100%, and 95.50%, respectively, for T. marneffei [132]. However, the Inh-ICT has some limitations on the relatively low diagnostic sensitivity and occasional ambiguity in the reading and interpretation of the observed results. The innovative sandwich ICT strip was created to improve the diagnostic efficiency of ICT strips by using a mannose binding lectin, which recognizes mannose residue called Galanthus nivalis agglutinin (GNA) or snowdrop lectin, in conjunction with MAb 4D1 [133]. The MAb4D1-GNA-based ICT showed specific binding activity with yeast phase antigen of T. marneffei, and it did not react with other common pathogenic fungal antigens. The diagnostic performance of the ICT was validated using 341 urine samples from patents with culture-confirmed T. marneffei infection and from a control group of healthy individuals and patients with other infections in an endemic area resulting 89.47% sensitivity, 100% specificity, and 97.65% accuracy. As a result, the T. marneffei ICT should be evaluated for clinical use in the context of rapid and affordable point-of-care diagnostic test to reduce the burden of talaromycosis mortality in patients in low-income countries.

4.4 Molecular diagnosis

The polymerase chain reaction (PCR) has been utilized effectively for the specific detection of many pathogenic fungi. The nucleotide primer PM2 and PM4 have been developed to amplify a 347 bp fragment of the internal transcribed spacer (ITS) element between 18 s rRNA and 5.8 s rRNA [134]. Novel oligonucleotide probes RRF1 and RRH1 were used in PCR southern hybridization format for the amplification of a 631 bp. fragment of the 18 s rRNA and then hybridized with a T. marneffei specific 15 oligonucleotide probes [135]. Further molecular diagnosis method was described; a one tube seminested PCR assay based on the 18 s rRNA region was developed to identify T. marneffei genome [136]. This method was useful and can detect T. marneffei DNA both from culture and clinical samples. An additional nested PCR test and a real-time PCR assay were used to detect T. marneffei in whole blood samples. Given the high sensitivity of nested PCR (82%) and real-time PCR (91%), the combination of these two PCR methods provides an interesting alternative for identifying T. marneffei DNA in whole blood samples [137]. However, these methods need the clinician establishing a hypothesis before to examination. T. marneffei is an uncommon pathogen in non-HIV individuals, particularly in areas outside of Southeast Asia where detection and therapy of talaromycosis may be restricted. Next-generation sequencing (NGS) based on metagenomics has recently been used to successfully diagnose disseminated T. marneffei infection. Under these conditions, the use of mNGS enabled for the rapid and accurate diagnosis of T. marneffei without the requirement for specified problematic pathogens, which is a proven advantage in talaromycosis diagnosis, resulting in improved individual patient treatment [138, 139]. At the time, the cost is the major hindrance to its wide usage. If the cost of NGS is reduced further and expertise is made more widely available, it will be an effective instrument in the repertoire for laboratory diagnosis of T. marneffei infection.

5. Conclusion

Talaromyces (previously Penicillium) marneffei causes a life-threatening invasive mycosis both in immunocompromised and immunocompetent individuals living in tropical and subtropical Asia. T. marneffei can adapt and express many virulence factors to survive inside hosts and then infect in those patients. The current understanding of the dynamic interaction between T. marneffei and its mammalian hosts emphasizes the role of virulence factors, such as adhesion to host tissue, dimorphic switching, oxidative responses, heat-shock protein, and melanin, which allowed the pathogen to evade host immune cells. Diagnosis of talaromycosis is frequently delayed which can result in unnecessary antibiotic use, unnecessary hospital admissions, and increased morbidity and mortality. Conventional methods of diagnosis have relied on the culture or examination of fungi; however, the time required to obtain results from culture and the lack of sensitivity of visual inspection tests can make them inconvenient. Thus, rapid diagnosis frequently based on antigen testing can help with the identification of talaromycosis.

Conflict of interest

The author declares no conflict of interest.