Cryptococcus and Cryptococcosis in Human and Animal Model: An Overview

Eriques Gonçalves da Silva¹, Diniz Pereira Leite-Jr.^1,2, Margareth Léa da Silva Maia¹, Sandra Marilia de Souza Silva³, Francisco de Assis Baroni⁴, Marcos Ereno Auler⁵, Amanda Latércia Tranches Dias⁶, Débora Moreira¹, Rinaldo Ferreira Gandra⁷, Regina Teixeira Barbieri Ramos¹, André Gasparetto⁸, Elisangela Santana de Oliveira Dantas⁹, Valéria Tognato Costa¹, Claudete Rodrigues Paula^1*

¹School of Dentistry, University of São Paulo (USP), São Paulo, Brazil.
²Specialized Medical Mycology Center, Federal University of Mato Grosso (UFMT), Cuiabá, Brazil.
³Federal University of ABC (UFABC), Santo André, Brazil.
⁴Rural Federal University of Rio de Janeiro (UFRRJ), Rio de Janeiro, Brazil.
⁵Faculty of Pharmacy, University of the Western Center of Paraná (UNICENTRO), Campus Cedeteg, Guarapuava, Brazil.
⁶Federal University of Alfenas (UNIFAL), Alfenas, Brazil.
⁷University Hospital, State University of West Paraná (UNIOESTE), Cascavel, Brazil.
⁸Department of Dentistry, State University of Maringa (UEM), Maringá, Brazil.
⁹Institute of Biosciences, Federal University of Mato Grosso (UFMT), Cuiabá, Brazil.

DOI: 10.4236/aim.2020.104014                

Abstract

Background: Yeasts from the genus Cryptococcus (species C. neoformans and C. gattii) are opportunistic pathogens that afflict humans with low immunity caused by diseases such as AIDS or by the usage of immunosuppressive drugs. It is a fatal disease that has been studied around the whole world and its most fatal form is the brain cryptococcosis. Purpose: The present review describes the disease from pathogen isolation, the various clinical presentations of the disease, the most important virulence factors of yeast in human and animal model and their clinical issues. Methods: On this review, several published studies about the disease are presented. Results: Numerous researches have been done worldwide in order to find a kind of therapy that is more effective against the disease. Amphotericin B, in all forms is still the drug of choice in the treatment of the cryptococcosis. Fluconazole, as well as voriconazole in combination with amphotericin B, is recommended in the cases of treatment failure. Conclusion: This study presented has elucidated a little more about the disease. Further studies should be conducted to find more diagnoses that are accurate as well as more effective treatments for eradicating the disease. In this study, the bibliographic survey makes reference to the world literature; with regard to ecology, taxonomy, mains factors related to virulence, the clinical manifestations, the action of antifungal drugs and histopathological analysis used in an animal model, were the objectives deleterious aspects of this study, thus informing, in a simple way, the importance of this microorganism for research and researchers working with this global disease, called Cryptococcosis.

Keywords

Cryptocococcus, Cryptococcosis, Animal Model, Cryptococcus neoformans, Cryptococcusgattii, Virulence Related Factors

1. Introduction

1.1. Isolation of Yeast into the Environment

There has been significant progress in the ecological distribution, as well as in the population genetics, in molecular epidemiology and in the clinical characteristics of Cryptococcus genus [1]. Church towers in metropolitan areas are large habitats of pigeons, thus facilitating the spread of microorganism propagules [2] [3].

Cryptococcus neoformans are usually found in places of a high concentration of people such as, urban areas [2] [4]. Trees of the genus Eucalyptus spp., as well as pigeons present in some specific places, such as nursing homes and hospitals, should be considered main risk factors for the immunocompromised population [5].

1.2. Genus Cryptococcus

There are now hundreds of emerging pathogenic fungi. Cryptococcus neoformans and Cryptococcus gattii, are currently two of the main opportunistic/pathogenic species for humans [6] [7]. C. neoformans and C. gattii cause infections in humans and animals, and the yeast is acquired through infective particles of the environment. Hollow decaying trees and soil are taken as the ecological niche of yeast [8] ; as well as other possible sources and microhabitats of infection [9].

1.3. Cryptococcus neoformans and Cryptococcus gattii

Various researches reveal that C. neoformans in immunocompromised individuals causes meningoencephalitis, which can lead to death. C. gattii affects immunocompromised and immunocompetent patients [10]. Several molecular biology techniques have been used for the genetic identification of C. neoformans and C. gattii with the aim of comparing the groups.

Some of these techniques highlight polymerase chain reaction (PCR) fingerprinting, amplified fragment length polymorphisms (AFLP), microsatellite typing [11] ; study of the length of restriction fragment polymorphism (RFLP) [12] and more recently, whole genome sequencing (WGS) [13]. Based on capsule structural and differences techniques these genus yeasts can be classified into Cryptococcus neoformans species complex (serotype A, VN I) and Cryptococcus gattii species complex: C. gattii sensu stricto (VGI), Cryptococcus deuterogattii (VGII), Cryptococcus bacillisporus (VGIII), Cryptococcus tetragattii (VGIV) and Cryptococcus decagattii (VGIII/VGIV) [14] [15] [16].

1.4. Virulence

The ability to cause cryptococcosis, comes from the capacity of these yeasts to reproduce itself and grow inside the host, which offers conditions for the microorganism to survive in its interior [17]. Some issues as polysaccharide capsule, melanin production, urease production, and phospholipases are the main virulence factors of C. neoformans and C. gattii, although the yeast is considered an accidental pathogen since it does not complete its life cycle in the host [18]. One of the important facilitating attributes for the colonization of pathogenic fungi is the body temperature responsible for infection in humans [17]. The main difference among C. neoformans and C. gattii species and other fungal pathogens is the capsule that surrounds the cell’s surface.

This capsule is composed of complex polysaccharide polymers (mannan, xylose, glucuronic acid) as well as the cell wall that is associated with the capsule, which is a matrix composed of alpha and beta glucans, chitin, chitosan and mannoproteins [19]. While the molecular structure of melanin is still controversial, some significant studies have been carried out to understand the particular aspects related to macro and microstructure in the last decades [20]. According to some authors, urease is an important role in ensuring the use of urea produced by the host and used by the yeast as a nutrient [21] [22].

Urease is responsible for the dissolution of the microvessels of the central nervous system (CNS) [23]. Phospholipase is highly likely to be another enzyme responsible for the pathogenicity of yeast [24], being one more crucial factors in the virulence of the microorganism [25].

1.5. Diagnosis of Cryptococcosis

Cryptococcosis is related to death in immunocompromised patients. In recent years many researches and clinical trials have led to improvement, such as disease prevention. An accurate diagnosis, and correct therapy have collaborated in the treatment of this disease [26]. One of the main components of the C. neoformans and C. gattii capsule is glucuronoxylomannan (GXM), detected by the antigen agglutination test (CALAT) [27].

Particularly useful in the detection of Cryptococcus species the Film Array multiplex PCR panel, is a promising platform for a rapid diagnosis of the disease, predicting the sterility of the culture, as well as differentiating IRIS in positive recurrences among people with recurrent symptoms. Otherwise, a less costly method as well as higher velocity and greater variety of species is the polymorphism size analysis (FSP) that analyzes the size of the ITS1 and ITS2 regions [28] (Table 1).

1.6. Disease

Cryptococcosis is an opportunistic mycosis that affects immunosuppressed humans and animals; this disease is acquired by inhalation of encapsulated yeast as well as by basidiospores found in the environment [29] [30].

1.7. Humans

The most common manifestation is the involvement of the central nervous system, but pulmonary disease also occurs [31], in immunosuppressed patients, especially with the progression of acquired immunodeficiency (AIDS) and is related to deaths [28].

1.8. Clinical Presentation of Cryptococcosis

Cryptococcus spp., is a major cause of meningitis around the world. Cryptococcosis meningitis is more common in adult patients infected with the HIV virus [32]. Pulmonary cryptococcosis can manifest itself in several ways: subclinical, granulomatous, infection of the respiratory tract with isolated pulmonary nodule or even acute respiratory insufficiency that can lead to death of the patient [33]. Some reports indicate that Cryptococcosis of the central nervous system and

Table 1. Molecular characterization, virulence and diagnosis.

pulmonary infection; are common in patients who express the disseminated disease [34]. The fungus can lead the patient to extensive fibrosis in the subarachnoid space to find small veins, inducing venous congestion, causing cerebral infarction, and thus may be fatal [35].

1.9. Animal Model

The infection of central nervous system in the murine model is responsible for the death of the animals [36]. The choice of the ideal model depends on the research objectives. In animals, studies have evaluated C. neoformans infection in mammals [37]. These yeasts are found in silkworm, fruit fly, nematode and larvae [38] [39] [40] [41].

Invertebrate models such as silkworm, pneumatoid, fruit fly, and larvae have been used in experiments in the study of this disease [41]. The use of an animal model is very convenient in the study of the pathogenicity of microorganism, as well as understanding the evolution of the disease [42]. C57BL/6 mouse is an excellent model used to study the inflammatory response caused by C. gattii as it is more susceptible to disease due to its lower inflammatory response [43]. In severe SCID (Combined Immunodeficiency Disorder) animal models, combined immunodeficiency is very useful for the therapeutic and immunological study that simulates immunodeficient patients [36] [44] [45] [46].

2. Histopathologic Analysis

2.1. Brain Tissue—Mouse Model

Histological study of brain tissue in immunodeficient mice showed on the first day right after inoculation, the presence of edemas and, congested vessels. On the second day, liquefaction area and focal necrosis appeared and intercalated with fragments of necrotic cells after the 5 days. The yeast are visualized in the meninges on the seventh day and this condition which persisted for up to 15 days from the initial of inoculation, C. neoformans was also observed in the brain parenchyma (Figures 1(A)-(J)).

2.2. Lung Tissue—Mouse Model

In pulmonary tissue, focal areas of bleeding and mild neutrophilic inflammatory infiltrate into the alveolar wall are observed one day after inoculation, progressing to pulmonary congestion. On the 11th day of infection, edema, congestion, hemorrhage, diffuse and focal neutrophilic infiltrate, as well as intense amount of C. neoformans interspersed with necrotic cell fragments (Figures 2(A)-(D)).

2.3. Immunology

Evidence shows that C. neoformans can damage host cells and tissues, although this yeast is not considered a cytotoxic pathogen [47]. Studies have shown that the male mouse strain type is useful for studying brain infection after fungemia, also arguing that the main immune cells are dendritic cells and resident alveolar

Figure 1. Histological analysis of brain tissue, of immunocompetent mice (image (A)-(J)). (I) Histopathology of brain tissue in immunodeficient murine model, 5 days after inoculation, (A) presenting a discrete area of liquefaction necrosis, focal HE, 400×, (B) C. neoformans displayed, interspersed with fragments of necrotic cells, 7 days after inoculation, PAS, 400×. (II) Histopathology of brain tissue in immunodeficient murine model at 11 days after the inoculation; (C) presenting large areas of necrosis associated with liquefaction of cavitation’s in the parenchyma, HE, 400×; (D) quantify of intense C. neoformans displayed inside the cavities, PAS, 400×. (III) Histopathology of brain tissue in immunocompetent murine model. (E) 24 hours after inoculation presenting edema, HE, 200×, (F) 48 hours after inoculation vessel congested, HE, 200×. (IV) Histopathology of brain tissue in immunocompetent murine model. (G) C. neoformans observed on meninges, 7 days after inoculation, HE, 200×, (H) mononuclear inflammatory infiltrate in the 9th day after inoculation, HE, 500×. (V) Histopathology of brain tissue in immunocompetent murine model, 15 days after inoculation. (I) C. neoformans in the meninges, HE, 200×, (J) C. neoformans in the parenchyma, HE, 500×. (VI) Histopathology of brain tissue in immunocompetent murine model, 15 days after inoculation. (L) C. neoformans in the meninges, HE, 200×, (M) C. neoformans in the parenchyma, HE, 500×. Author: Prates, R.A.

macrophages [48].

After being recognized and phagocytosed by effector cells, such as neutrophils, alveolar macrophages and dendritic cells, alveolo and distal airways provide conditions for the yeast to reproduce outside of these associated cells or after recognition by the respiratory epithelium [49].

The main routes for entry of C. neoformans into the host are the lungs, where the organism escapes from the organ intracellularly within of the macrophage or through other phagocytes. This yeast migrates out of the lungs with a mechanism called a “Trojan horse”, or via transcytosis. These cells go directly to the affected organs through respiratory etalial cells [50]. The blood-brain barrier (BBB) is responsible for invasion in the central nervous system by yeast through two mechanisms: transcytosis (yeast carried in endothelial cells) and a mechanism known as a trojan horse (yeast transported within of the macrophage) [51].

Cryptococcus is an intracellular pathogen that survives by hosting cells and reproducing inside infected macrophages [52]. The immune response in cryptococcosis is mediated by macrophages and helper T cells (Th1 and Th2) [53] [54]. IL-4 is responsible for the regulation of B-cells for the secretion of IgG1 and interferon-y antibodies through the stimulation of IgG2a antibodies, making

Figure 2. Histological analysis of lung tissue, of immunocompetent mice (image (A)-(D)). (I) Histopathology of lung tissue in immunodeficient murine model. (A) 1 day after inoculation showing focal areas of hemorrhage and discreet neutrophilic inflammatory infiltrate in the alveolar wall, (B) 5 days after inoculation with mild pulmonary congestion, HE, 400×. (II) Histopathology of lung tissue in immunodeficient murine model 11 days after inoculation. (C) Presenting edema, congestion hemorrhage diffuse and focal neutrophilic infiltrate, HE, 400×, (D) Intense quantify of C. neoformans observed in the capillaries of the alveolar wall, PAS, 1000. Author: Prates, R.A.

one of the Th1 or Th2 isotopes an indicator of the response [55] [56].

2.4. Treatments

Amphotericin B associated with flucytosine has been used to treat cryptococcosis. According to studies, fluconazole has a fungistatic effect, although it has been used as an alternative for the treatment of amphotericin and flucytosine. Regarding the authors, the drug does not effectively eliminate the fungus and has been associated with clinical relapse, as well as increasing the risk of drug resistance [57] [58]. When resources are limited, Amphotericin B associated with flucytosine over a period of two weeks, has been effective in treating this disease [59].

The treatment of choice in disseminated cryptococcosis is Amphotericin B associated with intravenous flucytosine. Amphotericin B deoxycholate associated with flucytosine and administered intravenously for a period of at least two weeks, may be a second therapeutic option, however, in HIV-positive patients as well as, transplanted, it is recommended to administer the combination of these drugs for a longer period (four to six weeks) [60]. The most effective therapy, which decreases the toxicity of these associated drugs, such as the encapsulation of Amphotericin B and fluconazole in fibrin microspheres, has been used successfully by bringing these associated drugs into the correct target [59]. Voriconazole associated with Amphotericin B in murine model, with fluconazole resistant strains is effective in the treatment of experimental cryptococcosis, significantly reducing the isolation of yeast in the lungs and brains of these animals, as well as increasing their survival [44]. The production of pro-inflammatory cytokines mediated by amphotericin B and voriconazole is dependent on the concentration administered [46].

3. Conclusion

Cryptococcosis is a fatal disease; many studies have been conducted with the aim of discovering a more accurate diagnosis as well as a more effective treatment for this disease. The review presented aims to elucidate and contribute a little more about it. Further studies should be performed to find more accurate diagnoses as well as more effective treatments for the eradication of this disease. This survey seeks to bring to light some factors related to virulence, taxonomy, ecology and action to antifungal drugs and the diverse clinical and histopathological manifestations of this fungal entity called Cryptococcus, where they were elaborated in an animal model, aiming to bring information in a simple way to researchers who develop research with this microorganism.

Research Involving Human Participants and/or Animals

N/A.

Informed Consent

N/A.

Authors’ Contributions

EGS prepared the study, carried out and was involved in writing the manuscript. CRP participated in the review of the manuscript and was the chief researcher. DPLJ collaborated in the final revision of the manuscript. All other authors were involved in the examination of biological materials, data analysis and the writing of the manuscript. All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare that there is no conflict of interest.

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