Invasive fungal infections and the management in immunocompromised conditions

ABSTRACT

Invasive fungal infections, being opportunistic, lead to severe disease in immunocompromised hosts with an increase in morbidity and mortality. The most common causative agents include Candida, Mucorales, Aspergillus, Cryptococcus, and Pneumocystis species. The common risk factors associated with invasive fungal infections are uncontrolled diabetes mellitus leading to hyperglycemia, neutropenia, immunosuppressive treatment like corticosteroids, and primary and secondary immunodeficiency disorders, especially acquired immunodeficiency syndrome. Various molecular therapeutic agents used for targeted therapy are also associated with an increase in opportunistic infections as they involve many signaling pathways related to immunity against pathogens. Prompt diagnosis of diseases caused by these invasive fungal infections is necessary because immunosuppression increases the risk of fatality, and treatment should be initiated immediately based on susceptibility and recommendations.

Introduction

Invasive fungal infections are opportunistic, leading to mild or no disease in immunocompetent hosts, but may infect and produce severe disease in immunosuppressed individuals. Many nonpathogenic fungi may result in significant morbidity and mortality in immunodeficient people.[1] Candida and Aspergillus species are the most common invasive fungal infections in immune-deficient hosts, followed by an increase in infections caused by hyaline and dematiaceous filamentous fungi also. The health sector faces new challenges due to emerging fungi like Candida auris, which causes multi-drug-resistant infections. Despite pronounced advances in antifungal therapy, due to the emergence of drug-resistant strains, the presence of severe underlying diseases, and the absence of appropriate research evaluating treatments, the patient outcome is poor with high morbidity and mortality. Neutrophils and macrophages are the main innate immune mediators against fungi.[2,3]

Literature Review

The most common risk factor associated with invasive fungal infections is uncontrolled diabetes mellitus (DM), followed by other causes of immunosuppression like corticosteroid therapy and neutropenia.[4] The risk of mycoses in persons with DM (PWD), which is one of the most prevalent noncommunicable diseases in India, increases to 1.38 times compared to nondiabetics. Etiological agents commonly causing invasive fungal infections in DM are Candida albicans, Cryptococcus, Aspergillus, Mucor, Rhizopus, and Fusarium.[5]

In the past few decades, the incidence and prevalence of invasive fungal diseases have increased markedly throughout the world due to the acquired immunodeficiency syndrome (AIDS) pandemic, increased use of myeloablative chemotherapy or immunosuppressive drugs, and solid organ or hematopoietic stem cell transplantation (HSCT). Recently, newer population groups at risk have appeared, like patients with severe respiratory virus-associated mold disease, coronavirus disease of 2019 (COVID-19), and immune-targeted therapy for inflammatory, autoimmune, or neoplastic conditions. Table 1 summarizes the risk factors, clinical presentation, and treatment recommendations for the most common opportunistic fungal infections.[2]

Table 1.

Clinical presentation and treatment of the most prevalent opportunistic human fungal infection

Fungal Infection	Fungal morphotype	Clinical presentation	Predisposed population	Treatment
Mucocutaneous candidiasis	Yeast with pseudo hyphae	Oropharyngeal, esophageal candidiasis, vaginal candidiasis	Corticosteroid or antibiotic use, HIV infection	Topical or oral azoles; echinocandins (for azole-resistant Candida strains)
Invasive candidiasis	Yeast with pseudo hyphae	Candidemia, intra-abdominal candidiasis, disseminated candidiasis	Neutropenia, critical illness, corticosteroid use, SOT, low birthweight premature neonates, abdominal surgery	Echinocandins
Invasive aspergillosis	Filamentous mould, hyphae	Pulmonary or disseminated aspergillosis, allergic bronchopulmonary aspergillosis	Corticosteroid use, neutropenia, HSCT, SOT, post-influenza or COVID-19, ibrutinib therapy	Voriconazole (first line), Itraconazole, Isavuconazole or posaconazole; corticosteroids; removal of the cause of immunosuppression
Zygomycosis	Filamentous mould, hyphae	ROCM, pulmonary, abdominopelvic, gastrointestinal, primary cutaneous, and disseminated zygomycosis	DM, Corticosteroid use, neutropenia, HSCT, SOT, post-influenza or COVID-19,	Amphotericin B with surgical debridement
Cryptococcosis	Yeast	Pneumonia, CNS or disseminated cryptococcosis	HIV infection, corticosteroid use, HSCT, ibrutinib therapy	Amphotericin B (with 5-flucytosine for CNS disease) followed by fluconazole; removal of cause of immunosuppression
Pneumocystosis	Cysts and trophozoites	Pneumonia or Disseminated infection	HIV infection, corticosteroid use	Trimethoprim– sulfamethoxazole; removal of cause of immunosuppression

DM: Diabetes mellitus; CNS: central nervous system; HSCT: hematopoietic stem cell transplantation; SOT: solid organ transplantation; ROCM: rhino-orbito-cerebral mucormycosis

Various extracellular fungi bind to dectin receptors on dendritic cells and induce IL-6 and IL-23, eliciting strong TH17 responses leading to the recruitment of neutrophils and monocytes, which destroy the fungi. Hence, neutropenic patients are prone to opportunistic fungal infections. Intracellular fungal infections elicit protective TH1 responses, which might result in tissue injury by granulomatous inflammation. Fungi may also stimulate defensive and distinct antibody responses.[3]

Targeted therapy by molecular therapeutic agents directed against intracellular signaling pathways has been developed for treating many inflammatory and malignant diseases, which are more effective and confer less nonspecific cytotoxicity compared to conventional chemotherapy.

However, due to the involvement of the signaling pathways in producing a cellular response to human pathogens, including fungi, the use of these agents has been associated with opportunistic infections (OI). Table 2 depicts the association of various molecular therapeutic agents with the OI.[2,6] Pattern recognition receptors sensing fungi mainly include C-type lectin receptors (CLRs), which play an important role in the immune response against fungi and have become an upcoming area of research interest.[7] Patients with various inborn immune diseases, like inherited deficiency of CARD9, the CLR adaptor protein, are prone to severe mucocutaneous and invasive fungal infections, as shown in Table 3.[2]

Table 2.

Association of immune-targeted therapy with human fungal infections

Name of the drug	Molecular target	Indications	Fungal infection susceptibility
Dasatinib	BCR-ABL, PDGFR, c-kit, c-src, Lck, Yes, Fyn, EphA2,	Ph-positive CML/ALL (imatinib-resistant)	PJP
Ruxolitinib, tofacitinib, baricitinib, upadacitinib, fedratinib	JAK1/2/3	MF, PV, GvHD, RA, IBD	Aspergillosis, invasive candidiasis, cryptococcosis, PJP, histoplasmosis, coccidioidomycosis, talaromycosis
Ibrutinib, zanubrutinib	BTK	CLL, MZL, MCL, WM, steroid-refractory GvHD	Aspergillosis (with CNS involvement), zygomycosis, cryptococcosis, PJP, blastomycosis, histoplasmosis, Fusariosis
Idelalisib	PI3Kδ	Refractory CLL, FL	PJP, aspergillosis
Infliximab, Etanercept,	TNF	RA, AS, psoriasis, IBD	PJP, histoplasmosis, coccidioidomycosis, blastomycosis
Adalimumab, Golimumab	TNF	RA, AS, psoriasis, IBD	Mucosal candidiasis, invasive candidiasis, aspergillosis, phaeohyphomycosis
Alemtuzumab	CD52	MS, CLL	Mucosal candidiasis, cryptococcosis, PJP
Ixekizumab, Secukinumab	IL-17A	Psoriasis, AS, IBD	Mucosal candidiasis
Brodalumab	IL-17RA
Ustekinumab	IL-12p40
Risankizumab, Guselkumab, Tildrakizumab	IL-23p19

AS: ankylosing spondylitis; RA: rheumatoid arthritis; TNF: tumor necrosis factor; IBD: inflammatory bowel disease; MS: multiple sclerosis; ALL: acute lymphoblastic leukemia; BCR-ABL: B-cell receptor-Abelson murine leukemia viral oncogene homolog 1 fusion protein; BTK: Bruton tyrosine kinase; CLL: chronic lymphocytic leukemia; CML: chronic myeloid leukemia; CNS: central nervous system; FL: follicular lymphoma; MZL: marginal zone lymphoma; PJP: Pneumocystis jirovecii pneumonia; GIST: gastrointestinal stromal tumor; GvHD: graft-versus-host disease; IL-2 interleukin-2; JAKs 1/2/3: Janus kinases 1/2/3; Lck: lymphocyte-specific protein tyrosine kinase; MCL: mantle-cell lymphoma; WM: Waldenström macroglobulinemia. MDS: myelodysplastic syndromes; MF: myelofibrosis; MPD: myeloproliferative disorders; PCP: Pneumocystis pneumonia; PDGFR: platelet-derived growth factor receptor; Ph: Philadelphia chromosome; PI3Kδ: phosphatidylinositol-4,5-bisphosphate 3-kinase delta isoform; PV: polycythemia vera; Yes: Yamaguchi sarcoma virus oncogene homolog

Table 3.

Association of inborn errors of immunity to fungal infections

Gene mutation	Predominant expression	Fungal infection susceptibility
CARD9	Myeloid phagocytes, Low in epithelial cells	CMCC, CNS candidiasis, aspergillosis, skin mucormycosis, phaeohyphomycosis, onychomycosis, deep dermatophytosis
IL12RB1	Lymphoid and myeloid cells	CMCC, cryptococcosis, histoplasmosis, coccidioidomycosis, paracoccidioidomycosis
IL17RA	Myeloid cells and epithelial cells	CMCC
IL17RC	Epithelial cells	CMCC
STAT1	Broad expression	Invasive candidiasis, CMCC, aspergillosis, mucormycosis, PJP, cryptococcosis, histoplasmosis, coccidioidomycosis
STAT3	Broad expression	CMCC, aspergillosis, cryptococcosis, PJP, histoplasmosis, dermatophytosis, gastrointestinal tract coccidioidomycosis, skin fusariosis
CYBB, CYBA, NCF2, NCF1	Myeloid phagocytes	Invasive candidiasis, aspergillosis,
AIRE	mTECs and eTACs	CMCC
GATA2	Neutrophils, Low in mononuclear phagocytes and T cells	Aspergillosis, cryptococcosis, PJP, histoplasmosis, coccidioidomycosis, blastomycosis
IFNGR1	Broad expression	Histoplasmosis, coccidioidomycosis

CMCC: chronic mucocutaneous candidiasis; CNS: central nervous system; eTAC: extrathymic AIRE-expressing cell; mTEC: medullary thymic epithelial cell; PJP: Pneumocystis jirovecii pneumonia

Invasive Fungal Infections in Immunocompromised Hosts

Candidiasis

Candidiasis, the most common fungal infection in immunosuppressed conditions, is an opportunistic fungal infection caused by species of the genus Candida, a yeast-like fungi.[1] It may present in humans as superficial cutaneous or mucocutaneous infections or invasive candidiasis in the form of candidemia, peritonitis, endophthalmitis, endocarditis, and disseminated candidiasis. 90% of patients presenting with fungemia are caused by Candida species, with a mortality rate of 40% to 80%.[8]

Mechanisms protecting the host against candidiasis include intact mucocutaneous barriers, neutrophils, monocytes, phagocytes, complements, and immunoglobulins. Hence, any wound, burn, ulceration, intravenous or Foley’s catheter, central intravascular access device, parenteral hyperalimentation, gastrointestinal tract surgery, broad-spectrum antibiotics, granulocytopenia, corticosteroid therapy, chronic granulomatous disease, myeloperoxidase deficiency, hypocomplementemia, and hypogammaglobulinemia make the patient prone to candidiasis. It has also been associated with premature birth, uncontrolled DM, bone marrow or solid organ transplantation, solid tumors, hematologic malignancies, and recent chemotherapy or radiation therapy.[9]

Around 70–80% of cases of invasive candidiasis are due to Candida glabrata and Candida albicans. In the past few decades, there has been a shift in the Candida species causing bloodstream infections from Candida albicans to non-albicans Candida. The incidence of Candida glabrata is increasing throughout the world and shows decreased susceptibility to azoles and polyenes, along with 20% of the cases associated with fluconazole resistance. Candida krusei is resistant to ketoconazole and fluconazole and shows decreased susceptibility to all other antifungals, especially amphotericin B and itraconazole. Candida lusitaniae is an uncommon Candida species that is sensitive to azoles and echinocandins but shows amphotericin B resistance. Candida parapsilosis can be isolated from hospitalized patients and cases having vascular catheter prosthetic devices. Candida tropicalis has led to candidemia in leukemic or post-bone marrow transplant patients.[8,10]

Diagnoses and treatment

Clinical, epidemiological, and laboratory findings should be integrated to diagnose candidiasis. Patients having fever with persistent leukocytosis even after treatment with broad-spectrum antibiotics associated with persistent neutropenia or any other abovementioned risk factor should be suspected to have systemic candidiasis. Early empirical antifungal therapy should be started in such cases for effectiveness.[10]

Superficial candidiasis on histopathological examination shows epidermal spongiotic changes with irregular acanthosis and inflammation. The stratum corneum and upper layers of epidermis can characteristically show the presence of neutrophils. There can be the presence of spongiform pustulation, which is a small collection of neutrophils. Candida can be identified as spores and pseudo hyphae forms of fungi and confirmed on Periodic acid Schiff (PAS) and Gomori Methenamine silver (GMS) stains [Figure 1]. Spongiform pustulation can also be seen in impetigo, subcorneal pustulosis, pustular psoriasis, and acute generalized subcorneal pustulosis conditions.[11] In lesions at uncommon locations like laryngeal candidiasis, the possibility of candidiasis should be considered clinically in patients with predisposing risk factors.[12]

Figure 1.

Candidiasis showing the presence of yeast and pseudo hyphae forms of candida (400X magnification) (a) Periodic acid Schiff stain (b) Gomori methenamine silver stain

Germ-tube formation, which is hyphae production from yeast cells after incubation of 2–3 hours, can morphologically identify Candida albicans or Candida dubliniensis. CHROMagar Candida and biochemical assays API20C and API32C can identify Candida species. The Candida albicans peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) test can identify Candida albicans in 24–48 hours. Recent modifications of this test can be used for concurrent identification of Candida albicans or Candida glabrata.[10,13] The gold standard test for candidemia is blood culture, which has been simplified by automated identification systems and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). It has a low sensitivity of 21–71% but helps in identifying species and antifungal susceptibility.[14] Antifungal susceptibility testing has been standardized using the Clinical Laboratory Standards Institute (CLSI) micro broth dilution or disk diffusion methodology, which helps in taking treatment decisions, especially for patients having advanced HIV disease with antifungal-refractory oral or esophageal candidiasis.[15] Among the nonculture Candida detection assays, the 1,3 β-D-Glucan (BDG) assay, an amebocyte lysis assay, is the most commonly used test, having 92% sensitivity, 81% specificity, and a high negative predictive value, used to exclude candidiasis and avoid inessential treatment. The Candida mannan assay, Candida heat-labile antigen assay, D-arabinitol assay, enolase assay, and FungiTell assay can also be used.[14] Molecular tests like polymerase chain reaction (PCR) assay and DNA probes are still in the investigational phase.[15]

The latest treatment recommendations include the echinocandins caspofungin, micafungin, and anidulafungin, along with voriconazole, posaconazole, and amphotericin B in many conditions. The Infectious Diseases Society of America (IDSA) recommends echinocandins as the initial therapy for treating candidemia and switching to azoles after 5–7 days, based on the susceptibility of the isolate, negative fungal blood culture, and patient stability. Therapy should usually be continued for 14 days after the blood culture becomes negative.[16]

Zygomycosis

Fungi in the orders Mucorales and Entomophthorales lead to infections known as zygomycosis. Due to the predominance of Mucorales infections in humans, commonly this group of diseases is also known as mucormycosis.[17] These pathogens are opportunistic and saprophytic and the third most common cause of invasive fungal infection in immunosuppression, especially in post-stem cell or solid organ transplant recipients, patients with hematologic malignancies, and rarely immunocompetent hosts.[17] Other predisposing factors include uncontrolled DM, metabolic acidosis, severe and prolonged neutropenia, steroid use, extensive skin injury, penetrating trauma, burns, HIV infection, deferoxamine therapy, history of voriconazole therapy, chemotherapy, peritoneal dialysis, renal failure, intravenous drug use, and postsplenectomy.[18] Zygomycosis is most commonly associated with uncontrolled DM in India, whereas in developed nations, hematological malignancies and recipients of solid organ transplantation are the most common associations. The incidence of zygomycosis in India is not known as there is no population-based study, but the estimated prevalence is around 70 times higher in India than that reported throughout the world.[19]

Rhizopus of the Mucoraceae family is the most commonly isolated species from cases of human zygomycosis, of which the most prevalent is Rhizopus arrhizus (Rhizopus oryzae). Rhizopus arrhizus-induced zygomycosis has an acute presentation, and despite early management, it can be lethal. These species are angio-invasive and diffusely spread in the major blood vessels, leading to ischemia, necrosis, infarction, and extensive tissue destruction in the neighboring tissues, ensuing generation of black pus. The overall mortality rate of zygomycosis is 40–50%, which increases to 70–80% in PWD and 85% in rhino cerebral zygomycosis.[1,4,18]

Depending upon the localization, zygomycosis can clinically present as rhino-orbit-cerebral mucormycosis (ROCM), pulmonary, abdominopelvic and gastrointestinal, primary cutaneous, and disseminated zygomycosis or can have uncommon presentations like endocarditis, osteomyelitis, pericarditis, and renal involvement.[18,19] The most common presentation in India is ROCM, followed by pulmonary and cutaneous involvement, while it is pulmonary type in the developed countries.[19] PWD usually presents with ROCM, and 67% of ROCM cases are seen in PWD.[20] Pulmonary mucormycosis is not common in DM, but it might present with complications like endotracheal involvement, vocal cord palsy, thickening of the posterior tracheal band, mediastinal or hilar lymphadenopathy, atelectasis, cavitary lesion, and effusion.[21]

Diagnosis and treatment

Effective management of zygomycosis depends upon early diagnosis along with identification of the extent of invasion. Radiological imaging and endoscopy of sinuses followed by microbiological and histopathological examination of the tissue are the pillars of diagnosing zygomycosis.[22]

Histopathological examination of tissue biopsy will show characteristic broad, nonseptate, ribbon-like fungal hyphae with right-angle branching, which can rarely be pauciseptate [Figure 2]. This fungus will infiltrate the affected tissue layers and blood vessels. There might be the presence of thrombosis, necrosis, and hemorrhage. Vascular and perineural invasion are commonly seen in invasive mucormycosis.[23,24] Many conditions may clinically and radiologically present as a necrotic lesion in the subglottis. These differential diagnoses include secondary tracheal wall ischemic injury caused by prolonged intubation; inflammatory conditions like Wegener’s granulomatosis and sarcoidosis; infectious lesions like bacterial, tuberculosis, aspergillosis, and mucormycosis; and neoplasms like chondrosarcoma, adenoid cystic carcinoma, and lymphoproliferative disorders. Hence, histopathological examination of the biopsy from the lesion is important for precise diagnosis, identifying invasion of mucor in the tissue, vessels, and nerves.[25]

Figure 2.

Broad, nonseptate hyphae of Mucor having 90-degree angle branching (400X magnification) (a) Hematoxylin and Eosin stain (b) Periodic acid Schiff stain

Potassium hydroxide mount, Calcofluor white examination, and culture can be done for diagnosis of zygomycosis.[22] Fungal culture is necessary to identify the genus, but as Mucorales do not grow well in culture media, PCR in fresh tissue samples is more reliable than tissue or blood culture. New molecular and antigenic tools are required for the early and accurate diagnosis of mucormycosis and its therapeutic monitoring.[24]

According to the current guidelines, a combined medical and surgical treatment is recommended for the best outcome.[4] The main antifungal drug for zygomycosis is liposomal amphotericin B, along with surgical debridement of the affected area. Administering granulocyte-macrophage colony-stimulating factor improves the number and function of neutrophils, and IFN-γ ameliorates the function of monocytes, macrophages, and neutrophils.[4,25] If the immunosuppressed condition is not cured, even surgical debridement will not suffice, and repeated debridement will be indispensable and might even evolve into disseminated disease. However, in localized diseases with underlying DM, effectively controlling blood glucose levels along with antifungal therapy, even without surgical debridement, might be effective.[26]

Aspergillosis

Aspergillus is a ubiquitous hyaline mold having more than 100 species, of which Aspergillus fumigatus and Aspergillus niger and, less often, Aspergillus flavus and Aspergillus clavatus cause disease in humans. The transmission to the human host occurs via inhalation of fungal spores.[27] Protective host mechanisms against Aspergillus spores start with the mucous layer and the cilia in the respiratory tract. Phagocytosis occurs through macrophages and neutrophils, which might be inhibited by corticosteroid administration or toxic products produced by various Aspergillus species. Proteins like 1,3 BDG on the fungal cell wall stimulate major immune effector pathways, which attract phagocytes and activate alveolar macrophages. The released cytokines attract neutrophils, which release NADPH-dependent ROS and kill the invasive hyphae. Primary and secondary immunodeficiency diseases may also cause neutropenia or neutrophil dysfunction.[28,29]

In humans, Aspergillus predominantly involves the lungs, presenting with allergic bronchopulmonary aspergillosis (ABPA), chronic necrotizing pulmonary aspergillosis (CNPA), aspergilloma, or invasive aspergillosis. In immunosuppressed hosts, there is vascular invasion, which may lead to the presence of infarction, hemorrhage, and necrosis of lung tissue. It might become lethal due to angioinvasion or may present as endophthalmitis, endocarditis, rhinosinusitis, wound, skin, or cerebral infections and abscesses in the myocardium, liver, spleen, kidney, soft tissue, and bone. Fungal endocarditis most commonly occurs due to Candida, followed by Aspergillus.[30] Hence, immunocompromised states due to underlying lung diseases like cavitary lesions, asthma, chronic obstructive pulmonary disease, and cystic fibrosis; DM; immunosuppressive drug therapy; prolonged steroid use; leucopenia; and primary or secondary immunodeficiencies predispose the patient to invasive aspergillosis.[5,30]

Diagnosis and treatment

Radiological imaging, histopathological examination, serological markers, and culture can correctly diagnose aspergillosis. In a necrotic mass that is neither clinically nor radiologically suspected to be due to fungal infection, histopathological examination of the tissue biopsy remains the cornerstone of identifying aspergillosis. Aspergillus hyphae are characteristically septate, with branching at 45° angles that may be associated with acute inflammatory infiltrate, eosinophils, granulomas, blood vessel invasion, necrosis, and tissue destruction. In severe infections or in areas of high oxygen tension, conidia or fruiting bodies, specific for Aspergillus, can be identified, which develop from mycelia, comprising a vesicle and either one or two layers of phialides [Figure 3]. The hyphae and fruiting bodies are highlighted by PAS or GMS stains. Fusariosis and pseudallescheriosis have a similar hypha and hence require culture for confirmation. However, fruiting bodies are specific for aspergillosis.[29,31]

Figure 3.

(a and b) Mixed opportunistic fungal infection with fruiting bodies of Aspergillus and a few broad, aseptate Mucor hyphae (Hematoxylin and Eosin stain, 400X magnification)

Criteria laid down for diagnosing and treating ABPA include (1) clinical worsening of the patient, (2) total serum IgE level more than 1000 IU/mL or greater than 2 times elevation from baseline, (3) positive Aspergillus serology tests like Aspergillus precipitins or Aspergillus-specific IgG or IgE, and (4) radiologically new lung infiltrates on chest X-ray or CT scans.[32] Definitive diagnosis of invasive aspergillosis, CNPA, or aspergilloma requires the presence of aspergillus in tissue. In a patient who is clinically and radiologically showing pulmonary infiltrates, morphological identification of Aspergillus fungi on tissue biopsy or Calcofluor or a positive culture growth from sputum, tissue biopsy, or bronchoalveolar lavage (BAL) fluid should indicate commencement of therapy immediately, especially after bone marrow transplantation, as the sputum culture result has a 95% positive predictive value for invasive aspergillosis. However, a negative fungal culture does not rule out pulmonary aspergillosis as only 8–34% and 45–62% of cases show aspergillus growth on sputum and BAL fluid culture, respectively, out of those ultimately proven to be invasive aspergillosis in tissue biopsy or autopsy.[33]

Serological testing by galactomannan and 1,3 BDG assays, which are components of the cell wall, are approved by the Food and Drug Administration (FDA). The probable diagnosis of invasive aspergillosis can be considered without an invasive line of action in patients with its risk factors, congruous radiology, and two successively positive galactomannan assays. Another scenario establishing probable diagnosis of invasive aspergillosis in clinically suspicious patients is the isolation of Aspergillus species on sputum or BAL culture.[34] Real-time PCR assays from blood and BAL are useful for early diagnosis of invasive aspergillosis reliably in high-risk populations. Species-specific real-time PCR assays test the presence or absence of the common Aspergillus fumigatus and the amphotericin B-resistant Aspergillus terreus.[35]

The preferred treatment for invasive aspergillosis, according to IDSA guidelines, is triazoles like voriconazole and posaconazole. Liposomal amphotericin B should be reserved for use when alternative agents are unavailable. Based on the site of disease, degree of immunosuppression, and clinical improvement, the treatment should be given for 6–12 weeks.[36] Caspofungin, anidulafungin, and FK463 are echinocandin glucan synthesis inhibitors that have a narrower gamut of activity and should be used only when Aspergillus is known to cause the infection.[37] In CNS aspergillosis, neurosurgery is more advantageous than isolated medical treatment.[38]

Cryptococcosis

Infection by Cryptococcus neoformans, an encapsulated yeast, may manifest as innocuous airway colonization and asymptomatic with only a positive skin test; localized cutaneous infection; meningitis; meningoencephalitis; or disseminated disease. Host immunity is the most important deciding factor in the development, diagnosis, and prognosis of cryptococcosis.[39] In immunocompetent hosts, cryptococcosis either is asymptomatic or presents with minimal insignificant symptoms. However, it can persist in tissue dormancy for a long time and reactivate to produce disease whenever there is immunosuppression, like lymphopenia during the progression of HIV infection to 50–100 CD4 cells/μl, which is associated with an increased risk of cryptococcosis.[1] The two most common predisposing factors are HIV infection and corticosteroid use, like in post-transplant recipients or patients with autoimmune diseases.[40] Other important risk factors include DM, lymphomas, and chronic leukemias.[41]

As the Th1 response is protective against cryptococcosis, it is hypothesized that hosts who present with disseminated cryptococcosis in the absence of any risk factor might have genetic susceptibility due to some unrevealed abnormalities in their immune responses.[42] A shift in the predominant immune response to the Th2 type is indicated in view of defective production of IFN-γ and TNF-alpha but not IL-10 in some cases of cryptococcosis.[43] While treating CNS cryptococcosis, IFN-γ increases and CSF yeast count decreases, depicting an increased Th1 response.[44]

The genus Cryptococcus has more than 50 species, of which the predominant human pathogens are only Cryptococcus neoformans and Cryptococcus gattii, having 5 serotypes based on antigenic specificity of the capsular polysaccharide: serotypes A, D, and AD of Cryptococcus neoformans and serotypes B and C of Cryptococcus gattii. Throughout the world, in immunocompromised hosts, including HIV-positive patients, mostly Cryptococcus neoformans serotype A leads to cryptococcosis. Cryptococcus gattii mostly infects immunocompetent hosts, has a slow response to treatment, and is predisposed to develop intracerebral masses known as cryptococcomas. Infected donor organs can also transmit cryptococcus through organ transplantation.[45]

Diagnosis and treatment

Cerebrospinal fluid (CSF) routine and microscopic examination, staining, culture, and immunodiagnostic tests have to be performed for diagnosing cryptococcal meningitis. Total WBC count and glucose levels are low, with normal or increased protein levels.[46] It can be diagnosed by its morphology, biochemical tests, and culture growth at 37°C. The yeast spores after inhalation accumulate in the pulmonary alveoli, wherein glucosylceramide synthase is required for their survival extracellularly, unlike inside alveolar macrophages after phagocytosis. The capsule of the yeast is antiphagocytic, which blocks phagocytic recognition and inhibits leukocyte chemotaxis. Both cellular and humoral host responses are activated, leading to increased T-helper cell activity, skin test conversion, reduced tissue viable organisms, increased anticryptococcal antibodies, and soluble anticryptococcal factors. The patient may present with an asymptomatic pulmonary infection developing to meningitis later or isolated lung involvement in the form of pneumonia, ill-defined mass lesions, nodules, and occasionally pleural effusion.[1,39]

On histopathological examination, characteristically, there is a cystic cluster of Cryptococcus neoformans yeast with an absence of well-defined inflammatory response or granulomas usually. There is usually absent or minimal necrosis or organ dysfunction until disease advancement. The surrounding inflammatory reaction varies with the patient’s immune status and stage of the lesion. Immunocompetent patients usually show a brisk granulomatous response wherein the organisms are difficult to identify without special stains, whereas immunocompromised hosts show minimal inflammation and edema around the abundant yeast forms. The yeast forms are usually widely separated due to thick mucoid capsules. PAS stain highlights the yeast, and mucicarmine stains in the typical gelatinous capsule as bright pink.[43] Encapsulated yeasts can also be identified by GMS, alcian blue, toluidine blue, or methylene blue stains. Fontana-Masson stain can identify melanin production. CSF can show the presence of 5–20 um budding or single yeasts on India ink examination [Figure 4] in around 80% of HIV-positive cases having cryptococcal meningoencephalitis and around 50% of non-HIV-infected cases. This is basically due to a large number of yeasts in CSF, between 106 and 107 CFU of yeasts/ml in HIV-positive cases. Calcofluor and GMS stains can identify this yeast in tissue biopsy. Quantitative CSF yeast counts have an excellent predictive value for outcome and prognosis but are still done only for research purposes. Cryptococcus neoformans and Cryptococcus gattii can be differentiated by culture, molecular, and antibody methods, but at present, it has epidemiological significance only.[47]

Figure 4.

Yeast forms of cryptococcus in CSF (400X magnification). (a) India ink preparation showing encapsulated yeast. (b) Gram stain showing a cluster of yeast forms

Serum cryptococcal antibodies are measured only in epidemiological studies of exposure, and their presence may indicate a good prognosis. Detection of cryptococcal capsular polysaccharide antigen in serum or CSF by latex agglutination or ELISA has a sensitivity and specificity of more than 90% and is used for diagnosis followed by cautious repeat testing or confirmation by culture. The incidence of developing cryptococcosis is very high in patients with isolated cryptococcal polysaccharidemia and negative culture. CSF examination is recommended in these cases, followed by empirical antifungal therapy even if the CSF is negative.[47] A lateral flow test is upcoming, which is a cheap, simple dipstick test and can use finger-prick blood or urine.[48] The yeast can easily be grown on standard culture media in 2–10 days and can also be isolated from blood culture systems. Disseminated cryptococcal infection should have a positive blood culture or a positive culture from at least two different sites. The presence of encapsulated yeasts in CSF, tissue biopsy, sputum, blood, or urine is considered significant, even in asymptomatic cases, and should be investigated for disseminated disease by culture and antigen detection.[46]

The therapeutic goal of cryptococcal infections in HIV-positive patients having a CD4 count of less than 200 cells/μl is to control the acute infection, followed by lifelong suppression of Cryptococcus neoformans, which might be discontinued in HIV-infected cases who have effectively completed an initial course of therapy and have remained asymptomatic with a reversal CD4 count to greater than 200 CD4 cells/μl for more than 6 months. However, if the CD4 count reduces again to less than 200 cells/μl, suppressive therapy should be restarted.[49]

Pneumocystosis

Pneumocystis, a genus of unicellular fungi, depicts airborne transmission and is found as the trophozoite form, usually existing in clusters; the sporozoite or precystic form; and the cyst containing several intracystic bodies or spores. Pneumocystis carinii pneumonia, now known as Pneumocystis jirovecii pneumonia (PJP), is the most common opportunistic infection in HIV-positive cases and may become life-threatening.[50,51] PJP can also present as immune reconstitution syndrome.[52,53] Persons predisposed to PJP include those with primary immune deficiencies, those who are severely malnourished, those on prolonged immunosuppressive treatment including post-transplant recipients, or those having any hematologic or nonhematologic malignancies. HIV-infected persons with a CD4+ cell count less than 200/μl and not receiving PJP prophylaxis or having any other opportunistic infections are also at high risk for PJP.[1]

Diagnosis and treatment

Radiologically, PJP characteristically presents as a bilateral interstitial pneumonia with diffuse patchy consolidative and ground-glass opacities.[54] Morphological identification of the organism in sputum, BAL specimens, and transbronchial or open lung biopsy specimens is required for definitive diagnosis of PJP.[53]

Special stains like GMS, toluidine-blue O, calcofluor white, and Gram–Weigert stain the cell wall of the cysts, while Giemsa, Diff-Quik, and Wright stains identify the sporozoites and trophozoites. Direct immunofluorescent antibodies are monoclonal antibodies to pneumocystis used for immunofluorescent staining on induced sputum or BAL and are the diagnostic method of choice. On histopathology, PJP typically shows the presence of foamy eosinophilic exudates within the alveoli. Occasionally, a granulomatous inflammation can be seen, usually with necrosis. Granulomatous PJP comprises pneumocystis organisms, usually trophozoites that do not stain with silver stains, inside the granulomas, along with epithelioid histiocytes surrounded by a rim of lymphocytes. The cyst counts increase when there is macrophage dysfunction due to CMV coinfection or GCSF deficiency.[54] On electron microscopy, alveolar-capillary permeability is increased.[1] However, serological tests like 1,3-BDG are also sensitive for Candida, Aspergillus, and Pneumocystis.[55] LDH levels rise proportionately to the level of lung injury, which decreases with effective treatment. Persistent elevation indicates failure of therapy and poor prognosis.[1] Active pneumocystis infection can be differentiated from mere colonization by real-time quantitative pneumocystis PCR assays but is still not used routinely.[56] The role of the noninvasive blood cell-free DNA PCR assay is also under investigation in immunocompromised patients. Next-generation sequencing can identify multiple infections like CMV and PJP from a single specimen.[54]

In critically ill patients with high risk factors and suspicion of PJP, therapy should be immediately started even without reaching a definite diagnosis. Although PJP is a fungal pneumonia, it does not respond to antifungal therapy. Disease intensity based on the alveolar-arterial gradient determines the therapy. Primarily antibiotics are recommended for mild, moderate, and severe PJP. Trimethoprim-sulfamethoxazole is as effective as intravenous pentamidine and better than other treatment modalities. In HIV-infected cases with severe PJP, corticosteroids can be added to initial treatment. Cessation of smoking and chemoprophylaxis can be used as prophylactic treatment.[57]

Conclusion

Debilitated patients with immunosuppression are the usual hosts for a more diverse range of opportunistic fungal infections having an inherent low virulence. These infections are having an increase in incidence associated with an increase in the burden of uncontrolled DM, cancer, and AIDS, as well as the use of post-transplant chemotherapy, multiple antibiotics, corticosteroids, and immunosuppressives. Management of these OIs should be prompt and standardized with newer diagnostic methods and treatment guidelines to decrease mortality in immunosuppressed individuals. Many lesions may clinically and radiologically present as masses in uncommon locations. Clinically, all differential diagnoses should be taken into consideration. Histopathological and microbiological examination is the keystone for identifying and managing these opportunistic fungal infections.

Ethical statement

The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to. Ethical approval was waivered as this is a review article with no original research data and no patient identification details.

Consent

No patient details were used for this research.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.