1651CHAPTER 210 Ebolavirus and Marburgvirus Infections

laboratory diagnosis is performed with patient samples inactivated in

mobile field “glove boxes” by on-site personnel trained in the safe use of

diagnostic assays adapted for field use in lower-containment settings.

Consequently, diagnostic samples should be collected and processed

with great caution and with the use of appropriate PPE and strict

barrier techniques. With adherence to established biosafety precautionary measures, samples should be sent in suitable transport media

to national or international WHO reference laboratories. Acute-phase

blood/serum is the preferred diagnostic specimen, because it usually

contains high titers of filovirions and filovirion-specific antibodies.

The current assay of choice for the diagnosis of filovirus infection

is reverse-transcription polymerase chain reaction (RT-PCR) targeting

one or more filovirus genes; a typical detection limit is 1000–5000

PFU/mL of serum, depending on the assay. Safe, rapid-turnaround,

and standardized in-field PCR-based approaches (e.g., the Cepheid

GeneXpert platform) are increasingly deployed during outbreaks.

Antigen-capture enzyme-linked immunosorbent assays (ELISAs) for

the detection of filovirus genomes and filovirion proteins are in development and may be useful as rapid point-of-care diagnostic tests in

the future. Direct IgM capture, direct IgG capture, or IgM-capture

ELISA is used for the detection of filovirion-targeting antibodies from

patients in the later stages of disease (i.e., those who have been able

to mount a detectable antibody response), including survivors. All of

these assays can be conducted on samples treated with guanidinium

isothiocyanate (for RT-PCR), cobalt-60 irradiation (for ELISA), or

other effective measures that render filoviruses noninfectious. Virus

isolation in cell culture and plaque assays for quantification or diagnostic confirmation are relatively easy but must be performed in maximalcontainment laboratories. If available, electron microscopic examination of inactivated samples or cultures can further support the diagnosis because filovirions have unique filamentous shapes (Fig. 210-2).

Formalin-fixed skin biopsy samples and possibly skin swabs can be

useful for safe postmortem diagnoses. In-field (or near in-field) rapid

genome sequencing was first deployed to inform classic epidemiology

in Western Africa during the 2013–2016 EVD outbreak and is likely

to become a mainstay of outbreak control and response, even in challenging settings.

TREATMENT

Filovirus Infections

Treatment of patients with suspected or confirmed filovirus infection should be administered by health care workers who are using

appropriate PPE and who have been well trained in the complex

care of the FVD patient under restrictions appropriate for infection

prevention and control (see “Control and Prevention,” below).

Treatment of FVD has historically been entirely supportive (and

even that therapy has been limited by resource constraints) as

the efficacy and safety of specific antiviral countermeasures had

not been rigorously studied outside of animal models of disease.

The 2013–2016 EVD outbreak in Western Africa highlighted the

need to conduct rigorous, feasible, and ethically acceptable clinical research in the outbreak setting. Building on challenges and

lessons learned in that setting, the first-of-its-kind Pamoja Tulinde

Maisha (PALM) randomized clinical trial was conducted during

the 2018–2020 EVD outbreak in the Democratic Republic of the

Congo and identified two therapeutics based on Ebola virus–

specific mAbs to improve survival rates. mAb114 (ansuvimab-zykl)

and REG-EB3 (atoltivimab, maftivimab, and odesivimab-ebgn)

were subsequently approved for the treatment of EVD in PCRconfirmed adults (including pregnant women) and children. Both

are administered via carefully monitored single dose infusions to be

initiated as soon as possible after diagnosis. In addition, the PALM

trial demonstrated the will and capacity to deliver more advanced

supportive and critical care accompanying specific therapeutics in

the outbreak setting. Although evidence is lacking, consensus treatment strategies include those generally recommended for severe

septicemia/sepsis/shock (Chap. 304) and should be applied with an

emphasis on standard approaches—i.e., monitoring and response to

respiratory dysfunction (e.g., oxygen), circulatory dysfunction (e.g.,

intravascular fluid repletion and vasopressor support), and CNS

dysfunction (e.g., ruling out of reversible causes, notably hypoglycemia)—as well as the detection and management of acute kidney

injury, hemorrhage, electrolyte derangements, and nutritional status and the prevention and treatment of secondary or co-infections.

Pain management and administration of antipyretics, antiemetics,

and antidiarrheal agents may be considered. Crucial strategies to

improve outcomes in the most severely ill FVD patients include

preventing organ dysfunction and providing safe and effective

temporary organ support (e.g., mechanical ventilation and renal

replacement) in order to expand the window for administration of

medical countermeasures and development of effective endogenous

immune responses.

■ COMPLICATIONS

Even in patients who have initial virologic or clinical improvement,

complications in the second or third weeks of illness may include

secondary infections, persistent renal dysfunction, neurologic compromise (e.g., Ebola-virus-related meningoencephalitis, cerebrovascular

events, seizures), cardiac dysfunction (e.g., myocarditis, pericarditis),

and venous thrombosis. Pregnancy and labor cause severe and frequently fatal complications in filovirus infections due to clotting factor

consumption, fetal loss, and/or severe blood loss during birth.

A number of sequelae have been self-reported or historically

described in survivors of FVD, including prolonged and sometimes

incapacitating arthralgia and myalgia, asthenia, alopecia, visual problems (including uveitis), hearing loss, memory loss and neurocognitive

dysfunction, mental health conditions (anxiety, depression, posttraumatic

stress disorder), and reproductive problems. Well-controlled observational studies of EVD survivors were first conducted in the aftermath of

the 2013–2016 West African EVD outbreak. Compared with their closecontact controls, Liberian EVD survivors had an increased incidence

of headache, arthralgia and myalgia, memory loss, fatigue, and urinary

frequency as well as abnormal results in abdominal, chest, neurologic,

musculoskeletal, and ocular exams. Of individual and public health

significance is the potential for persistence of filoviruses in immuneprivileged tissue compartments (and their associated fluids) in FVD

survivors, most commonly in the semen of male survivors (with the rare

but documented potential for sexual transmission and reignition of outbreaks) and rarely in the CNS (causing recrudescent meningoencephalitis), the eye (causing recrudescent uveitis), and the placenta (causing

transmission or placental insufficiency). True relapses that resemble

the course of primary FVD are extremely rare but have been described.

■ PROGNOSIS

Among the most severe of acute viral diseases in humans, FVD generally has a poor prognosis, although with much greater heterogeneity

than was historically assumed; i.e., the 90% case–fatality rate ascribed

for many decades to EVD has required revision. With an incomplete

evidence base, the outcome probably depends on factors that include the

particular filovirus causing the infection (Fig. 210-3), host factors (age,

immune status, unknown host genetic factors), virus exposure route and

dose, viral load, the presence and severity of organ dysfunction, and—

critically—the availability of filovirus-specific countermeasures and

requisite supportive care. Continued advances in the latter countermeasures and supportive care will likely result in improved survival

rates in EVD, but the long-term health and well-being of and survival

outcomes for FVD survivors are unknown. It is also uncertain how

increased access to filovirus-specific therapeutics and life-saving

support will impact filovirus persistence in survivors; short- and longterm surveillance will be necessary to avoid individual consequences

(e.g., relapse, recrudescent inflammatory syndromes) and public health

consequences (e.g., reignition of outbreak transmission chains in the

peri-outbreak or post-outbreak period).

Although remarkable progress has been made at the human EVD

bedside, the same cannot yet be said for disease caused by filoviruses

other than Ebola virus, either in acute disease or in convalescence.

1652 PART 5 Infectious Diseases

■ CONTROL AND PREVENTION

Prevention of filovirus exposure in nature is difficult because the

ecology of the viruses is not completely understood. To prevent marburgvirus infection, the most useful advice to people entering or living

in areas where Egyptian rousettes can be found is to avoid direct or

indirect contact with these animals. Prevention in nature is more difficult in the case of pathogenic ebolaviruses, largely because definite

reservoirs have not yet been pinpointed. EBOD outbreaks have been

associated not so much with bats as with hunting or consumption of

nonhuman primates. The mechanism of introduction of ebolaviruses

into nonhuman primate populations, if it occurs at all, is unclear. (Only

one ebolavirus, Taï Forest virus, has unequivocally been detected in

wild nonhuman primates.) Therefore, for now, to prevent ebolavirus

infection, the only advice that can be offered to travelers and locals

is to avoid contact with bushmeat, nonhuman primates, and bats. In

any setting, the early local involvement of medical anthropologists is

strongly advised to ensure proper communications and explanations

that are not perceived as threatening or patronizing.

Biomedical prevention strategies have historically been limited to

tried-and-true pillars of outbreak control, centering on the identification and isolation of cases, contact tracing, ensuring that health

care workers and other response personnel have appropriate training

and capacity in infection prevention and control, and preventing

high-risk transmission events. Measures aimed at preventing and

controlling infection, including relatively simple barrier nursing

techniques, vigilant use of appropriate PPE, quarantine, and contact

tracing, usually effectively terminate or at least contain FVD outbreaks. Isolation of infected people and their contacts and avoidance

of direct person-to-person contact without appropriate PPE usually

prevents further spread, as the virions are not transmitted through

droplets or aerosols under natural conditions. Typical protective gear

sufficient to prevent filovirus infections consists of disposable gloves,

gowns, and shoe covers and a face shield and/or goggles. If available,

N-95 or N-100 respirators may be used to further limit infection risk.

Positive-air-pressure respirators should be considered for high-risk

medical procedures, such as intubation or suctioning. Medical equipment used in the care of an infected patient, such as gloves or

syringes, should never be reused. Because filovirions are enveloped,

disinfection with detergents (e.g., 1% sodium deoxycholate, diethyl

ether, or phenolic compounds) is relatively straightforward. Bleach

solutions are recommended at 1:100 for surface disinfection and

1:10 for application to excreta or corpses. Whenever possible, potentially contaminated materials should be autoclaved, irradiated, or

destroyed.

Emerging from research conducted during the 2013–2016 EVD

outbreak in Western Africa, a vaccine based on a recombinant vesicular

stomatitis Indiana virus expressing Ebola virus glycoprotein (rVSVZEBOV/Ervebo) was the first filovirus vaccine approved for use in

the United States and the European Union. It is now widely deployed

in a reactive-ring vaccination strategy, targeting close contacts and

their contacts in EVD outbreak settings, and also used for vaccination

of health care workers. Development and evaluation of other vaccine

candidates continue toward complementary preventive approaches for

non-outbreak or peri-outbreak settings, with emphasis on the durability

of immune responses and increases in preventive breadth toward other

filoviruses.

Even in the absence of high-level evidence, expert consensus

informs the targeted use of Ebola-virus–specific vaccine or postexposure prophylaxis to prevent infection or disease in health care

workers considered to have had a high-risk Ebola virus exposure (e.g.,

after needlestick injury). For male survivors, abstinence from sexual

activity with a partner for at least 12 months after disappearance of

clinical signs is recommended, unless testing proves semen to be free

of filoviruses. (The use of condoms is generally recommended for all

sexual activities.) Reproductive tract and CNS tissues, including ocular

tissues and fluids from survivors, should be handled with appropriate precautions until demonstrated to be filovirus-free. The role of

filovirus-specific therapeutics in the prevention or treatment of filoviral persistence is unclear.

■ FURTHER READING

Cnops L et al: Essentials of filoviral load quantification. Lancet Infect

Dis 16:e134, 2016.

Dudas G et al: Virus genomes reveal factors that spread and sustained

the Ebola epidemic. Nature 544:309, 2017.

Hoenen T et al: Therapeutic strategies to target the Ebola virus life

cycle. Nat Rev Microbiol 17:593, 2019.

Jacob ST et al: Ebola virus disease. Nat Rev Dis Primers 6:13, 2020.

Kuhn JH et al: Filoviridae, in Fields Virology, Vol 1, 7th ed, PM

Howley et al (eds). Philadelphia, Wolters Kluwer/Lippincott Williams

& Wilkins, 2020, pp 449–503.

Matz KM et al: Ebola vaccine trials: Progress in vaccine safety and

immunogenicity. Expert Rev Vaccines 18:1229, 2019.

Mulangu S et al: A randomized, controlled trial of Ebola virus disease

therapeutics. N Engl J Med 381:2293, 2019.

Regules JA et al: A recombinant vesicular stomatitis virus Ebola vaccine. N Engl J Med 376:330, 2017.

Section 16 Fungal Infections

211

DEFINITION AND ETIOLOGY

In recent decades, human fungal infections have dramatically increased

worldwide as a result of the AIDS pandemic, the widespread use of

antibacterial agents, and the introduction of cytotoxic agents and

precision medicine biologics for the treatment of autoimmune and

neoplastic diseases and for use in patients undergoing solid organ

transplantation or hematopoietic stem cell transplantation. Moreover,

of great concern has been the recent rise in fungal infections caused

by drug-resistant species, such as azole- and/or echinocandin-resistant

Candida glabrata and Candida auris and azole-resistant Aspergillus

fumigatus. Among the ~5 million fungal species, only a few cause

human infections (Table 211-1).

Fungal infections are classified as mucocutaneous and deep organ

infections on the basis of anatomic location and as endemic and

opportunistic infections on the basis of epidemiology. Mucocutaneous

infections can cause serious morbidity but are rarely fatal. Deep organ

infections cause severe illness and often carry a high mortality rate. The

endemic mycoses are caused by fungi that are not part of the normal

human microbiota but are environmentally acquired. The opportunistic mycoses are caused by fungi (Candida, Aspergillus) that often are

components of the human microbiota and whose ubiquity in nature

renders them easily acquired by immunosuppressed hosts (Table 211-1).

Opportunistic fungi cause serious infections when impaired host

immune responses allow the organisms to transition from commensals to invasive pathogens. Endemic fungi typically cause self-limited

disease in immunocompetent hosts but severe illness in immunosuppressed patients.

Fungi are morphologically classified as yeast, mold, and dimorphic.

Yeasts are seen as round single cells or budding organisms. Molds grow

as filamentous forms called hyphae both at room temperature and in

tissue. Aspergillus, Mucorales, and dermatophytes that infect skin and

nails are mold fungi. Variations exist within this classification system.

For instance, when Candida infects tissue, both yeasts and filamentous forms (pseudohyphae) may be present (except in the cases of

Pathogenesis, Diagnosis,

and Treatment of

Fungal Infections

Michail S. Lionakis,

John E. Edwards Jr.

1653CHAPTER 211 Pathogenesis, Diagnosis, and Treatment of Fungal Infections

TABLE 211-1 Major Fungal Infections, Associated At-Risk Patient Populations, and Diagnostic Tests

INFECTION (MOST COMMON

FUNGAL GENERA AND SPECIES) CLINICAL SYNDROME(S) RISK FACTOR(S) DIAGNOSTIC TEST(S)

Mold (Filamentous) Fungi

Aspergillosis

(Aspergillus fumigatus, A. terreus,

A. flavus, A. niger, A. nidulansa

)

Pneumonia or disseminated

infection

ABPA

Keratitis

Neutropenia, glucocorticoids,

HSCT, post-influenza or

COVID-19, BTK inhibition

Atopic individuals

Direct inoculation

Culture of BAL fluid: low sensitivity, nonspecific (colonization,

contamination)

Histologic examination of tissueb

: acute-angle septate hyphae

Biomarkers: GM (BAL > serum); serum BDG (nonspecific)

Mucormycosis

(Rhizopus, Rhizomucor, Mucor,

Cunninghamella, and Lichtheimia

spp.)

Sinopulmonary infection

Rhinocerebral infection

Necrotizing skin infection

Neutropenia, HSCT

Diabetic ketoacidosis

Direct inoculation (e.g., tornado

victims)

Culture of BAL fluid or sinus tissue: very low sensitivity

Histologic examination of tissue: ribbon-like aseptate hyphae

Biomarkers: Negative

Fusariosis

(Fusarium solani, F. oxysporum)

Pneumonia or disseminated

infection

Keratitis

Neutropenia

Direct inoculation

Culture of tissue or blood: one of the few molds recovered

from blood

Histologic examination of tissue: acute-angle septate hyphae

Biomarkers: GM can be positive; BDG (nonspecific)

Scedosporiosis

(Scedosporium apiospermum)

Pneumonia or disseminated

infection

Neutropenia, glucocorticoids,

HSCT

Culture of BAL: low sensitivity, nonspecific (colonization,

contamination)

Histologic examination of tissue: acute-angle septate hyphae

Biomarkers: BDG can be positive

Phaeohyphomycosis

(Cladophialophora, Alternaria,

Phialophora, Rhinocladiella,

Exophiala, and Exserohilum spp.)

Sinopulmonary, CNS, or

disseminated infection

Skin infection

Allergic sinusitis

HSCT, neutropenia,

glucocorticoids, healthy

individuals (for CNS)

Direct inoculation

Atopic individuals

Culture of ordinarily sterile site

Histologic examination of tissue: cell walls may appear

dark brown or golden on H&E; Fontana-Masson may stain

fungal melanin

Dermatophytosis

(Trichophyton, Microsporum, and

Epidermophyton spp.)

Skin and nail infections Healthy individuals Culture or microscopic examination of scrapings or clippings:

chains of arthrospores (diagnostic)

Eumycetoma

(Madurella mycetomatis)

Skin and subcutaneous

infections

Healthy individuals Culture and macroscopic and histologic examination of grains

harvested from biopsy or aspiration

Yeast Fungi

Mucosal candidiasisc

(Candida albicans, C. glabrata)

Oropharyngeal or

esophageal candidiasis

Vulvovaginal candidiasis

AIDS, glucocorticoids

Antibiotic use

Culture of mucosal surfaces

Histologic examination of esophageal tissue or wet

preparation (10% KOH) of vaginal discharge: yeast and/or

pseudohyphae

Invasive candidiasisc

(C. albicans, C. glabrata,

C. parapsilosis, C. tropicalis, C. auris)

Candidemia

Disseminated infection

(spleen, liver, kidney, eye,

heart, CNS)

Critical illness (ICU)

Neutropenia, glucocorticoids

Culture of blood: low sensitivity

Histologic examination of tissue: yeast and/or pseudohyphae

Biomarkers/other tests: BDG (nonspecific); T2 magnetic

resonance in whole blood

Cryptococcosis

(Cryptococcus neoformans, C. gattii)

Pneumonia

Osteomyelitis

Meningoencephalitis

AIDS, glucocorticoids

Sarcoidosis

AIDS, AAbs to IFN-γ

or GM-CSF, BTK or JAK

inhibition

Culture of CSF, BAL fluid, blood

Microscopic examination of tissue or

CSF: encapsulated yeast (GMS, India ink, mucicarmine stain)

Biomarkers: Cryptococcus Ag (serum, CSF) is sensitive

and specific

Trichosporonosisd

(Trichosporon asahii, T. mucoides,

T. asteroides)

Superficial skin infection

(white piedra)

Disseminated infection

(skin, eye)

Healthy individuals

Neutropenia, glucocorticoids,

HSCT, SOT

Culture of tissue or blood

Histologic examination of tissue: yeasts, hyphae,

and arthroconidia

Biomarkers: BDG can be positive

Endemic Dimorphic Fungi

Histoplasmosis

(Histoplasma capsulatum, H. duboisii

[in Africa])

Self-limited pneumonia

Disseminated infection

(liver, bone, bone marrow)

Fibrosing mediastinitis

Healthy individuals

AIDS, SOT, glucocorticoids,

AAbs to IFN-γ, JAK or TNF-α

inhibition

Culture of blood or tissue: low sensitivity; weeks needed

for growth

Histologic examination of tissue: yeast with narrow-based

budding

Other tests: Histoplasma Ag (urine > serum > BAL); BDG can

be positive; serology (CF) can be useful in non-AIDS patients

Blastomycosis

(Blastomyces dermatitidis,

B. gilchristii)

Pneumonia

Disseminated infection

(skin, bone, mucosal

surfaces, genitourinary tract)

Healthy individuals

AIDS, glucocorticoids,

TNF-α inhibition

Culture of BAL or tissue: low sensitivity; weeks needed for

growth

Histologic examination of tissue: yeast with broad-based budding

Other tests: serology (CF, ID) has low sensitivity; Blastomyces Ag

test cross-reacts with other endemic fungi; GM can be positive

Coccidioidomycosis

(Coccidioides immitis, C. posadasii)

Self-limited pneumonia

Disseminated infection

(CNS, bone)

Healthy individuals

AIDS, glucocorticoids,

TNF-α inhibition

Culture is diagnostice

Histologic examination: spherules

Other tests: serology (CF, ID); Coccidioides Ag test can be

useful in CNS infection; BDG can be positive

(Continued)

1654 PART 5 Infectious Diseases

TABLE 211-1 Major Fungal Infections, Associated At-Risk Patient Populations, and Diagnostic Tests

INFECTION (MOST COMMON

FUNGAL GENERA AND SPECIES) CLINICAL SYNDROME(S) RISK FACTOR(S) DIAGNOSTIC TEST(S)

Paracoccidioidomycosis

(Paracoccidioides brasiliensis,

P. lutzii)

Pneumonia

Disseminated infection

(skin, bone, mucosal

surfaces)

Healthy individuals

AIDS, glucocorticoids

Culture of tissue: active disease; several weeks

needed for growth

Histologic examination of KOH preparations or tissue: yeast

with budding in steering-wheel pattern

Other tests: serology (ID, CF); Paracoccidioides Ag test

Sporotrichosis

(Sporothrix schenckii)

Lymphocutaneous infection

(ascending lymphangitis)

Disseminated infection

Direct inoculation

AIDS, glucocorticoids

Culture of tissue (diagnostic)

Histologic examination: cigar-shaped yeast, often with

surrounding asteroid body

Talaromycosis

(Talaromyces marneffei)

Pneumonia

Disseminated infection

(skin, bone, mucosal

surfaces)

Healthy individuals

AIDS, glucocorticoids,

AAbs to IFN-γ

Culture of tissue (diagnostic)

Histologic examination of tissue: yeasts with transverse septa

Biomarkers: GM is often positive

Adiaspiromycosis

(Emmonsia crescens, E. parva)

Pneumonia Occupational dust exposure Culture: nonculturable

Histologic examination: thick-walled adiaspore within

granuloma

Emergomycosis

(Emergomyces africanus,

E. pasteurianus)

Disseminated infection

(lungs, skin)

AIDS, SOT Culture of infected tissue

Histologic examination of tissue: yeast with narrowbased budding

Biomarkers: Histoplasma Ag can be positive

Chromoblastomycosis

(Fonsecaea pedrosoi,

F. monophora)

Skin and subcutaneous

tissue infections

Healthy individuals Culture of infected tissue

Histologic examination of scrapings (KOH) or tissue (GMS):

sclerotic bodies (pathognomonic)

Other Fungi

Pneumocystosisf

(Pneumocystis jirovecii)

Pneumonia

Disseminated infection (eye,

CNS, skin, gastrointestinal

tract)

AIDS, glucocorticoids,

BTK inhibition

AIDS

Culture: nonculturable

Histologic examination (gold standard): special (GMS, DiffQuik) or immunofluorescence stains

Biomarkers/other tests: BDG (nonspecific); BAL fluid PCR

(sensitive; can be positive in colonized individuals)

a

A. nidulans is seen almost exclusively in chronic granulomatous disease. b

GMS or PAS stains. c

Some Candida species form pseudohyphae. d

Trichosporon species are

yeast-like fungi that also generate septate hyphae and arthroconidia. e

Coccidioides is a laboratory hazard. It is important to notify the microbiology laboratory if this

infection is suspected. f

Pneumocystis is present in cyst and trophozoite forms.

Abbreviations: AAbs, autoantibodies; ABPA, allergic bronchopulmonary aspergillosis; Ag, antigen; BAL, bronchoalveolar lavage; BDG, β-D-glucan; BTK, Bruton’s tyrosine

kinase; CF, complement fixation; CNS, central nervous system; CSF, cerebrospinal fluid; GM, galactomannan; GM-CSF, granulocyte-macrophage colony-stimulating factor;

GMS, Gomori methenamine silver; H&E, hematoxylin and eosin; HSCT, hematopoietic stem cell transplantation; ICU, intensive care unit; ID, immunodiffusion; IFN-γ,

interferon γ; JAK, Janus kinase; KOH, potassium hydroxide; PAS, periodic acid–Schiff; PCR, polymerase chain reaction; SOT, solid organ transplantation; TNF-α, tumor

necrosis factor α.

C. glabrata and C. auris, which form only yeasts in tissue); in contrast,

Cryptococcus exists only in yeast form. Dimorphic is the term used to

describe fungi that grow as yeasts or large spherical structures in tissue

but as filamentous forms at room temperature in the environment

(Table 211-1).

Patients acquire deep organ infection by molds and endemic dimorphic fungi via inhalation. Skin dermatophytes are primarily environmentally acquired, but human-to-human transmission may also occur.

The commensal Candida invades deep tissues from sites of mucosal

colonization, usually in the gastrointestinal tract.

In this chapter, we outline general principles of immunology,

diagnosis, and treatment related to the most common human fungal

infections.

■ PATHOGENESIS

In the past decade, our understanding of fungal recognition pathways

and of tissue-specific innate and adaptive antifungal host defense mechanisms has markedly expanded. A major breakthrough has been the

discovery and functional characterization of the C-type lectin receptor/

spleen tyrosine kinase/caspase recruitment domain–containing protein 9

(CLR/SYK/CARD9) signaling pathway, which mediates fungal polysaccharide recognition and orchestrates proinflammatory mediator

production, leukocyte recruitment, inflammasome activation, and

Th17 cell differentiation upon fungal invasion. Human inherited

CARD9 deficiency causes severe mucocutaneous and invasive fungal

disease and is the only known primary immunodeficiency to feature

fungus-specific infection susceptibility without a predisposition to

other infections, autoimmunity, allergy, or cancer. Notably, CARD9-deficient patients develop infections by certain fungi in certain tissues,

including (1) chronic mucocutaneous candidiasis linked to defective

interleukin (IL) 17 responses; (2) infections of the central nervous

system (CNS) caused by Candida (but also by Aspergillus and phaeohyphomycetes) and linked to impaired microglial-neutrophilic responses;

and (3) deep dermatophytosis. Thus, the clinical use of SYK inhibitors

for autoimmunity and cancer may cause opportunistic fungal disease.

Human inherited deficiency of Toll-like receptor (TLR) signaling does

not lead to spontaneous fungal disease, yet polymorphisms in TLR

pathway molecules may increase the risk of fungal disease in critically

ill or immunosuppressed persons, and TLR stimulation may boost

protective CLR immunity, as has been shown with the TLR7 agonist

imiquimod in chromoblastomycosis.

The development of clinically relevant animal models of mycoses

and the phenotypic characterization of fungal infections that develop

in patients with primary immunodeficiencies and in recipients of

immune pathway–targeting biologics have led to the delineation of

fungus-, cell-, and tissue-specific requirements for antifungal host

defense (Fig. 211-1).

At the mucosal interface, IL-17-producing lymphoid cells play a

critical role in protection by driving epithelial cell production of antimicrobial peptides that restrict mucosal Candida invasion. Indeed,

AIDS patients are at risk for mucosal—but not invasive—candidiasis.

Concordantly, inherited deficiency of IL-17 signaling caused by mutations in IL17F, IL17RA, IL17RC, or TRAF3IP2 (encoding the IL-17

receptor adaptor ACT1) or pharmacologic inhibition of IL-17 signaling

(Continued)

1655CHAPTER 211 Pathogenesis, Diagnosis, and Treatment of Fungal Infections

IL6R

IL23R

CXCR2

CXCR2

NADPH

NADP+

O2

–

H2O2

SOD

e–

CXCL1

GM-CSF

IFN-λ

Aspergillus

conidia

Phagosome

Multilobed

nucleus

CXCL2

Blood

Lung

RORγT

γδ T cell

AAbs

AAbs

IL-22

IL-17RA IL-17RC IL-22R1 IL-10Rβ

IL-17F

IL-17A

IL-17A/IL-17F

ACT1

Epithelial cells

Macrophage

Th17 cell Neutrophil Th1 cell

Neutrophil

Nucleus

Nucleus

TYK2

TYK2

JAK2

JAK2

17RA IL-17RC IL-22R1 IL-10

ACT1

Ep c al itheliap thelial cells

Candida

yeast and

pseudohyphae

Nucleus

GM-CSF

IFN-γR1

IFNγ

IFNγ

JAK2

JAK1

JAK2

STAT4

STAT1

STAT1

STAT4

TYK2

IL12Rβ1 IL12Rβ2

AAbs

IFN-γR2 TNFα

IL-12

IL-12

Yeast

(Histoplasma,

Cryptococcus)

Phagosome

IFNγ

STAT3

STAT3

p22phox

gp91phox

p67phox p47phox

p40phox

FIGURE 211-1 Host defense against fungi. Left: Production of IL-17A, IL-17F, and IL-22 by Th17 cells, Tc17 cells, γ δ T cells, and innate lymphoid cells confers protection from

mucosal Candida invasion. STAT3 promotes Th17 differentiation via RORγt induction. IL-17A and IL-17F bind to IL-17RA and IL-17RC on epithelial cells and signal via ACT1

to produce antimicrobial peptides that inhibit fungal growth. IL-22 binds to its receptor on epithelial cells and activates STAT3 to mediate epithelial proliferation and repair.

Middle: Activation of CXCR2+

 neutrophils recruited from blood in the Aspergillus-infected lung enables assembly of the five subunits of NADPH oxidase and superoxide

generation that promotes fungal killing. Production of reactive oxygen species by neutrophils is facilitated by recruited monocyte-derived and plasmacytoid dendritic cells

via type I and type III IFNs and GM-CSF. Right: The interaction of Th1 cells with macrophages is protective against intramacrophagic endemic dimorphic fungi, Pneumocystis,

and Cryptococcus. Upon fungal uptake, macrophages produce IL-12 that binds to its receptor on T cells and activates STAT4, with consequent release of IFN-γ. IFN-γ binds to

its receptor on macrophages and activates STAT1, thereby enabling fungal killing. TNF-α and GM-CSF are also critical for macrophage activation. AAbs, autoantibodies; IL,

interleukin; IFN, interferon; JAK, Janus kinase; GM-CSF, granulocyte-macrophage colony-stimulating factor; NADPH, nicotinamide adenine dinucleotide phosphate; RORγt;

RAR-related orphan receptor γ; SOD, superoxide dismutase; STAT, signal transducer and activator of transcription; TNF, tumor necrosis factor; TYK2, tyrosine kinase 2.

by biologics that target IL-12p40, IL-23p19, IL-17A, IL-17A/IL-17F, or

IL-17RA causes mucosal—but not invasive—candidiasis. Other conditions that underlie a predisposition to chronic mucocutaneous candidiasis include primary immunodeficiencies due to mutations in STAT3,

STAT1, DOCK8, JNK1, IRF8, RORC, and CARD9, all of which impair

Th17 cells, as well as autoimmune polyendocrinopathy–candidiasis–

ectodermal dystrophy (APECED) and thymoma, which feature autoantibodies to IL-17A, IL-17F, and IL-22. Of note, vaginal candidiasis

(unlike oropharyngeal and esophageal candidiasis) develops in the

setting of antibiotic treatment, not AIDS; this observation underscores

the role of the microbiota in fungal control at the vaginal—but not the

oral—mucosa.

On the other hand, neutrophils—but not lymphocytes—are critical

for control of invasive infections caused by Aspergillus (and other

inhaled molds) and Candida (Fig. 211-1). Indeed, patients with

chemotherapy-induced neutropenia and patients undergoing allogeneic hematopoietic stem cell transplantation are at risk for invasive

aspergillosis and candidiasis. Both oxidative and nonoxidative burst–

dependent effector mechanisms are operational within neutrophils

for fungal killing. Inherited deficiency in neutrophil superoxide

generation due to mutations in the five subunits of the nicotinamide

adenine dinucleotide phosphate (NADPH) oxidase complex causes

chronic granulomatous disease, a prototypic primary immunodeficiency that carries a lifetime risk for invasive aspergillosis of ~40–50%;

infrequently (i.e., in <5% of cases), chronic granulomatous disease

predisposes to invasive candidiasis. The unexpected development of

invasive mold infections in recipients of Bruton’s tyrosine kinase (BTK)

inhibitors has recently uncovered the critical role of BTK in promoting

myeloid phagocyte-dependent antifungal effector functions.

Moreover, host defenses against fungi that reside within macrophages, such as Cryptococcus, Pneumocystis, and endemic dimorphic

fungi, depend on the interplay of interferon γ (IFN-γ)–producing

lymphoid cells and IL-12-producing macrophages that enable intramacrophagic fungal killing (Fig. 211-1). Indeed, AIDS patients and

those receiving glucocorticoids, which affect lymphocytes and macrophages both quantitatively and qualitatively, are at risk for severe

infections by these fungi. Accordingly, inherited impairment of the

IL-12/IFN-γ signaling axis caused by mutations in IL12RB1, IFNGR1,

IFNGR2, STAT1, IRF8, or GATA2 underlies susceptibility to severe

infection by intramacrophagic fungi (and other intramacrophagic

pathogens, such as mycobacteria and salmonellae). In addition, the

IFN-γ-targeting monoclonal antibody emapalumab, JAK inhibitors

1656 PART 5 Infectious Diseases

that block IFN-γ-dependent cellular responses, and autoantibodies

to IFN-γ predispose to infection with intramacrophagic fungi, as do

biologics targeting tumor necrosis factor α (TNF-α) and autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF).

The latter predisposing factors reveal the central role of these two

Th1-associated cytokines—TNF-α and GM-CSF—in macrophage

activation.

Taken together, these observations show that the cellular and

molecular factors that drive protective antifungal immune responses

vary greatly with the anatomic site of the infection, the offending

fungus, and the patient population (Table 211-1). The growing body

of data on human immunologic responses to fungi holds promise in

informing precision medicine strategies for risk assessment, prophylaxis, immunotherapy, and vaccination of vulnerable patients.

■ DIAGNOSIS

The diagnostic modalities used for various fungal infections are

outlined in Table 211-1 and are detailed in the chapters on specific

mycoses that follow in this section. Definitive diagnosis of a fungal

infection requires histopathologic identification of the fungus invading

tissue with parallel culture of the fungus from the specimen. Certain

fungi have distinctive morphologic features that facilitate diagnosis

(Table 211-1). The stains most often used to identify fungi are periodic acid–Schiff and Gomori methenamine silver. Candida, unlike

other fungi, is visible on gram-stained tissue smears. Hematoxylin and

eosin stains define accompanying histologic features of fungal disease

(granuloma formation, angioinvasion, necrosis) but are insufficient to

reliably identify fungi in tissue. A positive India ink stain of cerebrospinal fluid (CSF) is diagnostic for cryptococcosis. Most laboratories use

calcofluor white staining coupled with fluorescence microscopy to

identify fungi in fluid specimens. A positive fungal culture of blood or

tissue may signify either a patient’s colonization or lab contamination

instead of true infection, with the most likely scenario depending on

the fungus and the anatomic site. In blood, Candida can be detected

with any of the widely used automated blood culture systems, but

the lysis-centrifugation technique increases the sensitivity of blood

cultures for both Candida and other less common fungi (e.g., Histoplasma). Matrix-assisted laser desorption/ionization time-of-flight

mass spectrometry (MALDI-TOF-MS) is now used extensively for

detection and speciation of fungi recovered from culture.

The several available fungal-antigen and serologic tests vary in sensitivity and specificity. The most reliable of these tests are the antibody

to Coccidioides, Histoplasma antigen, and cryptococcal polysaccharide

antigen. Serologic tests are also available for other endemic dimorphic

fungi (Table 211-1). The galactomannan test—especially in the bronchoalveolar lavage fluid—is useful for the diagnosis of aspergillosis;

however, false-negative results are common, particularly in patients

receiving antifungal prophylaxis, and false-positive results may occur

with other fungal infections. The β-glucan test has a high negative

predictive value for invasive candidiasis but lacks specificity. T2

magnetic resonance is now approved by the U.S. Food and Drug

Administration (FDA) for detection of Candida in blood. Several

polymerase chain reaction and nucleic acid hybridization assays exist

for fungal detection but are not standardized and are not widely used

in the clinic.

■ ANTIFUNGAL DRUGS

This section provides a brief overview of available agents for the treatment of fungal infections. Drug regimens and schedules are detailed

in the chapters on specific mycoses that follow in this section. Since

fungal organisms, like human cells, are eukaryotic, the identification

of drugs that selectively kill or inhibit fungi but that are not toxic to

human cells poses challenges. Indeed, far fewer antifungal than antibacterial agents have been introduced into clinical medicine.

Early initiation of appropriate antifungal therapy is a critical determinant of favorable outcome, as has been shown for candidemia,

aspergillosis, and mucormycosis. In addition, source control of the

infection is important—e.g., with removal of the central venous catheter in candidemia, drainage of abdominal abscesses in intraabdominal

candidiasis, and surgical debridement of sinus tissue in mucormycosis. Moreover, an essential factor in a favorable prognosis in

patients with opportunistic mycoses is the achievement of immune

reconstitution—e.g., with neutrophil recovery, tapering of glucocorticoids or other immunosuppressive drugs, or initiation of combination

antiretroviral therapy in AIDS.

■ AMPHOTERICIN B

The advent of amphotericin B (AmB) in the 1950s revolutionized the

treatment of deep-seated mycoses. Before the availability of AmB,

cryptococcal meningitis and other disseminated fungal infections were

nearly always fatal. AmB remains the broadest-spectrum antifungal

agent. Its fungicidal mechanism of action involves direct binding to

ergosterol and intercalation into the fungal cell membrane, which leads

to osmotic cell lysis. AmB remains the preferred antifungal agent for

the treatment of mucormycosis and fusariosis and for induction therapy for cryptococcal meningitis and disseminated infections caused

by endemic dimorphic fungi. However, AmB has several limitations,

including lack of an oral formulation and significant toxicity, primarily

renal and infusion-related (fever, chills, thrombosis). The introduction of lipid AmB formulations has ameliorated these toxicities, and

the lipid formulations have largely replaced the original deoxycholate

formulation in resource-rich settings. In developing countries, AmB

deoxycholate is still widely used because of the high cost of the lipid

formulations. The two lipid formulations commonly used in the clinic

are liposomal AmB and AmB lipid complex, which exhibit comparable

efficacy, toxicity, and tissue penetration profiles.

■ AZOLES

Azoles offer important advantages over AmB, such as the availability of

oral and IV formulations and a lack of renal toxicity. The mechanism of

action of azoles involves inhibition of lanosterol 14α-demethylase and

ergosterol synthesis in the fungal cell membrane, with a consequent

accumulation of toxic sterol intermediates and growth arrest. Unlike

AmB, azoles are considered fungistatic.

Fluconazole Fluconazole plays an important role in the treatment

of several fungal infections. Its major advantages are the availability of

oral and IV formulations, a long half-life, penetration into most body

fluids (ocular fluid, CSF, urine), and minimal toxicity. This drug rarely

causes liver toxicity; high doses may result in alopecia, dry mouth,

and a metallic taste. Notably, the administration of even low doses of

fluconazole to pregnant women for the treatment of vaginal candidiasis

was recently linked to miscarriage and stillbirth. Fluconazole has no

activity against molds and most endemic dimorphic fungi and is less

active than the newer azoles against C. glabrata and C. krusei.

Fluconazole is the preferred agent for the treatment of coccidioidal

meningitis, although relapses may occur despite therapy. Fluconazole

is also used as consolidation and maintenance therapy for cryptococcal

meningitis and for the treatment of mucosal candidiasis. It is used for

treating candidemia in patients who are not critically ill or immunosuppressed; in these patients, fluconazole was found to be as efficacious

as AmB. Because of increasing rates of azole-resistant Candida strains,

many clinicians opt to initiate therapy with an echinocandin, which is

replaced by fluconazole once a susceptible Candida species is recovered. Fluconazole is effective as prophylaxis in recipients of high-risk

liver and allogeneic bone marrow transplants, although many centers

now use posaconazole in neutropenic patients, given its added spectrum against molds. Fluconazole prophylaxis in leukemic patients, in

AIDS patients with low CD4+ T-cell counts, and in patients on surgical

intensive care units is controversial.

Itraconazole Itraconazole is available in oral (capsule, suspension)

and IV formulations and has broader antifungal activity—i.e., against

molds and endemic dimorphic fungi. Itraconazole is the drug of choice

for mild to moderate histoplasmosis and blastomycosis and has also

been used to treat chronic coccidioidomycosis, phaeohyphomycosis,

sporotrichosis, and mucocutaneous mycoses such as oropharyngeal candidiasis, tinea versicolor, tinea capitis, and onychomycosis.

Although it is approved by the FDA for use in febrile neutropenic

1657CHAPTER 211 Pathogenesis, Diagnosis, and Treatment of Fungal Infections

patients, most centers now use newer azoles in such patients. Disadvantages of itraconazole include its poor CSF penetration, the use of

cyclodextrin in its oral suspension and IV formulation, and its variable

level of absorption in the capsule form, which requires monitoring of

blood levels in patients receiving capsules for disseminated mycoses.

Itraconazole is a potent CYP3A4 inhibitor; this characteristic leads to

significant drug interactions. The drug causes hepatotoxicity and cardiac toxicity that may manifest as congestive heart failure.

Voriconazole Voriconazole is also available in oral and IV formulations, has far broader antifungal activity than fluconazole (including C. glabrata, C. krusei, Aspergillus, Scedosporium, and endemic

dimorphic fungi—but not Mucorales), and penetrates into most body

fluids (ocular fluid, CSF). It is the preferred agent for the treatment of

aspergillosis and also has been used to treat scedosporiosis and as stepdown (but not primary) therapy for coccidioidomycosis, blastomycosis, and histoplasmosis. Voriconazole is considerably more expensive

than fluconazole, and, as with itraconazole, its use is associated with

numerous interactions with drugs typically used in patients at risk for

fungal infections. Hepatotoxicity, visual disturbances, and skin rashes

(including photosensitivity) are relatively common, and long-term use

requires skin cancer surveillance. A unique toxicity of voriconazole

among azoles is fluorosis-associated periostitis. It is crucial to monitor

drug levels because (1) voriconazole is metabolized in the liver by

CYP2C9, CYP3A4, and CYP2C19; and (2) human genetic variation

in CYP2C19 activity exists and can lead to significant interpatient

variability in drug levels. Dosages should be reduced in patients with

hepatic, but not renal, failure; however, because the IV formulation is

prepared in cyclodextrin, it should be given with caution to patients

with severe renal failure.

Posaconazole Posaconazole has broader activity than voriconazole, including activity against Mucorales. Both oral (suspension,

tablet) and IV formulations are available. Posaconazole is approved by

the FDA for antifungal prophylaxis in neutropenic leukemic patients

and allogeneic hematopoietic stem cell transplant recipients as well as

for treatment of oropharyngeal candidiasis, including infections refractory to fluconazole or itraconazole. Posaconazole has been reported to

be effective salvage therapy for aspergillosis, mucormycosis, fusariosis,

cryptococcosis, histoplasmosis, and coccidioidomycosis, although

controlled clinical trials are lacking. The tablet formulation is not hampered by the suboptimal absorption that occurs with the suspension;

the tablet also results in higher and more reliable blood levels of the

drug. Posaconazole is less hepatotoxic than voriconazole and does not

cause the skin, visual, or bone toxicity that occurs with voriconazole.

However, the use of posaconazole is linked to significant P450-related

drug interactions.

Isavuconazole The newest azole, isavuconazole, is available in oral

and IV formulations and has broad antifungal activity similar to that of

posaconazole. Isavuconazole is approved by the FDA for treatment of

aspergillosis (on the basis of a randomized controlled trial that found

it noninferior to voriconazole) and mucormycosis (on the basis of an

open-label, noncomparative trial of 37 patients). Future experience

will definitively determine its place in the antifungal armamentarium.

Isavuconazole appears to be less hepatotoxic than voriconazole; it does

not cause skin or visual toxicity, and it causes fewer P450-associated

drug interactions than voriconazole.

■ ECHINOCANDINS

The echinocandins include the FDA-approved drugs caspofungin,

anidulafungin, and micafungin, which are available solely as an IV

formulation and inhibit β-1,3-glucan synthase, an enzyme that is

crucial for fungal cell-wall synthesis but is not a constituent of human

cells. The three echinocandins have comparable efficacy, toxicity, and

tissue penetration profiles; are fungicidal for Candida and fungistatic

for Aspergillus; and have no activity against other molds, Cryptococcus, or endemic dimorphic fungi. Their most common use to date

is in candidal infections. These drugs offer three major advantages:

minimal toxicity, minimal drug interactions, and activity against all

Candida species. The minimal inhibitory concentrations (MICs) of

echinocandins are higher against Candida parapsilosis than against

other Candida species, but the higher MICs do not translate into less

clinical efficacy against this species.

In controlled trials, caspofungin was as efficacious as AmB against

candidemia and invasive candidiasis and as efficacious as fluconazole

against candidal esophagitis. Caspofungin has also been efficacious

as salvage therapy for aspergillosis. Anidulafungin is approved by the

FDA as therapy for candidemia in nonneutropenic patients and for

Candida esophagitis, abdominal infection, and peritonitis. In controlled trials, anidulafungin was noninferior and possibly superior to

fluconazole against candidemia and invasive candidiasis and was as

efficacious as fluconazole against candidal esophagitis. Micafungin is

approved by the FDA for the treatment of candidal esophagitis and

candidemia and for antifungal prophylaxis in hematopoietic stem

cell transplantation. Moreover, micafungin yielded favorable results

when used for the treatment of invasive aspergillosis and candidiasis

in open-label trials.

■ FLUCYTOSINE (5-FLUOROCYTOSINE)

Flucytosine use has diminished as newer antifungal drugs have been

developed. Its mechanism of action involves intrafungal conversion

to 5-fluorouracil, which inhibits fungal DNA synthesis. The use of

flucytosine in combination with AmB as induction therapy for cryptococcal meningitis is based on the drugs’ synergistic interaction and

favorable flucytosine CSF penetration that promotes a rapid decline

of the cryptococcal burden in the CSF. Flucytosine is also used in

combination with AmB for the treatment of candidal meningitis and

endocarditis, although comparative trials with AmB monotherapy are

lacking. Flucytosine monotherapy is not recommended as it is associated with the development of resistance. Flucytosine can cause bone

marrow suppression and liver toxicity, which are intensified when the

drug is used with AmB.

■ GRISEOFULVIN AND TERBINAFINE

Historically, griseofulvin was used for ringworm infection. Terbinafine,

which inhibits squalene epoxidase and ergosterol synthesis, is now

used for onychomycosis and ringworm infection and is as effective as

itraconazole and more effective than griseofulvin in both conditions.

Although active against other fungi, terbinafine penetrates poorly into

tissues beyond the skin and nails and therefore is not preferred for systemic mycoses. Terbinafine carries a risk for hepatotoxicity.

■ TOPICAL ANTIFUNGAL AGENTS

A detailed discussion of topical agents for mucocutaneous mycoses is

beyond the scope of this chapter; the reader is referred to Chap. 219

and the dermatology literature. Azoles such as clotrimazole, miconazole, and ketoconazole are often used topically to treat common

cutaneous mycoses as well as oropharyngeal and vaginal candidiasis.

In vaginal candidiasis, oral fluconazole given once has the advantage of

not requiring repeated intravaginal application. The polyenes nystatin

and AmB have also been used topically for oropharyngeal and vaginal

candidiasis. Agents from other classes that are used to treat these conditions include ciclopirox, haloprogin, terbinafine, naftifine, tolnaftate,

and undecylenic acid.

■ FURTHER READING

Bennett JE: Introduction to mycoses, in Mandell, Douglas, and Bennett’s

Principles and Practice of Infectious Diseases, 9th ed, JE Bennett et al

(eds). Philadelphia, Elsevier Saunders, 2020, pp 3082–3086.

Lionakis MS, Levitz SM: Host control of fungal infections: Lessons

from basic studies and human cohorts. Annu Rev Immunol 36:157,

2018.

Pappas PG et al: Clinical mycology today: A synopsis of the mycoses

study group education and research consortium (MSGERC) second

biennial meeting, September 27–30, 2018, Big Sky, Montana, a proposed global research agenda. Med Mycol 58:569, 2020.