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INFECTIOUS DISEASE BACTERIOLOGY IMMUNOLOGY MYCOLOGY PARASITOLOGY VIROLOGY

PORTUGUESE

 

MYCOLOGY - CHAPTER   THREE  

YEASTS  

Dr Errol Reiss Ph.D.
Research Microbiologist (retired)
Centers for Disease Control and Prevention
Atlanta, Georgia, USA

Dr Errol Reiss' contribution to this Section is written in his private capacity. No official support or endorsement by the Centers for Disease Control and Prevention, Department of Health and Human Services is intended nor should be inferred.

Dr Art DiSalvo
Emeritus  Director, Nevada State Laboratory
Emeritus Director of Laboratories, South Carolina Department of Health and Environmental Control

 

 

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Figure 1
Brewer's yeast (also known as Baker's yeast) with bud and bud scars (Saccharomyces cerevisiae).
 © Dennis Kunkel Microscopy, Inc.  Used with permission
This chapter covers Candida and Cryptococcus species. Information about other medically important yeasts and Pneumocystis is addressed in the General Reference (Reiss et al., 2012.)

Yeast, defined: a unicellular fungus that reproduces by budding or fission (fig 1.)

Figure 2
Candida albicans - yeast and hyphae stages. A yeast-like fungus commonly occuring on human skin, in the upper respiratory, alimentary & female genital tracts. This fungus has a dimorphic life cycle with yeast and hyphal stages. The yeast produces hyphae (strands) and pseudohyphae. The pseudohyphae can give rise to yeast cells by apical or lateral budding. Causes candidiasis which includes thrush (an infection of the mouth and vagina) and vulvo-vaginitis.
© Dennis Kunkel Microscopy, Inc.  Used with permission

CANDIDIASIS

The genus Candida is comprised of more than 314 genetically diverse yeast species but fewer than 20 are associated with the disease, candidiasis (Daniel et al. 2014.) The five most common causes are: Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei  (fig 2.). Candida albicans (fig 3, fig 4) is an endogenous yeast-like fungus, the most common species in human infections. Found worldwide, it is carried by 30 to 50% of normal humans in the mouth, gut, and vagina. C. albicans is present as a commensal but may convert to a pathogen depending on the immune and/or endocrine status of the human host (See Table1: Risk Factors).

Morphology

Candida albicans is “polymorphic” growing as budding yeast cells, pseudohyphae (a transitional form), and true hyphae. Morphology is under genetic control, and to maintain the yeast form C. albicans needs a transcription repressor, NRg1 (Lu et al., 2014.) [pop-up here]

More Information


The yeast form is 10-12 µm diam., Gram-positive, and grows overnight on most bacterial and fungal media. C. albicans produces germ tubes (fig 5, fig 6 ) In the laboratory, pseudohyphae develop terminal round chlamydospores. The production of germ tubes and chlamydospores are important characters to identify C. albicans in the lab. Infected tissue shows evidence of a mix of yeast forms, pseudohyphae, and true hyphae (fig 7, fig 8, fig 9, fig 10 ). Another medically important species, Candida glabrata grows only in the yeast form.

Clinical scientists, infectious disease physicians and research microbiologists should be aware of 2 important findings affecting naming of fungi, including yeasts, and the discovery of their “genetic” or “sister” species:

  • The “One fungus, One name” rule. Rules of fungal taxonomy were revised in 2011 at the 18th International Botanical Congress in Australia. The new “International Code of Nomenclature for Algae, Fungi and Plants” states, as of 2013:
    • Separate names for asexual forms (anamorphs) will end.
    • The goal is to use only a single name for a species. The appropriate single name will be determined according to scientific accuracy and also to established medical usage so as to avoid or minimize confusion by clinicians.
  • The discovery of previously unrecognized “genetic” or “sister” species by molecular phylogenetic methods, (DNA fingerprinting and DNA sequence analysis.) This topic is covered with respect to yeasts, in Merseguel et al., 2015, in which both the identity of genetic species and the acceptable names for individual species are discussed, such as the discovery the newly emerging Candida species, C. auris (see 2017 update, below.) An example is Candida dubliniensis, a genetic “sister” species related to Candida albicans. The identity of C. dubliniensis eluded mycologists until relevant molecular methods became available.



Candidiasis

Candidiasis may be hospital acquired or acquired in the community (as shown in Table 1.) Infections with Candida usually occur when a patient has disruptions in innate or T-cell immunity, normal microbiota, or normal physiology. Immunosuppressive drugs for cancer chemotherapy or for maintenance of an organ transplant can result in persistent granulocytopenia. Granulocytes are an important aspect of host defense against invasive candidiasis.

Prolonged antibiotic therapy destroys the balance of normal microbiota in the intestine allowing overgrowth of endogenous Candida. Immune-normal individuals are at risk for candidiasis after invasive procedures such as cardiac and intra-abdominal surgery and the use of central venous catheters which disrupt normal anatomic barriers.

The establishment of infection with Candida species appears to be a property of the host - not the organism. As the host becomes more debilitated, the risk of invasive disease increases. Humans with persistent or profound neutropenia are at risk for invasive multisystem candidiasis, whereas patients whose immunodeficit is in the T-cell limb of the immune response are more likely to develop mucosal candidiasis.


Clinical Forms

  • Oral candidiasis (oropharyngeal candidiasis) or “thrush” is seen as white patches on the mucosae of the mouth including the tongue and throat (fig 12 .) The affected area may be inflamed causing difficulty swallowing. Cracking and inflammation may occur around the mouth, referred to as oral cheilitis. Another clinical form is denture stomatitis. Oral candidiasis may spread to the esophagus (esophagitis.) Although many people harbor Candida species, oral candidiasis is typically found in patients whose T-cell mediated immunity is depressed including people infected with HIV and in 9 to 31% of persons living with AIDS. Other risk factors for oral candidiasis are diabetes, wearing dentures, or the use of systemic corticosteroids. CDC estimates that between 5 and 7% of neonates develop oral candidiasis. Untreated oral candidiasis in an immunosuppressed patient can lead to serious invasive disease.
  • Genital or vulvovaginal candidiasis (commonly known as “yeast infection”). Symptoms are genital itching, a burning sensation and vaginal discharge in women. Less frequently men may have symptoms on the penis: an itching rash. Most women will have at least once episode of vulvovaginal candidiasis. Women are more at risk of the infection if they are pregnant, diabetic, use broad spectrum antibiotics, or systemic corticosteroids.
  • Invasive candidiasis and candidemia is a serious disease initiated when Candida, normally on the skin or on the mucosal lining of the gastrointestinal tract, enters the bloodstream (candidemia) and then can disseminate to other organs. Symptoms include fever and chills that do not respond to antibiotics.
     

Table 1: Risk factors for Invasive candidiasis

Nonneutropenic Patients Anatomical disruption: cardiothoracic or intra-abdominal surgery, open wounds, burns
Intensive care unit stay
Intravascular catheters
Total parenteral nutrition
Drug treatment: antibiotics
 
Neutropenic Cancer Patients Mucosal ulceration (gastrointestinal tract)
Intravascular catheters
Cytotoxic and immunosuppressive drugs, glucocorticoids*

* More Information

Community factors Hemodialysis
Diabetes
 

Candidemia is the most common fungal bloodstream infection in hospitalized patients. In critically ill patients crude mortality due to invasive candidiasis or candidemia is in the range of 40%- 60%, reflecting in part the contribution of the underlying condition. In a French study, the case-fatality ratio was 45.9%. The attributable mortality of invasive candidiasis was reported to be between 5% and 71%. This wide range reflects how difficult it is to assess whether critically ill patients died either with or from fungal infections (Delaloye and Calandra, 2014.)

Other clinical forms of candidiasis

The following forms of candidiasis are complications of hematogenous spread:

  • Bone and joint

  • Chronic hepatosplenic

  • Endocarditis as a result of hematogenous seeding of a prosthetic or damaged heart valve, myocardium, or pericardial space.

  • Endophthalmitis

  • Renal

  • Skin lesions

Chronic mucocutaneous. A rare genetic deficiency in T-cell responsiveness to Candida.

CNS infection may occur as a result of hematogenous spread, or neurosurgery and ventriculoperitoneal shunts

Peritonitis in the setting of chronic ambulatory peritoneal dialysis, GI surgery.

Urinary tract infections- involving an indwelling bladder catheter or diabetes.


Therapy

  • Oral candidiasis. Treatment for the oral form in adults consists of clotrimazole troches or miconazole mucoadhesive buccal tablets. Oral fluconazole is indicated for moderate or severe forms. Itraconazole or voriconazole are reserved for fluconazole refractory disease. HIV-positive persons with oral candidiasis should receive effective antiretroviral therapy. Oropharyngeal candidiasis in these patients is treated with oral fluconazole suspension or tablets, itraconazole solution, or topical treatment with clotrimazole lozenges or miconazole mucoadhesive tablets. Esophageal candidiasis requires systemic therapy with oral fluconazole. Itraconazole solution or oral or IV voriconazole are recommended for fluconazole-refractory esophagitis.

  • Infants with thrush benefit from oral miconazole gel or nystatin oral suspension. Therapy for immunocompromised children consists of oral fluconazole.

  • Vulvovaginal Candidiasis. Topical antifungal agents are recommended for uncomplicated vulvovaginitis, or a single 150 mg oral dose of fluconazole.

  • Candidemia and Invasive Candidiasis. Central venous catheters should be removed as early as possible in the course of candidemia when the source is presumed to be the catheter, and this decision should be made according to the clinical status of the patient. Drug therapy alone will not suppress the fungus if the foreign body remains in the host.
    Non-neutropenic Patients. An echinocandin (caspofungin, micafungin, or anidulafungin) is recommended as initial therapy. Fluconazole is an acceptable alternative in patients who are not critically ill and who are considered unlikely to have a fluconazole-resistant Candida species. Empiric therapy for suspected invasive candidiasis in nonneutropenic patients in the ICU, or prophylaxis for prevention of same in the ICU are discussed by Pappas et al., 2016.

  • Neutropenic Patients. An echinocandin is recommended as initial therapy. Lipid formulation of Amphtotericin B is an effective alternative but is less attractive because of potential toxicity. In neutropenic patients sources of candidiasis other than a central venous catheter predominate, such as the gastrointestinal tract. Catheter removal should therefore be considered on an individual basis.

  • Among patients with suspected or proven azole- or echinocandin-resistant Candida infections therapy with the lipid form of Amphotericin B is recommended. Voriconazole is recommended as step-down therapy for C. krusei infections.

  • Transition from initial therapy with an echinocandin or with amphotericin B to fluconazole depends on clinical stability of the patient, susceptibility tests to show the isolate is susceptible to fluconazole, and when repeat blood cultures remain negative.

  • Dosage regimens, duration of therapy, and the role of dilated ophthalmologic exams, follow-up blood cultures, G-CSF mobilized granulocyte infusions, or therapy of other forms of candidiasis including neonatal forms of this disease can be found in Pappas et al., 2016.


Laboratory Diagnosis

Once isolated from clinical specimens the different Candida species are identified by classical methods: morphology and biochemical reactions. Diagnosis of candidemia is by culture of the organism from blood. Specimens sent to the lab depend on the clinical presentation and may include: blood, vaginal discharge, urine, feces, nail clippings or material from cutaneous or mucocutaneous lesions. Candida species (other than C. albicans) account for an increasing number of nosocomial infections. Speciation is important because there is significant antifungal resistance among the different species. Blood culture methods, MALDI-TOF, and multiplex PCR automated instruments to diagnose candidemia are discussed in the Introduction Sub-Section of Medical Mycology. These advances have improved the time to an identification so as to start antifungal therapy earlier, increasing chances for a successful result.


2017 Updates

Emerging pathogenic yeast - Candida auris and a note about Candida krusei

In 2009, a new Candida species was named Candida auris, after its isolation from the ear of a woman in Japan. Later, review of culture collections found an earlier strain of this yeast dated to 1996 in South Korea (CDC Candida auris website.) This emerging yeast is often multidrug resistant and has caused outbreaks in healthcare settings. Click on pop-up to see taxonomic status of C. auris.

 

Epidemiology

By 2016, C. auris was identified in several countries in various regions. Single cases of C. auris were reported from Germany, Japan, Kuwait, and Norway. Multiple cases were reported from Canada, Colombia, India, Israel, Kenya, Oman, Pakistan, Panama, South Korea, South Africa, Spain, the United Kingdom, the United States (primarily from New York City metropolitan area and New Jersey) and Venezuela. In some countries, transmission of C. auris occurred in more than one hospital. U.S. cases of C. auris occurred in patients who had recent stays in healthcare facilities in India, Pakistan, South Africa and Venezuela (CDC.)

C. auris has gone, in a few years, from an unknown pathogen to cause 40% of invasive candidiasis in some facilities. Hospital records in the U.S.A. indicated very few cases before 2016, suggesting that C. auris may still be new in the United States (but see below on difficulty in making an accurate identification.) Health departments and healthcare facilities are alerted to report cases, particularly among patients with extensive healthcare exposure, including in long-term care facilities.

Results of genome sequencing of C. auris isolates showed no single strain of C. auris was spreading world-wide. Instead, isolates fall into 4 clades along geographic lines. Possibly, distinct strains emerged nearly simultaneously in 4 different world regions. The cause of such a development remains speculative, including the possibility that environmental change may have led to its emergence, followed by transmission in healthcare settings.

Biology. Compared with C. albicans, C. auris does not produce pseudohyphae, hyphae or chlamydospores, has reduced ability to adhere to catheter material, and forms thinner biofilms (Larkin et al., 2017.) C. auris strains (6/16 isolates) possessed phospholipase activity and 64% (9/14 isolates) tested positive for secreted proteinase (Larkin et al., 2017.) C. auris isolates are positive for assimilation of N-acetylglucosamine, succinate, and gluconate, whereas negative results are recorded for its nearest neighbors C. haemulonii and C. duobushaemulonii (Kathuria et al., 2015.) C. auris isolates show multidrug resistance against fluconazole and amphotericin B, with some isolates also having high MIC values for voriconazole and itraconazole (Larkin et al., 2017.) Additionally, caspofungin, micafungin, and anidulafungin were effective in vitro against the tested isolates but C. auris may acquire resistance to these echinocandins while on treatment. As of September 2017, at least 5 patients in the U.S.A. had echinocandin-resistant isolates (CDC C. auris website.)

The apparent rarity of C. auris diagnosis in the U.S. may be underestimated because of unreliable identification (Mizusawa et al, 2017.) To improve diagnosis CDC assembled a panel of C. auris, C. haemulonii, C. duobushaemulonii, Saccharomyces species, Kodamaea ohmeri, Candida krusei and C. lusitaniae. Mizusawa et al., using existing test methods and databases, sought to identify C. auris in the panel. The API20c AUX kit, a venerable biochemical kit for yeast identification, misidentified C. auris as Rhodotorula glutinis, a strange outcome since the latter is a pigmented yeast whereas C. auris is not (that yeast owes its name to the anatomic location from which it was originally isolated: the ear canal and not from the symbol “Au” for the element, gold.) Other yeast identification formats VITEK 2, YST BD, Phoenix yeast identification system and MicroScan misidentified C. auris as C. haemulonii, C. catenulata, C. famata, C. lusitaniae, C. guilliermondii, or C. parapsilosis. Because of the lack of C. auris entries in FDA approved libraries, it was not identified by commercial MALDI-TOF MS systems. The take-home from this analysis is: C. auris cannot be reliably identified by standard biochemical identification methods and that MALDI-TOF MS databases need updating.

2017 Update

Candida krusei is Pichia kudriavzevii
C. krusei is less well studied than the other Candida species, particularly in relation to virulence (reviewed by Turner and Butler, 2014.) Since the advent of “One Fungus, One Name” rule, do not be surprised if this fungus is referred to by the teleomorph (sexually competent) name: Pichia kudriavzevii (Kurtzman et al. 2011, reviewed in: www.sciencedirect.com/topics/immunology-and-microbiology/candida-krusei ) The carriage of C. krusei in healthy individuals is usually very low, except in some populations such as Wayampi Amerindians, where it appears to originate from food or the environment. The incidence of C. krusei in infection is also low, yet the yeast is of considerable concern because of its relative resistance to azoles and other antifungal drugs.

 

candid.jpg (40233 bytes) Figure 3
 Oval budding yeast cells of Candida albicans. Fluorescent antibody stain. 

CDC/Maxine Jalbert, Dr. Leo Kaufman. lek1@cdc.gov 
thrush.jpg (60483 bytes) Figure 4
Oral thrush.
CDC

candi3.jpg (42054 bytes) Figure 5
Gross pathology of rabbit kidney lesions due to experimental Candida albicans infection. Rabbit was cortisone-treated.
  CDC

candid5.jpg (84477 bytes) Figure 6
Sputum smear from patient with pulmonary candidiasis. Gram stain.
CDC 

candi4.jpg (123774 bytes) Figure 7
Histopathology of Candida albicans infection. Methenamine silver stain. Pseudohyphae and true hyphae.
ASCP 
 


candid6.jpg (166827 bytes) Figure 8
Histopathology of Candida esophagitis. Methenamine silver stain (digitally colorized).
  CDC

candid2.jpg (17510 bytes)  Figure 9
Candida albicans showing germ tubes. Calcofluor white stain in peptone medium. Germ tube production is a diagnostic feature of C. albicans. 
CDC/Mercy Hospital, Toledo, OH/Dr. Brian Harrington 

candid4.jpg (29923 bytes) Figure 10
Candida albicans showing germ tube production in serum. Gram stain. 

CDC/Dr. Lucille K. Georg 

 candida200.jpg (25184 bytes)  Figure 11
Gram-stain of vaginal smear showing Candida albicans epithelial cells and many gram-negative rods. (1,000X oil) 
© Danny L. Wiedbrauk, Warde Medical Laboratories, Ann Arbor, Michigan and The MicrobeLibrary

 

Figure 12
Encapsulated pathogenic yeast fungus (Cryptococcus neoformans). A yeast-like fungus that reprouces by budding. A acidic mucopolysaccharide capsule completely encloses the fungus. It can cause the disease called cryptococcosis; especially in immune deficient humans, such as in patients with HIV / AIDS. The infection may cause meningitis in the lungs, skin or other body regions. The most common clinical form is meningoencephalitis. It is caused by inhaling the fungus found in soil that has been contaminated by pigeon droppings.
 
© Dennis Kunkel Microscopy, Inc.  Used with permission

crypto1.jpg (71375 bytes)  Figure 13
Cryptococcosis of lung in patient with AIDS. Histopathology of lung shows widened alveolar septum containing a few inflammatory cells and numerous yeasts of Cryptococcus neoformans  
CDC/Dr. Edwin P. Ewing, Jr. epe1@cdc.gov 

crypto2.jpg (103360 bytes) Figure 14
Cryptococcosis of lung in patient with AIDS. Mucicarmine stain. Histopathology of lung shows widened alveolar septum containing a few inflammatory cells and numerous yeasts of Cryptococcus neoformans. The inner layer of the yeast capsule stains red.
CDC/Dr. Edwin P. Ewing, Jr. epe1@cdc.gov 

Figure 14a
Life cycle of Cryptococcus gatti.
CDC

Figure 14b
A lung lesion tissue specimen, with morphology associated with the disease cryptococcosis due to the infiltration of Cryptococcus. sp. fungal organisms.
CDC

Figure 14c
Global burden of HIV-related cryptococcal meningitis
CDC: Adapted from BJ Park et al., AIDS 2009;23:525-530



 Figure 14d
Causes of death in sub-Saharan Africa, excluding HIV/AIDS
CDC: Adapted from BJ Park et al., AIDS 2009;23:525-530



 


 

CRYPTOCOCCOSIS

Cryptococcus neoformans and C. gattii (the Cryptococcus species complex)

Disease Definition

Exposure to cryptococci is common but disease is uncommon and linked to host factors. The portal of entry is the respiratory system. The patient acquires the infection from the environment after inhaling airborne desiccated yeast cells and/or basidiospores which then lodge in the lungs. Cryptococcosis manifests most commonly as meningitis but many cases of pulmonary disease have also been recognized. Another frequent site of dissemination is the skin. Most infections are, however, asymptomatic. The disease is not contagious.
Etiologic Agents. Members of the Cryptococcus species complex include Cryptococcus neoformans and C. gattii which are very distinctive yeasts. The yeast cells are spherical, 3 - 7 µm diam., with narrow-based buds and surrounded by a polysaccharide capsule (fig 13). They are classed in the Basidiomycota and occur as two species complexes.

Background

C. neoformans was formerly considered a single asexual species with 4 capsular serotypes (A,B,C,D.) Then, in 1970, a strain with distinct elliptical shaped yeast cells from an African patient was named Cryptococcus neoformans var. gattii.

The observation of a sexual cycle by K.J. Kwon-Chung (1975) led her to describe the sexual forms as Filobasidiella neoformans and Filobasidiella bacillispora. Next, in 2002 C. neoformans var. gattii was raised to species level. The sexual species F. neoformans corresponds to asexual C. neoformans species (serotypes A and D), and F. bacillispora corresponds to C. gattii (serotypes B and C). Because the rule “One Fungus-One Name” was adopted, the genus name “Filobasidella” no longer applies. Turning to C. neoformans, phylogenetic analysis asserts that serotype A is C. neoformans whereas serotype D is referred to as C. deneoformans as the two species differ at ~10% of nucleotide positions. Current consensus is there are two species complexes: Cryptococcus neoformans species complex and the C. gattii species complex.

The status of C. gattii is unexpectedly genetically diverse. Outbreaks of cryptococcosis in new geographic locations over the last 2 decades stimulated further molecular studies of C. gattii.



Phylogenetic Studies and Molecular Subtyping

Molecular typing analysis of C. gattii revealed intra-species genetic diversity consisting of four distinct genetic groups: VGI-VGIV. This nomenclature is used to apply to the global population structure of C. gattii and enables tracing of virulent strains. From a practical standpoint, it is not possible to identify these genetic species in the laboratory since, at present, there are no certain biologic differences and no differences in the rDNA genes commonly used for rapid DNA testing. Consequently the recommended guideline, for the time being, is to use “Cryptococcus neoformans species complex” and “C. gattii species complex.” (Kwon-Chung et al., 2017)

In summary, the current concept of population structure of the Cryptococcus neoformans and C. gattii species complexes is: C. neoformans (molecular types VNI and VNII); C. deneoformans (VNIV), C. gattii (VGI), C. bacillisporus (VGIII), C. deuterogattii (VGII), C. tetragattii (VGIV), C. decagattii (rare).



Geographic Distribution/Ecologic Niche

The geographic distribution of cryptococcosis is world-wide (fig 14.)


Cryptococcus neoformans

The ecological niche of C. neoformans is pigeon and chicken droppings. Although this yeast is easily recovered from pigeon droppings, a direct epidemiologic link has yet to be established between exposure to pigeon droppings and a specific human infection. Disease production is probably a property of the host -- not the organism. The source of human infection is not clear. This organism is ubiquitous, especially in areas such as abandoned buildings contaminated with pigeon droppings.

Cryptococcus gattii

This is found in soil and is associated with several species of trees in tropical and sub-tropical regions of the world. Recently, possibly as a result of climate change, C. gattii infections were identified in the Pacific Northwest of the U.S.A. and western Canada especially on Vancouver Island (see Epidemiology Highlight, below and fig 15.)


Incidence/Prevalence

An estimated 220,000 cases of cryptococcal meningitis occur among people with HIV/AIDS worldwide each year, resulting in nearly 181,000 deaths. Most cases occur in sub-Saharan Africa. In the U.S.A. estimates of cryptococcosis are incomplete because the disease is only reportable in a few states. Results from surveillance in two U.S. locations in the year 2000 indicated an annual incidence of cryptococcosis among persons with AIDS was between 2 -7 cases per 1,000, with an overall incidence of 0.4 to 1.3 cases/100,000 cases (CDC 2017.)
C. gattii infections are worldwide, with cases of occurring in Papua New Guinea, Australia, and South America. C. gattii infections have also occurred in British Columbia, Canada since 1999 and in the U.S. Pacific Northwest since 2004 (96 cases reported to CDC during December 2004 – July 2011). Nearly all C. gattii cases in the U.S. are from Oregon, Washington, and California with a small number in other states.https://www.cdc.gov/fungal/diseases/cryptococcosis-gattii/statistics.html - three


Epidemiology highlight

Emergence of Cryptococcus gattii in the Pacific Northwest (Acheson et al., 2017)

The ecology of C. gattii remained unknown until its discovered association with Eucalyptus trees in Australia in 1990. Cryptococcosis caused by C. gattii was considered mainly a tropical and subtropical disease until it emerged in 1999 on Vancouver Island, British Columbia, spreading into the Pacific Northwest. Genomic studies traced the outbreak genotypes, VGIIa and VGIIb, to an ancestral C. gattii strain from the Amazon rainforest, spreading from there to different parts of the world. Vancouver Island has one of the highest annual incidences of C. gattii human infections in the world. From 1999 to 2015, 393 cases were reported in British Columbia (Acheson et al., 2017.) The fungus infected immunocompetent individuals, including local residents, visiting tourists, wild and domestic animals. The search for its ecologic niche began in 2001. Cases were clustered along the east side of Vancouver Island in the rain shadow, where the flora and soil are unique to the Coastal Douglas Fir and Western Hemlock biogeoclimatic zone of dry summers (average 17.6 ◦C), with mild winters rarely below freezing. Its emergence in this temperate area suggests the fungus expanded its ecological niche. Whether C. gattii was recently introduced to Canada or existed undetected for years is not clear but a match between a clinically isolated 1970’s Seattle strain and the 1999 Vancouver Island outbreak VGII strain may suggest the latter.



Risk Groups/Risk Factors

As with other fungal infections, people at most risk of cryptococcosis include people living with AIDS, and patients receiving immunosuppressive therapy for cancer or for retention of haemopoietic stem cell or solid organ transplants.


Transmission

The primary source of Cryptococcus neoformans is soil mixed with excreta of the common pigeon, Columba livia but pigeons, themselves, are not infected. Cryptococcus gattii is found in the decaying hollows of certain tree species. Once inhaled, desiccated yeast cells and/or basidiospores of cryptococci germinate forming dividing yeast cells. They can then disseminate from the lungs to other parts of the body via the bloodstream, sometimes inside macrophages. The appearance of symptoms usually occurs several months (average six to seven) after breathing in spores but, in some cases, several years of latency pass before symptoms are observed.


Determinants of Pathogenicity

The polysaccharide capsule is antiphagocytic, may suppress T-cell function, and is considered a virulence factor. C. neoformans also produces an enzyme, phenoloxidase, involved in melanin production, another virulence factor. The two species complexes’ ability to grow at 37oC differs from the other saprobic Cryptococcus species, even those with polysaccharide capsules.


Clinical Forms

Subclinical disease and latency

The initial exposure may be many years before the manifestation of disease, with the yeast being sequestered during this time. When disease occurs it may be subacute or chronic. In addition to causing meningitis, C. neoformans may also infect lungs (figs 16, 17, 18) and disseminate to the skin. The disease in the lungs and skin is characterized by the formation of a granulomatous reaction with giant cells.

As with other fungal diseases, there has been an increase in the recognition of pulmonary infection. Pulmonary cryptococcosis is accompanied by malaise (fever and headache), cough, shortness of breath, and chest pain. The yeast may also form a mass in the mediastinum called a cryptococcoma. The presence of Cryptococcus gattii can lead to the growth of cryptococcomas in various parts of the body.

Cryptococcal meningitis

he fungus can spread from the lungs to the nervous system, including the brain causing meningoencephalitis. According to the CDC, there is a long latent time (two to fourteen months) between exposure and the manifestation of symptoms. The potentially fatal meningoencephalitis caused by C. neoformans has a prolonged evolution over several months. Patients’ symptoms may begin with vision problems and headache and, in the absence of specific antifungal therapy, progress to delirium, nuchal rigidity, then coma and death.

Symptoms include:

  • Fever
  • Headache
  • Neck pain
  • Nausea
  • Light sensitivity
  • Confusion



Therapy

The patient requires treatment by antifungal agents for up to six months. The drugs of choice to treat severe cryptococcosis are i.v .amphotericin B in combination with 5-fluorocytosine (5-FC). 5-FC is an oral drug. These two drugs are synergistic and their association is advantageous. Stepdown therapy is the use of oral fluconazole. In milder cases, or in resource-poor areas, fluconazole or itraconazole is used as monotherapy.


Laboratory Detection, Recovery, Identification

The alert physician will consider cryptococcosis in the differential diagnosis and order a lumbar puncture. The CSF is analyzed for its characteristic chemistry (elevated protein and decreased glucose), cells (usually monocytes), and evidence of an encapsulated yeast. The latter is observed microscopically in an India ink prep, (fig 19) or by a serologic assay for the capsular polysaccharide of C. neoformans. The India Ink test demonstrates the capsule of this yeast, whereas the lateral line assay for antigen (see below) is more sensitive and specific such that a decreasing antigen titer indicates a good prognosis, while an increasing titer has a poor prognosis. When you consider cryptococcosis, think of capsules and CNS disease.

Clinical material sent to the lab is CSF, biopsy material, and urine (for some unexplained reason the organism can be isolated from the urine in both the CNS and systemic infections). This yeast will grow overnight on bacterial or fungal media at 37°C. but growth is a little slower at room temperature. In culture, the organism grows as creamy, white, mucoid (because of the capsule) colonies. Growth in culture is usually visible in 24 to 48 h. As the culture ages, it turns brown due to melanin produced by the enzyme phenoloxidase. After growth in the laboratory, microscopic morphology is part of the laboratory identification of Cryptococcus species.

Microscopy

The yeast is a round encapsulated single cell with a narrow based bud. Overall size including the capsule may be 15 - 20 µm diameter with a cell diameter of 7 µm. Yeast cells vary considerably in size and shape. Identification is based on physiologic reactions. The API20c profile is useful, and the urease test is positive. Although they are white yeasts, C. neoformans and C. gattii produce a brown colony effect when grown on Niger seed agar or by using caffeic acid disks. These tests are discussed in detail in Reiss et al., 2012.

Histopathology

Pathologists use a mucicarmine stain, which stains the capsule, to identify the organism in tissue sections (fig 16). There is usually little or no inflammatory response. The Direct Fluorescent Antibody test identifies the organism in culture or tissue section causing the yeast cell wall to stain green.

Serologic assays. Tests for antibodies

To test the patient's serum there are three serologic tests: The Indirect Fluorescent Antibody test, the Tube Agglutination test for antibody, and antigen detection in CSF or serum.


Tests for Antigen

The latex agglutination test, a legacy test originated in the 1960’s (Bloomfield et al., 1963), is being replaced by the IMMY CrAg LFA (Cryptococcal Antigen Lateral Flow Assay). It is a rapid, immunochromatographic dipstick test for the qualitative and semiquanitative detection of cryptococcal polysaccharide antigen (IMMY, Inc., Norman OK.) The CrAg LFA is sensitive and FDA cleared for both Cryptococcus neoformans and C. gattii. It does not require pretreatment of CSF by boiling or proteinase pretreatment of serum. In the semiquantitative form two-fold serial dilutions are set-up in individual tubes. A video of the method of procedure is at : http://www.immy.com/products/lateral-flow-assays/crag-lfa/#1473450453921-a2843b7f-7b86

As the patient improves, the antigen titer will also decrease.

Preemptive antifungal treatment can prevent or detect a significant proportion of cryptococcal meningitis cases at an early stage because cryptococcal antigen can be detected in blood before development of clinical disease. The CrAg lateral flowassays that can be used for diagnosis at the point of care. CrAg screening has been adopted into policy by over 20 countries in Africa,

CGB medium

To distinguish Cryptococcus gattii from Cryptococcus neoformans, the organisms are grown on the differential medium: canavanine-glycine-bromothymol blue agar (CGB.) C. neoformans and C. gattii differ in their ability to assimilate nitrogen. L- Canavanine is structurally related to L-arginine, but when incorporated into proteins in place of arginine, there is loss of function. C. gattii is resistant to canavanine. C. neoformans is susceptible to canavanine or, if not, this species does not assimilate glycine and the medium remains yellow. On CGB medium C. gattii produces glycine decarboxylase and uses glycine as a carbon and nitrogen source. The ammonia released when glycine is cleaved causes an increase in pH turning the indicator bromothymol blue from yellow to blue. Given C. gattii's increasing profile worldwide as a pathogen and its documented spread to nontraditional areas of endemicity, CGB agar is an inexpensive and convenient way to screen for emergence in patients.

 


 

 


Figure 15
C. neoformans: India Ink preparation
Dr Arthur DiSalvo

crypto3.jpg (99057 bytes) Figure 16
Cryptococcosis of lung in patient with AIDS. Methenamine silver stain. Histopathology of lung shows numerous extracellular yeasts of Cryptococcus neoformans within analveolar space. Yeasts show narrow-base budding and characteristic variation in size.
CDC/Dr. Edwin P. Ewing, Jr. epe1@cdc.gov

 

 

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