MYCOLOGY - CHAPTER  FIVE  

FILAMENTOUS FUNGI 

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

 

  Fig.1
A. fumigatus microscopic morphology

Microscopic morphology of A. fumigatus shows uniseriate conidial heads. Conidiophores are short, smooth-walled, with conical terminal vesicles, supporting a single row of phialides on the upper 2/3 of the vesicle. Conidia are extruded singly from phialides forming chains which usually are disrupted in preparation of slides. Conidia are round and green. Source: #041 (Kaminski library)

Fig. 2.
A. flavus microscopic morphology

Conidial heads are biseriate but with some phialides borne directly on the vesicle. Conidiophores are hyaline and coarsely roughened. Conidia are globose to subglobose, pale green and spiny.
Source: E. Reiss

Fig.3
Microscopic morphology of A.niger

A. niger on Czapek dox agar. Dark-brown to black conidial heads.

Source: Geraldine Kaminski Library of Medical Mycology #044

SYSTEMIC MYCOSES CAUSED BY OPPORTUNISTIC HYALINE MOLDS

ASPERGILLOSIS 

Introduction/Disease Definition

Aspergilli produce a wide variety of diseases. Like the mucormycetes, they are ubiquitous in nature and play a significant role in the degradation of plant material as in composting. Similar to Candida species and the mucormycetes they rarely infect immune-normal hosts. These molds are distributed world-wide and are commonly found in soil, food, paint, heat-ventilation-air conditioning systems including those in hospitals.

Aspergillosis includes a broad range of clinical forms including:

• Allergic bronchopulmonary aspergillosis (ABPA).
• Chronic necrotizing pulmonary aspergillosis
• Colonizing aspergilloma in pre-existing pulmonary cavities, and paranasal sinuses
• Endocarditis
• Intoxication by ingestion of peanuts or corn contaminated with aflatoxin (A. flavus )
• Invasive pulmonary aspergillosis (IPA) in compromised hosts
• Keratitis (post-traumatic)
• Otomycosis
• Primary cutaneous aspergillosis (host compromised)

Aspergilli grow as hyaline molds with septate hyphae of uniform diameter that branch at acute angles. Colony colors vary according to the species (see illustrations). The conspicuous microscopic feature is the sporing (conidial) head consisting of a conidiophore terminating in a bulbous vesicle containing one (monoseriate) or two (biseriate) rows of phialides from which round conidia are extruded. The conidia remain attached in columns but are easily dispersed in air currents. In tissue sections Aspergillus hyphae stain poorly with H&E, requiring PAS or GMS for good visibility. Exposure to air spaces in lungs or sinuses may result in formation of conidial heads in situ. Aspergillus hyphae have a predilection for the elastic lamina of blood vessels causing hemorrhage, thrombosis, and infarcts.

Diagnosis

The most serious clinical form, IPA, is diagnosed by a combination of radiographic findings and laboratory tests. Diagnostic imaging of pulmonary lesions using CT scan facilitates further testing. Blood cultures are often negative. Sputum cultures and bronchoalveolar lavage fluid are relevant for direct examination and culture. Invasive lung biopsy and histopathology are additional tools. Galactomannan antigenemia in IPA is measured by EIA. Once a culture is growing, morphologic criteria are very useful, and genetic ID may be used if an unusual Aspergillus species is suspected. The specific methods are covered in more detail in the Section: Laboratory Detection, Recovery, and Identification

Etiologic Agents

There are more than one hundred species of aspergilli. The most common etiologic agents of aspergillosis in the U.S.A. are:

• Aspergillus fumigatus (Fig. 1)

• A. flavus (Fig. 2)

• A. niger (Fig. 3)

Geographic Distribution/Ecologic Niche

A fumigatus is world-wide in distribution. It is thermotolerant, growing up to 45^oC, and is found in composts, wood chips, hay, where it has a role as a cellulose decomposer. A. flavus is a plant pathogen causing post-harvest infestation of corn, peanuts.

Epidemiology, Incidence, and Prevalence

Compared with the general population, the incidence of IA and mucormycosis is much higher in patients with hematologic malignancies (HM), hematopoietic stem cell transplants (HSCT), or with solid organ transplants (SOT).  Hospital discharge summary data in the U.S. from 2000-2013 for patients with these conditions found a total of169,110 IA cases, for a rate change / million persons over the 13 y period from 32.8 to 46.0, increasing annually by 3% (Vallabhaneni et al., 2017). This increase may be explained by an increased number of HSCT and SOT procedures.  With respect to HSCT as a category, a significant decline occurred in the IA rate (-4.6%). Among HM patients the IA the rate did not change significantly. Among SOT recipients, IA rates increased 4.1%. 

Advances in diagnostics for aspergillosis, (the galactomannan serum assay, chest computed tomography) have increased diagnosis. On the other hand, increased awareness and the use of anti-mold prophylaxis suggest a real decline or stabilization in IA occurred in HSCT and HM patients. 

Mortality for IA is 20%–50% depending on the underlying condition. But Vallabhanen et al., 2017 found that in-hospital deaths occurred in only 16% of IA (higher in HSCT patients.) Others reported only 40% of invasive fungal infections are diagnosed pre-mortem. There was no difference in fungal disease-free survival with voriconazole when compared with fluconazole among HSCT recipients, but there was a trend toward lower invasive fungal infections, fewer IA infections, and less frequent need for empiric antifungal therapy in the voriconazole arm.  This underscores that prophylaxis should be used to prevent IA in HM and HSCT patients during high-risk periods. Prophylaxis may prevent IA but leaves patients susceptible to mucormycosis because of the greater resistance of Mucorales to azoles. Among SOT patients IA increased, with lung transplantation carrying the highest risk.

Aspergilloma is a fungus ball that develops due to the formation of hyphae inside a preexisting lung cavity (e.g. tuberculosis, sarcoidosis), usually in the upper lobe. CT scan of lung (axial view) Yellow circle indicates cavity containing fungus ball and small crescent of air above it. The mycologic term for it is a mycetoma.

Case courtesy of Dr Domenico Nicoletti, https://radiopaedia.org/cases
 

Summary of Risk Factors for Invasive Aspergillosis

Persons receiving cancer chemotherapy, HSCT for hematologic malignancy, other stem cell transplant recipients, patients with solid tumors, or those receiving immunosuppressive therapy for maintenance of SOTs where two of three criteria are satisfied:

• <1000 PMN/ µL for 1 wk or more

• Cytotoxic drug therapy

• High dose prolonged corticosteroid therapy or prolonged therapy with other immunosuppressive agents.
   

Other factors: Defective neutrophil function (e.g.: chronic granulomatous disease), IV drug use, underlying lung disease including tuberculosis.

Transmission

Aspergillus conidia, often present in hospital air, are small enough to reach the paranasal sinuses and alveoli. Conidia can circulate through air supply ducts in hospitals and enter chest wounds during surgery. Aspergillosis is not observed in patients housed in laminar air flow rooms.

Determinants of Pathogenicity

Many microbial factors of Aspergillus fumigatus are implicated in pathogenesis. A full treatment of the following can be seen in “Medical Mycology” on CRteacher2.com

 Conidia of small size with rapid germination

Rapid growth of hyphae and temperature tolerance

Adherence of hyphae to host tissues

Secretion of damaging metabolites, e.g., gliotoxin

 Siderophores that enable mold to grow in an iron-poor environment

Production of elastase, proteinases, hemolysin, and phospholipase C contribute to pathogenesis.

Clinical Forms

The spectrum of clinical types of pulmonary aspergillosis includes:

ABPA.  Allergic hypersensitivity to A. fumigatus. Symptoms vary from mild respiratory distress to alveolar fibrosis.  Occurring in up to 2.5% of adults with asthma; it is also common in cystic fibrosis patients .Symptoms are episodes of feeling unwell, coughing and wheezing. Some patients cough up sputum plugs containing fungal hyphae. If untreated in the long term ABPA can lead to permanent lung damage (fibrosis or bronchiectasis). Treat with steroids by aerosol or mouth (prednisolone), especially during attacks. Itraconazole is useful in reducing the amount of steroids required.    

Aspergilloma. Fungus ball is characteristically seen in old cavities of TB patients, or in the paranasal sinuses. These are recognized by x-ray or CT scan (Fig. 4), because the lesion (a colony of mold growing in the cavity) is shaped like a half-moon (semi-lunar growth). Patients may cough up fungal elements because the organism frequently invades the bronchus. Chains of conidia are sometimes seen in the sputum. Treatment is reserved for when there is pulmonary hemorrhage requiring surgery to remove the fungus ball and cavity. Debridement of paranasal sinuses may be indicated to relieve symptoms or hemorrhage.  

Chronic necrotizing pulmonary aspergillosis. These patients may have underlying structural lung disease and are treated with low dose corticosteroids. It is chronic, progressing with infiltrates, fungus balls, but does not progress to vascular invasion or dissemination. Antifungal therapy is required. 

Invasive pulmonary aspergillosis (IA or IPA). Aspergillosis is primarily a pulmonary disease accompanied by fever, pulmonary infiltrates, pleuritic chest pain and hemoptysis, progressing with aggressive tissue invasion and hematogenous dissemination from the lung to other organs including brain, heart, osseous, aural, and cutaneous lesions.

 

a: A. fumigatus
b. A. flavus
c. A. terreus
d. A. niger

Source: Reiss, Shadomy, Lyon, 2011

Fig. 6 Aspergillosis histopathology lung

Histopathology—aspergillosis, lung. GMS stain, The septate hyphae are of similar diameter and form acute angle (dichotomous) branching, i.e., a single hypha branches into two even hyphae, and then the mycelium continues branching in this fashion.

Reproduced from Chandler FW and JC Watts 1987 Pathologic diagnosis of fungal infections. ASCP Press, Chicago.303 p. with permission from the American Society for Clinical Pathology

Therapy
 

Guidelines for diagnosis and management of aspergillosis were updated in 2016 (Patterson et al., 2016). Herein we summarize recommended therapy for invasive aspergillosis. Information about therapy for other forms of aspergillosis are found in that update.

• Prophylaxis. Posaconazole, voriconazole, and/or micafungin are recommended during prolonged neutropenia for those who are at high risk for IA.  Prophylaxis with caspofungin is also probably effective. Prophylaxis with itraconazole is effective, but therapy may be limited by absorption and tolerability. 

• Empiric antifungal therapy is recommended for high-risk patients with prolonged neutropenia who remain persistently febrile despite broad-spectrum antibiotic therapy.  Options include a lipid formulation of AmB, an echinocandin, or voriconazole.

•   Primary therapy. Triazoles are preferred to treat and prevent IA in most patients. Alternative therapies include liposomal AmB, isavuconazole. Early initiation of therapy with voriconazole is warranted in patients strongly suspected of IA while a diagnosis is evaluated. Treatment of IA should be continued for at least 6–12 wks, depending on the level and length of immunosuppression, site of disease, and evidence of clinical improvement. Patients with successfully treated IA who require further immunosuppression, are indicated for prophylaxis to prevent recurrence.

• The use of serum or BAL fungal biomarkers such as galactomannan or (1→3)-β-D-glucan to guide antifungal therapy in asymptomatic or febrile high-risk patients can reduce unnecessary antifungal therapy.

Emerging resistance to azole antifungal agents in strains of Aspergillus fumigatus (Walker et al., 2018). Infections with strains of A. fumigatus resistant to all azole antifungal agents have become common in Western Europe and are emerging in the U.S.A.  Pan–azole-resistant A. fumigatus strains with mutations in the cyp51A gene are associated with higher rates of treatment failure and death. These mutations are linked to use of azole fungicides in agricultural and environmental applications and not to prior azole ther­apy, because patients with these infections frequently lack a history of clinical exposure to azoles. A survey of 1584 U.S. infectious disease physicians was conducted in June 2016.  Pan–azole-resis­tant isolates were reported by 4 (8%) of 51 physicians in the South, 7 (11%) of 63 in the Midwest, 2 (5%) of 37 in the Northeast and 3 (4%) of 70 in the Western U.S. These findings underline the importance of broader susceptibility testing.

Laboratory Detection, Recovery, and Identification

Direct examination

Specimens for direct examination include: sputum, nasal scrapings, bronchoalveolar lavage fluid, fine needle aspirates, thorascopic biopsy, and other biopsy materials. Hyphal elements appear under KOH prep (with or w/o Calcofluor white fluorescent brightener). They are seen as hyaline, closely septate, dichotomously branched at acute angles. Bloody or mucoid specimens should be treated with Mucolyse®. Such findings prompt further steps to identify the causative agent.

Culture

Media for growth include SDA-Emmons agar, Czapek-Dox (not in general clinical use), and Malt extract agar (often used in air sampling devices). On SDA-Emmons agar conidial structures form in 48-72 h at 30-37^oC. The colony begins as a dense white mycelium which later, based on conidial color, assumes a variety of colors, according to species, Tease mounts are the first choice for microscopy, but if they are not revealing then slide cultures will show the asexual reproductive structures, the sporing heads. Hyphae are colorless, of even diameter, septate, and branch at acute angles. Species differentiation is based on the appearance of the conidiophores and colony color.  

Morphology of major Aspergillus spp.

Colony Morphology (Fig. 5)

Microscopic Morphology (Figs. 1-3, with explanations)

Genetic Identification: PCR on blood or serum specimens

This research is focused to detect Aspergillus DNA directly in clinical specimens such as whole blood, plasma, or serum. Blood cultures of Aspergillus are rarely positive, however, the presence of free or phagocytosed fragments of rDNA facilitates this approach. PCR from blood or serum has moderate diagnostic accuracy as a screening test for IA in high-risk patients. A negative test allows the diagnosis to be excluded with moderate to high confidence. Consecutive positives show good specificity in diagnosis of IA, prompting radiologic and other tests or pre-emptive therapy when clinical suspicion is high.   

In a proof of principle report, the ITS2 subregion of rDNA had enough diversity to facilitate probe design to identify 7 major Aspergillus spp. Post-amplification utilized real time analysis LightCycler® or the Luminex® assay (Etienne et al., 2009). 

Clinical validation of whole blood extraction of DNA was reported by Rogers et al, 2013.  Three mL blood samples from proven or probable IA patients were processed by red cell lysis, white cell lysis, bead beating, and purification using a Roche Diagnostics kit. PCR utilized two different targets: either 28S rDNA or ITS rDNA in two different medical centers. The incidence of IA was 20% (center 1) and 25% (center 2) in allo-SCT patients, compared with 3% (center 1) and 20% (center 2) in acute leukemia patients. Results in patients with proven or positive IA, where two consecutive PCR tests were positive, both ITS and 28S based tests showed 34% and 47% sensitivity and 88% and 74% specificity, respectively,  from center 1 and center 2. Positive predictive value of both tests was in the 31-32% range, whereas negative predictive value was between 77- and 93%. Collating tests from two different medical centers is difficult with different treatment plans and variations in sample preparation.

 Whole blood processing is labor-intensive whereas use of serum or plasma is less laborious and is only marginally less sensitive than whole blood. Plasma   avoids loss of DNA from trapping in clots. Tips and tricks and detailed sample prep protocols are found in Barnes et al., 2018. The most common PCR targets in rDNA are 18S, 28S, and ITS regions; best specificity is obtained using 28S rDNA. The qPCR assays should utilize a probe; increasing cost and complexity but increasing test specificity. Targeting the Aspergillus genus is most reliable for detecting A. fumigatus at low DNA concentrations. A DNA calibrator for assessing Aspergillus is optimal for assays targeting serum or plasma, facilitating inter-lab comparisons and quality control.

 The take-home message is that PCR tests have moderate sensitivity, good specificity, and moderate to high negative predictive value. When PCR results are combined with results of serum galactomannan tests, positive and negative predictive values increase.
 

Histopathology 

Formalin-fixed, paraffin-embedded tissue (FFPE) can provide a rapid, presumptive diagnosis of aspergillosis. (Guarner and Brandt, 2011). Standard H&E stains fungal elements but with low contrast between fungi and tissue cells. Silver stain (e.g.: Gomori methenamine silver [GMS]) or Periodic acid Schiff stain are relevant for a presumptive diagnosis. Aspergillus spp. appear as thin, 3- to 12-µm diam., septate, acute-angle or dichotomously branched hyphae of regular width (Fig. 6). Vesicles with conidia may be found if fungi are exposed to an air space in cavitary lesions or sinuses. A hallmark of infection with aspergilli (and for mucormycetes) is invasion of the elastic lamina of blood vessels creating hemorrhage, thrombosis, and infarction of surrounding tissue. Tell-tale histopathologic signs of individual species are A. niger infection, wherein calcium oxalate crystals may be deposited and for A. terreus which produces round or pear-shaped accessory conidia directly along the lateral hyphae.

In ABPA, there are thick, tenacious mucous plugs in normal, fibrotic, or bronchioectatic airways. Microscopically, the mucus contains inflammatory cells (eosinophils, lymphocytes, and macrophages). In allergic fungal rhinosinusitis histopathology reveals scattered hyphae in the allergic mucin without fungal tissue invasion. Fungus balls (aspergillomas) in chronic pulmonary aspergillosis consist of hyphae enmeshed in necrotic material. Cultures of the allergic mucin of patients with allergic fungal rhinosinusitis yield in addition to Aspergillus spp., melanized molds such as Bipolaris spicifera, and Curvularia lunata.

 Pitfalls in morphologic diagnosis are that some tissue sections with septate acute-angle-branching hyphae turn out after culture to be Scedosporium spp, or Fusarium spp. Thus efforts have been made to develop PCR methods to extract and amplify the small amounts of DNA available. Laboratory developed procedures are having some success using actual tissue specimens from confirmed cases (Salehi et al. 2016).  Real-time quantitative PCR (qPCR) targeting the ITS2 subregion of rDNA uses an automated EZ1 extraction instrument (Qiagen Inc.). Sections of paraffin-embedded tissues are digested with proteinase K, then processed by automated nucleic acid extraction and the Qiagen DNA tissue kit. Real-time PCR is performed using a Roche LightCycler 480 Instrument. Five Aspergillus species were detected with specific probes:   A. fumigatus, A. terreus, A. niger, A. flavus, and A. nidulans/A. versicolor. Other hyalohyphomycetes detected were Fusarium spp., Scedosporium apiospermum, S. aurantiacum, and Lomentospora prolificans. Of the mucormycetes, specific probes detected Rhizopus microsporus, R. oryzae, Mucor, Cunninghamella bertholletiae, Lichtheimia, Syncephalastrum, and Rhizomucor species. The qPCR procedure resulted in a sensitivity of 64% for identification of fungi in FFPE tissues. Among 59 qPCR-positive specimens, there was agreement between PCR and histopathology in 47 specimens (80%).

Serology
Platelia Aspergillus EIA (acquired by Bio-Rad, Inc.) is a kit to detect galactomannan (GM) antigenemia in invasive aspergillosis, GM is a heat-stable cell wall antigen. Patient serum specimens giving a positive test should be re-confirmed by re-testing the same sample.  In one study, serum specimens from hematology-oncology patients included 27 cases of IA. The sensitivity, specificity of the Platelia Aspergillus EIA were, 92.6%, 95.4% (respectively). In 25 patients tested at intervals, the test became positive with respect to first clinical suspicion from -23 to +4 days.  (A false-positive rate of 7.4%)  (Maertens et al, 1999). 

Diagnosis of allergic bronchopulmonary aspergillosis may be assisted by employing an agar gel immunodiffusion test. There may be 1 to 5 precipitin bands. Three or more bands usually indicate increasingly severity of the disease. This test is available as a service from MiraVista Laboratories, Mayo Medical Laboratories. Kits to detect fungal antibodies including Aspergillus are available from Immunomycologics, Inc.

 

Selected References

 

Fig.7 Mycotic thrombus, brain mucormycosis, PAS stain. Source: E. Reiss

Fig. 8. Rhizopus oryzae

a. Sporangium with sporangiospores

b. sporangiophore

c. rhizoids. Source: E. Reiss
Simple or branched sporangiophores are long (up to 1.5 mm) arising from stolons opposite rhizoids, often in groups of three or more. Sporangia are round with a flattened base, up to 175 µm diam., containing many spores. Columellae soon collapse to an umbrella-like shape after spore release.

Fig. 9. Apophysomyces elegans.
 

Mature sporangium of Apophysomyces elegans with the distinctive funnel shaped apophysis. Colonies are fast growing, white, becoming brownish with age, composed of broad, pauci-septate hyphae. Sporangiophores are unbranched tapering towards the apophysis. Sporangiophores are at right angles from the aerial hyphae, anchored by rhizoids. Sporangia are small and pear-shaped. Columellae are rounded and the apophyses are funnel or bell-shaped. Sporangiospores are brown colored in mass. Good growth at 26C, 37C and 42C.

Source: #565. Geraldine Kaminski Library of Medical Mycology.

MUCORMYCOSIS 

Disease Definition

Mucormycosis* is an acute inflammation of soft tissue, usually with fungal invasion of the blood vessels (angioinvasion) which can rapidly progress to fatal disease in susceptible human hosts. Mucormycosis is caused by several species in the order Mucorales, subphylum Mucoromycotina, (e.g.: Rhizopus oryzae, Lichtheimia (formerly Absidia), Rhizomucor).  Mucormycetes are ubiquitous in the environment and rarely cause disease in an immune-normal host. Rhinocerebral mucormycosis can occur in patients with diabetic ketoacidosis. Other underlying conditions which cause susceptibility are:  hematologic malignancy, complications of organ or stem cell transplants, severe burns, or intravenous drug use. Post-traumatic mucormycosis (traffic accidents, natural disasters) is another clinical form. 

(*formerly zygomycosis)

Typical case .An uncontrolled diabetic patient comes to ER (may be comatose depending on the state of diabetes) and a cotton-like growth or eschar is observed on the roof of the mouth or in the nose. These are the hyphae of the organism. If untreated, the patient will die within a few hours or days. What do you do to help this patient first? Controlling the diabetic state is most important before administering amphotericin B (AmB) or liposomal AmB. These fungi have a tendency to invade blood vessels (particularly arteries) and enter the brain via the blood vessels and/or by direct extension through the cribiform plate (Fig. 7). This is why they cause death so quickly.

Diagnosis

Direct microscopic examination of tissue scrapings, sinus aspirates, bronchoalveolar lavage fluid, biopsy of necrotic tissue, accompanied by culture (these are rapid growing molds.) There are no available serologic tests. Detection of Mucorales DNA in blood is an emerging technology. See “Laboratory Detection…”.

Etiologic Agents

The common genera and species causing mucormycosis are (alphabetic order not in order of prevalence:

Lichtheimia, Mucor circinelloides, Mucor spp. Rhizomucor, Rhizopus oryzae (Fig. 8).

Notable outbreaks of post traumatic mucormycosis have been caused by Apophysomyces. (Fig. 9) See “Epidemiologic Highlight”  

Geographic Distribution/Ecologic Niche

These fungi are found world-wide, commonly in soil, food, organic debris etc. They are seen on decaying fruit and vegetables in the refrigerator and on moldy bread. Their spores become airborne and may be inhaled, alight on open wounds, or are ingested with contaminated fruit or bread.

Epidemiology 

Prevalence

Mucormycosis-related hospitalizations in the U.S. were estimated for the period January 2005-June 2014 (Kontoyiannis et al., 2016).  Prevalence for this period was 0.12/10,000 discharges. A total of 555 hospitalizations for mucormycosis were recorded among 47 million inpatients. A total of 23% of these were dead at discharge; 30-37% of patients were readmitted within 1-3 mo, and the average cost of hospital stay was $112,419. There was no clear trend in changes of prevalence across the years. The most common antifungal drugs used were AmB lipid complex, followed by liposomal AmB, and posaconazole. The low prevalence, high clinical and economic burden, and because common antifungal therapies (fluconazole and voriconazole) are not active against Mucorales, indicate the need for active surveillance and prophylactic treatment in susceptible patients.

 

Epidemiologic Highlight

Cutaneous mucormycosis following 2011 tornado in Missouri

 

Risk Groups/Factors

• Deferoxamine therapy. The increased risk of mucormycosis in patients in renal failure receiving deferoxamine iron chelation therapy is explained because deferoxamine acts as a siderophore for Mucorales, supplying previously unavailable iron to the fungi. Iron liberated from deferoxamine is likely transported into the fungus by high-affinity iron permease (Ibrahim et al., 2008).

• Diabetic ketoacidosis. The decrease in pH increases the availability of free iron, an essential growth factor for Mucorales.

• I.v. drug abuse. Approximately thirty cases of isolated cerebral mucormycosis in immunocompetent individuals were reported; most patients were injection drug users. In contrast to the presentation in immunocompromised patients, mucormycetes show a remarkable predilection for the basal ganglia and thalamus in the immunocompetent patient (Carpenter et al., 2007).

• Immunosuppression. Results from organ or stem cell transplantation, hematologic and other malignancies, HIV, high dose corticosteroids, or neutropenia.

• Malnutrition as a contributing factor.

• Severe burns, Penetrating injury following accidents. Intact skin is a barrier to entry of Mucorales spores. Breakdown of the skin by burns or penetrating wounds exposes the patient to infection, which can progress as necrotizing cutaneous mucormycosis.

• Voriconazole antifungal therapy carries a risk of breakthrough mucormycosis, because Mucorales are not susceptible to that antifungal agent.
   

 

Transmission

Sporangiospores of Mucorales are carried on air currents and inhaled from the environment, or alight on open wounds and burns. As frequent saprobes on fruit and bread, the sporangiospores may also be ingested.
 

Determinants of Pathogenicity

Pathogenic factors include iron utilization at acidic pH, and production of lipase. Bacterial endosymbionts of Rhizopus species produce toxins implicated in pathogenesis. Further information is provided in Reiss et al., 2011.
 

Clinical Forms

Rhino-cerebral mucormycosis presents as rhinosinusitis, sinusitis, rhino-orbital, or rhinocerebral disease.  Disease progresses from necrosis or eschar in the nasal cavity or on the palate, trigeminal and facial cranial nerve palsy, paralysis of eye muscles with loss of vision.  Intracranial complications include  abscesses and cavernous and sagittal sinus thrombosis

Pulmonary mucormycosis includes fever unresponsive to antibiotics, cough, pleuritic chest pain, and dyspnea. Invasion of blood vessels can lead to fatal hemoptysis. Characteristics of pulmonary mucormycosis and aspergillosis are indistinguishable.

Cutaneous mucormycosis is acquired by direct spore inoculation or via burns or traumatic wounds. Erythema and induration of the skin typically progresses to necrosis with a black eschar

Gastrointestinal mucormycosis is rare, only a fraction of cases are diagnosed ante mortem. Risk factors include extreme malnutrition, prematurity, immunosuppression.

Rhizopus stolonifer

Source: Microbiology and our life, Google +https://plus.google.com/
105505628269180107992/posts/
Phz78h1SzSA?pli=1

Fig. 11 Histopathology. Lichtheimia infection of lung. Gomori Methanamine silver (GMS) stain

Typical mucormycete hyphae in tissue also shows a sporangium of Lichtheimia species.

Source Geraldine Kaminski Library of Medical Mycology #590

Therapy

Success in mucormycosis requires a combination of: early diagnosis, restoration of euglycemia, reversal of other risk factors, including immunosuppression (if possible), prompt start of antifungal therapy, and surgical debridement (as needed). Delay in using an amphotericin-B based drug (>6 d after diagnosis) may double mortality at 12 wks in patients with diabetes (of all types) (Sun and Singh 2011).

Mortality in the 1990’s declined from previous levels to 40%, but outcomes are poor, especially in stem cell transplant patients with disseminated disease where mortality remains in the 90-95% range (Sun and Singh 2011). AmB based agents are preferred for mucormycosis. All-cause mortality varies from 39% to 75% in patients receiving lipid formulations of AmB as primary or salvage therapy.  Liposomal AmB delivers improved response and survival compared with AmB deoxycholate. Echinocandins may have a role as combination therapy with AmB.

Posaconazole is the first azole with broad activity against Mucorales. Monotherapy with posaconazole is not recommended, instead it is used as salvage therapy. Isavuconazole is a newer triazole approved in the U.S. for treatment of mucormycosis, and when AmB is not feasible. Recently, studies reported cases of breakthrough mucormycosis and other invasive fungal infection in patients receiving isavuconazole as prophylaxis or treatment (Sipsas et al, 2018).

Laboratory Detection, Recovery, and Identification

Culture

A rapid growing, loose, white mold is visible within 24 to 48 hours. With age, and the formation of sporangia, the colony becomes dark gray. Called “pin molds” because the sporangia containing dark spores are visible to the unaided eye (Fig. 10). The same appearance occurs in tissue sections. Species are identified by their pattern of sporulation in culture. 

Medium for growth. Emmons-modified Sabouraud Dextrose agar (SDA-Emmons) with antibiotics is the medium of choice. Mucorales grow rapidly with antibiotics and without antifungal agents such as cycloheximide. Mucorales also grow on medium containing the fungicide Benomyl (10 µg/mL). Some Mucorales grow at elevated temp. so that cultures are best grown at 25 ^oC, 35 ^oC, 40 ^oC and 45^oC. Please see Reiss et al., for a temperature tolerance table for the Mucorales (Table 17A.2) (Reiss et al., 2011)

Failure to grow from autopsy and pre mortem specimens may occur because grinding tissues may kill their sparsely septate hyphae. Failure to sporulate of some Mucorales species, such as Apophysomyces, may require special conditions. Ways to overcome these obstacles are discussed in Reiss et al., 2011.

 

Microscopic morphology of selected Mucorales

Features common to the microscopic morphology of Mucorales are that the hyphae are wide (10-15 microns), ribbon-like and pauci-septate. The only septae separate the sporulating structures from the vegetative hyphae. Asexual spores are produced in sporangia borne on sporangiophores. At the base of the sporangia is a bulbous enlarged area, the columella.The sporangiophores of some species are anchored by rootlike structures: the rhizoids.

Apophysis: Applies to the funnel-shaped swelling of a sporangiophore, immediately below the columella, seen in some Mucorales.

Stolons. A hypha that grows horizontally along the surface of the substrate and from which sporangiophores and rhizoids arise. 

Rhizopus oryzae (syn. R. arrhizus) Fig. 8. (with description)

Apophysomyces elegans Fig. 9 (with description)

 

Histopathology

Tissue grinding disrupts delicate Mucorales hyphae. Mincing or use of Seward Stomacher® is recommended. Fungal silver stain (GMS) is most useful and readily demonstrates the hyphae in tissue which are large (5-15 µm diam.) hyaline, non- or pauci-septate. Short hyphal elements are seen near or in blood vessels (Gade et al., 2017). Tissue is invaded in many directions resulting in bizarre, varicose shapes, wrinkled, or collapsed. In cross section they may resemble yeast cells. (Fig. 11)
 

Selected References

 

Fig.13A Microscopic morphology of Fusarium solani : macroconidia

Species of Fusarium typically produce both macro- and microconidia. Macroconidia are formed after 4-7 days from short multi-branched conidiophores, They are 3- to 5- septate (usually 3- septate), fusiform, cylindrical, often moderately curved.

Source: Geraldine Kaminski library of mycology # 468

Fig. 13B. Microscopic morphology of Fusarium solani: microconidia

In F. solani, microconidia are usually abundant, cylindrical to oval, 1- to 2-celled and formed from long lateral phialides

Source: Geraldine Kaminski library of mycology # 467

FUSARIUM MYCOSIS

Introduction/Disease Definition

This is a large genus of plant pathogens also causing disease in humans especially in Immuncompromised patients. Clinical forms include paronychia, sinusitis, pulmonary, and extra-pulmonary disseminated infections. In immune normal patients: locally invasive disease-- mycotic keratitis (at risk are contact lens wearers) and endophthalmitis, onychomycosis, burn infections, sepsis in peritoneal dialysis patients.  

Etiologic Agents

Major pathogens are Fusarium solani and F.  oxysporum. Each existing as a species complex.  

Geographic Distribution/Ecologic Niche

World-wide, all climatic zones in soil, plant debris, on plants.  Dispersal of conidia occurs in air and in water (including hospital water supply, especially F. oxysporum.

Incidence

Fusarium species are the most frequent mold cause of mycotic keratitis and are second to Aspergillus as cause of invasive mycosis in stem cell transplant (SCT) patients. Rates vary widely among institutions.

Risk Groups/Factors

• Onychomycosis-- walking barefoot 

• Keratitis-- Soft contact lens wearers, field-workers exposed to potential eye injury

• Abdominal sepsis-- Peritoneal dialysis.

• Sino-pulmonary and disseminated– Prolonged, profound neutropenia, systemic corticosteroid therapy,  especially among patients with leukemia or those receiving stem cell transplants.
   

Transmission

Inhaled conidia including aerosols from hospital water supply or other water source; ingestion or injection through skin trauma.

Determinants of Pathogenicity

Produce mycotoxins but their role in disease is unknown. Angioinvasive tendency leading to thrombosis, infarcts.

Clinical Forms

In immune-normal persons: 

Nail infections, keratitis, and secondary infections in burn patients.

Keratitis

Fusarium spp. keratitis is a sight-threatening disease often affecting otherwise-healthy patients. The infection is difficult to treat because Fusarium spp. are highly resistant to most antifungals. Corneal infection may progress to endophthalmitis with poor visual outcome (Walther et al, 2017). Find the recommendations of the Centers for Disease Control for contact lens wearers at: https://www.cdc.gov/contactlenses/index.html

Fusarium spp keratitis is associated with contact lens wear, trauma, including surgery, and immunosuppressive disease or immunosuppressive medication. Common in tropical and subtropical countries where the main risk is traumatic damage to the cornea. Increased use of contact lenses poses a risk for Fusarium keratitis in urban areas and temperate climates. An international outbreak, (2005 to 2006) of keratitis in contact lens wearers was linked to decreased activity of antimicrobial alexidine against Fusarium spp. after heating the cleaning solution ReNu with MoistureLoc.   

Epidemiologic analyses are affected by the complex taxonomy of the genus Fusarium which consists of closely related species with similar morphology combined into 20 species complexes. Eye infections are predominantly caused by the Fusarium solani species complex (FSSC). But morphology is insufficient for differentiation which is reliant on molecular methods for species identification.

Members of the FSSC account for most keratitis infections world-wide, and require longer therapeutic courses, increased need for keratoplasty and even then are associated with poorer outcome than infections with other Fusarium spp.

Therapy

Invasive treatment of Fusarium keratitis and endophthalmitis is frequently necessary.

Outcome can be poor including the possibility of enucleations. Long delays before the correct diagnosis is a risk factor for poor outcome. Steroid use which suppresses inflammation may also contribute to delayed diagnosis due to intraocular spread.

Amphotericin B has the lowest MICs for Fusarium spp.  MICs for another polyene, natamycin, are higher but isolates with MICs of <16 µg/mL are considered susceptible to natamycin, because those concentrations are reached in the eye. Natamycin, due to its smaller molecular size, may more easily penetrate the eye than AmB.

Resistance to most antifungals is typical of Fusarium species which are innately resistant to echinocandins, and have high MICs with respect to azoles. All isolates of the FSSC had MICs for terbinafine of >32 µg/mL, whereas other Fusarium species had terbinafine MICs of < 8 µg/mL.  

Risk factors

• Use of soft contact lenses is possibly the most important risk factor

• Some lens disinfectant solutions used are not effective against Fusarium spp. especially when heated.

•  “No-rub” multipurpose contact lens solutions

• Higher frequency of silicone hydrogel lenses

• Overnight use and poor lens care

• Refilling bottles of disinfectant solution, 
   

In immunocompromised patients:  Hematogenously disseminated disease with skin lesions, Invasive sinusitis, nail infections with paronychia, and also pulmonary mycosis. Skin lesions are multiple, purpuric nodules with central necrosis.

Therapy

Deep-seated Fusarium spp. infections are refractory to treatment. The drug of choice for the treatment of invasive fusariosis is either voriconazole or liposomal amphotericin B. The outcome is usually poor, and largely dependent on the recovery of the immune status of the host, particularly from neutropenia. Natamycin drops for keratitis; nystatin cream for skin lesions.

Laboratory Detection, Recovery, and Identification

Skin lesions and blood cultures are the main specimens for diagnosis.

Biopsy of skin lesions for histopathology and culture; Histopathology reveals branching hyaline, septate hyphae, invading blood vessels.

In contrast to aspergillosis, blood cultures are frequently positive.

KOH preparations from nail clippings, skin scrapings, corneal specimens in cases of keratitis are useful. 

Culture

The genus Fusarium can be identified in culture by the production of hyaline, crescent, or banana-shaped, multicelled macroconidia but identification using morphology relies on few specific criteria (Tortorano et al., 2014) 

Colony morphology

Rapid growing, in a variety of colors: blue-green, beige, salmon, lavender, red, violet and purple (Fig. 12).

Microscopic morphology

Macro and microconidia are present. Macroconidia, sickle shaped with 3-5 septa and 14-80 µm in length, borne on single or branched conidiophores with phialides.   Microconidia are borne on long or short simple conidiophores, single or double celled, ovoid to cylindrical shaped in mucous balls or short chains (Figs 13A, 13B) 

Molecular ID of Fusarium species

Molecular methods overcome the difficulties of phenotypic identification. Phylogenetic species recognition is based on genealogic concordance of multilocus DNA sequence data and has identified > 69 clinically important Fusarium species (O’Donnell et al., 2010).   

Many cryptic species are in the Fusarium solani species complex (FSSC) and F. incarnatum-F. equiseti species complexes (FIESC). These two complexes contain at least 75 species, including 41 found in human and animal mycoses.  Multilocus DNA sequence data determines species boundaries accurately, including the epidemiologically important haplotypes such as the widespread F. oxysporum species complex 3-a (FOSC 3-a) and Fusarium solani complex FSSC 1-a and 2-d. The latter is common in hospital water systems.

O’Donnell et al. (2010) devised a three-locus DNA sequence database for 69 human opportunistic/pathogenic Fusarium species.

EF-1α, RPB1, and RPB2 are the 3 loci.  (RPB1 is the DNA-directed RNA polymerase largest subunit).   

• They established an internet-accessed database to accurately identify and place novel Fusarium isolates in their proper phylogenetic position. 

• This effort includes an archive of sets of isolates from Europe and the U.S.

• The Fusarium database is found at the Centraalbureau voor Schimmelcultures (CBS) Biodiversity Centre to facilitate global access 
   

A large scale genomic analysis of the human and plant pathogens in the genus Fusarium is in Geiser et al., 2013

MALDI-TOF MS species identification 

A database of spectra was constructed from 289 strains belonging to 40 Fusarium species (Triest et al., 2015). Depending on the score value using MALDI Biotyper 3.0 software a value of 2.0 as identification cutoff resulted in 82.8% correct identifications to the species level. Decreasing the cutoff from 2.0 to 1.4 resulted in rates of 91.0% correct species-level identifications.  Correct identifications increased to 97.0% when some Fusarium species complexes were taken as a whole. MALDI-TOF MS identification failed with only 4 Fusarium strains in that study (F. incarnatum, F. equiseti, F. sporotrichioides and F. sacchari). Antifungal susceptibility testing of the isolates suggested that discriminating between Fusarium species complexes may be unnecessary for members of the same species complex.  

Selected References

Fig. 14. Microscopic morphology Scedosporium apiospermum.

Microscopic morphology of Scedosprium apiospermum shows numerous, single-celled, pale-brown, broadly clavate to ovoid conidia, borne singly or in small groups on elongate, simple or branched conidiophores or laterally on hyphae.

Source: Geraldine Kaminski library of mycology 525.

PSEUDALLESCHERIA/SCEDOSPORIUM MYCOSIS

Introduction/ Disease Definition

Subcutaneous, sino-pulmonary, and pulmonary-disseminated infections caused by the molds in the Pseudallescheria boydii species complex and other Scedosporium species. Portal of entry is either respiratory via inhalation of conidia or by traumatic implantation of thorny plants and splinters. Eumycetoma is a special category of subcutaneous mycosis and is discussed separately

Diagnosis

Specimens including biopsy material, BAL fluid, bronchial washings, CSF, sputum, tracheal aspirates are examined in 10% KOH preps. Detection is improved with Calcofluor fluorescent brightener.  Gram-stained smears show acute angle branched, septate hyaline hyphae similar to that of other hyaline molds, but may show an irregular branching pattern. 
 

Etiologic Agents

Environmental molds:  Pseudallescheria boydii; Scedosporium apiospermum and minor species.  Scedosporium prolificans reclassed as Lomentospora prolificans is a melanized mold.

Geographic Distribution/Ecologic Niche

Worldwide agents of sino-pulmonary and pulmonary- disseminated mycosis in immunocompromised hosts.  Causes eumycetoma in N America. These species are isolated from agricultural soil, sewage, and polluted water.

Epidemiology

An emerging mycosis in immunocompromised hosts, third in prevalence behind aspergillosis and Fusarium mycosis. Approximately 1%-27% (depending on study design) of opportunistic mold mycoses are caused by non-Aspergillus species molds.

Risk Groups/Factors

Immune-normal host: occupational or recreational exposure to penetrating injuries, contact lens wearers, near-drowning accident victims, cystic fibrosis patients. Immunocompromised host: Patients with hematologic malignancy, stem cell- and organ transplant recipients.

Transmission

Traumatic implantation or inhalation of conidia, aspiration of conidia in water (near drowning accidents). 

Determinants of Pathogenicity

• Adventitious sporulation. Conidia produced in tissue by S. apiospermum and L. prolificans contribute to hematogenous spread. 

• Cell wall α-D-glucan of conidia and hyphae is recognized by Toll receptor 2 (TLR-2) leading to phagocytosis or endocytosis.  L. prolificans glycoproteins on conidia bind to epithelial cells.

• Enzymes.  Wall-associated alkaline- and acid phosphatases and a secreted metallopeptidase 

• Melanin.    L. prolificans is melanized, which protects the pathogen.

Clinical Forms

Fungal sinusitis, pneumonia, fungus ball in a preexisting pulmonary cavity, extrapulmonary dissemination, cerebral abscess. May be neurotropic with the CNS as a site of dissemination. Also a cause of Eumycetoma (See EUMYCETOMA sub-section, below)
 

Therapy

Pseudallescheria/Scedosporium species are less susceptible to AmB than Aspergillus fumigatus; voriconazole (VRC) is the preferred therapy; L. prolificans is refractory to most monotherapy, may be responsive to combination therapy.
 

Laboratory Detection, Recovery, and Identification

Specimens for direct examination and culture: grains, skin scrapings, exudates, sputum, sinus drainage, invasive biopsy including drainage from closed cerebral abscess; blood cultures. Growth is rapid on SDA. May demonstrate sexual reproduction: cleistothecia with ascospores after 2-3 weeks incubation, or after transfer to cornmeal agar. Asexual reproduction is enteroblastic with single large obovoid annelloconidia (Fig. 14). Sometimes conidiophores are in small clusters: synnemata.  Histopathology: difficult to distinguish from Aspergillus species. Sometimes chlamydospores and conidia are produced in vivo; L. prolificans gives a positive Fontana-Masson staining reaction in tissue for melanin.

Fig. 15
Chromoblastomycosis, foot. Source: CDC/Dr. Libero Ajello

Fig. 16. Histopathology. Sclerotic bodies (muriform cells) chromoblastomycosis

  Fig. 17 Microscopic morphology of Cladophialophora carrionii

Microscopic morphology of Cladophialophora carrionii (magnified 475X). Elongate conidiophores produce branched acropetal chains of smooth-walled conidia. A common cause of chromoblastomycosis particularly prevalent in arid and semi-arid areas, most often in tropical and subtropical zones.

Source: CDC/Dr. Lucille K. Georg

Fig. 18 Microscopic morphology Phialophora verrucosa

Conidia-laden conidiophores of a Phialophora verrucosa from a slide culture. Flask-shaped phialides, each lipped by a collarette. Each phialide terminates in a bundle of round, to ovoid conidia. Phialophora spp. are causative agents of both chromoblastomycosis, and phaeohyphomycosis.

Source: CDC/Dr. Libero Ajello

MYCOSES OF IMPLANTATION CAUSED BY MELANIZED MOLDS

CHROMOBLASTOMYCOSIS
 

Introduction/Disease Definition

This is a chronic, localized infection of subcutaneous tissues caused by several species of melanized fungi. The route of entry is usually by penetrating injury with a thorn or splinter containing the fungal elements resulting in lesions most often on the arm, foot or leg. Most aspects of this disease are reviewed in Queiroz-Telles et al., 2017.  

Diagnosis

Physical diagnosis depends on the presence of chronic cutaneous and subcutaneous nodules or plaques that enlarge over time to become verrucous (Fig. 15).   Laboratory diagnosis is facilitated by histopathology and culture. The tissue form is unique and diagnostic, consisting of a cluster of round, thick-walled cells that divide by internal cleavage planes (def:  sclerotic bodies, or muriform cells) (Fig. 16).  They appear in H&E stained tissue as brown or copper-colored, the so-called “copper pennies”. These cells occur in tissue of most if not all agents of chromoblastomycosis. Speciation is determined by the pattern of conidiogenesis of the mold form in slide cultures on SDA agar.

Etiologic Agents

The etiologic agents of chromoblastomycosis are septate, slow growing molds, darkly pigmented members of a single family the Herpotrichiellaceae. The most common agents are: 

• Fonsecaea pedrosoi   

• Cladophialophora carrionii (Fig. 17)  
  The above 2 species are the most common causes of chromoblastomycosis. C. carrionii occurs in semiarid areas, whereas Fonsecaea pedrosoi is associated with humid climates

• Phialophora verrucosa (Fig. 18)

•  Rhinocladiella aquaspersa is a less frequent causative agent.

Exophiala spp. are occasional causes of chromoblastomycosis but most often cause other infections. This genus produces a mucoid yeast form, but as the culture ages the mucoid appearance dries and becomes more hyphal.

Geographic Distribution/Ecologic Niche

The causative agents are saprobes located in soil and decaying vegetation, world-wide.
 

Epidemiology

Incidence and Prevalence

World-wide with increased prevalence in subtropics or tropics. May also occur in the southern U.S.A.

Risk Groups/Factors

 Barefoot labor in tropical or subtropical areas.
 

Transmission

There is no known person-to-person transmission.
 

Clinical Forms

These fungi induce a granulomatous reaction: pseudoepitheliomatous hyperplasia. The lesions, growing slowly over mo or years become cauliflower-like, with the affected limb becoming distorted by fibrosis and lymphedema (Fig. 15)
 

Therapy

Small early lesions can be removed surgically. Lesions in moderate to severe clinical forms are refractory and healing is very difficult, requiring long-term systemic antifungal treatment. The following methods are used in addition to antifungal agents:

Cryotherapy. Recommended for patients treated with antifungal agents resulting in decreased size of lesions. Liquid nitrogen is applied to small lesions; larger lesions receive cryotherapy in sections. The agent is applied with a cotton tip applicator or spray.

 Heat therapy. Heat above the max growth temp of the agents (42-46oC) applied for a period of 1 mo with a chemical pocket hand warmer in an occluded bandage for the entire day. Lesion size should subside after this period. Resolution of lesions has been seen within ~2 mo.

 Laser therapy. CO2 laser has been applied with good effect alone or combined with other treatment.

Photodynamic therapy (PDT). PDT combines visible light of a coherent wavelength to stimulate a photosensitizer leading to target cell damage.  PDT with a red light-emitting diode light combined with methylene blue photosensitizer was well-tolerated producing remission of lesions (Lyon et al., 2011).

 

Laboratory Detection, Recovery, and  Identification
 

Direct examination. Surface scrapings from the surface of warty lesions mounted in 10% KOH preps may reveal round brown sclerotic bodies (muriform cells) dividing by transverse septations.
 

Histopathology. Biopsy of skin lesions stained with H&E will also show the characteristic sclerotic bodies (Fig. 18).
 

Culture. Crust, exudates, part of minced biopsy specimens planted on SDA with antibiotics and cycloheximide. Small dark green to black colonies become evident after 10 d to 2 wks. Species identification is determined by slide culture on PDA.
 

In Vitro Antifungal Susceptibility

Itraconazole, posaconazole, voriconazole and isavuconazole show the best in vitro activity against agents of chromoblastomycosis, but amphotericin B, 5-fluorcytosine, fluconazole and echinocandins have limited activity.

First-Line Therapy

Itraconazole is standard therapy for chromoblastomycosis with cure rates ranging from 15-to 80%. An oral capsule form shows clinically significant activity against most chromoblastomycosis agents being more effective against C. carrionii than against F. pedrosoi.  Clinical and mycologic cure can be achieved with long term therapy for many mo. When lesion reduction has occurred cryosurgery can be applied to remove the remaining lesions. 

Terbinafine is the second most frequently used antifungal agent for chromoblastomycosis with cure rates after long-term therapy similar to those achieved with itraconazole. Terbinafine has fewer drug interactions than itraconazole and may also possess an antifibrotic effect.

Combination therapy, usually with itraconazole and terbinafine, has been used as the last option salvage therapy for refractory or advanced chromoblastomycosis or when monotherapy has failed. In vitro studies have not shown synergism or antagonism of this combination.

Among extended-spectrum triazoles, posaconazole has the best potential for treating chromoblastomycosis, including severe or refractory forms. Posaconazole has broad in vitro activity against most melanized fungi including causative agents of chromoblastomycosis and phaeohyphomycosis. In addition to oral suspensions   are newer delayed released tablets. Delayed-release posaconazole tablets may achieve higher average plasma concentrations than those achieved with the oral solution and are well-tolerated. Posaconazole oral solution has better pharmacodynamic and pharmacokinetic profiles than the itraconazole capsule formulation. 

Oral voriconazole has been used to good effect in a few cases of refractory disease.   Isavuconazole is very effective in vitro against melanized fungi, and is another therapeutic option. 

Selected References

See text for description. Photo credit: Hernández-Restrepo M, et al., 2018 (open access creative commons license)

PHAEOHYPHOMYCOSIS (Phaeo def: (Gr.: dark)

Phaeohyphomycosis includes 3 different clinical forms:

–        Cutaneous-subcutaneous cysts:  lesions are cystic, nodular, verrucous, ulcerated, or plaque types

–        Cerebral abscess and other deep-seated sites of infection

–        Fungal sinusitis

The disease is defined by the appearance of fungi in tissue (a histopathologic definition): 

–         It includes melanized molds and yeastlike relatives which grow in tissue or in the nasal sinuses as hyphae, or a mix of hyphae and yeast forms.

Clinical Forms and their Etiologic agents

Subcutaneous cyst form. Wangiella (=Exophiala) dermatitidis, Exophiala jeanselmei, Lomentospora prolificans. Less frequent agents are: Alternaria spp., Bipolaris spicifera (=Curvularia spicifera), Curvularia lunata, Exserohilum rostratum.

Brain abscess form: Cladophialophora bantiana (thermotolerant to 42-45oC)

Fungal sinusitis form (allergic fungal sinusitis): Bipolaris spicifera.

 

Therapy

Allergic fungal sinusitis: Surgery, steroids, itraconazole.

 Cerebral abscess: Surgery (if possible), extended spectrum azoles and liposomal amphotericin B, or echinocandin, possible role for 5-fluorocytosine.against Cladophialophora bantiana.

Subcutaneous cysts. Surgery, azoles

Epidemiologic Highlight

Epidemiologic Highlight: Largest outbreak of health care-associated infections ever reported in the U.S.

About Exserohilum rostratum

Ex­serohilum is a melanized mold classed in the Pleosporaceae, Pleosporales in a well-supported clade separate from related Curvularia and Bipolaris spp. Exserohilum may infect immunocompromised and immune-normal hosts with clinical forms ranging from cutaneous infections (phaeohyphomycosis) to disseminated disease, also including allergic fungal sinusitis.  E. rostratum is a plant pathogen affecting a range of species particularly grasses.

Growth is rapid on PDA plates (sporulation more intense on 15% V-8 juice agar.) E. rostratum grows on medium containing benomyl, 10 µg/mL.

Colony morphology

Deep brown surface and black reverse. Wooly or cottony aerial mycelia.

Microscopic morphology

Conidia are straight or slightly bent, ellipsoid to fusiform (some rostrate= beak-like), and typically with 7- 9 septa (some with 4-14) per conidium (Fig. 19) Conidia have a protruding, truncate hilum (Hernández-Restrepo et al., 2018). The septum above the hilum is thickened and dark.  End cells often paler than other cells (McGinnis et al., 1986).

The clinical relevance of MIC data for Exserohilum has not been established. Amphotericin B, itraconazole, and posaconazole were the most active drugs in vitro exhibiting MICs of <1 µg/ml, with both isavuconazole and voriconazole showing MICs below achievable serum trough levels (Chowdhary et al., 2015).

Selected References

 

A massive foot eumycetoma with multiple sinuses and discharge with black grains.

Source: posted to FigShare.com on 27.03.2015, 00:49 by Ahmed Fahal EL Sheikh Mahgoub Ahmed M. EL Hassan Manar Elsheikh Abdel-Rahman
Creative commons license.
 

EUMYCETOMA

Mycetomas are localized chronic granulomatous infections of the cutaneous and subcutaneous tissues, usually on the lower extremities (less commonly on the hand, chest or back). Infection initiates after a penetrating injury from plant material, a thorn or splinter, containing the fungus.  Slowly, over months and years, multiple granulomas and abscesses develop containing large aggregates of fungal hyphae (Fig. 20). (Fahal et al., 2015)

Fistulas form with draining sinus tracts discharging grains, which are compact masses of fungal hyphae embedded in a cement-like extracellular matrix. Fibrosis around the grains is often severe. Tumefaction (indurated swelling) of the limb ensues. Eventually the affected limb is deformed with destruction of muscle, fascia, and bone (osteomyelitis). Hematogenous or lymphatic spread from a primary focus to distant sites is very rare. Synonyms: Madura foot, anthill foot (arch.). Eumycetoma is caused by a variety of true fungi, whereas bacterial mycetoma is caused by actinomycetes.  Diagnosis of the etiologic agent, whether eumycetoma or actinomycetoma, is essential for patient management because the prognosis and therapy differs.  
 

Geographic distribution

Eumycetoma is endemic in the tropics and subtropics between latitudes 15°S and 30°N around the Tropic of Cancer (23.5^oN).  The major endemic area is in Sudan; other areas are Mexico, Central and S America, India, Indonesia, Pakistan, other African countries, the Middle East, occasionally in temperate zones including the U.S.A.

Etiologic agents

The number of fungi producing eumycetoma is upwards of 21-30 species; both hyaline and melanized molds may be involved. Madurella mycetomatis, occurring in the arid regions of Africa, accounts for most cases worldwide, causing > 70% of cases in Central Africa including the Sudan. Madurella grisea is a common etiologic agent in S America.  Leptosphaeria senegalensis and Leptosphaeria tompkinsii are common causes in W Africa. Pseudallescheria boydii and Scedosporium spp. are the most common causes in U.S.A. Major agents of actinomycotic mycetoma are Nocardia brasiliensis, Actinomadura madurae, Streptomyces somaliensis,

Therapy

Surgery and long-term oral azole and/or terbinafine therapy. Most patients with eumycetoma are treated with either ketoconazole or itraconazole (Welsh et al., 2014). Itraconazole 200–400 mg/d for 6 mo is used to create a fibrous capsule around the lesion, followed by wide local excision, continuing itraconazole, 200–400 mg/d, until cure is achieved. Cure is defined as disappearance of the mass, all sinuses, normal ultrasound, and negative mycology. The decision to stop therapy is determined by complete sinus healing, disappearance of the eumycetoma mass clinically and radiologically by CT scan or MRI, and absence of the infecting agent. Other antifungal agents used as second-line treatment include voriconazole and posaconazole.

Laboratory

Detection is by examination of grains, biopsies, curettage from sinuses, and culture, molecular identification: PCR of rDNA. Grains are seen in a variety of colors (white, brown, yellow, black)

The three most common etiologic agents are:

• Madurella mycetomatis (black grain)

• *Exophiala jeanselmei (black grain)

• * Pseudallescheria boydii (white grains) also Scedosporium spp.

*The most common in the US. These organisms are associated with the soil, thus you see many infections in the feet and legs.

Clinical specimens for diagnosis: are pus containing grains, white, yellow, brown, or black; and tissue for histology.

The color, size and texture of the grains aid in the diagnosis of mycetomas.  Eumycotic mycetoma grains are visible to the unaided eye and measure 0.2 mm to several mm diam and contain hyphae 2-6  µm or larger in diam. Grains containing actinomycetes, have bacterial elements <2 µm diam. Washed grains mounted in 10%KOH are examined microscopically for size of filaments, color.

Culture

The agents of eumycetoma are all filamentous fungi which require at least 7-10 days for visible growth on the culture media and then another several days for specific identification. These fungi are identified by their colonial morphology, conidia formation, and biochemical reactions. Medium for fungal growth is Emmons’ modified Sabouraud dextrose agar containing antibiotics but not cycloheximide. Cultures are held for 6-8 wks.

Histopathology

H&E is a good first choice stain because microscopic structure of grains is readily visible. Gomori methenamine silver (GMS) or PAS stain is applied for eumycetoma grains, whereas Brown and Brenn stain is used for actinomycotic mycetoma. The species of fungi cannot be distinguished in histopathologic tissue sections. Fungi appear as branched septate hyphae, often distorted in shape and with chlamydospores at the periphery of grains. Images of histopathologic sections may be found in Chandler et al., 1980.

Selected References

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