—  SPECIALTY CONFERENCE  —

Infectious Disease Pathology

Case 5 - Fungal Infections

Jeannette Guarner, Emory University, Atlanta, GA





Click on slide thumbnail images for an enlarged view.

If you have any difficulties viewing these slides, email the webmaster.



Clinical History
54 year old woman that is being worked up for a lung mass that was seen in a chest X-Ray.


Case 5 - Figure 1
Case 5 - Figure 2

Introduction:
Coccidioides immitis is endemic in the San Joaquin valley in California. Coccidioides posadasii is present in desertic regions of Northwest Mexico, Arizona and New Mexico, and desertic areas in Argentina and Paraguay. However, very little differences have been found in morphology or clinical presentation between the 2 species. There is a clear correlation between incidence of disease and environmental factors; coccidioidomycosis increases when there are rainy summers followed by dry winters, following earthquakes, or when humans settle and develop the previously mentioned desertic areas. In any of these instances, Coccidioides arthrospores are released in higher numbers than the usual baseline.

Humans inhale the arthroconidia, which in the lung are transformed into multinucleated spherical structures that contain hundreds of endospores. It is estimated that up to 60% of individuals exposed have no symptoms while the remainder may present with what appears to be an acute community acquired pneumonia. In those patients with acute pneumonia, the chest X rays show lobar infiltrates and adenopathy. Several erythematous rashes (multiforme, nodosum or toxic) are reflections of immune response to the acute infection. The majority of acute pulmonary infections resolve; however, in a minority of patients the infection may become chronic progressive ("Valley fever") showing either a cavity or nodule. Extrapulmonary disease can occur in certain risk groups including African Americans, Asians, pregnant women, patients with diabetes, or those patients receiving corticosteroids for a variety of conditions. The most common sites of dissemination include skin, lymph nodes, bones, and joints; nonetheless the most feared complication is extension to the central nervous system. Besides acquiring infection through the respiratory route, there are rare reports of direct inoculation of skin giving rise to primary cutaneous lesions or acquisition through transplanted organs.

Pathological/Microscopic Findings and any Immunohistochemical or Other Studies:
The spherules of various sizes (10 to 100 microns) with multiple endospores (2 to 5 microns) are characteristic of coccidioidomycosis and can be seen with routine H&E stain. The walls of some of the spherules may appear ruptured and the endospores spill into surrounding tissues. Active lesions contain multiple organisms while resolving or residual lesions usually show lower number of organisms. GMS highlights spherule walls and endospores. In contrast, reactions with PAS vary with age of the structures: young endospores and spherules stain strongly while staining fades as the organisms mature. Mycelia can be observed in cavitary lung or skin lesions. Sensitivity for histopathologic detection of Coccidioides is 84% and 75% for cytology.

Inflammatory reaction to endospores is predominantly neutrophilic while reaction to spherules is granulomatous. Thus, early in the infection the lesions tend to show pyogranulomas because the concentration of spherules and endospores is high. Lymphocytic clusters of B and T cells have been described around well formed granulomas with necrosis and appear to be an important response to coccidioidomycosis. Eosinophils can be abundant and can create the Splendore-Höeppli phenomenon (an intense rim of eosinophilic material around the fungal elements). When coccidioidomycosis is suspected it is important to look for spherules. This should be done by going deeper into the blocks or submitting/ staining other blocks. It also needs to be remembered that in immunosuppressed patients, more than one infection may coexist, thus in endemic areas Pneumocysitis and Coccidioides could be found in the same specimen.

In the United States, Coccidioides spp. are select agents that are governed by specific rules related to the possession, use, and transfer of these agents. This fungus grows easily in the laboratory (93% sensitivity) and the arthroconidia can be easily aerosolized, thus all work with cultures must be performed in a class II biological safety cabinet. Detection of antibodies to Coccidioides can be an important diagnostic tool. Today, IgM and IgG are generally measured using EIA and/or immunodiffusion; however, some laboratories continue to perform tube precipitin to measure IgM and complement fixation to measure IgG antibodies. False negative serology has been seen in up to 38% of patients with hematogenous infection and 46% of fatal cases. Detection of antigens in the urine using EIA has been shown to be present in 71% of patients with coccidioidomycosis but shows cross-reaction in 10% of patients with other endemic mycosis.

Differential Diagnoses:
Dimorphic fungi are defined as those that appear as yeast forms at 37°C but are molds when cultured at colder temperatures (usually 25°C). Coccidioides is a dimorphic fungus and, with a few exceptions, most of the fungi in the differential diagnosis are also dimorphic.

1. Rhinosporidium seeberi, a mesomycetozoan parasite that causes palate and nasopharyngeal polyps, is in the differential diagnosis of coccidioidomycosis because it produces large sporangia (some can be seen with the naked eye) with multiple internal endospores. R. seeberi has very similar morphology but its sporangia and endospores are bigger than Coccidioides spherules, and its inner sporangial wall stains with mucicarmine. It is important to remember that endospores outside spherules or young spherules without endospores can be confused with Blastomyces, Histoplasma, Emmonsia, Candida, Pneumocystis and other yeasts.

2. Blastomyces dermatitidis in tissue appear as yeasts that measure 8 to 15 microns in diameter, have thick refractile cell walls, and may show a single, broad-based bud. The yeasts can be observed in a variety of specimens including sputum, bronchoalveolar lavage, fine needle aspirates from lung, skin or other lesions, cerebrospinal fluid and surgical resections. The thick refractile cell wall of this organism gives the appearance of a space between the fungal cell contents and the surrounding tissue when hematoxylin and eosin (H&E) stain is used. Inside the cell wall, the multiple nuclei of the yeasts stain with hematoxylin. Occasionally, B. dermatitidis can show smaller yeast forms, the so called microforms. In addition, B. dermatitidis can be seen with a variety of routinely used preparations and stains such as KOH and Papanicolau. The contour of the yeast is best highlighted by staining the cell wall with fungal silver stains such as GMS or PAS. The inflammatory reaction accompanying the yeasts is primarily granulomatous with varying degrees of neutrophilic infiltrate, thus it has been described as pyogranulomatous inflammation. It needs to be remembered that blastomycosis can be concomitantly present with neoplasias and tuberculosis.

Few studies have systematically compared the presence of broad-based budding yeasts in histopathologic or cytologic specimens with culture or other diagnostic methods that would confirm the diagnosis of blastomycosis. A retrospective study of 53 patients showed that Coccidioides immitis, Candida albicans or Aspergillus were recovered from 4 (10%) pathologic specimens demonstrating broad-based budding yeasts in direct histopathologic examination. An earlier study of patients with blastomycosis commented that a high percent of their cultures were overgrown with Candida. This suggests that not all broad based-budding yeasts in the 8 to 15 micron size range are Blastomyces. Since histopathologic or cytologic results can usually be provided before the culture is available, there is pressure to use these results to guide treatment, particularly because B. dermatitidis can take up to 3 weeks to grow or may not grow at all from these specimens. Sensitivity of culture varies depending on the sample that was obtained, and may range from 62% to 100%. Diagnostic yield of histopathology will depend on the expertise present in the center where the patient is seen. Because of the possibility of histopathologic false positive results, pathologists should describe the yeast and budding pattern that is observed in the tissue specimen, and should list the yeasts that can have this morphology in the report comment field. In addition, alternative tests (urine or serum antigen detection or presence of antibodies in serum) may need to be menitioned in the comment, especially in cases from non-endemic areas or when the clinical picture is not typical.

3. Histoplasma capsulatum var. capsulatum in tissue is an oval 2 to 4 micron yeast that may show narrow-based buds. With H&E stain, the basophilic yeast cytoplasm is separated from the surrounding tissue by a clear zone corresponding to the cell wall. GMS and PAS stains highlight the cell wall. Because the yeasts are initially ingested by macrophages, they appear to be clustered and some authors have suggested this is an important diagnostic feature. This clustering within histiocytes and occasionally within neutrophils is the presentation of Histoplasma in fluids stained with Papanicolaou stain or blood smears stained with Giemsa. H. capsulatum var. duboisii is larger (8 to 15 microns in diameter) than var. capsulatum, and has similar morphology but is pigmented.

Few lung tissue samples of cases with acute pulmonary histoplasmosis have been studied and have shown nodular areas of parenchymal and vascular necrosis associated with lympho-histiocytic vasculitis. The histopathologic picture resembles lymphomatoid granulomatosis but scattered small granulomas with small yeasts in the parenchyma should suggest the diagnosis of histoplasmosis. Chronic lung infections that radiographically appear as coin lesions show typical granulomatous inflammation with central necrosis and calcified material. Yeasts are usually found in this necrotic calcified material, which can be lost during processing and cutting of the tissue. In immunosuppressed patients, sheets of macrophages filled with yeasts characterize disseminated disease. The collections of macrophages distort the organ architecture and produce necrotic areas. Because the morphology of H. capsulatum is not specific, it is important to perform clinico-epidemiological correlation.

Several fungi can be confused with H. capsulatum var. capsulatum when studying tissue sections: The small variant of B. dermatitidis (in these cases the presence of broad-based budding and seeking larger forms can be helpful in making the diagnosis of blastomycosis), capsule deficient cryptococci (in these cases size variation and looking for weakly positive mucicarmine-staining yeasts may suggest the diagnosis of cryptococcosis), endospores of Coccidioides spp. (looking for remnants of a ruptured spherule or an intact spherule is paramount for diagnosis of coccidioidomycosis), Pneumocystis jirovecii (these organisms lack budding and have a silver staining dot), Penicillium marneffei (show formation of a transverse septum rather than budding), lastly, Candida glabrata (may show more size variability than histoplasmosis). In addition, several protozoa can also show intracellular organisms of similar size including the agents of leishmaniasis, toxoplasmosis, and Chagas disease, that should be differentiated from histoplasmosis: The histopathologic difference between these organisms and Histoplasma is that H&E stains the entire protozoan and none show the halo produced by the fungal cell wall. Kinetoplasts (a distinct hematoxylin-stained bar to the side of the nucleus) should be observed if the patient has leishmaniasis or Chagas disease. Infected cells in toxoplasmosis and Chagas disease are somatic (cardiomyocytes, neurons) rather than histiocytes. In summary, definitive diagnosis of histoplasmosis can be difficult in tissue sections and, if cultures were not obtained in the tissue specimen, alternative testing should be considered. Pathologist should describe the yeast in the diagnosis field and in a comment suggest the different fungi that can have this morphology.

4. Another unusual fungus that is in the histopathologic differential diagnosis of coccidioidomycosis is Emmonsia crescens. This dimorphic fungus is inhaled or inoculated into the skin when humans are in close contact with rodent burrows and aerosolized conidia. The amount of conidia inoculated will determine the presentation: Small inocula are asymptomatic and present as walled off granulomas, while larger inocula can result in acute severe pulmonary disease. The disease is known as adiaspiromycosis or haplomycosis, due to the presence in tissue of adiaspores, large thick-walled structures formed by transformation of hyphae. Adiaspores are thick, double-walled spherules that measure 20 to 400 micron or more and are empty or contain eosinophilic hyaline globules. When stained with GMS the entire wall thickness stains and shows fenestrations. Adiaspores usually elicit a granulomatous inflammatory reaction and the differential diagnosis is frequently with helmiths.

5. In tissues Candida organisms appear as mats of yeasts measuring 3 to 5 microns in diameter intermingled with pseudohyphae also referred to as filaments. The filaments may show periodic constrictions. The organisms can be seen with H&E, GMS and PAS. The only Candida species that does not produce filaments is C. glabrata. Because of its inherent resistance to fluconazole, C. glabrata needs to be recognized. C. glabrata is the Candida spp. that can be confused with Histoplasma, Blastomyces, endospores of Coccidioides and Cryptococcus.

6. Atypical inflammatory reactions to Pneumocystis have been documented including interstitial pulmonary fiborisis, granulomas, hyaline membranes, and interstitial lung infiltrates. In these instances, it is difficult to suspect that Pneumocystis is present in the lesion. The usual histopathology of Pneumocystis in lung tissue sections stained with H&E is as foamy intraalveolar eosinophilic exudates with minimal inflammatory infiltrate. In Papanicolaou stained respiratory cytology specimens, the organisms blend into the mucous blue green background. GMS stain demonstrates that the foamy material in tissue sections or cytologic specimens corresponds to multiple organisms which are thin-walled spheres of 2 to 5 microns that have a large capsular dot. Collapsed organisms are usually found intermingled with intact organisms.

Conclusions
Fungal infections are becoming more frequent because of expansion of at-risk populations and use of treatment modalities that permit longer survival of these patients. Some of the changes in endemic fungal infections can be attributed to climate changes, extension of human habitats, ease of travel, and shifting populations. At-risk populations for opportunistic fungal infections or disseminated endemic fungal infections include patients who have received transplants, those prescribed immunosuppressive and chemotherapeutic agents, HIV-infected patients, premature infants, the elderly, and patients undergoing major surgery. Thus, a shift in the mycoses encountered in the healthcare setting has occurred with fungi previously considered non-pathogenic, including mucoraceous genera (formerly zygomycetes) and a variety of both hyaline and dematiaceous molds, now being commonly seen in immunocompromised patients. Diagnosis of infections outside the usual geographic or epidemiologic setting can be challenging. Furthermore, advances in diagnostic radiology have allowed greater ability to pursue specific diagnoses by collecting tissue biopsies from body sites formerly not available for histopathologic examination. In addition, the therapeutic armamentarium now available and the presence of resistance to different drugs by these fungi have compounded the diagnostic challenges. Nowadays, the use of fungal GMS cannot solve alone these challenges and newer diagnostic techniques may be required. However, histopathology continues to be a rapid and cost- effective means of providing a presumptive or definitive diagnosis of an invasive fungal infection while waiting for fungal culture results. Histopathology may be the only material available when no culture growth occurs or cultures were not ordered. Thus, microbiologists, pathologists and clinicians need to be aware of the limitations of tissue diagnosis, the pitfalls of morphologic diagnosis, and the tests that can be performed in tissue and other samples to make organism-specific diagnoses.

Retrospective studies that correlate culture results with histopathology and cytology showed that overall accuracy for microscopic morphologic techniques can vary from 20 to 80%. The lowest correlation has been reported for invasive septate molds. Even though GMS and PAS stains were used more frequently in the cases correctly diagnosed than those misclassified, special stains did not significantly improve pathologists' diagnostic capabilities. Misclassification of cases occurs when the pathologist has a false sense of ability to categorize fungal organisms by genus based on microscopic morphology alone, when inappropriate terminology is used such that other potential molds within a particular category are not included in the differential diagnosis, or when there is lack of knowledge of morphologic mimics of yeasts and hyphal forms. The misclassifications with greater potential for adverse consequences occurred when there were few, folded, fragmented and/or necrotic fungal elements in the specimen and the structures in hyphae could not be adequately categorized as septate versus pauciseptate hyaline molds. Therefore, clinicians need to be aware that misclassifications in histopathologic examination occur in at least 20% of cases, and pathologists need to give as much information as possible without overextending their diagnostic capabilities.

In order to avoid misclassifications, pathologists should describe the fungal elements observed in the sample and refrain from trying to offer a specific diagnosis. Pathologists need to remember that there are very few instances where morphologic characteristics are specific. Some groups have suggested the use of templates or synoptic reporting for the diagnosis of fungal infections. In these templates the diagnosis line includes a description of the yeast or mold, the presence or absence of fungal invasion of tissues and vessels, and the host reaction to the fungus (inflammation, necrosis, hemorrhage). In a second comment field, the pathologist should clearly state the most frequent fungi associated with that morphology as well as other possible organisms (fungi and parasites) that should be considered in the differential diagnosis. All pathology reports should also include a statement in the comment regarding the importance of correlating clinico- epidemiologic features and results of cultures and other laboratory tests that could aid in the diagnosis.

References:
  1. Guarner J, Brandt ME. Histopathologic diagnosis of fungal infections in the 21st century. Clin Micro Rev 2011; 24:247-80

  2. Adam RD, Elliott SP, and Taljanovicc MS. 2009. The spectrum and presentation of disseminated coccidioidomycosis. Am. J. Med. 122:770-777.

  3. Ampel NM. 2009. Coccidioidomycosis: A review of recenta dvances. Clin. Chest. Med. 30:241-251.

  4. Bariola J, Perry P, Pappas PG, et al. 2010. Blastomycosis of the central nervous system: A multicenter review of diagnosis and treatment in the modern era. C. Infect. Dis. 50:797-804.

  5. Brugiere O, Forget E, Biondi G, et al. 2009. Coccidioidomycosis in a lung transplant recipient acquired from the donor graft in France. Transplantation. 88:1319- 1320.

  6. Bueno-Fischer G, Mocelin H, Bittencourt-Severo C, et al. 2009. Histoplasmosis in children. Paed. Resp. Rev. 10:172- 177.

  7. Chandler F, and Watts JC. 1997. Histoplasmosis capsulate, p. 1007-1016. In Connor DH, Chandler FW, Schwartz DA, Manz HJ, and Lack EE (ed.), Pathology of infectious diseases, 1st ed., vol 2. Stamford, Appleton & Lange Co., Hong Kong.

  8. Durkin M, Connolly P, Kuberski T, et al. 2008. Diagnosis of coccidioidomycosis with use of the Coccidioides antigen enzyme immunoassay. Clin. Infect. Dis. 47:e69–73.

  9. Gupta N, Arora S, Rajwanshi A, et al. 2009. Histoplasmosis: cytodiagnosis and review of literature with special emphasis on differential diagnosis on cytomorphology. Cytopathol DOI:10.1111/j.1365- 2303.2009.00693.x.

  10. Hartel PH, Shilo K, Klassen-Fischer M, et al. 2010. Granulomatous reaction to Pneumocystis jirovecii: Clinicopathologic review of 20 cases. Am. J. Surg. Pathol. 34:730-4.

  11. Lemos LB, Guo M, and Baliga M. 2000. Blastomycosis: Organ involvement and etiologic diagnosis. A review of 123 patients from Mississippi. Ann. Diagn. Pathol. 4:391-406.

  12. Li L, Dial SM, Schmelz M, Rennels MA, and Ampel NM. 2005. Cellular immune suppressor activity resides in lymphocyte cell clusters adjacent to granulomata in human coccidioidomycosis. Infect. Immunol. 73:3923-3928.

  13. Luna M. 1997. Candidiasis, p. 953-964. In Connor DH, Chandler FW, Schwartz DA, Manz HJ, and Lack EE. (ed.), Pathology of infectious diseases, 1st ed., vol. 2. Stamford, Appleton & Lange Co., Hong Kong.

  14. Martynowicz M, and Prakash UBS. 2002. Pulmonary blastomycosis: an appraisal of diagnostic techniques. Chest. 121:768-773.

  15. McKinnell JA, and Pappas PG. 2009. Blastomycosis: New insights into diagnosis, prevention, and treatment. Clin. Chest. Med. 30:227-239.

  16. Miller MB, Hendren R, and Gilligan PH. 2004. Posttransplantation disseminated coccidioidomycosis acquired from donor lungs. J. Clin. Microbiol. 42:2347- 2349.

  17. Mukhopadhyay S, and Katzenstein ALA. 2010. Biopsy findings in acute pulmonary histoplasmosis: Unusual histologic features in 4 cases mimicking lymphomatoid granulomatosis. Am. J. Surg. Pathol. 34:541-546.

  18. Pappagianis D, and Chandler FW. 1997. Coccidioidomycosis, p. 977-987. In Connor DH, Chandler FW, Schwartz DA, Manz HJ, and Lack EE (ed.), Pathology of infectious diseases, 1st ed., vol. 2. Stamford, Appleton & Lange Co., Hong Kong.,

  19. Parish JM, and Blair JE. 2008. Coccidioidomycosis. Mayo. Clin. Proc. 83:343-349.

  20. Patel AJ, Gattuso P, and Reddy VB. 2010. Diagnosis of blastomycosis in surgical pathology and cytopathology: Correlation with microbiologic culture. Am. J. Surg. Pathol. 34:256-261.

  21. Sangoi AR, Rogers WM, Longacre TA, et al. 2009. Challenges and pitfalls of morphologic identification of fungal infections in histologic and cytologic specimens: A ten-year retrospective Rreview at a single institution. Am. J. Clin. Pathol. 131:364-375.

  22. Saubolle MA. 2007. Laboratory aspects in the diagnosis of coccidioidomycosis. Ann. NY. Acad. Sci. 1111:301-314.

  23. Schofield CM, Murray CK, Horvath EE, et al. 2007. Correlation of culture with histopathology in fungal burn wound colonization and infection. Burns. 33:341-3346. Infect. Dis. 49:1878-1882.

  24. Tarrand JJ, Lichterfeld M, Warraich I, et al. 2003. Diagnosis of invasive septate mold infections: A correlation of microbiological culture and histologic or cytologic examination. Am. J. Clin. Pathol. 119:854-858.

  25. Watts JC, and Chandler FW. 1997. Adiaspiromycosis, p. 929-932. In Connor DH, Chandler FW, Schwartz DA, Manz HJ, and Lack EE (ed.), Pathology of infectious diseases, 1st ed., vol. 2. Stamford, Appleton & Lange Co., Hong Kong.