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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2014  |  Volume : 57  |  Issue : 4  |  Page : 583-587
Diagnostic accuracy of morphologic identification of filamentous fungi in paraffin embedded tissue sections: Correlation of histological and culture diagnosis


1 Department of Pathology, Nizam's Institute of Medical Sciences, Hyderabad, Andhra Pradesh, India
2 Department of Microbiology, Nizam's Institute of Medical Sciences, Hyderabad, Andhra Pradesh, India

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Date of Web Publication11-Oct-2014
 

   Abstract 

Aims and Objectives: The aim was to investigate the correlation between histological and culture diagnosis of filamentous fungi. Materials and Methods: Tissue sections from biopsy samples stained with Hematoxylin and Eosin and special stains from samples of chronic invasive/noninvasive sinusitis and intracranial space occupying lesions during 2005-2011 diagnosed to have infection due to filamentous fungi were reviewed. The histopathology and culture diagnoses were analyzed for correlation and discrepancy. Results: There were 125 samples positive for filamentous fungi on biopsy. Of these 76 (60.8%) were submitted for culture and fungi grew in 30 (39.97%) samples. There was a positive correlation between histological and culture diagnosis in 25 (83.33%) samples that included Aspergillus species (16/19), Zygomycetes species (8/10) and dematiaceous fungi (1/1). The negative yield of fungi was more in Zygomycetes species (20/30) when compared to Aspergillus species (25/44). There was a discrepancy in diagnosis in 5/30 (16.67%) samples which included probable dual infection in two, and dematiaceous fungi being interpreted as Aspergillus species in three samples. Conclusion: Histopathology plays a major role in the diagnosis of infection due to filamentous fungi, especially when cultures are not submitted or negative. The discrepancy between histological and culture diagnosis was either due to dematiaceous fungi being interpreted as Aspergillus species or probable dual infection.

Keywords: Culture correlation, diagnostic accuracy, filamenatous fungi, histopathology

How to cite this article:
Challa S, Pamidi U, Uppin SG, Uppin MS, Vemu L. Diagnostic accuracy of morphologic identification of filamentous fungi in paraffin embedded tissue sections: Correlation of histological and culture diagnosis . Indian J Pathol Microbiol 2014;57:583-7

How to cite this URL:
Challa S, Pamidi U, Uppin SG, Uppin MS, Vemu L. Diagnostic accuracy of morphologic identification of filamentous fungi in paraffin embedded tissue sections: Correlation of histological and culture diagnosis . Indian J Pathol Microbiol [serial online] 2014 [cited 2019 Oct 21];57:583-7. Available from: http://www.ijpmonline.org/text.asp?2014/57/4/583/142673



   Introduction Top


With the increase in the incidence of fungal infections, there is a shift in the spectrum of etiological agents, causing invasive infections. Aspergillus species are other than Aspergillus fumigatus, and Zygomycetes species have emerged as important pathogens. Fungi previously considered nonpathogenic like hyaline and dematiaceous fungi are identified in invasive infections more frequently. [1],[2],[3],[4] Early and precise diagnosis is important for favorable outcome, especially in central nervous system (CNS) infections. [5] The diagnosis of CNS fungal infection depends on histology and culture studies. Utility of alternate methods of diagnosis is limited especially for filamentous fungi. Majority of CNS infections caused by filamentous fungi result from sinocranial spread. [6],[7],[8],[9],[10],[11] The etiological agents of fungal rhinosinusitis (FRS) include Aspergillus species, Zygomycetes species, hyaline and dematiaceous fungi. [7],[10],[11],[12] Though histopathology provides a rapid and cost effective means of providing presumptive diagnosis, culture studies are considered gold standard for the identification of etiological agents. [4] There may be overlap in the morphology of the stated fungi in tissue sections stained with Hematoxylin and Eosin (H and E), Gomori's methenamine silver (GMS), and periodic acid Schiff (PAS) causing limitation in diagnosis. Further, culture may not always be available or positive. Hence, the diagnosis on histopathology is important despite its limitations. [12],[13]

In this study, we studied the diagnostic utility of histologic diagnosis based on morphology of fungal forms in the invaded tissue and correlated with positive culture for filamentous fungi causing sinocranial and cerebral infections. The possible reasons for the discrepancy were analyzed.


   Materials and methods Top


This was a retrospective study conducted from 2005 to 2011. Biopsy/resection specimen's positive for filamentous fungi on histopathological examination, causing FRS with or without orbital/intracranial extension and intracranial space occupying lesions were included in the study. The demographic data, predisposing factors, clinical, and radiological features were collected from medical records. The FRS was classified according to the criteria proposed by deShazo et al. and Ferguson. [14],[15]

The histopathological diagnosis was reviewed with H and E, GMS and PAS stains. Masson-Fontana (MF) stain was done wherever necessary. Thin slender septate dichotomously branching at acute angle were classified as probable Aspergillus species. Broad, hyaline aseptate/pauci septate hyphae branching irregularly or at right angles were classified as Zygomycetes species; slender pigmented irregularly swollen septate hyphae along with yeast forms were classified as dematiaceous fungi. Pseudohyphae with yeast forms were classified as Candida species. [16] The microbiological records were verified for corresponding culture reports.


   Results Top


There were 125 samples where diagnosis of infection with filamentous fungi was made on histopathology in the study period. These were 87 males and 38 females (male:female = 2.3:1) with age ranging from 4 months to 84 years (median 47 years). The predisposing factors were identified in 30 (24%) patients which included diabetes mellitus, [17] systemic lupus erythematosus, [1] autoimmune disease, [1] postrenal transplant on steroid treatment [1] and chronic suppurative otitis media. [2] The clinical syndromes included chronic noninvasive FRS (mycetoma) in 3, allergic FRS in 17, chronic invasive FRS in 11, chronic invasive granulomatous FRS in 57, acute fulminant FRS in 30, and brain abscess/granuloma in 7. Tissue was not submitted for culture in 49/125 (39.2%) samples. Tissue for culture was submitted in 76/125 (60.8%) samples. Culture was negative in 46/76 (60.53%) samples. [Table 1] gives the age, gender, predisposing factors, clinical syndromes and culture characteristics of cerebral, and sinocranial fungal infections diagnosed on histopathology.
Table 1: Age, gender, predisposing factors, clinical syndromes and culture characteristics of patients with cerebral and sinocranial fungal infections

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Analysis of histopathological and culture results

Samples where tissues were not submitted for culture (49/125) were excluded from further analysis. [Figure 1] gives the results of histological diagnosis based on morphology of fungal forms, organism wise and positive culture. Fungal culture was positive in 30/76 (39.97%) samples. There was a positive correlation between histological and culture diagnosis in 25/30 (83.33%) samples. There was a positive correlation in 16/19 (84.21%) Aspergillus species, 8/10 (80%) Zygomycetes species, 1/1 dematiaceous fungus diagnosed on histology. One biopsy which was positive for Zygomycetes species on histology yielded both Rhizopus oryzae and Aspergillus flavus on culture. Only one dematiaceous fungus was identified on histology whereas culture yielded four dematiaceous fungi.
Figure 1: Histopathological and culture results of cerebral and sinocranial fungal infections

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There was misdiagnosis on histology in 5/30 (16.67%) samples. In two samples, Zygomycetes species were diagnosed on histology where culture yielded A. flavus. Review of the histological sections showed broad pauci septate fungi with angio-invasion and infarct with neutrophilic infiltrate consistent with Zygomycetes infection. This probably represents a dual infection where only one fungus was represented in either tissue or culture [Figure 2]. In three samples, probable Aspergillus species were diagnosed on histology where culture yielded dematiaceous fungi including two Alternaria species and one Bipolaris species. Review of the histological examination did not show melanin pigment or characteristic hyphal morphology on H and E [Figure 3]. However, MF stain showed positivity for melanin. Immunohistochemistry with polyclonal anti-Aspergillus antibody was negative in all three samples. [Table 2] gives the clinicopathological features, histological and culture characteristics of the discordant cases.
Figure 2: Gomori's methenamine silver (GMS) stained histological section showing broad aseptate hyphae characteristic of Zygomycetes species invading the vessel wall (GMS ×100). In this case culture grew Aspergillus falvus

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Figure 3: Histological section of a case of probable Aspergillus species diagnosed on histology with culture yield of dematiaceous fungus. (a) Granuloma with inset showing negative staining fungal hyphae (black arrow) within the cytoplasm of gaint cell (H and E, ×100, inset; H and E, ×400), (b) Gomori's methenamine silver (GMS) stain highlighting septate, slightly bulbous hyphae branching at acute angles (GMS, ×400) and (c) Masson-Fontana (MF) positivity for melanin within the fungal hyphae (black arrow) (MF, ×400)

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Table 2: Clinicopathological, histological and culture characteristics of discrepant cases

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In 46/76 (66.67%) samples, fungi failed to grow in culture though filamentous fungi were identified on histology. The negative cultures constituted 25/44 (56.82%) of Aspergillus species, 20/30 (66.67%) of Zygomycetes species, and 1/1 of Candida species diagnosed on histology.


   Discussion Top


Though culture studies are considered the gold standard for the identification of etiologic agents, they may not always be available or positive. [9],[11],[18] Moreover differentiating colonization and contamination from pathogens may be difficult. [4],[19] Accurate diagnosis of the etiologic agent is important as the in vitro susceptibility to antifungal agents of different species, and the emerging pathogens is variable. [4],[20]

Histopathology provides rapid and cost-effective means of providing diagnosis. [4] Histopathologic diagnosis of filamentous fungi depends on the morphology and tissue reaction. The tissue reaction depends on the type of fungus, site involved and immune status of the host. [16],[19] The morphology of the fungus may be distorted due to type of biopsy, crush artefacts and processing. [4] Though GMS and PAS are useful in delineating fungal morphology, they have limitations when the material is scanty, and the number of fungal elements is sparse. These limitations lead to misinterpretation and misdiagnosis on histopathology. [4] There are very few studies that correlated histopathology and culture diagnosis. [12],[13],[20],[21],[22] In the present study, we analyzed the correlation and discrepancy between histological and culture diagnosis.

In the present study, culture studies were requested in 76/125 (60.80%) samples where there was the presence of filamentous fungi on histology. Fungi were recovered in 30/76 (39.97%) samples. Lee et al. [23] reported 31% concomitant cultures and 43.4% recovery of fungi in culture in biopsies positive for filamentous fungi. [23]

[Table 3] gives the comparison of various series with the present study correlating histology and culture. There was a positive correlation between histopathology and culture diagnosis in 25/30 (83.33%) samples. Sangoi et al. [12] reported 79% correlation between morphology on histological/cytological samples and culture studies. Rickerts et al. [21] reported presence of hyphae in tissue in 27/58 (48%) of invasive mold infections. Aspergillus species was the most common organism identified in biopsy and culture in our study. This is in agreement with earlier studies. [7],[9],[10],[11],[12],[20],[21]
Table 3: Comparative analysis of various series correlating histology and culture diagnosis

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The fungi failed to grow in culture in 46/76 (66.67%) samples though filamentous fungi were identified in tissues. [20],[23] The failure to grow may be related to sampling error of tissue submitted to pathology and microbiology laboratories. The presence of dead hyphae or processing may also result in a negative culture. Several factors may influence the yield of fungi in culture which include suboptimal culture conditions, presence of necrosis, processing of tissue, and prolonged antifungal treatment. [4],[20],[21],[24]

The negative yield of culture was more with Zygomycetes species (20/30; 66.67%) when compared to Aspergillus species (25/44; 56.82%). Zygomycetes hyphae are very fragile as they are aseptate or pauci septate, which makes them liable to damage, as a result, of tissue manipulation. [25] This may explain the culture being negative. Recovery of Aspergillus species from infected tissues was reported to be low and was attributed to low density of organism, nonviable hyphae and culture conditions. [17],[20] Walsh et al. [17] suggested ways to improve culture positivity by not mincing tissue but homogenize, mimic in vivo conditions in culture and avoid adding cycloheximide to culture.

There was a discrepancy between histologic and culture diagnosis in 5/30 (16.67%) samples in our study. Lee et al. [23] reported discordance in 17% samples and Sangoi et al. [12] reported discrepancy in 21% samples. In two of our samples, Zygomycetes species were identified on histology whereas A. flavus was identified on culture. Aspergillus species show thin, septate, acute angle branching hyphae and Zygomycetes species show broad, hyaline, aseptate/pauci septate hyphae. In both of our samples, retrospective analysis showed Zygomycetes species only. The clinical syndrome was chronic invasive sinusitis. Similar situation was reported by Sangoi et al. [12] This may be attributed to sampling error or dual infection. Fungus present in abundance only gets diagnosed on histology. Sampling error, aggressive processing or tissue manipulation prior to antifungal treatment may also contribute to the result. [4] Processing artefacts leading to twisted and folded or fragmented hyphae may be misdiagnosed. [4] Both our biopsies showed lot of necrosis and neutrophilic infiltration. Misdiagnosis results in unnecessary drug exposure and/or delayed treatment. [26] In 3/5 samples, histologic diagnosis was probable Aspergillus species and culture grew dematiaceous fungi. Retrospective analysis of the biopsies did not reveal melanin pigment in the hyphae on H and E. This was due to misdiagnosis of all septate hyphae as due to probable Aspergillus species. Though the comment was added to correlate with culture studies, this was considered a major error. A study of 122 specimens by Lee et al. [23] showed concordance in 83% cases with septate acute angle branching to be Aspergillus species while hyaline septate fungi and dematiaceous fungi were recovered in culture. [23] MF stain for melanin was not found to be of diagnostic value in differentiating dematiaceous fungi from Aspergillus species. [4],[27] Culture is the only way to diagnose hyaline septate fungi, and histology cannot differentiate Aspergillus species from hyaline septate fungi and dematiaceous fungi with no identifiable pigment on H and E. [4],[28] In our series, we did not find hyaline septate hyphae.

In one sample, Zygomycetes species were seen on histology but culture yielded both R. oryzae and A. flavus. Histopathology and culture complement each other in diagnosing dual/mixed infections. [4] With the advent of polymerase chain reaction (PCR), it is realized that dual infections are more frequent than reported previously and may account for about 20% cases. [29]

The overall yield of fungi in culture in the present study was low. This was also reported in other studies. [12],[20],[21],[23] Rickerts et al. [21] in their study reported increase in the etiologic diagnosis from 63% using culture methods to 96% with the combined use of 2P PCR assays in patients with proven invasive mold infection. In our study, a significant number of samples were not submitted for culture and entire tissue was submitted in formalin. This was a major limitation for the diagnosis.

In countries like India, histopathology still plays a major role in the diagnosis of fungal infections, despite its limitation. Sangoi et al. [12] suggested a reporting template to avoid misinterpretation on morphologic diagnosis. They suggested that the report should include morphology of the fungi, along with tissue reaction to indicate invasion and suggest the most frequent fungi associated with the morphology as well as other possible fungi with similar morphology. The data presented in this study highlights the need for improvement in the awareness amongst physicians, improve the existing culture facilities and introduce molecular methods for diagnosis. Molecular profiling of fungi though ideal is not yet available for routine diagnostic use. There is a need to implement institutionalized template to histology reporting to avoid misinterpretation.

 
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Correspondence Address:
Sundaram Challa
Department of Pathology, Nizam's Institute of Medical Sciences, Hyderabad - 500 082, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.142673

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