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ORIGINAL ARTICLE Table of Contents   
Year : 2008  |  Volume : 51  |  Issue : 2  |  Page : 215-217
Autofluorescence: A screening test for mycotic infection in tissues

Department of Pathology, Sri Ramachandra Medical College and Research Institute, Porur, Chennai, India

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Fungal infection is a major health concern as the clinical features are not very distinctive. Lack of rapid diagnostic techniques results in delay in diagnosis, which may even culminate in a fatal outcome. The fact that many pathogenic fungal organisms autofluoresce in hematoxylin and eosin (H and E)-stained sections under ultraviolet illumination led us to evaluate the role of autofluorescence as a rapid screening technique for fungal infections. The aim of the present study was to assess the value of autofluorescence as a screening method for detecting fungi on tissue sections and to compare the results of autofluorescence with conventional histochemical stains for fungi. Hematoxylin and eosin-stained slides of mycotic lesions were examined under fluorescent microscope and the findings were compared with results of Gomori's methenamine silver and periodic acid-Schiff stains. We found fungal autofluorescence in 63 out of 64 cases studied, with a sensitivity of 97.8% and specificity of 100% in comparison with fungal stains. This was statistically significant (P < 0.05). We conclude that autofluorescence can be used as a rapid screening method for identification of fungi in tissue sections as it does not require any other specialized staining procedure

Keywords: Autofluorescence, fungus, tissue sections

How to cite this article:
Rao S, Rajkumar A, Ehtesham M, Prathiba D. Autofluorescence: A screening test for mycotic infection in tissues. Indian J Pathol Microbiol 2008;51:215-7

How to cite this URL:
Rao S, Rajkumar A, Ehtesham M, Prathiba D. Autofluorescence: A screening test for mycotic infection in tissues. Indian J Pathol Microbiol [serial online] 2008 [cited 2020 Nov 25];51:215-7. Available from: https://www.ijpmonline.org/text.asp?2008/51/2/215/41690

   Introduction Top

Fungal infections can occur in healthy individuals but are more common as opportunistic infections in debilitated patients whose normal defense mechanisms are impaired. A fatal outcome is possible in these patients, as fungal infection may remain undiagnosed for want of mycological diagnostic services. [1] In cases of systemic mycoses, an early diagnosis and treatment are therefore critical. [2] Diagnostic modalities available are histopathology, direct microscopic examination of clinical specimen, serology and culture. [3]

Serologic test for detection of fungal antibodies in the circulation is of limited value, especially in the acutely ill immunocompromised patients in whom antibody production is impaired. Sequential tests to detect rising antibody titer are needed to eliminate false-positive cases and these tests take about 2 to 3 weeks. Although serology is potentially valuable, time requirement and specificity present an obvious limitation. [4] Polymerase chain reaction (PCR) technology provides rapid diagnosis of fungal infections but has to be used cautiously to reduce false positivity. [5] The prolonged incubation time is a major limitation of fungal cultures as a diagnostic tool. Under these circumstances, a major responsibility lies in the hands of the pathologist to give an early diagnosis on tissue sections, especially when specimen has not been sent for culture.

Utilization of ultraviolet light in the diagnosis of fungal infection ranges from in vivo diagnosis of dermatophytic infection to in vitro tissue diagnosis of fungal infection by immunofluorescent techniques. However, immunofluorescence is of limited value as nonspecific staining interferes with interpretation.

Considering the above facts, the present study was designed to assess the value of autofluorescence as a preliminary screening procedure for the detection of fungi in tissue sections.

   Materials And Methods Top

This study was done at Sri Ramachandra Medical College and Research Institute, Chennai, India. Formalin-fixed paraffin-embedded hematoxylin and eosin-stained slides from 64 cases of fungal infections diagnosed from 2001 to 2006 were studied. Skin and subcutaneous fungal infections were excluded from this study as stratum corneum exhibits strong autofluorescence, which could interfere with the interpretation.

Hematoxylin and eosin (H and E)-stained sections were examined under Nikon fluorescent microscope without adding any immunoreagents at a wave length of 334 to 365 nm using dichroic mirror filters 40-455. The presence of fungal organisms exhibiting autofluorescence was recorded. An attempt was made to categorize the fungal organisms seen. Fifteen slides of normal areas from the same organ obtained from other surgical pathology specimens were included as control. The results of autofluorescence were compared with results of periodic acid-Schiff (PAS) stain with diastase and Gomori's methenamine silver (GMS)-stained sections.

To determine the significance of the test, the results were subjected to statistical analysis.

Observation and results

Sixty-three of the 64 cases showed positivity for fungus on autofluorescence [Table 1]. The most common site involved was sino-nasal tract (37.5%) [Graph 1]-[Figure 4]. The most common pathogen seen was Aspergillus (39.6%); the other fungi found were from Zygomycetes group (31.7%), candida (28.6%) and mixed infection consisting of Zygomycetes and Aspergillus (1.6%) [Graph 2]-[Figure 5].

One case diagnosed as candidal infection of the kidney failed to produce any autofluorescence. Aspergillus gave green autofluorescence highlighting even septations at higher magnification [Figure 1]. Zygomycetes colonies gave green autofluorescence, with background tissue giving a yellow or brownish fluorescence [Figure 2]. Candida showed green autofluorescence highlighting the budding yeast and pseudohyphal forms [Figure 3]. Zygomycetes and Candida gave stronger and brighter fluorescence than Aspergillus species. The control cases did not show any autofluorescence.

The autofluorescence test was 97.8% sensitive and 100% specific, with a p value of < 0.05 in comparison with GMS and PAS stains, which are considered to be the gold standard for detecting fungi. The degree of autofluorescence had no correlation with age of slide, as even a 4-year-old slide from the files gave strong fluorescence [Table 2].

There was 1 case of candida in the kidney, which stained well with GMS but remained unstained with PAS. In 4 cases of Zygomycetes and 3 cases of Aspergillus, we noticed that the fungal colonies were focally positive with PAS stain, though they stained well with GMS stain.

   Discussion Top

Histopathology remains one of the major diagnostic tools in mycology, especially when cultures are not available. [6] At times fungi cannot be visualized in H and E-stained sections and special stains become mandatory. [3] Gomori's methamine silver and PAS are special stains used by histopathologists for diagnosing fungal infection on tissue sections. [7]

In our study, autofluorescence was useful in identifying the species of fungi, in addition to its utility as a screening procedure. A similar finding was reported in the literature earlier. [8],[9] In the present study, the three autofluorescent fungal species identified were Aspergillus, Zygomycetes and candida. A green autofluorescence was noted in our study as compared to bright green-to-yellow green autofluorescence in earlier studies. [9],[10] This may be due to mild variation in the wave length, fixing method, or mounting medium used. [11]

All cases of Zygomycetes demonstrated bright green fluorescence, a finding which was in contrast to earlier studies, where they found them to be nonfluorescent. [9],[10] The results of PAS stain and autofluorescence correlated well. The cases which showed focal positivity and negativity by PAS stain also showed similar pattern on autofluorescence. It is an established fact that dead fungi fail to give PAS positivity. [12] Lack of autofluorescence may also be due to the same reason.

Our study found no correlation with age of slides, in contrast to the findings of Mann, who found inconsistent autofluorescence in older cases. [10] This may be due to differences in the tissue-processing methods used. In the present study, we also found that vascularized granulation tissue, bone, elastic lamina of artery and collagen gave intense background yellow fluorescence, in contrast to the green fluorescence provided by the fungal organisms - a finding in concordance with similar studies in the past. [9]

Gomori's methenamine silver was found to be more consistent in staining the fungal elements in comparison with PAS stain. GMS stains old and nonviable fungal elements more efficiently than PAS and the findings in our study confirm the same. [12]

To conclude, the technique of autofluorescence is a simple and quick procedure, which can be done on routinely prepared H and E-stained slide using fluorescent microscope without addition of any immunoreagents. Its high sensitivity and specificity make it an ideal screening method.

   References Top

1.Randhawa HS. Respiratory and systemic mycoses: An overview. Indian J Chest Dis Allied Sci 2000;42:207-19.  Back to cited text no. 1  [PUBMED]  
2.Davies SF. Diagnosis of pulmonary fungal infections. Semin Respir Infect 1988;3:162-71.  Back to cited text no. 2  [PUBMED]  
3.Safar A, Marsan J, Marglani O, Al-Sebeih K, Al-Harbi J, Valvoda M. Early identification of rhinocerebral mucormycosis. J Otolaryngol 2005;34:166-71.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]
4.Kaufman L. Immunohistologic diagnosis of systemic mycoses: An update. Eur J Epidemiol 1992;8:377-82.  Back to cited text no. 4  [PUBMED]  
5.Iyer RS, Pal RB, Patel RY, Banker DD. Polymerase chain reaction based diagnosis of systemic fungal infections and sensitivity testing of the fungal isolates. Indian J Med Microbiol 2002;20:132-6.  Back to cited text no. 5    
6.Schwartz J. The diagnosis of deep mycoses by morphological methods. Hum Pathol 1982;13: 519-33.  Back to cited text no. 6    
7.Bancroft JD. Marilyn gamble: Theory and practice of histological techniques. 5 th ed, Churchill Livingstone; 2002. p. 325-44.  Back to cited text no. 7    
8.Hettlich C, Kupper TH, Wehle K, Pfitzer P. Aspergillus in the Papanicolaou stain: Morphology, fluorescence and diagnostic feasiblility. Cytopathol 1998;9:381-8.  Back to cited text no. 8    
9.Graham AR. Fungal autofluorescence with ultraviolet illumination. Am J Clin Pathol 1983;79:231-4.  Back to cited text no. 9  [PUBMED]  
10.Mann JL. Autofluorescence of fungi: An aid to detection in tissue sections. Am J Clin Pathol 1983;79:587-90.  Back to cited text no. 10  [PUBMED]  
11.Graf B, Gobel UB, Adam T. Qualitative and quantitative studies of autofluorescence in fungi. Mycoses1998;41:39-46.  Back to cited text no. 11    
12.William G, Merz Roderick J, Hay. Topley and Wilson's Microbiology and Microbial Infections. Vol. 4. 10 th ed. Hodder Arnold; 2005. p. 121-43  Back to cited text no. 12    

Correspondence Address:
D Prathiba
Department of Pathology, Sri Ramachandra Medical College and Research Institute, Porur, Chennai - 600 116
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.41690

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2]

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