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Histopathology and Microbiology of Infective Keratitis

 
Introduction

The aetiology of most cases of keratitis remains unclear because the causative agents respond to broad-spectrum antibiotics. Problems occur when they become resistant to local therapies. Widespread use of broad spectrum antibiotics also increases the risk for fungal keratitis. Further diagnostic measures such as corneal scrapings or biopsies are then necessary. Culture and direct microscopic detection of causative organisms are the two important microbiological investigations that are widely used. Although culture of microbial pathogens is considered to be the gold standard, direct microscopic evaluation of smears provides immediate information about the causative organisms. Several techniques have been used for the direct microscopic identification of microbes from corneal scrapes: Gomori s methenamine silver, periodic acid- Schiff, calcofluor white and fluorescein-conjugated lectins yield accurate results. (1) . Cultures require a longer time depending on the organisms (24 h to 3 weeks) whereas examination of a smear can provide results in a short span of time, enabling the clinician to start empirical treatment. (1)

In some cases a therapeutic Penetrating Keratoplasty is warranted to salvage the eye
Valuable information can be obtained by examining the infected corneal tissue obtained.

We report a case series of 110 eyes in Sankara Nethralaya, a tertiary eye care center in Tamil Nadu who underwent therapeutic keratoplasty and histopathological and microbiological analysis of their infected corneal buttons

Aim

To identify the histopathological and microbiological characteristics of infected corneal buttons

Study Design

Retrospective case control study of 110 eyes in the last three years from our medical records
The histopathological and microbiological reports were collected and studied.  

Results


Of the 110 corneal buttons examined, the Mean age was 44.7 years and the male/female ratio was 2.44. 
In Microbiology,  some reports showed the identification of specific organisms like  Aspergillus ,  Fusarium  or  Microsporidiosis , some showed non specific organisms like  Fungi  or  Gram +ve bacteria  and some could not identify any organism. The histopathology reports showed the presence or absence of fungal or bacterial elements with comments on inflammation present in the tissues.
92 buttons could be satisfactorily identified with histopathology and microbiology. Of the remaining 18 buttons, 7 buttons were only identified on histopathology, 7 buttons were only identified on microbiology and 4 buttons gave no result with histopathology or microbiology

One button showed Fungal Keratitis on histopathological analysis, but was reported as bacterial keratitis on microbiology, one case of bacterial keratitis was correctly identified on histopathology and Bacillus cereus was identified on microbiology and one case of Microsporidial Keratitis was correctly identified on both Histopathology and Microbiology

Of the 99 specimens positive in microbiology, the organisms identified were Fusarium in 25, Aspergillus in 22, Curvularia in 2, Candida in 1, Paecilomyeces in 1, Microsporidia in 1 and in 47 buttons the organisms could not be identified, but was positive for non specific infection. 

Thus, Specific organisms where identified in 51/110 microbiological specimens and 48/110 showed non specific identification. 

Fungi and Hyphae were identified in 93 buttons on histopathology. 

Discussion

Most of the reports on the internet are microbiological studies of scrapings and smears with stains. Very few have a histopathological analysis. Review of literature on the role of corneal biopsy in the management of infective keratitis in limited.(2). The methodology of our literature review included seaching Pubmed for the terms  microbiology ,  histopathology  and  infective keratitis . Our search revealed that this is the largest series of buttons of infective keratitis analysed with histopathological and microbiological techniques. 
Problems occur when keratitis becomes resistant to local therapy or when there is progression despite microbiologically negative smears. For lack of sufficient and safe methods of obtaining corneal biopsies, severe keratitis is often followed by penetrating keratoplasty. (3)
Microbiology helps us to identify specific organisms by culture and their antibiotic sensitivities. But in some cases due to inadequate specimen acquisition a satisfactory identification is not possible. These cases may be diagnosed better on histopathological analysis. Histopathological analysis identified hyphae and fungi satisfactorily although the specific diagnosis of the organism was not done. Careful histopathological examination can reveal many important disease characteristics of fungal keratitis. The depth of penetration and orientation of hyphae can be determined. This will give us an idea of the virulence of the organism. Fungi can be seen invading the Descemet s membrane. Figures 1-4 show colour slit lamp pictures of the corneas in diffuse illumination, 5-8 show the histopathological specimens, 9, 10 show Gomori methanamine stain of fungal keratitis, 11 shows bacterial keratitis and 12 shows Gomorra methanamine stain of microsporidiosis. 
In a recent report by Sood et al(4), males were more affected than females. In our study the male/female ratio was 2.44. Staphylococcus and Aspergillus were the most common isolates in that study whereas in ours it was Fusarium and Aspergillus. Geographical variation persists in the etiology of microbial keratitis. (4)
A study by Alexandraxis et al in 2000(2) reports the use of corneal biopsy in keratitis non responsive to microbial therapy. 33 corneal specimens were examined and 27(82%) had a positive biopsy in either histopathological or microbiological study. Close to 80% were bacterial, 10% fungal and 10% parasitic. In another study by Xie et al (5), penetrating keratoplasty was done for unresponsive studies and the buttons were examined. In their study, 98 of 108 of the recipient corneas had PAS positive fungal hyphae in tissue sections; 97 of 108 were culture positive for various fungi, including Fusarium (63), Aspergillus (14), Candida (9), Penicillium (4), and seven cases in which septate hyphae were seen but identification of the organism was not conclusive.
Our study which used corneal buttons as specimens had 106/110 (96.36%) positive in either microbiology or histopathology. The 6 specimens that were negative in the Alexandrakis study underwent penetrating keratoplasty and the buttons were evaluated like our study. 5 out of 6 showed the presence of fungal hyphae. 
We identified Mcrosporidia in one case. Though not commonly reported, microsporidial keratitis may in fact occur more commonly than believed. (6) Apart from being subjective, detection of microsporidial spores in smears requires expertise and training. Culture of microsporidia requires access to tissue culture facilities which, apart from being expensive, are unable to identify species. Our histopathological analysis with Gomori Methanamine Stain has been shown in Fig.12. 


Conclusion

Histopathological analysis is quite effective in the diagnosis of infective keratitis. It should be performed for every case undergoing penetrating keratoplasty. This helps to guide prophylactic post operative regime. Histopathology can also be done on corneal biopsies of infective keratitis. This can be helpful in cases where microbiology yields no conclusive results and can also be helpful in identifying the depth of penetration of infection. 

References

1./ Bharathi MJ. Microbiological diagnosis of infective keratitis: comparative evaluation of direct microscopy and culture results. Br J Ophthalmol. 2006Jul.26;90(10):1271 6. 
2./ Alexandrakis G, Haimovici R, Miller D, Alfonso EC. Corneal biopsy in the management of progressive microbial keratitis. Am J Ophthalmol. 2000;129(5):571 6. 
3./ Kompa S, Langefeld S, Kirchhof B, Schrage N. Corneal biopsy in keratitis performed with the microtrephine. Graefes Arch. Clin. Exp. Ophthalmol. 1999Nov.;237(11):915 9. 
4./ Sood N, Mehta D, Tewari A, Vegad M. Epidemiological and microbiological profile of infective keratitis in Ahmedabad. Indian J Ophthalmol. 2012;60(4):267. 
5./ Xie L, Dong X, Shi W. Treatment of fungal keratitis by penetrating keratoplasty. Br J Ophthalmol. 2001Sep.;85(9):1070 4. 
6./ Joseph J, Sharma S, Murthy SI, Krishna PV, Garg P, Nutheti R, et al. Microsporidial keratitis in India: 16S rRNA gene-based PCR assay for diagnosis and species identification of microsporidia in clinical samples. Invest Ophthalmol Vis Sci. 2006Oct.;47(10):4468 73. 











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Youez - 2016 - github.com/yon3zu
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