• View in gallery

    (A) Slit-lamp photograph of the left eye at initial presentation-needs some description, (B) Gram stain from the first scrapings showing filamentous structures with chlamydospores (×100), (C) photograph from the culture specimens showing Fusarium spp. (×40), (D) slit-lamp photograph of the left eye at day 10 showing rapid worsening characterized by diffuse corneal involvement up to the limbus, (E) calcoflour white mount from repeat scraping showing coinfection of Fusarium and Acanthamoeba (×40), and (F) Histopathology of the corneal button showing fungal filaments and Acanthamoeba on special staining (Gomori’s methamine sliver staining) at ×20. This figure appears in color at www.ajtmh.org.

  • View in gallery

    (A) Calcofluor white stained smear of corneal scraping (×40) showing presence of both fungus (arrowhead) and Acanthamoeba (arrow), (B) Gram stained smear of corneal scraping (×100) showing both fungus (arrowhead) and Acanthamoeba (arrow), (C) Histopathology of the corneal button (×20) showing Fungal filaments (arrowhead) and Acanthamoeba (arrow) on special staining (Gomori’s methamine sliver staining). This figure appears in color at www.ajtmh.org.

  • 1.

    Gopinathan U, Garg P, Fernandes M, Sharma S, Athmanathan S, Rao GN, 2002. The Epidemiological features and laboratory results of fungal keratitis: a 10-year review at a referral eye care center in south India. Cornea 21: 555559.

    • Search Google Scholar
    • Export Citation
  • 2.

    Sharma S, Garg P, Rao GN, 2000. Patients characteristics, diagnosis and treatment of non-contact lens related Acanthamoeba keratitis. Br J Ophthalmol 84: 11031108.

    • Search Google Scholar
    • Export Citation
  • 3.

    Nunes TE, Brazil NT, Fuentefria AM, Rott MB, 2016. Acanthamoeba and Fusarium interactions: a possible problem in keratitis. Acta Trop 157: 102107.

    • Search Google Scholar
    • Export Citation
  • 4.

    Froumis NA, Mondino BJ, Glasgow BJ, 2001. Acanthamoeba keratitis associated with fungal keratitis. Am J Ophthalmol 131: 508509.

  • 5.

    Tien SH, Sheu MM, 1999. Treatment of Acanthamoeba keratitis combined with fungal infection with polyhexamethylene biguanide. Kaohsiung J Med Sci 15: 665673.

    • Search Google Scholar
    • Export Citation
  • 6.

    Slade DS, Johnson JT, Tabin G, 2008. Acanthamoeba and fungal keratitis in a woman with a history of intacs corneal implants. Eye Contact Lens 34: 185187.

    • Search Google Scholar
    • Export Citation
  • 7.

    Lee WB, Grossniklaus HE, Edelhauser HF, 2010. Concurrent Acanthamoeba and Fusarium keratitis with silicone hydrogel contact lens use. Cornea 29: 210213.

    • Search Google Scholar
    • Export Citation
  • 8.

    Lin HC, Hsiao CH, Ma DH, Yeh LK, Tan HY, Lin MY, Huang SCM, 2009. Medical treatment for combined Fusarium and Acanthamoeba keratitis. Acta Ophthalmol 87: 199203.

    • Search Google Scholar
    • Export Citation
  • 9.

    Lin HC, Chu PH, Kuo YH, Shen SC, 2005. Clinical experience in managing Fusarium solani keratitis. Int J Clin Pract 59: 549554.

  • 10.

    Gopinathan U, Sharma S, Garg P, Rao GN, 2009. Review of epidemiological features, microbiological diagnosis and treatment outcome of microbial keratitis: experience of over a decade. Indian J Ophthalmol 57: 273279.

    • Search Google Scholar
    • Export Citation
  • 11.

    Luna LG, 1968. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. New York, NY: McGraw-Hill.

  • 12.

    Bharathi MJ, Ramakrishnan R, Meenakshi R, Shivakumar C, Raj DL, 2009. Analysis of the risk factors predisposing to fungal, bacterial & Acanthamoeba keratitis in south India. Indian J Med Res 130: 749757.

    • Search Google Scholar
    • Export Citation
  • 13.

    Gupta N, Samantaray JC, Duggal S, Srivastava V, Dhull CS, Chaudhary U, 2010. Acanthamoeba keratitis with Curvularia co-infection. Indian J Med Microbiol 28: 6771.

    • Search Google Scholar
    • Export Citation
  • 14.

    Rumelt S, Cohen I, Lefter E, Rehany U, 2001. Corneal coinfection with Scedosporium apiospermum and Acanthamoeba after sewage contaminated ocular injury. Cornea 20: 112116.

    • Search Google Scholar
    • Export Citation
  • 15.

    Gussler JR, Miller D, Jaffe M, Alfonso EC, 1995. Infection after radial keratotomy. Am J Ophthalmol 119: 798799.

  • 16.

    Babu K, Murthy KR, 2007. Combined fungal and Acanthamoeba keratitis: diagnosis by in vivo confocal microscopy. Eye (Lond) 21: 271272.

  • 17.

    Hu J, Wang Y, Xie L, 2009. Potential role of macrophages in experimental keratomycosis. Invest Ophthalmol Vis Sci 50: 20872094.

 

 

 

 

Case Report: Corneal Coinfection with Fungus and Amoeba: Report of Two Patients and Literature Review

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  • 1 Jhaveri Microbiology Centre, Brien Holden Eye Research Centre, L. V. Prasad Eye Institute, Hyderabad, India;
  • 2 Tej Kohli Cornea Institute, L. V. Prasad Eye Institute, Hyderabad, India

We report two cases of corneal coinfection with Acanthamoeba and Fusarium sp. along with the review of published literature. A 35-year-old woman and 65-year-old man presented to the institute with corneal ulcer refractory for treatment with topical antibiotics. Microbiological examination revealed the presence of Acanthamoeba cysts along with septate, hyaline fungal filaments. After emergency therapeutic penetrating keratoplasty (TPK) in both, the corneal tissue was sent for histopathologic examination, which confirmed the presence of Acanthamoeba and fungal coinfection. One patient had a recurrence of fungal infection after TPK. In subjects with a rapid progression of mycotic ulcer, coinfection with other microorganisms including Acanthamoeba should be suspected. The two cases presented here emphasize the importance of microbiology in making prompt diagnosis and appropriate management of these cases at an early stage.

INTRODUCTION

Microbial keratitis has been reported to be due to infection with a wide range of organisms. There are regional variations in the predominance of different microbes, reflecting different patient population and climatic effects.1 Although fungal keratitis is more common in tropical and subtropical areas, Acanthamoeba keratitis rarely presents with a positive history of contact lens use in India and South Asia, and is more often associated with a previous history of trauma or exposure to contaminated water.2 Compared with bacterial keratitis, isolated infection with organisms such as Acanthamoeba and fungi tend to follow a more insidious course and may need a therapeutic surgical intervention. A complicating factor in the treatment of keratitis is the coinfection with microorganisms which may result in significant changes for both and increased virulence in the mammalian hosts.3 So far, 13 cases of coinfection of cornea with Acanthamoeba and fungus have been reported, and the use of contact lenses was involved in five of them.47 Treatment regimens have varied from the biguanides, polyhexamethylene biguanide (PHMB) and chlorhexidine, to the diamide propamidine, the aminoglycoside neomycin, and the antifungal clotrimazole. The drugs have been used in different combinations, with varying degrees of success.8 The standard medical treatment of fungal keratitis is the frequent use of topical natamycin 5%, but any delay in diagnosis usually leads to therapeutic penetrating keratoplasty (TPK).9 Mixed infections can be difficult to manage because Acanthamoeba and fungus are resistant to initial empirical antimicrobial agents.3 The purpose of this study was to report two patients of keratitis where microbiological examination of the corneal scrapings showed both Acanthamoeba and Fusarium sp., and the diagnosis was confirmed by histopathology of the corneal tissue after TPK. We also present the current literature that is available on this dual infection of the cornea.

METHODS

This study was conducted at the L. V. Prasad Eye Institute, Hyderabad, India, a tertiary eye care facility in South India. A retrospective, consecutive review of medical and microbiology records of all patients seen between January 2012 and December 2016 identified two cases having infection with fungus and Acanthamoeba. Demographic details, cause and duration of symptoms, presenting and final visual acuity, surgical interventions, and laboratory data of these patients were collected. Both patients had undergone complete eye examination under slit lamp biomicroscopy and indirect ophthalmoscopy following collection of history and demographic details.

The patients had undergone complete microbiological investigations as described by us earlier.10 In brief, corneal scrapings were obtained from the patients under topical anesthesia (0.5% proparacaine hydrochloride) using a sterile surgical blade no. 15 on a Bard Parker handle. The corneal scrapings were examined by direct microscopic examination after staining with calcofluor white and Gram stain. Culture included a variety of media such as blood agar (5% sheep blood), chocolate agar (5% sheep blood), brain heart infusion broth, Sabouraud dextrose agar, and non-nutrient agar with an overlay of Escherichia coli (not taken for case 2) for the possible growth of bacteria and/or fungus and/or Acanthamoeba. Incubation of the media and their interpretation were as per our protocol published earlier.10

Post–penetrating keratoplasty in both cases, half corneal button (fixed in 10% buffered formalin) was sent to the pathology laboratory for histopathology examination. Paraffin embedded tissue sections were stained with hematoxylin and eosin, periodic acid-Schiff, and Gomori’s methenamine silver stain (GMS) following standard procedures.11 The other half of the corneal buttons were sent in sterile containers to the microbiology laboratory for culture. Minced tissue was inoculated on blood agar, brain heart infusion broth, Sabouraud dextrose agar, and non-nutrient agar with E. coli.

Case 1.

A 35-year-old woman presented to the cornea clinic with pain and redness in the left eye since 7 days. She had no history of trauma or use of contact lenses. Visual acuity was 20/40. Slit-lamp examination revealed anterior- to mid-stromal corneal infiltrate measuring 7 × 5.5 mm (Figure 1A). Microbiological examination of the corneal scrapings showed fungal filaments with chlamydospores on calcofluor white stain (CFW) and Gram stain (Figure 1B) which later grew Fusarium sp. in culture (Figure 1C). She was treated with 5% Natamycin eye drops hourly and oral ketoconazole 200 mg twice a day. A rapid worsening of the symptoms and progression of the ulcer was noted over the next few days. Vision was reduced to light perception and slit-lamp examination revealed a full thickness infiltrate involving the whole of the cornea with melt and ectasia inferiorly (Figure 1D). In view of rapid worsening, the patient was taken for TPK and corneal scrapings were repeated. Microbiological smears of the second scrapings revealed both fungal filaments (Figure 1E, arrowhead) and Acanthamoeba cysts (Figure 1E, arrow) on CFW, and both grew in culture. Histopathological examination of the corneal specimen revealed plenty of fungal hyphae and double walled Acanthamoeba cysts in Gomori’s methenamine silver staining, suggestive of coinfection with fungus and Acanthamoeba (Figure 1F, arrow). The culture of the corneal button grew only Fusarium spp. Postoperatively, the patient received topical natamycin (5%) and 0.02% chorhexidine along with oral ketoconazole (200 mg, twice daily) The postoperative course was uneventful with no recurrences until 6 months of follow up.

Figure 1.
Figure 1.

(A) Slit-lamp photograph of the left eye at initial presentation-needs some description, (B) Gram stain from the first scrapings showing filamentous structures with chlamydospores (×100), (C) photograph from the culture specimens showing Fusarium spp. (×40), (D) slit-lamp photograph of the left eye at day 10 showing rapid worsening characterized by diffuse corneal involvement up to the limbus, (E) calcoflour white mount from repeat scraping showing coinfection of Fusarium and Acanthamoeba (×40), and (F) Histopathology of the corneal button showing fungal filaments and Acanthamoeba on special staining (Gomori’s methamine sliver staining) at ×20. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene 99, 3; 10.4269/ajtmh.18-0158

Case 2.

A 65-year-old farmer presented with pain and redness in the left eye since 3 months, following injury with stick. He had consulted at several places and his records revealed a nonresponse to therapeutic trial with topical antibacterials. In one of the records, he was diagnosed to have viral keratitis with secondary glaucoma. At presentation, visual acuity was light perception with inaccurate projection of light rays. On slit-lamp examination, the lids were edematous, conjunctiva was congested, cornea showed a full thickness total corneal infiltrate with hypopyon. The intraocular details of the eye could not be appreciated because of the hazy cornea. Therefore, B scan ultrasonography of the posterior segment of the eye was performed to rule out any intraocular extension of the infection. The test was unremarkable with no echos seen in the vitreous cavity of the eye, indicating no involvement of the vitreous or retina. Microbiological examination of the corneal scrapings showed septate hyaline fungal filaments along with Acanthamoeba cysts on a CFW (Figure 2A) and Gram stain (Figure 2B). The extensive corneal involvement necessitated immediate TPK, following which the patient was advised hourly topical 5% Natamycin, 0.02% PHMB, and 0.02% Chlorhexidine. Histopathological examination of the corneal specimen revealed extensive lamellar stromal necrosis with the coexistence of both empty cysts and branching hyphae on Gomori’s methenamine silver staining (Figure 2C, arrow). The culture of the corneal button grew Fusarium species. A month later, the patient developed a graft infiltrate with anterior chamber exudates. A clinical impression of recurrence of infection was made. Corneal scrapings from the infiltrate site revealed the presence of Gram-positive cocci and septate filamentous fungus on microbiological smear examination. Cultures grew Staphylococcus epidermidis and Fusarium sp. Treatment with natamycin was continued, however, he was lost to follow up.

Figure 2.
Figure 2.

(A) Calcofluor white stained smear of corneal scraping (×40) showing presence of both fungus (arrowhead) and Acanthamoeba (arrow), (B) Gram stained smear of corneal scraping (×100) showing both fungus (arrowhead) and Acanthamoeba (arrow), (C) Histopathology of the corneal button (×20) showing Fungal filaments (arrowhead) and Acanthamoeba (arrow) on special staining (Gomori’s methamine sliver staining). This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene 99, 3; 10.4269/ajtmh.18-0158

DISCUSSION

In the setting of recalcitrant corneal ulcers unresponsive to medical treatment, the differential diagnoses include polymicrobial infections, resistant organisms, atypical etiologic agents, coexisting ocular surface disorders, and systemic ailments such as diabetes mellitus.12 Combined fungal and Acanthamoeba keratitis is a rare entity and can be difficult to diagnose and manage.6 Of the 3,733 fungal corneal infections treated at our institute during the study period, coinfection with fungus and bacteria was observed in 267 cases, and two cases were identified to have a coinfection with fungus and Acanthamoeba. In the first case presented here (Case 1), the initial microbiology reports showed the presence of fungus alone with chlamydospores; however, microbiological evaluation of the second scrapings identified both fungus and Acanthamoeba cysts. It is possible that at the first instance the cysts were missed as they resemble the fungal chlamydospores and one may tend to identify the predominant organism. In the second case, however, in the initial microbiological investigation itself both fungus and Acanthamoeba infection was revealed. Both the patients here required a therapeutic keratoplasty, and one had a recurrence of fungal infection after keratoplasty. Several factors are implicated in poor response to medical management in coinfection with Acanthamoeba and fungus. Treatment failures and limitations of medical treatment even in isolated fungus and Acanthamoeba infections is well known. In our first case, there was a rapid worsening of the infiltrate over a few days, but the diagnosis of Acanthamoeba along with fungus was made only on repeat scrapings after worsening of symptoms. In the second case, the infection was long standing and had involved the entire cornea at the time of initial presentation which could account for the poor prognosis and the recurrence in the second case.

Literature search revealed 13 cases with concurrent Acanthamoeba and Fusarium keratitis (Table 1); all were initially misdiagnosed as bacterial or viral keratitis.48,1316 Of these cases, five had a history of use of contact lens48 whereas injury by foreign body was seen in two cases,5 including one from our series. Corneal trauma allows the microorganisms access to the stroma and deep epithelial layers. In most reported cases, the scrapings showed the presence of Acanthamoeba, and only after corneal perforation followed by subsequent TPK has the diagnosis of fungal corneal infection been discovered through corneal histopathology.47 Only one case established a diagnosis with the help of confocal microscopy.16 In both our patients, the direct examination of the corneal scrapings and histopathological examination of the corneal tissue were confirmatory of the diagnosis of Fusarium and Acanthamoeba infection. Molecular tests such as polymerase chain reaction would have been performed if required. However, they were not considered necessary in our patients. In addition, Lin et al.8 suggested that Acanthamoeba may progress faster because the fungi may serve as nutrient for the amoeba in mixed infections, whereas Hu et al.,17 suggested that as the amoebae and human macrophage share similar features, the accidental selection of fungal virulence traits by Acanthamoeba, may help Fusarium solani to escape or to survive in human macrophages, which may lead to worsening of keratitis. The delay in fungal diagnosis may represent the faster progression of Acanthamoeba keratitis in dual infections leading to corneal perforation.

Table 1

Details of Previous Reports on mixed infection of Acanthamoeba and fungal keratitis in the Literature (PubMed)

S. noReferenceOrganismNo of patientsRisk factorTreatmentOutcome (VA)
1Gussler et al. (1995)15Fusarium sp.1Radial keratotomy (tap water)PHMBTPK (20/20)
2Tien and Sheu (1999)5Mould3DM, mud-related trauma, and SCLNeomycin, metronidazole (case 1), amikacin, piperacillin (cases 2, 3), PHMB, fluconazole, TPKTPK (HM, 20∕100, 20∕20)
3Froumis et al. (2001)4Scedosporium apiospermum1 (two eyes)Contact lensCyclosporin A, Pred-Forte, propamidine, neomycin,TPK, lensectomy, vitrectomy (CF)
4Babu and Murthy (2007)16Fusarium sp.1AgriculuristNatamycin, propamidine, PHMB20/30
5Slade et al. (2008)6Fungus (HP section)1Intacs corneal implantsCycloplegicsTPK
6Lin et al. (2009)8Fusarium sp.2Contact lens and FB (case 2)PHMB, propamidine, clotrimazole, natamycin20/20, 20/200
7Gupta et al. (2010)13Curvularia sp.1Pond waterPropamidine isothionate, natamycin, fluconazole, neosporin cycloplegic eye dropsImproved
8Lee et al. (2010)7Fusarium solani1Silicone hydrogel contact lensPropamidine, chlorhexidine, neomycin/polymyxin BTPK twice (20/400)
10Current case 1F. solani1UnknownPHMB, chlorhexidine, natamycinTPK (CF)
11Current case 2Fusarium sp.1Injury-stickPHMB, chlorhexidine, natamycinTPK (LFU)

CF = counting fingers; DM = diabetes mellitus; FB = foreign body; HM = hand movements; HP = histopathology; LFU = lost to follow up; PHMB = polyhexamethylene biguanide; SCL = soft contact lens; TPK = therapeutic penetrating keratoplasty.

Regardless, coexistent atypical infectious keratitis with these two organisms, although extremely rare, resulted in severe keratolysis and necessitated TPK with poor visual outcomes like most of the cases described previously. Better visual outcomes are most often reported in cases where TPK could be avoided.8,13,16 It is probable that the infectious keratitis in our patients would have responded favorably if combined antifungal and antiamoebic treatments were instituted simultaneously in the early stages of the disease before complete corneal involvement. Specific diagnosis was not made and both our cases had not received specific therapy. Meticulous microbiological investigation of all cases with clinical diagnosis of microbial keratitis is recommended.

Acknowledgments:

We thank the Hyderabad Eye Research Foundation, Hyderabad, and B. Sreedhar Rao for helping with the histopathology sections.

REFERENCES

  • 1.

    Gopinathan U, Garg P, Fernandes M, Sharma S, Athmanathan S, Rao GN, 2002. The Epidemiological features and laboratory results of fungal keratitis: a 10-year review at a referral eye care center in south India. Cornea 21: 555559.

    • Search Google Scholar
    • Export Citation
  • 2.

    Sharma S, Garg P, Rao GN, 2000. Patients characteristics, diagnosis and treatment of non-contact lens related Acanthamoeba keratitis. Br J Ophthalmol 84: 11031108.

    • Search Google Scholar
    • Export Citation
  • 3.

    Nunes TE, Brazil NT, Fuentefria AM, Rott MB, 2016. Acanthamoeba and Fusarium interactions: a possible problem in keratitis. Acta Trop 157: 102107.

    • Search Google Scholar
    • Export Citation
  • 4.

    Froumis NA, Mondino BJ, Glasgow BJ, 2001. Acanthamoeba keratitis associated with fungal keratitis. Am J Ophthalmol 131: 508509.

  • 5.

    Tien SH, Sheu MM, 1999. Treatment of Acanthamoeba keratitis combined with fungal infection with polyhexamethylene biguanide. Kaohsiung J Med Sci 15: 665673.

    • Search Google Scholar
    • Export Citation
  • 6.

    Slade DS, Johnson JT, Tabin G, 2008. Acanthamoeba and fungal keratitis in a woman with a history of intacs corneal implants. Eye Contact Lens 34: 185187.

    • Search Google Scholar
    • Export Citation
  • 7.

    Lee WB, Grossniklaus HE, Edelhauser HF, 2010. Concurrent Acanthamoeba and Fusarium keratitis with silicone hydrogel contact lens use. Cornea 29: 210213.

    • Search Google Scholar
    • Export Citation
  • 8.

    Lin HC, Hsiao CH, Ma DH, Yeh LK, Tan HY, Lin MY, Huang SCM, 2009. Medical treatment for combined Fusarium and Acanthamoeba keratitis. Acta Ophthalmol 87: 199203.

    • Search Google Scholar
    • Export Citation
  • 9.

    Lin HC, Chu PH, Kuo YH, Shen SC, 2005. Clinical experience in managing Fusarium solani keratitis. Int J Clin Pract 59: 549554.

  • 10.

    Gopinathan U, Sharma S, Garg P, Rao GN, 2009. Review of epidemiological features, microbiological diagnosis and treatment outcome of microbial keratitis: experience of over a decade. Indian J Ophthalmol 57: 273279.

    • Search Google Scholar
    • Export Citation
  • 11.

    Luna LG, 1968. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. New York, NY: McGraw-Hill.

  • 12.

    Bharathi MJ, Ramakrishnan R, Meenakshi R, Shivakumar C, Raj DL, 2009. Analysis of the risk factors predisposing to fungal, bacterial & Acanthamoeba keratitis in south India. Indian J Med Res 130: 749757.

    • Search Google Scholar
    • Export Citation
  • 13.

    Gupta N, Samantaray JC, Duggal S, Srivastava V, Dhull CS, Chaudhary U, 2010. Acanthamoeba keratitis with Curvularia co-infection. Indian J Med Microbiol 28: 6771.

    • Search Google Scholar
    • Export Citation
  • 14.

    Rumelt S, Cohen I, Lefter E, Rehany U, 2001. Corneal coinfection with Scedosporium apiospermum and Acanthamoeba after sewage contaminated ocular injury. Cornea 20: 112116.

    • Search Google Scholar
    • Export Citation
  • 15.

    Gussler JR, Miller D, Jaffe M, Alfonso EC, 1995. Infection after radial keratotomy. Am J Ophthalmol 119: 798799.

  • 16.

    Babu K, Murthy KR, 2007. Combined fungal and Acanthamoeba keratitis: diagnosis by in vivo confocal microscopy. Eye (Lond) 21: 271272.

  • 17.

    Hu J, Wang Y, Xie L, 2009. Potential role of macrophages in experimental keratomycosis. Invest Ophthalmol Vis Sci 50: 20872094.

Author Notes

Address correspondence to Joveeta Joseph, Jhaveri Microbiology Centre, Brien Holden Eye Research Centre, L. V. Prasad Eye Institute, Banjara Hills, Rd. No 2, Hyderabad, Telangana 500034, India. E-mail: joveeta@lvpei.org

Ethical approval: Informed consent was obtained from all individual participants included in the study.

Authors’ addresses: Joveeta Joseph and Savitri Sharma, Jhaveri Microbiology Centre, L. V. Prasad Eye Institute, Hyderabad, India, E-mails: joveeta@lvpei.org and savitri@lvpei.org. Sunita Chaurasia, Tej Kohli Cornea Institute, L. V. Prasad Eye Institute, Hyderabad, India, E-mail: sunita@lvpei.org.

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