All six acanthamoeba patients were in between 28 to 58 years age group, 4 were males and 2 were females. All were from agricultural background and none of the patients wore contact lens. Four patients had history of trauma with vegetative matters. Patients presented late to the eye hospital and mean duration of having symptoms to presentation to Biratnagar Eye Hospital was 19 days (Standard deviation 7.78). Five patients had best corrected visual acquity (BCVA) of hand movement to perception of light vision at the time of presentation and one had 2/60 in the affected eye. The details of all the patients including demographic chracteristics, history of trauma, duration and vision at presentation have been summarized in Table1.
Table 1 Demographic and clinical profile of patients with Acanthamoeba keratitis
Case No
|
Month
|
Age/Sex
|
Occupation
|
Predisposing factors/traumatic agents
|
Vision at presentation
|
Duration of presentation
|
1
|
April 2019
|
47/F
|
House wife
|
H/o vegetative trauma 15 days back followed by use of tube well water in LE after trauma
|
LE: Hand Movement close to face
|
15 days
|
2
|
June 2019
|
28/M
|
Farmer
|
None
|
RE: PL, PR accurate in all quadrants
|
30 days
|
3
|
July 2019
|
32/M
|
Farmer
|
H/O vegetative trauma
|
RE-2/60
|
10 days
|
4
|
September 2019
|
32/F
|
House wife
|
None
|
LE- PL, PR accurate in all quadrants
|
25 days
|
5
|
November 2019
|
33/M
|
Farmer
|
H/O vegetative trauma
|
RE- HM close to face
|
20 days
|
6
|
December 2019
|
58/M
|
Farmer
|
H/O vegetative trauma
|
RE- HM close to face
|
12 days
|
Severe excruciating pain was the main complaint in two patients. However, ring infiltrate was the hallmark in all the patients [Fig. 1]. Based on the history and clinical findings, provisional diagnosis of Acanthamoeba keratitis was made in four patients. One patient was clinically suspected as fungal keratitis and the other as viral keratitis.
The details of the corneal ulcer, their size and the presence of hypopyon have been described in Table 2.
Table 2 Corneal findings of the patients
Case No
|
Details of ulcer
|
Size of the ulcer
|
Presence of hypopyon
|
|
Central+ Paracentral
|
Paracentral
|
2to 6 mm
|
More than 6 mm
|
Yes
|
No
|
1
|
✓
|
|
|
✓
|
✓
|
|
2
|
✓
|
|
|
✓
|
✓
|
|
3
|
|
✓
|
✓
|
|
|
✓
|
4
|
✓
|
|
✓
|
|
|
✓
|
5
|
✓
|
|
|
✓
|
|
✓
|
6
|
✓
|
|
|
✓
|
✓
|
|
Microbiological laboratory work-up
All patients underwent corneal scraping with 15-degree bard parker blade and scraped tissue was sent for Gram and KOH stain. It was also inoculated in Blood agar, Chocolate agar and Sabouraud Dextrose Agar (SDA). If the acanthamoeba cysts were identified in either Gram and KOH stain, then scraping was further inoculated in non-nutrient agar overladen with Escherichia coli.
Results of microbiological assessment-
Double walled cysts of Acanthamoeba were noted on 10% KOH mount [Fig. 2] in first scraping in four cases (Case 2,4,5 and 6) whereas only in second scraping in two cases. Gram stain revealed acanthamoeba cyst [Fig. 3] in one of the cases (Case 5). Culture of first four cases in the non-nutrient agar with E.coli showed clear/ feeding tracks along the lawn of E.coli which signifies the presence of migrating trophozoites that feed on the bacilli [Fig. 4]. Cysts isolated from culture showed inner polygonal and outer wrinkled wall [Fig. 5a] which was further highlighted with Giemsa stain [Fig. 5b]. The Blood agar, Chocolate agar and SDA cultures were negative in all cases.
Polymerase Chain Reaction (PCR) report: PCR testing facility is not available at our hospital and in Nepal for Acanthamoeba so a communication was made with Centre for Disease Control and Prevention (CDC), USA and samples of initial four patients were sent in a special carrier media, as per their instructions. All four samples were Acanthamoeba positive by a diagnostic real-time PCR. All 4 samples have been genotyped successfully and belong to genotype T4. Two of the strains appeared identical in Sanger sequences to each other, while the other two were unique. Sample of last two patients could not be sent, as they were lost for the follow-up. The laboratory findings are illustrated in Table 3.
Table 3 Microbiological and Polymerase chain reaction findings from the scrapped samples
Case No
|
1st Scraping
|
2nd Scraping
|
Culture
(Non Nutrient Agar with E.coli)
|
PCR
|
|
Gram
|
KOH
|
Gram
|
KOH
|
|
|
1
|
No organisms isolated
|
No organisms isolated
|
No organisms isolated
|
Cyst like structure resembling Acanthamoeba
|
In 2nd scrapping, Amoebal feeding tracks along the lawn of E.Coli noted after 4 days.
|
Positive
|
2
|
No organisms isolated
|
Cyst like structure resembling Acanthamoeba
|
|
|
Amoebal feeding tracks along the lawn of E.coli noted after 5 days.
|
Positive
|
3
|
No organisms isolated
|
No organisms isolated
|
No organisms isolated
|
Cyst like structure resembling Acanthamoeba
|
In 2nd scrapping, Amoebal feeding tracks along the lawn of E.coli noted after 5 days.
|
Positive
|
4
|
No organisms isolated
|
Cyst like structure resembling Acanthamoeba
|
|
|
Amoebal feeding tracks along the lawn of E.coli noted after 7 days.
|
Positive
|
5
|
Cyst like structure resembling Acanthamoeba
|
Cyst like structure resembling Acanthamoeba
|
|
|
Not done
|
Not done
|
6
|
No organisms isolated
|
Cyst like structure resembling Acanthamoeba
|
|
|
Not done
|
Not done
|
Treatment
All patients except case number 3 received amoebicidal treatment for the first time at Biratnagar Eye Hospital. Amoebicidal treatment was started with Chlorhexidine 0.02% eye drop prepared at the pharmacy of Sagarmatha Choudhary Eye Hospital. Initially the drug was started on half hourly basis and dose was reduced after 3 days to hourly basis, subsequently it was tapered to 2 hourly after one week. Supplementary treatment included treatment with Moxifloxacin eye drop, a combination of polymyxin B sulfate, neomycin sulfate and bacitracin. Case no 3 was continued on antifungal (Natamicin eye drop 5 % half hourly + Fluconazole eye drop 0.3% half hourly) and antibiotic (Moxifloxacin eye drop 0.5% half hourly) as the patient was already receiving the medication before he was diagnosed in our hospital and had the scarring started around the ulcer despite having thin cornea. This patient underwent cyanoacrylate glue application along with BCL. Gradually eye drops were tapered in all patients.
All patients showed early response to medication with dramatic reduction in pain. First four patients came for regular follow-up and showed a healing with corneal scarring. Case number 3 who received antifungal treatment only had gained BCVA of 6/9 whereas remaining 3 patients had vision of hand movement. Case 5 and 6 went to other eye hospitals to seek further opinion and were lost for the follow-up.