Abstract
Aims
To determine the sensitivity, specificity and predictive values of potassium hydroxide (KOH) wet mount, Gram stain, Giemsa stain and Kinyoun's acid‐fast stain in the diagnosis of infective keratitis.
Methods
A retrospective analysis of all patients with clinically diagnosed infective keratitis presenting between September 1999 and September 2002 was carried out. Corneal scrapes were taken and subjected to direct microscopy and culture.
Results
3298 eyes of 3295 consecutive patients with infective keratitis were evaluated, of which 1138 (34.51%) eyes had fungal growth alone, 1069 (32.41%) had bacterial growth alone, 33 (1%) had Acanthamoeba growth alone, 83 (2.5%) had mixed microbial growth and the remaining 975 (29.56%) had no growth. The sensitivity of KOH wet mount was higher (99.3%; 95% confidence interval (CI) 98.6 to 99.6) in the detection of fungi, 100% (95% CI 90.4 to 100) in the detection of Nocardia and 91.4% (95% CI 75.8 to 97) in the detection of Acanthamoeba) than that of Gram‐stained smears (89.2% (95% CI 87.3 to 90.8) in fungi, 87% (95% CI 73.0 to 94.6) in Nocardia and 60% (95% CI 42.2 to 75.6) in the detection of Acanthamoeba) in the detection of fungi, Nocardia and Acanthamoeba. 1764 of 3295 (53.54%) patients presented more than 7 days after onset of illness and 84.69% of the eyes had corneal ulcers with size >2 mm in diameter. Positivities of KOH (44.46%; p<0.001) and Gram‐stained smears (77.37%; p<0.001) were found to be higher among eyes with larger ulcers (>2 mm) than among eyes with smaller ulcers (<2 mm).
Conclusion
KOH smear is of greater diagnostic value in the management of infective keratitis, and it is recommended in all clinics without exception for establishing timely treatment.
Infective keratitis is an ocular emergency that requires prompt and appropriate management to ensure the best visual outcome for the patient. Without adequate treatment, corneal infection leads to blindness through corneal scarring and endophthalmitis.1 To minimise ocular morbidity, timely antimicrobial treatment must be initiated on the basis of clinical and microbiological evaluation.2,3 A clinical diagnosis of infective keratitis does not give an unequivocal indication of the causative organisms because a wide range of organisms can produce a similar clinical picture.4,5,6 Culture and direct microscopic detection of causative organisms are the two important microbiological investigations that are widely used. Although culturing 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,7 periodic acid‐Schiff,8 calcofluor white9 and fluorescein‐conjugated lectins10 yield accurate results, but are time consuming and require special infrastructures. In addition, the cost of each test makes them inapplicable in primary, secondary and even in most tertiary centres.
The conventional techniques, potassium hydroxide (KOH) wet mount, Gram stain and Giemsa stain, are widely used for the rapid detection of microbes7,11; however, owing to misinterpretation, presence of artefacts, and lack of detection of Candida and other yeasts, the sensitivity of these methods is highly variable.7,9,12,13,14,15 Thus, there is a need to study the efficacy of available direct microscopic techniques in the detection of microbes from corneal scrapes, thereby creating an awareness to establish a simple microbiological investigation in all ophthalmic clinics for timely treatment, and thereby preventing loss of vision. This study was conducted to evaluate all microbial keratitis treated at a tertiary eye care referral centre in south India. The aims of this investigation were to determine the sensitivity and specificity of KOH smear, Gram stain, Giemsa stain and Kinyoun's acid‐fast stain in the diagnosis of infective keratitis. We believe that these comprehensive data on direct microscopy will encourage ophthalmologists to carry out KOH smears even in small clinics.
Materials and methods
This retrospective study included all patients with clinically diagnosed infective keratitis presenting at Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Tirunelveli, south India over 3 years from September 1999 to September 2002. Corneal ulceration was defined as a loss of the corneal epithelium, with underlying stromal infiltration and suppuration associated with signs of inflammation with or without hypopyon.16,17 Patients with suspected or confirmed viral keratitis and healing ulcers were excluded, as were those with Mooren's ulcers, interstitial keratitis, sterile neurotropic ulcers and any ulcer associated with autoimmune conditions. A standardised protocol was followed for each patient, with corneal ulceration for the evaluation of microbiological features and clinical findings.16,17,18
Clinical procedures
All patients were examined using a slit‐lamp biomicroscope; the size of the epithelial defect after staining with 2% fluorescein was measured with the variable slit on the biomicroscope and recorded in millimetres. Using standard techniques,16,17,18 corneal scrape was observed under the magnification of a slit lamp or operating microscope after instillation of 0.5% proparacaine hydrochloride,18 using flame‐sterilised Kimura's spatula or a sterile Bard‐Parker blade (no 15). The spatula or blade was scraped over the surface of the suppurative area in a series of short, moderately firm strokes to sample both the leading edges and the base of each infiltrated area in one direction from the peripheral margins towards the centre of the suppurative area. The material obtained was initially smeared onto clean sterile labelled glass slides for 10% KOH wet mount, Gram stain, Giemsa stain and Kinyoun's acid‐fast stain for suspected actinomycete keratitis. The material obtained by the next scrape was inoculated directly onto the surface of solid media such as sheep's blood agar, chocolate agar, Sabouraud's dextrose agar, potato dextrose agar or non‐nutrient agar in rows of C‐shaped streaks, and also inoculated into the depth of liquid media such as brain heart infusion broth and thioglycollate medium. Rescrapes were taken after reflaming and cooling the spatula or using new sterile blades to obtain additional material from multiple areas of corneal suppuration.
Laboratory procedures
All inoculated media were incubated aerobically. The inoculated Sabouraud's dextrose agar was incubated at 27°C, examined daily, and discarded at 3 weeks if no growth was seen. The inoculated blood agar, chocolate agar, thioglycollate broth and brain heart infusion broth were incubated at 37°C, examined daily and discarded at 7 days if growth was not seen. The inoculated non‐nutrient agar plates were incubated at 37°C after overlaying with Escherichia coli broth cultures, were examined daily for the presence of Acanthamoeba species, and discarded at 3 weeks if there were no signs of growth.16,17,18 Microbial cultures were considered relevant if growth of the same organism was observed on more than one solid‐phase medium; if there was confluent growth at the site of inoculation on one solid medium; if growth of one medium was consistent with direct microscopy findings (ie, appropriate staining and morphology with Gram stain); or if the same organism was grown from repeated scraping.16,17,18 Pearson's χ2 test was used to carry out the statistical analysis wherever required and p value <0.05 was considered to be significant.
Results
A total of 3295 patients with clinical diagnosis of infective keratitis were evaluated at our institute, of which a single eye was infected in 3292 patients and both eyes were infected in 3 patients; thus, a total of 3298 eyes with corneal ulceration were studied. Of 3298 eyes, 1138 (34.51%) eyes had fungal growth alone, 1069 (32.41%) had bacterial growth alone, 33 (1%) had Acanthamoeba growth alone, 83 (2.5%) had mixed microbial growth and the remaining 975 (29.56%) had no growth. Table 1 presents the microbial growth pattern of corneal scrapes obtained from 3298 eyes of patients with infective keratitis. A total of 1216 bacterial isolates from 1151 eyes with keratitis and 1226 fungal isolates from 1220 eyes with keratitis were recovered. Tables 2 and 3 document the bacterial and fungal pathogens respectively recovered from patients with infective keratitis.
Table 1 Microbial growth pattern of corneal scrapes obtained from 3298 consecutive eyes with infective keratitis in south India.
| Sl no | Growth pattern | No of eyes (%) |
|---|---|---|
| 1 | Pure fungal growth | 1138 (34.50) |
| Single species of fungi | 1132 (34.32) | |
| Two species of fungi | 6 (0.18) | |
| 2 | Pure bacterial growth | 1069 (32.41) |
| Single species of bacteria | 1004 (30.44) | |
| Two species of bacteria | 65 (1.97) | |
| 3 | Pure protozoan (Acanthamoeba) growth | 33 (1) |
| 4 | Mixed fungal and bacterial growth | 81 (2.46) |
| Single species of fungi and single species of bacteria | ||
| 5 | Mixed Acanthamoeba and single species of fungal growth | 1 (0.03) |
| 6 | Mixed Acanthamoeba and single species of bacterial growth | 1 (0.03) |
| Eyes with microbial keratitis that showed positive cultures | 2323 (70.44) | |
| Eyes with microbial keratitis that showed negative cultures | 975 (29.56) |
Table 2 Bacterial pathogens isolated from corneal scrapes of 1151 eyes with infective keratitis treated at a tertiary eye care referral centre in south India.
| Sl no | Bacterial isolates | Pure isolates | Mixed with other bacteria | Mixed with fungal spp | Mixed with Acanthamoeba sp | Total no of bacterial isolates (%) |
|---|---|---|---|---|---|---|
| 1 | Gram‐positive cocci | 675 | 65 | 40 | 780 (64.14) | |
| Streptococcus pneumoniae | 417 | 7 | 14 | 438 (36.03) | ||
| Staphylococcus epidermidis | 155 | 43 | 24 | 222 (18.25) | ||
| Staphylococcus aureus | 36 | 10 | 0 | 46 (3.78) | ||
| Miccrococcus spp | 6 | 0 | 0 | 6 (0.49) | ||
| α‐Haemolytic streptococci | 46 | 5 | 2 | 53 (4.36) | ||
| β‐Haemolytic streptococci | 6 | 0 | 6 (0.49) | |||
| Non‐haemolytic streptococci | 9 | 0 | 9 (0.74) | |||
| 2 | Gram‐positive bacilli | 33 | 22 | 2 | 57 (4.69) | |
| Bacillus spp | 12 | 15 | 0 | 27 (2.22) | ||
| Corynebacterium spp | 21 | 7 | 2 | 30 (2.47) | ||
| 3 | Gram‐negative cocci and coccobacilli | 12 | 12 (0.99) | |||
| Moraxella spp | 9 | 9 (0.74) | ||||
| Neisseria spp | 3 | 3 (0.25) | ||||
| 4 | Aerobic actinomycetes | 39 | 7 | 46 (3.78) | ||
| Nocardia spp | 39 | 7 | 46 (3.78) | |||
| 5 | Gram‐negative bacilli | 245 | 36 | 39 | 1 | 321 (26.40) |
| Pseudomonas spp | 173 | 29 | 36 | 1 | 239 (19.65) | |
| Enterobacter spp | 26 | 5 | 3 | 34 (2.81) | ||
| Klebsiella spp | 10 | 2 | 12 (0.99) | |||
| Proteus spp | 6 | 6 (0.49) | ||||
| Alcaligens spp | 6 | 6 (0.49) | ||||
| Haemophilus spp | 6 | 6 (0.49) | ||||
| Acinetobacter spp | 6 | 6 (0.49) | ||||
| E coli | 4 | 4 (0.33) | ||||
| Serratia spp | 3 | 3 (0.25) | ||||
| Citrobacter spp | 5 | 5 (0.41) | ||||
| Total number of isolates (%) | 1004 (82.57) | 130 (10.69) | 81 (6.66) | 1 (0.08) | 1216 (100) |
Table 3 Fungal pathogens isolated from corneal scrapes obtained from 1220 eyes with infective keratitis treated at a tertiary eye care referral centre in south India.
| Sl no | Name of the fungal isolates | Pure isolates | Mixed with other fungal spp | Mixed with bacterial spp | Mixed with Acanthamoeba spp | Total number of fungal isolates (%) |
|---|---|---|---|---|---|---|
| 1 | Hyaline fungi | 840 | 9 | 41 | 1 | 891 (72.68) |
| Fusarium spp | 483 | 3 | 24 | 1 | 511 (41.68) | |
| Aspergillus spp | 292 | 4 | 9 | 305 (24.88) | ||
| Mucor spp | 6 | 6 (0.49) | ||||
| Rhizopus spp | 4 | 4 (0.33) | ||||
| Penicillium spp | 4 | 4 (0.33) | ||||
| Unidentified hyaline fungal species | 51 | 2 | 8 | 61 (4.97) | ||
| 2 | Dematiaceous fungi | 292 | 3 | 40 | 335 (27.32) | |
| Cladosporium spp | 73 | 1 | 7 | 81 (6.61) | ||
| Botryodiploidia spp | 53 | 4 | 57 (4.65) | |||
| Curvularia spp | 43 | 9 | 52 (4.24) | |||
| Biopolaris spp | 26 | 3 | 29 (2.36) | |||
| Exserohilum spp | 21 | 3 | 24 (1.96) | |||
| Alternaria spp | 11 | 2 | 13 (1.06) | |||
| Unidentified dematiacious fungal species | 65 | 2 | 12 | 79 (6.44) | ||
| Total number of isolates (%) | 1132 (92.33) | 12 (0.98) | 81 (6.61) | 1 (0.08) | 1226 (100) |
Analysis using culture as the gold standard showed that the sensitivity of the KOH wet‐mount preparation was higher (99.3% (95% confidence interval (CI) 98.6 to 99.6) in the detection of fungal filaments, 100% (95% CI 90.4 to 100) in the detection of Nocardia filaments and 91.4% (95% CI 75.8 to 97.8) in the detection of Acanthamoeba cysts) than that of Gram‐stained smears (89.2% (95% CI 87.3 to 90.8) in the detection of fungal filaments, 87% (95% CI 73.0 to 94.6) in the detection of Nocardia spp and 60% (95% CI 42.2 to 75.6) in the detection of Acanthamoeba cysts) in the detection of fungi, Nocardia and Acanthamoeba, whereas the specificity of Gram‐stained smears was 100% in the detection of fungi, Nocardia and Acanthamoeba. These results are summarised in table 4. The overall sensitivities of KOH smears (in the detection of fungus, Nocardia and Acanthamoeba) and Gram‐stained smears (in the detection of fungus, bacteria (including Nocardia) and Acanthamoeba) were 99.1% (95% CI 98.3 to 99.5) and 93.8% (95% CI 92.8 to 94.7), respectively (table 5).
Table 4 Correlation between direct microscopic (10% KOH wet‐mount preparation, Gram‐stained, Giemsa‐stained and Kinyoun's acid‐fast stained smears) detection and culture‐based diagnosis of fungus, bacteria, Nocardia and Acanthamoeba from corneal scrapes obtained from eyes (n = 3298) with infective keratitis.
| Sl no | Direct microscopic investigations | Results | No of eyes | Culture | Sensitivity (%) (95% CI) | Specificity (%) (95% CI) | Positive predictive value (%) (95% CI) | Negative predictive value (%) (95% CI) | |
|---|---|---|---|---|---|---|---|---|---|
| Positive | Negative | ||||||||
| 1 | Detection of fungal filaments in 10% KOH wet‐mount preparation | Positive | 1230 | 1211* | 19 | 99.3 | 99.1 | 98.5 | 99.6 |
| Negative | 2068 | 9† | 2059 | (98.6 to 99.6) | (98.5 to 99.4) | (97.6 to 99.0) | (99.1 to 99.8) | ||
| Total | 3298 | 1220‡ | 2078 | ||||||
| Detection of fungal filaments in Gram‐stained smear | Positive | 1088 | 1088§ | 0 | 89.2 | 100 | 100 | 94 | |
| Negative | 2210 | 132† | 2078 | (87.3 to 90.8) | (99.8 to 100.0) | (99.6 to 100) | (92.9 to 95.0) | ||
| Total | 3298 | 1220‡ | 2078 | ||||||
| 2 | Detection of bacteria in Gram‐stained smear | Positive | 1203 | 1151¶ | 52 | 100 | 97.6 | 95.7 | 100 |
| Negative | 2095 | 0 | 2095 | (99.6 to 100) | (96.8 to 98.2) | (94.3 to 96.7) | (99.8 to 100) | ||
| Total | 3298 | 1151¶ | 2147 | ||||||
| 3 | Detection of Nocardia spp in 10% KOH wet‐mount preparation | Positive | 46 | 46** | 0 | 100 | 100 | 100 | 99.4 |
| Negative | 3252 | 0 | 3252 | (90.4 to 100) | (99.99 to 100) | (75.9 to 100) | (99.1 to 99.6) | ||
| Total | 3298 | 46** | 3252 | ||||||
| Detection of Nocardia spp in Gram‐stained smear | Positive | 40 | 40†† | 0 | 87 | 100 | 100 | 99.8 | |
| Negative | 3258 | 6‡‡ | 3252 | (73.0 to 94.6) | (99.9 to 100) | (89.1 to 100) | (99.6 to 99.9) | ||
| Total | 3298 | 46** | 3252 | ||||||
| Detection of Nocardia spp in Kinyoun's acid‐fast stained smear | Positive | 40 | 40†† | 0 | 87 | 100 | 100 | 99.8 | |
| Negative | 3258 | 6‡‡ | 3252 | (73.0 to 94.6) | (99.9 to 100) | (89.1 to 100) | (99.6 to 99.9) | ||
| Total | 3298 | 46** | 3252 | ||||||
| 4 | Detection of Acanthamoeba spp in 10% wet‐mount preparation | Positive | 32 | 32§§ | 0 | 91.4 | 100 | 100 | 99.9 |
| Negative | 3266 | 3¶¶ | 3263 | (75.8 to 97.8) | (99.9 to 100) | (86.7 to 100) | (99.7 to 100) | ||
| Total | 3298 | 35*** | 3263 | ||||||
| Detection of Acanthamoea spp in Gram‐stained smear | Positive | 21 | 21††† | 0 | 60 | 100 | 100 | 99.6 | |
| Negative | 3277 | 14¶¶ | 3263 | (42.2 to 75.6) | (99.9 to 100) | (80.8 to 100) | (99.3 to 99.8) | ||
| Total | 3298 | 35*** | 3263 | ||||||
| Detection of Acanthamoeba spp in Giemsa‐stained smear | Positive | 16 | 16‡‡‡ | 0 | 45.71 | 100 | 100 | 99.4 | |
| Negative | 3282 | 19¶¶ | 3263 | (29.2 to 63.1) | (99.9 to 100) | (75.9 to 100) | (991 to 99.6) | ||
| Total | 3298 | 35*** | 3263 | ||||||
*Of 1211 eyes, 1129 eyes had fungal growth alone, 81 eyes had both fungal and bacterial growth, and 1 eye had both fungal and acanthamoebic growth.
†Fungal growth alone.
‡Of 1220 eyes, 1138 had fungal growth alone, 81 had both fungal and bacterial growth, and 1 eye had both fungal and acanthamoebic growth.
§Of 1088 eyes, 1006 had fungal growth alone, 81 eyes had both fungal and bacterial growth, and 1 eye had both fungal and acanthamoebic growth.
¶Of 1151 eyes, 1069 eyes had bacterial growth alone, 81 eyes had both bacterial and fungal growth, and 1 eye had both bacterial and acanthamoebic growth.
**Of 46 eyes, 39 eyes had Nocardia growth alone and 7 eyes had both Nocardia and other bacterial growth.
††Of 40 eyes, 39 eyes had Nocardia growth alone and 1 had both Nocardia and other bacterial growth.
‡‡Nocardia growth alone.
§§Of 32 eyes, 30 eyes had acanthamoebic growth alone, 1 eye had both acanthamoebic and fungal growth, and 1 eye had both acanthamoebic and bacterial growth.
¶¶Acanthamoeba growth alone.
***Of 35 eyes, 33 eyes had acanthamoebic growth alone, 1 eye had both acanthamoebic and fungal growth, and 1 eye had both acanthamoebic and bacterial growth.
†††Of 21 eyes, 19 eyes had acanthamoebic growth alone, 1 eye had both acanthamoebic and fungal growth, and 1 eye had both acanthamoebic and bacterial growth.
‡‡‡Of 16 eyes, 14 eyes had acanthamoebic growth alone, 1 eye had both acanthamoebic and fungal growth, and 1 eye had both acanthamoebic and bacterial growth.
Table 5 Overall efficacy of 10% KOH wet‐mount preparation and Gram‐stained smear in the detection of fungus, bacteria (including Nocardia) and Acanthamoeba from corneal scrapes obtained from eyes (n = 3298) with infective keratitis.
| Sl no | Direct microscopic investigations | Results | No of eyes | Culture | Sensitivity (%) | Specificity (%) | Positive predictive value (%) | Negative predictive value (%) | |
|---|---|---|---|---|---|---|---|---|---|
| Positive | Negative | ||||||||
| 1 | Detection of fungus, Nocardia and Acanthamoeba in 10% KOH wet‐mount preparation | Positive | 1308* | 1289† | 19 | 99.1 | 99.0 | 98.5 | 99.4 |
| Negative | 1990‡ | 12§ | 1978 | (95% CI 98.3 to 99.5) | (95% CI 98.5 to 99.4) | (95% CI 97.7 to 99.1) | (95% CI 98.9 to 99.7) | ||
| Total | 3298 | 1301¶ | 1997 | ||||||
| 2 | Detection of bacteria (including Nocardia), fungi and Acanthamoeba in Gram‐stained smear | Positive | 2352** | 2300†† | 52 | 93.8 | 93.9 | 97.8 | 83.9 |
| Negative | 946‡‡ | 152§§ | 794 | (95% CI 92.8 to 94.7) | (95% CI 92.0 to 95.3) | (95% CI 97.1 to 98.3) | (95% CI 81.4 to 86.2) | ||
| Total | 3298 | 2452*** | 846 | ||||||
*Of 1308 eyes, 1230 were found to be positive for fungus, 46 were positive for Nocardia and 32 were positive for Acanthamoeba in 10% KOH wet‐mount preparation.
†Of 1289 eyes, 1211 had fungal growth, 46 had Nocardia growth and 32 had Acanthamoeba growth.
‡Eyes negative for fungus, Nocardia and Acanthamoeba in KOH wet‐mount preparation.
§Of 12 eyes, 9 had fungal growth and 3 had Acanthamoeba growth.
¶Of 1301 eyes, 1220 had fungal growth, 46 had Nocardia growth and 35 had Acanthamoeba growth.
**Of 2352 eyes, 1088 were found to be positive for fungus, 1203 were positive for bacteria, 40 were positive for Nocardia and 21 were positive for Acanthamoeba in Gram‐stained smear.
††Of 2300 eyes, 1088 eyes had fungal growth, 1151 eyes had bacterial growth, 40 had Nocardia growth and 21 had Acanthamoeba growth.
‡‡Eyes negative for fungus, bacteria (including Nocardia) and Acanthamoeba in Gram‐stained smear.
§§Of 152 eyes, 132 eyes had fungal growth, 6 had Nocardia growth and 14 had Acanthamoeba growth.
***Of 2452 eyes, 1220 had fungal growth, 1151 had bacterial growth, 46 had Nocardia growth, 35 had Acanthamoeba growth.
The false‐negative rate of KOH smears was less (0.43% in the detection of fungi, 0% in the detection Nocardia and 0.09% in the detection of Acanthamoeba) than that of Gram‐stained smears (5.97% in the detection of fungi, 0.18% in the detection of Nocardia, 0.43% in the detection of Acanthamoeba) and Giemsa‐stained smears (0.58% in the detection of Acanthamoeba). The false‐positive rate of KOH smears was 1.5% in the detection of fungi and 0% in the detection of Nocardia and Acanthamoeba, whereas no false‐positive rate was noted for Gram‐stained smears.
Most of the patients (53.54%) presented more than 7 days after onset of illness, and 100% of the patients with Acanthamoeba keratitis reported after 15 days. In all, 2793 of 3298 (84.69%) eyes had corneal ulcer with size >2 mm in diameter at initial presentation, of which 596 (18.07%) were >6 mm in diameter. Table 6 shows the size of the corneal ulcers measured and the duration of the patients' symptoms before presentation.
Table 6 Size of corneal ulcers (n = 3298) and duration of symptoms before presentation, and microbial growth pattern of patients (n = 3295) with infective keratitis.
| Sl no | Demographic characters | Total no of cases (%) | Bacterial growth (%) | Fungal growth (%) | Acanthamoeba growth (%) | Bacterial and fungal (%) | Acanthamoeba and fungal (%) | Acanthamoeba and bacterial (%) | No growth (%) | |
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | Size of the corneal ulcer | <2 mm | 505 (15.31) | 109 (10.2) | 59 (5.19) | 0 | 0 | 0 | 0 | 337 (34.56) |
| 2–6 mm | 2197 (66.62) | 797 (74.55) | 785 (68.98) | 6 (18.18) | 17 (20.99) | 0 | 0 | 592 (60.72) | ||
| >6 mm | 596 (18.07) | 163 (15.25) | 294 (25.83) | 27 (81.82) | 64 (79.01) | 1 (100) | 1 (100) | 46 (4.72) | ||
| Total no of eyes studied | 3298 (100) | 1069 (100) | 1138 (100) | 33 (100) | 81 (100) | 1 (100) | 1 (100) | 975 (100) | ||
| 2 | Duration of presentationafter onset of illness | 1–3 days | 497 (15.08) | 247 (23.17) | 109 (9.58) | 0 | 0 | 0 | 0 | 141 (14.46) |
| 4–7 days | 1034 (31.38) | 331 (31.05) | 415 (36.47) | 0 | 5 (6.17) | 0 | 0 | 283 (29.02) | ||
| 8–14 days | 817 (24.8) | 264 (24.76) | 370 (32.51) | 0 | 19 (23.46) | 0 | 0 | 164 (16.82) | ||
| 15–29 days | 541 (16.42) | 105 (9.85) | 111 (9.75) | 2 (6.06) | 39 (48.15) | 0 | 0 | 284 (29.13) | ||
| 1–2 months | 225 (6.83) | 91 (8.54) | 69 (6.06) | 16 (48.48) | 12 (14.81) | 1 (100) | 1 (100) | 35 (3.59) | ||
| >2 months | 181 (5.49) | 28 (2.63) | 64 (5.62) | 15 (45.45) | 6 (7.41) | 0 | 0 | 68 (6.97) | ||
| Total no of patients studied | 3295 (100) | 1066 (100) | 1138 (100) | 33 (100) | 81 (100) | 1 (100) | 1 (100) | 975 (100) | ||
The rate of positivity of KOH smears for microorganisms was 44.6% among eyes with corneal ulcer size >2 mm in diameter and 13.06% among eyes with corneal ulcer <2 mm in size (p<0.001; odds ratio (OR) 5.33, 95% CI 4.0.4 to 7.04). Similarly, the incidence of Gram‐stained smear positivity for microorganisms was 77.37% among eyes with corneal ulcer size >2 mm in diameter and 29.91% among eyes with corneal ulcer <2 mm in size (p<0.001; OR 8.02, 95% CI 6.46 to 9.95). Table 7 documents the size of the corneal ulcers evaluated and the microbiological correlation.
Table 7 Size of corneal ulcers evaluated in 3298 eyes and microbiological correlation.
| Sl no | Size of corneal ulcer | Positive for fungus | Positive for bacteria including Nocardia | Positive for Acanthamoeba | Positive for Nocardia | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| KOH +ve | Gram +ve | Culture +ve | KOH and culture +ve | KOH and Gram +ve | Gram and culture +ve | Gram +ve | Culture +ve | Gram and culture +ve | KOH +ve | Gram +ve | Culture +ve | KOH and culture +ve | KOH and Gram +ve | Gram and culture +ve | KOH +ve | Gram +ve | Culture +ve | KOH and culture +ve | KOH and Gram +ve | Gram and culture +ve | ||
| 1 | <2 mm | 61 | 15 | 59 | 50 | 15 | 15 | 136 | 109 | 109 | 0 | 0 | 0 | 0 | 0 | 0 | 5 | 0 | 5 | 0 | 0 | 0 |
| 2 | 2–6 mm | 806 | 718 | 802 | 802 | 719 | 719 | 826 | 814 | 814 | 5 | 0 | 6 | 5 | 0 | 0 | 23 | 22 | 23 | 22 | 22 | 22 |
| 3 | >6 mm | 363 | 355 | 359 | 359 | 355 | 355 | 241 | 228 | 228 | 27 | 21 | 29 | 27 | 21 | 21 | 18 | 18 | 18 | 18 | 18 | 18 |
| Total | 1230 | 1088 | 1220 | 1211 | 1088 | 1088 | 1203 | 1151 | 1151 | 32 | 21 | 35 | 32 | 21 | 21 | 46 | 40 | 46 | 40 | 40 | 40 | |
Discussion
Methods for rapid detection of microbial agents and confirmation of clinical diagnosis are extremely important in the management of infective keratitis. The common laboratory techniques for identifying microbial agents causing corneal infections are culture and direct microscopic smear examinations of the corneal scrapes.16,19 In addition, molecular diagnosis of pathogenic agents is a newer technology for accurate identification of the causative agents20 but is inapplicable to all patients with corneal ulcer, as it is more expensive. Although cultures require a longer time depending on the organisms (24 h to 3 weeks), examination of a smear can provide results in a short span of time, enabling the clinician to start empirical treatment.21 At this study centre, we regularly carry out 10% KOH wet‐mount preparation, Gram‐stain procedure, and culture for all clinically diagnosed cases of infective keratitis. In addition, if required, Geimsa and Kinyoun's acid‐fast staining procedures are also carried out for suspected cases. In this analysis, observations of smears and cultures of corneal scrapes obtained from all 3298 eyes highlight the value of the traditional method of KOH wet‐mount preparation in the diagnosis of fungal, Nocardia and acanthamoebic keratitis. The overall sensitivity of KOH smears achieved in this study was higher than the sensitivity reported by other previous reports. Sharma et al9 reported 81.2% sensitivity and 83.8% specificity of KOH wet‐mount preparation in the detection of fungal filaments. Hagan et al21 reported 80% sensitivity and 93% specificity of KOH preparation in fungal detection.
The size of the corneal ulcers, scraping technique, amount of the scraped material and microscopic observation of the scraped materials are some of the factors that contributed to this. Generally, corneal ulcers with large sizes will provide enough material for microbiological investigation, both for smears and cultures, and preparation of additional slides and inoculation of more than one culture media are also possible. In our centre, most patients (84.69%) with corneal ulcers presented with large ulcers (>2 mm in diameter) owing to late presentation. In this study, there is a marked association between the smear positivity and size of the corneal ulcer. Larger ulcers (>2 mm in diameter) provided a higher positivity rate in both KOH and Gram‐stained smears than ulcers <2 mm in diameter, and thus the rate of positivity of smears increased according to the size of the corneal ulcers. Inappropriate previous medical treatment before presentation to our institute,16 use of non‐prescription drugs sold over the counter, use of traditional eye medicines,16 non‐availability of proper health eye care and poor socioeconomic status19 are the important factors responsible for the late presentation of patients with corneal ulcers to tertiary eye centres.16 This leads to increase in the size and severity of the ulcers, providing enough material with good microbial load for microscopy and culture. Hence, these factors may be responsible for increased sensitivity of the KOH smears in this study.
However, the value of 10% KOH wet‐mount preparation in the diagnosis of fungal keratitis lies in its ability to clear the scrapes of cellular debris, thereby rendering hyphal fragments more refractile on microscopic examination. The staining quality of Gram stain is often variable, hyphal elements often appear as linearly stained precipitates, and it is usually not possible to determine whether they are coenocytic or septate. If the stained smear of scrape from an ulcer is thick, the hyphae will be interspersed throughout the necrotic tissue and their identification may be difficult or impossible.22 Similarly, in KOH smears, Nocardia was easily recognised as very fine, intertwined, narrow, delicate, branching filaments, whereas in Gram‐stained smears, Nocardia appeared as Gram‐positive, beaded, coccoid and thin branching filaments, and were difficult to identify. In Kinyoun's acid‐fast stained smears, Nocardia appeared as thin, pink, branching filaments on a blue background.23 Although 10% KOH smear is a sensitive method for the detection of Nocardia, Kinyoun's acid‐fast stain helps to differentiate Nocardia spp from Actinomyces spp by its acid‐fastness.23
In the diagnosis of bacterial keratitis, the sensitivity of Gram stain (100%) obtained in this study was higher than that reported by Sharma et al11 in early keratitis (36%) and also in advanced keratitis (40.9%). Asbell and Stenson24 reported 67.0% sensitivity of Gram stain in the detection of bacteria in the US, and Dunlop et al25 reported 62.0% detection in Bangladesh. The results of this analysis indicate that Gram stain has a vital role in the diagnosis of bacterial keratitis. Similar to fungi and Nocardia detection, the sensitivity of 10% KOH smears (91.45%) was higher than that of Gram‐stained smears (60%) and Giemsa‐stained smears (45.71%) in the detection of Acanthamoeba. Sharma et al26 reported 87.1% sensitivity of KOH with calcofluor white preparation in the detection of Acanthamoeba cysts in Hyderabad. In the 10% KOH wet‐mount preparation, Acanthamoeba was recognised in the form of double‐walled cysts, having an outer wrinkled wall (ectocysts) and an inner polygonal, stellate wall (endocyst).27 The clarity of the cysts in the preparation was remarkable. However, in older KOH preparations, slight disintegration was noted in the cyst morphology. The staining quality of Gram‐stained smears is often variable. In Gram‐stained smears, the structure of the cyst was not as remarkable, and resembled tissue macrophages, mononuclear cells or degenerative epithelium.
Although the rate of positivity of Gram‐stained smears seemed to be higher than that of KOH smears in the detection of microorganisms, the overall analysis shows that the sensitivity of KOH smears was markedly higher than that of Gram‐stained smears in the detection of fungi, Nocardia and Acanthamoeba. If we carry out KOH smears alone, we can easily classify corneal ulcers based on the result of KOH smears as fungal, Nocardia or Acanthamoeba, where the smear would be positive for these organisms, or other than these three groups where it would be negative, and this is helpful to institute early, specific treatment. Thus, a KOH smear is of greater diagnostic value in the management of infective keratitis, and so the simple, sensitive technique of KOH wet mounting is recommended in all clinics without exception for establishing timely treatment. This study reports on examination of smears of corneal scrape results for fungi, bacteria, Nocardia and Acanthamoeba. Gram stain was found to be very dependable for making decisions in the treatment of bacterial keratitis, thereby ensuring a place for empirical treatment in suspected cases of bacterial keratitis. KOH wet‐mount preparation was highly reliable in confirming the diagnosis of fungal keratitis, Nocardia keratitis and Acanthamoeba keratitis.
Footnotes
Competing interests: None declared.
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