Role of Liquid Culture Media in the Laboratory Diagnosis of Microbial Keratitis




Purpose


To determine whether liquid culture media are helpful in the diagnosis of infectious keratitis.


Design


Retrospective noncomparative case series.


Subjects and Methods


This is a retrospective review of microbiology records of 114 corneal scraping samples from infectious keratitis patients. Samples were processed by corneal smear microscopy (potassium hydroxide with calcofluor white and Gram stains) and culture examination (5% sheep blood agar, sheep blood chocolate agar, Sabouraud dextrose agar, brain heart infusion, thioglycolate broth, and Robertson’s cooked meat broth. Cases where at least 1 liquid medium was taken were included in the study and all cases were required to have significant growth in culture as per the institutional criteria. Results of smear examination and culture growth were analyzed.


Results


Out of 114 cases, 44 (38.59%) were bacterial, 62 (54.38%) fungal, and 8 (7.01%) were mixed (bacteria + fungus) infection. Thirty-eight out of 44 cases of bacterial keratitis (86.36%) were diagnosed by solid media alone (criterion 1) and 6 of 44 (13.63%) required liquid media for diagnosis ( P < .001). In fungal keratitis, 61 of 62 cases (98.38%) were diagnosed using solid media alone (criterion 1) while 1 case required liquid media for diagnosis. In mixed infection, none of the cases required liquid media for diagnosis of fungal component; however, all 8 cases required liquid media for establishing bacterial component.


Conclusions


Liquid culture media increase the chance of isolation of bacteria in pure bacterial and/or mixed infection; however, their role in isolating fungus is limited. Owing to overlap in clinical diagnosis of bacterial and fungal keratitis, we recommend inclusion of both solid and liquid culture media in the laboratory diagnosis of nonviral keratitis.


Infectious keratitis is an important clinical entity that can potentially threaten vision. It is defined as corneal epithelial defect associated with stromal infiltrate caused by various micro-organisms, the most common and important being bacteria and fungi. It is one of the leading causes of blindness in developing countries. Accurate and rapid identification of the micro-organism is required for successful treatment of the disease. Smear and culture examination of the corneal sample is considered the gold standard for identification of the offending agent, which helps in guiding appropriate therapy to the patient. The American Academy of Ophthalmology recommends blood agar, chocolate agar, Sabouraud dextrose agar, and thioglycolate broth as standard media to be included. Although the recommendation is to collect the corneal scraping on both solid and liquid media, there is an ongoing debate in the literature on the actual clinical relevance of using multiple culture media. Added to that, there seems to be reluctance among ophthalmologists to treat microbial keratitis patients based on laboratory investigation results. Nearly half of the corneal ulcer patients in southern California were treated empirically without culture examination. Noncompliance to the recommended guidelines was said to be attributable to the high success rate of empirical treatment (>90%), cost of culture examination, and common failure of culture to identify the cause. To balance between the recommended guidelines and actual community practice, many authors have recommended modified culture techniques in microbial keratitis. Schonheyder and associates suggested that the broth culture technique may replace the standard technique of direct plate culture. Others have concluded that blood or chocolate agar alone is a reasonable alternative for standard culture methods.


Our institutional protocol, apart from direct microscopic examination of corneal scrapings stained with potassium hydroxide with calcofluor white and Gram stain, consists of inclusion of blood agar, chocolate agar, Sabouraud dextrose agar, brain heart infusion broth, thioglycolate broth, Robertson’s cooked meat broth, and non-nutrient agar for the investigation of nonviral corneal ulcers. We believe that this repertoire allows detection of bacteria (aerobic/anaerobic), fungi, or parasites associated with nonviral keratitis. However, we are aware that it is not always possible to inoculate corneal samples in all these media and liquid media are either inoculated towards the end or are sacrificed. This prompted us to determine retrospectively whether liquid culture media were helpful in the diagnosis of infectious keratitis, and we report the results in this communication.


Subjects and Methods


Microbiology records of 114 patients seen in the cornea service of the L.V. Prasad Eye Institute between January 1, and June 30, 2011 were retrospectively analyzed. The retrospective evaluation of microbiology records was approved by the ethics committee of the institute (Hyderabad Eye Research Foundation; Ethics Ref No. LEC 04-13-38; LVPEI-B-35-2013). These patients were diagnosed to have infectious keratitis and had been subjected to corneal smear microscopy (stained with potassium hydroxide with calcofluor white and Gram stain) and culture examination of the corneal scrapings. Type of media and the sequence of inoculation included 5% sheep blood agar, 5% sheep blood chocolate agar, Sabouraud dextrose agar (SDA), brain heart infusion (BHI), thioglycolate broth (THIO), Robertson’s cooked meat broth (RCM), and non-nutrient agar (NNA). The corneal scraping sample was obtained on a slit-lamp microscope with no. 15 blade attached to a Bard Parker handle after instillation of 1 drop of topical proparacaine (0.5%). Care was taken to collect the scraping directly from the corneal infiltrate without coming in contact with the conjunctiva and lids.


All media were incubated at 36°C ± 1°C except chocolate agar (5% CO 2 at 36°C ± 1°C) and SDA (27°C). Positivity of growth in different media and results of smear examination were noted. Table 1 shows the distribution of type of media that were taken for the 114 cases. Cases where at least 1 liquid medium was taken were included in the study and all cases included in the study were required to have significant growth in culture. Significance of the culture was based on: confluent growth (10 or more colonies) on the inoculum of 1 solid medium (criterion 1), and/or growth in more than 1 medium (criterion 2), and/or growth in 1 medium consistent with direct smear result (criterion 3). All media were observed daily for growth for 2 weeks. Time taken for growth was noted. Identification of bacterial colonies was done after consideration of colony characteristics, Gram reaction, morphology, and results of biochemical tests. API system (bioMérieux, Paris, France) was used for bacterial identification. Fungal species were identified by observing the rate of growth, color, consistency, texture, and characteristic microscopic features. Statistical analysis was done using online statistical software (QuickCalcs, GraphPad software Inc, San Diego, California, USA). Fisher exact test was used to calculate the P value and P < .05 was considered to be statistically significant.



Table 1

Types of Media Used for Inoculating Corneal Scrapings of 114 Patients With Nonviral Microbial Keratitis







































Media Inoculated Bacterial Keratitis N = 44 Fungal Keratitis N = 62 Mixed Infection N = 8
Blood agar 42 55 8
Chocolate agar 41 59 8
Sabouraud dextrose agar 42 59 8
Brain heart infusion broth 42 58 8
Thioglycolate broth 38 49 7
Robertson’s cooked meat broth 29 45 5




Results


Applying the criteria for significance of the culture results, 44 of 114 cases (38.59%) were bacterial, 62 (54.38%) were fungal, and 8 (7.01%) were mixed bacterial and fungal. Among the 44 bacterial cases analyzed, 38 (86.36%) showed confluent growth on at least 1 solid medium. In 32 of 42 cases (76.19%), growth was seen in blood agar, in 28 of 41 (68.29%) growth was seen in chocolate agar, and in 22 of 44 cases (50.00%) growth was seen in both media. The average duration for growth in blood and chocolate agar was 2 days (range 1-7) and 1 day (range 1-5), respectively. Whereas BHI showed growth in 26 of 42 cases (61.90%), thioglycolate was positive in 20 of 38 (52.63%) and RCM in 21 of 29 (72.41%). The average time for growth in liquid media was 2 days. Staphylococcus species was the most common micro-organism isolated, followed by Streptococcus pneumoniae ( Table 2 ). Criterion 1 was applicable in 38 of 44 cases (86.36%) that showed confluent growth in solid media. Samples from 2 cases (4.54%) showed growth in 2 different liquid media and were diagnosed by criterion 2 while in 4 cases (9.09%) criterion 3 was applied as the culture in 1 liquid medium was consistent with smear results of the corneal scraping. Among these 6 bacteria, 4 showed growth in RCM, 3 in BHI, and 2 in THIO ( Table 3 ). Thus, 6 of 44 (13.63%) samples of bacterial keratitis required liquid media in order to establish the diagnosis. Applicability of criterion 1 was significantly higher than criteria 2 and 3 combined ( P < .001, Fisher exact test).



Table 2

Types of Bacteria and Fungi Isolated From Corneal Scrapings of 44 Patients With Bacterial Keratitis and 62 Patients With Fungal Keratitis

























































































Bacterial Isolates (N = 44) Number Fungal Isolates (N = 62) Number
Staphylococcus species 10 Unidentified dematiaceous fungus 11
Staphylococcus aureus 5 Unidentified hyaline fungus 3
Streptococcus pneumoniae 8 Fusarium species 7
Alpha-hemolytic streptococci 1 Fusarium solani 8
Corynebacterium striatum 1 Aspergillus flavus 9
Corynebacterium propinquum 2 Aspergillus terreus 1
Corynebacterium species 3 Aspergillus fumigatus 8
Haemophilus parainfluenza 1 Fonsacea pedrosi 2
Enterobacter species 1 Curvularia lunata 3
Enterobacter cloacae 2 Curvularia clavata 1
Escherichia coli 1 Candida species 2
Micrococcus species 1 Cylindrocarpon destructans 3
Bacillus species 1 Penicillum species 1
Klebsiella pneumoniae 2 Acremonium species 1
Pseudomonas aeruginosa 3 Cladosporium species 1
Achromobacter xylosoxidans 2 Wangiella jeanselmei 1


Table 3

Type of Microorganisms Isolated From 6 Cases of Pure Bacterial Keratitis That Showed Growth Exclusively in Liquid Media Along With Their Smear, Culture Report, and Time of Growth
































Organism Smear Culture Medium
Staphylococcus species Gram-positive cocci RCM (1)
Staphylococcus species Negative BHI (1), THIO (2)
Streptococcus pneumoniae Gram-positive cocci BHI (2)
Enterobacter cloacae Negative BHI (2), RCM (1)
Pseudomonas aeruginosa Gram-negative bacilli RCM (2)
Pseudomonas aeruginosa Gram-negative bacilli RCM (1), THIO (1)

BHI = brain heart infusion; RCM = Robertson’s cooked meat broth; THIO = thioglycolate broth.

Values in parentheses indicate the time in days required for the growth of bacteria.


Among the 62 cases of fungal keratitis, culture was positive in solid media in 61 of 62 cases (98.38%), blood agar was positive in 51 of 55 (92.72%), chocolate agar in 50 of 59 (84.74%), and SDA in 47 of 59 (79.66%). The average duration for growth was 3 days (range 1-11 days) in blood/chocolate agar and 2 days (1-5 days) in SDA. Fungal growth was seen in liquid media in 52 of 62 cases (83.87%), BHI was positive in 42 of 58 (72.41%), thioglycolate broth in 16 of 49 (32.65%), and RCM in 25 of 45 (55.55%). The average time for growth in all 3 liquid media was 2 days. Unidentified dematiaceous fungus was the most common fungus, followed by Aspergillus flavus ( Table 2 ). Sixty-one out of 62 samples (98.38%) were diagnosed by criterion 1. None of the cases was diagnosed using criterion 2 and only 1 case (1.61%) fell within criterion 3 for diagnosis using liquid media.


Details of 8 cases with mixed growth of fungi and bacteria are shown in Table 4 . All 8 cases showed fungal growth in solid media. None of the 8 cases required liquid media for diagnosis of fungal component. In 6 of 8 cases (75%) bacteria were diagnosed using liquid media alone (criteria 2 and 3) and 2 cases required both solid and liquid media (criterion 2). BHI showed growth of bacteria in 7 of 8 cases, RCM in 3 of 8, and THIO in 2 of 8 cases. In all 8 cases (100%) bacterial growth was seen in liquid media. The most common bacteria found in mixed infection was Staphylococcus species and most common fungus was Aspergillus ( Table 4 ).



Table 4

Type of Micro-organisms Isolated From 8 Cases of Mixed-Infection Keratitis (Bacteria + Fungus) With Their Smear, Culture Report, and Time of Growth



































































Patient No. Organism Isolated in Mixed Infection (Bacteria + Fungus) Result of Microscopy (KOH + CFW Stain) Positive for Fungal Growth Result of Microscopy (Gram Stain) Positive for Bacterial Growth
1 Unidentified dematiaceous fungus
Staphylococcus species
Corynebacterium species
Fungus BA (1), CA (1), SDA (2) GPC BHI (1)
2 Acremonium species
Staphylococcus species
Negative SDA (3) GPC BHI (1)
3 Curvularia lunata
Staphylococcus species
Fungus BA (1), CA (1), SDA (1) Negative BHI (1), RCM (1)
4 Unidentified hyaline fungus
Corynebacterium propinquum
Fungus BA (1), SDA (1) Negative BHI (2), RCM (2)
5 Aspergillus fumigatus
Staphylococcus species
Corynebacterium species
Fungus RCM (1), SDA (1), BHI (1) Negative BA (1), THIO (1)
6 Unidentified hyaline fungus
Staphylococcus aureus
Fungus BA (2), SDA (2) Fungus BHI (2), BA (2)
7 Aspergillus flavus
Staphylococcus species
Fungus BA (1), CA (1), THIO (1), SDA (1) Fungus RCM (1), BHI (1)
8 Candida species
Staphylococcus species
Negative BA (1), CA (1) Negative BHI (1), THIO (1)

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Jan 9, 2017 | Posted by in OPHTHALMOLOGY | Comments Off on Role of Liquid Culture Media in the Laboratory Diagnosis of Microbial Keratitis
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