Microbiological Analysis of Ophthalmic Solutions

ABSTRACT:

Microbial corneal infection is the most serious complication of contact lens wear. A contaminated contact lens solution and accessories can act as a reservoir for microorganisms that could potentially compromise contact lens wear and lead to sight threatening adverse events. The rate of lens case solutions contamination is commonly over 50%. The aim of our research study to isolate microorganisms from contact lens storage solutions and to characterize and identified according to their colonial morphology, cultural characteristics and biochemical test.  We take different samples of lens care storage solution from asymptomatic patients. Culture samples on blood agar and macconkey agar for gram positive and negative bacteria, SDA for fungal isolation and PAS (pages amoeba saline) for Acanthamoeba isolation. And we found 100% lens care storage solutions contaminated from microorganisms. In which our result shows that 50% samples were contaminated with gram positive bacteria while 50% samples were contaminated with gram negative bacteria, and 67% Fungus were present. Acanthamoeba were not isolated in any tested sample, may be because it rarely isolated invitro from samples. Hence it is summarized that it is extremely important to maintain high level of contact lens hygiene and that you follow all the cleaning procedures. Proper care should be taken by contact lens users, to maintain high level of hygiene, proper changing of lens care solution after some time.

Key words: contact lenses, Acanthamoeba, hygiene

 INTRODUCTION

Ocular infections rate increases dramatically nowadays and one of the most important factor is the increased use of contact lenses. Contact lenses (CLs) are now worn by millions of people worldwide and by about 1.65 million people in the UK.  The wearing of CLs causes changes in the cornea in terms of structure, turnover, tear production and oxygen and carbon dioxide levels. These changes in themselves can produce problems and may also exacerbate pre-existing conditions, and results in different types of ocular infections

Approximately 6% of CL wearers per year will develop some problem associated with their use, although the majority of these problems will be fairly minor. A recent study has found that they account for 9.1% of the referrals into the eye casualty unit. There is increasing evidence that bacterial biofilms play a role in a variety of ocular infections. Bacterial growth is characterized as a biofilm when bacteria attach to a surface and/or to each other. Microbial keratitis is a potentially blinding disease that is rare in normal eyes unless associated with contact lens (CL) wear. 

One of the study shows that During a nine-year period ending in 1977, we scraped and cultured 663 corneal ulcers. Of these cases of keratitis, 238 were bacterial infections, 133 were fungal, and 292 were culture-negative. Pseudomonas was the predominant bacterial organism, and Fusarium was the most common fungus isolated. The clinical profile of patients with fungal keratitis differed from that of patients with Pseudomonas keratitis. Thirty-three percent of the patients with Pseudomonas keratitis were wearing contact lenses at the time of their infection, and the remainder had a high incidence of predisposing ocular conditions 

Infection is the most frequent cause of keratitis. Bacteria, viruses, fungi, and parasitic organisms may all infect the cornea, causing infectious or microbial keratitis.

The spectrum of causative organisms in all microbial keratitis varies by climate and predisposing factor. In general, Gram-positive bacteria are more frequently recovered in temperate climate regions, and Gram-negative bacteria and fungi in tropical or sub-tropical climates.^9,10,11  Fungi account for 5–40% of culture proven infections.

Pseudomonas aeruginosa is the most commonly recovered causative organism in contact lens-related disease, followed by Gram-positive bacteria, fungi and Acanthamoeba.

The lens, storage case, and ocular environment may offer a suitable survival niche for this environmental organism. P. aeruginosa can adhere to and colonise lens materials during wear and survive in contact lens storage cases. About 80 to 90 percent of contact lens-related eye infections are bacterial. Many involve common and tough-to-treat bacteria known as staphylococcus aureus. But the most severe may be pseudomonas aeruginosa, a fast-growing bacterial infection that can lead to a hole in your cornea. Fungus was also reported to cause serious eye infections. In 2006, a major fungal eye infection outbreak occurred in the United States. Infection of Fusarium keratitis was observed among contact lens users.  Acanthamoeba eye infections in contact lens wearers are rare but serious, and they often start because of improper lens handling and poor hygiene  because contact lenses may be an important vehicle for the transfer of amoebae from contaminated contact lens cases to the cornea. Viruses also known to cause serious eye infections they may be transmitted from contact lenses of diseased patient, Herpes and adenoviral infections can occur during CL wear, Although HIV has been isolated from ocular tissues, tears and soft CLs used by patients with AIDS, there are no documented cases of HIV transmission through contaminated tears or CLs. HSV keratitis is a major cause of blindness worldwide.

Because contact lenses may be an important vehicle for the transfer of microorganisms from contaminated contact lens solutions to the cornea, we undertook a research study of the microbiological analysis of the contact lens solutions from asymptomatic daily wear cosmetic contact lens wearers. Particular note was made of the lens hygiene systems being employed and their relative effectiveness in preventing or inhibiting microbial colonization of the contact lens solutions.

MATERIAL AND METHOD:

Sample collection

To isolate microorganisms from contact lens storage cases, we take different samples of lens care storage cases solution from asymptomatic patients. Sample was collected by swabbing on different media as according to the requirement.

Bacterial Isolation

Using a standard 5, ul bacterial loop, the lens case solution was cultured onto 5% Columbia blood agar and MacConkey agar plates and incubated in air at 30°C for 3 days. After incubation all lactose fermenting and non-lactose fermenting Gram negative bacilli were identified to the genus level using a series of manual biochemical tests." These tests included; Oxidase, motility, oxidation/fermentation, citrate, methyl red, indole, Voges-Proskauer, and growth on Macconkey agar. Catalase was assayed qualitatively using hydrogen peroxide as outlined in the Manual of Clinical Microbiology.'1 Gram positive bacteria were identified by their characteristic colonial morphology and Gram stain appearance. No attempt was made to further speciate bacteria.

 Fungal Isolation

A 0.5 ml aliquot of the lens case solution was cultured onto an SAC slope (Sabouraud dextrose agar+chloramphenicol 0. 1%+gentamicin 0'4%) and incubated in air at 27°C. Cultures were incubated for 14 days before being discarded as negative. Positive cultures were identified by microscopic and macroscopic morphology.

 Amoebal Isolation

A 1 0 ml aliquot of the contact lens case solution cultured on a Pages amoeba saline (PAS) agar plate.PAS  which had previously been spread with a lawn of heat killed (65°C/30 minutes) Escherichia coli.1I The plates were incubated in a humidified chamber at 30°C for up to 7 days, and examined every 48 hours. Cultured amoebae were identified as either Acanthamoeba or other free living amoebae by morphology of cyst, flagellate, and trophozoites stages.

RESULTS

Microbiological analysis of contact lenses showed increased level of microbial contamination. ALL the samples were contaminated with microbes except amoeba. Out of 6 samples, 3 were contaminated with gram positive bacteria(33.3% streptococcus, 16% staphylococcus, 16% bacillus) while other three samples were contaminated with gram negative bacteria(33.3% pseudomonas aeruginosa, 16%  Klebsiella). Fungus was isolated in four out of six samples (50% Aspergillus niger, 16% Aspergillus flavus, 16% Aspergillus fumigatus, 16% sporangium). Acanthamoeba were not isolated in any tested sample, may be because it rarely isolated invitro from samples.

Table 1: Morphological, Cultural & Biochemical Characteristics of the isolated gram positive bacteria

number of sample	colonial characteristics on BA	Cultural characteristics	gram R/C	catalase	Coagulase	growth on MSA	Identified organism
SAMPLE#1	Gray white colonies with beta hemolysis	Cocci in chains	+ve	-ve		-	Streptococcus
SAMPLE#2	Large round yellow colonies with beta hemolysis/ and gray colonies	1.Cocci in clusters/ 2.Cocci in chains	+ve	1.+ve 2. -ve	1. +ve 2. -ve	1.-ve 2.+ve	1.Staphylococcus 2.Streptococcus
SAMPLE#3	Large colonies with beta hemolysis	Scattered rods	+ve	+ve	-	-	Bacillus

Table 2: Morphological, Cultural & Biochemical Characteristics of the isolated gram negative bacteria

Number of samples	Colonial morphology	Cultural characteristic	Gram R/C	TSI	citrate	urease	oxidase	Identified organism
Sample#4	Small round non-lactose fermenting colonies	Scattered rods	-ve	K/A	+ve	-ve	+ve	Pseudomonas aeruginosa
Sample#5	Small round non-lactose fermenting colonies	Scattered rods	-ve	K/A	+ve	-ve	+ve	Pseudomonas aeruginosa
Sample#6	Large gummy lactose fermenting colonies	Rods	-ve	A/A	+ve	+ve	-ve	Klebsiella

  

Table: 3 macroscopic and microscopic characteristics of isolated fungal species

Number of sample		Macroscopic characteristics	Microscopic characteristics	Fungus identified
Sample#4		Black colonies	Dark brown round conidia	Aspergillus niger
Sample#3		1.Black colonies 2.velvety white colony	1.dark brown conidial heads 2. sporangiospores, branched hyphae	Aspergillus niger/mucor
Sample#5		Black colonies	dark brown conidial heads	Aspergillus niger
Sample#6	Powdery / smoky green		1.Conidial heads & short conidiophores 2. septate hyphae & long conidiophores		Aspergillus fumigates/ Aspergillus flavus

DISCUSSION

Corneal infection is the most common vision threatening complication of contact lens wear. Organisms isolated from contact lens associated corneal ulcers have often been shown to be identical to those isolated from the contact lens case, making the contaminated contact lens solution a possible replenishable source of pathogenic microbes. In spite of the apparent adherence to recommended cleaning and disinfecting regimes, a significant degree of microbial contamination of contact lens solutions  was found in this study.

In our research a significant degree of microbial contamination of contact lens storage solution was found in this study. Many of the contaminants identified were potential pathogens and as such should have been prevented by the disinfectant regimen used. In our research study different bacterial strains (100%) and fungal strains (80%) isolated and protozoa were not isolated in our study. This survey highlights that there is a need for improvement in contact lens storage solution hygiene. Current contact lens disinfection methods do not appear to be providing a desirable level of Microbial protection. It was reported in the previous study that most common microbial contaminant isolated in descending prevalence were bacteria (78%)>fungi (24%)>protozoa (20%). Nearly all contact lens cases had mixed microbial populations. The most common bacterial contaminants isolated were non-fermentative Gram negatives followed by coliforms, other Gram negative, and Gram positive organisms (Trevor B Gray et al., 1995).

In our research study highly pathogenic organisms were isolated, Pseudomonas can be spread from the hands in the lens care solution that gets contaminated and is not properly cleaned causes ophthalmic infections. Streptococcus was also isolated, Streptococcal infections are an important cause of corneal ulcers, endophthalmitis, conjunctivitis, and dacryocystitis. Staphylococcus is also highly pathogenic bacteria cause’s ophthalmic infections. There are a number of ways to get staph and strep infections once you have a tear or injury in your cornea. Infection spread when eye comes into contact with a contaminated object (such as contaminated water or a dirty contact lens). Bacillus is also pathogenic isolate; the pathogencity of the gram positive spore forming bacilli for the eye was first reported in 1980 cause’s infection of cornea also Bacillus endophthalmitis is a highly explosive infection of the eye that commonly results in rapid inflammation and vision loss.

In fungal isolation most commonly Aspergillus species were isolated from contact lens storage solution samples. In Mycotic keratitis Two basic forms have been recognized: that due to filamentous fungi (especially Fusarium and Aspergillus), these highly pathogenic isolates from our samples may cause severe eye infections.

 Persistent microbial contamination of contact lens storage solutions is common and is associated with microbial keratitis and sterile corneal infiltrates. The single best way to avoid eye infections is to follow proper lens care guidelines as prescribed by the eye care professionals. In particular, including a "rub and rinse" step in the lens cleaning process, minimizing contact with water while wearing contact lenses and replacing the lens case frequently can help reduce the risk of infection.

CONCLUSION:

We conclude from our research study that the contact lens case solutions are the single most important potential reservoir for contact lens contamination leading to infection. Contact lenses and the solutions used with them are medical devices and are regulated by the Food and Drug Administration; therefore, it is extremely important that patients maintain regular appointments to ensure they are receiving clinical guidance from their eye doctor based on individual eye health needs. Clean and safe handling of contact lenses is one of the most important measures contact lens wearers can take to protect their sight. Exercising optimal care and hygiene with contact lenses can keep the eyes healthy. Our research study found that contact lens wearers who have poor hygiene habits also have increased bacterial contamination in their contact lens cases. Bacterial And fungal contamination was observed in several cases. Colonization of the lens storage case by pathogenic micro-organisms predisposes lens wearers to microbial or sterile keratitis.

Recommendations to contact lens wearers

From this and previous data, the authors suggest the following measures should result in less contact lens case and contact lens solution  contamination; thereby possibly reducing the risk

of microbial keratitis, proper use of disinfectant solutions, disinfection of the contact lens case, wash  hands before applying lens to the eye, homemade saline never be used, use ophthalmic solution of known high quality brand, and change it regularly.