Conjunctival Flora Before and After Application of 5% Povidone-Iodine Solution

Conjunctival flora is attached with protein links to conjunctival cells. For this reason, the resident flora, Corynebacteria and Staphylococcus epidermidis are almost always present in normal conjunctives of any age, and depuration mechanism like tears, blinking or even the antibiotic do not remove it [1]. Staphylococcus epidermidis, or other coagulasa negative Staphylococcus, Corynebacteria or Propionibacterium acnes resident in normal conjunctiva and eye lids are named as frequent post-surgical endophthalmitis cause [2], pathogen bacteria as Staphylococcus aureus and Streptococcus pneumoniae are also related to acute and severe endophthalmitis, reason why it is very important to diminish the conjunctival alive bacteria previous to cataract or other ocular surgery and since 1985 Apt had proposed the use of antiseptic solutions like povidone-iodine in ocular surface. [3]. Some years later, Speaker proved in one comparative study the efficiency of antisepsis using this solution with a statistical significance less endophtalmitis as surgical risk in anterior segment ocular surgeries [4]. The use of drops of aqueous 5 % solution of povidone-iodine (polivinil pirrolidonil iodine solution) is manadatory in all ocular surgeries and its efficacy has been proved and published [5] even when not all bacteria species are diminished to 0 CFU.


Introduction
Conjunctival flora is attached with protein links to conjunctival cells. For this reason, the resident flora, Corynebacteria and Staphylococcus epidermidis are almost always present in normal conjunctives of any age, and depuration mechanism like tears, blinking or even the antibiotic do not remove it [1]. Staphylococcus epidermidis, or other coagulasa negative Staphylococcus, Corynebacteria or Propionibacterium acnes resident in normal conjunctiva and eye lids are named as frequent post-surgical endophthalmitis cause [2], pathogen bacteria as Staphylococcus aureus and Streptococcus pneumoniae are also related to acute and severe endophthalmitis, reason why it is very important to diminish the conjunctival alive bacteria previous to cataract or other ocular surgery and since 1985 Apt had proposed the use of antiseptic solutions like povidone-iodine in ocular surface. [3]. Some years later, Speaker proved in one comparative study the efficiency of antisepsis using this solution with a statistical significance less endophtalmitis as surgical risk in anterior segment ocular surgeries [4]. The use of drops of aqueous 5 % solution of povidone-iodine (polivinil pirrolidonil iodine solution) is manadatory in all ocular surgeries and its efficacy has been proved and published [5] even when not all bacteria species are diminished to 0 CFU.

Problem statement
Prevention of an endophthalmitis event as post surgical complications in cataract or other ocular surgeries is very important for the eye preservation for a good visual acuity and integrity. The purpouse of this survey is to determine the efficacy of 2 drops of 5% povidone-iodine aqueous solution applied in conjunctival sacs 1 to 2 minutes before every cactarat surgery for to reduce conjunctival alive flora in 100 patients, and to know which bacteria are susceptible or resistent and remain alive in conjunctival sacs despite the antiseptic method.

Application area
In all ocular surgeries and applicaton of intravitreal injections.

Research course
This is a prospective, comparative and linear study in 100 patients that were submitted to cataract surgery.

Method used
In each patient immediately before the surgery was taken one sample (A) without antibiotic medical treatment of conjunctival sacs in the eye that will be submitted to surgery with a cotton swab for quantitative and qualitative determination of aerobic and anaerobic bacteria, mesured in count forming unit (CFU), and a second sample (B) 2 minutes after the application of 5% povidone-iodine aqueous solution and wash out with sterile saline solution. All patients were attended in an eye care hospital in México City, Asociación para Evitar la Ceguera en México "Dr. Luis Sánches Bulnes" IAP. Collection of each specimen was performed by completely rotating the cotton swab through the lower conjunctival sac from the temporal to nasal conjunctival zone. We took care not to touch the eye lid margin or lashes. Immediately the cultures for aerobica and anaerobic bacteria were made by direct inoculation in 5% blood sheep agar with brain heart agar base, exposed on one swab side, then it was rotated 180 grades and inoculated on chocolate agar enriched plate media using the other swab side, by this technique bacteria collected in samples A and B have been distribuited equally in both solid culture media. After that, the cotton swab tip was put in thioglicolate broth an incubated at 37 centigrade for 7 days. Blood agar plates were incubated for aerobic, hemolyitic and microaerophylic bacteria for 48 hours in a candle jar (5% CO2) , and chocolate agar plates were incubated for anaerobic bacteria in anaerobic bags for 7 days in 37 Centigrades incubator. The enriched thioglycolate broth with 1% hemin and 0.001% vitamine K [6], was used for detection of even small amounts of living anaerobic, or microaerobic fastidious bacteria and incubated 7 days at 37C, and subcultured for anaerobic bacteria. Multiple bacterial species were counted, selected and identified each one in the plates of blood agar and chocolate agar and expresed in CFU [7]. The same technique for the cultures was used for samples B, after two minutes of the action of antiseptic solution, and wash away with steril saline solution. We used the sample A taken without any medical treatment as comparative sample, and samples B as problem sample for statistical calculations. Aerobic and anaerobic bacteria were identified with semi-automated techniques by Crystal ® system (BBL Meryland USA) Gram stain, oxidase, catalase and indol reaction

Status
Inclusion criteria: 1. All patients that will be subbmitted to cataract surgery in a period of one month attended in Hospital para Evitar la Ceguera en México "Dr. Luis Sánchez Bulnes" IAP in surgeries performed by Anterior Segment Service 2. Patients of any age that were submitted to cataract surgery.

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Exclusion criteria: 1. Patient that have been used topical antibiotic drops for prophylactic treatment before surgery.

Results
In the conjunctivas of 19 patients, sample A and B were negative for cultures of aerobic and anaerobic bacteria, 81 patients sowed 26 bacterial species.
The total CFU including all aerobic and anaerobic bacterial species isolated in all the samples A before the application of 5% povidone iodine solution was 5,701 and were diminished to 193 CFU including aerobic and anaerobic bacteria, with statistical significance calculated by Fisher method, of x= 0.033 as is shown in

Further research
The contamination of aquous humor in anterior chamber by periocular flora from conjunctiva, Meibomian and Zies glands, or lids, was demonstrated by Saint-Blancart even with the application of topical antibiotic drops before the surgery [8] and the confirmation that are the same Staphylococcus epidermidis from conjunctival flora was maden by rPCR methods by pulse fields in gel [2]. In our survey the most numerous bacterial colonization was Staphylococcus epidermidis and Micrococcus luteus isolated in conjunctiva before the antiseptic solution application and remained some CFU from both species in the patients conjunctivas after the ansiseptic application, as it is described by Bausz [9]. We found Corynebaterium diphteriae in normal conjunctivas before the antiseptic application (sample A), with no clinical significance, because theydid not have in the bacterial cells DNA the structural Gen of Fago B that make it sinthetize its harmful toxin. Rothia dentocariosa were found in the sample A in two patients, and remained in one patient after the antiseptis method, this anaerobic bacteria, has been related as corneal pathogen [10] or as post-surgical endophtalmitis cause [11] in our patients did not have clinicals significance.
Survival bacterial in presence of universal antiseptic solution as 5% povidone-iodine mesured in comparative application methods [12] are indicative of some physical protective mechanism for bacteria as mucus, or tears proteins that capture the iodine molecules and its remaind in Henle conjunctival crypts, factors that have not been well studied.
There is no total conjunctiva surface asepsia using topical antibiotic drops alone or iodine compound as demonstrate Inoue. [13] For this reason there are some antiseptic proposal using topical antibiotics drops in conjunctiva during three days before the anterior segment intraocular surgery [14] reaching an important reduction of bacterial CFU in the conjunctival surface and using 5% povidone iodine solution before the ocular surgeries. Has been sugested the use of polyhexamethyl-biguanida as preoperative antiseptic for cataract surgery with equal microbicidal efficacy that povidone-iodine [15], this application have not further demonstrative studies. The comparison between povidone -iodine 16 times diluited from 10% concentration and 0.05% solution of chlorexidine gluconate, povidone iodine solution showed superior disinfectant effect. [16]

Conclusion
This survey demonstrate the effectivness of 5% povidone-iodine aqueous solution for the erradication of pathogenic bacteria like Streptococcus pneumoniae, Staphylococcus aureus, Actinomyces pyogenes, A meiyeri, and A odontolyticus in conjuntival surface. The largest population of Staphylococcus epidermidis and Micrococcus luteus in conjuntival isolated in sample A remained in low quantities in sample B with statistical significance. There were two patients with colonization after antiseptic technique method used as described, with a different bacterial genus in sample A that in sample B, the colonization was caused by Acinetobacter lwoffi and Enerobacter faecalis.
With the exception of Micrococcus luteus and Corynebacterium xerosis others bacterial species were present in conjunctiva after the antisepsis in very low quantities as 1 or 2 colonies.