**20. Practical strategies for coinfection diagnosis**

Analysis of drug interactions with simultaneous application is still developing. Modified methods have been proposed related to more accurate isobolographic analysis, and *in vitro* models approach physiological conditions. Animal models have also been used. This area will

An appreciation for the fact that in nature bacteria adhere to many abiotic or biotic surfaces, embedded in an extracellular matrix, and form communities known as "biofilms" has emerged over the past few decades [90]. Biofilm formation conferred on individual bacteria the ability to collaborate and to adapt to a range of harsh environmental conditions and, perhaps most of all, to evade predation by phagocytic microbes. The formation of a biofilm provides a microbe with a small measure of control over the local environment, including fluctuations in

Advances in medical biofilm research have led to the understanding that biofilms represent the prevalent form of bacterial life during tissue colonization and may occur in more than 80

Members of a biofilm community, which can be of the same or multiple species, show varying stages of differentiation and exchange information, metabolites, and genes with each other. As a result, members of the biofilm community are in a diversity of physiologies influenced by the unequal sharing of nutrients and metabolic by-products, which results in subpopula‐ tions with increased tolerance to antimicrobials and environmental stresses, the host immune

Canonically, biofilm development has been grouped into five stages that are reflective of conditions in many, but not all, biofilms: (1) reversible aggregation of planktonic cells on a surface, (2) irreversible adhesion, (3) formation of microcolonies, (4) biofilm maturation, and (5) detachment and dispersion of cells [99]. The events that are of special significance for ocular infections and the treatment of biofilm infections will be discussed in greater detail, while the reader is referred to several excellent reviews for details on other biofilm-related

The biofilms involve the production of an extracellular matrix (ECM) that embedded the cells and, in some cases, binds the cells together and that can be composed of polysaccharides, lipopolysaccharides, proteins, or extracellular DNA [10]. This process may be active or passive, in that cells on the surface of an adherent colony that are lysed by the ejection of neutrophil antimicrobial factors may encase and protect siblings below in a matrix consisting simply of cell lysate. Whatever the nature of the matrix, its chemical and physical properties contribute to the differentiation of cells within the encased population, a process that can protect the bacteria from the action of antimicrobial agents, host immune responses, bacteriophages, and

temperature, pH, ultraviolet light, starvation, and exposure to toxic agents [91, 92].

revolutionize therapeutic interventions.

% of microbial infections in the body [93].

subjects [19, 100,101].

phagocytic amoeba [19].

system, and predatory microorganisms [19, 94, 95, 96, 97, 98].

**19. Biofilms**

132 Advances in Common Eye Infections

The two leading causes of vision impairment worldwide are uncorrected refractive errors and cataract. Measures for managing those eye abnormalities frequently include the use of contact lenses and the placement of intraocular lenses and have enhanced the quality of life of millions of patients. Although the use of such devices is the great importance for correction of a variety of visual aberrations, these devices also provide a new surface on which many microbial pathogens can form biofilms (Table 2). As a result, device-related ocular infections are an important limitation of the success of such procedures. Moreover, many infections progress to secondary permanent sequelae that may lead to poor visual outcomes and occasionally loss of sight, such as acute bacterial endophthalmitis or corneal ulceration.

In all infection diseases, not only ocular infection, it is important to make sure of the micro‐ biological diagnosis, especially when the coinfections are a large percentage of the total infections. The results will provide a report on the distribution and trends in microbiological and antibiotic sensitivity patterns that will affect the patient's treatment and prognosis. We have developed simple and practical strategies in each phase for ocular infection diagnosis, including the coinfections summarized in Figure 1.


**Table 2.** Biofilm-associated infections of the eye

A successful microbiological study consists in a correct identification, but, it begins since the pre-analytic phase, where the ophthalmologist plays an important role, so that, in our laboratory, we have improved an initial lesson to emphasize two principal things. The first thing is awareness of the importance to take the ocular sample before the intensive topical antibiotic treatment. It allows us to have greater chance of bacterial growth, although it has been described that scraping may accelerate disease resolution by enhancing antibiotic penetration and the therapeutic debridement of the necrotic tissue [108]. The second thing is that during the lesson we teach to the ophthalmologist the properly way to select, collect, and transport the sample to optimize the analysis and interpretation. For the collection, we prepared kits with all the necessary to take the sample for a molecular and microbiological diagnosis; the kit contains chocolate agar (ChA), Columbia agar (CA), and Brain-Heart Infusion (BHI); these are enrichment mediums for the exigent bacteria growth, like *Streptococ‐ cus* spp. and *Kocuria* spp.; the kit also contains Sabouraud dextrose agar (SDA), for fungi growth; different types of applicators (cotton, alginate, and rayon), a glass slide for the frotis, and finally a transport media for the molecular diagnosis are also included. On the other hand, we have accord with the ophthalmologist the conditions for the sample collection and storage that are summarized in Table 3, and especially, we have established the sequence for seeded the sample because of the small amount of material and small numbers of organisms obtainable from the eye: one swab for ChA, CA, and BHI, another swab for SDA, and the frotis for Gram, Wright, and Calcofluor stain. In conclusion, the pre-analytic phase is a continuous team work between the ophthalmologist and the laboratory staff.

A successful microbiological study consists in a correct identification, but, it begins since the pre-analytic phase, where the ophthalmologist plays an important role, so that, in our laboratory, we have improved an initial lesson to emphasize two principal things. The first thing is awareness of the importance to take the ocular sample before the intensive topical antibiotic treatment. It allows us to have greater chance of bacterial growth, although it has been described that scraping may accelerate disease resolution by enhancing antibiotic penetration and the therapeutic debridement of the necrotic tissue [108]. The second thing is that during the lesson we teach to the ophthalmologist the properly way to select, collect, and transport the sample to optimize the analysis and interpretation. For the collection, we prepared kits with all the necessary to take the sample for a molecular and microbiological diagnosis; the kit contains chocolate agar (ChA), Columbia agar (CA), and Brain-Heart Infusion (BHI); these are enrichment mediums for the exigent bacteria growth, like *Streptococ‐ cus* spp. and *Kocuria* spp.; the kit also contains Sabouraud dextrose agar (SDA), for fungi growth; different types of applicators (cotton, alginate, and rayon), a glass slide for the frotis, and finally a transport media for the molecular diagnosis are also included. On the other hand, we have accord with the ophthalmologist the conditions for the sample collection and storage that are summarized in Table 3, and especially, we have established the sequence for seeded the sample because of the small amount of material and small numbers of organisms obtainable from the eye: one swab for ChA, CA, and BHI, another swab for SDA, and the frotis for Gram, Wright, and Calcofluor stain. In conclusion, the pre-analytic phase is a continuous team work

*Staphylococcus* spp. Punctual plugs

*Staphylococcus* spp., *P. aeruginosa,* and *M. chelonae* Lacrimal intubation devices

**Biofilm localization**

Intraocular lens posterior capsule

Contact lens

Corneal stroma (crystalline keratopathy)

Scleral buckles

between the ophthalmologist and the laboratory staff.

**Disease Main causative agents of infection**

**Endophthalmitis** Coagulase-negative staphylococci and

Fungi and

**Scleral buckle infection** Gram-positive cocci and nontuberculous *Mycobacterium* spp.

**Table 2.** Biofilm-associated infections of the eye

**Keratitis**

134 Advances in Common Eye Infections

**Lacrimal system infections** **and/or found in the biofilms**

*Acanthamoeba* spp. less frequently

bacilli and yeasts less frequently

*Staphylococcus aureus* and other staphylococcal species, *Pseudomonas aeruginosa* and *Serratia* spp.

Viridans group Streptococci. Gram-negative

**Periorbital infections** *Staphylococcus* spp. and mixed species biofilms Sockets and orbital plates

*Propionibacterium acnes*

**Figure 1.** Practical strategies for coinfection diagnosis during the three analytic phases. (I) The pre-analytic phase, when the ophthalmologist is training for selecting, collecting, and transporting the sample, plays an important role. (II) The analytic phase with microbiological and molecular techniques. In the microbiological diagnosis, the laboratory staff's experience is important to discern a pure from a mixed culture; molecular techniques are used to determine non-cultivable microorganisms. (III) The post-analytic phase, the final result in which the partnership between the ophthalmologist and the laboratory staff is reflected in the best outcomes for patients.

In the analytic phase, the sample could be processed by microbiology or by molecular techniques. About the microbiological diagnosis, the agar plates are checked every day, looking for bacterial or fungal growth. We have implemented a prolonged microorganism cultured of up to 15 days, because most of the hospital population includes multi-treatment patients, so that the microorganisms begin to grow until a week of incubation. Most of the times, the microorganisms involved in a coinfection are closely interacting, being impossible the identification in the automatized system (Vitek 2C, bioMérieux, France). The use of simple and classical microbiological techniques has allowed us to separate these interactions, for


Chocolate agar (ChA), Columbia agar (CA), Brain-Heart Infusion (BHI), Sabouraud dextrose agar (SDA), 37 °C for bacteria growth, 28 °C for fungi growth, and 4 °C for sample conservation

**Table 3.** Conditions for the sample collection and storage from ocular infections

example, the sonication (physical separation technique based on ultrasonic waves) and the use of selective media as MacConkey agar (MCk) and mannitol-salt agar (MSA) seeded by a perfect open streak for a good separation of the microorganisms, for positive and negative Gram bacteria, respectively. Talking about the fungi infections, the good sample collected by the ophthalmologists has been sufficient for a fungi growth and a direct observation of the macromorphology and micromorphology for the identification. However, the molecular techniques have revolutionized the ocular infection and coinfection diagnosis; these techni‐ ques are more sensitive, specific, and rapid and impact in the best outcome for the patient. The molecular techniques consist in the amplification of conserved regions of the different microorganisms involved in ocular infection, for example, Gram (+)/Gram (−) bacteria; Generic Fungi; herpes viruses I, II, and zoster; Cytomegalovirus; *Chlamydia* sp.; adenovirus; *Mycobac‐ terium tuberculosis* complex (MTC) and no *Mycobacterium tuberculosis* complex (NTC)*; Toxo‐ plasma gondii*; and *Acanthamoeba* spp. by polymerase chain reaction (PCR). The PCR helps us for the identification of coinfection caused not only bacteria-bacteria or bacteria-fungi but also coinfection caused by viruses and parasites with bacteria or fungi.

Finally, the post-analytic phase consists of the interpretation of the results. Most laboratories do not report *Staphylococcus epidermidis* and *Staphylococcus aureus*, because they are part of the ocular surface microbiota; however, the laboratory staff of ocular microbiology knows that these microorganisms can be involved directly in the ocular infection, and these two micro‐ organisms have been reported as the microorganisms most frequently isolated in infectious keratitis [109, 110]. It is important to consider the risk factor associated before deciding whether the microorganisms isolated are responsible for the infection or are a contamination.

In conclusion, the diagnosis of infectious disease is best achieved by applying in-depth knowledge of medical and laboratory science by integrating a strategic view of host-parasite interactions. Clearly, the best outcomes for patients are the result of strong partnerships between the clinician and the laboratory specialist [111].
