**2. Microarrays**

As we described before, the PCR have several advantages over the culture of to identify mi‐ croorganisms from infection. However, the disability to work with different genomes at the same time and the obtaining product with the same molecular size, make the PCR not the better method for diagnosis. Therefore, there have been developed new methods of diagno‐ sis that not only reduce the time process; also they have more sensibility and specificity [14].

That is the case of the DNA microarrays, also called biochip, DNA chip, or gene array, which are defined as an orderly arrangement of samples gene for matching known and un‐ known DNA samples based on base-pairing rules and automating the process of identifying the unknowns and they were created by Brown P.O. y Botstein D. in 1999. An experiment with a single DNA chip can provide to researchers information on thousands of genes si‐ multaneously, a dramatic increase in throughput. Microarray-based technology, with its ad‐ vantage of highly parallel detection, has been applied to both population profiling and to functional studies of complex microbial communities in the environment [15, 16]. In addi‐ tion, several studies have reported the use of PCR-amplified genomic fragment sequences as probes.

In 2005, Yeo *et al.,* reported an outbreak of acute hemorrhagic conjunctivitis in Singapore [11]. Patients were diagnosed clinically with acute hemorrhagic conjunctivitis and it was identified by PCR the presence of an enterovirus and molecular typing confirmed a variant of coxsackievirus A24 (CA24v). Full-length genome sequencing results showed that CA24v virus was responsible for the outbreak and it was evolved from virus emerged 40 years ago.

For identification by PCR/ESI-MS using oligonucleotides specific for bacterial groups rather than to a particular species, although variable regions are amplified between species and strains. Additionally, there are species-specific oligonucleotides used as primers that target genes for antibiotic resistance or some pathogenic characteristics [12]. Subsequent to amplifi‐ cation, amplicons are subjected to mass spectrometry and the pattern obtained is compared with those in the databases. The ability to identify an organism without prior knowledge of the Gram, or group of microorganisms is another advantage [12], since the stains are not re‐ quired or previous isolates that provides fast trial and will always be possible to identify the microorganisms. This technology will be improving the identification of microorganism in ocular infections, it takes some advantages as certainty and specificity, and however the cost

Kaleta *et al.,* designed a study to evaluate the feasibility of the use of PCR/ESI-MS to identify microorganisms directly from blood culture bottles in the clinical microbiology laboratory [12]. The high concordance of the results of this technique with those of standard methods, particularly at the genus level, demonstrates that PCR/ESI-MS technique is capable of rapid‐ ly evaluating clinically complex specimens providing information as to the selection and ad‐

About eye microorganisms, Pedreira *et al.,*evaluated the efficacy of a prophylactic regimen of daily topical 0.5% moxifloxacin and 5% povidone-iodine in patients with Boston type I. The patients with the prophylactic regimen were sampled and analyzed by standard culture methods and by PCR/ESI-MS [13]. The molecular diagnostic approach using PCR/ESI-MS yielded data comparable with those obtained using standard microbiologic techniques. Be‐ cause of the high throughput nature and rapid results, the method might be a useful surveil‐

As we described before, the PCR have several advantages over the culture of to identify mi‐ croorganisms from infection. However, the disability to work with different genomes at the same time and the obtaining product with the same molecular size, make the PCR not the better method for diagnosis. Therefore, there have been developed new methods of diagno‐ sis that not only reduce the time process; also they have more sensibility and specificity [14]. That is the case of the DNA microarrays, also called biochip, DNA chip, or gene array, which are defined as an orderly arrangement of samples gene for matching known and un‐

*1.1.3. PCR coupled to mass spectrometry using electrospray ionization (PCR / ESI-MS)*

is the major disadvantage.

4 Common Eye Infections

ministration of targeted antibiotics.

lance tool in patients with Boston type I.

**2. Microarrays**

The gene arrays can be classified as macroarrays or microarrays, depending on the size of the sample spots. Macroarrays contain sample spot sizes of about 300 microns or larger and can be easily imaged by existing gel and blot scanners. The sample spot sizes in microarray are typically less than 200 microns in diameter and these arrays usually contain thousands of spots; microarrays require specialized robotics and imaging equipment.

There are several steps in the design and implementation of a DNA microarray experiment, as is shown in Figure 1.

**Figure 1.** Design and implementation of DNA microarray experiment

It is important to mention that the microarrays made with cDNA are called spotted arrays because the probes are created *in vitro* and the robot put the spots on the microplate. Instead of the microarrays with oligonucleotides which are created *in situ* [17]. Recent studies have used synthesized oligonucleotides as probes because of their flexibility in design and prepa‐ ration; with intensive specificity evaluation applied to the probe design criteria [18].

Both macroarrays and microarrays can have two application forms for the DNA microarray technology: the identification of sequence (gene/gene mutation) and determination of ex‐ pression level of genes.

The determination of expression level of genes the microarrays can study the transcriptome or the proteome. For the transcriptome microarrays the probes consist on cDNA that hybrid‐ ize with the mRNA of the cell. By the other hand, the proteome microarrays can use proteins as probe or the antibody making the antigen-antibody reaction. One example of this micro‐ array is the peptide microarray analysis of *in silico*-predicted epitopes for serological diagno‐ sis of *Toxoplasma gondii* infection in humans [19].

About the identification of gene sequence, microarray should have genomic DNA as probe of a specific chromosome, specifically all the genes that compose the chromosome. Or when a microarray only has a gene with one or more different nucleotides called Single Nucleo‐ tide Polymorphism (SNP) can detect a gene mutation [20].

These microarrays are used to determinate the cancer progression; all the changes on these gene are important to establish a clinical forecast [20]. Such microarrays have been used for the detection of specific bacteria [22, 23], species determination [24], and screening of envi‐ ronmental sequences related to a certain function within a community [25, 26].

Chin-I *et al.,* coupled 16S rDNA PCR and DNA hybridization technology to construct a microar‐ ray for simultaneous detection and discrimination of eight fish pathogens (*Aeromonas hydrophi‐ la, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae* and *Vibrio anguillarum*) commonly encountered in aquaculture. The array comprised short oligonucleotide probes complementary to the poly‐ morphic regions of 16S rRNA genes from the target pathogens. The results showed that each probe consistently identified its corresponding target strain with 100% specificity [27].

Yu-Cheng *et al.,* designed the DNA probes and PCR primers for the detection of *Listeria mon‐ ocytogens, Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli* O157:H7*, Salmonella spp., Vibrio parahaemolyticus, Streptococcus agalactiae*and *Pseudomonas fluorescens* by using two sets of multiplex PCR, followed by a chromogenic macroarray system, these organisms in milk or other food products could be simultaneously detected [28].

An example of microarray designed for infection diagnosis is a microarray developed by Uchida *et al.,* for the direct detection of pathogens in osteoarticular infections by polymerase chain reaction amplification and microarray hybridization [29].

And finally, and the most interesting DNA microarray used for the endophthalmitis diagno‐ sis is the one developed by Tsutomu *et al.* They used 13 samples of vitreous fluid (VF) ob‐ tained from 13 patients during vitrectomy. Vitreous fluids from three patients with suspected endophthalmitis and ten controls without infection were subjected to testing for the presence of bacteria and fungi in culture tests, polymerase chain reaction (PCR) analysis, and DNA microarray analysis. The DNA microarray contained the spots for 16S rDNA, var‐ iable and conserved areas for bacteria, and the 18S rDNA for fungi. No control sample was positive for bacteria or fungi in the culture test, PCR, or microarray analysis. Specimens from two patients (Cases 1 and 2) with suspected endophthalmitis were positive for bacteria in PCR, and a specimen from one patient (Case 3) was positive for fungi in PCR. *Klebsiella pneumonia* (Case 1), *Streptococcus agalactiae* (Case 2), and *Candida parapsilosis* (Case 3) in the PCR-positive specimens were identified by DNA microarray analysis within 24 hours. Cul‐ ture results were also positive for *K. pneumonia* in Case 1, *S. agalactiae* in Case 2, and *C. para‐ psilosis*in Case 3, but required 3 to 4 days to obtain [30].

For infection diagnosis, microarray analysis is complementary to routine cultures for identi‐ fying causative microorganisms and is likely to be a useful tool in patients who require rap‐ id diagnosis and early treatment.
