ES2932995A1 - Eye parasite detection system - Google Patents

Abstract

Ocular parasite detection system. The invention makes it possible to detect parasites such as Pseudomonas and Staphylococcus, as well as all the species belonging to the Acanthamoeba genus, so that it consists of a control support on which a colloidal solution of monodisperse gold nanoparticles is established. This solution presents a color that changes when it comes into contact with the parasite's RNA, so that the system can be implanted in different types of support in a simple and economical way, such as lens cases, disposable strips for direct application on the ocular surface etc. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

Eye parasite detection system

TECHNIQUE SECTOR

The present invention consists of the development of an early detection system for Acanthamoeba spp. and other eye parasites based on a colorimetric change through the use of gold nanoparticles.

BACKGROUND OF THE INVENTION

Eye infections can be caused by a wide variety of microorganisms: fungi, bacteria, viruses or parasites that can cause cases of conjunctivitis, endophthalmitis, uveitis, keratitis.

Infections can occur due to exogenous microorganisms when the eye is damaged, allowing it to enter the eye, or microorganisms usually present can be the origin of ocular infections, especially if the defensive systems are altered by the presence of contact lenses, operations surgical, or other invasive techniques.

Microbial keratitis is one of the most dangerous pathologies in contact lens wearers. Bacteria of the genus Pseudomonas spp. specifically Pseudomonas aeruginosa are the main causes followed by Staphylococcus spp. Infections caused by Acanthamoeba spp and fungi represent a smaller proportion, but are more serious. Fungi are opportunistic germs that can affect the cornea causing fungal keratitis. They are mainly produced by filamentous fungi (Fusarium solani and Aspergillus) and Candida albicans yeasts. Its presence increases in the case of contact lenses since they adhere easily.

The free-living amoebas belonging to the genus Acanthamoeba are unicellular organisms, specifically protozoa whose biological cycle has two stages: the trophozoite, which is the active phase in which the amoeba can replicate, and the cyst, which is the latent phase that the microorganism acquired when environmental conditions are not favorable. These amoebas can live in multiple environments: soil, dust, air, and water.

In addition, under the appropriate conditions, it can behave as a parasite in different hosts. There are other eye parasites such as Toxoplasma gondii, Demodex sp among others, but Acanthamoeba spp is the only one related to the use of contact lenses and the one with the highest incidence in our country.

In humans, Acanthamoeba can cause various pathologies. At the ocular level and in relation to the present invention, it is responsible for amebic keratitis (AK), a pathology that affects the corneal surface. The most common species that can cause this pathology are Acanthamoeba castellani and Acanthamoeba polyphaga.

Acanthamoeba keratitis presents with pain, photophobia, epithelial defect, edema, and if not treated properly, it can cause complete loss of vision.

There are different risk factors for the development of keratitis, among which are trauma, damage to the ocular surface and the use of contact lenses (CL). There are numerous studies that demonstrate a relationship between the use of contact lenses and the development of QA, becoming considered as its main risk factor. Its use during the bath, poor hygiene practices, as well as cleaning with tap water increase the risk of infection.

The incidence of this pathology is difficult to estimate precisely due to regional variations. Approximately 5% of contact lens-associated keratitis is caused by Acanthamoeba.

P. aeruginosa is a versatile gram-negative bacterium found in a wide range of environments, but it also acts as an opportunistic pathogen, causing numerous types of infection. It is responsible for most bacterial keratitis, especially in contact lens wearers.

The associated symptoms include acute pain, redness, and photophobia. A corneal ulcer with mucopurulent discharge forms on the surface that may progress to corneal perforation. P. aeruginosa is particularly virulent and rapidly evolving, and can cause loss of the eyeball. This bacterium adheres to contact lenses, and is resistant to cleaning with maintenance solutions. Bacteria are responsible for most cases of microbial keratitis, between 70% and 90% according to different studies.

Regarding the diagnosis of infections caused by the different pathogens, it is important that it be early, being a key factor so that the infection can be treated on time and thus avoid a worse prognosis. It is important to make a good differential diagnosis between herpetic, fungal, parasitic or bacterial keratitis.

In vivo diagnosis can be made by analyzing the cornea with confocal microscopy. It is a non-invasive, highly sensitive technique. In the case of Acanthamoeba keratitis, it only reliably detects cysts and cannot be used to make a definitive diagnosis. In addition, to carry it out, a high-performance team and qualified personnel are needed, which is why it is not always accessible.

For in vitro diagnosis, there are several methods. On the one hand, the polymerase chain reaction (PCR) which has high sensitivity and is fast. However, especially in the case of LC users with Acanthamoeba keratitis the result may be positive even in the absence of pathogen.

On the other hand, a sample can be obtained by corneal scraping or biopsy for microbiological plate cultures. This technique is traditionally considered the reference test for the detection of these pathogens, however, it is a process that can take up to 3 weeks.

The presence of these pathogens can also be verified by histopathological analysis of corneal scrapings that can be stained using different stains. The sensitivity of this method is lower than the previous ones.

Currently, the complexity in the diagnosis of the different types of keratitis makes it a slow process that makes it difficult to resolve keratitis.

Regarding current treatments, topical antibiotics remain the first-line treatment for bacterial keratitis. Infections caused by P.aeruginosa must be treated with combination drug therapy because the bacterium creates resistance quickly. In some cases improvement has also been seen with the use of corticosteroids, although controversy remains.

Treatment of fungal keratitis often has worse outcomes than bacterial keratitis, and there is little evidence to guide treatment. Antifungals are used as topical treatment. Some of them, however, have poor eye penetration. New generations of antifungals seem to have better action results, such as Voriconazole, which can also be administered orally.

Current treatment options for Acanthamoeba keratitis use a wide variety of actives. Treatment usually begins with the use of biguadines, generally combined with diamidine. It is currently the only therapy that has shown effectiveness against Amoeba cysts in vitro. Antiamoebic antibiotics are used as drugs, and sometimes the use of topical corticosteroids can be recommended to reduce severe inflammation. In cases in which the infection penetrates deeply into the cornea, the only effective option is keratoplasty.

Some surgical treatments such as corneal cryotherapy, amniotic membrane transplantation, seem to be effective in AK, restoring the integrity of the ocular surface.

Other treatments such as crosslinking seem to have a possible effect in the treatment of AK, microbial and fungal keratitis.

The search for faster and more effective treatments continues to be the subject of numerous studies.

On the other hand, the growing use of nanoparticles in the field of biomedicine for the diagnosis and treatment of diseases means that their use has also been tested in the treatment against Acanthamoeba, against fungal and bacterial keratitis.

In vitro experiments show that cobalt nanoparticles have amebicidal activity against A. castellanii as well as titanium oxide (TiO2) nanoparticles that have an inhibitory effect against the growth of Acanthamoeba. The conjugation of gold nanoparticles with certain drugs or compounds such as chlorhexidine gluconate or cinnamic acid have shown their effectiveness against A. castellana. However, its use has not been studied for the detection of Acanthamoeba or for the rest of ocular pathogens.

Noble metal nanoparticles such as gold nanoparticles (AuNPs) have excellent physical, chemical and biological properties. One of the properties that make them especially interesting is the surface plasmon resonance (LSPR). This surface plasmon is produced by the collective oscillation of the free electrons in the gold conduction band when excited by an incident light beam of a certain frequency. When the frequency of the incident radiation coincides with the resonant frequency of the oscillation of the electrons, an absorption phenomenon occurs. Gold nanoparticles are spheres whose absorption band is within the visible spectrum. Its diameter is generally between 2-50nm, as its size increases, the absorption band moves to longer wavelengths, varying the color of the colloidal suspension.

Therefore, the phenomenon of the surface plasmon band is influenced by the size and shape of the nanoparticles, as well as by other factors such as the solvent, concentration, spatial distribution, and the distance between particles, among others. This interesting property is the reason why they can be used as colorimetric sensors.

Since the diagnosis of Acanthamoeba keratitis and other ocular parasites is a complex and often slow process and there is no defined standard treatment, the creation of an early detection method is essential to minimize the severity of the pathology.

For this reason, the present invention describes a system for the detection of these ocular pathogens, paying special attention to those associated with the use of contact lenses, specifically in the detection of the presence of Acanthamoeba and Pseudomonas aeruginosa.

EXPLANATION OF THE INVENTION

The present invention consists of a detection system for the presence of Acanthamoeba created with gold nanoparticles. The system allows you to confirm the presence of said pathogen with a visible color change of the sample.

The system to establish the detection is through the RNA recognition of the amoeba. Detection consists of the use of a messenger RNA molecule to detect RNA sequences of the parasite. To carry out the in vitro experiments, the RNA sequence of the amoeba was requested to be complementary to DNA, since RNA is easily degraded. The method used consists in contacting the Acanthamoeba RNA with nanoparticles that have oligonucleotides attached to them. The oligonucleotides attached to the gold nanoparticles each have a sequence complementary to the sequence of one of the Acanthamoeba RNA parts to be detected. The union of the Acanthamoeba DNA sequence with the complementary oligonucleotides attached to the gold nanoparticles is known as hybridization, which causes an aggregation of the gold nanoparticles.

The development of the detection system is based on the localized surface plasmon resonance or LSPR of the previously mentioned gold nanoparticles. Depending on the size of the nanoparticles, the color of the nanoparticle colloidal suspension varies. In this case, the color of said suspension is red, but when the hybridization of the oligonucleotides in the nanoparticles with the nucleic acid takes place, a color change of the suspension occurs due to the aggregation of the gold nanoparticles. The absorption band shifts to the right and a visible color change is produced indicating the presence or absence of Acanthamoeba in our sample.

The use of this system in a lens case for the detection of Acanthamoeba spp. and other infectious pathogens is straightforward. Nanoparticles can be attached to the base of the contact lens case. In this way, in the event that the contact lenses are contaminated with the amoeba, it will remain in the contact lens maintenance fluid found in the storage case, thus producing a color change to purple-bluish. at the base of the case of the lens holder indicating its presence.

This method to detect Acanthamoeba and other bacteria based on observing a color change with the naked eye is cheap, fast, and does not require specialized equipment. This makes it suitable for use in research laboratories, ophthalmology clinics, hospitals, opticians, etc.

The invention is applicable to the detection of Pseudomonas and Staphylococcus, as well as all species belonging to the Acanthamoeba genus including Acanthamoeba astronyxis, A. castellanii, A commandoni, A culbertsoni, A. divionensis, A. griffini, A. hatchetti, A. healyi, A. jacobsi, A. lenticulata, A. lugdunensis, A. mauritaniensis, A. palestinensis, A. pearci, A. polyphaga, A. pustulosa, A. quina, A. rhysodes, A.royreba, A. terrcola, A. triangularis and A. tubiashi.

DESCRIPTION OF THE DRAWINGS

To complement the description that is going to be made below and in order to help a better understanding of the characteristics of the invention, according to a preferred example of its practical embodiment, a set of drawings is attached as an integral part of said description. where, with an illustrative and non-limiting nature, the following has been represented:

Figure 1 shows a schematic diagram of the model for binding probes 1 and 2 (DNA) to the nanoparticles and the target. The distance of the selected sequences is 18nm, so the size of the Np must be greater than this to promote aggregation.

Figure 2 shows the color change that can be seen when hybridization occurs, with the synthetic target ( Acanthamoeba DNA) and as a consequence, the aggregation of gold nanoparticles. The result is after 10min of adding the target to the solution.

Figure 3 shows the color change of the colloidal solution of gold nanoparticles when detecting the presence of Acanthamoeba. The sample used is a solution with Acanthamoeba extracted directly from cultures carried out. Hybridization occurs, and as a consequence, the aggregation of the AuNPs obtaining a visible color change.

Figure 4. Absorption spectrum of gold nanoparticles after the addition of Acanthamoeba (represented as a dashed line) showing the change in the absorption peak produced by their aggregation.

Figure 5. Illustration of how the lens case works as a storage device. detection. On the left we have the case with the bottom surface with functionalized nanoparticles. If the contact lenses are not contaminated the color seen is red. On the right we have the case when the contact lenses are contaminated with the pathogen, producing a color change at the bottom of the case to purple, confirming its presence.

PREFERRED MODE OF EMBODIMENT OF THE INVENTION

The nanoparticles used in the invention are gold nanospheres. The size of the nanoparticles is 15nm and 70nm, with a size between 15 and 35nm being preferable.

The method used to synthesize gold nanoparticles is based on citrate reduction, according to known processes.

A colloidal solution of stable monodisperse gold nanoparticles with a size that can be between 1nm and 150nm is obtained, in this case 25nm was chosen. Suitable gold nanoparticles are also commercially available.

Characterization of the nanoparticles:

Localized surface plasmon resonance or LSPR of the gold nanoparticles results in a strong absorbance band in the visible region (500 nm-600 nm), which can be measured by UV-Vis spectroscopy.

The final concentration of gold (1) nanoparticles was calculated using the Beer-Lambert Law.

In order to obtain more concentrated nanoparticles, the solution is centrifuged. For the selection of the nanoparticle size to be used, the length of the recognition sequence (Acanthamoeba RNA = target) that we used was considered. The size of the gold nanoparticles (1) must be greater than the length of the target (2) (in this case, 18 nm) to obtain the plasmon coupling effect, all as shown in figure 1.

Regarding the functionalization of the nanoparticles, the nanoparticles were functionalized with oligonucleotides modified with thiol groups at their 3' and 5' ends to allow ligation. Half of the beads are functionalized with thiol-terminated single-stranded DNA with a complementary base sequence to one end of the target; the other half is similarly functionalized, but with a complementary base sequence at the other end of the target. The thiol binds to gold.

The DNA base sequence is different. A theoretical oligonucleotide/particle ratio was considered in accordance with existing relative publications.

The following table details the values for two selected sizes.

Functionalization with a first probe.

The objective of this method is to decrease the repulsion of charges with the addition of salt, which is added slowly to maintain the stability of the gold nanoparticles.

For this, a solution containing gold nanoparticles (10.85 nM), SDS (0.1%) and PB (0.01 M) was prepared, the first probe was added until reaching a final concentration of 44 ^M, with a RATIO of 4055 oligonucleotides / nanoparticles. . The mixture of oligonucleotides and gold nanoparticles was incubated at room temperature for 20 min. To improve the binding of the oligonucleotide to the nanoparticle surface, salt was added to the nanoparticle solution with oligonucleotides. The NaCl solution (2 M), SDS (0.01%) and PB (0.01 M) were added progressively in steps of: 1, 1, 2, 2 and 4 ^L reaching a final NaCl concentration of 0. 0.2 M. After each addition, the mixture was sonicated for 10 seconds followed by a 20 minute incubation period at room temperature and 350 rpm. The final solution was incubated for 12 hours to allow time for binding.

To remove excess oligonucleotides, the solution is centrifuged and redispersed in 0.01% SDS.

As a control, the same solution is prepared without DNA.

Functionalization with a second probe.

The basis of this method is the protonation of the DNA bases to reduce their repulsion with the gold nanoparticles, for which a pH regulation protocol was carried out.

A citrate buffer solution (9.2mM) (PH: 3) and DNA 2 (6.6uM) was prepared. The solution was sonicated for 3min before adding the gold nanoparticles reaching a final concentration of 4.6nM and a Ratio =1442. The final solution was incubated for 2 hours in the thermomixer: 22 °C and 350rpm.

To remove excess DNA, the sample is centrifuged and redispersed in 10mM PB.

As a control, the same solution without DNA is prepared.

Hybridization.

Hybridization occurs when Acanthamoeba genetic material binds to complementary probe sequences that are each already attached to a gold nanoparticle. The Acanthamoeba sequence to be detected was selected according to the existing literature and was obtained from the commercial company Stab-Vida. Hereinafter referred to as "target".

The target is attached at one end to the first probe together with the functionalized nanoparticle and at the other end it is attached to the second probe, producing the aggregation of the gold nanoparticles due to this binding. A significant change is then produced in the dielectric properties of the medium surrounding the nanoparticles and a visible color change from red to purple is detected.

Nanoparticles functionalized with DNA 1 and DNA 2 are mixed in the presence of salt to promote aggregation with the target. The hybridization rate can be increased by increasing the NaCl concentration.

A mixture is prepared with the nanoparticles functionalized with the two types of oligonucleotides reaching a final concentration of 1nM. The concentration of nanoparticles is also an adjustable factor, depending on their size, and influences the ease of detecting visible color change.

To help binding with the target, salt (2M NaCl+10mMPB) is added until reaching a final concentration of 0.25M. The salt concentration is selected based on the concentration of gold nanoparticles. Finally, the target is added, reaching a determined minimum final concentration to observe the color change of the solution.

This same process is tested with real samples of Acanthamoeba. For this, monoaxenic cultures of the amoeba were carried out. Once extracted, functionalized nanoparticles were added to the solution. Instead of the synthetic target, the sample with real amoebas was added, producing a visible color change. Depending on the amoeba concentration, the change can be detected after 5 minutes.

The same technique used works with other Pseudomonas and Staphylococcus RNA sequences.

The system thus described can be integrated into a lens-holder case (3), with maintenance solution, like the one shown in figure 5, in which an area (4) is defined through which the previously described reaction can be visualized. before contact lenses that have been in contact with the pathogen, or it can be integrated into disposable strips with the solution described, which are applied directly to the ocular surface.

Claims (3)

1a.- Detection system for ocular parasites, parasites such as Pseudomonas and Staphylococcus, as well as all species belonging to the Acanthamoeba genus, characterized in that it consists of a control support on which a colloidal solution of monodisperse gold nanoparticles is established. , whose coloration is susceptible to change upon contact with the parasite's RNA. 2.- Eye parasite detection system, according to claim 1, characterized in that the control support is integrated into the base of a contact lens case. 3.- Eye parasite detection system, according to claim 1, characterized in that the control support is integrated into disposable strips for direct application to the ocular surface. 4 a- Eye parasite detection system, according to claim 1, characterized in that the gold nanoparticles materialize in gold nanospheres, with a size between 15nm and 70 nm.