Acanthamoeba spp. - amoebic keratitis (AK: Amebic keratitis) and granulomatous amoebic Encephalitis (GAE: Granulomatous Amebic Encephalitis): Microscopic examination; Isolation in culture; Molecular diagnosis (PCR); Molecular species identification (sequencing).

Information 09/12/2016.

Acanthamoeba spp. is a protozoan very ubicuitario free life in nature that can be found in habitats as diverse as natural freshwater, water taps, swimming pool water, earth (soil), sediments of oceans, air conditioning units, liquid maintenance contact lenses, dialysis units waters, ..., so are considered the most prevalent environmental protozoa. It has two life stages: trophozoites with active binary division develop pseudopods spinous hyaline, and cysts develop resistance when temperature conditions, pH and change dryness. They described 15 different genotypes (T1 to T15), become important when considering the sensitivity of molecular diagnostic tests. The most prevalent genotype in human infections is T4.

Several species of Acanthamoeba spp. We found causing severe cases of brain infection called granulomatous amoebic encephalitis (GAE: Granulomatous Amebic Encephalitis), a process that occurs in immunocompromised individuals with underlying conditions are difficult to diagnose, leading to a chronic course, which is often fatal and often diagnosed postmortem necropsy deceased patient but which have been currently described cases in which it has been able to make diagnosis with molecular methods applied to samples of cerebrospinal fluid before death, which can begin treatment. In addition, he is responsible for amoebic keratitis cases (AK: Amebic keratitis) that can cause blindness, affects young healthy individuals, particularly users of contact lenses.

The incidence of amoebic keratitis has increased over the past 30 years, calculated an incidence of 2-20 cases per million contact lens wearers, than in the US It is calculated corresponding to 10% of the population. Outbreaks have been associated with the use of batches of liquid solutions for maintenance of contact lenses were contaminated with Acanthamoeba spp. There are isolated cases of individuals who have undergone corneal trauma, washing contact lenses with tap water or other home preparations, or swimming or taking showers with wearing contact lenses. Amoebic keratitis is the acute, painful infection with nonspecific initial symptoms as a disproportionate eye pain, photophobia, redness, tearing, usually unilateral ocular involvement, but there are written cases of bilateral involvement. Patients suffering from corneal epithelium erosion irregularities and edema, accompanied by a radial infiltrate perineural, similar to that observed in Pseudomonas aeruginosa infections. Epithelial denudation and necrosis one stromal appears in subsequent stages also are frequent secondary bacterial infections, which complicates the diagnosis. Clinically it may be confused with Herpes simplex virus infection or fungal (fungal keratitis).

These processes appear to occur in several stages: adhesion of amoebae to host cells, cell invasion and tissue degradation. In cases of chronic (GAE) amoebic encephalitis amoebas would access the nervous system by hematogenous spread from a primary location in the lung or skin. In amoebic keratitis, amoebic environmental would be fixed to the eye tissue damaged corneal surface and invade the corneal stroma. Amoebas they segregate proteinases and serine proteases and have such binding ligands such as adhesion proteins ( "Laminin binding protein" and "Mannose Binding Protein). It has been shown in vitro that exposure of mannose amoebae trophozoites induces cytopathic release factors and lysis of corneal epithelial cells. This cytopathic activity can be inhibited by molecules that inhibit serine proteases, and also inhibiting fixation exogenous mannose.

Possible methods of diagnosis:

• Microscopic examination: microscopic examination can be done in fresh or stained smears. For its realization requires prepare smears on glass slides from injuries. For the test can be used fresh KOH solution. For examination of extensions can be used staining (Giemsa, hematoxylin-eosin, PAS, immunoperoxidase staining or fluorescent calcofluor white). These stains the most used are Giemsa which allows observing trophozoites or cysts, or staining calcofluor white to facilitate visualization of the cysts. Cysts polyhedral or stellate morphology are easier to observe that the trophozoites may be confused with inflammatory cells degenerate. This staining can be performed on extensions that have been previously stained with Giemsa or hematoxylin-eosin.

• Crops: can be isolated in culture in nutrient agar media not coated with a non - capsulated bacteria as Escherichia coli or Enterobacter aerogenes, but requires an incubation of days or weeks before discarding his presence watching crops periodically with an inverted microscope. Likewise, samples may be inoculated in cell cultures, which causes destruction of cells. The problem of crops is that samples obtained by scraping lesions must be processed immediately, so is not suitable for these samples should be sent when a distant laboratory. If crops are useful when it comes to perform detection on the solutions keeping contact lenses, which may be sent to a distant laboratory.

• Molecular diagnosis: currently the most recommended method for speed and specificity, and not subject to the limitations of sensitivity and conservation of other procedimintos. It can be done with corneal scrapings, but has shown it can also be performed in tears, which is not required to use an invasive method. It can also be performed with Dacron swabs impregnated in lesions with solutions keeping contact lenses, or cerebrospinal fluid in cases of granulomatous amoebic encephalitis. It should be noted that some drugs used in the treatment or used as local anesthetics may inhibit PCR testing, so samples must be taken prior to use.

Tests in IVAMI:

• Molecular diagnostic PCR.

• Microscopic examination by Giemsa staining or calcofluor white.

• Agar culture media with bacteria (not axenic cultures) or cell lines.

Samples recommended:

• Molecular diagnosis.

• • Dacron swab impregnated corneal injury. In the case of the ideal sample keratitis corneal scrapings is performed by a specialist (ophthalmologist). This sample has difficulty in transportation and is more suitable to prepare smears or crops grown directly on the patient. In this case, we recommend impregnating swab small diameter nylon or dacron. They do not use swabs with wooden stems, as they may have inhibitory substances of molecular testing.

• • Toruna impregnated Dacron tears (not alginate or agodón) in keratitis cases when it can not be or is not due to perform a corneal scraping.

• • A liquid obtained by eye puncture if intraocular involvement. When there endophthalmitis and puncture, vacuum syringe with very thin needle (tuberculin type) is performed. If the sample is imperceptible recommend sending the syringe into a sterile tube to wash our laboratory the bore of the needle or the center itself sample collection deposit a few drops (3-4) of sterile physiological solution in a sterile plastic vial (Eppendorf tube), and aspire and expel the wash liquid with the line path of the needle. If there is a greater volume than can be deposited in a vial, deposit the extracted volume

• • Liquid contact lens care. In the case of suspected infection through contact lenses, it is recommended to send both the lenses and the liquid where they are.

• • Cerebrospinal fluid if granulomatous amoebic encephalitis suspected.

• Microscopic examination:

• • Extension (smear) on a glass slide prepared in the query by the ophthalmologist, exudate obtained from the injury.

• cultivation:

• • Samples of liquid obtained by ocular swab or to be received in the laboratory within a short time period puncture, so it is not recommended.

Preservation and shipment of sample:

• Cooling, when the arrival time to the laboratory is less than 24 hours.

• Shipping container refrigerated within biological safety, and within isothermal containers with cold accumulator (freezable pack -frigolín-).

Delivery time :

• Less than 24 hours.

Cost of the test:

• Consult ivami@ivami.com