RU2623069C1 - Method for determination of prescriptions to conduct counter-herpetic therapy in case of chronic central bacterial corneal ulcers - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: presence of DNA of human herpesviruses: HSV1,2, VEB, CMV, HHV-6 is determined in the scrapes from the cornea obtained from the central zone of the ulcer and along its periphery and in blood plasma. In the presence of DNA of at least one virus in the scrape and/or in the blood plasma, anti-herpetic systemic and/or local therapy is additionally performed.

EFFECT: method provides complete accelerated epithelization of ulcer with prevention of perforation, intense corneal opacities, preservation and restoration of visual functions, prevention of torpid current of the corneal ulcer and exclusion of the need for surgical treatment.

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Description

The present invention relates to medicine, in particular to ophthalmology, and is intended to determine the indications for local and / or systemic antiherpetic treatment of central bacterial corneal ulcers with a protracted course.

Bacterial corneal ulcers can lead to corneal perforation, endophthalmitis, and eye loss. Ulcers can be localized in any part of the cornea, but more often occur in the central zone (over 70%), where the infection is more difficult, more difficult to treat, and scarring in this area always leads to vision loss [Maychuk Yu.F., Kononenko L. BUT. Fluoroquinolones antibiotics in the treatment of corneal ulcers caused by Pseudomonas aeruginosa when wearing contact lenses. Cataract and refractive surgery. 2003; T. 3. No. 1. S. 55-60]. Corneal blindness in developed countries ranges from 6.3% to 23.2% [Nurozler A.V. Results of therapeutic penetrating keratoplasty // Jpn. J. Ophthalmol. - 2004. - Vol. 48, No. 4. - P. 368-371].

In the acute stage of purulent corneal ulcer, the use of local and systemic antibacterial drugs is an indisputable fact [L. Kovaleva, E. Vakhova. Algorithms for pharmacotherapy of bacterial corneal ulcers. Effective pharmacotherapy. 2013. No. 23. S. 46-48].

At the stage of the reparative period, a bacterial corneal ulcer, cleansed of purulent necrotic masses, can acquire a torpid lingering character, accompanied by a persistent ulcer, and presents great difficulties in treatment. It is known that the outcome of such diseases is corneal throat with gross changes in the anterior segment of the eye, requiring reconstructive end-to-end keratoplasty (UPC). Various degenerative-dystrophic processes often lead to perforation or pronounced thinning of the cornea, when the only means of saving the eye and restoring vision is SKP [Slonimsky A.Yu. Possibilities of reconstructive end-to-end corneal transplantation for various pathologies of the anterior segment of the eye and an approach to solving the main postkeratoplastic problems.: Abstract of thesis. for the degree of Doctor of Medical Sciences: 14.00.08 // Aleksey Yurievich Slonimsky. - M., 2004. - 44 p.].

The incidence of complications in the form of a corneal abscess, endophthalmitis, descemetocele or corneal perforation varies from 5 to 15% with a bacterial ulcer. Severe complications pose the need for surgical treatment.

In such conditions, laboratory etiological diagnosis is of particular importance. Currently, methods such as immunofluorescence studies of conjunctival scrapings for the herpes simplex virus (MFA) have become traditional; microbiological examination (smears and crops). However, judging by the above statistics on the insufficiently high result of treatment, these methods are clearly insufficient.

The insufficient effectiveness of diagnosis and treatment causes an urgent need for modern, more accurate and informative methods of laboratory diagnostics, which allow one to find out and reliably confirm the etiology of a prolonged corneal ulcer in the early stages of the disease, identify mixed forms of infection, and prescribe etiotropic and pathogenetic therapy in a timely manner.

Currently, clinical practice has accumulated data that allow us to talk about the characteristic signs of herpetic and bacterial corneal ulcers. In some cases, this suggests the etiology of the disease during biomicroscopy and immediately begin etiotropic therapy until the results of laboratory tests are obtained [Maychuk Yu.F. Viral eye diseases. M .: Medicine, 1981. 272 p .; Kovaleva L.A., Vakhova E.S. Pharmacotherapy Algorithms for Bacterial Corneal Ulcers // Effective Pharmacotherapy. 2013. No. 23. S. 46-48.].

However, the described clinical signs are not always pathognomonic, and the etiological diagnosis according to the clinical picture is not always informative, it often depends on subjective factors and specialist qualifications, on the current epidemiological situation, on the individual characteristics of the patient’s body, on the accompanying ophthalmic and somatic diseases, on the duration of the course ulcers, from joining a secondary, including bacterial infection. Often this leads to errors in the etiological diagnosis of corneal ulcers and unjustified pharmacotherapy: prolonged and unsystematic use of antibiotics, corticosteroids and antiviral drugs causes an imbalance in the microflora of the conjunctival cavity, the resistance of microorganisms to antibiotics increases significantly, which entails a long and difficult course of the inflammatory process, treatment.

In this regard, it is necessary to use laboratory diagnostic methods, among which the most traditional are immunofluorescence studies of scrapings from the conjunctiva to the herpes simplex virus. A known method of the same purpose, using fluorescent antibodies in the diagnosis and treatment of herpetic keratitis [Zaitseva N.S., Kasparov A.A., Muravyova T.V. et al. The method of fluorescent antibodies in the diagnosis and treatment of herpetic keratitis. Bulletin of Ophthalmology; 1973. No. 6, S. 48-51]. The immunofluorescence method (the method of fluorescent antibodies) has a number of significant advantages for the diagnosis of the acute active stage of the disease: high sensitivity, speed of response; however, after the start of treatment and as the process becomes chronic, its effectiveness decreases significantly (5-6 times). This method is taken as the closest analogue.

The present invention is to develop an adequate method for determining the indications for the implementation of antiherpetic therapy as part of the complex treatment of central bacterial corneal ulcers with a protracted course.

The technical result of the proposed method is a full-fledged accelerated epithelization of an ulcer with the prevention of perforation, intense clouding of the cornea, preservation and restoration of visual functions, prevention of the torpid course of corneal ulcers and eliminating the need for surgical treatment.

The technical result is achieved by detecting human herpes virus DNA (HHV) in the blood and / or scraping from the entire area of the ulcer, including the edges.

The method allows to determine the feasibility of including antiherpetic therapy in the complex treatment of central bacterial corneal ulcers, reducing the time of epithelization and patient disability.

In recent years, PCR, which allows the determination of the genetic material of infectious disease pathogens (DNA, RNA), has been widely used in the diagnosis of inflammatory eye diseases. Compared with traditional diagnostic methods, PCR is highly sensitive (reveals the minimum amount of nucleic acid) and specificity. PCR research belongs to the category of the most accurate diagnostic methods for determining the genomes of microorganisms in humans. The reliability of such studies reaches 95-99%. [Slepova O.S., Svetlova E.V., Kovaleva L.A., Makarov P.V., Kugusheva A.E., Denisova E.V., Vakhova E.S., Zakharova G.Yu., Kondratieva Yu.A., Andryushin A.E., Demkin V.V. Studies of human herpes simplex virus type 6 and other herpes viruses that cause eye diseases by polymerase chain reaction // Issues of Virology. 2015.V. 60. No. 6. S. 45-48]. In this work, it was shown that keratoplasty results are affected not only by the presence of herpes viruses in the tissues of the eye, but also in the blood plasma.

Systemic (blood) or local (scraping from corneal ulcer) detection of HSV DNA in patients with bacterial corneal ulcer raises the question of whether antiviral drugs should be included in the treatment algorithm of this group of patients.

Studies were conducted among 42 patients with protracted bacterial ulcers. In 36 patients, HSV 1,2, VEB, and HHV-6 DNA were detected, which, after cleansing the ulcer surface from purulent-necrotic masses and discontinuation of traditional antibacterial therapy, carried out systemic and / or local antiherpetic therapy. Antiviral treatment contributed to the acceleration of repair and eliminated the need for surgical treatment in comparison with the group that did not receive antiviral treatment, consisting of 32 patients in whom the bacterial ulcer, cleansed of purulent-necrotic masses on the background of traditional antibacterial therapy, assumed the character of a pure persistent corneal ulcer.

Material for PCR testing is corneal cells and blood. To this end, corneal cells are scarified from the entire area and edges of the ulcer to obtain stromal and epithelial cells preserved along the edges of the ulcer. Superficial scarification is carried out without going deeper into the stroma, and without affecting the thickness of the cornea in the ulcer area, in order to avoid additional trauma to the cornea.

Virus DNA can be detected both in the blood and in a scraping from the cornea, or it can be found in the blood and not in the scraping, and vice versa. Similar results are explained by the fact that in different phases of herpesvirus infection the virus may be in the blood (viremia), but more often it is present at the site of inflammation (cornea). It is this fact that explains the need for the appointment of systemic and local antiherpetic therapy.

Material from the patient was examined in real-time PCR. When setting up PCR, the iQ iCycler real-time amplifier with a fluorescent label (BioRad, USA), PCR box (Cyclotemp JV RTS), a household refrigerator, disposable dishes, and disposable gloves were used. Arrangement of PCR.

DNA is isolated from the samples using the kits for isolating “RealBest DNA extraction 1”, “RealBest DNA extraction 2”, “RealBest DNA extraction 3” in accordance with the instructions for use of the kit. Each group of images passing through the DNA isolation procedure should contain a positive control sample (FFP) and a negative control sample (FAC), which are a component of the DNA isolation kit.

Before starting work, the reagent kits are removed from the refrigerator, the finished reaction mixture for PCR (HRS) in the package (without opening) is kept at T 18-25 ° C for at least 30 minutes. Then open the package and cut off with the knife the required number of test tubes with GCS together with the film covering them (according to the number of test samples, including 1 DEC and 1 DEC).

In each test tube, 50 μl of the appropriate solution of the isolated DNA are pipetted with a separate tip with a filter without capturing the precipitate and tightly closed. The tubes are placed in an amplifier and the amplification program is run in accordance with the instructions for the iQ iCycler amplifier (BioRad, USA) for real-time recording of fluorescent signals.

When using the kit with the iQ iCycler amplifier and the Real-Best Diagnostics program (Vector-Best CJSC), analysis and recording of the results (positive - contains DNA of the pathogen or negative - does not contain DNA of the pathogen) are performed automatically.

The method is as follows: in a patient with a prolonged course of a bacterial ulcer, scraping is taken from the entire area and edges of the corneal ulcer and blood for PCR. Before taking material from the corneal ulcer, generally accepted epibulbar anesthesia is required, after installing the anesthetic under magnification on a slit lamp, corneal cells are scarified from the entire area and edges of the ulcer using a disposable sterile scalpel, the resulting material is placed in disposable plastic sterile tubes. Blood sampling is carried out from the cubital vein into a test tube, the resulting serum is poured into aliquots into sterile disposable plastic tubes. In the scraping from the cornea obtained from the entire area of the ulcer, including its edges, and in the blood plasma, the presence of human herpes virus DNA is determined: HSV 1.2, VEB, CMV, HHV-6. In the presence of DNA of at least one virus in scraping and / or in blood plasma, antiherpetic systemic and / or local therapy is additionally performed.

Clinical examples

Example 1

Patient K., born in 1985.

I was hospitalized in the Department of Infectious and Allergic Eye Diseases of the Moscow Scientific Research Institute of GB named after Helmholtz in 2015 With a diagnosis of OS - Pseudomonas corneal ulcer of mild severity (after wearing soft contact lenses). OU - moderate myopia.

Against the background of wearing soft contact lenses, the OS turned red, photophobia, lacrimation appeared, and visual acuity decreased. On the first day of the disease, she visited an ophthalmologist at the place of residence, keratitis OS was diagnosed, treatment was prescribed: Diphenhydramine + Interferon alpha-2a, Dexamethasone, Solcoseryl. On day 16 of the disease, pain appeared, profuse purulent discharge, OS vision was significantly reduced. The patient turned to the SRI them. Helmholtz on the 18th day of the disease.

On admission: complaints of pain in the OS, photophobia, lacrimation, purulent discharge from the OS, decreased visual acuity.

Visual acuity at admission:

Vis OD = 0.2; (-) 3.5 = 1.0 OS = 0.01; does not correct.

The treatment was carried out with eye drops of Levofloxacin in the regime of forced installations in the conjunctival cavity for two days with a ten-hour night break. On the first day of treatment, the first hour of installation was carried out every 5 minutes, starting from the second hour - every two hours, on the second day - every three hours. On the third day of treatment, they switched to generally accepted treatment regimens, since the corneal ulcer was cleansed of purulent-necrotic masses, and the destruction of the corneal stroma completely stopped. The disappearance of Pseudomonas aeruginosa in smears and crops was observed after the first day of treatment.

After cleansing the surface of the corneal ulcer from purulent-necrotic masses, the surface area of the ulcer did not decrease, and there was no epithelization. The use of reparative agents for 4 days: Dexpanthenol 3 times a day, and glycosaminoglycans sulfated 3 times a day, did not improve the clinical picture, the corneal ulcer took on a torpid course.

In a scraping from the cornea obtained from the central zone of the ulcer and along its periphery, the presence of HSV1,2 DNA was detected, and in the blood - the presence of HSV1,2 and HHV-6 DNA. In this regard, antiherpetic drugs were added to the complex therapy: eye drops Diphenhydramine + Interferon alpha-2a 3 times a day, eye ointment Acyclovir 3 times a day, systemically: inside Valaciclovir 500 mg 2 times a day, for 5 days.

The use of antiherpetic drugs led to complete epithelialization of corneal ulcers within 5 days. Thanks to the use of the complex treatment regimen: the use of antibacterial treatment in combination with antiherpetic drugs, a non-intense surface clouding has formed on the site of the resorbed corneal ulcer, which does not affect the visual acuity of the left eye.

The patient was discharged on the 13th day of treatment with recovery.

Visual acuity at discharge:

Vis OD = 0.2; (-) 3.5 = 1.0 OS = 0.2; (-) 3.5 = 1.0.

Example 2

Patient F., born in 1996.

I was hospitalized in the Department of Infectious and Allergic Eye Diseases of the Moscow Scientific Research Institute of GB named after Helmholtz in 2015. With a diagnosis of OS - Staphylococcal corneal ulcer of moderate severity. Uveitis with hypopion.

Before contacting the Research Institute of GB them. Helmholtz was treated on an outpatient basis and in a hospital at the place of residence. Against the background of acute respiratory viral infections, the OS reddened, photophobia, lacrimation appeared, and visual acuity decreased. On the third day of the disease, she went to the ophthalmologist at the place of residence, keratitis OS was diagnosed, treatment was prescribed: Polyadenyl acid + Polyuridyl acid, Indomethacin, Dexpanthenol.

Against the background of outpatient treatment, no improvement was noted, in this regard, on the 8th day from the onset of the disease, the patient was hospitalized in the ophthalmology department of a multidisciplinary hospital in the community with a diagnosis of OS - Keratitis. The treatment was carried out: topically - Benzyldimethyl-myristoylamino-propylammonium, Polyadenyl acid + Polyuridyl acid, Indomethacin, Dexamethasone, Solcoseryl, systemically - Gentamicin and Ascorbic acid intramuscularly.

Against the background of inpatient treatment, ulcer epithelization was absent, on day 21 of inpatient treatment (28 days from the onset of the disease), pain appeared, moderate purulent discharge, OS vision was significantly reduced. The patient turned to the SRI them. Helmholtz on the 38th day of illness.

On admission: complaints of pain in the OS, photophobia, lacrimation, purulent discharge from the OS, decreased visual acuity.

Visual acuity at admission:

Vis OD = 1,0 OS = correct light projection; does not correct.

The treatment was carried out with eye drops of Levofloxacin 4 times a day, eye ointment Colistimetate sodium + Tetracycline + Chloramphenicol 3 times a day. On the fourth day of treatment, the corneal ulcer cleared of purulent-necrotic masses, and the destruction of the corneal stroma completely stopped. The disappearance of Staphylococc aureus in smears and cultures was noted after the first day of treatment.

After cleansing the surface of the corneal ulcer from purulent-necrotic masses, the surface area of the ulcer did not decrease, and there was no epithelization. The use of reparative agents for 5 days: Solcoseryl 3 times a day and Glycosaminoglycans sulfated 3 times a day, intramuscular use of Solcoseryl solution 2.0 ml 1 time per day, did not improve the clinical picture, corneal ulcer took on a torpid course.

In scraping from the cornea obtained from the central zone of the ulcer and along its periphery, the presence of HSV1,2 and HHV-6 DNA was detected, and in the blood - the presence of HHV-6 DNA. In this regard, on the 10th day of inpatient treatment, antiherpetic drugs were added to the complex therapy: eye drops Diphenhydramine + Interferon alpha-2a 4 times a day, eye ointment Acyclovir 3 times a day, systemically: inside Valacyclovir with 500 mg 2 times a day, within 5 days.

The use of antiherpetic drugs led to complete epithelialization of corneal ulcers within 5 days. Thanks to the use of the complex treatment regimen: the use of antibacterial treatment in combination with antiherpetic drugs, a surface clouding has formed on the site of the resorbed corneal ulcer, slightly reducing visual acuity in the left eye.

The patient was discharged on the 15th day of treatment with recovery.

Visual acuity at discharge:

Vis OD = 1.0 OS = 0.7; does not correct.

Example 3

Patient K., born in 1966.

He was hospitalized in the Department of Infectious and Allergic Eye Diseases of the Moscow Scientific Research Institute of GB named after Helmholtz in 2015. With a diagnosis of OS - Pseudomonas corneal ulcer of a severe degree. Uveitis with hypopion.

Before contacting the Research Institute of GB them. Helmholtz was treated on an outpatient basis and in a hospital at the place of residence. Against the background of wearing soft contact lenses, the OS turned red, photophobia, lacrimation appeared, and visual acuity decreased. On the fifth day of the disease, he visited an ophthalmologist at the place of residence, keratitis OS was diagnosed, treatment was prescribed: Diphenhydramine + Interferon alpha-2a, Dexamethasone, Solcoseryl.

Against the background of outpatient treatment, no improvement was noted, in this regard, on the 21st day from the onset of the disease, the patient was hospitalized in the ophthalmology department of a multidisciplinary hospital in the community with a diagnosis of OS - Keratitis. The treatment was carried out: topically - Benzyldimethyl-myristoylamino-propylammonium, Polyadenyl acid + Polyuridyl acid, Dexamethasone, Solcoseryl, systemically - Acyclovir, Ofloxacin.

Against the background of inpatient treatment, ulcer epithelization was absent, on day 13 of inpatient treatment (34 days from the onset of the disease), pain appeared, profuse mucopurulent discharge, OS vision sharply decreased from 0.4 to light projection. The patient turned to the SRI them. Helmholtz on the 35th day of the disease.

On admission: complaints of pain in the OS, photophobia, lacrimation, mucopurulent discharge from the OS, decreased visual acuity.

Visual acuity at admission:

Vis OD = 1,0 OS = correct light projection; does not correct.

The treatment was carried out with eye drops of Levofloxacin 4 times a day, eye ointment Colistimetate sodium + Tetracycline + Chloramphenicol 3 times a day, Gentamicin - parabulbar. On the fifth day of treatment, the corneal ulcer cleared of purulent-necrotic masses, and the destruction of the corneal stroma completely stopped. The disappearance of Pseudomonas aeruginisae in smears and crops was observed after the first day of treatment.

After complete relief of the bacterial infection and purification of the surface of the corneal ulcer from purulent necrotic masses, the surface area of the ulcer did not decrease, and there was no epithelization. The use of reparative agents for 6 days: Solcoseryl 3 times a day, and glycosaminoglycans sulfated 3 times a day, intramuscular use of a solution of Solcoseryl 2.0 ml 1 time per day, did not improve the clinical picture, the corneal ulcer took a torpid course.

In a scraping from the cornea and in the blood, the DNA of herpes viruses was not found. In this regard, on the 12th day of inpatient treatment, the patient received a soft contact lens (MKL) on the cornea, continuing complex conservative therapy. Antiherpetic drugs were not prescribed. The use of MCL on the background of intensive conservative therapy led to complete epithelization of the corneal ulcer within 10 days, but intense opacification formed on the site of the epithelialized ulcer.

The patient was discharged on the 22nd day of treatment with recovery.

Visual acuity at discharge:

Vis OD = 1.0 OS = 0.08; does not correct.

Thus, the proposed method allows to prevent the development of a protracted torpid nature of the course of bacterial corneal ulcers, intense clouding of the cornea, perforation of the cornea, to preserve and restore visual functions, to avoid the need for surgical treatment (amnioplasty and / or end-to-end keratoplasty).

Claims (1)

A method for determining indications for antiherpetic therapy for central bacterial ulcers of the cornea with a protracted course, characterized in that the presence of human herpes viruses DNA is determined in a scraping from the cornea obtained from the central zone of the ulcer and along its periphery, and in blood plasma: HSV1,2, VEB, CMV, HHV-6, and in the presence of DNA of at least one virus in scraping and / or in blood plasma, antiherpetic systemic and / or local therapy is additionally performed.