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W2044299024 abstract "Purpose: Herpes stromal keratitis is a serious condition and the most frequent cause of unilateral blindness. The real-time PCR is an accurate and fast diagnostic method for an analysis of infectious agents causing keratitis and keratouveitis. The aim of the study was to assess the relationship between clinical symptoms, treatment efficacy monitoring and viral quantity in corneal swabs determined by quantitative real-time PCR method. The real-time PCR method was used as well for the detection of other viral eye pathogens. Methods: A total of 212 patients (136 men and 76 women) suspect of having herpes simplex virus (HSV) keratitis or keratouveitis were included in the study. The detection and quantitative analysis of the viral DNA were performed using the EliGene HSV1 RT kit, and the result was correlated with the clinical picture of the disease. The patients were routinely treated with acyclovir applied locally or, alternatively, in systemic administration. In a case of acyclovir treatment resistant keratitis, the patients were treated with local ganciclovir (Virgan gel ophth 0.15%). Results: A total of 636 analyses of the viral DNA were performed; 85 patients were positive for HSV1 (198 detected). There were 16 acyclovir resistant cases of keratitis (14%). Conclusions: The real-time PCR appears as a fast and accurate method for an exact identification of the viral DNA in patients with herpes stromal keratitis. The introduction of the quantification is important for the treatment evaluation and for the specification of a so-called acyclovir resistant keratitis. A long-term systemic administration in maintenance doses may lead to the resistance and repeated, frequent relapses of the disease. The Polymerase Chain Reaction (PCR) is a powerful molecular biological tool for the analytic quantification of nucleic acids from infinitesimal starting quantities. It is possible to determine quantities of DNA in studied samples from several hundreds of DNA molecules to hundreds of millions of DNA molecules by DNA quantification by the real-time PCR methods. For the DNA detection of pathogenic organisms, only few microlitres of samples (e.g. tears) can be used in the case that the total volume of sample includes more than 10 DNA molecules that is the lower limit of sensitivity of the real-time PCR method. Viral infections meet these criteria, and low sample volumes can be used. The real-time PCR (TaqMan chemistry) is a variant of PCR in which the amplification reaction is performed using probes with attached fluorescent dye (reporter) and quencher. Herpes simplex virus type 1 is a prevalent viral pathogen infecting 60–90% of the adult world population and is a leading infectious cause of corneal blindness in the developed world (Whitley & Roizman 2001). Corneal infection with HSV-1 represents a significant clinical problem for its potential to cause blindness. It involves a two-step process of infection and re-infection. Initially, HSV-1 replicates in the mucous membranes of the mouth or corneal epithelium, where sensory and autonomic nerve terminals are infected by HSV1. The virus is then transported by axons in a retrograde direction up to sensory ganglion cell bodies. Although many ganglion neurons support a lytic infection, a subpopulation of neurons supports an HSV1 infection in which the virus remains in a latent stage. The second phase of corneal infection arises from viral reactivation in the latently infected ganglion cells of the trigeminal ganglion. After anterograde transport to the eye, new virus can be found in tears and in the corneal epithelium and stroma. With repeated reactivation cycles, the corneal stroma becomes progressively more scarred, with resulting decrease in the vision and other ocular complications including the glaucoma, iritis and cataract, and even necrotizing retinitis (Kaufman 1978). In general, diagnosis of HSV1 is based on clinical findings or done by standard laboratory methods of antigen detection and virus isolation from clinical samples. Until today, PCR has not been commonly used in ophthalmology for the diagnosis of infectious external-surface diseases, keratitis and infectious anterior and posterior uveitis. Current treatment includes local application of antiviral medication (acyclovir, ganciclovir), which is supported, in the case of recurrent and severe herpetic infections, with a systemic administration of antiviral agents (acyclovir in oral or parenteral form). Current dosage recommendations in the maintenance treatment, acyclovir 200 mg b.i.d., for several months do not necessarily protect from the relapse. On the basis of encouraging preliminary results (Hlinomazová et al. 2008); we decided to use the real-time PCR method for the detection and the quantification of HSV1 from corneal swabs and to study the relationships between clinical symptoms, HSV1 quantities and the treatment by antiviral agents. The possibility of the usage of the real-time PCR method for the detection of other viral eye pathogens was also examined. This study was approved by the Medical Ethics Committee of the Faculty Hospital Brno. An appropriate informed consent was obtained from each subject before the collection of specimens. Totally, 212 patients (76 women and 136 men) with suspect HSV1 keratitis or keratoconjunctivitis were involved in the study. The mean age of patients was 42 ± 9.2 with a range from 4 to 82 years. The patients with bacterial infections (verified by cultivation) were excluded from the study because bacterial infection affects the clinical symptoms and than can lead to false results. Clinical examinations were performed using the Hogan’s classification. Samples were collected at the time of first visit and than on days 7, 14, 21 and 28 until a negative PCR result was obtained. The patient was sampled three times at each session, to obtain relevant quantities of DNA. Samples were collected by flocked swabs (Ultra minitip flocked swab; Copan Diagnostics, Murrieta, CA, USA). Swabs were partly used for the taking of sample of tears and partly for whipping up the infected parts of the cornea that were observed under slit lamp microscopy. After collection, the swabs were submerged in storage solution (EliDNA Store kit; (ELISABETH PHARMACON, Brno, Czech Republic)). The storage solution, according to manufacturer’s instruction manual, allows storage and transportation of the viral DNA at room temperature for at least 7 days. Samples were transported under the above-described conditions to the laboratory and were analysed within 2 days. Patients were treated by locally applied acyclovir five times a day for 7–10 days (Zovirax, ung. ophth. 3%, GlaxoSmithKline, Middlesex, UK); patients with keratouveitis received combined local and systemic therapy of acyclovir (Herpesin; Pliva-Lachema, Brno, Czech Republic) 400–800 mg five times a day every 4 hr with a night pause for 10–30 days. If DNA concentration measured by the PCR method was unchanged after 1 week of the treatment with acyclovir, the keratitis was assessed as acyclovir-resistant keratitis and ganciclovir (Virgan gel ophth. 0,15%; Laboratoires THEA Clermont Ferrand, France) were used for subsequent treatment. The DNA was extracted from flocked swabs using the UltraClean Tissue DNA Isolation Kit (MoBio, Carlsbad, CA, USA). Storage solution and swabs were vortexed with TD1 buffer (the part of isolation kit), and the obtained solution was loaded onto a silica spin filter and washed by buffers according to the instruction manual. The DNA was diluted in 50 μl of elution buffer. Extracted DNA was stored at 4°C for maximum 2 days and after the real-time PCR analysis the rest of DNA was frozen at −20°C. All samples were analysed by the real-time PCR detection kit EliGene HSV1 RT (ELISABETH PHARMACON) that is in vitro medical diagnostic device intended for usage on the Applied Biosystems Real Time 7300 instrument (ABI7300, Applied Biosystems, Foster City, CA, USA). This kit allows an absolute quantitative analysis of the PCR product using EliDNA HSV1 QRT Standard (ELISABETH PHARMACON). For the absolute quantification of viral DNA, the above-mentioned kits were used in this study according to the instruction manual of the manufacturer. Internal controls that are a part of the EliGene HSV1 RT kit were used for a control of DNA isolation and subsequent PCR reaction. The manufacturer determined the sensitivity of the EliGene HSV1 RT Kit to be 10 genomic DNA added to the amplification mix. The EliDNA HSV1 QRT Standard allows a setting up of the calibration line of the real-time instrument in a range from 2.5 × 103 to 2.5 × 106 of DNA particles in 5 μl of the isolated DNA. For PCR diagnostics of HSV2, VZV, adenovirus and EBV, the EliGene HSV2 RT Kit, EliGene VZV RT Kit, EliGene Adenovirus RT Kit and EliGene EBV Kit were used, respectively. Except the EliGene EBV Kit (for nested PCR detection), all used kits are intended for in vitro diagnostics and for the usage on the ABI7300 instrument. All manufacturers’ recommendations in the instruction manuals were followed up including the usage of internal controls. A random blind test was used for the detection of potential false positive results. Time to time, Physicians at the Department of Ophthalmology leaved clean swabs between clinical samples. In the laboratory, no false positive result was observed. The sensitivity of above-mentioned methods was examined by positive control samples that we created in our laboratory by cloning into plasmid. The DNA fragment of given pathogen (HSV1, HSV2 and VZV) that is used for real-time PCR detection was included into cloning plasmid vector pGEM (R) – T Easy Vector System I (Promega, Madison, WI, USA) and after cloning pure plasmid DNA was isolated by UltraClean 6 Minute Mini Plasmid Prep Kit (MoBio, USA). The quantity of DNA was determined by spectrophotometer (NanoPhotometer Implen, Germany), and the number of plasmid DNA was calculated. Prepared plasmid positive control was diluted on 1000, 10 000, 100 000 and 1 million copies, and the absolute quantification was examined by above-mentioned kits. In the comparison with quantities obtained for quantification controls manufactured by ELISABETH PHARMACON, we obtained same quantitative results. DNA of all samples for all tested pathogens did undergo the confirmation by different PCR methods (nested PCR) in the Department of clinical microbiology that is part of University Hospital Brno. All positive samples were confirmed by second PCR method as positive and all negative samples were confirmed by second PCR method as negative. Totally, 636 real-time PCR analyses (according to 7 days repetitions) of HSV1 DNA were performed in 212 patients. Among these analyses, 85 HSV1 positive patients (totally 198 of positive results according to 7 days repetitions of positive patients’ analyses) were identified. The clinical examination of positive patients was made according to Hogan’s criteria, and patients were classified into clinically pathological groups (Table 1). In 18% of the patients, another viral infection was found using the PCR: adenoviruses (18), VZV (18), EBV (1), HSV2 (2) (see Table 2). In 42 patients (19%) no infection was detected with the PCR method. These cases were either primarily incorrect working diagnoses (35 patients – see Table 3) or false negative results of the PCR (seven patients). When other diagnostic tests, including confocal microscopy, Schirmer test I, II and the test of corneal sensitivity (Cochet–Bonnet esthesiometer) were used, these diagnoses were reassessed. The seven negative results determined by the PCR were placed among false negative results because all the seven superficial keratitis cases without stromal impairment had responded well to acyclovir. The Department of clinical microbiology obtained same negative PCR results for these seven samples. In total, 212 patients were tested; 85 of them were positive, 120 negative, seven false negative and no sample was false positive. The quantitative analysis of viral DNA with the PCR method was performed repeatedly, in the course of 7 days, in patients positive for HSV1. The clinical picture of the disease was monitored after antiviral treatment initiation and the quantity of viral DNA was evaluated in association with healing processes in the cornea during time. The results are summarized in Table 4. The results show that in 69 patients after the local acyclovir treatment initiation the quantity of viral DNA diminished approximately 100-fold within 1 week. All patients with response to acyclovir were successfully treated after 4 weeks. When we evaluated 16 (14%) patients with persistent positive results retrospectively, we came to the conclusion that these patients did not respond to the treatment by viral DNA reduction – their positivity value remained stable, in average 7000 viral particles in 1 μl of the isolated DNA sample (see Table 5). These patients were assigned as acyclovir resistant and were treated with local ganciclovir. In all patients who experienced a reduction in quantification, clinical picture improved as well; in all cases it corresponded with the initial severity. Sixteen acyclovir resistant patients (14%) were found in the group of patients. The diagnostic procedure of this clinical-pathological category is essential for further development of the disease in individual patients. Our sample included three primary infections with no reaction to acyclovir and 13 patients repeatedly treated with antiviral agents of different kinds without any convincing effect. All 16 patients were successfully treated after 4 weeks of the ganciclovir treatment. Relapse rate within 12 months in these patients was 4 ± 0.8 in average. After ganciclovir treatment the relapse rate within 12 months was only 0.9 ± 0.5. The traditional cultivation method is a standard technique of the detection of the aetiological agent (virus, bacteria or fungi) of infectious keratitis. Its disadvantages are, however, low sensitivity (false negative results of cultivation, impossibility to detect latent viral infections) and also a longer time until the result is obtained. A molecular technique – PCR – seems to be a new additional method, in some cases indispensable for clinical ophthalmology. The DNA diagnostics implementation both in superficial and in stromal cases of keratitis is substantial for specification of the aetiology of infection and for targeted treatment. Recently, because of a number of factors (lifestyle diseases, frequent antibiotics use, increase in immunodeficiency cases), there have been changes in clinical picture of the superficial keratitis presenting now as atypical epithelial keratitis which is difficult to diagnose aetiologically (Kaye et al. 1991; Fukuda et al. 2008). As early as in 1985, Saiki described the contribution of the polymerase chain reaction to the diagnostics of viral diseases of the eye. In the last 10 years, laboratory methods of viral DNA detection have experienced a huge progress. There has been a changeover from a common PCR with a sensitivity of approximately 100 genomic DNAs to nested PCR methods with a sensitivity of 1–10 genomic DNAs. Nowadays, clinical laboratories proceed generally to real-time PCR methods, which have, if a correct design is used, same or even better sensitivity than the nested PCR. Unlike the nested PCR, which is a two-step method and possesses a risk of cross-contamination, the real-time PCR is done in one step without opening the microtubes, which considerably reduces the possibility of false positive results. The real-time PCR significantly increases the success of herpetic viruses detection compared to traditional techniques. It has also overcome the limits of the conventional PCR. Stránská et al. (2004) suggest that higher concentration of viruses is needed for positive results of cultivation in the comparison with the real–time PCR. Easty et al. (1987) described the most successful isolation of HSV1 from scarified cornea. Nowadays, the majority of clinical laboratories have significantly reduced the use of virus cultivation. This time-consuming, expensive and methodologically difficult method has made place for DNA diagnostic methods, which are not only faster and more sensitive but have also other significant advantages. Our results show that cultivation of viruses was inappropriate under given conditions. A sample of 20 randomly selected patients with a positive PCR result for HSV1 was assessed once more using the cultivation and it was negative in all cases. Kaye et al. (2000) evaluated in their study the sensitivity and specificity of various methods of HSV1 detection in cornea of patients who had undergone perforating keratoplasty. The methods used were cultivation, PCR, immunohistochemistry (IHC) and in-situ hybridization (ISH). These techniques detect different components of the HSV1. Cultivation is a standard way of detection in active infection only. The authors concluded that both techniques (PCR and IHC) appear very sensitive and their combination is able to increase their specificity up to 97%. Viral DNA detection with the PCR is very accurate for both acute and latent infections. Our study supports the hypothesis that real-time PCR sensitivity for the detection of HSV1 corneal infection is higher than 90%. Only seven patients (3.3%) in our study responded to acyclovir treatment when PCR results were negative. We suppose that these seven real-time PCR negative samples obtained no viral particles. It is necessary to say that we wiped cornea by swab and we do not used scarification of cornea. It seems like in the minimum of HSV1 positive corneas, the virus is not present in tears and corneal surface but probably in stroma that could lead to PCR negative results. The introduction of quantification of viral DNA from a sample is of an essential importance for the treatment efficacy monitoring and sometimes for the diagnostics of acyclovir resistant cases of keratitis and keratouveitis. The ratio of clinically inaccurate diagnoses in our study favours the necessity of DNA analysis of standard samples in superficial keratitis resistant to standard treatment. Koizumi et al. (1999) did not find any correlation between the clinical manifestation of atypical keratitis and PCR diagnostic results in these cases. A detailed analysis of the reasons of false diagnoses in our sample showed that the clinical picture was misleading mainly in suspect metaherpetic keratitis. Our experience is that this is mainly a matter of precise methodology of the sample collection with viral DNA collection into stabilizing solution, which enables a detection of low concentrations of viral DNAs in the sample. Simultaneously, a control group undergone a sample collection with sterile dry cotton swab into a physiological solution; negative results were found in 20 of these control collections. It shows the key importance of viral DNA collection into a stabilizing buffer (see the Materials and Methods paragraph). Another contribution of the real-time PCR, and the one we consider the most important, is the possibility of the treatment efficacy monitoring. The real-time PCR enables quantification of the amount of viral DNA in a clinical sample. Collection of samples from tears and cotton swabs is often discussed for the possible collection-associated imprecision when quantifying DNA. As it has been shown a sample recollection within several minutes in the same patient does not affect the results of quantification. The recollection can only lead to a 2-3-fold different amount of viral particles at the most, which is not detectable with the real-time PCR quantification. The real-time PCR shows approximately 5-fold and more differences, which are also clinically relevant and essential as this study showed. It is recommended and suitable to continue a maintenance treatment with systemic acyclovir 200 mg b.i.d. for 2–6 months if the keratitis relapses (Wilhelmus et al. 1998). Inefficacy of this treatment in above shown 18% of the patients (6 out of 32 patients on systemic acyclovir) remains a problem. We suppose that this dosage leads to insufficient therapeutic drug level and thus to resistance to the drug. The coincidence of two adenoviruses and HSV in a summer time during an epidemic of keratoconjunctivitis remains a curiosity. Uchio et al. (2000) demonstrated that 4.3% of the patients with epidemic keratoconjunctivitis were positive for HSV1 (in a cell culture). In our group of patients the coincidence of the two different viral corneal infections was found using the PCR in 13 patients (15.2%). The question is which infection was the initial one. The results of this extensive study show that DNA diagnostics of viral keratitis using the real-time PCR method is a fast and reliable laboratory technique, which enables an exact diagnostic assessment in diagnostically complex patients. A recently introduced technique of viral DNA quantification is in the same time an exact indicator of the treatment efficacy, and it enables to diagnose cases of acyclovir resistant keratitis or keratouveitis without the necessity of viral DNA sequencing. The sample collecting method as introduced seems to be careful enough without the necessity of cornea scarification, leading thus to a physical destruction by means of repeated sample takings. Sample collection into stabilizing solution, which preserves the viral DNA, significantly increases sensitivity of this technique. Third of patients with moderately positive results on the beginning of the treatment (5 × 104–5 × 102 viral particles/5 μl of DNA sample) were later observed as acyclovir resistant. We did not observe any acyclovir resistant patients with initial higher or lower quantity of HSV1 DNA by real-time PCR analysis. The quantity of 5 × 104–5 × 102 viral particles/5 μl of sample could be seen as positive predictor of higher risk of acyclovir resistant keratitis or keratoconjunctivitis. We observed that real-time PCR quantification of HSV1 in ocular samples can clearly show the acyclovir resistance after 1 week of the treatment. This project was supported by the Internal Grant Agency of the Ministry of Health of the Czech Republic – IGA MZ CR Nr. NR8507-3/2005 and by project MPO TIP: FR-TI1/478." @default.
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W2044299024 title "The treatment of HSV1 ocular infections using quantitative real-time PCR results" @default.
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