Herpes simplex keratitis journal,product presentation video ideas,alternative medicine definition of health hazard,chances of getting herpes with no symptoms 8dpo - Plans Download

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A 44-year-old man presented to the emergency department with 2 days history of redness, photophobia, tearing, foreign body sensation and blurred vision in his left eye. Keratitis is a condition in which the eye's cornea, the front part of the eye, becomes inflamed. Herpes zoster keratitis, associated with Herpes zoster ophthalmicus, which is a form of Shingles.
Amoebic infection of the cornea is a serious corneal infection, often affecting contact lens wearers.[3][4] It is usually caused by Acanthamoeba. Exposure keratitis — due to dryness of the cornea caused by incomplete or inadequate eyelid closure.
Contact lens acute red eye (CLARE) — a non-ulcerative sterile keratitis associated with colonization of Gram-negative bacteria on contact lenses. In addition, contact lens wearers are typically advised to discontinue contact lens wear and replace contaminated contact lenses and contact lens cases. Aciclovir is the mainstay of treatment for HSV keratitis and steroids should be avoided at all costs in this condition. A nanoparticle formulation reduces the corneal toxicity of indomethacin eye drops and enhances its corneal permeability. Pediatric microbial keratitis in taiwan: clinical and microbiological profiles, 1998-2002 versus 2008-2012. Jetton JA1, Ding K, Kim Y, Stone DU.Author information 1Departments of *Ophthalmology, Dean A.
Up until now a total of 37 cases of canaliculus stenosis in the course of primary herpes simplex infection have been reported. Freeman et ala€‰4 never found total occlusion of the canaliculi in acutely herpes simplex infected rabbits. This recent issue is free to all users to allow everyone the opportunity to see the full scope and typical content of BJO. Don't forget to sign up for content alerts so you keep up to date with all the articles as they are published. Answer: The central round epithelial defect with heaped up margins in the setting of a past history of herpes zoster ophthalmicus is highly suggestive of a neurotrophic epithelial defect.
Answer: A history of herpes simplex keratitis or herpes zoster on the same side of the neurotrophic epithelial defect is common. Answer: Mild cases with some preservation of corneal sensation may be treated with antibiotic ointments, artificial tears, and frequent follow-up.
Except where otherwise noted, this work is licensed under Creative Commons Attribution-NonCommercial 4.0 International License. Background: PCR is a molecular technique for herpes simplex virus (HSV) detection that can cause life-threatening infections such as encephalitis and keratitis.
Objectives: The purpose of this study is the design of competitive IAC for PCR diagnosis of HSV, which in fact is the main cause of keratitis and viral encephalitis in developed countries.
Materials and Methods: Composite primers for PCR amplification of Leishmania major kDNA (kinetoplast DNA) were designed and optimized to use as IAC-HSV. Conclusions: Indeed, using other DNA as an IAC is expected to detect false-negative results and amplification of the DNA is the key tool to examine the accuracy of amplification and detection steps. Herpes simplex virus (HSV) is responsible for a variety of serious diseases such as infection of cornea (keratitis) leading to corneal blindness and perilous infection of central nervous system such as encephalitis, which is the most common form of sporadic viral encephalitis in western countries (1, 2).
The purpose of this study was to design a competitive IAC for PCR diagnosis of HSV, which is the main cause of keratitis and viral encephalitis in developed countries. A total of 105 cerebrospinal fluid (CSF) samples from patients with central nervous system diseases (suspected of HSV, cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV), and human herpes virus 6 (HHV-6) (15, 16) and 78 samples from corneal epithelium (suspected of HSV, EBV, and CMV) of patients with herpetic dendritic keratitis and uveitis (17, 18) were obtained from the Virology Department of the Pasteur Institute in Iran (Tehran) that were collected from different hospitals (unknown). Other species of Herpesviridae genomic DNA, such as VZV, EBV, CMV, and non-herpes genomic DNA including, Hepatitis B virus Salmonella Spp Mycobacterium tuberculosis (all were obtained from Pasteur Institute of Iran, Tehran), human genome and HSV as positive control were amplified by PCR with HSV primers in described PCR condition to test PCR specificity for the target site of primers. Colonies containing inserted plasmid (pIACHSV) were extracted and purified by an AccuprepA® Plasmid Extraction Kit (Cat. A total of 105 CSF and 78 keratitis specimens were obtained from the Virology Department of Pasteur Institute in Iran (Tehran).
A 10-fold serial dilution of HSV genomic DNA was used to find the range of sensitivity in PCR assay. After obtaining the expected 662 bp PCR amplicon, the ideal concentration of IAC was optimized and there could be no inhibition of amplification of target DNA. The sensitivity of PCR detection for HSV in the presence of IAC was examined by mixing different concentrations of the HSV template in the presence of IAC DNA. A total of 105 CSF and 78 keratitis specimens were examined by both culture and PCR assay to demonstrate the comparative diagnostic results. PCR assay was carried out for CSF and keratitis specimens in the presence of IAC to confirm the exact function and performance of IAC.
In our investigation, the specificity of the primer set was examined against other species of Herpesviridae and non-HSV genomic DNA. Mare NjoyaDepartment A of ophthalmology, Faculty of medecine of Rabat, University of Mohammed V, Rabat, Morocco. Please consider expanding the lead to provide an accessible overview of all important aspects of the article. Viral infection of the cornea is often caused by the herpes simplex virus which frequently leaves what is called a 'dendritic ulcer'. Bacterial infection of the cornea can follow from an injury or from wearing contact lenses.
Infectious keratitis can progress rapidly, and generally requires urgent antibacterial, antifungal, or antiviral therapy to eliminate the pathogen. Others may result in perforation of the cornea, (an infection inside the eye), or even loss of the eye. The stenosis was in the mid portion of the canaliculi starting at about 5 mm from the punctae.
In fact, Coster and Welham1 demonstrated, using electron microscopy, in biopsy specimen particles with a size and morphology compatible with HSV. Other associated conditions that need to be considered in the differential diagnosis include topical anesthetic abuse and crack cocaine abuse.
However, the main issues, false-negative results causing by PCR inhibitors, of this technique that reduce PCR efficiency.
IAC-HSV amplified in a non-stringent condition, ligated into pTZ57R plasmid vector, and transformed into Escherichia coli JM207 and then cloned. This internal amplification control is applicable for early reliable diagnosis of HSV in different loads of virus in different specimens. The above-mentioned issues provide extreme necessity for a rapid, reliable, and specific diagnostic approach.
Specimens were used for comparison tests and examinations of the performance of constructed IAC. Primary amount of IAC was measured by a NanoDrop spectrophotometer, A260 nm, Eppendorf, Germany. In addition to other PCR reagents, each reaction was contained 1 AµL of IAC in a final volume of 25 AµL.


Two expecting signals for HSV and IAC DNA were observed at 454 bp and 662 bp, respectively. It can be seen from the CSF specimens that the PCR assay generated 21 positive results; however, in the culture method, only 10 positive results had been achieved. Figure 4 shows there is an inhibited specimen, which indicates that one or more factors have not functioned correctly in the course of extortion or any other possible problems. Although most of the employed DNA in the specificity test was DNA HSV-dependent, still no signal was observed that shows the precision and specificity in primer sequences (Figure 2).
Role of specific innate immune responses in herpes simplex virus infection of the central nervous system. Reactivation of latent virus (under fever, injury or surgery, etc.) in the ophthalmic branch of the TG may lead to structural damage of the cornea. The bacteria involved are Staphylococcus aureus and for contact lens wearers, Pseudomonas aeruginosa. With proper medical attention, infections can usually be successfully treated without long-term visual loss. It is known that decreasing direct cell stimulation and reducing the amount applied via increasing bioavailability are useful for improving these issues.
Conditions affecting the fifth cranial nerve, such as neurosurgery, cerebellopontine angle tumors, and pontine stroke, can also result in corneal anesthesia and epithelial breakdown. To overcome this problem, a competitive internal amplification control (IAC) was constructed for conventional PCR using the PCR-cloning technique. Therefore, a suitable method of detection should provide the results in the shortest possible time and highest accuracy to inform the respective physician or patient (3).Although viral culture is the gold standard for detection of HSV DNA, PCR techniques are more sensitive and the results are available in less than 4 hours (4).
CSF specimens were obtained by subsequent lumbar punctures and aqueous samples from uveitis were obtained by anterior chamber paracentesis. Genomic DNA (HSV-1, KOS strain) was obtained from the Pasteur Institute of Iran (Tehran) to use as a positive control.
3112, Pioneer, Korea) according to the manufacturera€™s instructions and the extracted plasmid was kept at 4A°C. For more confirmation of IAC, the PCR assay was performed for all specimens with an HSV thermal profile as described above. VERO cells (National Cell Bank, Pasteur Institute of Iran, Tehran) were added to RPMI medium (Sigma Co., USA), which was supplemented with penicillin G (10000 U), streptomycin (100 Aµg), and 10% FBS. The resulting 662 bp fragments of DNA were cloned and transformed into a host and the new recombinant plasmids, which are known as pIACHSV, were then extracted. As a result, the lowest responsible concentration of IAC that could produce a distinct signal on the gel was 1000 plasmids (NanoDrop spectrophotometer, A260 nm, Eppendorf, Germany), which is preferred as an ideal concentration of IAC to be used per reaction (Figure 3).
In the case of the keratitis specimens, 15 positive results were achieved by PCR and 10 by culture. In addition, it can be seen that there is a true-negative results for which only the IAC signal was produced.
HandorDepartment A of ophthalmology, Faculty of medecine of Rabat, University of Mohammed V, Rabat, Morocco. Epithelial dendritic lesions represent the most frequent type of HSV keratitis and are caused by the virus replication in the corneal surface.2 There are several antiviral agents that are effective against HSV such as acyclovir, valacyclovir or ganciclovir.
Recently, PCR has been developed extensively for virus infection diagnosis by many laboratories. HSV-1 (KOS strain) was cultured using VERO cell line (National Cell Bank, Pasteur Institute of Iran, Tehran) on RPMI medium (Sigma Co, USA) supplemented with penicillin G (10000 U), streptomycin (100 Aµg), and 10% FBS in 1 L of RPMI medium for examination as a positive control. PCR products were electrophoresed and visualized on 1.5% agarose (stained with SYBR Green, CinnaGen, Iran) and markers SM1108 (50-1500 bp, MBI Fermentas, USA) were used.
The concentration of IAC remained constant at 1000 plasmids within a broad range of HSV DNA concentrations without the affection of the target signal. IMCnano eye drops are tolerated better by a human cornea epithelial cell line (HCE-T) than commercially available NDSAIDs preparations (IMC, pranoprofen, diclofenac, bromfenac and nepafenac eye drops), and corneal wound healing in rat eyes with debrided corneal epithelium instilled with IMCnano eye drops is significantly better than that of eyes instilled with commercially available IMC eye drops. This technique has beneficial characteristics including reliability, speed, and sensitivity that are vital for an ideal diagnostic assay. Subsequent to the observation of 70a€“90% cytopathic effects viruses were harvested and kept at 4A°C until used.
Genomic DNA of other species of HSV and non-HSV DNA were used to test the specificity of the HSV PCR assay. Therefore, the sensitivity of PCR and culture in the case of CSF specimens were 100% and 27.6%, respectively.
However, in the case of presence of a very low amount of target DNA, no signal may be seen in the results. An IAC is a non-target DNA sequence, which is present in the tube reaction along with HSV DNA that will be amplified simultaneously in the PCR assay under the same condition. LezrekDepartment A of ophthalmology, Faculty of medecine of Rabat, University of Mohammed V, Rabat, Morocco.
In addition, the accumulation of IMC in HCE-T cells treated with the IMCnano eye drops for 30min was 19.9% that of the accumulation from commercially available IMC eye drops. In spite of its outstanding advantages, PCR has some problems such as false-positive and -negative results (5). Finally, PCR analysis for each specimen with IAC included negative and positive controls were performed as a referee to confirm the results (20).
Figure 2 shows no signal appeared due to the absence of homologous target sequences (All data not shown).
However, the sensitivity of PCR and culture in the case of keratitis specimens were 100% and 66.66%, respectively. It should be mentioned that IAC should have unique characteristics such as stable, non-infectious, easily produced, and standardized (4).As already mentioned in the previous section, there are two major approaches for IAC construction competitive and noncompetitive approach (21). On the other hand, the corneal penetration of IMC from IMCnano eye drops was significantly greater than in the case of the commercially available IMC eye drops in both in vivo and in vitro studies using rabbit corneas.
Clearly, by using a negative control (external control) during the PCR assay a false-positive result can be resolved. Obviously, constructed composite primer was only used to make IAC and for final detection of target DNA, only HSV primers used. In a noncompetitive approach, there are two different set of primers for IAC and target DNA.
HafidiDepartment A of ophthalmology, Faculty of medecine of Rabat, University of Mohammed V, Rabat, Morocco. Taken together, we hypothesize that a nanoparticle formulation reduces the corneal toxicity of IMC eye drops, probably because the accumulation of IMC from IMCnano eye drops in the eye is lower than that from commercially available IMC eye drops.
However, the major drawback of PCR is in producing false-negative results that remain unresolved. Therefore, two PCR reactions will be carried out in one PCR tube with the same thermal profile. In addition, the nanoparticle formulation may allow a decrease in the amount of IMC used due to the increase in bioavailability, resulting in reduced drug toxicity. False-negative results are obtained from the malfunction of thermal cycler, imprecise extraction, insufficient Taq DNA polymerase, and other inhibitory substances that the exact act of these inhibitors are largely unknown (6).


The optimization of IAC with this method seems to be complicated due to the extensive range of annealing temperatures for IAC and target as well as the increase of risk to produce nonspecific signals due to the use of two different set of primers (22). SabraneDepartment A of ophthalmology, Faculty of medecine of Rabat, University of Mohammed V, Rabat, Morocco. These findings provide significant information that can be used to design further studies aimed at developing less toxic eye drops. Although several extraction protocols and commercial kits have been developed to remove the probable PCR inhibiting substances from the specimen (7) and they seem to be ineffective for inhibitory removal that leads to a decrease of PCR sensitivity.In order to enhance the certainty in negative PCR results, the presence of internal amplification control (IAC) is required (8, 9).
On the other hand, the competitive method has only one set of primers for IAC and target DNA. IAC is a DNA sequence that is added to the sample and will be amplified with the target DNA simultaneously.
DaoudiDepartment A of ophthalmology, Faculty of medecine of Rabat, University of Mohammed V, Rabat, Morocco. Although IAC is commonly used for the detection of availability of PCR inhibitors (10), few technical problems were observed during IAC construction. The main drawback of this method is the decrease of sensitivity of PCR due to the enhancement or reduction of amplification in one of the products (IAC or target DNA) that can lead to a false-negative result.
Here a simple method for IAC construction with the least practical difficulties was used because of the above-mentioned requirements. Thus, the most critical factor to overcome this issue is to determine the proper concentration of IAC. Therefore, utilizing an IAC DNA in each PCR reaction that simultaneously amplified with viral target DNA with the same thermal profile used as an effective tool to overcome false-negative results in the PCR diagnosis of HSV.Few studies have been carried out on the subject of HSV internal control. In this way, IAC is able to compete with the DNA target to bind with the primers reasonably (23).
Burggraf and Olgemoller (11) constructed a single-stranded oligonucleotide as an internal control for a newly designed real time PCR for rapid detection of HSV. The amplified internal control was detected by the same probe that detects the amplified target. However, even low concentrations of these inhibitors can cause the target DNA signal to disappear (especially in low concentration of target DNA). Phenotypic and genotypic characterization of acyclovir-resistant corneal HSV-1 isolates from immunocompetent patients with recurrent herpetic keratitis.
In another study, heterologous DNA was used to establish a multiple internal control DNA contained five IAC for five competitive Light Cycler-PCR assays including HSV IAC by using hybridization probes (12).
On the other hand, when the amount of target DNA is high, a low concentration of IAC will be eliminated due to the presence of competition during the amplification process (14).
The current investigation develops a simple and accurate path for IAC construction that has never been used by real-time and conventional PCR for HSV diagnosis.In this study, IAC was designed in a competitive way by employing composite primer technique. Therefore, by using the constructed IAC, an accurate and reliable approach was designed to examine the PCR amplification steps for HSV diagnosis, which is a common routine assay for all diagnosis laboratories (14). Composite primers were made by introducing HSV primer sequences to the 5a€™ ends of kDNA primer sequences. The resulting composite primers have 5a€™ over-hanging ends that are complementary to HSV primers. The selected primers for IAC are relatively short, which eliminate the risk of obtaining nonspecific bands on the gel (Figures 3 and 4). The employed gene for IAC in our study has a broad spectrum annealing temperature and, as a result, is insensitive to thermal profile (14). Furthermore, utilizing a cloning technique provides a limitless source of IAC as a recombinant DNA that can be easily used in diagnostic laboratories. Moreover, the difference in size of IAC fragments and HSV DNA is suitable and easy to detect.
It is necessary to know that the presence of two sets of primer sequences in one reaction will increase the possibility of nonspecific band production.
Therefore, we used the composite primer technique to have only one set.When the target signal was observed, it meant that the inhibited IAC amplification has occurred due to the high concentration of target DNA. Consequently, with the amplification of the target DNA, the amplification of IAC is not mandatory. If neither IAC nor the target DNA have generated a signal, it can be concluded that at least one inhibitor was present in the PCR assay (24). The obtained results from the culture method indicated that PCR is a much more sensitive technique for HSV diagnosis in comparison with the viral culture method and would be considered a reference technique (25, 26) regardless of whether the samples were obtained from CSF or keratitis.In the recent years, IAC was constructed for various pathogens by means of different DNAs (27, 28). The gap between the two fragments (IAC and target DNA) should be relatively high and be easily distinguishable (28). In the present investigation, the size difference in the constructed IAC fragments and HSV DNA is about 200 bp, which can be easily distinguished.
Also, in different studies (23, 29), IAC were constructed for real-time PCR diagnosis for different pathogens by using single stranded oligonucleotides.
However, we designed an IAC for general PCR and by using non-target gene (kDNA f Leishmania), which would be used for construction of IAC for many other target pathogens that PCR diagnosis was due to a broad annealing temperature.
By using introduced IAC, which is applicable easily with broad range of annealing temperature, to establish a new reliable method to have the least false-negative results for this kind of diagnosis.The methodology is simple and should enable laboratories to conveniently complement their existing panel of PCR assays with such an IAC. Until now, the concentration of IAC and target sequence was considered a critical parameter for the detection limit of a PCR assay.
The demonstrated stability in the current study makes this DNA suitable for the construction of IAC in other pathogenic DNA, a fact that can also be applicable for other molecular based viral detection systems. Moreover, the higher amount of target DNA present in the samples, the lower was the increase of the IAC specific amplification signals on the gel. As a result, during the complementing of PCR assay with competitive IAC, the amount of IAC plays an important role that can avoid the competitive inhibition of the respective virus-specific PCR and loss of assay sensitivity. It can be seen that due to the competition between the two DNAs in the sample reaction, a high concentration of IAC in the sample of reaction is not influenced by feeble and least inhibitors. Therefore, determination of an optimized amount of IAC in each PCR reaction is vital (21, 30, 31) that, in fact, can be a limitation of this study that can be improved with further investigation.
Since the introduced IAC may not express all inhibitory factors in HSV diagnosis and it might just explain some of them.In conclusion, if a nucleic acid amplification method is to be a highly reliable diagnostic test for pathogens, it must include internal control. Here, a novel IAC with a competitive approach was constructed for HSV diagnosis, which was in an agreement and not a reflection with the HSV thermal profile. It can be a sensitive tool to obtain reliable results for HSV diagnosis with a PCR assay, which is a cost effective, simple, and accurate method.
The use of IAC DNA may streamline assays and may avoid the risk of handling errors in HSV detection due to the addition of only one IAC to the various samples.



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