Multiplex Polymerase Chain Reaction For Detection Of Herpes Virus In Cornea Donor And The Study On Virus Transmission After Keratoplasty

Herpetic ocular disease is a main cause of blindness through out the world. Keratoplasty is the useful therapy to regain visual acuity. However the risk of viral transmission via corneal transplantation is increased at same time. Three parts of studies were performed to investigate the value of multiplex polymerase chain reaction (PCR) in diagnosing herpes virus infection of ocular viral disease and screening eye bank donor. The rabbit model was established to discuss the transmission of herpes virus between host and donor after penetrating keratoplasty (PKP).1. The utility of multiplex PCR in the diagnosing of ocular viral disease.Herpes simplex virus type 1(HSV-1), type 2 (HSV-2), cytomegalovirus (CMV), and varicella-zoster virus (VZV) belong to the same herpes family, the Herpesviridae, that cause ocular disease. They are difficult to differentiate by clinical findings alone. Additionally, the different structure of these viruses results in the differentiation in response to the anti-virus therapy. An accurate and rapid identification of these viruses is important to avoid incorrect diagnosis and to initiate early treatment. Traditional laboratory diagnostic techniques, including virus isolation, detection of antigen and antibody, all suffer from one or more limitation such as slowness and insensitivity. The technique of PCR has been used to detect viral nucleic acid, and the amplification of different sequences has been conventionally performed by separate PCR reactions for different types of viruses. It is a time consuming and costly technique and may be impossible if the amount of the clinical specimen is limited. More recently multiplex PCR has been reported. It has advantage of combining primers to detect several viruses in one test simultaneously.Ninety-four specimens, collected from 73 patients with clinically suspected herpes virus ocular infection, were tested for HSV-1, HSV-2, CMV and VZV by multiplex and uniplex PCR. The sensitivity and specificity were compared to virus isolation. The primers were constructed according to the entire DNA sequence of the glycoprotein D of these viruses. The primers were selected carefully to avoid co-reaction and restriction enzyme HinfI and HapII cutting sites were involved in the fragments of HSV-1 and HSV-2 PCR products. The reliability was confirmed according to the distinct digest patterns. The multiplex PCR products of these four viruses are 208bp,276bp,392bp and 355bp respectively, it is easy to make diagnosis by naked eye. Humanβ-actin gene was selected as an internal control to detect occasional false negative results, in order to avoid the failure in the DNA extraction procedure or the presence of DNA amplification inhibitors.The sensitivity of multiplex PCR was established using DNA extracts of serial 10 fold/two fold dilutions of standard strains of viruses. Virus titers of the standard strains were performed by the plaque assay in appropriate cell lines using culture supernatant. The sensitivity (detection limit) of uniplex PCR for HSV-1, HSV-2, CMV and VZV was 4, 4, 6 and 4 PFU/ml while that of multiplex PCR was 4, 4, 12 and 4 PFU/ml respectively. The sensitivity of multiplex and uniplex PCR for detection of HSV-1, HSV-2 and VZV were found to be the same. But there was two folds decrease in detection limit of CMV by multiplex PCR. The specificity was confirmed by negative multiplex PCR results of all negative control, including 30 patients with non-viral disease and 20 health volunteers.Forty one (57 specimens) out of 73 patients (94 specimens) were PCR positive (41/73, 56.2%) and 32 patients (37 specimens) were PCR negative (32/73, 43.8%) by both multiplex and uniplex PCR. Thirty patients (30/41, 73.2%) were HSV-1 positive and 11 patients (11/41, 26.8%) were VZV positive. The ratio was higher than virus isolation (7/37, 18.9%). One patient without anti viral treatment was positive for VZV at 4th day by virus isolation, indicated that the anti viral therapy probably affected the detection of virus. We suggested the specimens should be taken before the initiation of therapy.There were no CMV and VZV DNA found in our study and this was probably explained by different addiction of herpes virus to tissue. HSV-1 and VZV were always said to cause skin and ocular surface disease. But HSV-2 was said to usually cause genital lesions or led to acute retinal necrosis syndrome as CMV. The samples in this study were all from skin and ocular surface but not aqueous humor and vitreous, so the positive results were all HSV-1 and VZV. The further study will be done to confirm the specificity of multiplex PCR to detect HSV-2 and CMV with intraocular specimens.These results demonstrate that under the optimal reaction condition: 1.5mM MgC₂, 200uM dNTPs, 12.5pmol each primers and 35 cycles at 56℃annealing temperature, the multiplex PCR is as reliable as uniplex PCR. The four pairs of primers for HSV-1, HSV-2, CMV and VZV work well together. This method enables us to screen four pathogens simultaneously from one specimen, thus time saving and cost-effectiveness.2. The utility of multiplex PCR in eye bank cornea donorThe keratoplasty is a main therapy for blindness caused by corneal diseases. But the risk of viral transmission through PKP was also increased. Especially the herpes viruses were reported to establish a lifelong cycle of latency in cornea and trigeminal ganglion (TG) without any clinical presentation. So the clear cornea donor observed by slit lamp does not really indicate the health donor. The virus latent in the cornea or TG can be reactivated by stimuli such as grafting procedure itself, the application of local immunosuppressant and corticosteroids after operation. The recurrence of corneal herpetic infection may lead to primary graft failure (PGF).According to the results of PCR technique, 2³8% of organ culture donor corneas contained HSV DNA. But the limited cornea donor was not suitable for several detections. Multiplex PCR was used in this study to detect HSV-1, HSV-2, CMV and VZV from 64 eye bank cornea donors and 59 recipient cornea buttons obtained during PKP. Two out of 64 (2/64, 3.1%) eye bank donors were HSV-1 positive by multiplex PCR. Although the ratio was less than the other studies, the results implied the health cornea donor might serve as a reservoir of latent herpes viruses. Sever endothelial necrosis and reduce of endothelial cell were observed when these two infected donors cultured from 5 to 7 days. HSV-1 DNA was detected in the media in 10 days after tissue culture. These results were the same as other's findings: the herpes virus latent in cornea can be reactivated and replication during culture. There were 18 out of 59 patients were diagnosed with viral keratitis clinically. Among them, 14 patients were HSV-1 positive and 4 patients were VZV positive by multiplex PCR. The results further confirm the clinical diagnoses. Interestedly, HSV-1 was detected from a patient who was diagnosed by fungal keratitis. The results implied the co-infection of virus and fungal although the co-infection was rarely in ocular infection. But if it were, multiplex PCR will be very suitable for detecting several pathogens from only one specimen.All PKP performed with these donors had an uncomplicated course. But one patient of them who clinically diagnosed with HSK before PKP suffered epithelial lesion and edema in corneal grafts rim six months after operation. It was difficult to get diagnosis according to the atypical symptom, HSV-1 DNA was found in tear sample and corneal swab by multiplex PCR. The immediate anti-viral treatment was given and the patient recovered soon. This result demonstrated that atypical herpetic keratitis after PKP could not be ignored. We contributed the situation to the recurrence of herpetic infection and the recipient could have been the source of virus. Although the pathological button was removed during PKP and the source of virus was prevented, virus remained in corneal rim and TG still could be reactivated by stimuli,such as transplantation procedure, the application of local immunosuppressant and corticosteroids after operation. The viruses perhaps migrated via regenerated corneal nerve endings or cell-to-cell transmission.Multiplex PCR is a rapid and sensitive technique suit for corneal donor screen. The results of detection for HSV-1, HSV-2, CMV and VZV can be acquired within several hours before operation. Our study demonstrates HSV latency in corneal donor, and reactivation of HSV is suspected to be the cause of both PGF and atypical corneal lesion. In order to avoid graft failure the laboratory test should be done to initiate anti-viral therapy in time. 3. Experimental study on herpes virus transmission after PKPHuman being is a natural host of herpes virus and more and more investigations show that cornea is another reservoir of herpes virus besides TG. If the cornea donor latent with herpes virus was used for keratoplasty, the virus could reactivate by operating procedure and the application of local immunosuppressant and corticosteroids after operation. But some investigations suggested recipients could have been the source of virus. Although HSV-1 was considered as a main cause of PGF, the course of viral migration between donor and host remained unclear.In this study, twenty eyes from 10 rabbits were inoculated with HSV-1, HSV-2, CMV and VZV simultaneously and animal model of virus latency was established. PKP was performed between 7 herpes virus latent rabbits and 7 naive rabbits. Ninety days after PKP, the rabbits were induced to reactivate by transcorneal epinephrine iontophoresis. Two of 7 eyes of Group 1 (naive rabbits receive grafts from latently infected rabbits) were suffered PGF in 5 and 28 days after PKP respectively. HSV-1 and VZV DNA were detected in tear samples respectively by both viral isolation and multiplex PCR. This found indicated PGF probably caused by VZV besides HSV-1. The remained 5 eyes in Group 1 were found HSV-1 or VZV DNA in cornea grafts. Furthermore, HSV-1 and VZV were also detected in recipient cornea rims of 3 eyes especially in graft-host junction, and in corresponding TG of 2 eyes respectively. In Group 2 (latent infected rabbits received naive grafts),HSV-1 and VZV DNA were found in both recipient cornea rims and corresponding TG of all 7 eyes, but 4 grafts after induced.We hypothesized the latent herpes virus in donor graft could have been reactivated and undergone retrograde migration into the recipient cornea rim and corresponding TG after induced. Again, regenerated corneal nerve endings or cell-to-cell infection could serve as the conduit for this retrograde transmission. Moreover reactivating latent virus traveled from latent neuronal sites, such as TG or cornea, into cornea graft via trigeminal nerves. Axoplasmic flow within the sensory and autonomic nerves probably served as the conduit for this viral migration. The virus could migrate into the naive corneal grafts through nerve sprouts or by cell-to-cell infection. Our date provided important evidence to support the possibility of herpes virus corneal latency. The cornea may serve as a reservoir of latent virus and as a source of reactivation.To the best of our knowledge, this is the first time to inoculate animal with 4 viruses simultaneously, but HSV-2 and CMV DNA were not found. The absence of CMV DNA in cornea reflects its lymphotropic and leucotropic nature while HSV-2 most often causes genital lesion. On the contrary, HSV-1 and VZV are both dermatropic and neurotropic viruses. Our data lends greater weight to the finding of HSV-1 and VZV DNA and points to a keratotropic feature of these two viruses. Although 4 viruses inoculated at same time we did not found co-infection in this study. The reason was suspected as follow: Is there competition when 4 viruses involved combine with receptor on the surface of target cell? In this study, HSV-1 positive (9 eyes) is significant more than VZV positive (2 eyes). Once the viral infection emerged, the immunity reaction may raise the immunogenic to other viruses; Even if the co-infection existed, the sample content was too low, or one of the viral DNA content was lower comparatively to be tested. So we miss the co-infection in this study. Further study should be done to confirm this hypothesis.According to our experimental results, both cornea and TG are important latent sites for herpes virus. The latent viruses in rabbit eyes can be reactivated by adrenergic induction, and transmit from donor-to-host or host-to-donor via regenerate nerve endings or cell-to-cell after PKP. VZV can also cause PGF besides HSV-1. Viral infection could not be excluded when atypical cornea lesion present after PKP. Especially for the recipient with herpetic infection history, the anti-viral treatment should be given together with application of local immunosuppressant and corticosteroids to avoid the recurrence of viral infection from host-to-donor. We consider it is necessary to screen the latent viral infection of cornea donor before keratoplasy.