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Analysis of the Polymerase Chain Reaction in the Detection of Herpesvirus DNA From Fixed and Stained Tissue Sections
Abstract
The polymerase chain reaction (PCR) is a molecular diagnostic technique that has been applied to many infectious processes. Stained and unstained Tzanck smears, vesicle fluid swabs, and crusts have all been used as the source for template DNA for the PCR to document evidence of herpes simplex virus and varicella-zoster virus infection. Thirty-five cases with histologic evidence of acute herpesvirus infection were retrieved from archival tissue blocks that were up to 5 years old. Paraffin and hematoxylin-eosin-stained tissue sections obtained from routinely prepared glass slides from these cases were then examined for herpesvirus DNA using the PCR.
The PCR-detected herpesvirus DNA from 34 (97.1%) of 35 paraffin tissue samples. Herpes simplex virus and varicella-zoster virus DNA were detected in eight and 26 of these cases, respectively. For hematoxylin-eosin-stained tissue samples, PCR detected herpesvirus DNA sequences in 16 (45.7%) of 35 cases. Herpesvirus DNA was isolated from paraffin tissue sections and recently prepared hematoxylin-eosin-stained tissue samples obtained from archival tissue blocks that were up to 5 and 2 years old, respectively.
The PCR can detect herpesvirus DNA in extremely high yield from unstained paraffin-embedded tissue samples with histologic evidence of acute herpesvirus infection that are up to 5 years old. Herpesvirus DNA can also be identified in approximately 50% of these cases from hematoxylin-eosin-stained tissue sections obtained from routinely prepared glass slides.
... Besides, we used PCR-based techniques that, to date, represent the most sensitive methods to detect DNA or RNA presence in biological samples. PCR techniques for viral nucleic acids detection in paraffin-embedded specimens used in this study are widely replicated and they were shown being very reliable even many years after fixation [41][42][43]. ...
Background:
Infliximab is used for refractory Crohn's disease but there are concerns regarding long-term safety. Recently, JC-polyomavirus (JCV) was studied after 3 cases of progressive multifocal leukoencephalopathy (PML) were found after treatment with natalizumab. The aim of this study was to investigate the short-term effect of infliximab on reactivation of several harmful latent viruses.
Methods:
Sixty consecutive patients scheduled for infliximab induction course were prospectively enrolled. Blood samples were taken before each infliximab infusion at 0, 2, 6, and 14 weeks. Specific polymerase chain reaction (PCR) analyses were performed to detect JCV, Epstein-Barr virus (EBV), human herpes virus-6, (HHV-6), -7, -8, and cytomegalovirus (CMV).
Results:
Indications to infliximab were luminal and fistulizing disease in 49 and 15 cases, respectively. Clinical improvement and remission were achieved in 54 (90%) and 39 (65%) of patients, respectively, at 6 weeks. No patient was JCV-positive at any timepoint. EBV serology was positive for 59/60 patients (98%); EBV-PCR tests were transiently positive (>40 copies/10(5) Peripheral blood mononuclear cells, PBMC) in 4 (7%) patients after infliximab, but in each case were negative at subsequent timepoints. All patients were negative for HHV-6, -7, and -8 at all timepoints. CMV serology was positive in 42 patients (70%), but no CMV-PCR-positive patient was observed. There was no association between concomitant treatments or clinical characteristics and viral status.
Conclusions:
Our results support the safety of short-term infliximab treatment with respect to latent virus reactivation. The long-term effects of infliximab, particularly for the issue of lymphoproliferative disorders, warrants further studies with larger populations, but so far data are reassuring.
... This histologic picture is difficult to distinguish from that of other types of viral hepatitis, such as hepatitis B, and from that of cytomegalovirus [54] infections. Specific diagnosis can be made by culture of the virus or identification of virus-specific DNA by PCR [55]. Group 2 Granulomatous Disorders (Recently Recognized Causal Agents) Cat-Scratch Disease Cat-scratch disease or fever is also known as benign lymphoreticulosis or regional granulomatous lymphadenitis [56]. ...
Granulomatous disorders are frequently due to a wide variety of infections. Over the past decade advances in molecular diagnostic
techniques have allowed identification of organisms involved in granulomatous disorders that previously were of unknown etiolgy.
On the basis of currently available information, granulomatous infections can now be classified in three categones. Group
1 infections are due to a well-recognized organism. Group 2 comprises infections due to organisms that have been recently
identified in granulomas by molecular methods but are not readily isolated by conventional microbiological techniques. Group
3 consists of disorders for which the causal organisms have not yet been identified but are strongly suspected; further advances
in diagnostic techniques will lead to reclassification of some of these disorders as group 2. This review describes the etiology,
histopathologic features, and classification of granulomatous disorders, with an emphasis on those of groups 2 and 3.
... Besides, we used PCR-based techniques that, to date, represent the most sensitive methods to detect DNA or RNA presence in biological samples. PCR techniques for viral nucleic acids detection in paraffin-embedded specimens used in this study are widely replicated and they were shown being very reliable even many years after fixation [41][42][43]. ...
Herpesviridae infection or spread may be a hazard in immunodepressed patients. In the field of inflammatory bowel disease, refractory severe ulcerative colitis is a challenging condition, closely associated to immunosuppression both for inanition due to the disease activity and for immunosuppressive treatments. Cytomegalovirus (CMV) has been proposed as a major cause of refractoriness, while other Herpesviridae may be a risk factor in the long-term follow-up.
To evaluate the positivity rates of CMV, Epstein-Barr (EBV) and Human herpes virus-8 (HHV8) in a consecutive group of ulcerative colitis patients who underwent colectomy for refractoriness to medical treatment compared to a control group, using state of the art methods.
Colonic specimens from 24 consecutive patients with ulcerative colitis submitted to colectomy for refractoriness and from 20 controls (submitted to colectomy for colorectal cancer) were studied. Standard histology and immunohistochemistry (IHC) for CMV and specific polymerase chain-reaction (PCR) for CMV, EBV and HHV8 were carried out.
Regarding CMV, 1 case (4%) was positive at histology and IHC, whereas 3 cases (13%) were positive at PCR, compared to none in the control group (p=0.239). For EBV 2 cases (8%) and 2 controls (10%) were positive at PCR. None of the cases or of controls was positive for HHV8. The only clinical characteristic independently associated to CMV positivity was the white blood cell count at admission, higher among CMV positive patients (p<0.001). At the end of the post-surgery follow-up (median 7.3 years) none of the CMV positive cases experienced pouchitis, compared to 3/21 (14%) of the CMV negative cases (p=1.000).
Our data suggest that CMV is uncommon (13%), even though PCR techniques, considered to be the most sensitive tools, were used for virus detection and the study population is made by highly selected patients with definite refractoriness. EBV and HHV8 may represent a theoretical risk of immunosuppressive therapy because of their potential role as cancer triggers; however in our study, results seem to be reassuring that UC patients undergoing immunosuppressive therapy are not exposed to an excessive risk of viral infection.
Zoster and postherpetic neuralgia (PHN) are common conditions that have a significant impact on quality of life. Patients over the age of 50 years and those who are immunocompromised are at increased risk. The risk to the fetus is felt to be negligible with localized zoster in pregnancy. In cases in which the clinical diagnosis is unclear, immunofluorescent staining should be considered. Early antiviral therapy should be initiated in patients over the age of 50 years, those who are immunosuppressed, those with severe acute zoster and those with ophthalmic zoster. PHN can be difficult to treat; studies have shown that valacyclovir or famciclovir started within 72 h of rash onset, amitriptyline started within 48 h of rash onset and epidural local anesthetic with steroid given within the first 2 months may decrease the incidence of PHN. Pregabalin, narcotics, the lidocaine patch, topical capsaicin and intrathecal local anesthetic and steroid may help zoster-associated pain. A new zoster vaccine looks promising; a large double-blind study showed that vaccination halved the incidence of zoster and, in those who developed zoster, it reduced the incidence of PHN by two-thirds at 3 months and by three-quarters at 6 months.
The polymerase chain reaction (PCR) enables rapid and sensitive detection of VZV and HSV DNA and its efficiency depends mainly on the choice of the primers. Primers should hybridize to conserved DNA sequences within the viral genomes in order to avoid unreliable amplification due to DNA sequence variation between different strains. The aim of the study was to design and to evaluate a general primer pair which permits fast and reliable detection of HSV and VZV. The genes UL 15 of HSV and UL 42 of VZV share the highest degree of homology within the two genomes. We designed a primer pair (GPHV-RU) which hybridizes to these genes. The genetic variability of amplified sequences from clinical specimens was analyzed by restriction enzyme cleavage analysis and by temperature gradient SSCP analysis (TG-SSCP). PCR with GPHV-RU amplified viral sequences from all analyzed specimens (25 x VZV, 10 x HSV-1, 5 x HSV-2) obtained from patients with clinical evidence of HSV or VZV infection. Restriction enzyme cleavage analysis with Hpa II further permitted reliable distinction between VZV, HSV-1, and HSV-2. Analysis of the heterogeneity of the amplified sequences by restriction enzyme cleavage and by TG-SSCP demonstrated no variability between the analyzed clinical specimens of VZ and of HSV-2 and only one differing TG-SSCP-pattern within the HSV-1 isolates. The results suggest that detection of HSV and VZV using the new primer pair GPHV-RU should give reliable results as the amplified sequences show little genetic variability within clinical isolates of HSV-1/2 and VZV.
The aim of this study was to identify the source of surgical biopsy tissue in paraffin block and histochemically stained biopsy section on a slide suspected to be mislabeled. Commercially available polymerase chain reaction (PCR)-based human lymphocyte antigen (HLA) DQA1 and Polymarker kits (Roche Molecular Systems, Branchburg, NJ, U.S.A.). were used to generate DNA profiles from biopsy tissue material in paraffin block and on histological slide, and the reference blood sample was collected from the patient. The source of biopsy tissue was detected by HLA DQA1 and polymarker profiling. Profiles obtained from biopsy material were consistent with those obtained from reference blood sample. The PCR-based DNA profiling techniques can determine the source of minute tissue in paraffin block and stained tissue sections on slides routinely prepared for diagnosis of carcinoma.
Varicella-zoster virus (VZV) and herpes simplex virus (HSV) are human pathogens of significance involved in multiple diseases with either typical or atypical clinical features. In neonates and immunocompromised patients these alphaherpesviruses may cause life-threatening diseases such as encephalitis. Detection of VZV by virus culture is difficult. Polymerase chain reaction (PCR) is quicker and more sensitive and applicable in most clinical microbiological laboratories. Using degenerate primers, glycoprotein B (gB) DNA was amplified from all alphaherpesvirus field strains present in clinical samples. The amplification of gB allowed virus typing of VZV, HSV-1 and HSV-2 using restriction enzyme digestion of the PCR products. Degenerate primers can replace conventional primers in diagnostic PCR without loss of sensitivity and specificity.
We have characterized the susceptibility and genetic stability of varicella-zoster viruses (VZV) isolated from skin lesions of three patients with herpes zoster and six patients with varicella treated with conventional short-term acyclovir (ACV) administration. The susceptibilities to ACV of the serial isolates from the patients were examined, and there was no significant difference in the susceptibility to ACV among the isolates before and during the ACV treatment, indicating that conventional short-term ACV treatment did not generate ACV-resistant VZV infections. Polymerase chain reaction (PCR) analyses of these as well as seven thymidine kinase-deficient VZV strains derived from in vitro ACV treatment were carried out to examine their genomic stability. Five regions containing tandem direct reiterations (R1-R5) were amplified by PCR and compared, and the region containing the Pst I-site was also examined. PCR analyses demonstrated that the R1, R5 and the Pst I-sites were stable and useful in epidemiological studies even after ACV treatment. The R2, R3 and R4 sites were far less stable in these experimental conditions. In this paper we discuss the results of the PCR analyses with regard to the dynamics of VZV population in patients with VZV infection treated with conventional short-term ACV administration.
Formalin-induced DNA degradation was studied at different fixation times (3, 7, 16 and 32 days) each on 10 formalin fixed paraffin embedded tissues (FFPET) stored for 15 years at room temperature. The four different extraction protocols used in this study showed that Chelex100 extracts performed the best at 3 and 7 days of formalin fixation (DFF) (with regard to the quantity and the quality of the DNA). However, Qiamp extracts showed better results for long sized alleles, as well for single polymerase chain reaction (PCR) amplifications after 16 and 32 DFF, as for multiplex PCR at shorter fixation times. DNA degradation is expressed by the size of the amplified alleles, only 100 bp templates surviving after 32 DFF (AMG locus). Single locus amplifications (CD4 and FES/FPS alleles) performed better than multiplex PCR (ProfilerPlus), with nearly 100% positive results at 7 DFF. In both types of amplifications, the success rate decreased proportionally with the time of formalin fixation and, consequently, with the size of the required DNA template.
Herpes zoster is a common disease caused by the varicella-zoster virus. The use of virostatic agents as early as possible is necessary in shortening zoster-associated pain.
Rapid diagnosis is necessary for the optimal efficacy of antiviral therapy. The diagnosis in the early stage of infection is often difficult.
In the present study skin biopsies of patients with herpes zoster and unclear skin changes were analysed by detecting viral DNA using the polymerase chain reaction (PCR) in order to amplify open reading frames (ORF) 14, 29 and 63.
Varicella-zoster virus DNA could be detected with PCR of all three ORF not only from blisters but also from erythematous skin.
PCR is the method of choice for the viral diagnosis in herpes zoster before blister eruption.
DNA from archival Papanicolaou stained smears was successfully amplified using the polymerase chain reaction (PCR) to see if it could be used for retrospective genome studies such as detection of the presence of human papilloma virus (HPV) and changes in p53 gene expression. DNA was isolated and purified by treatment with proteinase K, phenol/chloroform, and isoamyl alcohol. Segments of the human beta actin and TGF beta 1 gene were amplified by PCR. Of all stains used in the preparation of Papanicolaou smears, only eosin was detectable as a greenish band in ethidium bromide treated DNA gels under ultraviolet illumination.
The polymerase chain reaction (PCR) was used to detect HSV DNA in genomic DNA extracted from skin biopsies obtained from healed skin of five patients with hyperpigmented macules following recurrent cutaneous HSV infections and from eight patients with HSV-associated erythema multiforme (EM). A 92-bp HSV-1 DNA fragment was found in all the skin biopsies from the site of recurrent HSV infection and in five of eight (62%) biopsies from the EM patients. Virus DNA was not found in tissues distant from the site of HSV recurrence or from a patient without a history of HSV infection. These findings confirm the presence of HSV in healed skin from the site of recurrent HSV disease and are consistent with the concept that HSV is involved in EM pathogenesis.
To compare Tzanck smears, viral cultures, and DNA diagnostic methods using the polymerase chain reaction (PCR) in detection of herpes simplex virus (HSV) or varicella-zoster virus (VZV) infection in clinically suspected cases.
A 12-month trial comparing PCR with viral cultures and Tzanck smears in patients with clinically suspected HSV or VZV infection.
Both ambulatory and hospitalized patients were recruited from a tertiary referral center and the Miami (Fla) Veterans Affairs Medical Center.
Convenience samples of patients clinically suspected to have HSV (n = 48) or VZV (n = 35). To be included in the final analysis patients needed to have a positive Tzanck smear, viral culture, or PCR result. Patients who were clinically suspected to have HSV but had VZV by viral culture or PCR were analyzed in the VZV group. Similarly, patients who were clinically suspected to have VZV, but had HSV by viral culture or PCR were analyzed in the HSV group. Seventy-seven patients were available for final analysis: HSV (n = 30), VZV (n = 32), and 15 control cases who did not have evidence of viral infection.
For HSV, PCR detected HSV DNA sequences in 73% of stained smears and 83% of unstained smears. For VZV infection, VZV DNA sequences were detected in 88% of stained smears and 97% of unstained smears. Viral DNA sequences were not detected in the 15 control cases. Viral cultures were positive in 83% and 44% of HSV and VZV cases, respectively. The Tzanck smear was positive in 60% and 75% of HSV and VZV cases, respectively.
PCR is a reliable method for detecting HSV and VZV DNA sequences from single stained and unstained Tzanck smears. It is clearly superior to viral culture in identifying VZV infection and is equivalent to conventional culture techniques in identifying cases of HSV.
Herpes simplex virus DNA was identified by the polymerase chain reaction with primers specific for the herpes DNA polymerase gene. A herpes-specific amplimer was detected in two of three cases in which the clinical and histologic features were inconclusive.
Sections from paraffin-embedded tissues of lesions interpreted as seborrheic keratoses localized to the pubic, genital, and crural regions were assayed for the presence of human papillomavirus types 6, 11, 16, 18, and 33 using DNA amplification followed by specific hybridization. Lesions with the histologic characteristics of condyloma were excluded from the study. Human papillomavirus DNA sequences were found in 24 (42%) of 57 seborrheic keratosis-like lesions from the genital region. No human papillomavirus DNA was detected in 27 control specimens that represented a variety of other processes occurring in this area. We conclude that human papillomavirus infection cannot be excluded in genital seborrheic keratoses.
The feasibility of detecting herpesvirus DNA in paraffin sections of routinely fixed and processed human necropsy brains by use of the polymerase chain reaction (PCR) was assessed. A 110 bp segment of the thymidine kinase gene of herpes simplex virus type 1 (HSV1) could readily be amplified in sections from the brains of six patients with acute HSV1 encephalitis but not from those of six patients with other neurological diseases, including varicella-zoster encephalitis and herpes simplex virus type 2 encephalitis. Primers suitable for amplifying c-myc were included in the PCRs for assessment of DNA preservation. This was found to be adequate to allow amplification of c-myc DNA in sections from all of the brains studied. The PCR provides a simple and specific means of detecting HSV1 DNA in routinely processed necropsy material.
Herpes-simplex-virus (HSV) DNA in cerebrospinal fluid was amplified by use of the polymerase chain reaction and identified by hybridisation to a specific oligonucleotide probe. Specimens of cerebrospinal fluid (CSF) from 4 of 4 patients with herpes simplex encephalitis were positive for HSV DNA, whereas CSF specimens from 6 patients with other central-nervous-system infections were negative. This technique may expedite diagnosis of herpes simplex encephalitis.
Laboratory tests for herpetic infections can be divided into (1) morphologic, (2) immunomorphologic, (3) serologic, and (4) virologic. Tzanck smears are easy to do, inexpensive, and compare favorably with cultures and immunofluorescence tests for specificity and sensitivity, but they require considerable experience to interpret accurately and they cannot differentiate between herpes type 1, herpes type 2, and varicella zoster infections. Biopsies are useful when clues to a diagnosis are being sought. Peroxidase-antiperoxidase and avidin-biotin tests present technical difficulties but interpretational difficulties are low and the results are available in a few hours. They can distinguish between herpes type 1, herpes type 2, and varicella zoster virus, as can immunofluorescence using monoclonal antibodies. Serologic tests are used primarily to distinguish between primary and recurrent herpes simplex infections. Virus isolation in tissue cultures is the gold standard for identifying herpes simplex virus but it is not 100% specific or 100% sensitive. Restriction endonuclease analysis identifies types and strains of virus by their deoxyribonucleic acid composition and it is very useful in epidemiologic studies. Ability to find virus by whatever method is influenced by the stage of the lesion. As lesions age, less infectivity and antigen result in less sensitivity of the tests.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme,
isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed
at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments
up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule
present only once in a sample of 10(5) cells.
The association between erythema multiforme (EM) and herpes simplex virus (HSV) infection has long been appreciated, although the exact role which HSV may play in the pathogenesis of this herpes-associated EM (HAEM), is unknown. Previous studies have suggested, but not definitively demonstrated, the presence of HSV in lesions of HAEM. The presence of HSV would support the hypothesis that an immune-mediated response directed against HSV-specific antigens in the skin is central to lesion development in HAEM. The purpose of this study was to examine lesions of EM for the presence of HSV DNA by using the polymerase chain reaction (PCR). In addition, in situ hybridization using an HSV-specific RNA probe was performed to further localize the HSV nucleic acids within the skin. DNA was extracted from formalin-fixed, paraffin-embedded specimens of cutaneous lesions of HAEM and also from EM for which no precipitating factor could be documented, otherwise known as idiopathic EM (IPEM). DNA from lesions of bullous pemphigoid served as a negative control. Using PCR to specifically amplify HSV sequences which might be present, and then performing Southern analysis, we demonstrated HSV DNA in 9/13 HAEM and 6/9 IPEM biopsies. No HSV was detected in six lesions of bullous pemphigoid. In situ hybridization of three cutaneous HAEM lesions using an 35S-labeled HSV-specific RNA probe localized the HSV nucleic acids predominantly to the epidermis. Three biopsies of chronic dermatitis, used as negative controls, did not demonstrate this specific hybridization. These findings confirm the presence of HSV in lesions of HAEM and are consistent with the concept of an HSV-specific immune-mediated pathogenesis for this disease. In addition, most cases of IPEM appear to be herpes associated despite the absence of clinically apparent HSV infection.
By means of a selective DNA amplification technique called polymerase chain reaction, proviral sequences of the human immunodeficiency
virus (HIV-1) were identified directly in DNA isolated from peripheral blood mononuclear cells (PBMCs) of persons seropositive
but not in DNA isolated from PBMCs of persons seronegative for the virus. Primer pairs from multiple regions of the HIV-1
genome were used to achieve maximum sensitivity of provirus detection. HIV-1 sequences were detected in 100% of DNA specimens
from seropositive, homosexual men from whom the virus was isolated by coculture, but in none of the DNA specimens from a control
group of seronegative, virus culture-negative persons. However, HIV-1 sequences were detected in 64% of DNA specimens from
seropositive, virus culture-negative homosexual men. This method of DNA amplification made it possible to obtain results within
3 days, whereas virus isolation takes up to 3 to 4 weeks. The method may therefore be used to complement or replace virus
isolation as a routine means of determining HIV-1 infection.