Detection of hepatitis "C" virus in formalin-fixed liver tissue by nested polymerase chain reaction.

Interpretation of antibody to hepatitis C virus (HCV) in patients with liver disease is difficult due to false-positive reactivity in some conditions. To evaluate the feasibility of HCV in archival material, HCV was sought in formalin-fixed, paraffin-embedded liver biopsy specimens. Nested polymerase chain reaction was used to detect hepatitis C virus in formalin-fixed, paraffin-embedded liver biopsy specimens after total RNA was extracted from tissue by proteinase K digestion and phenol/chloroform purification. The relative efficiency of amplification of HCV RNA from formalin-fixed material was estimated semiquantitatively by serial dilution of cDNA synthesised from RNA extracted from fresh and formalin-fixed sections from the same liver. Although HCV RNA could be detected in formalin-fixed liver tissue by nested PCR in 5/5 cases in which HCV was detected in serum, amplification was approximately 5-fold less efficient than when HCV was amplified from fresh tissue. Nevertheless, nested PCR of HCV from formalin-fixed liver tissue represents a useful technique in addressing some important questions related to the pathogenesis of liver disease.     

Demonstration of coxsackie virus RNA in formalin-fixed tissue sections from childhood myocarditis cases by in situ hybridization and the polymerase chain reaction

This is a combined study using in situ hybridization and the polymerase chain reaction to investigate the presence of Coxsackie virus RNA in formalin-fixed tissue from cases of childhood myocarditis. Of the ten cases studied, two were positive by both methods. The virus RNA was predominantly located in areas showing an inflammatory cell infiltrate and myofibre necrosis. These findings suggest that direct lytic infection of myocytes by virus is responsible for myocaiditis in these cases, rather than an autoimmune process, which has been suggested previously. The findings in one case, where the virus showed a marked sub-endocardial distribution, may have implications for the aetiology of endocardial fibroelastosis by confirming a viral tropism for this location. The techniques used in this study are easily repeatable and can be directly applied to look for viruses in a number of other diseases where a viral aetiology is suspected.     

石蜡包埋肝组织中丙型肝炎病毒的原位PCR检测

以丙型肝炎病毒（HCV）为研究对象探讨原位PCR这一新技术。用原位PCR和原位杂交检测石蜡包理肝组织中的HCV。7例肝硬变及8例肝细胞癌痛旁组织HCV的检出率分别为86％（6／7例）和50％（4／8例），明显高于同组原位杂交的42％（3／7例）和12．5％（1／8例），并且显示了良好的特异性。研究发现HCV主要分布于肝细胞浆中，并且在胆小管上皮细胞及单核细胞中也出现阳性信号。我们的研究表明原位PCR是一项敏感、特异的方法，它的广泛应用不仅可以提高丙型肝炎的诊断率，同时有助于其分子病理学的研究。     

聚合酶链反应标记轮状病毒地高辛素探针的初步应用

目的为探讨聚合酶链反应（ＰＣＲ）技术标记的地高辛素（ＤＩＧ）探针的特异性和敏感性。方法用聚合酶链反应技术制备地高辛素标记的人类轮状病毒（ＨＲＶ）ｃＤＮＡ探针，经ｃＤＮＡ－ＲＮＡ斑点杂交。结果该探针具ＨＲＶ特异性，可检出１０ｐｇ的ＲＮＡ。应用该项技术检测了１２０份婴幼儿腹泻粪样标本，其阳性率为６５％，明显高于ＰＡＧＥ方法（４９．１％）的阳性率。结论ＰＣＲ方法直接制备地高辛素标记的ｃＤＮＡ探针方便、快速、特异性好、标记率高。     

肝细胞肝癌组织中丙型肝炎病毒核心区基因及其表达 …

目的 采用多种方法检测丙型肝炎病毒（ＨＣＶ）核心区基因和表达产物在肝细胞肝癌（ＨＣＣ）组织中的分布及其在ＨＣＣ发生和发展中的作用。方法 对３９例ＨＣＣ患者进行免疫组化和原位杂匀的检测。ＩＳ－ＲＴ－ＰＣＲ法用于检测并定位ＨＣＶ－ＲＮＡ。微切组织的ＲＴ－ＰＣＲ法分别检测癌与癌旁组织中的ＨＣＶ－ＲＮＡ,以便能够控制扩增产物的来源。同时还进行 血清学ＥＬＩＳＡ和ＲＴ－ＰＣＲ的检测。结果 血清ＥＬＩＳＡ的阳     

Y-derived sequence detected in minute chromosomes by polymerase chain reaction and in situ hybridization

A 10-year-old girl and a 10-month-old girl, both with ambiguous genitalia, were found to have 45,X/46,X,mar and 45,X/46,X,r(?) mosaicism. The marker chromosomes in both girls were very small. Polymerase chain reaction, with synthetic oligonucleotide primers from Y-specific DNA sequences pY-80 and pY53.3 containing the sex-determining region Y(SRY), proved the marker chromosomes to contain the Y short arm material. In situ hybridization with probe pY-80 confirmed that the marker chromosomes included the Y short arms. These findings, together with ambiguous genitalia in the girls, indicate that the marker chromosomes include the testis-determining factor gene.     

Detection of herpes simplex virus DNA by in situ hybridization technique and polymerase chain reaction in Papanicolaou-stained cervicovaginal smears

Papanicolaou-stained cervicovaginal smears from six patients with herpes simplex virus (HSV) infection were destained and reprocessed by means of in situ hybridization (ISH) technique to demonstrate the presence of HSV DNA utilizing biotinylated probe. Positive results were obtained in all six cases with intense staining signal for the HSV DNA in the nuclei of cells having a ground-glass nuclear appearance as well as in multinucleated giant cells. Furthermore, a hybridization signal was also noted in smears that had been prepared as much as 3 yr previously. HSV type 2-specific antigen was confirmed in six destained smears by means of immunoperoxidase staining. Moreover, polymerase chain reaction (PCR) was also performed for four patients from Pap-destained cervicovaginal smears. Amplified HSV DNA was detected in all four cases as 106 basepair PCR products by polyacrylamide gel electrophoresis. The combined use of cytology and the ISH technique and PCR amplification was of great value for the rapid cytodiagnosis of genital infection of HSV.     

Determination of hepatitis C virus genotypes by melting-curve analysis of quantitative polymerase chain reaction products

Hepatitis C virus (HCV) is the major causative agent of non-A and non-B viral hepatitis. Factors associated with disease progression following HCV infection include the viral genotype, the patient's alcohol consumption and viral load. In this study, the COBAS AMPLICOR HCV MONITOR test, a commercially available quantitative assay for HCV RNA, was used for HCV genotyping analysis. Amplification products obtained from 100 HCV-positive cases were subjected to real-time polymerase chain reaction (PCR) typing using a single pair of fluorescence resonance energy transfer (FRET) probes and melting-curve analysis. Of 100 samples tested, two inhibited the PCR, two samples yielded discrepancies between our results and the reference laboratory results and the remaining samples provided correct typing. The present report suggests that HCV genotypes can be determined rapidly with FRET probes directly from COBAS AMPLICOR MONITOR test PCR products.     

Failure to detect hepatitis C virus (HCV) genome by polymerase chain reaction in human anti-HCV-positive intravenous immunoglobulins

The prevalence of HCV antibodies was determined by a second-generation ELISA and a four-antigen rccombinant immunoblot assay in nine intravenous immunoglobulin (IVIG) preparations commercially available in Italy. In addition, the clinical safety of six of them was ascertained by polymerase chain reaction (PCR) of HCV RNA and a prospective study in 14 patients with immunodeficiency disorders. Results indicated that all IVIG preparations were anti-HCV-positive. However, there were substantial variations in their anti-HCV antibody titres. The preparations retained IgG subelass reactivilies to HCV-associated structural (C223) and non-structural (C33c, C100-3) proteins. Our sensitive and specific PCR assay was unable to detect HCV RNA in the six preparations tested. Clinical surveillance of IVIG-trcated patients prospectivcly evaluated over a mean period of 83 months failed to delect clinical and/or biochemical evidence of hepatitis.     

Distribution of hepatitis B virus in the liver of chronic hepatitis C patients with occult hepatitis B virus infection

Although occult hepatitis B virus (HBV) infection (HBV-DNA in serum in the absence of hepatitis B surface antigen [HBsAg]) is common in chronic hepatitis C, its characteristics are not well known. In this work, the presence of HBV-DNA (by polymerase chain reaction; PCR) and its distribution (by in situ hybridization) in liver biopsies and peripheral blood mononuclear cells (PBMCs) from 32 patients with chronic hepatitis C and occult HBV infection and in 20 HBsAg chronic carriers were determined. The results showed that serum HBV-DNA levels were statistically lower (P = 0.001) in patients with occult HBV infection than in HBsAg chronic carriers. The HBV infection pattern in liver cells was identical between patients with occult HBV infection and those with chronic hepatitis B. However, the mean percentage of HBV-infected hepatocytes was significantly lower (P = 0.001) in patients with occult HBV infection (5 +/- 4.44%) than in HBsAg chronic carriers (17.99 +/- 11.58%). All patients with chronic hepatitis B have HBV-DNA in their PBMCs while this occurred in 50% of the cases with occult HBV infection. In conclusion, patients with occult HBV infection have a low number of HBV-infected hepatocytes and this fact could explain the lack of HBsAg detection and low viremia levels found in these cases.     

Unsuccessful effort to detect human papillomavirus DNA in urinary bladder cancers by the polymerase chain reaction and in situ hybridization

The association of human papillomavirus (HPV) with urinary bladder carcinogenesis is now a controversial issue. In order to certify the presence of HPV DNA in urinary bladder cancers, the polymerase chain reaction (PCR) using five primer sets for detecting various HPV types was used in this study as weli as in sltu hybridizetion (ISH) for HPV 16 and 18 detection. In the PCR study of 93 DNA samples extracted from formalin-fixed and paraffin-ernbedded urinary bladder cancem, no HPV DNA was detected in these tumor samples. The ISH study was also performed on the same tumor samples, but failed to demonstrate any HPV 16-or 18-positive signals in ail except one of the tumor samples. However, the FCR failed to demonstrate HPV 16 DNA even in the bladder cancer positive for HPV 16 DNA by the ISH. This ISH technique was able to demonstrate HPV 16 and 18 DNA in eight of 13 paraffinembedded cervical cancers, in which HPV 16 or 18 DNA had already been detected by the PCR. Our HPV study using PCR and ISH revealed that the HPV status of urinary bladder carcinomas was far different from that of cervical cancers.     

原位PCR技术检测石蜡包埋脑组织中人巨细胞病毒DNA

应用原位聚合酶链反应（ＩＳＰＣＲ）技术检测了２５例尸检畸形胎儿石蜡包埋脑组织中人巨细胞病毒（ＨＣＭＶ）ＤＮＡ，并与普通ＰＣＲ及原位杂交（ＩＳＨ）进行了比较。ＩＳＰＣＲ、ＰＣＲ及ＩＳＨ检测阳性率分别为４４％，３６％及２０％。与ＩＳＨ相比较，ＩＳＰＣＲ不仅检出阳性率高，而且信号强度增强。研究结果提示，ＩＳ－ＰＣＲ是诊断ＨＣＭＶ感染的快速、敏感、特异的实用方法。     

间接法原位PCR检测喉鳞癌组织HPV感染

建立稳定的原位ＰＣＲ方法，并探讨ＨＰＶ感染与喉鳞癌发生的关系。方法采用免疫组化、原位杂变、ＰＣＲ和间接原位ＰＣＲ技术，检测了５０例喉鳞癌中的ＨＰＶ感染情况。结果免疫组化衣壳抗原阳性者６例（１２％），原位杂交阳性者１３例（２６％），ＰＣＲ阳性者１０例（２０％），原位ＰＣＲ阳性者１７例（３４％），综合上述方法的检出率为４２％（２１例）。结论ＨＰＶ感染与喉癌有着明显的关系，间接原位ＰＣＲ在检测ＨＰＶ感染     

Comparison of the hypervariable region of hepatitis C virus genomes in plasma and liver

Nucleotide sequences of the hypervariable region of hepatitis C virus genomes obtained from plasma change rapidly during the course of in fection and are believed to play a part in immunological escape and consequently in the development of persistent infection. It is not known, however, whether these changes also occur in the liver. To clarify this aspect, RNA was extracted from the plasma and liver tissue of eight patients with chronic hepatitis C. After cDNA synthesis, DNA fragments that included the hypervariable region were amplified by the polymerase chain reaction. Consensus nucleotide sequences were determined directly from the polymerase chain reaction products by the dideoxy chain termination method. The diversity of the hypervariable region was analyzed further by the polymerase chain reaction-single strand conformation polymorphism analysis. Consensus nucleotide sequences of the hypervariable region were identical between the plasma and the liver in each patient. The polymerase chain reaction-single strand conformation polymorphism analysis showed multiple DNA bands that represented different hypervariable region sequences. Comparison of the single strand conformation polymorphism patterns revealed that the number, the mobility, and the density of bands were the same between the plasma and the liver. It is concluded that the population and the diversity of hepatitis C virus quasispecies as detected by the hypervariable region sequence are the same between the plasma and the liver despite rapid mutations, indicating that rapid changes in the population of hepatitis C virus quasispecies also occur in the liver. © 1995 Wiley-Liss, Inc.     

In situ hybridisation and in situ polymerase chain reaction detection of parvovirus B19 DNA within cells

Modification of an in situ polymerase chain reaction (ISPCR) technique is described for the detection of B19 parvovirus infection. Specific amplification of B19 DNA inside fixed cells was followed by hybridisation with a digoxigenin-labelled probe and then visualised by immunochemical reaction. The assay had higher sensitivity compared to direct in situ hybridisation and still allowed cellular localisation and characterisation of infected cells. This assay can be used as a confirmatory method for PCR in tissues and will allow further identification of tissues permissive for B19 parvovirus infection.     

Quantitative detection of hepatitis C virus RNA in urine of patients with chronic hepatitis C using a novel real-time PCR assay

Hepatitis C virus (HCV) RNA can be detected in body fluids such as urine. However, because of deficiencies in established isolation and detection methods, the actual prevalence and form of HCV RNA in the urine of patients with hepatitis C remain unclear. To more sensitively and accurately measure urine HCV RNA levels, a novel real-time PCR assay with a modified isolation method and short amplicon designed for short HCV RNA fragments was developed in this study. The limit of detection, precision, linearity, and specificity of the assay was evaluated and demonstrated high-quality performance. The prevalence of HCV RNA in the urine of viremic patients infected with HCV was 60% (36/60), as determined by a 62-bp assay. The HCV RNA detection rate and concentration were much lower with a 157-bp assay, and were undetectable with 222- and 304-bp assays. With the 62-bp assay, patients with detectable urine HCV RNA had significantly higher plasma HCV RNA levels ( P < 0.001), and plasma and urine concentrations were significantly positively correlated ( R ² = 0.708, P < 0.001). The method not only increased the detection rate of urine HCV RNA but also revealed the presence of short HCV RNA fragments in urine, indicating that urine from CHC patients with normal kidney function should not be infectious. In addition, it raised the possibility of urinary HCV RNA as a potential noninvasive marker for therapeutic monitoring of patients with hepatitis C.     

Intrahepatic expression of hepatitis C virus antigens in chronic liver disease

Localization of hepatitis C virus (HCV) antigens was studied in fresh frozen and formalin-fixed, paraffinembedded liver tissue by immunoperoxidase using monoclonal antibodies to nucleocapsid protein and polyclonal human immunoglobulin G purified from plasma containing antibodies to structural and non-structural antigens of hepatitis C virus. The results observed using monoclonal antibody to HCV core were similar to those of polyclonal IgG against HCV antigens in the majority of cases and both correlated well with HCV status as defined by ‘nested’ polymerase chain reaction. HCV antigens were detected in both hepatocytes and mononuclear cells. Using polyclonal human IgG, a small proportion of biliary epithelial cells were also positive in 6/29 patients. In most of the specimens examined, relatively few cells (1–5 per cent) were found to be positive for HCV antigens. The cryostat sections, using polyclonal IgG against HCV antigens, exhibited greater immunohistochemical staining, suggesting that the fixation and processing of the tissue may be a major factor in the conservation and the outcome of HCV antigen(s) findings. However, the results using monoclonal antibodies may reflect the specificity of antigen expression.