Quantitative analysis of cell-free Epstein-Barr virus DNA in the plasma of patients with peripheral T-cell and NK-cell lymphomas and peripheral T-cell proliferative diseases

BACKGROUND: The level of circulating EBV DNA is a prognostic marker in patients with some EBV-associated malignant diseases. OBJECTIVES: To investigate the presence and nature of Epstein-Barr virus (EBV) DNA in the plasma and to evaluate the correlation of plasma concentrations of EBV DNA with the EBV genomic status in peripheral blood T-cells and neoplastic cells and with the clinical outcome of patients with peripheral T-cell and NK-cell lymphomas (PTCL) and peripheral T-cell proliferative diseases (PTPD). STUDY DESIGN: EBV DNA in the plasma of 45 patients and 45 controls was measured using real-time PCR. The presence of the EBV genome in the isolated peripheral blood lymphocytes (CD3+ and CD3- cells) was analysed by PCR. Detection of EBV-encoded early RNA (EBER) in corresponding tumor tissues was carried out using in situ hybridization. DNase I digestion was applied to plasma samples to detect naked EBV DNA. RESULTS: Cell-free EBV DNA was detected in 32/38 (84%) of PTCL patients and 5/7 (71%) of PTPD patients, but not in the controls. Patients with EBV genome in peripheral blood CD3+ cells and EBV genome (EBER) in the tumor cells, compared to those without these findings, had significantly higher plasma EBV DNA levels. The majority of circulating EBV DNA molecules was naked form. The plasma EBV DNA levels were not related to survival. CONCLUSIONS: The concentration of EBV DNA in the plasma was not a prognostic marker in PTCL and PTPD patients.     

Rapid,sensitive, specific,and quantitative detection of human T-cell leukemia virus type 1 sequence in peripheral blood mononuclear cells by an improved polymerase chain reaction method with nested primers

Improving on the nested double polymerase chain reaction (PCR) described previously, we have developed a new two-step PCR (TS-PCR) method for detecting more specifically the human T-cell leukemia virus type 1 (HTLV-1) proviral sequences in peripheral blood mononuclear cells (PBMC). In our TS-PCR method, the point of modification is to use optimal concentrations of primers in the first amplification step in the range of 0.01–0.025 µM. This increases sensitivity and specificity enough to detect from 1 to 10⁵ copies of template DNA without radioisotopes. This method is rapid because of completion in 1 day and is also applicable for quantitative detection of clinical specimens. The data show that the quantitative detection of HTLV-1 proviral sequences by this method correlates with the anti-HTLV-1 antibody titers from serologic analysis of seropositive healthy carriers. Moreover, the TS-PCR method using each specific primer was also attempted for successful detection of other viral genomes; therefore, the principle of this method is widely suitable for routine detection of genomes in the basic and clinical microbiological fields.     

Polymerase chain reaction in the diagnosis of T-cell lymphoma in paraffin-embedded bone marrow biopsies: a comparative study

AIMS: In routine histological analysis of bone marrow biopsies, the distinction between reactive T-cell infiltrates and T-cell lymphoma can be difficult, even with the use of extensive immunohistochemistry. The aim of this study was to evaluate the diagnostic contribution of TCR-gamma gene rearrangement analysed by PCR. METHODS and RESULTS: The samples studied consisted of 46 paraffin-embedded bone marrow biopsies (diagnosis, staging and follow-up) from 26 patients with T-cell lymphoma. The bone marrow biopsies were categorized into three groups according to the morphological and immunohistochemical results. Group 1, positive for T-cell lymphoma (24 bone marrow biopsies), group 2, suspicion of T-cell lymphoma (15 bone marrow biopsies) and group 3, negative for T-cell lymphoma (seven bone marrow biopsies). DNA could be amplified in 45/46 bone marrow biopsies (98%). Clonal rearrangement was detected in 30/45 bone marrow biopsies tested (67%) including 15/24 bone marrow biopsies (62.5%) of group 1, 11/14 (78.5%) of group 2 and 4/7 (57%) of group 3. In total, PCR analysis supported a diagnosis of T-cell lymphoma in 15/45 bone marrow biopsies (33%), in which histological and/or immunohistochemical examination provided inconclusive evidence of malignancy. CONCLUSIONS: TCR-gamma PCR is a complementary tool for the assessment of T-cell lymphoma in bone marrow biopsies. Optimal evaluation of bone marrow biopsies requires an integrative approach of all available results from morphology, immunohistochemistry, molecular biology and clinical data.     

Vertical transmission of human T-cell leukemia virus type I (HTLV-I): Detection of proviral DNA in HTLV-I carrier gravida

The seroprevalence rate of human T-cell leukemia virus type I (HTLV-I) in pregnant women in the Osaka district was determined by enzyme-linked immunosorbent assay and Western blot analysis. Twenty-one (1.0%) of 2192 samples tested were positive for both assays and the seropositive parturients were found to be integrated with HTLV-I proviral DNA in their mononuclear cells by a DNA dot blot hybridization assay using HTLV-I DNA probe or by a selective DNA amplification technique using the polymerase chain reaction (PCR). On the other hand, proviral DNA was not detected in cord blood of the neonates born to the carrier mothers, indicating that transplacental infection of HTLV-I during pregnancy could be excluded. The results support the hypothesis that postpartum infection via breast milk plays a significant role among the possible perinatal transmission routes.     

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.     

Radioimmunoassay and enzyme-linked immunoassay of antibodies to the core protein (P24) of human T-lymphotropic virus (HTLV III)

Human T-cell lymphotropic viruses designated HTLV III or LAV are considered to represent the causative agent(s) of the acquired immunodeficiency syndrome (AIDS). Individuals who have been infected with these viruses may generally be identified on the basis of a positive serological test for antibodies against the protein components of these viruses. Purified viruses or viral proteins have been utilized for developing such tests. Since AIDS may be transmitted by blood transfusion and by blood products, screening of donors for antibodies to HTLV III/LAV has become a necessity. Such screening may be facilitated by the application of assays based on the use of crude virus-infected tissue culture media avoiding elaborate, expensive and potentially hazardous virus purification steps. Serum specimens were mixed with an appropriate dilution of an HTLV III-infected tissue culture-derived fraction, obtained by precipitation with polyethylene glycol 6000 and treatment with Tween 80 and tri-n-butylphosphate (to disrupt virus particles), and incubated with polystyrene beads coated with antibodies to HTLV III/LAV (anti-HTLV III). Subsequently, washed beads were incubated with either 125I- or beta-lactamase-labeled anti-HTLV III. The radioactivity or enzymatic activity associated with the beads was proportionate to the quantity of HTLV III antigen originally added to the beads. The presence of anti-HTLV III in serum specimens resulted in decreased antigen binding and thus in decreased radioactivity or diminished beta-lactamase activity associated with the beads. The test was specific for antibodies to the approximately equal to 24 kDa core protein of HTLV III. The prevalence of these antibodies (given in parentheses) in distinct populations was as follows: random blood donors (0.33%); hemophiliacs (36.4%); random homosexual males (25.1%); homosexual males preselected on the basis of positive markers for infection with hepatitis B virus (50%); and those with persistent lymphadenopathy (70%).     

Detection of herpes simplex virus type 1 DNA in bilateral human trigeminal ganglia and optic nerves by polymerase chain reaction

Herpes simplex virus type 1 (HSV-1) DNA was extracted from human trigeminal ganglia of 121 cadavers aged between 3 months and 94 years, and its PCR amplification was performed for the RL2 HSV-1 sequence using two pairs of primers. The HSV-1 DNA was detected in 74 of 121 patients (61%); 70/74 bilaterally, 3/74 only on the left side, and 1/74 only on the right side. Although the PCR-positive rate was significantly higher in advanced age, the correlation between the PCR-positive rate and gender was unclear. Additionally, HSV-1 DNA was not detected in any of the 50 optic nerve samples.     

Evaluation of clonal immunoglobulin heavy chain rearrangements in Hodgkin's disease using the polymerase chain reaction (PCR)

We have correlated histologic type of Hodgkin's disease, degree of Hodgkin and Reed-Sternberg cell infiltration, percentage of Hodgkin and Reed-Sternberg cell positivity for latent membrane protein, immunophenotype of Hodgkin and Reed-Sternberg cells, and immunoglobulin heavy chain (IgH) gene rearrangements detected by polymerase chain reaction (PCR) in 56 unselected Hodgkin's disease cases. Two protocols were used for amplification of IgH gene using Fr2 or Fr3 V-region primers, in conjunction with nested primers directed to the JH region. PCR products were run on polyacrylamide gels. Immunohistochemical studies were performed on paraffin sections using monoclonal antibodies for CD20 and latent membrane protein, and polyclonal antibody to CD3. Using both primer combinations we detected a definitive clonal band in 23.2% of the Hodgkin's disease cases. Clonal IgH rearrangements were detected in 23.6% of nodular sclerosis type and in 28.5% of mixed cellularity type. Using a highly sensitive method such as PCR, more than 20% of unselected cases of Hodgkin's disease were found to contain B-cell clonal proliferations, but there was no correlation between histological and immunological parameters and molecular analysis results.     

Detection of measles virus genomic sequences in SSPE brain tissue by the polymerase chain reaction

The polymerase chain reaction (PCR) was modified to detect RNA genomic sequences by generating cDNA copies of these sequences as a preliminary step. Oligonucleotide primer pairs complementary to sequences in each of the five major structural protein genes of the measles virus (nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, and hemagglutinin protein) were synthesized. PCR products were tentatively identified by visualization of bands of the appropriate size by ethidium bromide staining after gel electrophoresis, and identity was confirmed by subsequent restriction enzyme cleavage of the products at predetermined sites to yield fragments of predicted size. This method successfully amplified 400-500 base regions from each of these five genes in RNA extracts of wild measles virus cultured in Vero cells and in RNA extracted from most of the SSPE brain tissues tested, but not in RNA from any control brain tissues. Measles virus genome was detected in SSPE brain tissues stored frozen for as long as 27 years and formalin-fixed paraffin-embedded subacute sclerosing panencephalitis (SSPE) brain tissues as old as 9 years. This method provides a simple, rapid and highly sensitive means of detecting and identifying sequences of RNA genomes by PCR. The success of this method in detecting measles virus in SSPE brain tissue suggests that PCR is appropriate to investigate the possible presence of RNA viruses in other neurological disorders of unknown etiology.     

The polymerase chain reaction in the diagnosis of early mycosis fungoides

The earliest or patch stage of mycosis fungoides may present diagnostic difficulty both clinically and pathologically. The present study of the polymerase chain reaction (PCR) as a diagnostic tool in early mycosis fungoides was therefore undertaken, using a rapid PCR method for the detection of γ- and β-chain T-cell receptor (TCR) gene rearrangements in routine formalin-fixed, paraffin-embedded histological sections. Forty-two biopsies were studied from 26 patients with mycosis fungoides. Twenty-three skin biopsies with a clinicopathological diagnosis of early, or patch stage, mycosis fungoides were investigated. Of these, 18 (78 per cent) showed TCR-γ or both β- and γ-chain TCR gene rearrangements. TCR gene rearrangements were shown in seven of the 14 plaque stage lesions (50 per cent) and also in the single case of tumour stage disease. Where gene rearrangements were identified, these were identical in all biopsies from an individual patient, irrespective of the site of the lesion, the disease stage, or the time lapse between the biopsies. The PCR is therefore a highly sensitive technique, which can be performed on routine pathological material, in cases where the diagnosis of early mycosis fungoides cannot be made with certainty on conventional histopathological and immunohistochemical grounds. © 1997 John Wiley & Sons, Ltd.     

Detection of human herpesvirus 8 DNA sequences in peripheral blood mononuclear cells of children

Human herpesvirus 8 (HHV-8) DNA sequences were examined in peripheral blood mononuclear cell (PBMC) DNA samples of 56 children, 15 healthy adults, and 10 renal transplant patients by the polymerase chain reaction (PCR). The PCR amplification was carried out using the published KS330₂₃₃ primer pairs to amplify HHV-8 DNA sequences. The PCR-amplified products were confirmed by Southern blot hybridization with radiolabeled 233 bp HHV-8 DNA fragment, which was cloned and sequenced from the PCR-amplified product of Kaposi's sarcoma tissue. Six PCR-amplified product of four children and two renal transplant patients were cloned and sequenced. HHV-8 DNA sequences were detected in 36 of 56 (64%) normal children, in 12 of 15 (80%) healthy adults, and in all 10 renal transplant patients by Southern blot hybridization of PCR-amplified products. Six PCR-amplified products were confirmed by sequencing. These results suggest that HHV-8 infection is prevalent in the Japanese population with infection occurring in early childhood. J. Med. Virol. 53:81–84, 1997. © 1997 Wiley-Liss, Inc.     

Detection of enteroviral infection in myocardial tissues by polymerase chain reaction (PCR)

Objective: To determine if enteroviral infection is linked to myocarditis and dilated cardiomyopathy. Enteroviruses, especially coxsackieviruses, appear to be the most common agents of viral myocarditis.
Methods: We collected 53 endomyocardial biopsies and two autopsy specimens from 41 patients affected by myocarditis or dilated cardiomyopathy. The patients were diagnosed clinically, hemodynamically, virologically and histologically (Dallas classification). We tested for the presence of enteroviral sequences by PCR, using 5' non-coding (coxsackievirus B3, CB3, map position 450–474, 584–603) derived primers. Specificity was confirmed using the Southern blot. We used a fraction of CB3 acutely infected mouse myocardial tissue as a control.
Results: We detected enteroviral sequences in four patients with active myocarditis, borderline myocarditis or cardiomyopathy. The patient with active myocarditis had shown neutralizing antibodies in serologic analysis for coxsackievirus B3 and B5.
Conclusions: The data support a weak link of enteroviral infection to human myocarditis and dilated cardiomyopathy, at least when using a PCR assay on biopsies.     

Identification and typing of molluscum contagiosum virus in clinical specimens by polymerase chain reaction

A polymerase chain reaction (PCR) which enables the detection of molluscum contagiosum virus (MCV) genomes in either fresh or formalin-fixed clinical specimens is described. The primers used were designed to amplify a 167 bp region of the 3.8kbp HindIII fragment K of the MCV 1 genome. The ability of this PCR to detect three common MCV types (1, 1v and 2) in clini-cal specimens was confirmed using frozen extracts from 75 molluscum lesions, and digests of single sections of 11 formalin-fixed, paraf-fin-embedded lesions; all of which had been previously typed by Southern hybridisation. In addition, 2 specimens previously negative by hybridisation were shown to be positive for MCV DNA by PCR. Confirmation of the identity of the PCR products and distinction between the two major MCV types (MCV 1/1v versus MCV 2) was achieved by comparison of the results of cleavage with the restriction endonucleases HhaI and SacI. Sequencing of the PCR products revealed complete homology between MCV 1 and 1v, but minor nucleotide variations between MCV 1/1v and MCV 2 were identified. As well as providing a highly sensitive means of diagnosis, the technique may also prove useful for investigations into the pathogenesis, epidemiology and natural history of molluscum contagiosum infection. J. Med. Virol. 53:205–211, 1997. © 1997 Wiley-Liss, Inc.     

The use of modified primer competitors to enhance yields and specificity of HLA class I amplification by polymerase chain reaction (PCR)

Various PCR based techniques have been developed for genomic HLA typing. The fidelity of these techniques is highly dependent upon the specificity of the primers for the given HLA locus. Due to the high degree of homology between HLA class I loci, few primer sites that selectively amplify genes at a given HLA class I locus may be identified. To avoid coamplification of homologous loci, we designed and applied primer competitors for PCR amplification of HLA-A, -B and -C loci. Primer competitors identical to the 3' end of the specific primers and completely degenerate in the 5' end were designed and titrated into the respective HLA-locus PCR mixtures. We found that inclusion of primer competitors in the PCR reaction increased the specificity and yields of HLA class I amplifications, in particular when crude DNA preparation was used as template. This was particularly true for DNA preparations of low quality. The method described here may be useful for various protocols for downstream genomic typing of HLA-A, -B and -C alleles. In particular the method is useful when DNA is in scarce supply (i.e., for extensive PCR based allelic typing) or when high yields and locus specificity of amplicons are needed (i.e., sequencing-based typing).     

Indeterminate western blot patterns in a cohort of individuals at high risk for human immunodeficiency virus (HIV-1) exposure

Our objective was to map serial patterns of Western blot reactivity over time of a cohort of initially ELISA-negative, Western blot-indeterminate individuals from a high-risk group and to determine if these individuals were at increased risk of harboring occult HIV-1 infection. A 2-year prospective study used serial ELISA, two types of Western blot, immunologic profiles, HIV-1 culture, and analysis by polymerase chain reaction. Subjects were 20 ELISA-negative, Western blot indeterminate homosexual volunteers and 20 matched seronegative controls. Results showed that 19 of 20 study subjects completed a mean of 17.0 months of clinical and laboratory follow-up. Reactivities with p24 and/or with p55 were the two most commonly observed Western blot patterns, occurring in 70% of individuals. Specific Western blot reactivity was dependent upon the particular immunoblot preparation being used and varied considerably on a longitudinal basis. No individual pattern appeared predictive of an increased likelihood of subsequent seroconversion to HIV-1 relative to controls. By all other criteria including polymerase chain reaction analysis, samples from 17 of 19 individuals remained negative for HIV-1 at each time point. Two individuals evolved from an indeterminate to a positive Western blot and, simultaneously, from a negative to a positive polymerase chain reaction analysis, during follow-up. Our conclusions were as follows. ELISA-negative, Western blot-indeterminate individuals from a high-risk group show marked variability in immunoblot findings over time, and these patterns do not appear predictive of an increased likelihood of infection. Polymerase chain reaction analysis is clearly of value in providing confirmation of the low probability of infection in this group, although in patients who do become infected, detection by this test may not always precede diagnosis by serologic methods.     

非霍奇金淋巴瘤EB病毒特异DNA序列的检测

目的　探讨EB病毒 (EBV)与中国南方地区非霍奇金淋巴瘤 (NHL)的相关性 ,以及EBV与不同类型NHL的关系。方法　采用PCR技术 ,检测 2 0 6例石蜡包埋的NHL组织及 2 3例反应性增生的淋巴组织中的EBV特异DNA序列。结果　(1 ) 2 0 6例NHL组织中 ,94例PCR扩增出EBV特异的DNA序列 ,阳性率 4 5 6 % ;对照组反应性增生的淋巴组织 2 3例中 ,5例阳性 ,阳性率 2 1 7% ;两者差异有显著性 (P <0 0 5 )。 (2 ) 2 0 6例NHL中B NHL 1 2 8例 ,EBV阳性者 4 8例 ,阳性率 37 5 % ;T NHL 78例 ,EBV阳性者 4 6例 ,阳性率 5 9 0 %。两者差异有显著性 (P <0 0 5 )。结论　EBV与中国南方地区NHL ,特别是T NHL有一定的相关性