The use of ELISA and nonradioactive DNA hybridization assays for the detection of human cytomegalovirus

A hybridization assay using a biotinylated DNA probe was compared to both ELISA and direct isolation methods for detecting human cytomegalovirus (HCMV). The biotin labeled HCMV AD 169 HindIII-O-DNA fragment was used in a dot-blot assay to screen for the presence of HCMV in 186 urine specimens obtained from kidney transplant patients. The biotinylated HCMV HindIII-O probe could detect 3 log10 TCID50 units of HCMV. Urine specimens were also examined for the presence of HCMV by either ELISA or direct isolation of virus in tissue culture. The HindIII-O fragment detected 12 of 20 culture positive samples (sensitivity, 60%). There were 5 samples which were probe positive and cell culture negative (specificity, 97%). The ELISA assay also detected 12 of 20 culture positive samples (sensitivity, 60%). Eight samples were ELISA positive, cell culture negative (specificity, 95%). Seven specimens were positive by all three criteria. Five specimens which were both ELISA positive and probe positive were cell culture negative. The ELISA positive, probe positive, culture negative specimens originated from patients who gave a culture positive specimen within 10 days of the original sample. The combination of probe and ELISA assays detected 16 of the 20 culture positive specimens (sensitivity, 80%). The combined use of biotinylated DNA probes and ELISA allows the detection of HCMV in urine specimens with good sensitivity and specificity.     

Polymerase chain reaction assay for detection of human cytomegalovirus.

Direct detection of human cytomegalovirus (HCMV) from clinical specimens was examined by using the polymerase chain reaction (PCR) for amplifying HCMV DNA. The efficiency of the amplification reaction was examined by using three different buffers and concentrations of deoxynucleotide triphosphates. The PCR assay was most efficient with a reaction mixture containing 17 mM ammonium sulfate, 67 mM Tris hydrochloride (pH 8.5), 7 mM MgCl2, 10 mM 2-mercaptoethanol, 170 micrograms of bovine serum albumin per ml, and each deoxynucleotide triphosphate at a final concentration of 1.5 mM. After 35 cycles of amplification, 0.15 fg of a plasmid containing the cloned target gene (corresponding to approximately six gene copies) was detected. The PCR assay correctly identified all of 24 clinical isolates of HCMV. Virus in urine specimens could be disrupted by heating at 93 degrees C for 30 min. The viral DNA was amplified directly from 5 microliters of preheated urine, with no further treatment before amplification. We tested the PCR assay on urine specimens from patients who had undergone renal transplantation that had been screened for the presence of HCMV by enzyme-linked immunosorbent assay, hybridization assay, and direct virus isolation. Specimens that were positive by one or more of these assays were screened by PCR. HCMV was consistently detected by PCR in all specimens that were positive by at least one other test. No cross-reactivity to other herpesviruses or MRC-5 cellular DNA was observed.     

Direct detection of the human papovavirus BK in urine of bone marrow transplant recipients: comparison of DNA hybridization with ELISA

Urine specimens from bone marrow transplant (BMT) recipients and from controls were directly tested for BK virus (BKV) DNA sequences by dot hybridization and for BKV antigen by a double-antibody indirect ELISA. A total of 158 specimens from 55 BMT patients (57 collected prior to or at the time of transplantation and 101 in the posttransplant period) and single urines from 125 control subjects were examined by both methods. A molecularly cloned, 32P-labelled BKV probe was hybridized with urine sediments that were spotted directly on nitrocellulose filters and denatured in situ. BKV DNA sequences were detected in 1 (1.8%) pretransplant and 22 (21.8%) posttransplant urines of BMT patients, and in none of control urines. In ELISA of urine supernatants, BKV antigen was detected in 1 (1.8%) pretransplant and 21 (20.8%) posttransplant urines of BMT patients and in 1 (0.8%) of the control urines. The results of the two tests correlated as follows: 16 urines were positive and 253 urines negative by both methods; seven specimens were positive by DNA hybridization only and seven were positive by ELISA alone. Virus excretion in urine was demonstrated in 20 (36.4%) patients by DNA hybridization, in 19 (34.5%) patients by ELISA, in 15 (27.3%) patients by both methods, and in 24 (44%) patients by at least one of the two tests.     

Detection of CMV in urine: comparison between DNA-DNA hybridization, virus isolation, and immunoelectron microscopy

Rapid detection of CMV-DNA in urine specimens by dot-blot hybridization was compared to conventional virus isolation and to virus identification using solid-phase immunoelectron microscopy (SPIEM). To detect viral DNA, 32P-labeled EcoR1 J fragment of CMV-DNA was used as a probe in the hybridization assay. In addition, DNA extracted from infected human embryo fibroblasts (amplified DNA) was also hybridized to the same probe. Urine specimens were obtained from 10 renal transplanted patients, seven premature infants, three family members, and five children suspected of CMV infection. CMV was isolated from 10 urine specimens and SPIEM detected viral particles in nine specimens. Ten positive samples were identified as such by hybridization with DNA extracted directly from urine specimens, while hybridization with amplified DNA yielded 17 positives. Only in one urine specimen, positive by virus isolation and SPIEM, DNA was not detected by the hybridization assays. Elevated IgG or IgM-specific antibodies were found in 10 patients. Hybridization with amplified DNA proved the most sensitive and relatively rapid assay, as compared with direct DNA detection in urine, tissue culture isolation, SPIEM, or serologic tests.     

Sensitive non-isotopic DNA hybridisation assay or immediate-early antigen detection for rapid identification of human cytomegalovirus in urine.

A sensitive non-radioactive DNA hybridisation assay employing digoxigenin-labelled probes was compared with immediate-early antigen detection and conventional virus isolation for the identification of human cytomegalovirus (HCMV) in 249 urine samples. Of 44 specimens yielding HCMV by virus isolation, more were positive by DNA hybridisation (32; 73%) than by immediate-early antigen detection (25; 52%) (P = 0.05). The specificity of the hybridisation assay in 45 apparently falsely positive specimens was supported by detection of HCMV DNA in 40 of these specimens using the polymerase chain reaction. Many urine specimens may thus contain large amounts of non-viable virus or free viral DNA. Evaluation of various protocols for the extraction and denaturation of virus DNA prior to hybridisation showed that proteinase K digestion with phenol/chloroform extraction was the most sensitive and reliable procedure. We conclude that the non-radioactive DNA hybridisation assay described is a potentially valuable routine diagnostic test.     

Development and application of a novel multiplex polymerase chain reaction for semi-quantitation of human Cytomegalovirus in clinical specimens

Human Cytomegalovirus (HCMV) is an important cause of morbidity and occasional mortality in immunocompromised patients. The aims of the study were to develop and apply a multiplex PCR for semi-quantitation of HCMV in clinical specimens, compare its efficiency with pp65 antigenemia assay and real-time PCR. A multiplex PCR combining the primers targeting three regions of the HCMV genome, viz. the morphological transforming region II (mtr II), UL-83 and glycoprotein O (gO) genes for the detection of the genome of HCMV was standardized with HCMV AD169 strain. This was evaluated against pp65 antigenemia assay by applying it on 70 peripheral blood specimens obtained from 70 post-renal transplant recipients. The multiplex PCR and a real-time PCR were prospectively applied to 31 clinical specimens from 29 immunocompromised patients. The multiplex PCR was specific for HCMV. The level of antigenemia and the copy number of the viral DNA as estimated by real-time PCR in the samples positive for all the three targets was significantly higher than in those that were positive for only one or two of the targets. The multiplex PCR provides a simple and effective means of quantifying HCMV in clinical specimens with efficiency equivalent to the pp65 antigenemia assay and real-time PCR.     

Cytomegalovirus DNA detection in sera from patients with active cytomegalovirus infections.

Using a specific and sensitive polymerase chain reaction method, we detected reliably the presence of human cytomegalovirus (HCMV) DNA directly in serum samples collected at an early stage of HCMV infection, even before immunoglobulin M (IgM) antibodies were measurable. HCMV DNA was detected in serum from all patients with active HCMV infection; in 91% of these patients, HCMV DNA was found in the acute-phase serum. In 13 of 44 patients, HCMV DNA was found in serum before HCMV-specific IgM. For four kidney transplant recipients, the occurrence of HCMV DNA in serum, virus isolation from urine and leukocytes, and HCMV IgG and IgM serology were determined. We found a correlation between HCMV DNA in serum and positive virus isolation from leukocytes. In three of five congenitally infected infants, HCMV DNA and HCMV IgM were detected in the same sample. Two other infants were HCMV DNA positive, although no HCMV IgM antibodies were measurable. HCMV was found in urine from these infants either by virus isolation or with the polymerase chain reaction. Serum from one of the 22 healthy HCMV-seropositive blood donors was HCMV polymerase chain reaction positive.     

Feline leukaemia virus proviral DNA detected by polymerase chain reaction in antigenaemic but non-viraemic (‘discordant’) cats

Summary. A nested polymerase chain reaction (PCR) was established to detect exogenous feline leukaemia virus (FeLV) proviral DNA in feline peripheral blood leukocytes (PBL). The assay detected a single copy of plasmid DNA of an infectious molecular clone of FeLV subgroup A in the sample by ethidium bromide staining in agarose gels. The utility of the nested PCR in the diagnosis of FeLV infections was compared with the detection of FeLV p27 antigen by enzyme-linked immunosorbent assay (ELISA) and virus isolation (VI). FeLV genomes were detected by PCR in all 4 samples that were positive by ELISA and VI but in none of 7 samples that were negative by the two methods. FeLV genomes were found by PCR in 13 of 39 samples from cats that were antigenaemic but from which no virus was isolated (‘discordant’ cats). These results demonstrated that a proportion of discordant cats harboured FeLV genome in their PBL. Received June 29, 1996 Accepted August 29, 1996     

Analysis of human cytomegalovirus DNA in urines of newborns and infants by means of a new ultrarapid real-time PCR-system.

BACKGROUND: Amplification techniques such as PCR are becoming increasingly popular in the field of diagnosis of human cytomegalovirus (HCMV) also, thus substituting conventional techniques like the time consuming HCMV antigen or cell culture assays. Current PCR protocols however, are labor intensive, and moreover, the need for extensive postamplification manipulations increases the risk of false positive results due to contamination with amplified products. OBJECTIVES: to overcome these shortcomings, the new ultrarapid and semi-automated real-time LightCycler PCR-system (LC-PCR), which combines amplification and detection in a closed capillary system, was tested for its suitability in diagnosis of HCMV in urines. STUDY DESIGN: 73 urine samples from 64 newborns and infants suspected of having congenitally or postnatally acquired HCMV were tested with the LC-PCR and results were compared with those obtained in parallel with a conventional PCR-ELISA and the rapid shell vial assay for detection of HCMV early antigen (EA-assay). RESULTS: with these methods, 31 newborns/infants were found to be infected with HCMV. HCMV DNA was detected in 39 urines while the EA-assay was positive in 33 urines. All the EA positive samples were also positive for HCMV DNA. In the urines of the remaining 33 newborns (34 urine samples) neither HCMV DNA nor EA were detectable. The overall agreement of the two PCR tests was 100% while a 92% agreement was obtained between the PCR and the EA-assays. As the sensitivity of the three tests turned out to be quite similiar, the discrepancy observed in the positive rate between PCR and EA-assay is due to other factors which will be discussed in detail. However, while LC-PCR takes only about 2 h from sample preparation to result generation, the EA-assay, such as the conventional PCR-ELISA, needs 24-48 h. Furthermore, due to its capability to perform cycle-by-cycle monitoring, the LC instrument enables semi-quantitative analysis of HCMV viral-load. CONCLUSIONS: LC-PCR is a suitable new tool for routine analysis of HCMV in the urines of newborns and infants. Compared to the conventional PCR-ELISA a considerable increase in test rapidity and reliability is achieved without the need to sacrifice sensitivity.     

Low correlation of human cytomegalovirus DNA amplification by polymerase chain reaction with cytomegalovirus disease in organ transplant recipients

Seventy-five organ transplant recipients underwent prolonged virological and serological follow-up for early detection of human Cytomegalovirus (HCMV) infection after transplantation. HCMV DNA detection by nested polymerase chain reaction (PCR) and HCMV early structural antigen (pp65) detection were carried out in 576 peripheral blood leucocyte (PBL) samples. Furthermore, 563 blood specimens were investigated by a commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulins G, M, and A against HCMV structural antigens. In eight of nine symptomatic organ transplant recipients, HCMV DNA was detected in one or more consecutive blood samples. HCMV DNA PCR was also positive in one or more samples from eight patients who never developed HCMV-related symptoms. HCMV pp65 antigen was detected almost exclusively in PBL samples from organ transplant recipients suffering from HCMV disease. However, antigenaemia was not detected in four PCR positive patients presenting clinical signs attributable to HCMV infection. Two of the initially HCMV DNA positive samples were not confirmed by retesting and hybridisation. The results of the present study demonstrate that despite the high specificity of nested PCR, HCMV DNA may be detected in the absence of clinical symptoms attributable to HCMV infection. In asymptomatic reactivation, limited replication of viral DNA may be responsible for positive results of PCR without any clinical relevance. In this context, pp65-antigen detection from PBL seems to have a better prognostic value, but is not always detected when clinical symptoms are present. © 1994 Wiley-Liss, Inc.     

巨细胞病毒感染与儿童免疫性血小板减少性紫癜的关系探讨

目的探讨人巨细胞病毒（HCMV）感染与儿童免疫性血小板减少性紫癜（ITP）的关系。方法采集154例ITP患儿（ITP组）和50例健康儿童（对照组）的血清样本,用Real-time PCR检测HCMV DNA,ELISA方法检测HCMV IgM、IgG抗体;其中105例ITP患儿和50例健康对照儿童采集了尿液标本,用Real-time PCR检测HCMV DNA,并比较ITP组HCMV DNA阳性患儿与阴性患儿的血小板数量的差异。结果 ITP患儿血清HCMV DNA、HCMV IgM及IgG抗体和尿HCMV DNA阳性率均明显高于健康对照儿童,两组比较差异均有统计学意义（P〈0.01）;ITP组HCMV DNA阳性患儿的血小板数量（29.72±14.54）×109/L与阴性患儿（41.28±18.35）×109/L比较差异有统计学意义（P〈0.05）。结论儿童感染HCMV可能是发生ITP的重要致病因素之一,这对指导临床有效治疗及预防ITP的发生有着积极的意义。     

Quantitation of human cytomegalovirus in recipients of solid organ transplants by real-time quantitative PCR and pp65 antigenemia

Human cytomegalovirus (HCMV) infections and anti-HCMV treatment are usually monitored by measuring pp65 antigenemia. This method is time-consuming, labour-intensive and requires skilled operators. We have compared results obtained using real-time Light Cycler quantitative PCR (QPCR) and the pp65 antigen assay on serial samples collected from recipients of solid organ transplants. We collected 198 blood samples from 14 solid organ transplant recipients and assayed them for pp65 antigen and with Light Cycler PCR. HCMV DNA was extracted from leukocytes and measured using primers and probe located in the UL83 region. The quantity of HCMV DNA was calculated using a standard curve prepared from a plasmid containing the target sequence. There was a good correlation between the number of pp65-positive cells and the DNA copy number (r = 0.57, P < 0.0001). A clinical threshold of 50 positive polymorphonuclear leukocytes/200,000 cells was equivalent to two log10 genome copies per capillary by Light Cycler PCR. HCMV DNA was detected before pp65 antigen in three patients at a mean time of 10 days, whereas the two tests were positive simultaneously for eight patients. Both the pp65 antigen data and DNA copy number decreased over time during antiviral treatment, although the QPCR was positive 28.2 days after the pp65 antigen assay had become negative. The real-time Light Cycler quantitative PCR assay is a rapid and labour-saving technique. This molecular method could be useful for monitoring infections and antiviral treatment in recipients of solid organ transplants.     

Detection of human cytomegalovirus pp67 late gene transcripts in cerebrospinal fluid of human immunodeficiency virus type 1-infected patients by nucleic acid sequence-based amplification

This study examined the clinical correlation between the presence of human cytomegalovirus (HCMV) pp67 mRNA in cerebrospinal fluid (CSF) and active HCMV central nervous system (CNS) disease in patients with human immunodeficiency virus type 1 (HIV-1). In total, 76 CSF specimens collected from 65 HIV-1-positive patients diagnosed with HCMV CNS disease, other non-HCMV-related CNS diseases, or no CNS disease were tested for the presence of HCMV pp67 mRNA using the NucliSens cytomegalovirus (CMV) pp67 assay (Organon Teknika, Durham, N.C.). The results were compared to those of a nested PCR for the detection of HCMV glycoprotein B DNA and to those obtained by viral culture (54 samples). CSF specimens collected from patients without HCMV CNS disease yielded the following results: pp67 assay negative, 62 of 62 specimens; culture negative, 41 of 41 specimens; and PCR negative, 56 of 62 specimens (6 specimens were positive). CSF specimens collected from patients with HCMV CNS disease yielded the following results: pp67 assay positive, 9 of 13 specimens; PCR positive, 13 of 13 specimens; and culture positive, 2 of 13 specimens. After resolution of the discordant results, the following positive and negative predictive values (PPV and NPV, respectively) for the diagnosis of HCMV CNS disease were determined. The PPV for PCR, pp67 assay, and culture were 68.4, 100, and 100%, respectively, and the NPV for PCR, pp67 assay, and culture were 100, 97.0, and 82. 7%, respectively. The sensitivities for DNA PCR, pp67 assay, and culture for the detection of HCMV were 100, 84.6, and 18%, respectively, and the clinical specificities were 90.5, 100, and 100%, respectively. This study indicates that the detection of HCMV pp67 mRNA in CSF has good correlation with active HCMV CNS disease, whereas CSF culture is insensitive and qualitative DNA PCR may detect latent nonreplicating virus in CSF from patients without HCMV CNS disease.     

Detection of human cytomegalovirus in plasma of AIDS patients during acute visceral disease by DNA amplification.

By using the polymerase chain reaction (PCR) amplification procedure, 19 (83%) of 23 plasma specimens obtained from individuals with AIDS and human cytomegalovirus (HCMV) visceral disease were found to be positive for plasma viremia as detected by PCR (PV-PCR), whereas 78% of cultures of peripheral blood leukocytes from the same samples were found to be positive. All 11 specimens prospectively obtained from individuals with acute HCMV disease were positive by PV-PCR. Plasma specimens from patients who received ganciclovir therapy rapidly became both culture and PV-PCR negative, and there was an excellent correlation between the two procedures. DNA detected by PV-PCR was unaffected by filtering plasma through a 0.2-microns-pore-size filter, although a conserved cellular gene, HLA-DQ alpha, was undetectable by PCR following filtration. HCMV DNA in plasma could be quantitated by PV-PCR by using endpoint serial dilutions, with detectable virus being present in 10(1) to 10(-2) microliters of plasma. A low titer of infectious virus could be detected in 2 of 11 plasma samples. The detection of HCMV DNA in plasma by PV-PCR promises to be a useful procedure for monitoring patients with AIDS suspected of having impending, acute, or recurrent HCMV visceral disease and suggests an additional route by which virus may disseminate in the immunocompromised host.     

Diagnostic value of amplification of human cytomegalovirus DNA from gastrointestinal biopsies from human immunodeficiency virus-infected patients.

In order to assess the value of human cytomegalovirus (HCMV) DNA amplification of gastrointestinal biopsies, we studied 57 human immunodeficiency virus-infected patients with and without gastrointestinal HCMV diseases. After DNA extraction, a 406-bp fragment from the unique short region of the HCMV genome was amplified by 35 cycles of polymerase chain reaction (PCR) and semiquantified from 80 to 80,000 HCMV genomic copies. Among 12 non-AIDS patients, the PCR assay was negative for 11 of 12 duodenal and 8 of 8 colorectal samples. It was also negative for 28 of 31 duodenal and 12 of 15 colorectal samples from 31 AIDS patients without gastrointestinal HCMV diseases. Among 14 AIDS patients with gastrointestinal HCMV diseases, the PCR assay was positive for 12 of 12 patients with HCMV duodenitis and for 13 of 13 patients with HCMV colitis. Results were dichotomized between high and low HCMV-DNA copy numbers. For duodenitis, sensitivity was 92% and specificity was 100%. For colitis, sensitivity was 92% and specificity was 93%. Specificity and sensitivity were not influenced by shedding status for HCMV or by other gastrointestinal infections. HCMV DNA amplification of gastrointestinal biopsies is a sensitive and specific tool for the diagnosis of gastrointestinal HCMV diseases in AIDS patients.     

Enzymatic amplification of human cytomegalovirus sequences by polymerase chain reaction.

Polymerase chain reaction (PCR) amplification was used to detect human cytomegalovirus (HCMV) sequences. The fragments selected for amplification were fragments of 130 and 152 base pairs (bp) located at two opposite ends of HCMV strain AD169 EcoRI fragment D. Amplification of the 152-bp DNA was consistently greater than that of the 130-bp DNA. At the optimal Mg2+ concentration of 5 mM, specific PCR amplification of 152-bp DNA with Taq polymerase was sensitive; only one AD169-infected fibroblast cell or 0.01 pg of AD169 fragment D DNA was needed for detection. This specific amplification was also found with various clinical HCMV isolates and peripheral blood cells and urine from patients. In 37 urine samples analyzed simultaneously by PCR and by virus cultivation, identical results were found in 35 samples, while 2 scored positive only by PCR. This suggests that specific amplification of 152-bp DNA is sensitive and can be used for rapid detection of HCMV infections.     

Comparison of loop-mediated isothermal amplification, real-time PCR, and virus isolation for the detection of herpes simplex virus in genital lesions

This study compares herpes simplex virus (HSV) type-specific loop-mediated isothermal amplification (LAMP) with virus isolation and real-time PCR. Genital tract specimens were obtained from 25 patients with genital lesions; two swab samples were collected from the vulva and cervix of each patient, for a total of 50 specimens. After culturing, 10 of 50 (20%) samples were positive for HSV-1 and 12 of 50 (24%) samples were positive for HSV-2. None of the patients excreted both HSV-1 and HSV-2 virus. An original HSV type-specific LAMP assay (30 min reaction) was compared with virus isolation and HSV type-specific real-time PCR. Viral DNA was detected by LAMP in 9 of 10 HSV-1 isolated samples and 11 of 12 HSV-2 isolated samples. No viral DNA was detected in samples without virus isolation. Thus, if virus isolation was used as the standard method, the LAMP protocol was highly sensitive and specific. In comparing LAMP to real-time PCR, viral DNA was detected by the LAMP method in 9 of 12 HSV-1 DNA positive samples and 11 of 18 HSV-2 DNA positive samples. If real-time PCR was used as the standard method, then, sensitivity of the LAMP method (in particular, for HSV-2) was low. Taking this into consideration, the LAMP reaction was extended to 60 min. This led to an increase in sensitivity, resulting in an additional one and three samples testing positive for HSV-1 LAMP and HSV-2 LAMP, respectively, compared to the original LAMP protocol. Therefore, the sensitivity of the LAMP method increased to about 80%.     

Cytomegalovirus DNA detection of an immediate early protein gene with nested primer oligonucleotides.

A rapid and sensitive polymerase chain reaction (PCR) was developed to detect conserved sequences from the immediate early gene of human cytomegalovirus (HCMV). The primers sequences were from EcoRI J fragment of Ad169. The first primer set was selected to amplify a 242 bp fragment and the next primer set was nested within the first and amplified a 146 bp fragment. With the single PCR system it was possible to detect 100 fg HCMV DNA but with double PCR 5-10 fg were detectable. Specific amplification was seen in urines from patients with HCMV infections. 20 urine samples were analysed by single PCR, double PCR and virus cultivation. The double PCR was the most sensitive method. Urines from healthy seropositive persons and cells infected with other members of the herpes virus family were negative with all three methods. This suggests that specific amplification by double PCR is sensitive and can be used for rapid detection of HCMV DNA in cases with activated infection.     

PCR Method for Detection of Adenovirus in Urine of Healthy and Human Immunodeficiency Virus-Infected Individuals

Adenoviruses (AdV) cause diseases that range from localized, self-limited illnesses to fatal infections in immunocompromised patients. Culture is assumed to be sensitive but requires viable virus and up to 3 weeks for detection, and it can be inhibited by bacterial contamination. A new PCR method amplifying a region of the hexon gene was developed in order to detect AdV in urine more rapidly and with greater sensitivity than obtainable by culture technology. All 18 serotypes tested were detected. Quantitatively, with optimized urine processing, AdV PCR detected 0.2 PFU/ml (serotype 11) and 10 DNA copies/ml (serotype 2). Serially collected urine samples from human immunodeficiency virus (HIV)-infected patients with concurrent cytomegalovirus retinitis were divided into three groups: AdV culture-positive samples, AdV culture-negative or bacterially contaminated samples from patients with a history of AdV culture-positive urines, and AdV culture-negative samples from patients without a history of AdV culture positivity. Urine samples from healthy adults were also tested by culture and PCR to screen for asymptomatic shedding. Amplification was assessed with and without prior DNA purification. AdV was detected by PCR in 90% of culture-positive urines (100% of unclotted samples, e.g., those culture positive after storage for PCR testing), 71% of culture-negative or bacterially contaminated urines from AdV-infected patients, and 28% from AdV culture-negative patients. Healthy volunteers were culture negative for AdV, and 96% were PCR negative. The new AdV PCR method is rapid and sensitive and can detect viral DNA in samples for which culturing is problematic. The role of AdV replication during HIV infection merits further investigation with sensitive tools such as PCR.     

Detection of hepatitis B virus genome in hepatocellular carcinoma tissues with PCR-in situ hybridization

The detection is described of hepatitis B virus (HBV)-DNA in preserved hepatocellular carcinoma tissues, which were derived from 14 HBV-seropositive patients. Detection was by polymerase chain reaction (PCR) amplification of the target sequence, followed by specific localization of the PCR product with in situ hybridization. PISH (PCR-in situ hybridization) yielded strong positive signals in most of the tumor tissues despite very low copy numbers of chromosome-integrated HBV genome, whereas no signal was detected in control samples, indicating that the signals were specific for HBV. Positive signals were sometimes detected in cirrhotic nodules surrounding the tumor regions, indicating that HBV had infected non-transformed liver cells. HBV-DNA was detected in both nucleus and cytoplasm in some specimens, possibly representing HBV at different stages of the life cycle. In one case, a gradient of viral DNA was revealed, with the highest DNA signal centered at the site of viral antigen expression. Taken together, PISH is shown to be a highly sensitive molecular detection method that is capable of detecting the presence of a low copy number viral genome in situ.