Detection of cytomegalovirus DNA in human placenta

Although human cytomegalovirus (CMV) is one of the most common causes of viral intrauterine and perinatal infection, its distribution in the placenta is poorly understood. The purpose of this study was to determine the frequency of CMV DNA positivity in placentas, to demonstrate the localization of the viral genome, and to identify the clinical features related to placental CMV. A total of 254 placentas from 231 mothers were investigated, and the maternal serum CMV immunoglobulin antibodies were measured. Specimens from both the placental parenchyma and the placental membrane close to the ruptured site in each placenta were examined for the presence of CMV DNA using dot blot hybridization after PCR amplification. None of 57 placentas from seronegative mothers was positive for CMV DNA. Of 197 placentas from seropositive mothers, 60 (30.5%) had CMV DNA in either the parenchyma or the membrane by dot blot analysis. In situ hybridization was carried out on these 60 placentas, and the localization of the viral genome was established in 19; CMV DNA was localized mostly to the villi, including the mesenchyme and trophoblasts, extravillous trophoblasts, and decidual cells. The mean gestational age at delivery was significantly later in the CMV DNA-positive placentas than in the negative placentas (36.9 +/- 5.1 vs 34.7 +/- 6.2 weeks, P = 0.0059). CMV DNA was detected in only 6 of 33 placentas delivered in the second trimester, and all six were associated with either severe maternal nephritis or severe chorioamnionitis. These results suggest that the CMV genome is common in placentas at later gestational ages and in those of earlier gestational ages with certain maternal complications.     

Detection and quantitation of hepatitis E virus in human faeces by real-time quantitative PCR

Hepatitis E Virus (HEV) is the causative agent of an acute and self-limited form of hepatitis. The virus is transmitted by the faecal-oral route and is a major cause of viral hepatitis in much of the developing world where it causes sporadic infections and large-scale epidemics. A simple and rapid protocol for the measurement of HEV faecal shedding by a real-time polymerase chain reaction (PCR) with the SYBR Green method on a LightCycler instrument, is described. After only 3h the real-time quantitative PCR method detected 10 molecules of HEV cDNA fragment per reaction tube and showed a high linear dynamic range of quantitation (10-10(6) molecules of cDNA/reaction) with a good correlation (r = -1.00). Its specificity was confirmed by assay in human faecal samples.     

Rapid detection and quantitation of human cytomegalovirus in urine through DNA hybridization

We detected cytomegalovirus DNA in clinical urine specimens after immobilization on nitrocellulose filters and hybridization with a radioactively labeled, cloned fragment of cytomegalovirus DNA. We accomplished the specific detection and quantitation of viral DNA within 24 hours with 39 urine specimens from nine patients with cytomegalovirus viruria, mostly at a tissue-culture infective titer of 10(3) per milliliter or higher. None of 57 urine specimens from 21 patients that were culture-negative for cytomegalovirus gave false-positive results. Analysis of specimens from patients with cytomegalovirus viruria showed a correlation of the infective titer with the intensity of DNA hybridization (r = 0.77). Hybridization of sequential urine specimens from a patient undergoing treatment with interferon for cytomegalovirus retinitis revealed quantitative variations in hybridizable viral DNA over a period that correlated with clinical findings. This assay can be useful in the selection of patients for antiviral therapy and for the assessment of its efficacy.     

Detection of human cytomegalovirus DNA in human tonsillar lymphocytes

The human cytomegalovirus (HCMV) was first isolated in cell cultures from the oropharynx, which is thought to be a site of primary infection. Although HCMV can be recovered from the oropharynx during reactivation phases, its exact site of latency is not known. In the present study we demonstrated evidence suggesting the presence of latent HCMV in this anatomic region--in the palatine tonsils. Samples from 30 tonsils obtained by tonsillectomy were screened for the presence of HCMV. Out of the 30 tonsil donors, 23 were seropositive for HCMV. Three methods were used in attempts to demonstrate HCMV's presence in the tonsils: (1) viral isolation attempts on various cell cultures, (2) immunohistochemical staining--immunoperoxidase method--designed to detect viral antigens, and (3) DNA dot hybridization with a HCMV-DNA probe designed to detect viral DNA. Neither infectious HCMV nor other viruses were isolated in cell cultures. No viral antigens were detected by immunoperoxidase staining in the tonsillar tissue. Four out of the 30 tonsils studied were found to contain viral DNA. In one case in which the tonsillar mononuclear (MN) fraction was separated from the polymorphonuclear (PMN) fraction, only the first fraction contained the viral DNA.     

Detection of cytomegalovirus DNA in human specimens by LightCycler PCR

Detection of human cytomegalovirus (CMV) DNA in clinical specimens is considered a cornerstone in the diagnosis of CMV disease. The aim of this study was to evaluate a newly designed LightCycler-based quantitative CMV PCR. Specimens of human origin (n = 200) were tested using the LightCycler PCR, the quantitative COBAS AMPLICOR CMV MONITOR (CACM) assay, and a qualitative in-house PCR assay for the presence of CMV DNA. Samples that were reactive in at least two of the three assays were considered CMV DNA positive (n = 95 [47. 5%]), while samples that were nonreactive in two of the three assays were considered CMV DNA negative (n = 105 [52.5%]). Using the LightCycler assay, CMV DNA was detected in 91 of the 95 CMV DNA-positive human specimens (sensitivity, 95.8%; 95% confidence interval [CI], 89.6 to 98.8) and in 1 of the CMV DNA-negative specimens (specificity, 99%; 95% CI, 94.8 to 99.8). Results of CMV load determination as assessed by both quantitative test systems were correlated (r = 0.73; P < 0.0001; 95% CI, 0.61 to 0.81). Results for undiluted samples containing a high CMV load were more accurate with the LightCycler test than were results obtained with the CACM test, which underestimated the viral load of samples containing high DNA copy numbers. The high level of sensitivity, specificity, accuracy, and rapidity provided by the LightCycler technology are favorable for the use of this system in the detection of CMV DNA in clinical specimens.     

Detection of human cytomegalovirus DNA by real-time quantitative PCR

A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA. This assay was used to demonstrate a higher CMV DNA load in plasma of bone marrow transplant patients than in that of blood donors. The CMV load was higher in CMV antigen-positive patients than in antigen-negative patients.     

Comparative quantitation of human cytomegalovirus DNA in blood leukocytes and plasma of transplant and AIDS patients.

A new method for the quantitation of human cytomegalovirus (HCMV) DNA was used to determine the levels of viral DNA in parallel in 120 blood leukocyte (leukoDNAemia) and plasma (plasmaDNAemia) samples from 8 heart or heart-lung transplant patients and 17 AIDS patients with disseminated HCMV infection. PlasmaDNAemia was consistently associated with leukoDNAemia in both groups of patients. However, at least in the transplant patients, plasmaDNAemia was not necessarily associated with clinical symptoms, appearing later and disappearing earlier than leukoDNAemia during the course of infection. Quantitative mean levels of leukoDNAemia were mostly higher than those of plasmaDNAemia in both transplant and AIDS patients. However, in the absence of antiviral treatment, plasmaDNAemia levels were significantly higher in AIDS patients than in transplant recipients, whereas leukoDNAemia levels were not significantly different between the two groups of patients. A significant correlation was found between leukoDNAemia and plasmaDNAemia in AIDS patients, as well as in transplant recipients, although to a lesser degree. However, from a diagnostic standpoint, quantitative determination of plasmaDNAemia appears to represent a much less sensitive parameter than that of leukoDNAemia (or antigenemia) for monitoring HCMV infections and antiviral treatment.     

Detection and quantitation of human papillomavirus DNA in peripheral blood mononuclear cells from blood donors

Human papillomavirus (HPV) is the etiological agent of cervical cancer. Also, HPV has been associated with anogenital cancer, oropharyngeal cancer, genital warts, and other dermatological diseases. HPV infects epithelial cells and their replication is closely linked to epithelial differentiation. The presence of HPV DNA in peripheral blood mononuclear cells (PBMC) has been reported in some patients with head and neck cancer, cervical cancer, and other genital diseases. However, the presence of HPV DNA in blood in asymptomatic subjects is still unresolved. The objective of this study was to evaluate the presence of HPV DNA in PBMC from asymptomatic blood donors. Blood samples were collected from 207 healthy Chilean blood donors. Genomic DNA was extracted from PBMC and HPV DNA detection was performed by real-time quantitative polymerase chain reaction assays with GP5+/6+ primers. HPV typing was carried out by genetic sequencing of a 140 to 150 bp fragment of the L1 gene. HPV DNA was detected in 6.8% (14/207) of blood donors. Single HPV infections were detected in seven blood donors. High-risk HPV was found in 6.3% (13/207) of cases: nine blood donors were infected with HPV-16, five with HPV-18, two with HPV-51, and one case was infected with either 32, 33, 45, 59, 66, 70, or 82. The median viral load value was 21.3 copies/mL blood or 13.4 HPV (+) cells per 10 ⁴ PBMC. These results show that HPV DNA is present in PBMC from healthy blood donors and it suggests that blood could be a new route of HPV dissemination.     

链置换式扩增检测羊水中巨细胞病毒DNA

目的：介绍一种简便快速准确检测羊水中ＣＭＶ－ＤＮＡ的改良ＰＣＲ－链置换式扩增用于诊断胎儿先天感染ＣＭＶ。方法，将组成套式ＰＣＲ的外内两对引物按照一定比例（外：内＝１：５０－１００）加在同一试管中一次扩增羊水和胎儿组织中ＣＭＶ－ＤＮＡ。结果：９０例异常孕产史的孕妇羊水检测ＣＭＶ－ＤＮＡ，阳性率为３８．９％（３５／９０），其中合并染色数目异常２例（４７，ＸＹＹ和４７，ＸＸ，＋２１）（已引产）核型及染色     

Detection of porcine anelloviruses in pork meat and human faeces

Torque teno viruses (TTV) are icosahedral, single-stranded circular DNA viruses infecting several vertebrate species. Currently, these viruses are considered non-pathogenic although they are suggested to be co-factors in several diseases. Recently single-stranded circular DNA viruses have been found in human faeces. Considering the consumption of pork meat products and the ubiquitous nature of swine TTV (Torque tenosus virus, TTSuV), the human population is frequently exposed to these viruses. To determine if TTSuVs could be delivered through food, human faecal samples were analysed for their presence. Indeed, the results of this study show that up to 25% of faecal samples were positive for known TTSuVs by PCR and sequencing. Additionally, all commercially available pork products purchased in Spanish supermarkets contained DNA of TTSuV.     

A highly sensitive assay for detection and quantitation of human cytomegalovirus DNA in serum and plasma by PCR and electrochemiluminescence

We describe a diagnostic PCR assay (D-PCR) and a quantitative PCR assay (Q-PCR) for the detection of human cytomegalovirus (CMV) in plasma and serum. In the D-PCR, DNA was purified from plasma or serum together with internal control (IC) DNA, which monitored both DNA extraction efficiency and PCR efficiency. DNA was subjected to PCR with a single primer pair, and the amount of PCR products was determined by electrochemiluminescence (ECL) in the QPCR System 5000 (Perkin-Elmer) after hybridization with Tris (2,2'-bipyridine) ruthenium (II) chelate-labeled probes. The lower limit of sensitivity of the D-PCR was reached at about 25 CMV particles/ml. Even with extremely low DNA inputs (four molecules of IC DNA/200 microl of plasma), very high yields (near 100%) were reached. DNA extracted from specimens that were CMV positive by the D-PCR was subsequently used in the Q-PCR, which was similar to the D-PCR. The viral load was calculated directly from the ratio of CMV and IC signals obtained by ECL. The Q-PCR assay is quantitative in the range of 100 to 150,000 copies of CMV/ml, independent of the anticoagulant. Interassay variation, intra-assay variation, and interspecimen variation were about 25%, suggesting that the Q-PCR will reliably detect fourfold differences in viral load. Comparison of paired serum and plasma specimens from CMV-infected individuals showed that serum CMV loads were frequently more than 10-fold lower than plasma CMV loads.     

Detection of cytomegalovirus DNA in short term cultures

Detection of human cytomegalovirus (CMV) by in situ DNA hybridisation six days after incubation of human diploid fibroblasts (ISDH-6) was evaluated prospectively in 205 urine samples, obtained from 57 kidney transplant and 17 bone marrow transplant recipients. The results were compared to those of conventional virus isolation (CVI) and the detection of CMV early antigens after one day of cultivation (EA-1). Of 42 samples positive for CMV by at least one of these methods, 40 (95%) were detected with ISDH-6. Thirty-five (83%) and 34 (81%) positive samples were found with CVI and EA-1, respectively. These data indicate that ISDH-6 is a sensitive method for detection of CMV. It can be used as a rapid and sensitive alternative to CVI in combination with EA-1.     

Detection of human cytomegalovirus DNA in various blood components after liver transplantation

The quantification of human cytomegalovirus (HCMV DNA) by real-time PCR is currentlya primary option for laboratory diagnosis of HCMV infection. However, the optimalsample material remains controversial due to the use of different PCR assays. Toexplore the best blood component for HCMV DNA surveillance after livertransplantation, whole blood (WB), serum (SE), and plasma (PL) specimens werecollected simultaneously from targeted patients and examined for HCMV DNA using onecommercially available assay. The HCMV DNA-positive rate with WB (16.67%) was higherthan that with either SE or PL (8.33%, both P<0.01). Quantitative DNA levels in WBwere of greater magnitude than those in SE (WB-SE mean log-transformed difference,0.99; 95%CI=0.74-1.25; P<0.0001) and PL (WB-PL mean log-transformed difference,1.37; 95%CI=1.07-1.66; P<0.0001). Dynamic monitoring revealed that HCMV DNA in WBwas positive sooner and had higher values for a longer period of time during therapy.With earlier positive detection, higher sensitivity, and yield of greater viralloads, WB compared favorably to SE or PL and hence is recommended as the superiormaterial for HCMV DNA surveillance after liver transplantation. In addition, infantrecipients require more intensive monitoring and prophylactic care because of theirhigher susceptibility to primary HCMV infection.     

Electron microscopy of human cytomegalovirus DNA

Summary Human cytomegalovirus DNA molecules isolated in an intact form from purified virions, were studied by electron microscopy. The viral DNA molecules were found to be linear and their average length 55.8±1.4 µm corresponding to a molecular weight of 107±2.7 × 10⁶ daltons.With 2 Figures     

Congenital human cytomegalovirus infection: value of human cytomegalovirus DNA quantification in amniotic fluid

A quantitative PCR assay (RS Elosa CMV, Lambdatech) was used to quantitate HCMV DNA in maternal amniotic fluid of 12 fetuses with congenital infection (group 1) and of 10 fetuses without congenital infection (group 2). HCMV detection was performed for both groups using culture and qualitative PCR. Histologic examinations of fetal tissues and placenta were carried out for 9 patients from group 1. The amniotic fluid viral loads were negative in all patients of group 2. In group 1, all viral loads were high (from 1.105 to > 107 cop/mL) and no difference was observed between symptomatic and asymptomatic foetuses. Further evaluation on larger samples is needed to define more precisely the pronostic value of HCMV DNA quantification in amniotic fluid.     

Development of a human DNA quantitation system

The AluQuant Human DNA Quantitation System has been developed for human-specific quantitation of forensic samples. This system uses probes specific to repetitive genetic elements allowing quantitation without target amplification. Target immobilization is unnecessary with employment of solution hybridization. The AluQuant Human DNA Quantitation System uses a series of enzymatic reactions to produce a luminescent signal proportional to the quantity of human DNA present. This report demonstrates a range of quantitation from 0.1-50 ng of human DNA. Signal from non-human DNAs tested was insignificant and addition of non-human DNAs into a human sample did not alter quantitation. Lastly, the system was unaffected by degradation of sample through sonication. The AluQuant Human DNA Quantitation System is a simple and sensitive method for quantitating the concentration of human DNA in forensic samples.     

Detection of human cytomegalovirus in glioma tumor tissues

A brain tumor that develops from glial cells is called a glioma. About half of all primary brain tumors form from glial cells. Gliomas are divided into subgroups, depending on the origin of the glial cells. Detection of human cytomegalovirus in tumor tissues of glioma patient was performed for the first time in Iran. These data show an association between human cytomegalovirus (HCMV) and malignant gliomas and suggest that HCMV may play an active role in glioma pathogenesis. Cytomegalovirus surgical biopsy specimens of glioma tissues (n?=?28) and non-tumor brain tissues (n?=?8) were obtained. Total DNA of specimens was extracted, and then, extracted DNA was evaluated by polymerase chain reaction for evidence of HCMV nucleic acids. Here, we show that a high percentage of malignant gliomas are infected by HCMV. Seventy-five percent of grade 4 glioma, 57 % of grade 3 glioma, and 33 % of grade 2 glioma were HCMV positive. All specimens of grade 1 glioma and control tissues were HCMV negative. The relationship between the grade of glioma and the presence of HCMV is significant (P value?=?0.035). Fisher exact test was used for statistical analysis.     

Size and complexity of human cytomegalovirus DNA.

Human cytomegalovirus DNA (Davis strain) has a molecular weight of approximately 150 × 10⁶, as determined by velocity sedimentation in sucrose gradients, by contour measurements of the molecules in the electron microscope, and by renaturation kinetics. Molecules of only one size class, 150 x 10⁶ daltons, were observed.