Real-time quantitative PCR for human herpesvirus 6 DNA

The diagnosis of human herpesvirus 6 (HHV-6) infection represents a complex issue because the most widely used diagnostic tools, such as immunoglobulin G antibody titer determination and qualitative DNA PCR with blood cells, are unable to distinguish between latent (clinically silent) and active (often clinically relevant) infection. We have developed a new, highly sensitive, quantitative PCR assay for the accurate measurement of HHV-6 DNA in tissue-derived cell suspensions and body fluids. The test uses a 5' nuclease, fluorogenic assay combined with real-time detection of PCR amplification products with the ABI PRISM 7700 sequence detector system. The sensitivity of this method is equal to the sensitivity of a nested PCR protocol (lower detection limit, 1 viral genome equivalent/test) for both the A and the B HHV-6 subgroups and shows a wider dynamic range of detection (from 1 to 10(6) viral genome equivalents/test) and a higher degree of accuracy, repeatability, and reproducibility compared to those of a standard quantitative-competitive PCR assay developed with the same reference DNA molecule. The novel technique is versatile, showing the same sensitivity and dynamic range with viral DNA extracted from different fluids (i.e., culture medium or plasma) or from tissue-derived cell suspensions. Furthermore, by virtue of its high-throughput format, this method is well suited for large epidemiological surveys.     

Real-time PCR determination of human herpesvirus 6 antiviral drug susceptibility

A quantitative real-time PCR assay was developed to determine the antiviral drug susceptibility of human herpesvirus 6 (HHV-6). After short-term culture of the virus, HHV-6 isolates’ susceptibility to the antiviral ganciclovir (GCV) was determined by measuring the HHV-6 variant B (HHV-6B) DNA levels in culture supernatants and infected cells using real-time PCR. A total of 12 well-characterized GCV-sensitive or -resistant strains and clinical isolates were used. This new assay with real-time PCR readout permitted the rapid (3 days), objective, and reproducible determination of HHV-6 drug susceptibilities with no need for stringent control of the initial multiplicity of infection. Furthermore, the real-time PCR assay results showed good correlation (r_s = 0.95) with those from the conventional TCID₅₀ (50% tissue culture infecting dose) reduction assay (TRA). Thus, the real-time PCR assay described in this report was found to be a suitable quantitative method for determining the susceptibility of HHV-6 to antiviral drugs. It is faster and simpler than the TRA, and it is amenable to use in the routine diagnostic virology laboratory.     

The evidence of human herpesvirus 6 infection in the lymph nodes of Hodgkin's disease

Human herpesvirus 6 (HHV-6), the causative agent of exanthem subitum, has been implicated in other diseases. Recently HHV-6-specific sequences have been detected by Southern blot analysis and polymerase chain reaction in the lymph nodes of three patients with Hodgkin's disease. The pathological localization of HHV-6, however, is still unknown. In order to study the pathological role of HHV-6 in Hodgkin's disease, we investigated, by immunohistochemical and molecular methods, two lymph node biopsies taken from a 7-year-old boy with Hodgkin's disease during the course of disease evolution. Although the histopathological findings of the first biopsy differed from those of the second, HHV-6 antigens and sequences could be detected in both lymph nodes by immunohistochemistry and in situ hybridization, respectively. HHV-6 was localized in macrophages, predominantly in lymphoid follicles, but not in ReedSternberg cells. Antibody titres to HHV-6 were consistent with reactivation of latency. Neither cytomegalovirus nor Epstein-Barr virus was present. Our data suggest a role for HHV-6 in the pathogenesis of Hodgkin's disease.     

Frequent shedding of human herpesvirus 6 in saliva

We have previously reported the isolation of HHV-6 from saliva samples. Because these isolations were made in PHA-stimulated lymphocytes from healthy adults, which may occasionally contain endogenous HHV-6, it was desirable to repeat this work using cord blood lymphocytes. In this study 18 isolations of viruses provisionally characterized as HHV-6 were made from 19 saliva samples by centrifugally enhanced inoculation into PHA-stimulated cord blood lymphocytes. HHV-6 was not found in 10 pernasal aspirates, 50 endocervical swabs, or 30 male urethral swabs. It is concluded that HHV-6 is usually present in the saliva of most adults and that this affords a possible explanation of the high infection rate with this virus in young children.     

Simultaneous quantification of Epstein-Barr virus, cytomegalovirus, and human herpesvirus 6 DNA in samples from transplant recipients by multiplex real-time PCR assay

We developed a multiplex real-time PCR assay using 6-carboxyfluorescein, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, and carbocyanine 5-labeled probes to simultaneously quantify Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6) DNA. When previously tested and stored DNA samples were examined, results of the multiplex real-time PCR assay were as sensitive and specific as those of a single real-time PCR assay. The multiplex assay was used to quantify the EBV, CMV, and HHV-6 DNA in 46 transplant recipients. A total of 303 whole-blood and plasma specimens were collected and analyzed. According to the results of the multiplex assay, the detection rates for viral DNA in whole blood and plasma were 23.8% and 5.9% for EBV, 11.2% and 5.3% for CMV, and 12.5% and 2.0% for HHV-6, respectively. All forms of viral DNA were detected more frequently in whole blood than in plasma. During the symptomatic period, EBV DNA was detected in all whole-blood specimens but not in all plasma specimens. Furthermore, the EBV DNA load in whole blood was higher during the symptomatic period than during the asymptomatic period, whereas the EBV DNA load in plasma was similar for both periods. These results demonstrate that whole blood is more suitable for the quantification of EBV DNA in transplant patients. However, a cutoff value with clinical relevance still needs to be determined.     

Real-time PCR quantification of human cytomegalovirus DNA in amniotic fluid samples from mothers with primary infection

A real-time PCR assay was developed to quantify human cytomegalovirus (HCMV) DNA in amniotic fluid (AF) samples collected from 30 pregnant women with primary HCMV infection as detected either from HCMV-immunoglobulin G (IgG) seroconversion or by the presence of HCMV-specific IgG and IgM associated with a low IgG avidity. Clinical information available for each case included ultrasonographic examination and fetal or newborn outcome. HCMV infection of fetuses or newborns was confirmed for the 30 studied cases. AF samples were subdivided into three groups. In group A (n = 13), fetuses presented major ultrasound abnormalities, and pregnancy was terminated. In group B (n = 13), fetuses had normal ultrasound findings, the pregnancy went to term, and the newborns were asymptomatic at birth. In group C (n = 4), fetuses had no or minor ultrasonographic signs, and pregnancy was terminated. The HCMV DNA load values in AF samples were significantly higher in group A (median, 2.8 x 10(5) genome equivalents [GE]/ml) than in group B (median, 8 x 10(3) GE/ml) (P = 0.014). Our findings suggest that HCMV load level in AF samples correlates with fetal clinical outcome but might also be dependent on other factors, such as the gestational age at the time of AF sampling and the time elapsed since maternal infection.     

Multicenter comparison of PCR assays for detection of human herpesvirus 6 DNA in serum

Human herpesvirus 6 (HHV-6) is a ubiquitous virus with which infections have been associated with pathologies ranging from delayed bone marrow engraftment to a variety of neurological diseases. The lack of a standardized assay that can be used to detect and estimate HHV-6 DNA contents in various clinical specimens can lead and has led to discordant results among investigators and on the potential association of HHV-6 to diseases. To identify the most reliable and sensitive assays, an identical set of 11 coded serum samples spiked with various quantities of the HHV-6A variant (range, 4 to 400,000 genome copies/ml) was sent to eight independent laboratories around the world. Each laboratory was asked to estimate the HHV-6 DNA content by use of its own protocols and assays. Among the various assays, three TaqMan-based real-time PCR assays yielded quantities that were closest to the quantity of HHV-6 that had been spiked. To provide better homogeneity between the results from the different laboratories working on HHV-6, we propose that investigators interested in quantifying HHV-6 in clinical samples adopt one of these assays.     

Human herpesvirus 6 DNA in peripheral blood cells and saliva from immunocompetent individuals.

Human herpesvirus 6 (HHV-6) genome equivalents were quantitated in peripheral blood mononuclear cells (PBMCs) and saliva from 20 healthy individuals by the polymerase chain reaction (PCR). Nineteen of 20 subjects (95%) harbored HHV-6 DNA: 18 (90%) had HHV-6 in their PBMCs and 18 had HHV-6 in their saliva. Quantitative PCR revealed HHV-6 DNA levels ranging from negative to 4,000 HHV-6 genome equivalents per 10(6) PBMCs and from negative to 200,000 HHV-6 genome equivalents per ml of saliva. Longitudinal saliva samples from 15 HHV-6-seropositive subjects revealed salivary HHV-6 DNA persistence in 13 subjects. HHV-6 antibodies were detected in 17 of 19 subjects, with titers ranging from 1:400 to 1:51,200 (geometric mean titer, 1:2,500). Antibody titers did not correlate with HHV-6 DNA levels in PBMCs or saliva (P = 0.27 and P = 0.44, respectively). One subject with persistent HHV-6 DNA lacked detectable HHV-6 antibodies. The high prevalence of HHV-6 DNA in PBMCs and saliva supports the concept that HHV-6 exists at these sites in normal individuals.     

Frequent isolation of human herpesvirus 7 from saliva samples

Human herpesvirus 7 (HHV-7) was isolated frequently from saliva specimens. The isolation rates were 81% (13/16) in adults, 70% (7/10) in children over 1 year old, and none (0/7) in children less than 1 year old, respectively, indicating that infection of HHV-7 occurs during early infancy and the virus shedding rate after infection is very high. Human herpesvirus 6 (HHV-6) was not isolated from saliva specimens although some studies on isolation of HHV-6 from saliva was reported previously. © 1993 Wiley-Liss, Inc.     

Multicenter comparison of PCR assays for detection of human herpesvirus 8 DNA in semen

Reported prevalences of human herpesvirus 8 (HHV-8) (Kaposi's sarcoma-associated herpesvirus) in semen have ranged widely. This is possibly due to differences in assay sensitivity, geographic or population-based differences in the true presence of the virus in semen, and PCR contamination. This study assessed interlaboratory sensitivity and reproducibility in the analysis of blinded experimental panels, each consisting of 48 specimens and being composed of semen specimens from different healthy artificial-insemination donors (n = 30) and human immunodeficiency virus (HIV)-infected patients (n = 7) plus positive (n = 4) and negative (n = 7) controls. The experimental panels analyzed in each laboratory were identical except for being independently coded. Of 10 experiments done in five laboratories, 5 experiments from three laboratories had evidence of PCR contamination; all instances of contamination were in the context of nested PCR procedures. In the experiments with no false-positive results, HHV-8 DNA was detected in three (8%) of the 37 semen specimens (two from artificial-insemination donors and one from an HIV-positive patient) but in only 3 (1.6%) of the 184 PCRs in which these specimens were analyzed. This suggests that HHV-8 DNA is present in semen at concentrations that can be too low to allow its consistent detection. This study emphasizes the importance of performing blinded, multi-institution experiments to provide a coherent basis for comparing results and to motivate standardization of methods.     

Calibrated real-time PCR assay for quantitation of human herpesvirus 8 DNA in biological fluids

Accurate laboratory tests for the diagnosis of active human herpesvirus 8 (HHV-8) infection are becoming essential to study the pathogenesis of HHV-8-associated tumors and for the clinical management of HHV-8-infected individuals. We have developed a highly sensitive, calibrated quantitative real-time PCR assay for the measurement of cell-free HHV-8 DNA in body fluids, based on the addition of a synthetic DNA calibrator prior to DNA extraction. The calibrator controls each sample for the presence of PCR inhibitors, determines a cutoff value of sensitivity for negative samples, and normalizes positive samples for the efficiency of DNA recovery. The assay shows a wide dynamic range of detection (between 1 and 10(6) viral genome equivalents/reaction) and a high degree of accuracy even in the presence of high amounts (up to 1 micro g) of human genomic DNA. Moreover, the assay has a very high sensitivity (lower detection limit, 10 genome equivalents/ml) and a high degree of reproducibility and repeatability with a coefficient of variation (CV) of <15 and 23%, respectively. Furthermore, the use of the calibrator improves the accuracy of quantitation and decreases the intersample variability (CV, 9 and 6%, respectively). The sensitivity and specificity of the assay were tested with a series of clinical specimens obtained from patients affected by various HHV-8-related diseases, as well as from a wide number of controls. In conclusion, our calibrated real-time PCR assay provides a reliable high-throughput method for quantitation of HHV-8 DNA in clinical and laboratory specimens.     

Comparison of a microtiter plate system to Southern blot for detection of human herpesvirus 8 DNA amplified from blood and saliva

The recent discovery of human herpesvirus 8 (HHV-8) as the etiologic agent of Kaposi's sarcoma (KS) has led to the interest in the development of PCR for this virus that is accurate, rapid, and convenient. We developed a sensitive PCR assay for HHV-8 with microtiter plate detection of amplimers. DNA was purified from white blood cells and saliva from HIV-infected men with and without Kaposi's sarcoma and one-step PCR was undertaken with primer sets specific for the N-terminal region of the glycoprotein B gene and open reading frame (orf) 26 of HHV-8. PCR was performed on 40 clinical specimens, followed by Southern blot and microtiter plate detection of amplimers. Results from the two methods of detection were nearly identical. Sensitivity for both methods based on serial dilution of a known standard was five to ten copies of HHV-8 per 400 ng of cellular DNA. In conclusion, microtiter plate detection of HHV-8 PCR amplimers is as sensitive and specific as Southern blot with much faster turnaround time at comparable cost, and utilizes common laboratory equipment.     

Demonstration of the human herpesvirus 6-induced DNA polymerase and DNase

Infection of HSB-2 cells with human herpesvirus 6 (HHV6) results in an approximately 51-fold increase in the level of DNA polymerase activity and a 4.44-fold increase in the level of DNase activity when compared to mock-infected cells. There was no increase in thymidine kinase, uracil-DNA glycosylase, or deoxyuridine triphosphate nucleotidohydrolase activities in the infected cells. The HHV6-induced DNase and DNA polymerase activities could be distinguished from their normal cellular counterparts on the basis of immunological specificities and in the case of DNA polymerase based upon differences in electrophoretic migration. Serological studies also demonstrated reactivity of the antisera not only for HHV6 but also for Epstein-Barr virus.     

Real-time PCR for detection of Brucella spp. DNA in human serum samples

Presented here are the results of an evaluation of an in-house real-time PCR assay for the rapid and specific diagnosis of human brucellosis. The assay was based on direct amplification from serum samples of a 169-bp portion of bcsp31, a gene found in all Brucella species and biovars. Species specificity and selectivity of this real-time PCR assay were evaluated using genomic DNA from 15 Brucella strains and 42 non-Brucella strains, and the results were 100%. Among 17 culture-proven brucellosis patients, sera from 11 gave a positive amplification signal, corresponding to a sensitivity of 64.7%. In contrast, negative results were obtained for all sera from 60 control patients, corresponding to a specificity of 100%. The results indicate this test is well adapted for definite confirmation of brucellosis cases, when Brucella cultures remain sterile and serological tests demonstrate the presence of cross-reacting antibodies against Brucella sp. and Yersinia enterocolitica O:9 antigens.     

Detection of human herpesvirus-6 DNA in peripheral blood and saliva

Saliva and peripheral blood samples from 20 healthy adults were examined for the presence of human herpesvirus-6 (HHV-6) DNA sequences using the polymerase chain reaction. Eighteen out of 20 whole saliva samples contained detectable HHV-6 genomes. The majority of peripheral blood samples were also positive; however, the results suggest that only a rare cell in the peripheral blood is infected. Serological studies did not reveal any correlation between HHV-6 antibody titre and the ability to detect HHV-6 DNA. The data indicate that HHV-6 genomes persist in the peripheral blood and oropharynx or salivary glands of most healthy individuals following primary infection.     

Detection of HPV-16 DNA by PCR in histologically cancer free lymph nodes from patients with cervical cancer.

The prognostic value of detection of human papillomavirus (HPV) type 16 DNA in histologically cancer free lymph nodes was assessed in left obturator lymph nodes from cervical cancer patients with HPV-16 positive primary tumours. HPV-16 DNA was detected by polymerase chain reaction in 12 of 35 patients with histologically cancer free lymph nodes. Of these 12 patients, only one developed a recurrence, suggesting HPV-16 DNA detection in cancer free lymph nodes has no prognostic value.     

Ontogeny of human fetal lymph nodes

Developing lymph nodes from 30 human embryos and fetuses with crown-rump lengths (CRL) of 18 mm (5.6 wk) to 245 mm (26 wk) were examined by light microscopy. The nodes were embedded in araldite, and the sections examined were approximately 1 μ in thickness. The development of nodes was divided into three stages: 1. the lymphatic plexus and connective tissue invagination (30 mm to 67 mm CRL); 2. the early fetal lymph node (43 mm to 95 mm CRL); and 3. the late fetal lymph node (CRL greater than 75 mm). The lymphatic plexus was formed by connective tissue invaginations and bridges which divided a lymph sac into a meshwork of channels and spaces. Connective tissue invaginations were endothelially-lined and were surrounded by lymphatic space. Reticular cells, macrophages, and blood vessels were found in these invaginations. Early fetal lymph nodes were formed from invaginations when the cellular density and lymphocyte content increased. The lymphatic space surrounding the early node was the developing subcapsular sinus. With further development the early node became packed with lymphocytes, increasing the cellular density and size of the node. The connective tissue surrounding the subcapsular sinus condensed to form the capsule. Afferent lymphatic vessels pierced the capsule. Capillaries, veins, postcapillary venules, and occasional arteries were found in early and late nodes.