Development of multiplexed real-time quantitative polymerase chain reaction assay for detecting human adenoviruses.

Adenoviruses (AdVs) have been associated with a wide variety of human disease and are increasingly recognized as viral pathogens that can cause significant morbidity and mortality in immunocompromised patients. Early detection of AdV DNA in plasma and sterile fluids has been shown to be useful for identifying patients at risk for invasive AdV disease. Because of the large number of existing Adv types, few real-time quantitative AdV polymerase chain reaction (PCR) assays published effectively cover all AdV types. We designed a series of AdV PCR primers and probes and empirically multiplexed them into 2 separate real-time PCR assays to quantitatively detect all 49 serotypes of human AdV (types 1-49) available from American Type Culture Collection. We then subsequently multiplexed all the primers and probes into 1 reaction. The sensitivity of these assays was determined to be less than 10 copies per reaction (500 copies/mL plasma). In a retrospective evaluation, we detected all 84 clinical AdV isolates isolated in cell culture from patients undergoing hematopoietic stem cell transplantation between 1981 and 1987. Prospective analysis of 46 consecutive clinical samples submitted for AdV testing showed greater sensitivity and equal specificity of the AdV PCR than viral culture. This real-time PCR assay allows rapid, sensitive, and specific quantification of all currently defined AdVs into either 2 or 1 multiplex assay for clinical samples.

Quantitative real-time polymerase chain reaction for detection of adenovirus after T cell-replete hematopoietic cell transplantation: viral load as a marker for invasive disease.

BACKGROUND: The value of adenovirus plasma DNA detection as an indicator for adenovirus disease is unknown in the context of T cell-replete hematopoietic cell transplantation, of which adenovirus disease is an uncommon but serious complication. METHODS: Three groups of 62 T cell-replete hematopoietic cell transplant recipients were selected and tested for adenovirus in plasma by polymerase chain reaction. RESULTS: Adenovirus was detected in 21 (87.5%) of 24 patients with proven adenovirus disease (group 1), in 4 (21%) of 19 patients who shed adenovirus (group 2), and in 1 (10.5%) of 19 uninfected control patients. The maximum viral load was significantly higher in group 1 (median maximum viral load, 6.3x10(6) copies/mL; range, 0 to 1.0x10(9) copies/mL) than in group 2 (median maximum viral load, 0 copies/mL; range, 0 to 1.7x10(8) copies/mL; P<.001) and in group 3 (median maximum viral load, 0 copies/mL; range 0-40 copies/mL; P<.001). All patients in group 2 who developed adenoviremia had symptoms compatible with adenovirus disease (i.e., possible disease). A minimal plasma viral load of 10(3) copies/mL was detected in all patients with proven or possible disease. Adenoviremia was detectable at a median of 19.5 days (range, 8-48 days) and 24 days (range, 9-41 days) before death for patients with proven and possible adenovirus disease, respectively. CONCLUSION: Sustained or high-level adenoviremia appears to be a specific and sensitive indicator of adenovirus disease after T cell-replete hematopoietic cell transplantation. In the context of low prevalence of adenovirus disease, the use of polymerase chain reaction of plasma specimens to detect virus might be a valuable tool to identify and treat patients at risk for viral invasive disease.

Polymerase chain reaction diagnosis of primary human herpesvirus-6 infection in the acute care setting.

OBJECTIVE: To evaluate the potential utility of identifying primary human herpesvirus (HHV)-6 infection in an emergency department setting by determining the frequency of HHV-6 viremia, diagnostic testing, and empiric treatment of serious bacterial infection (SBI) in HHV-6 viremic children, and concurrent SBI and HHV-6 viremia. STUDY DESIGN: Children under age 2 years and who had a blood specimen taken for evaluation of fever were tested for HHV-6 by polymerase chain reaction (PCR). HHV-6 viremia was defined as detection of HHV-6 DNA in acute plasma. RESULTS: A total of 32 of the 181 subjects (18%) had HHV-6 viremia. Children with HHV-6 viremia frequently underwent procedures for diagnosis and empiric treatment of SBI: 60% had bladder catheterizations, 6% had lumbar punctures, 47% had radiographs, 32% received empiric antibiotics, and 34% were hospitalized. Four of the 32 children with HHV-6 viremia (12.5%) were diagnosed with SBI, although none had a positive culture of blood or cerebrospinal fluid. CONCLUSIONS: Rapid diagnosis of HHV-6 viremia may not serve to adequately differentiate infants with and without SBI in acute care settings. Although no children with HHV-6 viremia had bacteremia or meningitis, it appears that additional criteria are needed to increase the specificity of HHV-6 PCR testing before withholding evaluation for SBI.

Clinical outcomes of human herpesvirus 6 reactivation after hematopoietic stem cell transplantation.

BACKGROUND: Although human herpesvirus 6 (HHV-6) is known to reactivate during hematopoietic stem cell transplantation (HSCT), the clinical significance of this finding is controversial. METHODS: We used a quantitative PCR test for HHV-6 to assay plasma samples prospectively collected from a cohort of 110 allogeneic HSCT recipients to evaluate the clinical effects of HHV-6 infection. A retrospective review of medical records was performed to determine clinical end points. RESULTS: HHV-6 reactivation occurred in 52 (47%) of the 110 subjects. Factors that increased the risk of subsequent HHV-6 reactivation were hematologic malignancy that occurred at a time other than the first remission (adjusted P = .002), a mismatch in the sexes of donor and recipient (adjusted P=.05), younger age (adjusted P = .01), and the receipt of glucocorticoids (adjusted P = .06). HHV-6 reactivation was associated with subsequent all-cause mortality (adjusted hazard ration [HR], 2.9; 95% confidence interval [CI], 1.1-7.5), grade 3-4 graft-versus-host disease (GVHD) (adjusted HR, 4.9; 95% CI, 1.5-16), a lower probability of monocyte engraftment (adjusted HR, 0.42; 95% CI; 0.22-0.80), a lower probability of platelet engraftment (adjusted HR, 0.47; 95% CI, 0.21-1.1; P = .05) and a higher platelet transfusion requirement (adjusted P = .02). A higher level of HHV-6 DNA was associated with subsequent central nervous system (CNS) dysfunction (HR, 21; 95% CI, 1.8-249). CONCLUSIONS: HHV-6 reactivation is common after allogeneic HSCT and is associated with subsequent delayed monocyte and platelet engraftment, increased platelet transfusion requirements, all-cause mortality, grade 3-4 GVHD, and CNS dysfunction.

A population-based study of primary human herpesvirus 6 infection.

BACKGROUND: Serologic studies indicate that human herpesvirus 6 (HHV-6) infects 90 percent of children by two years of age. Little is known about the acquisition, virologic course, and clinical manifestations of HHV-6 infection. METHODS: We prospectively studied a cohort of 277 children from birth through the first two years of life to define the pattern of acquisition of HHV-6. The children's saliva was tested weekly for HHV-6 DNA with the use of the polymerase chain reaction. Parents maintained a daily log of signs and symptoms of illness in their children. RESULTS: Primary HHV-6 infection occurred in 130 children, with cumulative percentages of 40 percent by the age of 12 months and 77 percent by the age of 24 months. The peak age of acquisition was between 9 and 21 months. The acquisition of HHV-6 was associated with female sex (adjusted hazard ratio, 1.7; 95 percent confidence interval, 1.2 to 2.4) and having older siblings (adjusted hazard ratio, 2.1; 95 percent confidence interval, 1.4 to 2.9). Among 81 children with a well-defined time of acquisition of HHV-6, 93 percent had symptoms, and 38 percent were seen by a physician. None had seizures. As compared with children who had other illnesses, those with primary HHV-6 infection were more likely to have fever (P=0.003), fussiness (P=0.02), diarrhea (P=0.03), rash (P=0.003), and roseola (P=0.002) and were more likely to visit a physician (P=0.003). CONCLUSIONS: The acquisition of HHV-6 in infancy is usually symptomatic and often results in medical evaluation. Roseola occurs in a minority of patients, and febrile seizures are infrequently associated with primary HHV-6 infection. Older siblings appear to serve as a source of HHV-6 transmission.

Human herpesvirus 6 infection in febrile infants ninety days of age and younger.

BACKGROUND: The importance of human herpesvirus 6 (HHV-6) as a pathogen in febrile infants