Decreased HHV-6 IgG in Alzheimer's Disease.

Human herpesviruses have previously been implicated in the pathogenesis of Alzheimer's disease (AD) but whether they are causal, facilitating, or confounding factors is yet to be established. A total of 50 AD subjects and 52 non-demented (ND) controls were analyzed in a multiplex assay for IgG reactivity toward herpes simplex virus (HSV), varicella zoster virus (VZV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6). The HHV-6 IgG reactivity was significantly lower in AD subjects compared to ND controls, whereas there were no differences in HSV, VZV, or CMV antibody levels between the groups. Analysis of peripheral blood mononuclear cells with a subtype-specific HHV-6 PCR revealed no signs of reactivation, as AD and ND subjects presented with comparable HHV-6 DNA levels in PBMCs, and all positive samples were of subtype B. Whether HHV-6 is a factor in AD remains to be elucidated in future studies.

Epidemiologic characteristics and seasonal distribution of human metapneumovirus infections in five epidemic seasons in Stockholm, Sweden, 2002-2006.

The presence of human metapneumovirus (hMPV) was analyzed retrospectively by reverse transcriptase-polymerase chain reaction (RT-PCR) in five epidemic seasons, in Stockholm, 2002-2006. The occurrence of hMPV was compared with five common respiratory viruses; respiratory syncytial virus, influenza A virus, influenza B virus, parainfluenza virus and adenovirus. With a detection rate of 2.9% (n = 143/4,989) in nasopharyngeal samples over the whole period, hMPV was the fourth most common respiratory virus after RSV, influenza A and parainfluenza virus. hMPV genotype A dominated over genotype B, out of 91 genotyped virus samples 87 belonged to genotype A and four belonged to genotype B. Approximately 50.3% (n = 72/143) of the hMPV positive patients were <3 years, 49.7% (71/143) were > or =3 years and 38,5% (n = 55/143) were <1 year. The relative frequencies of hMPV infections in the three age groups were 2.8% (72/2,579), 2.9% (71/2,410) and 2.6% (55/2,122), respectively. This age distribution differed from RSV, influenza A, B and parainfluenza virus. hMPV epidemics peaked in March, not coincident with RSV or parainfluenza virus. In successive epidemic seasons, large outbreaks of hMPV alternated with small outbreaks in a regular, biannual pattern. Large hMPV virus epidemics were anticyclical to large RSV epidemics. It is concluded that the epidemiology of hMPV differs markedly from other common respiratory viruses.

Risk factors for the development of cytomegalovirus disease after allogeneic stem cell transplantation.

BACKGROUND AND OBJECTIVES: Cytomegalovirus (CMV) disease remains an important complication of allogeneic stem cell transplantation (SCT). We studied viral load kinetics and correlated the viral load and other transplant factors with the development of CMV disease. DESIGN AND METHODS: We studied 162 consecutive patients who were CMV seropositive or had CMV seropositive donors. Quantification of CMV DNA was performed by real-time polymerase chain reaction. RESULTS: CMV DNA detected was detected in 105 of the 162 patients. The mean peak viral loads were similar at first and subsequent reactivations. The serologic status of the donors and recipients prior to SCT significantly influenced the viral load. The cumulative incidence of CMV disease was 1.8% at 100 days and 6.3% at 365 days after SCT. The peak viral load were higher in patients who developed CMV disease than in patients without CMV disease (log10 3.5; SE +/- 0.26/200,000 cells vs. log10 2.7; SE +/- 0.09/200,000 cells; p=0.02). However, in multivariate analysis, only acute graft-versus-host disease (GVHD) grade II-IV and a graft from a CMV-negative donor to a CMV-positive patient were significant risk factors for CMV disease. In patients who required more than one course of pre-emptive therapy, acute GVHD and the rate of decrease in viral load during first pre-emptive therapy were significant risk factors for subsequent development of CMV disease. INTERPRETATION AND CONCLUSIONS: A decrease in viral load during pre-emptive therapy is an important factor for later development of CMV disease.

Discrimination of lamivudine resistant minor HIV-1 variants by selective real-time PCR.

A selective real-time PCR (SPCR) method was developed and evaluated for discrimination of resistance mutations in minor human immunodeficiency virus type 1 (HIV-1) populations, using the M184 mutation site as a model system due to its high clinical importance and the low genetic barrier to its development. The method enabled detection of minor viral populations down to 0.1%, and the relative proportions of different quasispecies could be easily displayed using cycle threshold (C(t)) values. An excellent concordance was found when the assay was compared with direct sequencing and cloning results. The impact of mismatch between virus and primer/probe sequences was evaluated, showing that 3' end mutations in the selective downstream primers were very disruptive and that 5' end polymorphisms in the probe area were directly fatal, while mutations in the middle or the 3' end of the probe were less disruptive. These effects were compensated by introducing wobble bases to accommodate the mutations. This sensitive and reliable point-mutation assay, analyzing M184I/V and other important mutations, will be fruitful in gaining new scientific knowledge about the kinetics of resistance mutations in minor viral populations of HIV-1 infected patients at failure of antiretroviral therapy.

Comparison of a duplex quantitative real-time PCR assay and the COBAS Amplicor CMV Monitor test for detection of cytomegalovirus.

A duplex quantitative real-time PCR (qPCR) assay was designed to detect both the polymerase gene (pol) and the glycoprotein gene (gB) of cytomegalovirus (CMV). The detection limit of the qPCR was determined to be 1 to 3 copies/reaction and the linear measure interval was 10(3) to 10(8) copies/ml. The qPCR system was compared to the COBAS Amplicor CMV Monitor test (COBAS) by an analysis of 138 plasma samples. Both systems detected CMV in 71 cases and had negative results for 33 samples. In addition, 34 samples were positive by qPCR and negative by the COBAS assay, but in no case was the COBAS result positive and the qPCR result negative. Thus, qPCR detected 48% more positive cases than the COBAS method. For samples with > or = 10(5) copies/ml by qPCR, a saturation effect was seen in the COBAS assay and quantification required dilution. Copy numbers for pol and gB by qPCR generally agreed. However, the reproducibility of qPCR assays and the need for an international standard are discussed. Discrepant copy numbers for pol and gB by qPCR were found for samples from two patients, and sequence analysis revealed that the corresponding CMV strains were mismatched at four nucleotide positions compared with the gB fragment primer sequences. In conclusion, a duplex qPCR assay in a real-time format facilitates quantitative measurements and minimizes the risk of false-negative results.

A real-time TaqMan PCR for routine quantitation of cytomegalovirus DNA in crude leukocyte lysates from stem cell transplant patients.

A real-time TaqMan PCR based on the cytomegalovirus (CMV) polymerase (pol) gene was developed for quantitation of CMV DNA in crude peripheral blood leukocyte (PBL) lysate from stem cell transplantation (SCT) patients. The dynamic range of the assay was between 10 and 4x10(6) copies. Both intra- and inter-assay variability were well within +/-0.25 log10 S.D. Thus, a pooled PBL sample that was used as positive control in 57 consecutive TaqMan PCR runs over 7 months showed a stable CMV quantity (4.12+/-0.13, log10 mean+/-S.D.). The sensitivity of the pol TaqMan PCR was validated by parallel analysis of 177 PBL samples with a nested PCR. The use of crude PBL lysate as PCR input did not cause PCR inhibition. We demonstrated further the clinical utility of the newly developed TaqMan PCR by monitoring changes in CMV levels in eight patients receiving antiviral therapy. This TaqMan PCR was highly sensitive, reproducible, and stable and has served a useful tool for monitoring CMV DNA levels in large number of clinical samples in a routine diagnostic setting for over 1 year.