Differential kinetics of human cytomegalovirus load and antibody responses in primary infection of the immunocompetent and immunocompromised host.

The comparative long-term kinetics of human cytomegalovirus (HCMV) load and HCMV-specific antibody responses in the immunocompetent and immunocompromised solid-organ transplanted host during primary HCMV infection was investigated. In total, 40 immunocompetent subjects and 17 transplanted patients were examined for viral load as well as for IgG antibody responses to HCMV glycoproteins gH/gL/pUL128L, gH/gL and gB, and neutralizing antibodies in ARPE-19 epithelial cells and human fibroblasts. In parallel, the CD4(+) and CD8(+) HCMV-specific T-cell responses were determined by cytokine flow cytometry. Transplanted patients reached significantly higher viral DNA peaks, which persisted longer than in immunocompetent subjects. The ELISA-IgG responses to the pentamer, gH/gL and gB were significantly higher in primary infections of the immunocompetent until six months after onset, with the two antibody levels then overlapping from six to 12 months. Antibody levels neutralizing infection of epithelial cells were significantly higher in transplanted patients after six months, persisting for up to a year after transplantation. This trend was not observed for antibodies neutralizing infection of human fibroblasts, which showed higher titres in the immunocompetent over the entire one-year follow-up. In conclusion, in immunocompromised patients the viral load peak was much higher, while the neutralizing antibody response exceeded that detected in the immunocompetent host starting six months after onset of follow-up, often concomitantly with a lack of specific CD4(+) T cells. In this setting, the elevated antibody response occurred in the presence of differentiated follicular helper T cells in the blood, which decreased in number as did antibody titres upon reappearance of HCMV-specific CD4(+) T cells.

Clinical evaluation of the Roche Elecsys CMV IgG Avidity assay.

Congenital cytomegalovirus (CMV) infection has potentially severe consequences in newborns. The testing of pregnant women for CMV-specific antibodies may be useful for the identification of women at risk of transmitting the infection to the fetus. The determination of CMV IgG avidity helps to establish the timing of infection as IgG avidity matures during the course of infection. This study examines the performance of the Elecsys CMV IgG Avidity assay using preselected samples from patients at different phases of CMV infection. The Elecsys CMV IgG Avidity assay was tested at three sites using sequential samples from patients with recent primary CMV infection, as well as single samples from patients with recent primary or past CMV infection. The Elecsys assay discriminated well between early (low avidity) and late (high avidity) phases of infection in sequential serum samples. Overall, 98.8% of low-avidity samples corresponded to infection onset <180 days before sampling and 77.8% of all high-avidity results corresponded to infection onset >90 days before sampling. The assay's sensitivity was 90-97%, with specificity ranging from 89 to 100%, depending on the consideration of gray-zone avidity values. Single samples from recent primary or past infection showed similar distributions of avidity results. The Elecsys CMV IgG Avidity assay results are in agreement with preselected samples from patients with primary or past CMV infection, showing that the test is an adequate predictor of the phase of infection.

Clinical evaluation of new automated cytomegalovirus IgM and IgG assays for the Elecsys(®) analyser platform.

Cytomegalovirus (CMV) is a leading cause of physical and neurological abnormalities in newborns. Hence, the diagnosis of CMV infection in pregnant women is necessary in order to allow appropriate management of their pregnancy. New assays have been developed for the Roche Elecsys® immunoassay platform that detect CMV-specific immunoglobulin (Ig)M and IgG, with the IgM assay designed to target IgM produced at the start of infection rather than IgM persisting later in infection. This study aimed to evaluate the performance of the new assays compared with other commercial kits widely distributed in laboratories. The performance of the Elecsys and comparator CMV IgM and IgG assays was assessed using 967 preselected patient samples characterised by CMV infection status, as well as being compared using 1,668 unselected clinical samples. The Elecsys CMV IgM and IgG assays performed consistently with comparator assays using the preselected samples. The Elecsys CMV IgM assay showed improved sensitivity compared with the Enzygnost® assay in primary infection (91.2 % vs. 79.4 %) and improved specificity over the Architect® assay in potentially cross-reacting samples (94.1 % vs. 82.4 %). The Elecsys IgM assay reported fewer positive results in the later stages of CMV infection compared with ETI-CYTOK-M ELISA, while the Elecsys IgG assay reported slightly fewer negative results in the early stages of infection compared with ETI-CYTOK-G ELISA. There was good agreement between Elecsys and comparator assays using unselected clinical samples (range 90.4-99.4 %). The Elecsys CMV IgM and IgG assays compare well with routinely used assays and are suitable for clinical use.

Kinetics of effector functions and phenotype of virus-specific and γδ T lymphocytes in primary human cytomegalovirus infection during pregnancy.

The T-cell response to human cytomegalovirus (HCMV) primary infection was analyzed in 27 pregnant women during the first year after primary HCMV infection. Pregnant women with remote HCMV infection were enrolled as controls. Interferon-γ-producing T cells were readily detected at levels comparable (CD4(+)) or higher (CD8(+)) than controls, whereas the CD4(+) and CD8(+) lymphoproliferative response as well as IL-2 production was significantly reduced with respect to controls for at least 9 months after infection. In addition, CD45RA re-expression as well as cytotoxic T lymphocyte activity and perforin expression were the major components of the adaptive CD4(+) and CD8(+) T-cell immune response, while Vδ2(-) γδ T-cell expansion in response to HCMV infection followed kinetics similar to that of CD8(+) T cells. Reduced CD45RA re-expression directly correlated with HCMV transmission to the fetus, thus providing an important prognostic parameter.

Human cytomegalovirus tropism for endothelial/epithelial cells: scientific background and clinical implications.

Human cytomegalovirus (HCMV) has been routinely isolated from and propagated in vitro in human embryonic lung fibroblast (HELF) cell cultures, while in vivo it is known to infect predominantly endothelial and epithelial cells. In recent years, genetic determinants of the HCMV tropism for endothelial/epithelial cells were identified in the UL131A/UL130/UL128 locus of HCMV genome of wild-type strains. UL131A-UL128 gene products form a complex with glycoprotein H (gH) and L (gL) resulting in a gH/gL/UL131A-UL128 complex that is required for HCMV entry into endothelial/epithelial cells. In contrast, virus entry into fibroblasts has its genetic determinants in the complex gH/gL/gO (or gH/gL). During primary HCMV infection, the neutralising antibody response measured in endothelial cells (EC) is potent, occurs very early and is directed mostly against combinations of two or three gene products of the UL131A-128 locus. On the contrary, neutralising antibodies measured in fibroblasts appear late, are relatively weak in potency and are directed against gH and gB. The T-cell immune response to UL131A-UL128 gene products remains to be investigated. Recently, a role has been proposed for neutralising antibody in conferring prevention/protection against HCMV infection/disease in pregnant women with primary HCMV infection. However, the level of cooperation between humoral immunity and the well-established T-cell protection remains to be defined.

Prognostic markers of symptomatic congenital human cytomegalovirus infection in fetal blood.

OBJECTIVE: To identify fetal cord blood prognostic markers of symptomatic congenital human cytomegalovirus infection (HCMV). DESIGN: Retrospective observational study. SETTING: Fetal medicine unit in Milan and Medical virology unit in Pavia, Italy. POPULATION: HCMV-infected and -uninfected fetuses of mothers with primary HCMV infection during the period 1995-2009. METHODS: Overall, 94 blood samples from as many fetuses of 93 pregnant women experiencing primary HCMV infection were examined for multiple immunological, haematological and biochemical markers as well as virological markers. Congenital HCMV infection was diagnosed by detection of virus in amniotic fluid, and symptomatic/asymptomatic infections were determined by ultrasound scans, nuclear magnetic resonance imaging, histopathology or clinical examination at birth. Blood sample markers were retrospectively compared in symptomatic and asymptomatic fetuses with congenital infection. MAIN OUTCOME MEASURES: A statistical analysis was performed to determine the value of each parameter in predicting outcome. RESULTS: Univariate analysis showed that most nonviral and viral markers were significantly different in symptomatic (n = 16) compared with asymptomatic (n = 31) fetuses. Receiver operator characteristics analysis indicated that, with reference to an established cutoff for each marker, the best nonviral factors for differentiation of symptomatic from asymptomatic congenital infection were ß(2) -microglobulin and platelet count, and the best virological markers were immunoglobulin M antibody and DNAaemia. ß(2) -Microglobulin alone or the combination of these four markers reached the optimal diagnostic efficacy. CONCLUSIONS: The determination of multiple markers in fetal blood, following virus detection in amniotic fluid samples, is predictive of perinatal outcome in fetuses with HCMV infection.

Circulating endothelial giant cells permissive for human cytomegalovirus (HCMV) are detected in disseminated HCMV infections with organ involvement.

Giant cells fully permissive for human cytomegalovirus (HCMV) were found to circulate, at a variable proportion, in peripheral blood of 21 out of 25 immunocompromised patients with disseminated HCMV infection. Circulating endothelial giant cells (EGC) were identified by a specific monoclonal antibody of endothelial origin and shown to express immediate-early, early, and late viral proteins. Immunostaining patterns of different viral proteins were comparable to those detected in vitro in cultured human umbilical vein endothelial cells. EGC counts > 10 were associated with high levels (> 100) of HCMV viremia and antigenemia, as well as with an overt clinical syndrome in transplanted patients, and to an untreated long lasting organ localization in AIDS patients. On the other hand, EGC counts were < 10 during disseminated HCMV infections of both transplant recipients with no apparent organ syndrome and AIDS patients with recent organ involvement. In tissue sections from AIDS patients, infected endothelial cells were found to progressively enlarge till detaching from the small vessel wall and entering blood stream. HCMV-infected EGC represent a new systemic parameter suitable for the diagnosis of HCMV organ involvement and for the study of the pathogenesis of disseminated infections.

In vitro generation of human cytomegalovirus pp65 antigenemia, viremia, and leukoDNAemia.

Immunocompromised patients with disseminated human cytomegalovirus (HCMV) infection have circulating PMN carrying HCMV pp65 (antigenemia), infectious virus (viremia), and viral DNA (leukoDNAemia). Because HCMV does not fully replicate in PMN, it is generally hypothesized that virions and viral materials are taken up by phagocytosis from fully permissive HCMV-infected endothelial cells. However, no experimental evidence has ever been provided for these PMN-endothelium interactions. PMN from 11 donors were cocultured with endothelial cells infected with an endothelium-adapted HCMV strain and with human fibroblasts infected with low-passaged clinical and laboratory-adapted HCMV strains. pp65-positive PMN were detected after coculture with both HCMV-infected endothelial and fibroblast cells, provided that wild and not laboratory-adapted strains were used. In addition, cocultured PMN carried infectious virus as demonstrated by virus isolation and presence of complete virus particles by electron microscopy. Moreover, high levels of viral DNA were consistently detected by quantitative PCR in cocultured PMN. Thus, we have generated in vitro the three most important viral parameters detected in patients with disseminated HCMV infection (antigenemia, viremia, and leukoDNAemia). The failure of laboratory-adapted HCMV strain to induce this phenomenon demonstrates that important modifications have occurred in attenuated viral strains affecting basic biological functions.

Detection and pathogenicity of human metapneumovirus respiratory infection in pediatric Italian patients during a winter--spring season.

BACKGROUND: Some diagnostic, epidemiological and clinical features of the recently discovered human metapneumovirus remain to be investigated. OBJECTIVES: To study the best approach for the diagnosis of human metapneumovirus infections by both conventional and molecular methods, along with the human metapneumovirus circulation rate in northern Italy and the severity of human metapneumovirus respiratory infections in a pediatric patient population. STUDY DESIGN: Nasopharyngeal aspirates (NPA) were taken from 306 pediatric patients during the winter-spring season 2003-2004, and examined for conventional respiratory viruses by direct fluorescent staining and cell culture, while human coronavirus and human metapneumovirus were sought by RT-PCR. RESULTS: RT-PCR detected human metapneumovirus in 40/306 (13.1%) children positive for respiratory viruses, with an incidence intermediate between that of respiratory syncytial virus (58 patients, 18.9%) and that of influenzavirus infections (29 patients, 9.5%). Phylogenetic analysis showed cocirculation of both human metapneumovirus types (A and B) as well as their relevant subtypes (A1-A2 and B1-B2). Clinically, human metapneumovirus was found to be second to human respiratory syncytial virus alone, as a cause of respiratory tract infections, while duration of virus excretion appeared to correlate with severity of infection, and virus load in NPA with the stage of respiratory infection. CONCLUSION: (i) Human metapneumovirus is a major viral pathogen in the Italian pediatric patient population; (ii) the severity of lower respiratory tract infections approaches that of human respiratory syncytial virus; (iii) there are preliminary indications that the duration of virus excretion may reach 2-3 weeks and that the level of viral load in NPA correlates with the clinical stage of human metapneumovirus infection.

Primary human cytomegalovirus infections: kinetics of ELISA-IgG and neutralizing antibody in pauci/asymptomatic pregnant women vs symptomatic non-pregnant subjects.

BACKGROUND: Human cytomegalovirus infections are mostly asymptomatic in infants and young children, while they are often associated with overt clinical symptoms in adults. OBJECTIVES: To verify whether the antibody response to HCMV is more potent in symptomatic non-pregnant adults as compared to asymptomatic/paucisymptomatic pregnant women. STUDY DESIGN: Overall, 36 consecutive pregnant women with primary HCMV infection were compared with 10 consecutive symptomatic non-pregnant subjects with primary HCMV infection and overt clinical symptoms. Levels of IgG antibody responses to HCMV-infected cell lysate and the pentamer gH/gL/pUL128L, gH/gL and gB HCMV glycoprotein complexes as well as neutralizing antibodies preventing infection of epithelial cells (ARPE-19) and human embryonic lung fibroblast (HELF) cells were compared at intervals of 1-30, 31-60, 61-90, 91-180 and 181-360 days after onset of infection. In parallel, viral load was quantified by real-time PCR. RESULTS: In symptomatic non-pregnant subjects, the IgG responses to HCMV lysate as well as to gH/gL and ARPE-19 neutralizing antibodies were significantly higher from 31 to 60 through 180 days after infection onset. In the same patients, the IgG antibody responses to the pentamer and HELF-neutralizing antibody were significantly higher starting 90 days post-infection. CONCLUSIONS: The presence of overt clinical symptoms is associated with a significantly higher antibody response (concomitantly with a higher viral load) in non-pregnant subjects with symptomatic primary HCMV infection as compared to pregnant women with paucisymptomatic/ asymptomatic primary infection (and lower viral load).

A Nutritional Approach to the Prevention of Cancer: from Assessment to Personalized Intervention.

Among lifestyle factors, nutrition is one of the most important determinants of health, and represents a pivotal element of cancer risk. Nonetheless, epidemiological evidences of the relationship between several cancers and specific foods and nutrients is still inadequate, and solid conclusions are missing. Indeed, caloric restriction without malnutrition is associated to cancer prevention. Food may be also the primary route of exposure to contaminants such as metals, persistent organic pollutants, and pesticides. Exposuredisease associations and the interplay with genetic susceptibility requires further studies on genetic variation, environment, lifestyle, and chronic disease in order to eliminate and reduce associated health risks, thus contributing to improve health outcomes for the population. A primary nutritional approach for Active and Healthy Ageing (AHA) has been developed by the Nutrition group of the European Innovation Partnership (EIP) on AHA. The working group on lifestyles of the Italian Ministry of Health has developed a comprehensive approach to adequate nutrition using a consensus methodology to collect and integrate the available evidences from the literature and from the Italian experiences at the regional level, to raise the interest of other experts and relevant stakeholders to outline and scale-up joint strategies for a primary nutritional approach to cancer prevention.

Ganciclovir resistance as a result of oral ganciclovir in a heart transplant recipient with multiple human cytomegalovirus strains in blood.

BACKGROUND: The emergence of a ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) strain in a heart transplant recipient (HTR) coinfected by multiple HCMV strains was investigated. METHODS: A HTR with primary HCMV infection was treated with three induction courses of intravenous GCV followed by a 2-month maintenance treatment with oral GCV. HCMV antigenemia, viremia, and leukoDNAemia levels were monitored. GCV susceptibility was analyzed by an immediate-early antigen plaque reduction assay and by a rapid screening assay performed using peripheral blood leukocytes (PBL) as viral inoculum. The viral population in blood was investigated by restriction analysis of multiple genome regions. UL97 and UL54 genes were sequenced in parallel in both HCMV isolates and the relevant PBL samples. A rapid molecular assay for detection and quantitation of the GCV-resistant mutant was developed. RESULTS: The emergence of a GCV-resistant UL97 mutant (Cys-607 --> Tyr) was responsible for treatment failure during oral GCV therapy. The genetic analysis of the HCMV population showed the sequential appearance in blood of two unrelated strains (referred to as A and B). Strain A most likely derived from the transplanted organ and strain B from a subsequent blood transfusion. The resistant variant (Br) emerged from strain B and became predominant "in vivo" under the GCV pressure. However, after foscarnet administration, the resistant mutant disappeared in viral isolates, whereas it was still present as a minor proportion in PBL samples. CONCLUSION: (a) Oral GCV may select resistant strains in transplanted patients; (b) results of the rapid screening assay were clinically useful for shifting to an alternative treatment, thus avoiding the appearance of HCMV disease; (c) virus isolation may not be the most reliable approach to detection of HCMV drug-resistant strains; (d) a novel molecular assay for rapid detection of UL97 Cys-607 --> Tyr mutation directly in clinical specimens was developed, allowing earlier "in vivo" detection of the resistant mutant.

Human cytomegalovirus (HCMV) leukodnaemia correlates more closely with clinical symptoms than antigenemia and viremia in heart and heart-lung transplant recipients with primary HCMV infection.

BACKGROUND: In the last few years, human cytomegalovirus (HCMV) viremia, pp65 antigenemia, and leuko- and plasma-DNAemia have been developed to quantitate virus in blood of immunocompromised patients with HCMV infection. However, thus far, no conclusive studies have been performed to define the correlation of each of the different assays with clinical symptoms in primary HCMV infections. METHODS: This correlation was investigated in a population of 20 heart and heart-lung transplant recipients with primary HCMV infection using standardized virological methods. RESULTS: Median peak HCMV viremia, antigenemia, and leukoDNAemia levels were 110 (0-2,000) p72-positive fibroblasts, 450 (27-2,000) pp65-positive leukocytes, and >10,000 (1,358-10,000) genome equivalents (GE) in the 14 symptomatic patients and 18 (1-130) p72-positive fibroblasts, 86.5 (5-350) pp65-positive leukocytes, and 248 (10-863) GE in the six asymptomatic patients, respectively. The difference was statistically significant for antigenemia (P=0.009) and leukoDNAemia (P<0.0001). However, on an individual basis, unlike viremia and antigenemia, all DNA peaks of the 6 asymptomatic patients were below the DNA range of the 14 symptomatic patients (<1,000 GE), while all the 14 symptomatic patients had DNA peaks higher than those of asymptomatic patients (>1,000 GE). Follow-up confirmed these results, showing that 1,000-2,000 GE was the threshold zone for emergence of clinical symptoms. Symptoms were never observed in patients with secondary DNA peaks, except for one patient suffering from an HCMV organ localization (HCMV gastritis). CONCLUSIONS: LeukoDNAemia is the viral parameter of choice for monitoring of primary HCMV infections and antiviral treatment in heart and heart-lung transplant recipients. In this patient population, antigenemia-guided preemptive therapy could be replaced by leukoDNAemia-based antiviral therapy.

Pathogenesis of human cytomegalovirus infection and cellular targets.

In acquired immunodeficiency syndrome patients with human cytomegalovirus (HCMV), disseminated infection, and end-organ disease, autopsy findings show a generalized HCMV infection of endothelial cells. On the other hand, immunocompromised transplanted patients show presence of virus and virus products in peripheral blood leukocytes (PBL), when affected by a disseminated HCMV infection. All diagnostic assays are based on the detection of virus and viral components in PBL or whole blood, including polymorphonuclear leukocytes and monocytes. The interplay between endothelial cells and leukocytes represents the pathogenetic basis for all clinical syndromes originating during disseminated HCMV infections and is the trigger for the transmission of HCMV from mother to fetus during primary infections of pregnant women. The two biologic properties of endothelial cell tropism and leukocyte (polymorphonuclear- and monocyte-) tropism are shared by all recent clinical HCMV isolates, whereas they are missing in laboratory-adapted strains. The potential role of HCMV in the pathogenesis of atherosclerosis both in the immunocompetent (after angioplasty) and the heart transplant patient is receiving support from seroepidemiologic findings, in vivo animal models, in vitro data, and also some clinical observations. The interaction of endothelial cells and leukocytes with subsequent spreading of infection to smooth muscle cells may be a major pathogenetic mechanism at the basis of this important vascular disease.

Herpes simplex virus-1-specific human cytotoxic T lymphocytes are induced in vitro by autologous virus-infected mononuclear cells.

A new technique for in vitro activation of cytotoxic T lymphocytes (CTLs) specific for herpes simplex virus type 1 (HSV-1) is described. Autologous phytohemagglutinin (PHA)-activated, HSV-1-infected peripheral blood mononuclear cells (PBMC) were used, after fixation with 1% paraformaldehyde, to activate virus-specific CTLs in short-term cultures. The same unfixed PBMC were used as target cells in the cytotoxicity assay. By using this technique high levels of HSV-1-specific cytotoxic activity (50.06 +/- 16.76% at 30:1 effector:target ratio) were repeatedly obtained in 24 experiments using PBMC from 16 HSV-1 antibody-positive healthy donors, while no cytotoxic activity was observed using PBMC from 3 HSV-1 antibody-negative donors. HSV-1-induced CTLs were shown to be virus-specific as they did not lyse autologous, PHA-activated PBMC infected with influenza A virus or autologous Epstein-Barr virus (EBV) lymphoblastoid cell line (LCL), while they were able to lyse both HSV-1-infected, autologous PHA-activated PBMC and EBV-LCL. HSV-1-specific cytotoxicity was mediated by T lymphocytes, since depletion of CD3-positive cells from the effector population completely removed the killing of HSV-1-infected target cells. CD8-positive CTLs were primarily involved in the killing of HSV-1-infected targets since depletion of CD8-positive cells caused a strong reduction of virus-specific cytotoxic activity while elimination of CD4-positive lymphocytes increased killing capacity. Finally, this technique has proven to be highly reproducible, easy to perform, and thus suitable for clinical investigations.

Constitutive expression of human cytomegalovirus (HCMV) glycoprotein gpUL75 (gH) in astrocytoma cells: a study of the specific humoral immune response.

The humoral immune response to gpUL75 (gH) was determined in different groups of human cytomegalovirus (HCMV) infected subjects using a full-length glycoprotein constitutively expressed in an astrocytoma cell line. The recombinant molecule consisted of two distinct isoforms resembling the authentic protein of infected cells. Separated from the interactions of other viral gene products gH failed to form an oligomeric complex, thus exhibiting exclusively epitopes present on the monomer. Ninety five percent of serum samples from latently-infected healthy adults revealed the presence of gH-specific IgG. Moreover, examination of sequential sera from immunocompromised and immunocompetent individuals undergoing active HCMV infection demonstrated that antibodies to gH occurred in most cases simultaneously with those to the abundant surface antigen gpUL55 (gB) and at similar titres. Appearance of this response was correlated with a considerable increase of the virus-neutralizing activity and most likely associated with restriction of viral dissemination during subsequent viremic episodes. Together, these results suggest that glycoprotein H of HCMV is like gB, a highly immunogenic component of the infectious particle.

Use of the human cytomegalovirus (HCMV) antigenemia assay for diagnosis and monitoring of HCMV infections and detection of antiviral drug resistance in the immunocompromised.

BACKGROUND: Quantification of viral load in blood has proven to be helpful in the follow-up of disseminated HCMV infections in immunocompromised patients. OBJECTIVES: (i) To describe the antigenemia assay and its optimization and (ii) to analyze the use of the antigenemia assay for the diagnosis and monitoring of HCMV infections and for the detection of treatment failures. STUDY DESIGN: This article is intended to give an overview of our experience in the use of the antigenemia assay. RESULTS AND CONCLUSIONS: In solid organ transplant recipients and patients with AIDS, HCMV symptomatic infections mostly appear when antigenemia values are > 300 pp65-positive PBL/2 x 10(5) examined. To avoid the appearance of overt HCMV disease antiviral treatment could be administered when antigenemia levels are > 100 pp65-positive PBL/2 x 10(5) examined. Bone marrow transplant recipients show symptomatic HCMV infections when antigenemia values are > 100 pp65-positive PBL/2 x 10(5) examined. This group of patients should be treated when antigenemia levels are < 10 pp65-positive PBL/2 x 10(5) examined due to the higher mortality rate associated with HCMV complications. A decrease in antigenemia levels during therapy indicates a good response to antiviral drug, while stable or increasing values document a treatment failure and the emergence of drug-resistant HCMV strains.

A comparison of methods for detecting adenovirus type 8 keratoconjunctivitis during a nosocomial outbreak in a Neonatal Intensive Care Unit.

BACKGROUND: An outbreak of epidemic keratoconjunctivitis (EKC) due to adenovirus (Ad) type 8 and involving 14 members of the hospital staff and 33 neonates admitted to the Neonatal Intensive Care Unit of the local University Hospital occurred between September and December 2000 in Pavia, Italy. The outbreak was preceded by an outbreak of EKC within the community. OBJECTIVE: To compare the performance of conventional virus isolation on cell cultures, direct detection of Ad antigens in conjunctival cells by a direct fluorescent assay (DFA) and Ad DNA detection in conjunctival swabs by polymerase chain reaction (PCR) for diagnosis of adenoviral conjunctivitis. STUDY DESIGN: Of conjunctival swabs collected from 47 patients, all were tested by virus isolation, 43 by direct Ad antigen detection, and 37 by Ad DNA detection. Direct Ad antigen detection was carried out by DFA using a group-specific monoclonal antibody. Detection and subgrouping of Ad DNA by nested PCR was performed using two sets of primers complementary to hexon and fiber genes, respectively. RESULTS: Ad was detected in 24/47 (51.1%), 21/43 (48.8%), and 23/37 (62.1%) samples by virus isolation, direct antigen detection and PCR, respectively. Overall, 30/47 (63.8%) samples were Ad-positive. Of 37 specimens tested in parallel by all three methods, Ad was detected by at least one of the three techniques in 26/37 (70.3%). All Ad isolates were identified as serotype 8 by neutralization, while all PCR-positive samples were identified as belonging to subgroup D. No other virus was isolated from any conjunctival swab. Time required for test completion was 9.6 (4-20) days for virus isolation, 1-2 h for DFA and 24 h for PCR. CONCLUSIONS: DFA was a sensitive and rapid assay but results depend on the quality of sample and the expertise of the observer. PCR was the most sensitive assay, although it takes longer to perform and requires dedicated facilities; thus, it could be restricted to DFA-negative samples. Virus isolation is still useful from an epidemiological point of view.

HCV genotyping by three methods: analysis of discordant results based on sequencing.

BACKGROUND: Correct genotyping of hepatitis C virus (HCV) RNA-positive serum samples may have important clinical and therapeutic implications. OBJECTIVES: Three methods were compared to improve accuracy of HCV genotyping. STUDY DESIGN: A panel of 144 HCV RNA-positive sera prospectively tested by a modified Okamoto's type-specific reverse transcription-nested polymerase chain reaction (RT-nPCR) (Okamoto H, Tokita H, Sakamoto M, Kojima M, Iizuka H, Mishiro S. J Gen Virol 1993; 74: 2385-2390) was retrospectively analyzed by two recently described methods which were reported to identify all HCV types and the majority of HCV subtypes: (i) a restriction fragment length polymorphism (RFLP) analysis of PCR products amplified from the 5' untranslated region (5'UTR) of the viral genome (Pohjanpelto P, Lappalainen M, Widell A, Asikainen K, Paunio M. Clin Diagn Virol 1996; 7: 7-16); and (ii) a type-specific RT-nPCR relevant to the core region (Ohno T, Mizokami M, Wu R, Saleh M, Ohba K, Orito E, Mukaide M, Williams R, Lau J. J Clin Microbiol 1997; 35: 201-207). The panel (according to results given by the modified Okamoto's method) consisted of: (i) 105 sera belonging to five different HCV subtypes; (ii) 20 specimens containing a mixture of > or = 2 genotypes; and (iii) 19 untypeable clinical samples. RESULTS: There was agreement of the three methods for 78/144 (54.2%) blood samples, whereas discordant results were obtained for the remaining 66 samples, 56 of which could be typed by sequencing. Of these, 51 (91.7%) were correctly typed by RFLP, 37 (66.0%) by Ohno's and 27 (48.2%) by the modified Okamoto's procedure. The overall genotyping sensitivity of each method over the total number of 134 samples whose genotype was ascertained, was 96.2% for RFLP, 85.8% for Ohno's and 78.3% for the modified Okamoto's procedure. CONCLUSIONS: RFLP analysis, notwithstanding some limitations in subtyping efficiency of genotype 1 samples, appears superior to the two RT-nPCR methods because: (i) it is able to type a larger number of samples; (ii) it is more efficient in identifying genotypes 2a/c, which are widespread in Italy; (iii) it is highly sensitive (together with Ohno's method) in recognizing genotypes 3 and 4.

Diagnostic and prognostic value of human cytomegalovirus load and IgM antibody in blood of congenitally infected newborns.

BACKGROUND: Diagnosis of congenital human cytomegalovirus (HCMV) infection relies on virus isolation from urine collected in the first 3 weeks of life. However, very little is known about the presence, levels and duration of HCMV pp65 antigenemia, viremia and DNAemia in congenitally infected newborns. OBJECTIVES: To investigate the diagnostic and prognostic value of HCMV load determination in blood of newborns/infants with congenital HCMV infection. STUDY DESIGN: HCMV pp65 antigenemia, viremia and DNAemia were investigated in 116 sequential peripheral blood leukocytes (PBL) samples from 41 newborns/infants with congenital HCMV infection and in 34 PBL samples from 34 uninfected newborn. Virus-specific IgM were determined in parallel on 145 sequential serum samples. RESULTS: Compared to virus isolation from urine, sensitivities of DNAemia, antigenemia, viremia, and IgM determination were 100, 42.5, 28.2, and 70.7%, respectively. Specificity was 100% for all assays. Antigenemia, viremia and DNAemia levels were significantly higher and persisted longer in newborns with symptomatic infection compared to subclinically infected babies, whereas no difference was observed for virus-specific IgM antibody between the two groups. CONCLUSIONS: (i) determination of viral DNA in blood at birth appears to be a sensitive and specific marker for diagnosis of congenital HCMV infection; (ii) significantly higher levels of HCMV load were detected in infants with symptomatic HCMV infection; and (iii) virus clearance from blood occurs spontaneously both in symptomatic and subclinically infected infants. However, the process takes longer in infants presenting with symptoms at birth.