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.

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.

Human cytomegalovirus UL131A, UL130 and UL128 genes are highly conserved among field isolates.

Coding sequences of the UL131A, UL130, and UL128 genes of human cytomegalovirus (HCMV) were found to be highly conserved among 34 field isolates from pregnant women with primary HCMV infection and their fetuses or newborns, as well as from solid organ transplant recipients and patients with AIDS. No strain clustering was observed. In contrast, sequencing of UL55 (gB coding gene) allowed the 34 isolates to be clustered into 4 genotypes. The conservation of the UL131A-UL128 locus is consistent with the conclusion that the three encoded proteins are all essential for growth of HCMV in endothelial cells and virus transfer to leukocytes.

Changing circulation rate of human metapneumovirus strains and types among hospitalized pediatric patients during three consecutive winter-spring seasons. Brief report.

From 2001 through 2004, 808 pediatric patients admitted to hospital because of acute respiratory infections were examined for presence of respiratory viruses by either direct fluorescent staining using monoclonal antibodies or RT-PCR during three consecutive winter-spring seasons. On the whole, 336 (42%) patients were detected as positive for one or more respiratory viruses. The most widely circulating virus was human respiratory syncytial virus (hRSV) infecting 50% of positive patients, followed by human metapneumovirus (hMPV) found in 13% of patients, and then by influenza virus type A, human parainfluenzaviruses and coinfections. Significant variations in the circulation rate of hRSV, hMPV and influenzavirus type A were observed during the individual seasons. In addition, the circulation rates of the different types of hMPV changed yearly. In 2001-2002 and 2002-2003 hMPV circulated at a significant lower proportion than hRSV, while in 2003-2004 the circulation rates of the two viruses were closer. In conclusion, the 4 hMPV subtypes circulated yearly in Northern Italy flanking hRSV as major respiratory pathogens in the infantile patient population.

Congenital rubella infection following rubella outbreak in northern Italy, 2002: need for an effective vaccination programme.

Presented here are the details of a rubella outbreak that occurred in 2002 in the Lombardy region of northern Italy followed by a discussion of rubella vaccination policy in this country. From 13 maternal cases of rubella infection, congenital rubella infection was diagnosed in three fetuses and three newborns. Of the three infected fetuses, one was aborted and two died in utero, while of the three infected newborns, two were born with severe disease and one was subclinically infected. Follow-up revealed that one of the two symptomatic newborns had died at 4 months of age with disseminated rubella infection, while the other suffered from bilateral blindness and deafness and was severely retarded at 15 months of age. The remaining infant remained asymptomatic at 14 months. Congenital rubella remains a serious health problem in Italy and a successful vaccination strategy is required.

Rubella control in Italy.

In Italy, rubella vaccination has been recommended since 1972 for pre-adolescent girls, and since the early 1990s for all children in the second year of life. Nevertheless, coverage in children from 12 to 24 months of age is suboptimal (i.e., 56% in 1998, 78% in 2003), with wide variations among regions. As a result, rubella is still circulating in Italy, and in 1996 the percentage of women susceptible to rubella between 15 and 39 years of age was >5%. Congenital rubella syndrome (CRS) was a notifiable disease between 1987 and 1991, with a range of 8-76 cases reported annually. Since 1992, national incidence data are no longer available, but local reports show that CRS cases are still occurring. Nationwide, coordinated and uniform actions are needed to control CRS effectively. For this reason, the National Plan for the Elimination of Measles and of Congenital Rubella has recently been launched. This plan includes strategies aimed at increasing MMR vaccination coverage in children and specific control measures for congenital rubella control, i.e., improving the vaccination of susceptible women of childbearing age, and reintroducing national surveillance of CRS.

Optimized detection of respiratory viruses in nasopharyngeal secretions.

Nasopharyngeal secretions (NPS) from 121 (110 pediatric) patients with acute respiratory infections were examined for respiratory virus detection by: i) conventional virus isolation in cell cultures (CC) using HEp-2, LLC-MK2, and MDCK cells; ii) rapid virus isolation using shell vial cultures (SVC) of a mixture (MIX) of mink lung epithelial cells (Mv1Lu) and human lung carcinoma (A549) cells in comparison to LLC-MK2 and MDCK cells; iii) direct fluorescent antibody (DFA) assay on NPS cells. A pool of monoclonal antibodies (MAbs) to influenzavirus A and B, parainfluenzavirus types 1 to 3, adenoviruses and respiratory syncytial virus (RSV), as well as single MAbs to the same viruses, were used for virus identification in all three procedures. Results on 101 NPS examined in parallel showed a sensitivity of 89.5%, 73.7%, and 81.6% for CC, SVC, and DFA, respectively, with the relevant negative predictive values of 94.0%, 86.3%, and 90.0%. Specificity and positive predictive values were 100%. However, the combination of DFA and SVC gave best results in terms of sensitivity (94.7%) and negative predictive value (95.5%). Use of the new MIX cell culture system in the SVC procedure enhanced virus detection, while use of the MAb pool allowed prompt identification of negative samples and saving of reagents and time for all three procedures. The combination of DFA and SVC allows diagnosis of the large majority of viral respiratory infections within 48h, while conventional virus isolation on CC may be limited to laboratories involved in research and epidemiological studies.

Declining levels of rescued lymphoproliferative response to human cytomegalovirus (HCMV) in AIDS patients with or without HCMV disease following long-term HAART.

OBJECTIVE: To investigate the lymphoproliferative response (LPR) to human cytomegalovirus (HCMV) in two groups of AIDS patients undergoing long-term highly active antiretroviral therapy (HAART): group 1 ( n = 22) with nadir CD4(+) cell count <50/microl and no HCMV disease; group 2 ( n = 16) with <50/microl CD4(+) T-cell count and HCMV disease. All patients had previously undergone antiretroviral monotherapy or dual therapy before initiating HAART. STUDY DESIGN AND METHODS: The two groups of patients were tested prospectively for CD4(+) T cell count, HIV RNA load, HCMV viremia, and LPR to HCMV at baseline, and then after 3 and 4 years of HAART. A control group of 13 recently diagnosed treatment-naive AIDS patients with CD4(+) T-cell counts <100/microl was also investigated. RESULTS: No LPR to HCMV was found in any of the treatment-naive patients nor in any patient of the two groups examined at baseline, when HCMV viremia was 13.6% in the patient group without disease and 87.5% in the group with disease ( p <.0001). After 3 years of HAART, the frequency of patients who recovered an LPR to HCMV was not significantly different (81.8% in the group without HCMV disease, and 68.7% in the group with HCMV disease), whereas, compared with baseline, the HIV load decreased and the CD4(+) T-cell count increased significantly and to a comparable extent in the two groups of patients. In addition, the frequency of patients with HCMV viremia, although reduced, became comparable in both groups. After 4 years of HAART, the frequency of responders to HCMV without and with HCMV disease dropped to comparable levels (50.0 vs. 56.3%, respectively) in association with high median CD4(+) T-cell counts and low median HIV RNA plasma levels. In parallel, the frequency of patients with HCMV viremia did not change significantly. In addition, after between 3 and 4 years of HAART, although the frequency of stable responders and nonresponders remained unchanged (50%) in both groups, most of the remaining patients showed declining levels of responsiveness to HCMV. Although some patients from both groups were found to have CD4(+) T-cell counts >150/microl in the absence of LPR to HCMV, thus suggesting dissociation of specific and nonspecific immune reconstitution, a significant correlation was found between CD4(+) T-cell count and LPR to HCMV (r = 0.44; p <.001). From a clinical standpoint, anti-HCMV therapy could be safely discontinued in 8 patients with HCMV retinitis showing CD4(+) T-cell counts >150/microl, recovery of HCMV LPR, and no HCMV viremia. CONCLUSIONS: Declining levels of the previously recovered LPR to HCMV are often observed after long-term HAART. However, because the role of LPR in the evolution of HCMV infection and disease during HAART remains to be defined, the clinical impact of the declining LPR to HCMV must still be clarified in long-term prospective studies.

Comparative analysis of human cytomegalovirus-specific CD4(+) T-cell frequency and lymphoproliferative response in human immunodeficiency virus-positive patients.

Evaluation of human cytomegalovirus (HCMV)-specific T-helper immunity could contribute in optimizing anti-HCMV therapy in human immunodeficiency virus (HIV)-infected patients. Testin the lymphoproliferative response (LPR) is the standard technique used to evaluate T-helper response, but its use in the routine diagnostic laboratory setting can be problematic. The most promising new alternative technique is the determination of HCMV-specific CD4(+) T-cell frequency by flow cytometry detection of intracellular cytokine production after short-term antigen-specific activation of peripheral blood mononuclear cells. HCMV-specific LPR and CD4(+) T-cell frequency were compared in a group of 78 blood samples from 65 HIV-infected patients. The results showed concordance in 80.7% of samples. In addition, comparative analysis of sequential blood samples from 13 HIV-infected patients showed that while in about half of patients the T-helper HCMV-specific immune response remained stable during highly active antiretroviral therapy (HAART), in the other half declining levels of the HCMV-specific CD4(+)-mediated immune response were determined by either one or both assays. In conclusion, our data suggest that the determination of HCMV-specific CD4(+) T-cell frequency can be considered a valuable alternative to the LPR test for the detection of HCMV-specific T-helper response in HIV-infected patients. It could facilitate wider screening of anti-HCMV T-helper activity in HIV-infected patients, with potential benefits for clinicians in deciding strategies of discontinuation or maintenance of anti-HCMV therapy.

Human cytomegalovirus immediate-early messenger RNA in blood of pregnant women with primary infection and of congenitally infected newborns.

Human cytomegalovirus (HCMV) immediate-early messenger RNA (IEmRNA) in sequential blood samples from 32 pregnant women with primary infection and from 14 congenitally infected newborns was qualitatively investigated by nucleic acid sequence-based amplification. IEmRNA was detected in 100%, 75%, 36.3%, 22.2%, and 0% of samples collected 1, 2, 3, 4-6, and >6 months after onset of primary HCMV infection, respectively, showing 83.7% sensitivity and 92.2% specificity, compared with results of quantitative DNAemia (detection of viral DNA in blood). In infected newborns, IEmRNA was positive in 100% of samples collected 1-7 days (median, 1.5 days) and in 46.4% of samples collected 27-260 days (median, 88 days) after birth, showing 75.7% sensitivity and 100% specificity, compared with DNAemia results. IEmRNA was not detected in HCMV-immune individuals with remote or recurrent HCMV infection or in uninfected newborns. IEmRNA determination appears to be a valuable tool for early diagnosis of both primary and congenital HCMV infection.

Human cytomegalovirus replicates abortively in polymorphonuclear leukocytes after transfer from infected endothelial cells via transient microfusion events.

Using a recently developed model for in vitro generation of pp65-positive polymorphonuclear leukocytes (PMNLs), we demonstrated that PMNLs from immunocompetent subjects may harbor both infectious human cytomegalovirus (HCMV) and viral products (pp65, p72, DNA, and immediate-early [IE] and pp67 late mRNAs) as early as 60 min after coculture with human umbilical vein endothelial cells (HUVEC) or human embryonic lung fibroblasts (HELF) infected with a clinical HCMV isolate (VR6110) or other wild-type strains. The number of PMNLs positive for each viral parameter increased with coculture time. Using HELF infected with laboratory-adapted HCMV strains, only very small amounts of viral DNA and IE and late mRNAs were detected in PMNLs. A cellular mRNA, the vascular cell adhesion molecule-1 mRNA, which is abundantly present in both infected and uninfected HUVEC, was detected in much larger amounts in PMNLs cocultured with VR6110-infected cells than in controls. Coculture of PMNLs with VR6110-infected permissive cells in the presence or absence of RNA, protein, and viral DNA synthesis inhibitors showed that only IE genes were transcribed in PMNLs during coculture. Synthesis of IE transcripts in PMNLs was also supported by the finding that only the copy number of IE mRNA (and not the DNA or the pp67 mRNA) per infected PMNL increased markedly with time, and the pp67 to IE mRNA copy number ratio changed from greater than 10 in infected HUVEC to less than 1 in cocultured PMNLs. Fluorescent probe transfer experiments and electron microscopy studies indicated that transfer of infectious virus and viral products from infected cells to PMNLs is likely to be mediated by microfusion events induced by wild-type strains only. In addition, HCMV pp65 and p72 were both shown to localize in the nucleus of the same PMNLs by double immunostaining. Two different mechanisms may explain the virus presence in PMNLs: (i) one major mechanism consists of transitory microfusion events (induced by wild-type strains only) of HUVEC or HELF and PMNLs with transfer of viable virus and biologically active viral material to PMNLs; and (ii) one minor mechanism, i.e., endocytosis, occurs with both wild-type and laboratory strains and leads to the acquisition of very small amounts of viral nucleic acids. In conclusion, HCMV replicates abortively in PMNLs, and wild-type strains and their products (as well as cellular metabolites and fluorescent dyes) are transferred to PMNLs, thus providing evidence for a potential mechanism of HCMV dissemination in vivo.

Human cytomegalovirus immediate-early mRNA detection by nucleic acid sequence-based amplification as a new parameter for preemptive therapy in bone marrow transplant recipients.

Human cytomegalovirus (HCMV) infection was monitored retrospectively by qualitative determination of immediate-early (IE) mRNA by nucleic acid sequence-based amplification (NASBA) in a series of 51 bone marrow transplant (BMT) recipients. The qualitative results for IE mRNA obtained by NASBA were compared with those obtained by prospective quantitation of HCMV viremia and antigenemia and retrospective quantitation of DNA in blood (DNAemia) by PCR as well as by qualitative determination of late pp67 mRNA by NASBA. On the whole, of the 39 HCMV-positive patients (all asymptomatic), HCMV was detected in 14 (35.9%) by quantitation of viremia, 15 (38.5%) by detection of pp67 mRNA by NASBA, 32 (82.1%) by quantitation of DNAemia, and 33 (84.6%) by quantitation of antigenemia, while HCMV was detected in 38 (97.4%) patients by detection of IE mRNA by NASBA. In the immunocompetent host, IE mRNA was not detected by NASBA in 100 blood donors or during reactivated infections in 30 breast-feeding mothers. Likewise, NASBA did not detect IE mRNA in 56 solid-organ transplant recipients in the first 21 days after transplantation. By using NASBA for detection of IE mRNA as the reference standard for detection of HCMV infection in blood samples, the diagnostic sensitivities were 67.7% for quantitation of DNAemia, 59.0% for quantitation of antigenemia, 18.3% for detection of pp67 mRNA by NASBA, and 16.0% for quantitation of viremia. Specificities and negative and positive predictive values were >90.0, >70.0, and >80.0%, respectively, for all four assays. The mean times to first HCMV detection after bone marrow transplantation were 37.7 +/- 15.4 days for detection of IE mRNA by NASBA, 39.6 +/- 15.6 days for quantitation of antigenemia, 40.9 +/- 15.2 days for quantitation of DNAemia, and 43.7 +/- 16.3 or 43.7 +/- 17.5 days for quantitation of viremia and detection of pp67 mRNA by NASBA, respectively. On the whole, 31 BMT recipients received preemptive therapy by using confirmed antigenemia positivity as a cutoff, while 35 patients could have been treated by using NASBA positivity as a cutoff and 31 could have been treated by using quantitation of DNAemia as a cutoff. In single patients, IE mRNA was detected in every episode of active HCMV infection, mostly preceding and sometimes accompanying antigenemia and DNAemia, whereas pp67 mRNA was detected only concomitantly with the highest peaks of infection. HCMV IE mRNA detection may represent a useful parameter for initiation of preemptive therapy in BMT recipients.

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.

Diagnostic value of viral culture, polymerase chain reaction and western blot for HIV-1 infection in 218 infants born to HIV-infected mothers and examined at different ages.

In a prospective longitudinal 10-year (1988 to 1998) study, 308 sequential blood samples from 218 infants born to HIV-1 seropositive women were examined by blood culture, polymerase chain reaction (PCR) and Western Blot (WB) for HIV-1 infection within the first month of life (no. 47 specimens), at 2-6 (no. 125), 7-18 (no. 80), and > 18 (no. 56) months after birth. Clinical status at follow-up after the initial diagnosis of HIV infection was also evaluated. Vertically transmitted HIV infection was diagnosed in 45 children (24 children were diagnosed before 18 months of age), whereas 173 were found to be uninfected (transmission rate 20.6%). Sensitivities of viral culture, PCR and WB were 95.2%, 97.8%, 94.4%, and specificities were 99.5%, 97.6% and 20.7%, respectively. Thus, cumulative positive predictive values (PPV) of blood culture, PCR and WB were 97.5%, 88.2% and 23.4%, while negative predictive values (NPV) were 99.0%, 99.6% and 100.0%, respectively. In view of defining the optimal time of sampling for a correct diagnosis of HIV infection, a PPV of 100.0% was achieved earlier by viral culture (2-6 months of age) than by PCR (7-18 months of age). Meanwhile, a NPV of 100% was obtained earlier by PCR (within the first month of age) than by viral culture (2-6 months). These results indicate that a combination test strategy requiring two blood samples analyzed by viral culture and PCR may confirm or exclude HIV perinatal infection within the first 2 months of life rather than being delayed to later times. Clinical follow-up was performed in 35 children, of whom 7 developed a rapidly progressive disease, 23 showed a slow progression, while 5 children are still younger than 5 years and do not present severe clinical symptoms.

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.

Quantification of human cytomegalovirus DNA in amniotic fluid of mothers of congenitally infected fetuses.

A quantitative PCR assay was used to quantitate human cytomegalovirus DNA in amniotic fluid of mothers of 21 fetuses with congenital infection. Seven fetuses presented ultrasound abnormalities or were born with symptoms, whereas 14 fetuses were subclinically infected. Although the median DNA level was higher in symptomatic fetuses, the difference was not statistically significant (P = 0.09).

Clinical significance of expression of human cytomegalovirus pp67 late transcript in heart, lung, and bone marrow transplant recipients as determined by nucleic acid sequence-based amplification.

Human cytomegalovirus (HCMV) infection was monitored retrospectively by qualitative determination of pp67 mRNA (a late viral transcript) by nucleic acid sequence-based amplification (NASBA) in a series of 50 transplant recipients, including 26 solid-organ (11 heart and 15 lung) transplant recipients (SOTRs) and 24 bone marrow transplant recipients (BMTRs). NASBA results were compared with those obtained by prospective quantitation of HCMV viremia and antigenemia and retrospective quantitation of DNA in leukocytes (leukoDNAemia). On the whole, 29 patients were NASBA positive, whereas 10 were NASBA negative, and the blood of 11 patients remained HCMV negative. NASBA detected HCMV infection before quantitation of viremia did but after quantitation of leukoDNAemia and antigenemia did. In NASBA-positive blood samples, median levels of viremia, antigenemia, and leukoDNAemia were significantly higher than the relevant levels detected in NASBA-negative HCMV-positive blood samples. By using the quantitation of leukoDNAemia as the "gold standard," the analytical sensitivity (47.3%), as well as the negative predictive value (68. 3%), of NASBA for the diagnosis of HCMV infection intermediate between that of antigenemia quantitation (analytical sensitivity, 72. 3%) and that of viremia quantitation (analytical sensitivity, 28.7%), while the specificity and the positive predictive value were high (90 to 100%). However, with respect to the clinically relevant antigenemia cutoff of >/=100 used in this study for the initiation of preemptive therapy in SOTRs with reactivated HCMV infection, the clinical sensitivity of NASBA reached 100%, with a specificity of 68. 9%. Upon the initiation of antigenemia quantitation-guided treatment, the actual median antigenemia level was 158 (range, 124 to 580) in SOTRs who had reactivated infection and who presented with NASBA positivity 3.5 +/- 2.6 days in advance and 13.5 (range, 1 to 270) in the group that included BMTRs and SOTRs who had primary infection (in whom treatment was initiated upon the first confirmation of detection of HCMV in blood) and who presented with NASBA positivity 2.0 +/- 5.1 days later. Following antiviral treatment, the durations of the presence of antigenemia and pp67 mRNA in blood were found to be similar. In conclusion, monitoring of the expression of HCMV pp67 mRNA appears to be a promising, well-standardized tool for determination of the need for the initiation and termination of preemptive therapy. Its overall clinical impact should be analyzed in future prospective studies.

Standardization of the human cytomegalovirus antigenemia assay by means of in vitro-generated pp65-positive peripheral blood polymorphonuclear leukocytes.

We generated in vitro human cytomegalovirus (HCMV) pp65-positive polymorphonuclear leukocytes (PMN) resembling those detected in vivo, following cocultivation of PMN from healthy donors and wild-type HCMV-infected endothelial cells or fibroblasts. After purification, PMN are suitable for preparation of cytospots which can be used for the antigenemia assay. Cytospin preparations containing a predetermined number of in vitro-generated pp65-positive PMN were used to test some of the major parameters involved in performing the antigenemia assay. The results showed or confirmed that (i) formalin fixation followed by permeabilization is the best fixation procedure developed to date, (ii) the test performance levels provided by different pools of pp65-specific monoclonal antibodies may be significantly different, and (iii) long-term storage (for an unlimited time) is best achieved by keeping fixed slides at -80 degreesC, whereas short-term storage (for up to 1 month) is best achieved by keeping unfixed slides at room temperature. This finding signifies that slides can be shipped all over the world at room temperature. In conclusion, the newly developed procedure for in vitro generation of pp65-positive PMN will provide the basis for standardization of the HCMV antigenemia assay and development of quality control programs.