Neutralizing and ELISA IgG antibodies to human cytomegalovirus glycoprotein complexes may help date the onset of primary infection in pregnancy.

BACKGROUND: Definition of onset for primary human cytomegalovirus (HCMV) infection during pregnancy is critical for several reasons, including diagnosis of pre-conceptional infections and definition of gestational age at the time of infection. OBJECTIVE: To determine the onset of primary HCMV infection, differential kinetics of antibodies neutralizing infection of epithelial and fibroblast cells, as well as ELISA IgG antibodies to HCMV glycoprotein complexes (gC) gH/gL/pUL128L, gH/gL/gO, and gB were exploited and compared with conventional assays. STUDY DESIGN: In a series of 40 pregnant women with primary HCMV infection and ascertained HCMV-related mild clinical symptoms, the kinetics of different types of neutralizing and ELISA IgG antibodies were investigated with the aim of establishing criteria for dating the onset of primary infection in pregnant women without clinical symptoms. RESULTS: IgG antibodies to gB and gH/gL/pUL128L, as well as antibodies neutralizing infection of epithelial cells appeared early after infection onset (within 2-3 weeks) and increased rapidly, whereas antibodies to gH/gL/gO and antibodies neutralizing infection of fibroblasts appeared later (>30 days) and increased slowly. Both the conventional diagnostic assays (IgG, and IgM antibody, and IgG avidity index) and the novel assays for determination of antibody responses directed against HCMV gC allowed the definition of an algorithm indicating the onset of primary HCMV infection in asymptomatic women within a period of 1-2 months. CONCLUSION: New neutralization and ELISA IgG assays to HCMV gC provide additional tools for dating the onset of primary infection in pregnancy.

Virologic and immunologic monitoring of cytomegalovirus to guide preemptive therapy in solid-organ transplantation.

Control of human cytomegalovirus (HCMV) infection during the posttransplant period was investigated in 134 solid-organ transplant recipients by monitoring in parallel virologic and immunologic parameters for at least 1 year of follow-up. Virologic monitoring was achieved by determining HCMV DNAemia with real-time PCR, using the threshold of 300 000 DNA copies/mL blood as a cutoff for starting preemptive therapy. Immunologic monitoring included measurement of HCMV-specific CD4+ and CD8+ T cells by cytokine flow cytometry, using HCMV-infected dendritic cells as a stimulus. HCMV infection was diagnosed in 110 (82%) and required treatment in 49 (36%) patients. At 12 months after transplantation 'protective' immunity (≥0.4 CD4+ and CD8+ HCMV-specific T cells/µL blood) was achieved in 115/129 (89%) patients. During the entire study period, 122 patients reconstituting HCMV-specific CD4+ and CD8+ T-cell immunity at 60 days posttransplant onward were able to control HCMV infection, except for one patient who developed HCMV disease because of a rejection episode. Patients reconstituting HCMV-specific CD8+ only did not control HCMV infection. In conclusion, the presence of both HCMV-specific CD4+ and CD8+ T cells ≥ 0.4/µL blood appears to be protective against HCMV disease. This result does not apply to patients undergoing antirejection treatment, or reconstituting HCMV-specific CD8+ T cells only.

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.

Correlation of rhinovirus load in the respiratory tract and clinical symptoms in hospitalized immunocompetent and immunocompromised patients.

While human rhinoviruses (HRVs) are well accepted as a major cause of common cold syndromes (rhinitis), their role in the etiology of lower respiratory tract infections is still controversial, and their detection in asymptomatic patients is relatively common. The HRV pathogenic role in four groups of hospitalized patients (pediatric immunocompetent and immunocompromised patients, and adult immunocompetent and immunocompromised patients) was investigated by quantifying HRV load in nasopharyngeal aspirates or bronchoalveolar lavage samples by real-time reverse transcription PCR (RT-PCR). Real-time RT-PCR was performed in duplicate on all respiratory samples resulting positive by qualitative RT-PCR. In addition, molecular typing allowed detection of all known HRV species (A, B, and C). In immunocompetent pediatric patients HRVs were mostly associated with lower respiratory tract infections (in the absence of other viral agents) and wheezing, when viral load was > or =10(6) RNA copies/ml. In young immunocompromised patients (stem cell transplantation recipients), an inverse correlation between HRV persistence over time and time at which the infection occurred after transplantation was observed, whereas in adult immunocompromised patients (lung transplant recipients) HRVs could be detected at a medium-low level (<10(5) RNA copies/ml) in bronchoalveolar lavage samples taken routinely from asymptomatic patients. In conclusion, when detected at high viral load, HRVs may cause severe upper and lower respiratory tract infections, whereas when detected at a medium-low viral load, an event more frequent in immunocompromised subjects, they may represent only bystander viruses.

Preemptive therapy for systemic and pulmonary human cytomegalovirus infection in lung transplant recipients.

The incidence and treatment of both systemic and pulmonary human cytomegalovirus (HCMV) infection as well as HCMV-specific T-cell immune responses were investigated in 57 consecutive lung transplant recipients (LTR) by using as cutoffs for preemptive therapy: 300 000 DNA copies/mL whole blood for systemic infections and 100 000 DNA copies/mL bronchoalveolar lavage fluid for lung infections. Results showed that out of 29/57 LTR (50.9%) needing preemptive antiviral therapy, 15 (51.7%) reached the blood cutoff, 8 (27.6%) the pulmonary cutoff and 6 (20.7%) both the blood and the lung cutoff (3 simultaneously and 3 subsequently). Recovery of HCMV-specific T-cell immune responses was achieved much earlier for CD8+ than CD4+ T cells. However, protection from HCMV reactivation was conferred by the presence of both arms of the T-cell response. In two LTR reaching the pulmonary cutoff and not preemptively treated, a full HCMV-specific CD4+ and CD8+ T-cell response was associated with resolution of lung infection. Antirejection steroid therapy suppressed T-cell immune responses, thus facilitating HCMV reactivation. In conclusion, in LTR, monitoring HCMV infection in both blood and lungs, may improve preemptive therapy efficacy. In addition, monitoring the HCMV-specific T-cell immune response appears useful for predicting control of HCMV infection in the posttransplant period.

Clinical severity and molecular typing of human rhinovirus C strains during a fall outbreak affecting hospitalized patients.

BACKGROUND: The circulation rate and the clinical severity of infections caused by members of the new human rhinovirus C (HRV-C) species remain to be defined. OBJECTIVES: To investigate the epidemiologic and clinical impact of HRV-C strains in a fall outbreak interesting hospitalized patients. STUDY DESIGN: HRV species (A-C) were determined by phylogenetic analysis following amplification of two genome regions (5'NCR and VP4/VP2) by RT-PCR. HRV species were correlated with age, respiratory tract involvement, clinical symptoms, and HRV load in respiratory secretions. RESULTS: During the first week of the period October-November 2008, single HRV infections were associated with 95% of all respiratory syndromes affecting hospitalized patients. Then, HRV infections (single+coinfections) interested about 90% of positive samples until the end of October, when they declined in frequency until reaching about 30% at the end of November. Overall, 104 HRV strains were detected and, of these, 90 could be classified by phylogenetic analysis, as follows: 45 HRV-A, 12 HRV-B, 28 HRV-C, and 5 human enterovirus D strains. HRV-C identity was confirmed by detection of cis-acting replication elements (cre) in 23/23 strains. As for severity of respiratory syndromes, unlike HRV-A and HRV-B strains, HRV-C strains were responsible for a significantly higher rate (p<0.05) of lower respiratory tract infections in the pediatric as compared to adult patient population. CONCLUSIONS: HRV-C strains have been shown to circulate at a rate intermediate between HRV-A and HRV-B strains, showing a greater degree of clinical severity in the pediatric population.

Phylogenetic analysis of human coronavirus NL63 circulating in Italy.

BACKGROUND: Five known human coronaviruses infect the human respiratory tract: HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63 and HCoV-HKU1. OBJECTIVES: To evaluate the prevalence of HCoV-NL63 in hospitalized adult patients and to perform molecular characterization of Italian strains. STUDY DESIGN: HCoV-NL63 was sought by RT-PCR in 510 consecutive lower respiratory tract (LRT) samples, collected from 433 Central-Southern Italy patients over a 1-year period. Phylogenetic analysis was performed by partial sequencing of S and ORF1a. Additional S sequences from Northern Italy were included in the phylogenetic trees. RESULTS: HCoV-NL63 was detected in 10 patients (2.0%) with symptomatic respiratory diseases, mainly during winter. Phylogenetic analysis indicated a certain degree of heterogeneity in Italian isolates. The ORF1a gene clustering in phylogenetic trees did not match with that of the S gene. CONCLUSIONS: As observed by others, HCoV-NL63 is often associated with another virus. Phylogenetic characterization of HCoV-NL63 circulating in Italy indicates that this virus circulates as a mixture of variant strains, as observed in other countries.

Validation of a DNAemia cutoff for preemptive therapy of cytomegalovirus infection in adult hematopoietic stem cell transplant recipients.

A randomized trial comparing a DNAemia cutoff of 10 000 copies per ml whole blood and first pp65 antigenemia positivity for initiation of preemptive therapy of human cytomegalovirus (HCMV) infection in adult hematopoietic stem cell transplant recipients was completed. DNAemia was chosen for cutoff definition since it is more automatable and standardizable than antigenemia, and more closely reflects the actual viral replication. The primary end point of the study was to compare the number of patients treated in the two arms. A total of 83 patients (42 in the DNAemia, and 41 in the antigenemia arm) were enrolled in the study. The incidence of HCMV infection, as detected by the relevant randomization assay (76% in the DNAemia versus 85% in the antigenemia arm), was comparable in the two arms, whereas the number of patients treated was significantly lower in the DNAemia arm (63 versus 80%, P=0.02). A single patient in the DNAemia arm suffered from biopsy-proven HCMV gastric disease diagnosed in the absence of detectable virus in blood. The incidence of graft-versus-host disease, and transplantation-related mortality did not differ between the two arms. In conclusion, our study shows that the use of a cutoff significantly reduces the number of patients requiring antiviral treatment, thus sparing unnecessary drug administration.

Correlation of viral load as determined by real-time RT-PCR and clinical characteristics of respiratory syncytial virus lower respiratory tract infections in early infancy.

BACKGROUND: In infants hospitalized for a lower respiratory tract infection (RTI) caused by respiratory syncytial virus (RSV), the correlation between viral load (VL) and patient clinical characteristics remains to be defined. OBJECTIVES: To define this correlation. STUDY DESIGN: prospective study of 47 infants admitted to hospital in the period November 2006-May 2007 with a diagnosis of lower RTI. Nasopharyngeal aspirates (NPAs) were taken at admission, discharge, and at post-discharge control visits. VL was quantified by real-time RT-PCR for RSV subgroups A and B. RESULTS: Patients with bronchiolitis were compared with young patients with lower RTI other than bronchiolitis. Patients with bronchiolitis had a significantly lower age than patients with other syndromes, and a significantly longer duration of symptoms. Duration of hospitalization was not different in the two groups of patients, and was not related to RSV subgroup or viral coinfection. A sustained decrease in VL was observed in the general patient population between admission, discharge and post-discharge follow-up visits. CONCLUSIONS: (i) patients with bronchiolitis were significantly younger than patients with other lower RTIs; (ii) symptom duration was significantly longer in patients with bronchiolitis; (iii) RSV VL significantly decreased between admission and discharge.

Inconsistent responses of cytomegalovirus-specific T cells to pp65 and IE-1 versus infected dendritic cells in organ transplant recipients.

CD4(+) and CD8(+) T cells specific for human cytomegalovirus (HCMV) and two immunodominant HCMV antigens (pp65 and IE-1) were monitored in 20 solid organ transplant recipients undergoing primary (n = 4) or reactivated (n = 16) HCMV infection during the first year after transplantation by using as a stimulator either HCMV-infected autologous dendritic cells (DCs) or pp65- or IE-1 peptide mixtures. Turnaround times for test performance were 7 days for infected DCs and 24 h for peptides. Using infected DCs, HCMV-specific T-cell restoration occurred in all patients for CD8(+) and in 18/20 (90%) for CD4(+) T-cell subpopulations, resulting in virus clearance from blood. Using peptide mixtures, T-cell responses were less frequently detected. In detail, 14 (70%) patients showed pp65-specific CD8(+) T cells and 10 (50%) patients IE-1-specific CD8(+) T cells, whereas pp65-specific CD4(+) T cells were detected in 14 (70%) patients, and IE-1-specific CD4(+) T cells in three (15%) patients only. Protection from HCMV infection was associated with the presence of a HCMV-specific T-cell response directed against multiple viral proteins, but not against pp65 or IE-1 only. In conclusion, the use of pp65 and IE-1 peptide mixtures for rapid monitoring of HCMV-specific T-cell responses in solid organ transplant recipients underestimates the actual T-cell immune response against HCMV.

Control of human cytomegalovirus infection in patients infected with human immunodeficiency virus by high levels of specific CD8+ T-cells.

A new technique was used to simultaneously determine human cytomegalovirus (HCMV)-specific CD4(+) and CD8(+) T-cells in highly active anti-retroviral therapy (HAART)-naive and HAART-treated patients infected with human immunodeficiency virus (HIV). HIV-infected patients with HCMV infection, but without HCMV disease, showed low numbers of HCMV-specific CD4(+) cells and high numbers of CD8(+) T-cells, both before and during HAART. HIV-infected patients with HCMV disease had no HCMV-specific CD4(+) T-cells and extremely low levels of CD8(+) T-cells. Resolution of disease during HAART was associated with rescue of specific CD4(+) T-cells and a large increase in the specific CD8(+) T-cell count. Thus, HAART does not completely restore the normal immune function. In HIV-infected patients, sustained control of HCMV infection requires high frequencies of specific CD8(+) T-cells.

Monitoring of human cytomegalovirus-specific CD4 and CD8 T-cell immunity in patients receiving solid organ transplantation.

Absolute and human cytomegalovirus (HCMV)-specific CD4+ and CD8+ T-cell counts were monitored in 38 solid organ (20 heart, 9 lung and 9 kidney) transplant recipients during the first year after transplantation by a novel assay based on T-cell stimulation with HCMV-infected autologous dendritic cells. According to the pattern of T-cell restoration occurring either within the first month after transplantation or later, patients were classified as either early (n = 21) or late responders (n = 17). HCMV-specific CD4+ and CD8+ T-cell counts were consistently lower in late compared to early responders from baseline through 6 months after transplantation. In addition, in late responders, while HCMV infection preceded immune restoration, HCMV-specific CD4+ restoration was significantly delayed with respect to CD8+ T-cell restoration. The number of HCMV-specific CD4+ and CD8+ T-cells detected prior to transplantation significantly correlated with time to T-cell immunity restoration, in that higher HCMV-specific T-cell counts predicted earlier immune restoration. Clinically, the great majority of early responders (18/21, 85.7%) underwent self-resolving HCMV infections (p = 0.004), whereas the great majority of late responders (13/17, 76.5%) were affected by HCMV infections requiring antiviral treatment (p = <0.0001). Simultaneous monitoring of HCMV infection and HCMV-specific T-cell immunity predicts T-cell-mediated control of 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.

Incidence of human cytomegalovirus infection in patients with refractory solid tumors receiving nonmyeloablative allogeneic stem cell transplants versus recipients of standard SCT for hematologic malignancies.

Human cytomegalovirus (HCMV) infection is the most frequent infectious complication after conventional allogeneic stem cell transplantation (alloSCT). From December 1998 to December 2002, we prospectively monitored HCMV reactivation in 59 patients affected by solid tumors and undergoing nonmyeloablative alloSCT (NST). Patients were allografted from HLA-identical sibling donors after fludarabine/cyclophosphamide-based conditioning regimens. Seventeen (28.8%) of 59 patients presented with HCMV antigenemia, and 14 received ganciclovir, with successful HCMV clearance in all cases. No patient developed HCMV viremia or disease. The median time to HCMV reactivation was 54 days (range, 16-245 days) after NST. These patients were compared with a cohort of hematologic patients who were treated with conventional myeloablative alloSCT. Matching criteria included HCMV risk group, stem cell source, donor type, and age. In the myeloablative group, HCMV active infection was observed in 47 (85.4%) of 55 patients at a median time of 30 days (range, 13-64 days) after alloSCT, and HCMV infection occurred more frequently ( P < .001) and earlier ( P = .001) than in NST patients. Patients affected with solid tumors undergoing NST had a reduced and delayed incidence of HCMV active infection.

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.

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.