Evaluation of cytomegalovirus DNAaemia versus pp65-antigenaemia cutoff for guiding preemptive therapy in transplant recipients: a randomized study.

METHODS: A bicentre, randomized, prospective open-label study aimed at defining a DNAaemia versus antigenaemia cutoff for guiding preemptive therapy of human cytomegalovirus (HCMV) infections in solid organ transplant recipients (SOTR) was completed. Overall, 99 patients were enrolled in the DNAaemia arm and 101 patients in the antigenaemia arm. Patients were randomized to be monitored for HCMV infection in the blood by either assay. Antiviral treatment was started in both seropositive and seronegative patients when levels greater than 300,000 DNA copies/ml blood or 100 pp65-positive leukocytes in the relevant arm were reached. RESULTS: HCMV infection was detected in 81/99 (81.8%) patients in the DNAaemia arm and in 87/101 (86.1%) patients in the antigenaemia arm (P=ns). Antiviral treatment was given to 23/99 (23.0%) patients in the DNAaemia arm and 42/101 (41.0%) patients in the antigenaemia arm (P = 0.01). In the DNAaemia arm, antiviral therapy was significantly delayed and duration of the first course of treatment was significantly greater than in the antigenaemia arm. However, total duration of treatment was comparable in the two arms. No case of HCMV disease occurred in patients treated after reaching the relevant cutoff. However, four patients (three in the antigenaemia arm, and one in the DNAaemia arm) suffered from HCMV disease prior to reaching the relevant cutoff. CONCLUSIONS: Compared with antigenaemia, a single DNAaemia cutoff: (i) significantly reduces the number of patients requiring treatment; (ii) may be safely adopted to guide preemptive therapy of both primary and reactivated HCMV infections in SOTR; and (iii) does not significantly modify the overall duration of treatment.

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.

Monoclonal antibodies versus reverse transcription-PCR for detection of respiratory viruses in a patient population with respiratory tract infections admitted to hospital.

UNLABELLED: In the winter season 2001-2002, 239 nasopharyngeal aspirate and 15 bronchoalveolar lavage samples from 208 patients (135 pediatric and 73 adults, including 19 lung transplant recipients) admitted to hospital because of an acute respiratory tract infection were examined for rapid diagnosis of respiratory viruses by two diagnostic approaches: immunological, using specific monoclonal antibodies (MAb); and molecular, using specific reverse transcription (RT)-PCR assays. Both methods detected influenza viruses A (H1N1 and H3N2) and B, human parainfluenza virus types 1 to 3, human respiratory syncytial virus (hRSV) types A and B, and human adenoviruses. In addition, human coronavirus (hCoV) groups I (229E-like) and II (OC43-like), as well as the new human metapneumovirus (hMPV), types A and B, were searched for by RT-PCR alone. When results obtained by both methods were added, the overall percentage of patients positive for at least one respiratory virus peaked at 44.2%, involving 92/208 patients (81 pediatric, and 11 adults), while 116 patients (55.8%) were negative for any respiratory virus tested. The most common circulating virus was hRSV, infecting 54 (25.9%) patients (24 type A, and 30 type B strains), followed by hMPV, infecting 12 (5.8%) patients (7 type A and 5 type B strains). Coinfections by two respiratory viruses interested 11 (5.3%) patients, and 9 (81.8%) of these were infected by hRSV in association with another respiratory virus. In the great majority of infected children, hRSV and hMPV were associated with lower respiratory tract infections. In lung transplant recipients, viruses present in bronchoalveolar lavage appeared to be associated frequently with lower respiratory tract infections. IN CONCLUSION: the combination of immunological and molecular assays is the most sensitive approach to the diagnosis of respiratory viral infections; and infections caused by the less investigated hCoVs and hMPVs represent a fair proportion of respiratory infections.

Clinically-based determination of safe DNAemia cutoff levels for preemptive therapy or human cytomegalovirus infections in solid organ and hematopoietic stem cell transplant recipients.

Transplantation Centers using human cytomegalovirus (HCMV) antigenemia-based preemptive therapy will need to replace in the near future the antigenemia assay with a more standardized and automatable assay, such as a molecular assay quantifying HCMV DNA in blood (DNAemia). Thus, in view of replacing antigenemia with clinically safe cutoff values, DNAemia levels corresponding to antigenemia cutoffs guiding HCMV preemptive therapy were determined retrospectively in solid organ and hematopoietic stem cell transplant recipients (HSCTR) using an "in-house" quantitative PCR (QPCR) method. Since preemptive therapy had prevented appearance of HCMV disease in all patients tested, DNA cutoffs determined retrospectively had to be considered as safe clinically as antigenemia cutoffs used prospectively. However, in solid organ transplant recipients (SOTR), initiating preemptive therapy upon an antigenemia cutoff of 100 pp65-positive leukocytes, a DNAemia cutoff of 300,000 copies/ml blood had positive and negative predictive values of >90%, indicating that a DNAemia cutoff could achieve, in terms of prevention of HCMV disease, the same clinical results as the antigenemia cutoff. In HSCTR, initiating preemptive therapy upon first antigenemia positivity, a DNAemia cutoff of 10,000 copies/ml blood had a positive predictive value of >90%, indicating that the great majority of patients treated under the antigenemia guidance would have been treated also using this DNA cutoff. On the other hand, the negative predictive value of 28.6% indicated that two out of three HSCTR had been treated under the antigenemia guidance having the same levels of viral DNA as the untreated patients. The data suggest that a quantitative cutoff could be adopted as a guiding criterion for preemptive therapy also in HSCTR. Regression analysis allowed to determine the DNAemia (corresponding to QPCR) cutoff values for two commercial assays tested both in solid organ and HSCTR. Retrospective DNAemia cutoff values will be verified for safety in prospective trials.

In vitro model for the study of the dissociation of increasing antigenemia and decreasing DNAemia and viremia during treatment of human cytomegalovirus infection with ganciclovir in transplant recipients.

The paradox phenomenon (i.e., the dissociation of increasing antigenemia and decreasing DNAemia and viremia) that occurs during treatment of human cytomegalovirus (HCMV) infections with ganciclovir (Gcv), in transplant recipients, was investigated by use of an in vitro model for the study of interactions between polymorphonuclear leukocytes and endothelial cells. The paradox phenomenon was reproduced in vitro in the presence of Gcv and, to a much lesser extent, in the presence of cidofovir, but not in the presence of foscarnet. The pathogenetic basis for such a paradox response was found, by use of drug concentrations in the range of 90%-99% of the inhibitory dose, to rely on the partial synthesis of HCMV phosphoprotein 65. The opposite situation (i.e., the simultaneous increase of antigenemia, viremia, and DNAemia), which is observed in clinical conditions associated with inefficacy of treatment due to drug-resistant strains, was also reproduced in vitro by use of drug-resistant HCMV strains. The conclusion for clinicians is that antiviral therapy must be changed only in the latter case.

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.

Human cytomegalovirus pp67 mRNAemia versus pp65 antigenemia for guiding preemptive therapy in heart and lung transplant recipients: a prospective, randomized, controlled, open-label trial.

BACKGROUND: Preemptive therapy of human cytomegalovirus (HCMV) infections has gained popularity in transplantation centers. However, standardized protocols are not available. In particular, whether a qualitative molecular assay for detection of a late (pp67) HCMV mRNA represents a valuable alternative to quantitative antigenemia remains to be defined. METHODS: Overall, 82 heart (HTR) and lung (LTR) transplant recipients were randomized into two arms, where therapy was guided by qualitative pp67 mRNA NASBA (40 patients) or quantitative antigenemia (42 patients). In the NASBA arm, both primary and recurrent infections were treated upon first confirmed positive NASBA result. In the antigenemia arm, primary infections were treated upon first confirmed positive result, while recurrent infections were treated upon cutoff of 100 pp65-positive leukocytes. In both arms, therapy was stopped upon virus disappearance. Primary endpoint was duration of therapy. RESULTS: The number of treated/infected patients was significantly higher in the NASBA arm (25/30 vs. 15/39; P=0.015), as was the number of treated/relapsing patients (5/8 vs. 1/11; P=0.040), whereas the number of HCMV-infected/total number of patients was significantly higher in the antigenemia arm (39/42 vs. 30/40; P=0.026). Thus, in the NASBA arm, although the median duration of therapy was shorter compared to antigenemia (17 vs. 21 days, P>0.05), the overall number of days of therapy was significantly higher. No patient developed HCMV disease. CONCLUSION: pp67 mRNA NASBA can safely replace antigenemia, with some apparent advantages (semiautomation and objectivity of test results) and disadvantages (overtreatment of patients and greater duration of overall treatment).

Human cytomegalovirus immediate-early mRNAemia versus pp65 antigenemia for guiding pre-emptive therapy in children and young adults undergoing hematopoietic stem cell transplantation: a prospective, randomized, open-label trial.

In the search for better protocols of preemptive therapy of human cytomegalovirus (HCMV) infection in hematopoietic stem cell transplant (HSCT) recipients, we conducted a randomized trial comparing antigenemia with the nucleic acid sequence-based assay (NASBA) for determination of HCMV immediate-early messenger RNA (IEmRNA) as the guiding assay for initiation of pre-emptive antiviral treatment. In the IEmRNA arm, antiviral therapy was started upon IEmRNA positivity confirmed the following day, whereas in the antigenemia arm, therapy was started in the presence of either at least 2 pp65-positive leukocytes/2 x 105 examined or a single positive leukocyte confirmed the following day. In both arms, treatment was stopped upon 2 consecutive negative results. All patients were monitored for 3 months after HSCT. The primary end point of the study was duration of anti-HCMV therapy. On the whole, 80 children (41 in the IEmRNA and 39 in the antigenemia arm), recipients of transplants from either a relative or an unrelated donor, completed the study. No patient developed HCMV disease. In the IEmRNA arm, the incidence of HCMV infection was higher compared to the antigenemia arm (80% vs 51%, respectively, P =.0069), as well as the percentage of treated patients (66% vs 44%, respectively, P =.045). However, the percentage of relapses and treated relapses was comparable in the 2 arms. There was no significant difference in median duration of therapy per patient. Although these data indicate that IEmRNA determination does not offer advantages in terms of treatment duration, it can safely replace antigenemia, while semiautomation is the major advantage of the NASBA procedure.