Maria Torsellini

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

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. J. Med. Virol. 75:336–347, 2005.

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

Abstract 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.

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).

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.

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.

Diagnosis of human cytomegalovirus infections in the immunocompromised host

BACKGROUND In the last decade several major advances have been made in the rapid diagnosis of human cytomegalovirus (HCMV) infections and disease in immunocompromised patients both at the immunological and molecular level. OBJECTIVES The objective was to review in some detail the new diagnostic tools allowing determination and quantitation of HCMV infection in blood of transplanted and AIDS patients. STUDY DESIGN The determination and quantitation as well as the clinical significance of antigenemia, viremia, HCMV-infected circulating endothelial cells (CEC) and DNAemia will be discussed in view of the therapeutic management of HCMV disease. Levels of viremia represent the number of p72-positive cultured fibroblasts inoculated with 2 x 10(5)PBL, while levels of antigenemia represent number of pp65-positive PBL/2 x 10(5) PBL examined. The number of CEC is determined simultaneously and in parallel with antigenemia. DNAemia, both qualitative and quantitative, can be determined by polymerase chain reaction (PCR) per 1 x 10(5)PBL. The clinical utility of determining either immediate-early or late mRNA is still debated. RESULTS In solid organ transplant recipients mean levels of viremia of 100 and of antigenemia of 400 correlate with onset of clinical symptoms. The time between first HCMV positivity and the onset of symptoms (>/= 10 days), together with the observation that most patients with reactivated infection clear virus without treatment, allowed the establishment of an antigenemia cut-off of 100 for the initiation of treatment. On the other hand, seronegative recipients of solid organs from seropositive donors must be treated preemptively, i.e. at first appearance of HCMV positivity in blood. Due to the risk of early appearance of HCMV pneumonia, the same preemptive approach must be used in bone-marrow transplant recipients. In acquired immunodeficiency syndrome (AIDS) patients with HCMV infection/disease, general criteria for initiation of treatment are more difficult to establish and treatment must be maintained. CEC are detected only in untreated disseminated HCMV infections with organ involvement. Qualitative DNA determination is useful only in special cases, such as in aqueous or vitreous humor of AIDS patients with HCMV retinitis. Quantitative DNA levels obtained by PCR are much more helpful for diagnosing HCMV disease and establishing initiation of treatment. CONCLUSIONS New diagnostic procedures currently ensure fine monitoring of HCMV infections/diseases and evaluation of the effect of specific antiviral treatment in the immunocompromised host.

A 6-hour microneutralization assay for human cytomegalovirus antibody by using monoclonal antibodies

Abstract Human cytomegalovirus (HCMV) was quantified at 6, 24, and 96 h post infection by counting the number of infected cells which were stained by the immunoperoxidase technique using monoclonal antibodies reactive to different HCMV non-structural and structural proteins. A linear dose-effect relationship was found at the three indicated times. Three rapid neutralization (Nt) assays (one for each time) were developed, using the same virus inoculum. Human sera from individuals with primary, reactivated or remote HCMV infection gave comparable Nt antibody titers with the three assays. The 6-hour rapid Nt assay appears highly suitable for determination of HCMV immune status and provides results the same day of blood collection.

Subgrouping of human rotavirus strains by complement fixation, indirect double-antibody sandwich enzyme-linked immunosorbent assay and solid-phase immune electron microscopy

SummaryComplement fixation (CF), indirect double-antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) and solid-phase immune electron microscopy (SPIEM) were compared for their ability to subgroup 73 human rotavirus (HRV) strains from infants and young children with gastroenteritis admitted to one or the other of two different hospitals of Northern Italy. By both indirect DAS ELISA and SPIEM all 73 HRV strains were classified into one or the other of two subgroups. By CF only 67 strains could be subgrouped, as six HRV-positive stools showed anticomplementary activity which could not be eliminated. Indirect DAS ELISA required subgroup-specific, unabsorbed antisera from two different animal species. For SPIEM two antisera from a single animal species were needed, but they had to be absorbed with single-shelled bovine rotavirus for HRV subgrouping to be reliable. Indirect DAS ELISA appeared to be the technique most suitable for extensive application in epidemiological studies of HRV infections by different subgroups. However, SPIEM allowed rapid subgrouping of HRV in stool specimens showing anticomplementary activity in the CF test or non-specific reactions in the ELISA test. In one area of Northern Italy the prevalence of subgroup I HRV infections was 7.8 per cent, while in another it reached 68.1 per cent in the same period.

Characterization of rotavirus subgroup-specific monoclonal antibodies and use in single-sandwich ELISA systems for rapid subgrouping of human strains

SummaryTwo subgroup-specific monoclonal antibodies (MAb) raised in mice against group A human rotavirus were shown to react by immunoblotting with the trimeric form of VP6 of the homologous subgroup and successfully applied to development of new single-sandwich ELISA systems for rapid subgrouping of human strains. All of the 344 strains tested could be subgrouped, but for two of them prior propagation in cell cultures was required.