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This study compared the performance of the LIAISON®XL system of immunoglobulin (Ig) G and IgM immunoassays for the diagnosis of Toxoplasma gondii, cytomegalovirus (CMV), and rubella virus infections with that of the ARCHITECT system. Patient serum samples, previously screened and clinically diagnosed with T. gondii, CMV or rubella, were used to compare LIAISON®XL and ARCHITECT IgG and IgM immunoassays. LIAISON®XL Toxo and CMV IgG avidity assays were also compared with equivalent ARCHITECT assays and reference methods. Overall agreement between the LIAISON®XL and ARCHITECT assays was 99% and 92% for the Toxo IgG and IgM assays, respectively, 98% and 96% for the CMV IgG and IgM assays, respectively, and 93% and 98% for the rubella virus IgG and IgM assays, respectively. LIAISON®XL IgG Toxo and CMV avidity assays showed high concordance with the VIDAS® Toxo IgG avidity assay and an in-house CMV avidity assay (reference methods), and faster IgG avidity maturation in a larger number of samples collected months after the primary infection compared with equivalent ARCHITECT assays. LIAISON®XL assays for detection of anti-T. gondii, CMV and rubella virus IgG and IgM are at least equal to the competitor assays on the ARCHITECT platform.

Background: An incorrect definition of immune status to human cytomegalovirus (HCMV) can lead to incorrect management of pregnant women., Objectives: Aims of the study were: i) to describe 10 cases of unconfirmed HCMV IgG-seroconversion in pregnancy; ii) to develop a panel of confirmatory tests to define HCMV serostatus; iii) to investigate the frequency of false IgG-positive results in pregnant women screened with the LIAISON ® CMVIgGII automated assay., Study Design: Blood samples from 10 pregnant women referred for HCMV IgG-seroconversion were examined to confirm/exclude a primary infection. In addition, samples were tested for HCMV IgG by immunoblotting, neutralization assay, and ELISA against gB, gH/gL/pUL128L and gH/gL/gO recombinant glycoproteins. LIAISON ® CMVIgGII results obtained on 1158 pregnant women were reviewed and samples with low IgG titers were further investigated., Results: A primary infection was excluded in the 10 women referred for HCMV IgG seroconversion. None of them was confirmed to be IgG-seropositive. Of the 1158 women prenatally screened by LIAISON ® CMVIgGII, 678 (59%) were IgG-positive and, of these, 40 (5.9%) showed low levels of IgG (14-50 U/mL). Thirty-three women with low IgG-positivity were further tested by confirmatory tests and 11 (33.3%) were found to be non reactive to HCMV., Conclusions: At least 1.6% (11/678) women who tested positive with LIAISON ® CMVIgGII were found to be seronegative when tested with confirmatory tests. These women should be informed to reduce the risk of a primary HCMV infection. Furthermore, should a congenital infection occur in any of these women, a maternal non-primary infection could be erroneously diagnosed., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hepatitis B vaccine is the most effective strategy to control hepatitis B virus (HBV) infection and disease. It is considered that an anti-HBs (antibodies against HBV surface antigen) titer >10 mIU/ml, measured shortly after a complete vaccination schedule, provides protection against infection. Approximately 4-10% of healthy individuals fail to respond to 3-dose vaccination. Long-term HBV-specific memory T-cell response has not been fully investigated, mainly due to the lack of a suitable assay. We quantified HBV-specific expandable memory T cells by using a cultured IFN-γ enzyme-linked immunospot (ELISPOT) assay. Cultured ELISPOT response to an overlapping peptide pool representing the complete L (large) HBV envelope polypeptide was evaluated in 41 healthy subjects vaccinated 15-20 y earlier and 5 unvaccinated. Plasma samples were tested for anti-HBs. Vaccinated subjects had significantly higher HBV-specific T-cellular response than unvaccinated (p = 0.0002). HBV-specific T-cell response was mainly mediated by CD4 + T cells. No concordance was found between cultured ELISPOT and anti-HBs data in vaccinated subjects. Thirty-one (76%) vaccinated subjects were responders (anti-HBs >10 mIU/ml). Nineteen (46%) vaccinated subjects were considered to be responders in cultured ELISPOT. Twenty-two (54%) vaccinated subjects were considered non-responders in cultured ELISPOT; 5 of them (23%) were also humoral non-responders. About 12% of healthy HBV-vaccinated subjects are both humoral and cellular non-responders. Although the prognostic value of this assay has not been established in terms of predictability for susceptibility to de-novo HBV infection, ELISPOT data suggest that these subjects may be at risk for HBV infection and disease, especially health care workers.

Polyomaviruses KI (KIPyV) and WU (WUPyV) were described recently in children with acute respiratory disease. The pathogenic potential of these human viruses has not been determined completely, but a correlation between immunosuppression and virus reactivation has been suggested. In the present study, the association between KI/WUPyV infection and immunosuppression was investigated using sequential nasopharyngeal aspirates from asymptomatic adult hematopoietic stem cell transplant recipients. In parallel, an investigation on the WU/KIPyV prevalence in children with acute respiratory disease was also carried out. Two of the 126 samples obtained from the 31 hematopoietic transplant recipients were positive for KIPyV (1 sample, 0.79%) and WUPyV (1 sample, 0.79%). Both samples were obtained 15 days after allogeneic transplantation and virus persistence was not observed in subsequent samples. In symptomatic children, 7 of the 486 nasopharyngeal aspirates were positive for WUPyV (1.4%) and 1 for KIPyV (0.2%). Single polyomavirus infection was detected in four patients, whereas the remaining patients were co-infected with respiratory syncityal virus (three patients) or adenovirus (one patient). The results suggest that WU/KIPyVs have a limited circulation in Italy and a low pathogenic potential in young children. Brief and asymptomatic infection can occur in hematopoietic transplant recipients., ((c) 2009 Wiley-Liss, Inc.)

Sequential nasopharyngeal aspirates from patients without respiratory symptoms undergoing hematopoietic stem cell transplantation (HSCT) were tested for genomic RNA of human metapneumovirus (hMPV). Persistent hMPV infection was documented in most of them and confirmed by virus isolation. hMPV infection etiology was also evaluated during the same period in samples from pediatric patients with acute respiratory diseases (ARDs). Sequence analysis of hMPV in HSCT recipients documented infection by hMPV genotype A and strong interhost similarity; this pattern differs from that observed in pediatric patients with ARDs. The data indicate that HSCT recipients may frequently develop symptomless hMPV infection.

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 (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.

Fifty AIDS patients were investigated for human cytomegalovirus (HCMV) viraemia when potentially HCMV-related clinical symptoms or syndromes were observed. Nine patients underwent prolonged virologic follow-up, while 41 additional patients were examined only once or sporadically. Concentrated preparations of polymorphonuclear leukocytes (PMNL) from 153 blood samples were obtained for monitoring: (1) early virus isolation in cell cultures 24 h p.i. (viraemia); (2) early structural antigen detection in cytospin preparations (antigenemia); and (3) HCMV DNA in blood (DNAemia) through DNA amplification by the polymerase chain reaction (PCR). Viraemia and antigenemia were quantitated, whereas evaluation of DNAemia was only qualitative. A good correlation between levels of viraemia and antigenemia was consistently found except during ganciclovir treatment. HCMV-related clinical symptoms were observed when the number of infected PMNL was greater than 100 per 2 x 10(5) cells examined. All 56 blood samples positive for viraemia and antigenemia were also PCR-positive, whereas 44 samples (39 of which taken from patients with ascertained HCMV infection in blood) were positive by PCR only. Viraemia and antigenemia were often unrelated to HCMV organ syndromes, such as retinitis, in which only DNAemia was often detected. Prolonged ganciclovir treatment kept viraemia, antigenemia and even DNAemia at a low or negative level, yet drug discontinuation led to rapid progression of HCMV infection in blood. In addition, prolonged antiviral treatment could induce appearance of ganciclovir-resistant HCMV strains, requiring alternative foscarnet therapy. In conclusion, determination of viraemia and antigenemia appears essential for correct clinical management and antiviral treatment of disseminated HCMV infections in AIDS patients. However, PCR is the most sensitive method for diagnosis and monitoring of HCMV infections in blood at a pre-clinical stage.

We report the follow-up and treatment of HCMV infections in three patients with AIDS. The patients, affected by HCMV retinitis, have been followed 24, 12 and 6 months respectively. The antiviral treatment was based on the DHPG administration which was substituted in one case of resistance to DHPG with Foscarnet. In the follow-up period, virological tests have been performed to detect the presence of the HCMV antigenemia/viremia. The results show that, to avoid the progression of the retinitis, the antiviral treatment must not be stopped or discontinued. DHPG and Foscarnet were able to limit the infection to the eye and were well tolerated. In the HCMV-infected patients, the continuous monitoring of the antigenemia/viremia is of main importance to follow the clinical and therapeutical course of the disease.

Eighty-two HIV-1-seropositive subjects were examined for the presence and quantification of human cytomegalovirus (HCMV) in peripheral blood polymorphonuclear leukocytes (PMNL) by polymerase chain reaction, culture and immunofluorescence in order to investigate the relationship between viraemia and immunosuppression. Patients were divided into three groups: (1) asymptomatic subjects with greater than 400 x 10(6)/l CD4 lymphocytes (n = 30); (2) asymptomatic subjects with less than 400 x 10(6)/l of CD4 lymphocytes and zidovudine (n = 20), and (3) AIDS-related complex (ARC)/AIDS patients on zidovudine (n = 32). Evidence of HCMV infection in circulating PMNL was found in 15 out of 29 ARC/AIDS patients examined (51.7%), whereas no infection was detected among the 50 asymptomatic HIV-1-seropositive subjects. HCMV-related symptoms were found only where the number of infected PMNL was greater than 50 per 2 x 10(5) cells.

Fifty human cytomegalovirus (HCMV) isolates were recovered from different clinical specimens (buffy coat, throat washing and urine) obtained from fifty patients (23 AIDS patients, 20 heart transplant recipients, 1 bone marrow transplant recipient, 2 newborns with congenital HCMV infection and 4 immunocompetent individuals with acute HCMV infection). The isolates were previously identified by immunological methods and then examined for identification by the polymerase chain reaction. In parallel, reference HCMV strains as well as other human members of the Herpesviridae family (reference and wild strains) were examined as controls. Two pairs of primers relevant to the immediate-early and late regions of HCMV DNA, respectively, were used. The DNA amplification product was highly specific; in addition, all fifty HCMV isolates were amplified by both pairs of primers and thus identified as HCMV. These preliminary results show that selected pairs of primers are able to amplify DNA regions conserved enough to allow virus identification among a large number of HCMV strains. In addition, they are highly promising in view of the use of PCR for direct detection of HCMV DNA in clinical samples.

A nosocomial outbreak of rotavirus gastroenteritis involving 52 newborns occurred between June and September 1988 at the University Children's Hospital of Freiburg, Federal Republic of Germany. Stools from 27 representative patients were examined for rotavirus serotypes, using a monoclonal antibody-based enzyme-linked immunosorbent assay. The electropherotype was also examined by polyacrylamide gel electrophoresis of genomic RNA. As many as 18 patients were found to be infected by serotype 4, subtype 4B strain, and in all of them the same electropherotype was detected. Although rotavirus from the remaining nine patients could not be typed, the electropherotype in four was identical to that of the serotype 4, subtype 4B strain. Thus, most of the patients in the outbreak were infected by the same rotavirus strain. Retrospective epidemiological studies showed that the 4B strain began to circulate at the hospital in January 1988, whereas only rotavirus serotypes 1, 3, and 4A were detected in 1985-1987. The primary case of the outbreak was presumably a newborn with acute gastroenteritis, admitted to the hospital from a small maternity unit in the same urban area. During the outbreak, 12 of 44 healthy newborns in the nurseries of the Children's Hospital and other maternity hospitals were found to be asymptomatic rotavirus carriers, and in three of the newborns the same 4B strain was detected. This is the first reported outbreak caused by a serotype 4, subtype 4B strain.

An extended epidemiological survey on the circulation of the 4 established human rotavirus (HRV) serotypes in some European countries was carried out on 831 fecal strains collected from infants and young children with acute non-bacterial gastroenteritis during 1981-88. Typing was done by enzyme-linked immunosorbent assay and/or solid-phase immune electron microscopy using VP7 type-specific neutralizing monoclonal antibodies. Serotype 1 HRV strains were found to be largely predominant in this period both in Italy and other countries, whereas serotype 4 strains were less common. The number of strains of serotypes 1 and 4 circulating in Europe was equivalent only in 1983-84. Serotype 2 strains were significantly represented only in 1981-84, while strains of serotype 3 were nearly absent, since only 8 strains (2 of which belonged to subgroup I) were found during the entire study period. About 10% of strains could not be typed, while 9 strains exhibited dual VP7 reactivity and 6 were non-group A HRVs. These epidemiological findings must be taken into consideration when deciding strategies for preparing vaccines to be used in Europe.

Using murine monoclonal antibodies (MAbs) raised against the common antigen of group A rotavirus (RV), two single-sandwich ELISA systems were developed for detection of RV in stools: one using polyclonal antibody (PAb) as capture and a MAb as detector antibody (referred to as PAb-MAb assay); and the other based on the use of two different MAbs as capture and detector antibodies (referred to as MAb-MAb assay). In each single-sandwich ELISA system, samples and peroxidase-labeled MAb were incubated sequentially (two-step method) or simultaneously (one-step method). Using the two-step procedure on purified RV, 50 pg of protein was detected in the PAb-MAb as well as in the MAb-MAb assay, whereas the one-step method detected 0.4 ng and a conventional double-sandwich ELISA detected 3.2 ng of viral protein. Titration of RV samples from stools and cell cultures showed that single-sandwich ELISA titers were, on the average, 10-100-fold higher than those obtained by electron microscopy (EM), but 10-100-fold lower than those obtained by solid-phase immune EM (SPIEM). However, when 200 stool samples previously examined by EM or SPIEM were tested by the single-sandwich ELISA systems, specificity and sensitivity of these assays were 100%, and comparable to SPIEM. No false positive results were obtained when 54 samples of meconium and 91 stools from newborns in the first five days of life were tested. The two-step procedure appeared to be somewhat preferable over the one-step method, which, although faster, gave a marked prozone with a few samples in the MAb-MAb assay. The use of MAbs in rapid single-sandwich ELISA systems for RV detection in stools appears highly convenient, due to reliable results and short test performance times.

An outbreak of acute gastroenteritis, involving 30 infants and young children aged 2 months to 4 years, took place in a pediatric ward of the University Hospital of Pavia, Northern Italy, in the period from November 9 to December 1, 1986. Out of the 14 patients examined, ten were found to shed rotavirus with stools. All strains were characterized for serotype, using a monoclonal antibody-based enzyme-linked immunosorbent assay, and for electropherotype, by polyacrylamide gel electrophoresis of genomic RNA. It was shown that a single serotype 4 (subtype 4A) strain spread within the ward from a primary case to seven other patients. The remaining two patients were found to be infected by a serotype 1 strain that was circulating in the same area prior to the outbreak. The clinical symptoms were unusually severe, since significant dehydration was observed in four of the eight serotype 4 rotavirus-infected children. Previous epidemiological studies had shown that since 1983 serotype 4 strains had not been circulating in Pavia, and the electropherotype of the newly circulating serotype 4 strain was different from those observed in 1981-1983. Thus, the severity of the diarrheal disease appeared to be related to the circulation of both a new serotype and a new electropherotype.

Peripheral blood polymorphonuclear (PMN) cells from 35 immunocompromised patients (22 heart transplant recipients and 13 AIDS patients) and four normal subjects were tested for the presence of human cytomegalovirus (HCMV) immediate early antigen (IEA) (antigenemia) by indirect immunofluorescence (IFA) and IEA-specific monoclonal antibodies (MAb). PMN samples were tested in parallel for HCMV isolation (viremia) by using MAb to viral early antigens (EA) and the IFA technique 24-48 hr after inoculation onto human fibroblast monolayers. HCMV was isolated from 26 of 83 PMN samples examined: of these, 25 were also positive for HCMV IEA (96% sensitivity). Seven additional PMN samples negative for viral isolation resulted IEA-positive (87.7% specificity). Six of the seven discordant samples were taken from four patients during ganciclovir treatment. The transitory dissociation between positive HCMV antigenemia and negative viremia during antiviral treatment was followed, at the end of the therapy, either by virus clearance and disappearance of IEA-positive PMNs (one patient) or by reappearance of viremia (three patients). Among concordant positive samples, a significant correlation was observed between the number of IEA-positive PMN leukocytes and EA-positive nuclei of infected fibroblasts, when the same number of PMNs were used for both tests.