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.

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.

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.

The stereoselective targeting of a specific enzyme-substrate complex is the molecular mechanism for the synergic inhibition of HIV-1 reverse transcriptase by (R)-(-)-PPO464: a novel generation of nonnucleoside inhibitors.

The human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitor pyrrolopyridooxazepinone (PPO) derivative, (+/-)-PPO294, was shown to be active toward wild type and mutated HIV-1 RT and to act synergistically in combination with 3'-azido-3'-deoxythymidine (Campiani, G., Morelli, E., Fabbrini, M., Nacci, V., Greco, G., Novellino, E., Ramunno, A., Maga, G., Spadari, S., Caliendo, G., Bergamini, A., Faggioli, E., Uccella, I., Bolacchi, F., Marini, S., (1999) J. Med. Chem. 42, 4462-4470). The (+/-)-PPO294 racemate was resolved into its pure enantiomers, and the absolute configuration was determined by x-ray analysis. Only one enantiomer, (R)-(-)-PPO464, displayed antiviral activity against both the wild type and the K103N mutant HIV-1 RT and was found to interact exclusively with the reaction intermediate formed by RT complexed with both the DNA and the nucleotide substrates. Being the first compound of its class to display this behavior, (R)-(-)-PPO464 is the representative of a novel generation of nonnucleoside inhibitors. (R)-(-)-PPO464 showed significant synergism when tested in combination with other RT inhibitors and efficiently inhibited viral replication when tested against the laboratory strain HIV-1 IIIB or against either wild type or multidrug-resistant clinical isolates. Pharmacokinetic studies in mice and rats showed a more favorable profile for (R)-(-)-PPO464 than for the corresponding racemate. (R)-(-)-PPO464 was also found to easily cross the blood-brain barrier. The coadministration of the HIV-1 protease inhibitor ritonavir increased the bioavailability of (R)-(-)-PPO464, having little effect on its plasma and brain elimination rates.

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.

Analysis of HIV drug-resistant quasispecies in plasma, peripheral blood mononuclear cells and viral isolates from treatment-naive and HAART patients.

The pattern of HIV-1 reverse transcriptase and protease mutations conferring resistance to antiretroviral drugs was studied in five treatment-naive patients and five HIV-infected patients receiving HAART [two reverse transcriptase inhibitors + one protease inhibitor] for > or = 1 year. Direct sequencing was performed on plasma HIV RNA, HIV DNA from peripheral blood mononuclear cells (PBMCs), and RNA from viral isolates. In addition, reverse transcriptase and protease PCR products from PBMCs HIV DNA, plasma HIV RNA, and viral isolate RNA were cloned in a plasmid to study the quasispecies distribution of drug-resistance associated mutations. Direct sequencing of HIV DNA from PBMCs and HIV RNA from plasma and viral isolates did not show the presence of drug resistance associated mutations in both reverse transcriptase and protease of HIV from all five treatment-naive patients. On the contrary, mutation analysis obtained by cloning plasma HIV RNA and PBMCs DNA showed the presence of drug-resistance related mutations at a low frequency in both HIV enzymes of four out of five treatment-naive patients. On the other hand, direct sequencing of plasma HIV RNA showed the presence of several reverse transcriptase and protease mutations in all five treated patients. Mutation analysis performed by cloning PBMCs HIV DNA, and HIV RNA from plasma and viral isolates, revealed additional reverse transcriptase and protease changes compared to direct sequencing of the relevant biological samples. All the additional changes were observed in a minority of clones. In conclusion, the data suggest that less frequent drug-resistant viral variants not detected by direct sequencing of PBMCs, plasma samples, or viral isolates are present in both treatment-naive and treatment-experienced HIV patients. These findings may have important implications in the understanding of the selection process of drug-resistant variants under drug pressure.

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.

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).

Prenatal diagnostic and prognostic value of human cytomegalovirus load and IgM antibody response in blood of congenitally infected fetuses.

Human cytomegalovirus (HCMV) load and virus-specific IgM were quantified in blood of 36 fetuses from mothers with primary HCMV infection. Nineteen fetuses were congenitally infected and 17 were uninfected as diagnosed by virus isolation from and DNA detection in amniotic fluid. Sensitivity of antigenemia was 57.9%; of viremia, 55. 5%; of leukoDNAemia, 82.3%; and of IgM, 57.9%; specificity was 100% for all assays. When amniocentesis was performed, 4 HCMV-infected fetuses (group A) showed abnormal ultrasound and biochemical/hematologic findings, 8 (group B) had elevated gamma-glutamyl transferase values, and 7 (group C) had normal ultrasound and biochemical findings. Virus loads were higher in groups A and B than in group C. In group A, no pregnancy went to term, in group B, 3 of 6 newborns were symptomatic at birth, and in group C, the 6 newborns were subclinically infected. Taken together, virologic, laboratory, and ultrasound findings may contribute to a better prognostic definition of fetal HCMV infection.

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.

Rapid detection of enteroviral RNA in cerebrospinal fluid (CSF) from patients with aseptic meningitis by reverse transcription-nested polymerase chain reaction.

The aim of this study was to compare conventional enterovirus isolation with rapid detection of enteroviral RNA by a reverse transcription-nested polymerase chain reaction (RT-nPCR) method amplifying the 5' nontranslated region of the enteroviral genome in specimens from patients with aseptic meningitis. Reference enterovirus strains and clinical enterovirus isolates were analyzed to evaluate assay sensitivity and specificity. All known enteroviral serotypes tested, but one (echovirus type 22), were detected by RT-nPCR. A series of unrelated viral isolates as well as CSF samples from patients with meningitis/encephalitis or neurological syndromes unrelated to enterovirus infection were included as controls. A total of 47 specimens (31 CSF, 12 rectal swabs, 4 throat swabs) from 30 patients with aseptic meningitis were available for the study. Of the 31 CSF samples tested from 30 patients, 17 from 17 patients (54.8%) were positive by RT-nPCR, while only 10 from 10 patients (32.2%) were positive by culture. Thus, RT-nPCR allowed diagnosis of enterovirus meningitis in 7 additional patients compared to cell culture. The cytopathic effect was observed 5-15 days after inoculation of CSF specimens onto cell cultures, while direct detection of viral RNA in CSF samples by RT-nPCR permitted diagnosis of enteroviral meningitis within 1-2 days. On the whole, viral isolation was positive in 12/47 (25.5%) specimens, whereas viral RNA was detected by RT-nPCR in 11 additional samples (23/47, 48.9%). Specimens of the control group were consistently negative by both viral isolation and RT-nPCR. Restriction endonuclease analysis of PCR products (RFLP) was applied to differentiate poliovirus (PV) from non-polio enteroviruses (NPEV). All enterovirus strains detected in clinical samples (n = 23) were identified as NPEV by RFLP. Clinical isolates were typed by neutralization as echovirus type 30 (n = 6), while 6 were not typed. In conclusion, detection of enteroviral RNA in CSF by RT-nPCR allows: i) rapid diagnosis of enteroviral meningitis; ii) increased sensitivity with respect to virus isolation; iii) differentiation between PV and NPEV infections of the central nervous system.

Diagnostic significance and clinical impact of quantitative assays for diagnosis of human cytomegalovirus infection/disease in immunocompromised patients.

In recent years several assays have been developed for quantitation of human cytomegalovirus (HCMV) in blood of immunocompromised (transplanted and AIDS) patients. It is currently agreed that the only reliable indication of the degree of dissemination of HCMV infection/disease is the measurement of HCMV in blood. Diagnosis of HCMV end-organ disease (organ localizations) often does not benefit from quantitation of virus in blood, but requires detection and quantification of virus in samples taken locally. The most important and clinically useful diagnostic assays for HCMV quantitation in blood are: i) viremia, quantifying infectious HCMV carried by peripheral blood leukocytes (PBL); ii) pp65-antigenemia, quantifying the number of PBL positive for HCMV pp65 in the nucleus; iii) circulating cytomegalic endothelial cell (CEC) viremia (CEC-viremia) measuring the number of circulating CEC carrying infectious HCMV (during the antigenemia assay); iv) leuko- and plasma-DNAemia, quantifying the number of HCMV genome equivalents present in PBL or plasma, respectively, by quantitative polymerase chain reaction (Q-PCR). Other less widely used assays are: i) determination of immediate early and late gene transcripts (mRNA) to detect active viral infection; ii) in situ hybridization to detect viral nucleic acid (DNA or RNA) in tissue sections or cell smears; iii) in situ PCR to detect a low DNA copy number in single cells. Monitoring of HCMV infection/disease in transplant recipients and AIDS patients has established threshold values for different assays above which HCMV-related clinical symptoms are likely to appear. These values are approximately 10 for viremia, 100 for antigenemia and 1,000 GE for leukoDNAemia, and are valid for both solid organ and bone marrow transplant recipients as well as AIDS patients, whereas presence of even a single circulating CEC is sufficient to suggest the presence of a disseminated HCMV infection with potential organ involvement. Monitoring of antiviral treatment of HCMV infection/disease with either ganciclovir or foscarnet has aimed at keeping virologic parameters below the threshold values reported above. On the other hand, rising levels of the same virologic parameters during antiviral treatment have mostly revealed emergence of resistant HCMV strains to either ganciclovir (mutations in the UL97 or DNA polymerase gene) or foscarnet (mutations in the UL54 gene) or both drugs (double resistance with both types of mutations). Rapid assays for chemosensitivity testing of virus directly in clinical specimens have been developed to allow timely (4-6 days) detection of resistance to a drug and provide clinicians with the rationale for shifting to an alternative treatment.

Ganciclovir resistance as a result of oral ganciclovir in a heart transplant recipient with multiple human cytomegalovirus strains in blood.

BACKGROUND: The emergence of a ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) strain in a heart transplant recipient (HTR) coinfected by multiple HCMV strains was investigated. METHODS: A HTR with primary HCMV infection was treated with three induction courses of intravenous GCV followed by a 2-month maintenance treatment with oral GCV. HCMV antigenemia, viremia, and leukoDNAemia levels were monitored. GCV susceptibility was analyzed by an immediate-early antigen plaque reduction assay and by a rapid screening assay performed using peripheral blood leukocytes (PBL) as viral inoculum. The viral population in blood was investigated by restriction analysis of multiple genome regions. UL97 and UL54 genes were sequenced in parallel in both HCMV isolates and the relevant PBL samples. A rapid molecular assay for detection and quantitation of the GCV-resistant mutant was developed. RESULTS: The emergence of a GCV-resistant UL97 mutant (Cys-607 --> Tyr) was responsible for treatment failure during oral GCV therapy. The genetic analysis of the HCMV population showed the sequential appearance in blood of two unrelated strains (referred to as A and B). Strain A most likely derived from the transplanted organ and strain B from a subsequent blood transfusion. The resistant variant (Br) emerged from strain B and became predominant "in vivo" under the GCV pressure. However, after foscarnet administration, the resistant mutant disappeared in viral isolates, whereas it was still present as a minor proportion in PBL samples. CONCLUSION: (a) Oral GCV may select resistant strains in transplanted patients; (b) results of the rapid screening assay were clinically useful for shifting to an alternative treatment, thus avoiding the appearance of HCMV disease; (c) virus isolation may not be the most reliable approach to detection of HCMV drug-resistant strains; (d) a novel molecular assay for rapid detection of UL97 Cys-607 --> Tyr mutation directly in clinical specimens was developed, allowing earlier "in vivo" detection of the resistant mutant.

Improved prenatal diagnosis of congenital human cytomegalovirus infection by a modified nested polymerase chain reaction.

Two major variables may cause false-negative results in prenatal diagnosis of congenital human cytomegalovirus (HCMV) infection: sensitivity of the techniques(s) used; and time elapsed between maternal infection and antenatal testing. Previous results indicated that rapid HCMV isolation from amniotic fluid samples and viral DNA detection in amniotic fluid by nested polymerase chain reaction (nPCR) had comparable levels of sensitivity (69.2% and 76.9%, respectively). The nPCR protocol was reviewed following two additional false-negative antenatal diagnosis in a twin pregnancy during which two procedures were performed at 18 and 23 weeks of gestation, respectively. In the new assay, multiple (instead of single) and 100 (instead of 20) microliters amniotic fluid aliquots were individually amplified and tested by nPCR. By using this approach, low DNA levels (1-10 genome equivalents) were detected in 1-5/8 replicates of amniotic fluid samples taken from both twins during both procedures. In addition, viral DNA was detected in 5/6 replicates from two amniotic fluid samples still available from two previous false-negative cases. However, nPCR on multiple amniotic fluid replicates did not anticipate positive prenatal results in a retrospective case, which required two procedures for correct diagnosis and, when prospectively employed, did not avoid one additional false-negative prenatal diagnosis 8 weeks after maternal infection. Thus, delayed intrauterine transmission of the infection may be a potential cause of false-negative results. However, the combination of a very sensitive technique with appropriate timing of prenatal testing can substantially increase the reliability of prenatal diagnosis results.

Human cytomegalovirus (HCMV) leukodnaemia correlates more closely with clinical symptoms than antigenemia and viremia in heart and heart-lung transplant recipients with primary HCMV infection.

BACKGROUND: In the last few years, human cytomegalovirus (HCMV) viremia, pp65 antigenemia, and leuko- and plasma-DNAemia have been developed to quantitate virus in blood of immunocompromised patients with HCMV infection. However, thus far, no conclusive studies have been performed to define the correlation of each of the different assays with clinical symptoms in primary HCMV infections. METHODS: This correlation was investigated in a population of 20 heart and heart-lung transplant recipients with primary HCMV infection using standardized virological methods. RESULTS: Median peak HCMV viremia, antigenemia, and leukoDNAemia levels were 110 (0-2,000) p72-positive fibroblasts, 450 (27-2,000) pp65-positive leukocytes, and >10,000 (1,358-10,000) genome equivalents (GE) in the 14 symptomatic patients and 18 (1-130) p72-positive fibroblasts, 86.5 (5-350) pp65-positive leukocytes, and 248 (10-863) GE in the six asymptomatic patients, respectively. The difference was statistically significant for antigenemia (P=0.009) and leukoDNAemia (P<0.0001). However, on an individual basis, unlike viremia and antigenemia, all DNA peaks of the 6 asymptomatic patients were below the DNA range of the 14 symptomatic patients (<1,000 GE), while all the 14 symptomatic patients had DNA peaks higher than those of asymptomatic patients (>1,000 GE). Follow-up confirmed these results, showing that 1,000-2,000 GE was the threshold zone for emergence of clinical symptoms. Symptoms were never observed in patients with secondary DNA peaks, except for one patient suffering from an HCMV organ localization (HCMV gastritis). CONCLUSIONS: LeukoDNAemia is the viral parameter of choice for monitoring of primary HCMV infections and antiviral treatment in heart and heart-lung transplant recipients. In this patient population, antigenemia-guided preemptive therapy could be replaced by leukoDNAemia-based antiviral therapy.

Rising levels of human cytomegalovirus (HCMV) antigenemia during initial antiviral treatment of solid-organ transplant recipients with primary HCMV infection.

In 7 of 18 solid-organ transplant recipients with primary human cytomegalovirus (HCMV) infection, HCMV antigenemia levels were unexpectedly found to rise significantly (P = 0.018) during a mean time of 7.3 +/- 3.2 days after initiation of specific antiviral treatment, whereas corresponding levels of viremia dropped significantly (P = 0.043). Thus, shifting to an alternative antiviral drug based solely on increasing antigenemia levels is not justified in this group of patients.