Maurizio Zavattoni

Human Cytomegalovirus in Blood of Immunocompetent Persons during Primary Infection: Prognostic Implications for Pregnancy

Human cytomegalovirus (HCMV) was investigated in peripheral blood leukocytes (PBL) of 52 immunocompetent patients (40 pregnant women) with primary HCMV infection by quantitative determination of pp65 antigenemia, viremia, and leukoDNAemia. pp65 antigenemia was detected in 12 (57.1%) of 21, 4 (25%) of 16, and 0 of 10 patients examined 1, 2, and 3 months after onset, respectively. Viremia was detected in 5 (26.3%) of 19 patients during the first month only. LeukoDNAemia was detected in 20 of 20, 17 (89.5%) of 19, and 9 (47.3%) of 19 patients tested 1, 2, and 3 months after onset, respectively. Four (26.6%) of 15 patients were still DNAemia-positive at 4-6 months, whereas none were positive at >6 months. HCMV was not detected in PBL of 20 HCMV-immune donors or of 9 seropositive subjects with recurrent infection. Virus levels were low by all assays and did not correlate with clinical course of infection, intrauterine transmission, or severity of outcome. Invasive procedures in the presence of maternal leukoDNAemia did not seem to interfere with vertical transmission of HCMV infection.

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.

Diagnosis and Outcome of Preconceptional and Periconceptional Primary Human Cytomegalovirus Infections

Primary human cytomegalovirus (HCMV) infection occurring in pregnant women within 3 months before (preconceptional) or within 4 weeks after (periconceptional) the last menstrual period represents an as-yet-undefined risk to the fetus. One (9.1%) of 11 newborns born to 12 women with preconceptional infection was subclinically infected (1 aborted fetus was not examined for infection). Of 20 pregnancies in women with periconceptional infection, 7 were terminated before 12 weeks of gestation (aborted fetus was not examined), 1 was terminated at 23 weeks after prenatal diagnosis of congenital infection, and 12 continued to term. Of those 12, 3 resulted in newborns who were congenitally infected. Thus, in the periconceptional group, intrauterine transmission occurred in 4 (30.8%) of 13 pregnancies for which the virologic outcome was known. One newborn was symptomatic at birth, and disseminated HCMV infection was diagnosed in an aborted fetus. Periconceptional primary HCMV infection seems to bear a higher risk of unfavorable outcome than preconceptional infection, and counseling should be adjusted accordingly.

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 technique(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) μl 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. J. Med. Virol. 56:99–103, 1998.

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.

Role of prenatal diagnosis and counseling in the management of 735 pregnancies complicated by primary human cytomegalovirus infection: A 20-year experience

BACKGROUND The burden of congenital human cytomegalovirus (HCMV) infection is well recognized. However, screening for maternal infection remains controversial in view of diagnostic challenges, counseling difficulties, and absence of medical treatment. OBJECTIVE To assess the role of prenatal diagnosis and counseling in the management of pregnancy complicated by primary HCMV infection. STUDY DESIGN Retrospective study aimed at investigating diagnostic features, options, and pregnancy outcome in 735 women with primary HCMV infection over a period of 20 years (1990-2009). RESULTS Overall, 25.6% women were found to be seronegative before the actual pregnancy. However, none were informed about HCMV infection and potential prevention strategies. Diagnosis of primary HCMV infection was achieved by seroconversion in 44.4% cases and by different combinations of virus-specific IgM, low IgG avidity, and DNAemia in 43.9% cases. Non-specific symptoms and/or haematological/biochemical alterations were recalled by 73.5% women. The onset of infection could be established, and counseling adjusted accordingly in >90% cases. The overall rate of vertical transmission was 37.1%, ranging from 5.6% for preconceptional infections to 64.1% for third trimester infections. Amniocentesis was chosen by 43.1% women, whereas pregnancy termination was requested by 15.6%. CONCLUSIONS Reference virology centers and ad hoc trained and experienced physicians are required for accurate diagnosis of primary infection in pregnancy and ensuing counseling. Prenatal diagnosis has a central role in the management of pregnancies complicated by primary HCMV infection. HCMV-seronegative women should receive adequate information.

Development of Human Cytomegalovirus—Specific T Cell Immunity during Primary Infection of Pregnant Women and Its Correlation with Virus Transmission to the Fetus

OBJECTIVE We sought to study the development of human cytomegalovirus (HCMV)-specific T cell-mediated immune responses during primary HCMV infection in pregnancy. METHODS The HCMV-specific lymphoproliferative response (LPR) and intracellular cytokine (interferon [IFN]- gamma and interleukin [IL]-2) production were investigated during the first year after primary infection in 49 pregnant women and 9 nonpregnant control subjects. An HCMV-specific CD4(+) and CD8(+) T cell LPR was detected by the 5,6-carboxyfluorescein diacetate succinimidyl ester dilution method, and a cell-division index was calculated. RESULTS The CD4(+) T cell LPR developed slightly earlier than the CD8(+) T cell LPR. However, CDI values for both T cell subpopulations were lower than those of seropositive control subjects in both pregnant and nonpregnant individuals. During the first month after infection, IFN- gamma -producing CD4(+) and CD8(+) T cells were consistently observed, whereas IL-2-producing T cells were very rarely detected in blood. A correlation between the development of HCMV-specific LPR and virus clearance from blood was observed. A significantly delayed development of the CD4(+) T cell LPR was observed in infected mothers who transmitted virus to the fetus, compared with those who did not. CONCLUSIONS The development of adaptive T cell immunity after primary HCMV infection appears to be a complex and slow process until a memory T cell response develops. The T cell immune response appears to influence vertical HCMV transmission.

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.

Monitoring of ganciclovir sensitivity of multiple human cytomegalovirus strains coinfecting blood of an AIDS patient by an immediate-early antigen plaque assay

A plaque-reduction assay for chemosensitivity testing of human cytomegalovirus (HCMV) strains was developed based on early detection of viral plaques 96 h p.i. by a monoclonal antibody to the major immediate-early protein p72. Sequential HCMV isolates from an AIDS patient undergoing multiple courses of ganciclovir treatment during an 18-month follow-up were tested by the new assay, showing emergence of a ganciclovir-resistant strain. However, cloning of viral isolates and Southern blot hybridization analysis showed the simultaneous presence of three different HCMV strains in blood. Of these, the resistant strain was likely to be selected during prolonged maintenance antiviral treatment, emerging during full drug regimen, while the two sensitive strains reappeared in association with the resistant one following drug discontinuation. This finding was demonstrated by high levels of ID90 and ID99 in sequential mixed viral populations. The new plaque assay leads to reduction in time needed for chemosensitivity testing and permits rapid tracing of drug-resistant strains in a mixed viral population.

Quantitation of herpes simplex virus DNA in cerebrospinal fluid of patients with herpes simplex encephalitis by the polymerase chain reaction

BACKGROUND Previous studies have shown the diagnostic utility of qualitative detection of herpes simplex virus (HSV) DNA by the polymerase chain reaction (PCR) in cerebrospinal fluid samples (CSF) from patients with herpes simplex encephalitis (HSE). OBJECTIVES To determine whether quantitation of HSV DNA in CSF could be useful for monitoring efficacy of antiviral therapy and provide prognostic indications. STUDY DESIGN A quantitative PCR assay using an internal control for evaluation of PCR efficiency and detection of PCR inhibitors was developed and used for retrospective testing of 98 CSF samples from 26 patients with serologically diagnosed HSE during the period 1980-1995. RESULTS HSV DNA was detected in 36 CSF samples from 23 patients. PCR positivity was 100% for CSF samples collected within 10 days after onset, and 30.4 and 18.7% for samples collected 11-20 and 21-40 days later, respectively. The 3 PCR-negative patients had their first CSF collected 14, 16, and 28 days after onset, respectively. Three of 98 (3.1%) CSF samples were completely or partially inhibitory to PCR. Initial DNA levels were not significantly different in patients with HSE due to either primary or recurrent HSV infection. In addition, they were not related to severity of clinical symptoms nor were predictive of the outcome. A progressive decrease in viral DNA levels was observed both in patients who received acyclovir therapy and in a small number of untreated patients. CONCLUSIONS This study: (i) confirms the high sensitivity of PCR for the diagnosis of HSE; (ii) emphasizes the need for an internal control of amplification of achieve maximal sensitivity and perform reliable quantitation of viral DNA; and (iii) suggests that CSF might not be the best specimen to investigate in studies of the natural history of HSE.