Analysis of the humoral and cellular immune response after a full course of BNT162b2 anti-SARS-CoV-2 vaccine in cancer patients treated with PD-1/PD-L1 inhibitors with or without chemotherapy: an update after 6 months of follow-up.

BACKGROUND: The durability of immunogenicity of SARS-CoV-2 vaccination in cancer patients remains to be elucidated. We prospectively evaluated the immunogenicity of the vaccine in triggering both the humoral and the cell-mediated immune response in cancer patients treated with anti-programmed cell death protein 1/programmed death-ligand 1 with or without chemotherapy 6 months after BNT162b2 vaccine. PATIENTS AND METHODS: In the previous study, 88 patients were enrolled, whereas the analyses below refer to the 60 patients still on immunotherapy at the time of the follow-up. According to previous SARS-CoV-2 exposure, patients were classified as SARS-CoV-2-naive (without previous SARS-CoV-2 exposure) and SARS-CoV-2-experienced (with previous SARS-CoV-2 infection). Neutralizing antibody (NT Ab) titer against the B.1.1 strain and total anti-spike immunoglobulin G concentration were quantified in serum samples. The enzyme-linked immunosorbent spot assay was used for quantification of anti-spike interferon-γ (IFN-γ)-producing cells/106 peripheral blood mononuclear cells. Fifty patients (83.0%) were on immunotherapy alone, whereas 10 patients (7%) were on chemo-immunotherapy. We analyzed separately patients on immunotherapy and patients on chemo-immunotherapy. RESULTS: The median T-cell response at 6 months was significantly lower than that measured at 3 weeks after vaccination [50 interquartile range (IQR) 20-118.8 versus 175 IQR 67.5-371.3 IFN-γ-producing cells/106 peripheral blood mononuclear cells; P < 0.0001]. The median reduction of immunoglobulin G concentration was 88% in SARS-CoV-2-naive subjects and 2.1% in SARS-CoV-2-experienced subjects. SARS-CoV-2 NT Ab titer was maintained in SARS-CoV-2-experienced subjects, whereas a significant decrease was observed in SARS-CoV-2-naive subjects (from median 1 : 160, IQR 1 : 40-1 : 640 to median 1 : 20, IQR 1 : 10-1 : 40; P < 0.0001). A weak correlation was observed between SARS-CoV-2 NT Ab titer and spike-specific IFN-γ-producing cells at both 6 months and 3 weeks after vaccination (r = 0.467; P = 0.0002 and r = 0.428; P = 0.0006, respectively). CONCLUSIONS: Our work highlights a reduction in the immune response in cancer patients, particularly in SARS-CoV-2-naive subjects. Our data support administering a third dose of COVID-19 vaccine to cancer patients treated with programmed cell death protein 1/programmed death-ligand 1 inhibitors.

Immunogenicity and safety after the third dose of BNT162b2 anti-SARS-CoV-2 vaccine in patients with solid tumors on active treatment: a prospective cohort study.

BACKGROUND: Although a full course of coronavirus disease 2019 (COVID-19) vaccine is effective in cancer patients, the duration of the protection and the efficacy of a booster dose against the new variants remain unknown. We prospectively evaluated the immunogenicity of the third dose of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) BNT162b2 messenger RNA vaccine in cancer patients undergoing active treatment. PATIENTS AND METHODS: Patients with solid cancer, vaccinated with a booster dose during active treatment, were enrolled in this study. Patients were classified into SARS-CoV-2 naïve (without previous COVID-19 infection) and SARS-CoV-2 experienced (with previous COVID-19 infection). Neutralizing antibody (NT Ab) titer and total anti-Spike immunoglobulin G (IgG) concentration were quantified in serum. Heparinized whole blood samples were used for SARS-CoV-2 Interferon Gamma Release Assay (IGRA). The primary endpoint was to assess the increase of IgG antibody level between baseline and 3 weeks after the booster. RESULTS: One hundred and forty-two consecutive patients were recruited. In SARS-CoV-2-naïve subjects, the median level of IgG was 157 BAU/ml [interquartile range (IQR) 62-423 BAU/ml] at T0 and reached a median of 2080 BAU/ml (IQR 2080-2080 BAU/ml) at 3 weeks after booster administration (T1; P < 0.0001). A median 16-fold increase of SARS-CoV-2 NT Ab titer (IQR 4-32) was observed in naïve subjects (from median 20, IQR 10-40, to median 640, IQR 160-640; P < 0.0001). Median interferon-γ level at T1 was significantly higher than that measured at T0 in SARS-CoV-2-naïve subjects (P = 0.0049) but not in SARS-CoV-2-experienced patients. The median level of SARS-CoV-2 NT Abs was 32-fold lower against Omicron compared to the wild-type strain (P = 0.0004) and 12-fold lower compared to the Delta strain (P = 0.0110). CONCLUSIONS: The third dose is able to trigger both the humoral and the cell-mediated immune response in cancer patients on active treatment. Our preliminary data about the neutralization of the SARS-CoV-2 vaccine against variants of concern seem to confirm the lower vaccine activity.

Six-month humoral and cellular immune response to the third dose of BNT162b2 anti-SARS-CoV-2 vaccine in patients with solid tumors: a longitudinal cohort study with a focus on the variants of concern.

BACKGROUND: The role and the durability of the immunogenicity of the third dose of vaccine against COVID-19 variants of concern in cancer patients have to be elucidated. PATIENTS AND METHODS: We have prospectively evaluated the immunogenicity of the third dose of the SARS-CoV-2 BNT162b2 messenger RNA vaccine in triggering both humoral and cell-mediated immune response in patients with solid tumors undergoing active treatment 6 months after the booster. Neutralizing antibody (NT Ab) titers and total anti-spike immunoglobulin G concentrations were measured in serum. Heparinized whole blood samples were used for the SARS-CoV-2 interferon-γ release assay (IGRA). RESULTS: Six months after the third dose only two patients (2.4%) showed negative spike-specific immunoglobulin G antibody levels (<33.8 BAU/ml). The median level of SARS-CoV-2 NT Abs decreased and only 39/83 (47%) subjects showed maximum levels of NT Abs. T-cellular positive response was observed in 38/61 (62.3%) patients; the highest median level of response was observed 21 days after the third dose (354 mIU/ml, interquartile range 83.3-846.3 mIU/ml). The lowest median level of NT Ab response was observed against the Omicron variant (1 : 10, interquartile range 1 : 10-1 : 40) with a significant reduced rate of responder subjects with respect to the wild-type strain (77.5% versus 95%; P = 0.0022) and Delta variant (77.5% versus 93.7%; P = 0.0053). During the follow-up period, seven patients (8%) had a confirmed post-vaccination infection, but none of them required hospitalization or oxygen therapy. CONCLUSIONS: Our work highlights a significant humoral and cellular immune response among patients with solid tumors 6 months after the third BNT162b2 vaccine dose, although a reduction in neutralizing activity against Omicron was observed.

Early co-circulation of different clades of influenza A/H1N1v pandemic virus in northern Italy.

INTRODUCTION: The spatial diffusion over time of pandemic influenza A/HINI virus (A/HIN1v) was surveyed in Northern Italy (nearly 10 million inhabitants)from April to December 2009, and the molecular characteristics of circulating viruses were analyzed to identify the appearance of drift variants. About 45% of analyzed samples were laboratory-confirmed cases of A/HINlv. Sporadic cases occurred until the middle of June 2009, then, case numbers began to increase delineating distinct epidemiological phases of viral circulation. METHODS: RNA was extracted using RNeasy Mini kit (QIAGEN GmbH, Germany). Virological diagnosis of A/HINlv infection was carried out by real-time RT-PCR assay. Sequence analysis of hemagglutinin (HA) gene was performed through a RT-PCR assay specific for a 995 bp fragment (nt. 64-1,058) in the HAl domain. The nucleotide sequences were obtained by automated DNA sequencing. The HAl sequences were aligned with other sequences collected from GenBank database by ClustalX software. The multiple sequence alignment was used to perform a basic phylogenetic analysis and a phylogenetic tree from HA sequences was constructed. RESULTS: The HA gene sequences ofA/HINlv analyzed segregated into three genetically distinct clades and were characterized by the appearance of amino acid variations that were progressively fixed in the field viral population under scrutiny. CONCLUSIONS: These data suggest an early co-circulation of genetically distinct A/HNINv variants and emphasize the importance of a close molecular surveillance to detect rapidly the spread of new viral variants and to define their epidemiological impact.

SARS-CoV-2 viability on different surfaces after gaseous ozone treatment: a preliminary evaluation.

COVID-19 is a global health threat with a huge number of confirmed cases and deaths all over the world. Human-to-human transmission via respiratory droplets and contact with aerosol-infected surfaces are the major routes of virus spread. Because SARS-CoV-2 can remain in the air and on surfaces from several hours to several days, disinfection of frequently touched surfaces and critical rooms, in addition to observing individual hygiene tips, is required to reduce the virus spreading. Here we report on an investigation into the use of gaseous ozone as a potentially effective sanitizing method against the new coronavirus.

A snapshot of the immunogenicity, efficacy and safety of a full course of BNT162b2 anti-SARS-CoV-2 vaccine in cancer patients treated with PD-1/PD-L1 inhibitors: a longitudinal cohort study.

BACKGROUND: Very few cancer patients were enrolled in coronavirus disease-2019 vaccine studies. In order to address this gap of knowledge, real-world studies are mandatory. The aim of this study was to assess both humoral and cellular response after a messenger RNA vaccination schedule. PATIENTS AND METHODS: Eighty-eight consecutive cancer patients treated with programmed cell death protein 1/programmed death-ligand 1 inhibitors were enrolled from the beginning of the vaccination campaign for frail patients. Blood samples for humoral and cell-mediated immune response evaluation were obtained before vaccination (T0), before the second administration (T1) and 21 days after the second dose (T2). The primary endpoint was the evaluation of the percentage of participants showing a significant increase in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells, measured by an enzyme-linked immunospot assay, after the second dose of BNT162b2 vaccine. The proportion of patients who reached the primary endpoint is computed together with its exact binomial 95% confidence interval. RESULTS: In SARS-CoV-2-naïve subjects, spike-specific T-cell response was almost undetectable at T0 [median 0.0 interferon-γ (IFN-γ) spot forming units (SFU)/million peripheral blood mononuclear cell (PBMC) interquartile range (IQR) 0-7.5] and significantly increased at T1 and T2 (median 15.0 IFN-γ SFU/million PBMC, 25th-75th 0-40 versus 90 IFN-γ SFU/million PBMC, 25th-75th 32.5-224, respectively) (P < 0.001). Focusing on naïve and experienced SARS-CoV-2 subjects, no differences were reported both in terms of CD4- and CD8-specific T-cell response, suggesting that BNT162b2 is able to elicit both adaptive responses after complete vaccination schedule, regardless of previous SARS-CoV-2 exposure. The level of SARS-CoV-2 neutralizing antibodies was low at T1 in SARS-CoV-2-naïve subjects [median 1 : 5 (IQR 1 : 5-1 : 20)] but reached a significantly higher median of 1 : 80 (25th-75th 1 : 20-1 : 160) at T2 (P < 0.0001). Moreover, no COVID-19 cases were documented throughout the period of study. CONCLUSIONS: Our data have demonstrated that the administration of a full course of BNT162b2 vaccine elicited a sustained immune response against SARS-CoV-2 regardless of the type of cancer and/or the type of immune checkpoint inhibitors.

Severe acute respiratory syndrome coronavirus 2 RNA contamination of inanimate surfaces and virus viability in a health care emergency unit.

OBJECTIVES: To detect possible severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA contamination of inanimate surfaces in areas at high risk of aerosol formation by patients with coronavirus disease 2019 (COVID-19). METHODS: Sampling was performed in the emergency unit and the sub-intensive care ward. SARS-CoV-2 RNA was extracted from swabbed surfaces and objects and subjected to real-time RT-PCR targeting RNA-dependent RNA polymerase and E genes. Virus isolation from positive samples was attempted in vitro on Vero E6 cells. RESULTS: Twenty-six samples were collected and only two were positive for low-level SARS-CoV-2 RNA, both collected on the external surface of continuous positive airway pressure helmets. All transport media were inoculated onto susceptible cells, but none induced a cytopathic effect on day 7 of culture. CONCLUSIONS: Even though daily contact with inanimate surfaces and patient fomites in contaminated areas may be a medium of infection, our data obtained in real-life conditions suggest that it might be less extensive than hitherto recognized.

Correlation of viral load as determined by real-time RT-PCR and clinical characteristics of respiratory syncytial virus lower respiratory tract infections in early infancy.

BACKGROUND: In infants hospitalized for a lower respiratory tract infection (RTI) caused by respiratory syncytial virus (RSV), the correlation between viral load (VL) and patient clinical characteristics remains to be defined. OBJECTIVES: To define this correlation. STUDY DESIGN: prospective study of 47 infants admitted to hospital in the period November 2006-May 2007 with a diagnosis of lower RTI. Nasopharyngeal aspirates (NPAs) were taken at admission, discharge, and at post-discharge control visits. VL was quantified by real-time RT-PCR for RSV subgroups A and B. RESULTS: Patients with bronchiolitis were compared with young patients with lower RTI other than bronchiolitis. Patients with bronchiolitis had a significantly lower age than patients with other syndromes, and a significantly longer duration of symptoms. Duration of hospitalization was not different in the two groups of patients, and was not related to RSV subgroup or viral coinfection. A sustained decrease in VL was observed in the general patient population between admission, discharge and post-discharge follow-up visits. CONCLUSIONS: (i) patients with bronchiolitis were significantly younger than patients with other lower RTIs; (ii) symptom duration was significantly longer in patients with bronchiolitis; (iii) RSV VL significantly decreased between admission and discharge.

Circulating endothelial giant cells permissive for human cytomegalovirus (HCMV) are detected in disseminated HCMV infections with organ involvement.

Giant cells fully permissive for human cytomegalovirus (HCMV) were found to circulate, at a variable proportion, in peripheral blood of 21 out of 25 immunocompromised patients with disseminated HCMV infection. Circulating endothelial giant cells (EGC) were identified by a specific monoclonal antibody of endothelial origin and shown to express immediate-early, early, and late viral proteins. Immunostaining patterns of different viral proteins were comparable to those detected in vitro in cultured human umbilical vein endothelial cells. EGC counts > 10 were associated with high levels (> 100) of HCMV viremia and antigenemia, as well as with an overt clinical syndrome in transplanted patients, and to an untreated long lasting organ localization in AIDS patients. On the other hand, EGC counts were < 10 during disseminated HCMV infections of both transplant recipients with no apparent organ syndrome and AIDS patients with recent organ involvement. In tissue sections from AIDS patients, infected endothelial cells were found to progressively enlarge till detaching from the small vessel wall and entering blood stream. HCMV-infected EGC represent a new systemic parameter suitable for the diagnosis of HCMV organ involvement and for the study of the pathogenesis of disseminated infections.

In vitro generation of human cytomegalovirus pp65 antigenemia, viremia, and leukoDNAemia.

Immunocompromised patients with disseminated human cytomegalovirus (HCMV) infection have circulating PMN carrying HCMV pp65 (antigenemia), infectious virus (viremia), and viral DNA (leukoDNAemia). Because HCMV does not fully replicate in PMN, it is generally hypothesized that virions and viral materials are taken up by phagocytosis from fully permissive HCMV-infected endothelial cells. However, no experimental evidence has ever been provided for these PMN-endothelium interactions. PMN from 11 donors were cocultured with endothelial cells infected with an endothelium-adapted HCMV strain and with human fibroblasts infected with low-passaged clinical and laboratory-adapted HCMV strains. pp65-positive PMN were detected after coculture with both HCMV-infected endothelial and fibroblast cells, provided that wild and not laboratory-adapted strains were used. In addition, cocultured PMN carried infectious virus as demonstrated by virus isolation and presence of complete virus particles by electron microscopy. Moreover, high levels of viral DNA were consistently detected by quantitative PCR in cocultured PMN. Thus, we have generated in vitro the three most important viral parameters detected in patients with disseminated HCMV infection (antigenemia, viremia, and leukoDNAemia). The failure of laboratory-adapted HCMV strain to induce this phenomenon demonstrates that important modifications have occurred in attenuated viral strains affecting basic biological functions.

Phylogenetic characterization of Central/Southern European lineage 2 West Nile virus: analysis of human outbreaks in Italy and Greece, 2013-2014.

In recent years, West Nile virus (WNV) lineage 2 has been spreading and causing disease outbreaks in humans and animals in Europe. In order to characterize viral diversity, we performed full-length genome sequencing of WNV lineage 2 from human samples collected during outbreaks in Italy and Greece in 2013 and 2014. Phylogenetic analysis showed that these WNV lineage 2 genomes belonged to a monophyletic clade derived from a single introduction into Europe of the prototype Hungarian strain. Correlation of phylogenetic data with geospatial information showed geographical clustering of WNV genome sequences both in Italy and in Greece, indicating that the virus had evolved and diverged during its dispersal in Europe, leading to the emergence of novel genotypes, as it adapted to local ecological niches. These genotypes carried divergent conserved amino acid substitutions, which might have been relevant for viral adaptation, as suggested by selection pressure analysis and in silico and experimental modelling of sequence changes. In conclusion, the results of this study provide further information on WNV lineage 2 transmission dynamics in Europe, and emphasize the need for WNV surveillance activities to monitor viral evolution and diversity.

Human cytomegalovirus viraemia in HIV-1-seropositive patients at various clinical stages of infection.

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.

Clinical and virological monitoring of human cytomegalovirus infection in 294 heart transplant recipients.

Two hundred and ninety-four heart transplant recipients (HTR) were followed prospectively for a mean of 44.9 +/- 28.4 (range 1.0-100.8 months) after transplantation (tx). Immunosuppression was based on cyclosporine, azathioprine, and steroids, supplemented by a 7-day course of antithymocyte globulins. All patients were virologically monitored by inoculating aliquots of 2 x 10(5) peripheral blood polymorphonuclear leukocytes (PMNs) onto human embryonic lung fibroblasts monolayers grown in shell vials for early cytomegalovirus (CMV) identification and quantification (viremia). The same number of PMNs was cytocentrifuged onto glass slides for direct CMV pp65 antigen detection and quantification (antigenemia). Heparinized blood samples were collected weekly during the first 3 months following tx and at least twice a week if antigenemia and viremia levels were increasing. After 3 months, samples were collected if antigenemia and viremia persisted or when clinically indicated. The overall incidence of CMV infection was 53.4% (157/294). Only 32.4% (51/157) of the viremic patients required antiviral treatment because of symptomatic infection. Of the remaining 106 untreated CMV viremic HTR, 104 were asymptomatic while 2 had only mild clinical symptoms. The overall incidence of CMV infection in pre-tx CMV seropositive (CMV+) HTR was 50.9% (136/267); 75.7% (103/136) were asymptomatic and 24.3% (33/267); 75.7% (103/136) were asymptomatic and 24.3% (33/136) were symptomatic. The overall incidence of CMV infection in pre-tx CMV-seronegative (CMV-) HTR was 77.8% (21/27; P = 0.007 vs. seropositive HTR). Among 22 CMV- HTR with CMV+ donor, 20 (90.9%) had a CMV infection and all of them were symptomatic (versus 1 of 5 (20%) CMV- HTR with CMV- donor; P = 0.002, Fisher's exact test). The median numbers of circulating CMV-infected PMNs detected at the onset of clinical symptoms by the antigenemia and viremia assays were 385/2 x 10(5) and 100/2 x 10(5), respectively.

Search for Coxsackievirus B3 RNA in idiopathic dilated cardiomyopathy using gene amplification by polymerase chain reaction.

A polymerase chain reaction (PCR) amplification assay was developed to detect Coxsackievirus B3 ribonucleic acid (RNA) in blood and myocardial tissue of explanted hearts from 40 patients who underwent cardiac transplantation and in 1 normal heart. Twenty-one patients were affected by idiopathic dilated cardiomyopathy of different duration and 19 by coronary artery disease. Coxsackievirus B3 in vitro infected Vero cells and cells infected by related human enteroviruses (Coxsackievirus B2, B4, and poliovirus 1) were used as reaction controls. PCR was performed using 4 pairs of primers homologous to Coxsackie-virus B3 sequences. Three sets were located in regions of the genome conserved at nucleotide level between several enterovirus species (replicase gene, 5' noncoding region), while one was located in a Coxsackievirus B3-specific region (VP1 gene). Total RNA was prepared by acid guanidinium isothiocyanate extraction from tissue stored frozen at -80 degrees C. One microgram of total RNA was retrotranscribed with either antisense primer or with random hexanucleotide primers and then subjected to 40 cycles of amplification. PCR products were separated by electrophoresis on a 10% polyacrylamide gel, electrotransferred to a nylon membrane and then hybridized to oligonucleotide probes specific for the coxsackievirus B3 genome radiolabeled with radioactive isotope of phosphorous. All pairs of primers yielded specific amplification products when tested on Coxsackievirus B3-infected Vero cells, with a sensitivity of 1 infected cell out of 10(5) to 10(6) cells starting from 1 microgram total RNA. Primer sets for regions of Coxsackievirus B3 genome highly conserved between related enteroviral species gave positive amplification also when challenged with RNA from cells infected by Coxsackievirus B2, B4 and poliovirus 1.

Constitutive expression of human cytomegalovirus (HCMV) glycoprotein gpUL75 (gH) in astrocytoma cells: a study of the specific humoral immune response.

The humoral immune response to gpUL75 (gH) was determined in different groups of human cytomegalovirus (HCMV) infected subjects using a full-length glycoprotein constitutively expressed in an astrocytoma cell line. The recombinant molecule consisted of two distinct isoforms resembling the authentic protein of infected cells. Separated from the interactions of other viral gene products gH failed to form an oligomeric complex, thus exhibiting exclusively epitopes present on the monomer. Ninety five percent of serum samples from latently-infected healthy adults revealed the presence of gH-specific IgG. Moreover, examination of sequential sera from immunocompromised and immunocompetent individuals undergoing active HCMV infection demonstrated that antibodies to gH occurred in most cases simultaneously with those to the abundant surface antigen gpUL55 (gB) and at similar titres. Appearance of this response was correlated with a considerable increase of the virus-neutralizing activity and most likely associated with restriction of viral dissemination during subsequent viremic episodes. Together, these results suggest that glycoprotein H of HCMV is like gB, a highly immunogenic component of the infectious particle.

Use of the human cytomegalovirus (HCMV) antigenemia assay for diagnosis and monitoring of HCMV infections and detection of antiviral drug resistance in the immunocompromised.

BACKGROUND: Quantification of viral load in blood has proven to be helpful in the follow-up of disseminated HCMV infections in immunocompromised patients. OBJECTIVES: (i) To describe the antigenemia assay and its optimization and (ii) to analyze the use of the antigenemia assay for the diagnosis and monitoring of HCMV infections and for the detection of treatment failures. STUDY DESIGN: This article is intended to give an overview of our experience in the use of the antigenemia assay. RESULTS AND CONCLUSIONS: In solid organ transplant recipients and patients with AIDS, HCMV symptomatic infections mostly appear when antigenemia values are > 300 pp65-positive PBL/2 x 10(5) examined. To avoid the appearance of overt HCMV disease antiviral treatment could be administered when antigenemia levels are > 100 pp65-positive PBL/2 x 10(5) examined. Bone marrow transplant recipients show symptomatic HCMV infections when antigenemia values are > 100 pp65-positive PBL/2 x 10(5) examined. This group of patients should be treated when antigenemia levels are < 10 pp65-positive PBL/2 x 10(5) examined due to the higher mortality rate associated with HCMV complications. A decrease in antigenemia levels during therapy indicates a good response to antiviral drug, while stable or increasing values document a treatment failure and the emergence of drug-resistant HCMV strains.

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.

Quantitative systemic and local evaluation of the antiviral effect of ganciclovir and foscarnet induction treatment on human cytomegalovirus gastrointestinal disease of patients with AIDS. Italian Foscarnet GID Study Group.

In a group of 29 AIDS patients with biopsy-proven human cytomegalovirus (HCMV) gastrointestinal disease (GID), HCMV GID was shown to correlate mostly with systemic HCMV infection. The antiviral induction treatment (IT) with either ganciclovir (GCV) or foscarnet (PFA) caused a significant reduction in the level of HCMV antigenemia, viremia and leukoDNAemia, and a complete virus clearance or a sharp drop of viral load in the blood of 13/13 patients and in the gastrointestinal (GI) mucosa of 12/13 (92%) patients in the GCV arm, and in the blood of 13/14 (93%) patients and in the GI mucosa of 10/12 (83%) patients in the PFA arm of the study, respectively. Similarly, the clinical response was good in 13/15 (87%) patients in the GCV arm and in 13/14 (93%) patients in the PFA arm. In addition, the finding that 2/6 patients positive for HCMV isolated from both GI mucosa and blood prior to IT were still positive in the GI tract after IT, suggested that IT could be prolonged to clear the virus from GI tract. In conclusion, both GCV and PFA showed a remarkable systemic and local antiviral effect in the treatment of HCMV GID in AIDS patients.

Histopathologic and molecular profile of human cytomegalovirus infections in patients with heart transplants.

From November 1985 to December 1990, 2,552 endomyocardial biopsy specimens from 209 heart transplant patients were studied. Forty-four (21%) patients developed 45 episodes of major human cytomegalovirus infection (HCMV). Human cytomegalovirus infection was primary in 13 of 44 patients. Thirty-one patients developed episodes of recurrent major infection. One patient had both primary and recurrent infections. Conventional histopathologic and immunohistochemical study, in situ hybridization, and polymerase chain reaction were used to diagnose HCMV myocardial involvement on corresponding endomyocardial biopsy specimens performed during infection. Conventional morphologic study showed typical viral inclusion bodies in four biopsy specimens. Two cases had myocyte HCMV localization with necrotizing myocarditis, whereas two had endothelial cell involvement without any inflammatory reaction. In these four biopsy specimens, immunohistochemistry showed a higher number of infected cells than that recognized by conventional histopathologic study. In situ hybridization detected infected cells with no evidence of cytopathic effect. Polymerase chain reaction gave HCMV amplification products in two additional biopsy specimens otherwise interpreted as moderate and mild rejection, respectively. Therefore, 6 biopsies showed HCMV myocardial involvement (6 of 45; 13.3%): all were from patients with primary HCMV infection (6 of 13; 46%). None of 32 major recurrent infections showed any myocardial involvement. In conclusion, our study is the first to demonstrate that myocardial HCMV involvement preferentially occurs in primary infection and HCMV endothelial localization can be free from inflammatory reaction, whereas HCMV myocyte localization leads to necrotizing myocarditis. Polymerase chain reaction has a higher diagnostic sensitivity than in situ hybridization. However, polymerase chain reaction findings of HCMV DNA on otherwise negative endomyocardial biopsy specimens remains of questionable significance because polymerase chain reaction-positive biopsy samples do not necessarily indicate tissue infection. It is impossible to determine whether amplified sequences derive from circulating leukocytes or from tissue cells.