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BACKGROUND: SARS-CoV-2 infection is heterogeneous in clinical presentation and disease evolution. To investigate whether immune response to the virus can be influenced by genetic factors, we compared HLA and AB0 frequencies in organ transplant recipients and waitlisted patients according to presence or absence of SARS-CoV-2 infection. METHODS: A retrospective analysis was performed on an Italian cohort composed by transplanted and waitlisted patients in a January 2002-March 2020 time frame. Data from this cohort were merged with the Italian registry of COVID subjects, evaluating infection status of transplanted and waitlisted patients. A total of 56304 cases were studied with the aim of comparing HLA and AB0 frequencies according to the presence (n=265, COVID) or absence (n=56 039, COVID) of SARS-CoV-2 infection. RESULTS: The cumulative incidence rate of COVID-19 was 0.112% in the Italian population and 0.462% in waitlisted/transplanted patients (OR=4.2, 95%CI [3.7-4.7], P

Background Pneumocystis jiroveci pneumonia may be a life-threatening opportunistic infection in immunosuppressed solid organ transplant recipients. Despite effective treatment with high-dose trimethoprim-sulfamethoxazole and steroids, morbidity is often severe and lethality remains high. New therapeutic approaches are therefore warranted. Caspofungin, a beta-1,3-glucan synthesis inhibitor, has shown activity against the cyst forms of P. jiroveci in experimental animal models. We here report our preliminary clinical experience with caspofungin as an additional drug to the standard trimethoprim-sulfamethoxazole regimen. Methods Four solid organ transplant patients with severe hypoxemic P. jiroveci pneumonia were treated with the combination of trimethoprim-sulfametoxazole and caspofungin. In two cases, caspofungin was added as salvage treatment due to failure of trimethoprim-sulfametoxazole monotherapy. Results In these four patients, the use of caspofungin as an additional drug to the standard trimethoprim-sulfamethoxazole regimen led to a rapid improvement and a complete cure of pneumonia. No side effects or drug interactions were observed. Discussion This preliminary clinical experience suggests that the addition of caspofungin to trimethoprim-sulfamethoxazole, which is active against trophic forms, may provide a synergistic activity against P. jiroveci by fully inhibiting the organism life cycle.

Background. The risk of transmitting a hepatitis B virus (HBV) infection from donor kidneys with a past HBV serological profile may be negligible. Data on HBV transmission to kidney transplant recipients from donor organs that were anti-HBc/HBsAg - in Italy has not been previously reported. Anti-HBc testing in cadaver organ donors has been mandatory in Italy since 2002, when anti-HBc determinations were included in the National Guidelines for donor evaluation. Therefore, prior to that date kidney recipients from anti-HBc/HBsAg - donors can be identified retrospectively where stored serum is available for testing. Methods. The prevalence of anti-HBc Italian organ donors, the incidence of HBV transmission according to the recipients’ HBV status (vaccinated, recovered, or naive), and the clinical impact (5-year graft and patient survival rates) in the North Italy Transplant program was evaluated by retrospectively screening for anti-HBc antibodies in the sera of cadaver kidney donors used in transplants from 1997 to 1999. Results. Two hundred and ten donors were found to have been anti-HBc. At the time of the study, no active infection was observed in any of the 344 HBsAg - recipients, but 4/140 (2.86%) of the vaccinated recipients were found to have been anti-HBc/HBsAg - . None of these patients, however, had any biochemical or clinical history of HBV infection. Patient and graft survival rates of anti-HBc or anti-HBc - kidney recipients did not differ statistically. Conclusion. Kidney grafts from anti-HBc donors should be considered in all recipients because the benefit obtained from the transplantation out weighs the negligible risk of HBV transmission.

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).

Background. The aim of the study was to assess the incidence of cytomegalovirus (CMV) infection and disease in adult liver transplant recipients, using routine preemptive therapy guided by the pp65 antigenemia test. Methods. Antigenemia was monitored weekly after liver transplantation (OLTX) for the first 3 months, and once a month for another 3 months. CMV seronegative recipients were treated preemptively for the first positive antigenemia. Seropositive recipients were treated only when their antigenemia count reached a threshold of ≥100 positive cells per 200,000 leukocytes. Results. A total of 144 patients were included between June 1994 and April 1995, of which 137 (95%) were primary OLTX. The percentage of positive antigenemia and CMV disease was 55 and 8%, respectively. Seventy-eight (54%) patients were protocol-monitored for the entire follow-up (group 1) and received appropriate preemptive therapy, although 66 (46%) patients had protocol violation by having missed blood samples or blood drawn at unscheduled times (group 2). Using Cox's proportional hazards model, patients with a first antigenemia count of >11 leukocytes had a significantly higher rate of CMV disease compared to patients with an antigenemia count ≤11 leukocytes (RR=7.3, 95% confidence interval=2.2 to 24.5). In a multivariate Cox regression analysis, adjustments were made to control for: group 1 versus group 2, use of OKT3, and serology risk categories. This analysis showed that the relative rate of CMV disease was still significantly higher among patients with antigenemia count >11 leukocytes (adjusted RR=4.9, 95% confidence interval=1.3 to 18.1). The estimated cost of preemptive therapy was less than that of prophylaxis with i.v. (14-day course) or oral (90-day course) ganciclovir. Conclusions. Preemptive therapy guided by pp65 antigenemia is a useful and cost effective strategy for prevention of CMV disease.

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.

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 (P50.009) and leukoDNAemia (P 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

The present study aimed to investigate the relationship between acute rejection and human cytomegalovirus (HCMV) infection, as well as the coexpression of HLA-DR and immediate-early (IE) viral antigens, in 143 transbronchial biopsies and bronchoalveolar lavage fluids of 32 lung transplant recipients. We investigated the occurrence of morphologically overt viral infection with conventional histopathology, the expression of IE antigens with single labeling immunohistochemistry, the coexpression of IE antigens and HLA-DR molecules with double labeling techniques, and the presence of viral IE genes with polymerase chain reaction (PCR). Histopathologic study showed overt viral infections (12.6%) in 18 of the 143 biopsies ; 8 were in a context of pneumonia and 10 were localizations without surrounding inflammatory cells ; immunohistochemistry showed IE viral antigen expression in 31 (21.67%) ; PCR detected viral IE genes in 73/143 lavage fluids and biopsies (51%). The double labeling immunohistochemical technique showed that most IE antigen-expressing, noncytopathic cells were either HLA-DR negative in areas without infiltrates, or HLA-DR positive in those areas where inflammatory infiltrates were consistent, in the absence of viral cytopathy, with acute rejection. The results indicate that, in transplanted lung, the frequency of morphologically occult HCMV infection (as detected by immunohistochemistry and/or PCR) is much higher than that of morphologically overt viral infection. The occurrence of inflammatory infiltrates (consistent with acute rejection) around morphologically occult infected cells and the possible lack of inflammation around both early- and late-infected cells suggest that in biopsies with occult infection the infiltrates should be attributed to allograft reaction. This conclusion would be in keeping with the coexpression of HLA-DR and HCMV IE in infiltrate-rich biopsies that are consistent with acute rejection, as well as with the absence of HLA-DR expression in IE antigen-positive cells in infiltrate-free areas.

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.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.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, P0.05), the overall number of days of therapy was significantly higher. No patient developed HCMV disease.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).

Cytomegalovirus (CMV*), the first reported transplant-associated opportunistic virus (1), has remained the most frequent single cause or co-cause of infections throughout the ensuing three decades (2). Because prophylactic drug and hyperimmune globulin therapy has not been effective in these CMV-infected patients (2–4), Rubin (5) suggested withholding treatment until the phase of rapid viral replication (preemptive therapy). Early diagnosis of CMV infection, upon which this strategy depends, can be accomplished in the presymptomatic phase (6, 7) with a rapid quantitative direct demonstration of CMV antigen pp65 in cytospin preparations of peripheral blood leukocytes (PBL) (8, 9). This method has aided in the management of 20 CMV seronegative recipients of livers from seropositive donors, a circumstance that carries an 85–90% risk of virus transmission of CMV disease, presumably from migratory monocytes from the graft (10) that are known to harbor the latent virus in healthy seropositive individuals (11). The 20 recipients under tacrolimus/prednisone immunosuppression were followed for 308 ± 100.5 days (range, 63–520 days) after transplantation; a death at 63 days unrelated to CMV accounted for the only follow-up of 20 were considered positive. CMV IgG was also the routine test used for donor screening. Intravenous ganciclovir therapy (Cytovene; Syntex Pharmaceutical Ltd., Palo Alto, CA) was started only with the first detection of pp65 antigenemia, and continued until pp65 clearance. The ganciclovir dose was governed by creatinine clearance: >50 ml/min → 5 mg/kg of ganciclovir twice daily, 10–50 ml/min → 5 mg/kg every 24–48 hr, and