P. Grossi

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, Fishers 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.

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.

Guidance of ganciclovir therapy with pp65 antigenemia in cytomegalovirus-free recipients of livers from seropositive donors

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 <3 months. Heparinized blood samples were obtained by schedule during the first 3 months or when clinically indicated during this period and later. The pp65 antigen was detected in duplicate leukocyte spots (2 × 105 cells in 200 μl/glass slide) with the technique of Van der Bij et al. (8), using immunofluorescence (12) instead of immunoperoxidase staining (9). The anti-pp65 mouse monoclonal antibody was clone 1C3 (Biosoft, Argene, France). Fluorescein-isothiocyanate-labeled goat anti-mouse Fab2 fragment (Cappel, Durham, NC) was applied as the conjugate. Slides were counterstained with Evans blue dye and observed under 400 x magnification for the typical green nuclear fluorescence. A conventional shell vial culture assay was done on the same blood specimens. Anti-CMV antibodies (IgG and IgM) were measured before and after transplantation using a semiautomated immunofluorescence test (FIAX test system, Whittaker Bioproducts, Inc., Walkersville, MD). IgG titers >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 <10 ml/min → 5 mg/kg every 48–96 hr. As expected, 17 (85%) of the 20 patients had positive pp65 tests (CMV antigenemia) after a median posttransplant interval of 34 days (range, 9–83 days). Positive shell vial CMV cultures (viremia) were obtained at the same time as the first antigenemia diagnosis in only 5 of these 17 cases. Twelve of the 17 patients were asymptomatic when the first positive pp65 test was obtained, including a recipient who was diagnosed only 9 days after transplantation (Fig. 1). The pp65 levels were generally low: 20±28 (SD) per 2 × 105 leukocytes. However, 6 of the 12 had further rises in antigenemia for 1 to 12 days after therapy was started, to a median peak of73 stained PBLs (range, 29–446 PBLs), before antigenemia began to clear. An example is shown in Figure 1. Ganciclovir instituted at the time of the first antigenemia prevented clinical disease in 11 of the 12 infected patients; the exceptional patient had a transient fever. The five other patients had CMV hepatitis at the time antigenemia was detected 12–83 days after surgery (median, 34 days). The median number of pp65-positive cells was 475 (range, 177–872). Figure 1 Preemptive ganciclovir treatment guided with CMV antigenemia in an asymptomatic, previously seronegative liver transplantation recipient whose donor was CMV positive. Antigenemia was cleared with intravenous ganciclovir therapy in all 17 patients but recurred in 10 (59%) patients 5–174 days (median, 57 days) after the drug had been discontinued. Recurrence was independent of IgG conversion (P=0.26). The only patient who was symptomatic with recurrence had 430 pp65-stained cells, which cleared after resumption of ganciclovir. The nine asymptomatic patients had 3–101 pp65-stained cells. Ganciclovir retreatment promptly cleared the antigenemia in the recipient with 101 stained cells. Retreatment was withheld in the other eight patients whose samples had <100 stained cells, and the antigenemia resolved spontaneously in all. In these cases, the antigen assay was useful as a signal to begin preemptive antiviral treatment, as envisioned by Rubin (2), as a means to decide whether to reinstitute ganciclovir in the event of a recurrence (previously emphasized by The et al. [6]) and as a guide to individualize the duration of a ganciclovir course in all of these circumstances. The assay also can be an important differential diagnostic tool. The persistence of symptoms despite clearance of the CMV pp65 antigen is an unambiguous warning of co-infection by another pathogen. Conversely, failure of antigen clearance should raise suspicion of ganciclovir-resistant CMV mutants. The pp65 findings are specific. With a novel method for quantitation of CMV DNA in leukocytes, a highly significant correlation was demonstrated between leukoDNAemia and the presence and quantity of pp65 antigenemia (13). The sensitivity and quantitation nature of the pp65 test allowed new observations about response to therapy, such as the reason for increases in pp65 levels seen during the first few days after beginning ganciclovir in half of the preemptively treated patients. This may have reflected a delay in suppression of viral replication or, alternatively, phagocytosis by PBLs of degrading CMV matrix protein from lysed, infected leukocytes (14) or from the ballooned and infected endothelial cells that shed into the circulation (15).

Infectious complications in patients with the novacor left ventricular assist system

THE USE OF ventricular assist devices (VADs) as a bridge to transplantation has improved the quality of life and decreased the morbidity and mortality in patients awaiting transplantation. The success of VADs for longterm support in patients with heart failure has made the use of VADs for permanent cardiac assistance a promising therapy. However, infectious complications, often arising from the percutaneous driveline exit site, remain the single most important obstacle for widespread use of VADs. The incidence of infection following left VAD (LVAD) implantation has been reported to be 13% to 80%. According to a review of .2000 patients worldwide, clinically relevant infections occurred in 25% of LVAD recipients. Fortunately, these infections do not preclude heart transplantation. The aim of the present study was to evaluate the incidence of infectious complications and the impact of infection on outcome in a series of patients on circulatory mechanical assistance with the Novacor N100 LVAD (Baxter Healthcare, Inc, Novacor Division) at the Charles Dubost Cardiac Surgery Center, IRCCS San Matteo, University of Pavia, Italy.

Pneumocystis carinii pneumonia in heart transplant recipients.

SummarySeven cases ofPneumocystis carinii pneumonia (PCP) (two in 1988, three in 1989, one in 1990 and one in 1991) have been observed in a group of 241 heart transplant recipients tranplanted in Pavia, Italy, from November 1985 through December 1991. Median time to onset of symptoms was 100 days after transplantation (range 59–333 days). Diagnosis was achieved in all patients by cytological examination of bronchoalveolar lavage (BAL) fluid and/or transbronchial biopsy. Clinical and roentgenographic features were remarkably similar in all PCP-affected heart transplant recipients. A dry, persistent hacking cough associated with dyspnoea was consistently observed. Fever ranged from 37.6 to 39.4°C, median leukocyte count and median arterial oxygen saturation (SaO2) values were 7,300/mm3 (range 3,000–16,000/mm3) and 61% (range 49.3–93%), respectively. Median CD4+ count at the onset of symptoms was 211/mm3 (range 28–739/mm3). The only patient experiencing a recurrence of PCP had a CD4+ cell count of 28/mm3 at the end of treatment with trimethoprim-sulfamethoxazole (TMP-SMX). In all patients human cytomegalovirus was isolated from BAL fluids; however, treatment with TMP-SMX alone (20 mg/kg/day of TMP) was consistently followed by a complete recovery.ZusammenfassungZwischen November 1985 und Dezember 1991 wurden in Pavia, Italien, unter 241 Herztransplantatempfängern sieben Fälle vonPneumocystis carinii-Pneumonie (PCP) beobachtet. Zwei Fälle traten 1988, drei 1989 und einer 1990 auf. Bis zum Auftreten der Infektion vergingen nach der Transplantation im Median 100 Tage (Bereich 59–333 Tage). Bei allen Patienten wurde der Erregernachweis durch zytologische Untersuchung von Bronchoalvcolar-Lavage-Flüssigkeit und/oder transbronchial entnommenem Feinnadelbiopsie-Material gestellt. Die klinischen und radiologischen Bilder waren bei allen an PCP erkrankten Transplantatempfängern bemerkenswert ähnlich. In allen Fällen trat ein trockener Staccato-Husten mit Atemnot auf. Die Temperaturen lagen zwischen 37,6 und 39,4°C. Die Leukozytenwerte lagen im Mittel bei 7.300/mm3 (Bereich 3.000–16.000/mm3). Die mittlere arterielle Sauerstoffsättigung war auf 61% (Bereich 49,3–93%) erniedrigt. Bei Einsetzen der Symptomatik fanden sich mediane CD4+ - Zellzahlen von 211/mm3 (Bereich 28–739/mm3). Nur in einem Fall trat ein PCP-Rezidiv auf; bei diesem Patienten lagen die CD4+-Zellzahlen am Ende der Behandlung mit Trimethoprim-Sulfamethoxazol (TMP-SMX) bei 28/mm3. Bei allen Patienten wurde aus Bronchoalveolarflüssigkeit auch das Humane Cytomegalievirus isoliert. Dennoch trat schon unter Therapie mit TMP-SMX (20 mg/kg/Tag TMP) bei allen Patienten eine vollständige Heilung ein.

Prevalence and outcome of hepatitis B virus (HBV) infection following thoracic organ transplantation

Reactivation of hepatitis B virus (HBV) infection is known to occur in chronic HBsAg carriers who are immunosuppressed. From November 1985 through September 2000 a total of 786 recipients (160 female and 626 male, mean age 47.6, range 8-71 years) underwent thoracic organ transplantation (612 heart, 29 heart-lung, 70 double lung and 75 single lung) at our Institution. Twenty-one HbsAg positive, HBV-DNA negative patients underwent thoracic organ transplantation (15 heart and 6 lung). Nine (42.8%) patients died of HBV unrelated causes at a mean of 33.1 (median 17.6, range 0.167-107.3) months after transplantation, two are still alive with HBV-DNA negativity and 10 (47.6%) developed HBV reactivation, documented by HBV-DNA detection on peripheral blood, at a mean of 31.5 (median 20.7, range 0.133-129.5) months after transplantation. Of these 10 patients two died of decompensated liver disease before lamivudine was available at 20.9 and 40.9 months after transplantation, respectively. The other 8 patients underwent a liver biopsy and were treated with lamivudine at the dose of 100 mg/day. Furthermore five patients who tested negative for HBV markers and one who was HBsAb and HBcAb positive prior to transplantation were found HBV-DNA positive at a mean of 37.6 (median 25.1, range 9.5-120.6) months after transplantation and two of them were treated with lamivudine. Five of the 10 lamivudine treated patients cleared HBV-DNA from blood and the mean time to HBV-DNA clearance was 53.2 (range 31-83) days after the start of lamivudine treatment. Serum ALT returned to normal in 5/7 patients with pre-treatment liver function tests abnormalities after a mean of 123.6 (range 31-277) days of lamivudine treatment. No significant side effect was observed during the treatment that was given for a median of 296 days (range 2-908). Thoracic organ transplantation in HBsAg positive and HBVDNA negative recipients is followed by HBV reactivation in a high percentage of cases. However, the clinical outcome and the availability of lamivudine suggest not to exclude these patients from transplantation.

040 PROPIONIBACTERIUM ACNES PROSTHETIC VALVE ENDOCARDITIS: REPORT OF TWO CASES

Methods: We performed a retrospective study comprising 260 consecutive episodes of IE admitted in the University Hospital of Infectious Diseases Cluj-Napoca, Romania, during 200

Advances in Cytomegalovirus Diagnostic Testing and Their Implications for Management of Cytomegalovirus Infection in Transplant Recipients

Human cytomegalovirus (CMV) was the first “opportunistic” virus described in renal transplant recipients under azathioprine and prednisone (1). It has been the most frequent cause of infectious complications after whole organ transplantation with all subsequent immunosuppressive regimens (2-11). In seronegative recipients, latently infected allografts and leukocyte-containing blood products are documented means of virus transmission (12-16). Among pre-transplant CMV seropositive transplant recipients, CMV infection may occur after reactivation of latent infection or after reinfection (17). Coinfection with multiple strains has also been detected in immunocompromised individuals (18-20). These so called secondary infections occur in about 50% to 80% of pre-transplant seropositive transplant recipients but a smaller proportion of the patients, ranging from 10 to 40% according to the transplanted organ, develop a CMV disease (11). Active CMV infection occurs during the first three months after transplantation and may be accompanied by a broad spectrum of disease manifestations ranging from a mononucleosis like syndrome to severe pneumonia. In the scientific literature the definitions used for CMV infection, CMV viremia and CMV disease vary greatly. Since this can make comparison between different studies difficult, a consensus definition was achieved during the fourth and fifth CMV conference in Paris 1993 and Stockholm 1995 (21).

An intelligent system for monitoring infections in heart transplant recipients

A computer-based assistant for monitoring a patient’s clinical course requires the use of tools able to handle temporal issues. Thus, methodologies coming from two historically distinct worlds need to be combined: the traditional world of Data Base Management Systems (DBMS) and the world of Knowledge-Based Systems (KBS). This paper describes an intelligent system designed to assist the clinical staff in the management of a monitoring protocol of infections in heart transplant recipients. The system consists in a DBMS purposely oriented to the management of patient clinical data and of a KBS capable to reason on a large amount of data. Moreover, the system aims at providing a synthetic view of a patient’s clinical history and some diagnostic and therapeutic suggestions. The KBS retrieves findings stored in the data base and creates a complex taxonomy of objects representing a temporal network of significative events and episodes which occurred during the overall clinical patient history; then, from this temporal representation, it develops its reasoning based on the medical knowledge represented using frames and production rules.