Ilona Lewensohn-Fuchs

RESULTS OF DIFFERENT STRATEGIES FOR REDUCING CYTOMEGALOVIRUS-ASSOCIATED MORTALITY IN ALLOGENEIC STEM CELL TRANSPLANT RECIPIENTS

BACKGROUND Several preventive strategies against cytomegalovirus (CMV) disease have been developed during the last decade. These have frequently been used in combination, and it has been difficult to identify each strategys contribution. METHODS Risk factors for CMV disease, death in CMV disease and transplant-related mortality were analyzed in 584 patients, who underwent a total of 594 allogeneic bone marrow transplants. RESULTS The overall probability of CMV disease was 8.9%. No seronegative patient who had a seronegative marrow donor developed CMV disease. The corresponding probabilities for seronegative patients with seropositive donors, seropositive patients with seronegative donors, and seropositive patients with seropositive donors were 5.4%, 13.7%, and 11.7%, respectively. In multivariate Cox models, the use of preemptive antiviral therapy and being CMV-seronegative reduced the risk for CMV disease, CMV-associated death, and transplant-related mortality (TRM). Patients who received unrelated or mismatched family donor transplants had increased risks for CMV disease, CMV-associated death, and TRM. Older age was a significant risk factor for CMV disease and TRM. A total of 258 patients who were monitored by polymerase chain reaction for CMV DNA were analyzed separately to assess whether addition of another CMV preventive strategy could give benefit. Patients who received mismatched or unrelated donor transplants had increased risk for CMV disease, death in CMV disease, and TRM. High-dose acyclovir prophylaxis or addition of intravenous immune globulin had no influence. CONCLUSIONS Preemptive therapy based on polymerase chain reaction for CMV DNA was associated with reduced risks for CMV disease, CMV-associated death, and TRM, whereas other prophylactic modalities did not give additional benefit.

A real-time TaqMan PCR for routine quantitation of cytomegalovirus DNA in crude leukocyte lysates from stem cell transplant patients.

A real-time TaqMan PCR based on the cytomegalovirus (CMV) polymerase (pol) gene was developed for quantitation of CMV DNA in crude peripheral blood leukocyte (PBL) lysate from stem cell transplantation (SCT) patients. The dynamic range of the assay was between 10 and 4x10(6) copies. Both intra- and inter-assay variability were well within +/-0.25 log10 S.D. Thus, a pooled PBL sample that was used as positive control in 57 consecutive TaqMan PCR runs over 7 months showed a stable CMV quantity (4.12+/-0.13, log10 mean+/-S.D.). The sensitivity of the pol TaqMan PCR was validated by parallel analysis of 177 PBL samples with a nested PCR. The use of crude PBL lysate as PCR input did not cause PCR inhibition. We demonstrated further the clinical utility of the newly developed TaqMan PCR by monitoring changes in CMV levels in eight patients receiving antiviral therapy. This TaqMan PCR was highly sensitive, reproducible, and stable and has served a useful tool for monitoring CMV DNA levels in large number of clinical samples in a routine diagnostic setting for over 1 year.

Real-time monitoring of cytomegalovirus infections after stem cell transplantation using the TaqMan polymerase chain reaction assays.

BACKGROUND Real-time monitoring of cytomegalovirus (CMV) infections in transplant patients demands a rapid and high-throughput CMV DNA quantification method. METHODS TaqMan polymerase chain reaction assays based on CMV immediate early protein exon 4 and glycoprotein B were developed and were compared with a COBAS AMPLICOR CMV MONITOR (CMM) test for quantifying CMV DNA in peripheral blood leukocytes from seven stem cell transplant patients having received antiviral treatment. RESULTS There was a good correlation between the TaqMan assays and the CMM test for CMV DNA quantification. The throughput of the TaqMan assays was, however, about 3 times higher than that of the CMM test. The CMV DNA dynamics patterns determined by the TaqMan polymerase chain reaction were well in line with the outcome of the antiviral therapy. CONCLUSIONS The TaqMan assays may potentially serve as a useful tool for rapid quantification of CMV infections in stem cell transplant patients.

Influenza B in Transplant Patients

Six cases of influenza B occurred in transplanted patients in a period of 3 weeks. Three renal allograft recipients recovered within 5 days without antiviral therapy. Two allogeneic bone marrow recipients were treated with ribavirin inhalations during the leukopenic phase. Treatment was given until influenza B was no longer detected and fever disappeared after 5 and 6 days, respectively. Engraftment was not delayed and no side-effects were noted. One recipient of autologous marrow was treated for 2 days, but ribavirin was discontinued due to pleuritic pain. We conclude that influenza B can be spread by asymptomatic carriers in the nursing staff and in spite of reversed isolation with the use of gown, hand wash and gloves.

Cytomegalovirus DNA detection on Guthrie cards in patients with neonatal cholestasis

AIM To time the onset of cytomegalovirus (CMV) infection in patients (n=39) with CMV associated neonatal cholestasis by analysing CMV DNA on Guthrie cards sampled at 3 days of age. METHODS CMV infection was diagnosed by serology/urine isolation or by CMV DNA detection (polymerase chain reaction) in liver biopsy specimens. In order to time the infection dry blood filter paper discs were punched out from stored Guthrie cards. After phenol–chloroform extraction CMV DNA was detected by nested polymerase chain reaction. RESULTS All cards from control children (n=8) with congenital CMV tested positive; none of the negative controls (n=4) did so. Two of 39 cholestatic infants were CMV DNA positive; their mothers had serological signs compatible with infection during the second half of the pregnancy. All other cholestatic infants tested negative. CONCLUSIONS CMV DNA was not detected in most of the children using Guthrie cards, suggesting that infection developed at or soon after birth.

Leukocyte Depleted, Unscreened Blood Products Give a Low Risk for CMV Infection and Disease in CMV Seronegative Allogeneic Stem Cell Transplant Recipients with Seronegative Stem Cell Donors

Leukocyte depletion (LD) by blood product filtration has been shown to be similarly effective to the use of screened, CMV seronegative blood products to prevent CMV disease in CMV seronegative allogeneic stem cell transplant (SCT) patients with CMV seronegative donors. The aim of this retrospective study was to determine the risk for development of CMV infection requiring preemptive therapy and for CMV disease if unscreened products treated by prestorage LD is used. Forty-nine consecutive patients transplanted after June 1995 were included. As a control group, 33 patients transplanted from January 1992 to June 1995 in whom a combination of CMV seronegative and LD blood products were given. All patients were monitored weekly by a leukocyte-based PCR for CMV DNA detection. Preemptive therapy was initiated after two consecutively positive tests. No patient developed CMV disease in either group. CMV DNA was detected in 6/49 (p = NS) in the study group and in 3/33 patients in the historical control group. Two patients in the study group were given preemptive therapy compared to one patient in the control group. This study suggests that the risk for CMV disease and the need for preemptive therapy against CMV is low in CMV seronegative allogeneic SCT patients receiving grafts from CMV seronegative stem cell donors receiving LD blood products. Thus, this strategy can be safely used together with PCR monitoring and preemptive therapy.

Trends in seroprevalence of human papillomavirus type 16 among pregnant women in Stockholm, Sweden, during 1969–1989

To assess long‐term trends in the prevalence of oncogenic human papillomavirus (HPV) infection, we performed a cross‐sectional serosurvey of the seroprevalence of the major oncogenic HPV type, HPV16, among 3,512 pregnant women undergoing population‐based serological screening at the first trimester of pregnancy in the same catchment area in Stockholm, Sweden, during 1969, 1983 or 1989. The overall HPV16 seroprevalence rates were 16% in 1969, 22% in 1983 and 21% in 1989. Seroprevalence was significantly increased, comparing both 1969 vs. 1983 (p = 0.0005) and 1969 vs. 1989 (p = 0.008). By comparison, the previously reported herpes simplex 2 (HSV‐2) seroprevalence in the same women increased from 17% in 1969 to 32% in 1983 and 33% in 1989, whereas the seroprevalence rates of HSV‐1 were the same (69% in 1969, 63% in 1983 and 68% in 1989). Odds ratios for HPV16‐positive women to also be HSV‐2‐positive were 1.8 in 1969 (p < 0.005), 1.1 in 1983 (p = NS) and 1.0 in 1989. Our results suggest that both HSV‐2 and HPV16 became more generally spread in the Swedish population between 1969 and 1983 but that the spread has been stable during the 1980s. Int. J. Cancer 76:341–344, 1998.© 1998 Wiley‐Liss, Inc.

High‐dose acyclovir prophylaxis reduces cytomegalovirus disease in liver transplant patients

Cytomegalovirus (CMV) is still a major pathogen in liver transplantation (LTX). The clinical efficacy of prophylactic high‐dose acyclovir therapy (800 mg qid) was assessed for the prevention of CMV infection and disease in liver recipients. Fifty‐five patients were enrolled in a prospective, randomised, double‐blind and placebo‐controlled trial; 28 on acyclovir vs. 27 on placebo. The therapy was given for 12 weeks. The patients were followed for 24 weeks.

CMV PCR monitoring in leucocytes of transplant patients.

BACKGROUND The presumed latency of cytomegalovirus (CMV) in leucocytes and the sensitivity of the polymerase chain reaction (PCR) raise a question of its clinical value. OBJECTIVES To develop and standardize a CMV PCR as a diagnostic tool for CMV infection in solid organ and bone marrow transplant patients by comparing it to a likewise standardized isolation, rapid isolation and to clinical symptoms. STUDY DESIGN The material comprised 822 EDTA peripheral blood samples from 96 solid organ and 119 bone marrow transplant patients. One sample from each of 21 healthy bone marrow donors and 25 blood donors were used as controls. Two million leucocytes were lysed and one-tenth of a volume was used in a nested PCR employing immediate early gene primers. RESULTS The limit of detection was approximately 10 gene copies of a CMV DNA clone and 1 TCID(50) of extracted DNA from a cell suspension. The specificity was >/=0.99 when tested in CMV seronegative individuals. The positive and negative predictive values were 0.62 and 1.00, respectively. When PCR was compared to virus isolation/rapid culture in individual patients, PCR was positive more frequently in solid organ transplant patients than was CMV isolation/rapid culture, but the difference was not significant in bone marrow transplant patients. In isolation-positive patients, PCR became positive in samples taken 1-2 weeks earlier. In 54 solid organ transplant patients with PCR-positive samples, CMV-associated symptoms were present in 29/31 patients with CMV isolated from blood but in only 5/23 patients without viraemia. In 17 bone marrow transplant patients treated with ganciclovir, PCR became negative during or immediately after treatment in 14/20 (70%) episodes. This was true of 5/12 (42%) solid organ transplant patients. CONCLUSION Screening of transplant patients with CMV PCR can be standardized at a clinically relevant level so that antiviral therapy can be instituted early.

Congenital cytomegalovirus infection - a common cause of hearing loss of unknown aetiology.

Aim:  The aim of this study was to investigate the role of congenital cytomegalovirus (CMV) infection as a cause of various types of sensorineural hearing loss (SNHL) in a group of nonsyndromic children with otherwise unknown aetiology of hearing loss. Furthermore, the occurrence of combined congenital CMV infection and connexin 26 (Cx26) mutations was investigated.