Claudia C. Bauer

Emergence of Multiple Cytomegalovirus Strains in Blood and Lung of Lung Transplant Recipients

Background. Cytomegalovirus (CMV) is a major pathogen in lung transplant recipients (LTRs). The emergence of different CMV strains in lung and blood after transplantation has not yet been analyzed. Methods. In total, 75 serum and 91 broncheoalveolar lavage (BAL) samples obtained from 25 LTRs in the follow-up after transplantation were tested for the presence of different CMV strains. The gB, gN, and gO genes of the CMV isolates were analyzed by subtype-specific PCR, restriction fragment length polymorphism (RFLP), sequencing, and phylogenetic analysis. Results. Mixed CMV-strain populations were detected after cessation of antiviral prophylaxis in up to 80% and 90% of the patients’ BAL and serum, respectively, and this was independent of the CMV serostatus of donor and recipient. In five patients, the same single CMV strain was consistently detectable over at least 1 year in lung and blood, although in two of these cases donor and recipient had both been CMV-seropositive. Most CMV strains were distributed in the lung and blood compartment. Symptomatic CMV infection within the first year after transplantation was observed only in patients with mixed CMV-strain populations (P<0.05). Conclusion. Most LTRs harbor more than one CMV strain in their lung and blood compartment after cessation of prophylaxis, but the CMV strain distribution within and between the compartments varies between individuals and is not associated with the donor/recipient serostatus. The data further show that compartmentalization of CMV strains in lung versus blood seems to be a rare event and that the presence of mixed CMV-strain infections within the first year after transplantation may be disadvantageous for LTRs.

Relationship between Cytomegalovirus DNA Load in Epithelial Lining Fluid and Plasma of Lung Transplant Recipients and Analysis of Coinfection with Epstein-Barr Virus and Human Herpesvirus 6 in the Lung Compartment

ABSTRACT Cytomegalovirus (CMV) is a significant cause of morbidity and mortality in lung transplant recipients (LTRs). The aim of the present study was to elucidate the relationship between the CMV DNA load in the lung compartment and that in plasma. For CMV load determination, the level of CMV DNA in plasma and bronchoalveolar lavage (BAL) samples was measured in a total of 97 paired BAL and plasma samples obtained from 25 LTRs. The original virus concentration in the epithelial lining fluid (ELF) was calculated from the BAL samples by correcting for dilution using the urea dilution method. In addition, the load of Epstein-Barr virus (EBV) and that of human herpesvirus 6 (HHV-6) DNA also were determined in BAL samples, recalculated for their concentrations in the ELF, and compared with the CMV DNA load. CMV DNA was found more frequently and at significantly higher levels in the lung compartment than in plasma (P < 0.001, Wilcoxon test), and the CMV load in the ELF was associated with symptomatic CMV disease. EBV and HHV-6 were detected in 43.6% and 21.7% of the ELF samples, respectively. A statistically significant association was found between the CMV and EBV DNA loads in the ELF (P < 0.001; Spearmans rho = 0.651). Thus, in LTRs, determination of the CMV DNA load in the lung compartment may be advantageous compared to monitoring only viremia. The significant relationship between EBV and CMV DNA loads in the ELF of LTRs and its clinical impact require further investigation.

Virus Load Dynamics of Individual CMV-Genotypes in Lung Transplant Recipients With Mixed-Genotype Infections

Human cytomegalovirus (CMV) is a major cause of disease and transplant dysfunction in lung transplant recipients. Simultaneous emergence of more than one CMV‐genotype can occur, and appears to be disadvantageous for the patient. In this study, the dynamics of individual CMV‐genotypes in blood and lung was assessed within mixed CMV‐genotype populations emerging after lung transplantation. In 69 plasma and 76 bronchoalveolar lavage samples of 16 lung transplant recipients with mixed CMV‐genotype infections within the first year posttransplantation each of the major glycoprotein B (gB) and glycoprotein H (gH) genotypes was selectively quantified by genotype‐specific quantitative TaqMan assays. The data obtained revealed that individually different genotype dynamics occurred for the individual patients and that the relative levels of the genotypes to each other may change over time. The quantitative development was independent of the specific gB–gH‐genotype. In 10 of the 16 lung recipients the patients individual genotype composition was the same in blood and lung. Genotype development during the follow‐up was influenced by antiviral treatment. These data show for the first time that the CMV load used as diagnostic tool after transplantation is not always a constant entity but reflects the sum of the individual CMV‐genotype dynamics developing over time. J. Med. Virol. 80:1405–1414, 2008.

Association of cytomegalovirus DNA concentration in epithelial lining fluid and symptomatic cytomegalovirus infection in lung transplant recipients.

Background. The authors have investigated the effectiveness of virus detection from bronchoalveolar lavage (BAL) samples for the identification of symptomatic cytomegalovirus (CMV) infection in lung transplant recipients. Methods. Thus, 275 BAL samples taken from 105 lung transplant recipients during follow-up were analyzed by quantitative polymerase chain reaction (PCR) and virus isolation. Results. Quantitative PCR detected virus in all 24 BAL samples taken at onset of symptomatic disease, and virus culture in only 7 samples (29.2%). Twenty-three of 251 BAL samples (9.2%) were positive by PCR, although they were obtained in an asymptomatic phase. Quantitation of CMV DNA from BAL allowed no discrimination between symptomatic and asymptomatic infection in individual cases. However, when the urea dilution method was used to recalculate the CMV DNA concentration for the epithelial lining fluid (ELF) diluted in the BAL, a CMV DNA level of more than 104 copies/mL ELF was clearly associated with symptomatic disease. Conclusion. The CMV DNA level in ELF may be a marker for symptomatic CMV infection.

Clinical evaluation of a fully automated CMV PCR assay.

BACKGROUND There is a growing need for sensitive high-throughput cytomegalovirus (CMV) PCR tests due to the increasing number of immunocompromised patients requiring monitoring for active CMV infection. OBJECTIVES To compare the fully automated COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) (CAP/CTM) CMV test (this test is currently under development and not commercially available) for EDTA-plasma to the reference method COBAS(®) AMPLICOR CMV MONITOR. STUDY DESIGN A prospective feasibility study with parallel analysis of 433 EDTA-plasma samples from 277 patients on both systems was carried out after the analytical performance of the new system had been assessed. RESULTS The new system has a wide linear range from 2.0 to 7.3 log(10) CMV-DNA copies/ml EDTA-plasma and a detection limit of 46 copies/ml with excellent accuracy and precision. When testing clinical samples, the CAP/CTM CMV test compared extremely well with the COBAS(®) AMPLICOR CMV MONITOR (R(2)=0.93, p<0.001) with increased sensitivity and linear range. Discrepant samples all contained low titers of CMV-DNA. In two of the study patients, CMV-DNAemia was detected by the CAP/CTM CMV test up to eight weeks earlier than by COBAS(®) AMPLICOR CMV MONITOR. CONCLUSION An IVD/CE marked version of the CAP/CTM CMV test will enable laboratories to provide a sensitive, fully automated high-throughput CMV PCR.

An unrecognized epidemic of Mycoplasma pneumoniae infection in Vienna.

ZusammenfassungInfektionen mit Mycoplasma pneumoniae zeigen ausgeprägte epidemiologische Schwankungen, wobei alle 4 bis 7 Jahre eine 2- bis 10-fach erhöhte Inzidenz zu beobachten ist. Die regionale Epidemiologie von M. pneumoniae ist deshalb mit Hinblick auf die empirische antimikrobielle Therapie von ambulant erworbenen Infektionen des Respirationstrakts bedeutsam. Derzeit gibt es keine publizierten Daten zur Epidemiologie von M. pneumoniae Infektionen in Mitteleuropa. In dieser Studie wurden die Ergebnisse der Mykoplasmenserologie (Komplementbindungsreaktion) an der Klinischen Abteilung für Virologie am AKH-Universitätsklinikum Wien (2140 Betten, stationäre Aufnahmen: 94000/Jahr, ambulante Patientenfrequenz: 430000/Jahr) retrospektiv analysiert. Als Untersuchungszeitraum wurde 1/1995 bis 12/2004 gewählt (10 Jahre). Antikörpertiter gegen M. pneumoniae ≥ 1:64 wurden als Hinweis auf eine akute oder rezente Infektion gewertet. Die Anzahl der untersuchten Proben in den einzelnen Jahren zeigte nur geringe Schwankungen (Median: 2859 Proben/Jahr, Schwankungsbereich: 2257–3338). Im Median wurden pro Jahr 13 Patienten mit hochpositiver Mykoplasmenserologie registriert. Ein signifikanter epidemiologischer Gipfel zeigte sich im Jahr 2000 (43 Patienten), wobei eine steigende Inzidenz bereits im zweiten Halbjahr 1999 beobachtet werden konnte. Im Jahr 2001 war wieder die durchschnittliche jährliche Inzidenz erreicht. Ein Erfassungs-bzw. Meldesystem für Infektionen mit M. pneumoniae (d.h. Patienten mit erhöhten Antikörpertitern) könnte für Ärzte, die mit der Behandlung von Patienten mit respiratorischen Infektionen befasst sind, von Nutzen sein.SummaryMycoplasma pneumoniae infection shows epidemiological peaks with a 2- to 10-fold increased incidence every four to seven years. The regional epidemiology of M. pneumoniae infection is important with regard to empirical antibiotic treatment of community-acquired respiratory tract infections, which are the most common cause for visiting a physician. To date, no data on the epidemiology of M. pneumoniae in central Europe have been published. In the present study, the results of M. pneumoniae serology performed at the Clinical Division of Virology at Vienna General Hospital (a 2,140-bed university teaching hospital with an average of 94,000 admissions/year and 430,000 outpatient visits/year) in the 10-year period from January 1995 to December 2004 were analyzed retrospectively. Antibody titers ≥ 1:64 in complement fixation tests were considered indicative of acute or recent mycoplasma infection. The annual total number of serum specimens tested for anti-M. pneumoniae antibodies remained stable throughout the study period (median: 2859 samples/year, range: 2257–3338). The annual median number of patients with high M. pneumoniae titers was 13. A major epidemiological peak (43 patients) was observed in 2000, the epidemic starting in late 1999 and ending in 2001. A surveillance or reporting system for M. pneumoniae infections (i.e. positive serological results for M. pneumoniae) would be useful for physicians caring for patients with community-acquired respiratory tract infections.