Development and clinical application of a fully controlled quantitative PCR assay for cell-free cytomegalovirus in human plasma.

BACKGROUND: Cytomegalovirus (HCMV) disease continues to be a major problem in certain patient groups, including bone marrow transplant (BMT) recipients. The quantification of HCMV genome is clinically useful for the diagnosis of HCMV disease, for the virological surveillance of high-risk patients and for monitoring antiviral therapy. OBJECTIVES: To develop a novel, robust, and fully controlled PCR (qPCR) for the quantification of HCMV DNA in plasma samples and to demonstrate its clinical usefulness in the BMT setting. STUDY DESIGN: The newly developed HCMV qPCR employs cell culture-derived murine CMV as an internal control for both extraction and amplification. Following amplification using common primers, detection of both internal control and patient HCMV amplicons is by specific probes and a chemiluminescence microtitre plate system. Its performance was evaluated using the routine non-quantitative nested HCMV PCR on whole blood (NQPCR) and correlated with clinical events such as disease and antiviral therapy. RESULTS: A high level of concordance (85.1%) was found between the novel assay and the NQPCR, with the qPCR being slightly more sensitive. The samples giving discordant results generally had levels of HCMV DNA close to the limit of detectability or had been stored for prolonged periods. CONCLUSIONS: The use of plasma as an analyte by the newly developed assay avoids the detection of cell-associated virus. On the other hand, testing a comparatively large volume of plasma ensures that sensitivity is not compromised by not detecting cell-associated HCMV. In a small preliminary evaluation in BMT recipients, changes in HCMV 'viral load' correlated with initiation and discontinuation of antiviral therapy and were biologically plausible.

Molecular investigations of an outbreak of parainfluenza virus type 3 and respiratory syncytial virus infections in a hematology unit.

A large simultaneous outbreak of respiratory syncytial virus (RSV) and parainfluenza type 3 (PIV-3) infections occurred on an adult hematology unit. Implementation of enhanced infection control was complicated by cocirculation of the two different viruses, with prolonged viral shedding from infected patients, and placed great pressure on health care staff; of 27 infected hematopoietic stem cell transplant patients, 9 died, and the unit was closed for 2 months. Retrospective molecular investigation of the virus strains involved in the outbreak was performed by analyzing part of the fusion gene of PIV-3 and part of the glycoprotein gene of RSV. Reverse transcription-PCR on nasopharyngeal aspirates from patients infected before and during the simultaneous outbreak generated amplicons for sequence analysis. A single strain of RSV and a single strain of PIV-3 had spread from person to person within the unit; 7 patients were infected with RSV, 22 were infected with PIV-3, and 4 were infected with both viruses. The PIV-3 outbreak had started at the beginning of August 3 months before the RSV outbreak; it had arisen when PIV-3 was introduced from the community by a patient and passed to another patient, who became chronically infected with the identical strain and, in spite of being nursed in isolation, was most likely the source from which widespread infection occurred in November. Had these early cases been linked to a common PIV-3 strain at the time of diagnosis, enhanced infection control precautions might have prevented the eventual extensive spread of PIV-3, making it much easier to deal with the later RSV outbreak.

First reported outbreak of diarrhea due to adenovirus infection in a hematology unit for adults.

Numerous outbreaks of adenovirus infection from different types of health care settings, except a hematology unit, have been reported. This is the first report describing an outbreak of adenovirus infection causing diarrhea among adult hematopoietic stem cell transplant recipients. Six of 21 patients from the outbreak cohort were affected with diarrhea. Electron microscopy, cell culture, and direct DNA sequencing of amplicons generated from stool and blood samples were used to investigate this outbreak. Electron microscopy and cell culture detected adenovirus in stools from symptomatic patients. DNA sequencing of amplicons generated from stool samples confirmed nosocomial transmission of infection from a single index case. The outbreak strain was also detected in plasma of four of these patients, suggesting systemic infection. The outbreak strain was identified as type 12. Standard infection control measures were effective to control this outbreak.

Prenatal diagnosis of congenital rubella infection in the second trimester of pregnancy.

OBJECTIVES: This case report describes the clinical presentation, diagnosis and management of a case of acute rubella infection in the second trimester. The complex issues of prenatal diagnosis of a congenital rubella infection are discussed. METHODS: A 30-year-old woman presented with a fine macular rash at 15 weeks' gestation. Laboratory testing included maternal rubella-specific IgG and IgM detection (booking blood and acute-phase sample) together with measurement of IgG avidity. Prenatal diagnosis at 19 weeks (amniocentesis) and 23 weeks (amniocentesis and fetal blood) was done using a reverse-transcriptase polymerase chain reaction to detect rubella-specific RNA. The fetal blood sample was also tested for rubella-specific IgM. RESULTS: Maternal serological results confirmed an acute rubella infection at 15 weeks' gestation. Rubella-specific RNA and IgM were detected in the fetal blood taken at 23 weeks' gestation. However, no rubella RNA was detected in either of the amniotic fluid samples collected at 19 and 23 weeks. CONCLUSION: In second-trimester rubella where risk of fetal damage is low, prenatal diagnosis can identify the rubella-infected fetus, allowing the parents to make a more informed decision about their options. The optimal sample for prenatal diagnosis is fetal blood as no rubella-specific RNA was detected in the amniotic fluid.