Multiorgan WU Polyomavirus Infection in Bone Marrow Transplant Recipient.

WU polyomavirus (WUPyV) was detected in a bone marrow transplant recipient with severe acute respiratory distress syndrome who died in 2001. Crystalline lattices of polyomavirus-like particles were observed in the patient's lung by electron microscopy. WUPyV was detected in the lung and other tissues by real-time quantitative PCR and identified in the lung and trachea by immunohistochemistry. A subset of WUPyV-positive cells in the lung had morphologic features of macrophages. Although the role of WUPyV as a human pathogen remains unclear, these results clearly demonstrate evidence for infection of respiratory tract tissues in this patient.

Comparison of the Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa ProFlu+™ assay for detecting influenza and respiratory syncytial viruses.

The relative performance of 2 widely used reverse transcription polymerase chain reaction (RT-PCR) assays, the Focus diagnostics Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa Proflu+™ assay, was evaluated using 735 prospectively and retrospectively collected nasopharyngeal swab specimens. Overall, the assays showed positive and negative agreements of 100% and 99.7% for influenza A, 98.1% and 99.9% for influenza B, and 99.3% and 99.5% for respiratory syncytial virus. The relative analytical sensitivity of the 2 assays was also similar.

Influenza and respiratory syncytial virus detection in clinical specimens without nucleic acid extraction using FOCUS direct disc assay is substantially equivalent to the traditional methods and the FOCUS nucleic acid extraction-dependent RT-PCR assay.

In this study, we evaluated FOCUS diagnostic's Flu A/B & RSV direct kit (Direct Disc assay), designed to detect influenza (FLU) and respiratory syncytial viruses (RSV) directly in clinical specimens without nucleic acid extraction. This novel 'sample-to-answer', nucleic acid extraction-independent assay uses a unique disc to process, amplify, and detect viral targets in up to 8 specimens at a time. The performance of this assay for detecting FLU and RSV viruses was compared to the traditional methods (culture and/or direct florescent antibody testing) using 945 nasopharyngeal swab specimens. In addition, a total of 150 consecutive clinical specimens positive for FLU (FLU A=50, FLU B=50) or RSV (n=50) were tested in parallel using the novel Direct Disc assay and FOCUS diagnostic's nucleic acid extraction-dependent assay to assess their relative performance. Compared to the traditional methods, the overall (prospective+retrospective) positive/negative percent agreement was determined to be 96.6%/98.1% for FLU A, 98.4%/99.9% for FLU B, and 99.3%/98.8% for RSV. Compared to the nucleic acid extraction-dependent assay, the positive percent agreement was 90% (n=45/50) for FLU A, 92% (n=46/50) for FLU B, and 98% (n=49/50) for RSV. Overall, the Direct Disc assay showed good agreement with both traditional methods and nucleic acid extraction-dependent assay. Although we encountered some failures compared to the nucleic acid extraction-dependent assay, these limitations must be balanced against the substantial advantages of the extraction-free nature of this assay and rapid turnaround time.

Cache valley virus in a patient diagnosed with aseptic meningitis.

Cache Valley virus was initially isolated from mosquitoes and had been linked to central nervous system-associated diseases. A case of Cache Valley virus infection is described. The virus was cultured from a patient's cerebrospinal fluid and identified with real-time reverse transcription-PCR and sequencing, which also yielded the complete viral coding sequences.

Midtrimester fetal herpes simplex-2 diagnosis by serology, culture and quantitative polymerase chain reaction.

The acquisition of herpes simplex virus (HSV) in utero comprises a minority of neonatal herpes infections. Prenatal diagnosis is rare. We describe a midtrimester diagnosis of fetal HSV-2 infection. Ultrasound at 20 weeks for elevated maternal serum α-fetoprotein (MSAFP) showed lagging fetal growth, echogenic bowel, echogenic myocardium, and liver with a mottled pattern of echogenicity. Amniocentesis demonstrated normal karyotype, elevated AFP and positive acetylcholinesterase. Culture isolated HSV-2 with an aberrant growth pattern. Maternal serology was positive for HSV-2. Quantitative DNA polymerase chain reaction (PCR) showed 59 million copies/ml. Fetal autopsy demonstrated widespread tissue necrosis but only sparse HSV-2 inclusions. Fetal HSV-2 infection can be suspected when an elevated MSAFP accompanies ultrasound findings suggesting perinatal infection. Maternal HSV serology, amniotic fluid culture and quantitative PCR are recommended for diagnostic certainty and counseling.

Analytical and clinical sensitivity of the 3M rapid detection influenza A+B assay.

The performance of the 3M rapid detection influenza A+B (3M flu) assay was compared to the performance of other immunochromatographic assays. The clinical and analytical performance of the 3M flu assay was superior to that of BinaxNOW and Directigen EZ assays and equivalent to that of the QuickVue assay. The 3M flu assay offers an objective output and direct linkage to laboratory information systems.

Measles-mumps-rubella and varicella vaccine responses in extremely preterm infants.

OBJECTIVE: Extremely preterm infants mount lower antibody responses than term infants to several vaccines. The objective of this study was to measure the immunogenicity of measles-mumps-rubella and varicella vaccines in preterm and term children. METHODS: Immune status before immunization and immune response after immunization with measles-mumps-rubella and varicella vaccines at 15 months of age were compared in 32 infants, 16 of whom were preterm (< 29 weeks' gestation) and 16 of whom were term (> or = 37 weeks' gestation) at birth. Blood was drawn before vaccination and 3 to 6 weeks thereafter. Measles antibody was measured by plaque reduction neutralization assay. Mumps and rubella immunoglobulin G were measured in available sera by enzyme-linked fluorescent immunoassay. Varicella immunoglobulin G was measured in available sera by glycoprotein enzyme-linked immunosorbent assay. Values that were above or below the assay limits were assigned values double or half those limits, respectively. The primary outcome was the geometric mean antibody titer. RESULTS: Preterm children had lower mumps and rubella geometric mean titers than did term children before vaccine, and nearly all children were seronegative for each of the 4 vaccine antigens before immunization. Measles, mumps, rubella, and varicella geometric mean titers were similar between groups after vaccine. All children were seropositive for measles after vaccine, whereas 13 of 14 preterm and 11 of 13 term children were seropositive for mumps, 13 of 14 preterm and 13 of 13 term children were seropositive for rubella, and 11 of 16 preterm and 9 of 15 term children were seropositive for varicella. CONCLUSIONS: Preterm children mounted antibody responses that were similar to those of term children after measles-mumps-rubella and varicella vaccines at 15 months of age.

Sequence diversity in the glycoprotein B gene complicates real-time PCR assays for detection and quantification of cytomegalovirus.

Real-time quantitative PCR systems (Q-PCR) for the rapid detection and quantification of microorganisms in clinical specimens employ oligodeoxyribonucleotide primers and probes for specificity, which makes them vulnerable to false negatives caused by sequence diversity in the template. Schaade et al. (J. Clin. Microbiol. 39:3809, 2001) reported a sequence variant (C630T) in the cytomegalovirus (CMV) glycoprotein B (gB) gene that, although detectable in their Q-PCR assay, could not be accurately quantified. In an effort to evaluate the impact of CMV sequence variants in our patient population by use of a similar Q-PCR assay, we surveyed 54 isolates of CMV, each from a different patient. We detected evidence for the C630T variant in 4 of 54 (7.4%) patients. Furthermore, isolates from two additional patients were completely negative in the test. Sequencing of these false-negative isolates revealed multiple mutations within the probe hybridization sites. A Q-PCR that targeted the CMV polymerase gene instead of gB detected all 54 isolates. We suggest that Q-PCR assays for viral load be rigorously tested on large panels of viral isolates to assess the impact of sequence diversity on detection as well as quantification.

Practical and sensitive screening strategy for detection of influenza virus.

This study evaluated the performance of Directigen FluA combined with a 3-day flu screening culture for the detection of influenza virus. This abbreviated protocol was a useful and effective tool and resulted in a substantial reduction in time, effort, and money spent, while not compromising sensitivity of influenza virus detection.