Evaluation of serial urine viral cultures for the diagnosis of cytomegalovirus infection in neonates and infants.

Cytomegalovirus (CMV) is the most common cause of congenital infection worldwide. Urine viral culture is the standard for CMV diagnosis in neonates and infants. The objectives of this study were to compare the performance of serial paired rapid shell vial cultures (SVC) and routine viral cultures (RVC), and to determine the optimal number of cultures needed to detect positive cases. From 2001 to 2011, all paired CMV SVC and RVC performed on neonates and infants less than 100 days of age were recorded. Testing episodes were defined as sets of cultures performed within 7 days of one another. A total of 1264 neonates and infants underwent 1478 testing episodes; 68 (5.4%) had at least one episode with a positive CMV culture. In episodes where CMV was detected before day 21 of life, the first specimen was positive in 100% (16/16) of cases. When testing occurred after 21 days of life, the first specimen was positive in 82.7% (43/52) of cases, requiring three cultures to reach 100% detection. The SVC was more prone to assay failure than RVC. Overall, when RVC was compared to SVC, there was 86.0% positive agreement and 99.9% negative agreement. In conclusion, three serial urine samples are necessary for detection of CMV in specimens collected between day of life 22 and 99, while one sample may be sufficient on or before day of life 21. Though SVC was more sensitive than RVC, the risk of SVC failure supports the use of multimodality testing to optimize detection.

Comparison of the FDA-approved CDC DENV-1-4 real-time reverse transcription-PCR with a laboratory-developed assay for dengue virus detection and serotyping.

Dengue virus (DENV) is the agent of the most common vector-borne disease worldwide. Using 199 clinical samples collected from Nicaragua and Sri Lanka, a laboratory-developed DENV multiplex real-time reverse transcription-PCR (rRT-PCR) proved more clinically sensitive than the FDA-approved CDC assay for DENV serotypes 1 to 4 when measured against a composite reference standard, with sensitivities of 97.4% versus 87.1%, respectively.

Diagnosis of congenital CMV using PCR performed on formalin-fixed, paraffin-embedded placental tissue.

Congenital cytomegalovirus (CMV) infection may be asymptomatic until hearing loss manifests in childhood. Because diagnosis of congenital CMV requires viral detection within an infant's first 21 days of life, CMV polymerase chain reaction (PCR) on formalin-fixed, paraffin-embedded (FFPE) placental tissue provides a unique opportunity to identify congenital exposure in cases in which CMV is not initially suspected. To assess the utility of this approach, a database of all CMV cultures performed from July 2001 to March 2012 was used to identify infants in whom urine CMV cultures were obtained within 100 days of life. Corresponding placentas were then identified through the pathology database. The database was also queried to identify placentas in which CMV immunohistochemical analysis had been performed. CMV PCR was positive in FFPE placental tissue from 100% (5/5) of cases in which the first urine culture collected before the first 21 days of life was positive. Placentas from 20 infants with negative CMV urine cultures were CMV PCR negative. Interestingly, CMV was detected in 12.5% (1/8) of placentas in which the first CMV-positive urine culture was collected after the first 21 days of life. Furthermore, 4% (1/26) of placentas with chronic villitis by histology (no urine cultures available) were CMV PCR positive. In the 10 CMV PCR-positive placentas, including 3 cases of fetal demise, CMV immunohistochemistry was positive in just 6 cases. These results suggest that the confirmation of CMV exposure in utero by PCR of FFPE placental tissue provides a useful adjunct to histologic evaluation and may identify infants requiring close clinical follow-up.

Single-reaction, multiplex, real-time rt-PCR for the detection, quantitation, and serotyping of dengue viruses.

BACKGROUND: Dengue fever results from infection with one or more of four different serotypes of dengue virus (DENV). Despite the widespread nature of this infection, available molecular diagnostics have significant limitations. The aim of this study was to develop a multiplex, real-time, reverse transcriptase-PCR (rRT-PCR) for the detection, quantitation, and serotyping of dengue viruses in a single reaction. METHODOLOGY/PRINCIPAL FINDINGS: An rRT-PCR assay targeting the 5' untranslated region and capsid gene of the DENV genome was designed using molecular beacons to provide serotype specificity. Using reference DENV strains, the assay was linear from 7.0 to 1.0 log10 cDNA equivalents/µL for each serotype. The lower limit of detection using genomic RNA was 0.3, 13.8, 0.8, and 12.4 cDNA equivalents/µL for serotypes 1-4, respectively, which was 6- to 275-fold more analytically sensitive than a widely used hemi-nested RT-PCR. Using samples from Nicaragua collected within the first five days of illness, the multiplex rRT-PCR was positive in 100% (69/69) of specimens that were positive by the hemi-nested assay, with full serotype agreement. Furthermore, the multiplex rRT-PCR detected DENV RNA in 97.2% (35/36) of specimens from Sri Lanka positive for anti-DENV IgM antibodies compared to just 44.4% (16/36) by the hemi-nested RT-PCR. No amplification was observed in 80 clinical samples sent for routine quantitative hepatitis C virus testing or when genomic RNA from other flaviviruses was tested. CONCLUSIONS/SIGNIFICANCE: This single-reaction, quantitative, multiplex rRT-PCR for DENV serotyping demonstrates superior analytical and clinical performance, as well as simpler workflow compared to the hemi-nested RT-PCR reference. In particular, this multiplex rRT-PCR detects viral RNA and provides serotype information in specimens collected more than five days after fever onset and from patients who had already developed anti-DENV IgM antibodies. The implementation of this assay in dengue-endemic areas has the potential to improve both dengue diagnosis and epidemiologic surveillance.

A comparison of CMV detection in gastrointestinal mucosal biopsies using immunohistochemistry and PCR performed on formalin-fixed, paraffin-embedded tissue.

Cytomegalovirus (CMV) can precipitate and exacerbate gastrointestinal (GI) mucosal injury. The gold standard for CMV detection in formalin-fixed, paraffin-embedded (FFPE) tissue is immunohistochemistry (IHC). Although CMV polymerase chain reaction (PCR) on fresh tissue may be a valuable adjunct to IHC, its utility is unknown for FFPE tissues. We therefore evaluated quantitative, real-time CMV PCR in a total of 102 FFPE GI biopsy specimens from 74 patients with a history of hematopoietic stem cell or solid organ transplant, inflammatory bowel disease, human immunodeficiency virus infection, or unspecified colitis. CMV DNA was detected by PCR in 90.9% (30/33) of IHC-positive, 14.5% (8/55) of IHC-negative, and 20.0% (1/5) of IHC-equivocal FFPE tissues. Quantitation of CMV DNA copies normalized to ß-globin demonstrated a wide range of values (median 0.276; range, 0.0004 to 144.50). Importantly, 93.3% (14/15) of patients with IHC-positive, active colitis showed no evidence of CMV in matched concurrent, histologically normal biopsies tested by PCR. These results suggest that CMV PCR on FFPE GI biopsies complements IHC and has the potential to identify additional patients who may benefit from anti-CMV therapy.