A LightCycler and two TaqMan real-time PCR assays were evaluated against an older PCR with liquid-phase hybridization method for the detection of enterovirus RNA in 74 patient samples. The two-step LightCycler and the two-step TaqMan formats correlated well with each other (r 2 ؍ 0.90) and were equally sensitive compared to the liquid-phase hybridization method, whereas the one-step recombinant Tth DNA polymerase format was rather insensitive, detecting enterovirus RNA in only about one-half of those patient samples previously positive by liquid-phase hybridization. The two-step TaqMan method was optimized utilizing 10 l of cDNA and demonstrated the highest degree of analytical sensitivity among the methods evaluated in our study, being able to reproducibly quantify down to 510 copies of enteroviral RNA/ml of cerebrospinal fluid. This new assay can be performed in 4 h, is much less labor intensive, and showed less cross-reactivity with rhinovirus than the liquid-phase hybridization assay. Thus, the two-step TaqMan assay should prove useful in the diagnosis of enteroviral meningitis versus bacterial meningitis, thereby resulting in timely and appropriate clinical management that can amount to significant cost savings to the patient and health care system.Human enteroviruses are members of the family Picornaviridae, genus Enterovirus, and consist of more than 60 distinct serotypes. Most enteroviruses can be classified into one of five major groups, including coxsackieviruses A and B, echoviruses, enteroviruses, and polioviruses. Nonpolio enteroviruses represent the most common etiology of meningitis in the United States (22), accounting for more than 80% of all cases. Children, neonates, and immunocompromised individuals are particularly susceptible to enterovirus infection of the central nervous system (2,16,22). Though usually benign in nature, the clinical characteristics of enteroviral meningitis can be very similar to those of potentially life-threatening bacterial meningitis. Since bacterial meningitis requires hospital admission and aggressive treatment, the distinction of these entities is critical. Traditional culture-based methods for the detection of enterovirus in cerebrospinal fluid (CSF) are laborious, timeconsuming, and often insensitive. Thus, molecular assays are attractive in that they can allow rapid and accurate diagnosis, resulting in more appropriate and cost-effective treatment (20,28).Real-time PCR technology has provided an excellent platform for diagnostic PCR assays. Real-time PCR is quantitative and has low interassay and intra-assay variability (1,14,15,27). Recently, several groups have described real-time PCR methods for the detection of enterovirus in CSF (7,(17)(18)(19)(29)(30)(31). Based on bioinformatics analysis of published enterovirus sequences, we chose primers and probes to allow sensitive detection of these viruses. In this study, we evaluated three realtime PCR methods using these primers and probes on the TaqMan and LightCycler platforms. Our optimized method is more se...
Quantitative PCR is a diagnostic mainstay of clinical virology, and accurate quantitation of viral load among labs requires the use of international standards. However, the use of multiple passages of viral isolates to obtain sufficient material for international standards may result in genomic changes that complicate their use as quantitative standards. We performed next-generation sequencing to obtain single-nucleotide resolution and relative copy number of JC virus (JCV) clinical standards. Strikingly, the WHO international standard and the Exact v1/v2 prototype standards for JCV showed 8-fold and 4-fold variation in genomic coverage between different loci in the viral genome, respectively, due to large deletions in the large T antigen region. Intriguingly, several of the JCV standards sequenced in this study with large T antigen deletions were cultured in cell lines immortalized using simian virus 40 (SV40) T antigen, suggesting the possibility of transcomplementation in cell culture. Using a cutoff 5% allele fraction for junctional reads, 7 different rearrangements were present in the JC virus sequences present in the WHO standard across multiple library preparations and sequencing runs. Neither the copy number differences nor the rearrangements were observed in a clinical sample with a high copy number of JCV or a plasmid control. These results were also confirmed by the quantitative real-time PCR (qPCR), droplet digital PCR (ddPCR), and Sanger sequencing of multiple rearrangements. In summary, targeting different regions of the same international standard can result in up to an 8-fold difference in quantitation. We recommend the use of next-generation sequencing to validate standards in clinical virology.
The RAMP herpes simplex virus (HSV) culture confirmation test was compared with immunofluorescence (IF) staining with a specific HSV monoclonal antibody reagent for the detection of HSV in centrifugation culture. The RAMP test detected 47 of 57 IF-positive specimens (sensitivity, 88.6%) and agreed with 217 of 220 IF-negative specimens (specificity, 98.6%). The RAMP test can be performed in less than 15 min and gives an immediate visual result. However, the sensitivity and the false-positive and false-negative results need further investigation.
We evaluated a new membrane dot immunobinding assay (CMV-CUBE; Difco Laboratories) for the detection of cytomegalovirus (CMV) antibody in marrow transplant patients and donors. The CMV-CUBE assay was compared with a commercially available enzyme immunoassay (EIA; CMV STAT) and a latex agglutination (LA; CMVScan) test. Serum samples were collected from 311 transplant patients and donors prior to transplantation. A total of 164 serum specimens were positive for CMV antibody by one or more of the three assays, with 153 of 164 samples (93.3%) positive by aIl three tests. A total of 147 serum specimens were CMV antibody negative. CMV-CUBE detected 154 of 164 (94%) of the positive samples, EIA detected 160 of 164 (97.5%), and LA detected 157 of 164 (95.7%) CMV-positive samples. Compared with EIA, CMV-CUBE had a sensitivity of 95.6% and a specificity of 99.3%. Compared with LA, CMV-CUBE had a sensitivity of 97.5% and a specificity of 99.4%. CMV-CUBE is a simple and rapid visual assay which can be used for the qualitative detection of antibody to CMV in patient serum.
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