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Objectives. To define clinically relevant reference ('normal') values for cerebrospinal fluid (CSF) protein concentrations in pediatric patients who were evaluated for meningitis by traditional criteria and by enterovirus--polymerase chain reaction (EV-PCR). Design and Patients: A cohort of 906 consecutive pediatric patients to receive CSF analysis at St Louis Children's Hospital, St Louis, Mo, from June 1, 1998, to December 31,1998, was studied for clinical and laboratory data. Age-dependent CSF protein concentrations were then derived from a reference group of 225 patients in whom meningitis and other neurologic diseases were excluded by traditional clinical or laboratory criteria (excluding EV-PCR). Available CSF samples from 132 patients of the reference group were subsequently tested for EV-PCR. Results: In the reference group, the CSF protein concentration was highest and most variable in neonates, with a maximum of approximately 1.0 g/L. Cerebrospinal fluid protein concentration decreased rapidly to a nadir by 6 months and remained low throughout childhood, rarely exceeding 0.3 g/L and, finally, increasing in adolescence toward adult values. Enterovirus--polymerase chain reaction was positive in CSF of 11% of the reference group, with EV-PCR-positive patients having significantly higher CSF protein concentrations than EV-PCR-negative patients aged between 4 months and 14 years. Conclusions: Reference values for CSF protein exhibit a characteristic age dependence in pediatric patients. Continued standard use of adult reference values in the pediatric population is inappropriate. The unexpected finding of a positive EV-PCR in patients not diagnosed with meningitis by traditional criteria further emphasizes the importance of selecting the most clinically relevant reference group for age and other variables when defining normal laboratory values. Arch Pediatr Adolesc Med. 2000;154:827-831

ABSTRACTInfluenza A and B viruses and respiratory syncytial virus (RSV) are three common viruses implicated in seasonal respiratory tract infections and are a major cause of morbidity and mortality in adults and children worldwide. In recent years, an increasing number of commercial molecular tests have become available to diagnose respiratory viral infections. The Luminex Aries Flu A/B & RSV assay is a fully automated sample-to-answer molecular diagnostic assay for the detection of influenza A, influenza B, and RSV. The clinical performance of the Aries Flu A/B & RSV assay was prospectively evaluated in comparison to that of the Luminex xTAG respiratory viral panel (RVP) at four North American clinical institutions over a 2-year period. Of the 2,479 eligible nasopharyngeal swab specimens included in the prospective study, 2,371 gave concordant results between the assays. One hundred eight specimens generated results that were discordant with those from the xTAG RVP and were further analyzed by bidirectional sequencing. Final clinical sensitivity values of the Aries Flu A/B & RSV assay were 98.1% for influenza A virus, 98.0% for influenza B virus, and 97.7% for RSV. Final clinical specificities for all three pathogens ranged from 98.6% to 99.8%. Due to the low prevalence of influenza B, an additional 40 banked influenza B-positive specimens were tested at the participating clinical laboratories and were all accurately detected by the Aries Flu A/B & RSV assay. This study demonstrates that the Aries Flu A/B & RSV assay is a suitable method for rapid and accurate identification of these causative pathogens in respiratory infections.

ABSTRACTWe have developed and evaluated a real-time reverse transcriptase PCR (RT-PCR) assay for the detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO. This sequence, along with other GenBank sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP1 gene, with an analytic limit of detection of 4 copies per reaction, and it was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses.

ABSTRACTHigh-throughput multiplex assays for respiratory viruses are an important step forward in diagnostic virology. We compared one such assay, the PLx Multi-Code Respiratory Virus Panel (PLx-RVP), manufactured by Eragen Biosciences, Inc. (Madison, WI), with conventional virologic testing, consisting of fluorescent-antibody staining plus testing with the R-mix system and fibroblast tube cultures. The test set consisted of 410 archived respiratory specimens, mostly nasopharyngeal swabs, including 210 that had been positive by conventional testing for a balanced selection of common respiratory viruses. Specimens yielding discrepant results were evaluated using a panel of respiratory virus PCR assays developed, characterized, and validated with clinical specimens. PLx-RVP increased the total rate of detection of viruses by 35.8%, and there was a 25.7% increase in the rate of detection of positive specimens. Reference PCR assay results corroborated the PLx-RVP result for 54 (82%) of 66 discrepancies with conventional testing. Of the 12 specimens with discrepancies between PLx-RVp and the reference PCRs, 6 were positive for rhinovirus by PLx-RVP and the presence of rhinovirus was confirmed by nucleotide sequencing. The remaining six specimens included five in which the PLx-RVP failed to detect parainfluenza virus and one in which the detection of influenza A virus by PLx-RVP could not be confirmed by the reference PCR. Taking the results of the reference PCR assay results into account, the sensitivities of the PLx-RVP for individual viruses ranged from 94 to 100% and the specificities ranged from 99 to 100%. We conclude that PLx-RVP is a highly accurate system for the detection of respiratory viruses and significantly improves the rate of detection of these viruses compared to that by conventional virologic testing.

Studies have reported the presence of KI polyomavirus (KIPyV) and WU polyomavirus (WUPyV) in respiratory secretions of young patients. So far, evidence has not supported a link between infections with either virus and respiratory tract disease; however, there has not been a large comparison of KIPyV-infected patients to age-matched patient groups.

ABSTRACTThe MultiCode-PLx system (EraGen Biosciences, Inc., Madison, WI) for the detection of respiratory viruses uses an expanded genetic alphabet, multiplex PCR chemistry, and microsphere flow cytometry to rapidly detect and specifically identify 17 different respiratory viruses directly in clinical specimens. The MultiCode-PLx system was tested in parallel with direct fluorescent-antibody (DFA) staining and rapid shell vial culture (R-mix cells; Diagnostic Hybrids, Inc. Athens, OH) with 354 respiratory specimens from adult patients that were submitted to the clinical virology laboratory at the Emory University Hospital. Single-target PCRs were performed with retained samples to confirm the positive results obtained with the MultiCode-PLx system for viruses not covered by DFA and R-mix culture (metapneumovirus, coronaviruses [CoV], parainfluenza viruses 4a and 4b, and rhinoviruses) and to resolve any discrepancies between the DFA and R-mix culture and the MultiCode-PLx results for viruses common to both systems. Respiratory viruses were detected in 77 (21.8%) and 116 (32.7%) specimens by DFA and R-mix culture and with the MultiCode-PLx system, respectively. Among the viruses common to both systems, the MultiCode-PLx system detected significantly more influenza A viruses (P= 0.0026). An additional increased diagnostic yield with the MultiCode-PLx system resulted from the detection of human metapneumovirus (HMPV) in 9 specimens, human CoV (HCoV) in 3 specimens, and human rhinovirus (HRV) in 16 specimens. Also, two mixed viral infections were detected by the MultiCode-PLx system (HCoV OC43 and HRV infections and HMPV and HRV infections), but none were detected by DFA and R-mix culture. Single-target PCRs verified the results obtained with the MultiCode-PLx system for 73 of 81 (90.1%) specimens that had discordant results or that were not covered by DFA and R-mix culture. The MultiCode-PLx system provides clinical laboratories with a practical, rapid, and sensitive means for the massively multiplexed molecular detection of common respiratory viruses.

ABSTRACTWe performed a blinded study to compare repetitive-sequence PCR and multilocus sequence typing for genotyping hospital- and community-acquired methicillin-resistant Staphylococcus aureus(MRSA). The MRSA strains that were sequence type 8 (ST8), staphylococcal cassette chromosome mec(SCCmec) type IV, and Panton-Valentine leukocidin-positive clustered separately from those that were ST5 and SCCmectype II.

ABSTRACTDuring the 2001, 2002, and 2003 enterovirus seasons, we investigated the correlations between cerebrospinal fluid (CSF) nucleated cell counts and elevated CSF protein levels and the detection of enteroviral RNA by reverse transcription (RT)-PCR. Our objective was to determine if pleocytosis and/or elevated protein levels were predictive of positive RT-PCR results for enterovirus. We were also interested in determining if the presence of West Nile virus during the 2002 enteroviral season contributed to a change in these correlations. We found that in the group of patients aged >2 months, the absence of pleocytosis was highly predictive of a negative RT-PCR result. Elevated CSF protein level was not a good predictor of RT-PCR positivity for enterovirus and did not add to the diagnostic sensitivity or specificity of pleocytosis.

ABSTRACTPCR was used to amplify a 537-bp region of an Ehrlichia ewingiigene encoding a homologue of the 28-kDa major antigenic protein (P28) of Ehrlichia chaffeensis. The E. ewingii p28gene homologue was amplified from DNA extracted from whole blood obtained from four humans and one canine with confirmed cases of infection. Sequencing of the PCR products (505 bp) revealed a partial gene with homology to outer membrane protein genes from Ehrlichiaand Cowdriaspp.:p30of Ehrlichia canis(=71.3%),p28of E. chaffeensis(=68.3%), andmap1of Cowdria ruminantium(67.3%). The peptide sequence of the E. ewingiipartial gene product was deduced (168 amino acids) and the antigenicity profile was analyzed, revealing a hydrophilic protein with =69.1% identity to P28 of E. chaffeensis, =67.3% identity to P30 of E. canis, and =63.1% identity to MAP1 of C. ruminantium. Primers were selected from the E. ewingii p28sequence and used to develop a species-specific PCR diagnostic assay. The p28PCR assay amplified the expected 215-bp product from DNA that was extracted from EDTA-treated blood from each of the confirmed E. ewingiiinfections that were available. The assay did not produce PCR products with DNA extracted from E. chaffeensis-, E. canis-, or E. phagocytophila-infected samples, confirming the specificity of the p28assay forE. ewingii. The sensitivity of the E. ewingii-specific PCR assay was evaluated and determined to detect as few as 38 copies of the p28gene.

ABSTRACTBroad-range PCR primers were used to amplify part of thegroESLoperon of the canine pathogen Ehrlichia ewingii, recently recognized as a human pathogen, and the murine pathogen Ehrlichia muris. Phylogenetic analysis supported the relationships among Ehrlichiaspecies previously determined by comparison of 16S rRNA gene sequences. These sequences provide additional PCR targets for species for which few gene sequences have been determined.

Toxic-shock syndrome toxin-1 (TSST-1)-producing Stayliylococcirsatireus MN8 was grown in dialyzed beef heart infusion broth, or dialyzed beef heart infusion broth supplemented with 5 human blood or with 5 human semen, in the presence and absence of Today contraceptive sponges. Early growth of the organism was inhibited by the presence of the sponges in aerobic, static aerobic, and anaerobic cultures. In aerobic cultures that contained the sponge, minimal TSST-1 was detected by immunodiffusion assay, whereas in control cultures that did not contain the sponge, extensive toxin production was seen. In static and anaerobic cultures, the presence of the contraceptive sponge resulted in toxin levels less than or equal to the low levels seen in control cultures. These results suggest that Today cantraceptive sponges may inhibit TSST-1 production by S aureus.

Background: Centrifugation shell vial (SV) and conventional tube culture (TC) are the most common methods for detecting cytomegalovirus (CMV) viremia. Studies have indicated that SV is more sensitive than TC but at least one report suggested that TC was more sensitive. Because CMV in the blood is primarily associated with infected leukocytes, the number of leukocytes inoculated into the different culture systems could affect the sensitivities of the two systems. Objectives: To compare the sensitivities of SV and TC for detection of CMV viremia by inoculating equal numbers of leukocytes into paired SV cultures and TC cultures. Study design: Leukocytes from transplant recipients were isolated and counted. Equal numbers of leukocytes were then inoculated into each of two MRC-5 SV and into each of two MRC-5 TC. SV was considered positive when either one or both vials were positive, and TC was considered positive when either one or both tubes showed evidence of CMV cytopathic effect (CPE). Results: From a total of 434 specimens tested, 85 (19.6%) were positive by SV or TC. CMV was detected by SV in 75 (88%) of the positive specimens, compared to TC which was positive in 40 (47%) of the positive specimens. Conclusions: When equal numbers of leukocytes were inoculated into each system, SV had significantly greater sensitivity than TC for detecting CMV viremia. However, a small number of episodes of viremia were detected only by TC. Therefore, both methods should be used for maximum sensitivity.

ABSTRACTWe evaluated the artus RealArt Parvovirus B19 LC PCR reagent (artus biotech USA, San Francisco, Calif.) for real-time PCR detection of parvovirus B19 DNA by retesting 71 specimens previously submitted to our laboratory. The artus assay, which produces a quantitative result and provides an internal PCR control, appeared to be slightly more sensitive than our conventional qualitative PCR assay.

Blood samples collected from wild deer in Missouri in November of 2000 and 2001 were positive by PCR assays for Ehrlichia chaffeensis (50 of 217; 23%), Ehrlichia ewingii (44 of 217; 20%), and Anaplasma species (214 of 217; 99%). Nucleotide sequences of selected amplicons from the assay for anaplasma matched sequences of the white-tailed deer agent. Serologic analysis of 112 deer sampled in 2000 showed a very high prevalence of antibodies to E. chaffeensis (97 of 112; 87%) and a low prevalence of antibodies reactive with Anaplasma phagocytophila (2 of 112; 2%).

ABSTRACTBlood samples collected from wild deer in Missouri in November of 2000 and 2001 were positive by PCR assays for Ehrlichiachaffeensis(50 of 217; 23%), Ehrlichiaewingii(44 of 217; 20%), and Anaplasmaspecies (214 of 217; 99%). Nucleotide sequences of selected amplicons from the assay for anaplasma matched sequences of the white-tailed deer agent. Serologic analysis of 112 deer sampled in 2000 showed a very high prevalence of antibodies to E. chaffeensis(97 of 112; 87%) and a low prevalence of antibodies reactive with Anaplasmaphagocytophila(2 of 112; 2%).

Background:CMV disease is a major complication of allogeneic hematopoietic cell transplantation (HCT). Our widely used diagnostic modalities (H&E-based morphology and CMV IHC) are suboptimal in accuracy and frequently equivocal. Furthermore, current therapies for CMV disease are toxic and can cause serious complications such as prolonged neutropenia or progressive renal insufficiency. As a result, more accurate diagnosis that would help avoid potentially toxic treatments in patients who are truly negative for disease is highly desirable. The purpose of this study was to evaluate whether CMV PCR performed on formalin-fixed paraffin-embedded (FFPE) tissue offers additional diagnostic value to H&E/IHC in allogeneic HCT recipients.

ABSTRACTWe evaluated the DiaSorin DNA enzyme immunoassay (DEIA) kit for detection of enteroviral reverse transcription-PCR (RT-PCR) products amplified from cerebrospinal fluid. By use of an optical density of 0.05 as the absorbance cutoff, 35% of 198 specimens were PCR positive, whereas 16% were culture positive. DEIA was rapid and sensitive and can help implement enterovirus RT-PCR in clinical laboratories.

Herpes simplex virus type 2 (HSV-2) is known to cause aseptic meningitis, which can be recurrent. The diagnosis of HSV-2 infection is suggested when meningitis occurs simultaneously with genital lesions but may be obscure if genital lesions are not present or are not appreciated. Viral culture of the CSF is sometimes positive, but it may also be negative, especially in cases of recurrent disease. We report three cases of HSV meningitis in young women who did not have a history of genital herpetic lesions and for whom genital lesions were not noted on presentation. With use of the polymerase chain reaction (PCR), HSV DNA was detected in CSF from all three patients. The diagnosis of HSV meningitis was further confirmed by a positive culture of CSF in one patient's case and by demonstration of intrathecal synthesis of HSV antibodies in a second patient's case. The use of PCR can improve the recognition of HSV meningitis in adults presenting with aseptic meningitis, even in the absence of herpetic lesions.

Cytomegalovirus (CMV) DNA levels were measured by quantitative-competitive polymerase chain reaction (PCR) in weekly leukocyte samples from 50 renal transplant recipients, including 23 with symptomatic and 27 with asymptomatic CMV infection. Peak and week 4 CMV DNA levels were higher in symptomatic subjects (P = .07 and .02, respectively). In a logistic regression model, the logarithm of the week 4 level independently predicted symptomatic infection (odds ratio, 1.78 for a 1 log10 increase; 95% confidence interval, 1.14–2.78; P = .01). All subjects whose week 4 level exceeded 1000 copies/100,000 leukocytes developed symptoms. In subjects with adequate samples for analysis, CMV levels declined exponentially with ganciclovir treatment, with an average half-life of 3.3 days. Levels exceeding 10,000 copies were associated with prolonged time to clearing of CMV DNA. Potential clinical applications of quantitative CMV PCR include predicting occurrence of symptomatic first episodes after transplantation and individualizing duration of antiviral therapy.

A quantitative culture method was used to test serial blood specimens from 28 lung transplant recipients at risk of cytomegalovirus (CMV) infection (donor [D] or recipient [R] seropositive for CMV). Viremia occurred in 26 (93%) of 28 patients. Highest levels were seen when the donor was seropositive. The median of individual maximum levels was 2.13 infectious centers (ICs)/10s leukocytes for D+/R− patients (interquartile range [iqr], 0.12-21.77), 1.01 for D+/R+ (iqr, 0.32.32), and 0.10 for D−/R+ [iqr, 0.07-0.36; P = .030, Kruskal-Wallis test). Higher levels were seen in patients with biopsy-proven CMV pneumonitis compared with those with negative biopsies (mean, 0.24 [SD 0.51] ICs/105 leukocytes vs. 0.01 [SD 0.03]; P = .039, Wilcoxon test) and with symptomatic CMV episodes compared with asymptomatic episodes (median, 0.34 ICs/I0s [iqr, 0.11-0.61] vs. 0.08 ICs/105 [iqr, 0.03-0.13]; P = .045, Wilcoxon test). Further studies are required to determine whether quantification of CMV viremia by this method will be of practical value in the recognition of significant CMV infection in lung transplant recipients.