Incidence of respiratory viral infection in infants with respiratory symptoms evaluated for late-onset sepsis.

OBJECTIVE: To determine the frequency, etiology and impact of respiratory viral infection (RVI) on infants evaluated for late-onset sepsis (LOS), defined as sepsis occurring >72 h of life, in the neonatal intensive care unit. STUDY DESIGN: Prospective observational study conducted from 6 March 2014 to 3 May 2016 on infants evaluated for LOS. PCR viral panel performed on nasopharyngeal specimens among infants with clinical suspicion for RVI. Sequence analysis was performed to determine viral subtypes. Fisher's exact or χ2 tests were done to determine the impact of RVI. RESULTS: During the 26-month study, there were 357 blood cultures obtained for LOS evaluations, 29 (8%) had a respiratory virus detected. Only 88 (25%) of infants evaluated for LOS also had clinical suspicion for a respiratory viral infection. RSV (14 of 29; 48%) was the predominant virus detected. Almost all infants (13 of 14; 93%) with RSV required increased respiratory support. Antimicrobial therapy was withheld or discontinued on most infants with a virus detected (18 of 29; 62%) and in the majority where there was no confirmed bacterial co-infection (18 of 20; 90%). CONCLUSION: The incidence of RVI in infants being evaluated for LOS is about 8%. RVI should be considered in LOS evaluation to prevent unnecessary antibiotic therapy.

Comparison of three commercial RT-PCR systems for the detection of respiratory viruses.

BACKGROUND: Due to the insensitivity of rapid tests for respiratory viruses, nucleic acid amplification tests are quickly becoming the standard of care. OBJECTIVES AND STUDY DESIGN: The performance of the FilmArray Respiratory Panel (RP) and Verigene RV+ (RV+) were compared in a retrospective analysis of 89 clinical specimens previously determined to be positive for the following viruses by our test of record, Prodesse (Pro): influenza A (29, FluA), influenza B (13, FluB), respiratory syncytial virus (12, RSV), human metapneumovirus (10, hMPV), parainfluenza (14, PIV), and adenovirus (10, AdV). Samples positive for influenza A, B or RSV were tested by both methods, while the remainder were tested by RP only. True positives were defined as positive by two or more assays. RESULTS: Limit of detection (LOD) analyses demonstrated Pro had the lowest LOD for all FluA strains tested, PIV1, PIV2 and AdV; RV+ had the lowest LOD for FluB; and RP had the lowest LOD for RSV, PIV3 and hMPV. Of the 55 samples tested by RV+, all 54 true positive samples were positive by RV+. Of the 89 samples tested by RP, 85 of the 88 true positive samples were positive by RP. From these results, the overall sensitivities for influenza A, B and RSV were 100% and 98% for RV+ and RP, respectively. The overall sensitivity of RP for all viruses was 97%. CONCLUSIONS: In summary, these systems demonstrated excellent performance. Furthermore, each system has benefits which will ensure they will all have a niche in a clinical laboratory.

Premarket evaluations of the IMDx C. difficile for Abbott m2000 Assay and the BD Max Cdiff Assay.

Clostridium difficile-associated diarrhea is a well-recognized complication of antibiotic use. Historically, diagnosing C. difficile has been difficult, as antigen assays are insensitive and culture-based methods require several days to yield results. Nucleic acid amplification tests (NAATs) are quickly becoming the standard of care. We compared the performance of two automated investigational/research use only (IUO/RUO) NAATs for the detection of C. difficile toxin genes, the IMDx C. difficile for Abbott m2000 Assay (IMDx) and the BD Max Cdiff Assay (Max). A prospective analysis of 111 stool specimens received in the laboratory for C. difficile testing by the laboratory's test of record (TOR), the BD GeneOhm Cdiff Assay, and a retrospective analysis of 88 specimens previously determined to be positive for C. difficile were included in the study. One prospective specimen was excluded due to loss to follow-up discrepancy analysis. Of the remaining 198 specimens, 90 were positive by all three methods, 9 were positive by TOR and Max, and 3 were positive by TOR only. One negative specimen was initially inhibitory by Max. The remaining 95 specimens were negative by all methods. Toxigenic C. difficile culture was performed on the 12 discrepant samples. True C. difficile-positive status was defined as either positive by all three amplification assays or positive by toxigenic culture. Based on this definition, the sensitivity and specificity were 96.9% and 95% for Max and 92.8% and 100% for IMDx. In summary, both highly automated systems demonstrated excellent performance, and each has individual benefits, which will ensure that they will both have a niche in clinical laboratories.

Comparison of three real-time PCR for the quantification of polyomavirus BK.

BACKGROUND: Reactivation of latent polyomavirus BK is associated with nephropathy (PVAN) after renal transplantation. BK viral load determinations are a highly sensitive and specific method for predicting risk for PVAN. OBJECTIVES AND STUDY DESIGN: The performance of three real-time PCR for BKV DNA quantification (MultiCode(®)-RTx BK virus ASR [MC-RTx], MGB-Alert BKV ASR [MGB] and a laboratory developed assay [LDA]) were evaluated against a conventional PCR (test of record, TOR) in terms of linearity, dynamic range, and accuracy. RESULTS: The LOD (log(10) copies/ml) were 2.0, 2.0 and 3.0 for MC-RTx, MGB and LDA, respectively with a commercial plasma panel and 2.0, 2.6 and 3.5 with a urine panel. These assays demonstrated excellent linearity (r(2) = 1.0) and reproducibility (CV range = 0.7-20.4%, 0.9-13.2%, and 0.5-13%, respectively). In an analysis of 100 clinical specimens, all 76 samples defined as true positive for BKV DNA (positive by two or more methods or a recent history of positivity) were detected with MC-RTx, while only 64 were detected with MGB and 55 were detected with LDA. BKV DNA was not detected by any method in the true negative specimens. Based on these results, the sensitivities were 100% for MC-RTx, 84% for MGB and 72% for LDA. The greatest linear correlation with the mean concentration was observed with MC-RTx (r(2) = 0.96) with two samples (3%) with greater than 0.5 log(10) variance in quantification versus seven (11%) with MGB and ten (18%) with LDA. CONCLUSIONS: These real-time assays for BKV load demonstrated excellent performance characteristics, with the MC-RTx demonstrating the greatest sensitivity.

The impact of an enteroviral RT-PCR assay on the diagnosis of aseptic meningitis and patient management.

BACKGROUND: Enterovirus (EV) is a major cause of aseptic meningitis and non-specific febrile illness in children. Since the majority of patients are hospitalized for possible bacterial infection, a rapid test for the diagnosis of enteroviral meningitis (EVM) may reduce hospitalizations and unnecessary treatments. OBJECTIVE: To review the impact of an EV reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the diagnosis of EVM on patient management. STUDY DESIGN: CSF from 1056 patients admitted to the hospital between 1998 and 2001 was tested using EV RT-PCR. The results were correlated with CSF counts, diagnosis, test turnaround time (TAT) and length of hospital stay (LOS). RESULTS: EV RT-PCR was positive for 113 patients (11%). Of these cases, 92% occurred during the summer months and 77% were in children <19 years of age. Children <3 years old with EVM frequently had non-specific clinical findings and lacked pleocytosis. There was a significant correlation between decreasing LOS and TAT (r(2)=0.97, P<0.001). CONCLUSION: RT-PCR testing for EVM is an important tool to aid in the diagnosis of children with non-specific febrile illness. This test impacted patient management as measured by shortened patient stays, which should translate into significant health care savings.

A one-step RT-PCR assay using an enzyme-linked detection system for the diagnosis of enterovirus meningitis.

BACKGROUND: Enteroviruses are the most common cause of meningitis in the United States, with an estimated 50000-75000 cases each year. Enteroviral meningitis (EVM) is frequently a diagnosis of exclusion, as viral cultures lack sensitivity and require prolonged incubation periods. OBJECTIVE: To develop a sensitive and rapid test for the diagnosis of EVM. STUDY DESIGN: A rapid, one-step, reverse transcriptase-polymerase chain reaction (RT-PCR) was used in a prospective analysis of 160 patients who had cerebrospinal fluid (CSF) tested for enterovirus. RESULTS: Of the 160 patients, 14 were excluded due to missing CSF viral culture data. A total of 14 were CSF culture positive (10 with pleocytosis) and 19 were PCR positive (15 with pleocytosis). The ability to detect enterovirus by either culture or PCR correlated significantly with the white blood cell count in the CSF (P=0.001). Based on a clinical definition of enterovirus culture positive and pleocylosis: ten had definite EVM and 12 had probable EVM (pleocytosis without any other cause). Four had possible EVM (CSF culture positive without pleocytosis) and 18 had pleocytosis due to other causes. PCR was positive in all ten patients with definite EVM. Five out of 12 patients with probable EVM and three out of four patients with possible EVM. No patients with pleocytosis due to other causes were PCR positive and one patient that was defined as EVM negative (culture negative and no pleocytosis) was PCR positive. Overall, PCR was positive in 18 out of the 26 patients with a likelihood of EVM, while CSF culture was positive in only 14 cases. Our results demonstrated that RT-PCR enhances the sensitivity of enterovirus detection in CSF (69 vs. 54% for culture). CONCLUSION: The diagnosis of EVM is difficult to make clinically: the enhanced sensitivity and rapid turn around time of PCR will be of great clinical benefit.

Stimulation of lymphokines in Jurkat cells persistently infected with vaccinia virus.

The response of the human CD4+ T-cell line Jurkat to infection with vaccinia virus was investigated. Virus titers peaked approximately 3 to 4 days after infection, while cell growth paralleled that of uninfected cells, indicating that growth rates were not appreciably affected by viral infection. Results from plaque assays and fluorescence-activated cell sorter (FACS) analyses of virus antigens demonstrated that a persistent infection in which the percentage of infected cells and the virus titers fluctuated from passage to passage was established. Further characterization of the persistent infection revealed that the virus influences cellular functions. Induction of interleukin-2 (IL-2) and IL-2 receptor alpha (IL-2R alpha) in Jvac cells was shown by enzyme-linked immunosorbent assay and FACS analysis, respectively. Hybridization of cellular RNA with cloned probes confirmed the increased IL-2 expression and demonstrated that Jvac cells also expressed more IL-6 but not gamma interferon (IFN-gamma) or IL-1 beta. Dual-antibody staining and FACS analysis for vaccinia virus antigens and IL-2R alpha indicated that IL-2R alpha expression was restricted to the infected cells. Jvac cells were also resistant to superinfection, an additional proof that persistent infection elicited phenotypic changes in the cell population.

Activation of the human immunodeficiency virus type 1 long terminal repeat by vaccinia virus.

A variety of DNA viruses are known to activate gene expression directed by the long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1). In light of the proposed use of recombinant vaccinia virus for HIV-1 vaccines, evaluation of the role of vaccinia virus in HIV-1 activation is warranted. To investigate whether vaccinia virus induces HIV LTR-directed gene expression, transient expression assays in Jurkat cells persistently infected with vaccinia virus (Jvac) using plasmid DNA containing the LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were performed. CAT activity in Jvac cells was always recorded, although the level appears to fluctuate independently of virus titers. Dual intracytoplasmic staining and fluorescence-activated cell sorter analysis showed that CAT activity was expressed in the infected cells. CAT expression was not due to plasmid replication, since plasmid DNA extracted from Jvac cells 48 h after transfection was restricted only by enzymes which recognize methylated sequences, indicating a prokaryotic source for the DNA. These findings suggest that a factor(s) present in vaccinia virus-infected cells is capable of activating the LTR of HIV-1.

Evidence for polyclonal B cell activation as the mechanism for LCMV-induced autoimmune hemolytic anemia.

A docile substrain of lymphocytic choriomeningitis virus (LCMV) causes a persistent infection in adult C3HeB mice and induces a severe autoimmune hemolytic anemia (AIHA) which is maximal around three weeks post infection (PI). Evaluations of serum immunoglobulin levels of these mice demonstrated grossly elevated IgG2a levels along with increased IgG1 and IgG2b levels, suggesting that these animals also develop polyclonal B cell activation (PBA). Interestingly, LCMV-infected B10.BR mice did not demonstrate a marked hypogammaglobulinemia nor did they experience a severe hemolytic anemia. Although evaluations of the hematocrits indicated that these animals endure a mild anemia 21 days PI, a below normal reticulocyte count until day 18 PI suggests that there was a prolonged suppression in hematopoiesis. It is clear from RBC survival studies that there is not an accelerated rate of RBC elimination, as seen in infected C3H mice, demonstrating that the anemia in B10.BR mice is not due to a hemolytic process. These results imply a correlation between the development of PBA and AIHA, suggesting a cause and effect relationship.