Gary P. Leonardi

Comparison of conventional lateral-flow assays and a new fluorescent immunoassay to detect influenza viruses

Sofia, a novel, fluorescent lateral-flow immunoassay was compared with two conventional colorimetric assays, Quickvue Influenza A+B and Directigen FLU A+B, to identify influenza viral antigen from patient nasopharyngeal specimens. A total of 118 frozen original influenza-positive specimens and 57 prospective specimens were examined. Using rt-PCR as a referee assay, sensitivity values (%) for influenza A/B of 80.0/74.8, 73.3/59.3 and 73.3/40.7 were obtained using the Sofia, Quickvue and Directigen assays, respectively. All assays demonstrated reduced sensitivity for influenza B as compared with influenza A virus. With respect to the Sofia assay, the sensitivity of influenza B for the Directigen assay was significantly diminished. False positive results were not observed in the Sofia and Directigen assays. The Quickvue assay produced 3 false-positive results (2 influenza A and 1 influenza B) resulting in a specificity (%) of 96 and 98 for influenza A and B, respectively. Cross-reactivity to other respiratory viruses was not observed among immunoassays. A sensitivity rank (highest to low) of rt-PCR>culture>Sofia>Quickvue>Directigen was established using dilutions of influenza A and B. Sofia provides enhanced sensitivity and objective result interpretation over conventional colorimetric immunoassays.

Echovirus Type 30 Infection Associated with Aseptic Meningitis in Nassau County, New York, USA

An outbreak of echovirus type 30, primarily affecting infant under 1 year old, was documented in Nassau County, N.Y., USA. Twenty-four of the 40 infected patients were definitively diagnosed with aseptic meningitis. A positive correlation was found between the proportion of polymorphonuclear leukocytes and the overall cerebrospinal fluid white cell count.

Evaluation of respiratory syncytial virus (RSV) direct antigen detection assays for use in point-of-care testing

RSV infections cause lower respiratory tract infections and result in surges in physicians office, emergency department visits and hospitalizations, especially in infants and toddlers. Point-of-care (POC) testing reduces healthcare costs and permits informed decisions on treatment, however, optimal POC assays must be sensitive, easy to perform and provide rapid results. A prospective study tested 230 patient nasopharyngeal specimens using 4 RSV direct antigen detection assays (Directigen, Quickvue, Sofia and Veritor) and RT-PCR. A RSV dilution study was also performed to evaluate sensitivity. RSV fluorescent antibody testing in 46/230 patients was also evaluated. Sensitivity values obtained for the Sofia, Veritor, Directigen and Quickvue assays (%) were 85, 72.5, 70 and 57.5, respectively. Fluorescent result interpretation may account for Sofias enhanced sensitivity. Specificity (%) was 97-100 among assays. Sensitivity data were confirmed in the dilution studies. Fluorescent antibody testing demonstrated 64% sensitivity compared with RT-PCR. Objective result reporting, walk away testing and high sensitivity make the Sofia a valuable choice for POC testing. Veritors sensitivity may also render it acceptable in POC. Lack of objective results by Directigen and the poor sensitivity observed by Quickvue may preclude their value in diagnostic testing.

Comparison of the Sofia and Veritor Direct Antigen Detection Assay Systems for Identification of Influenza Viruses from Patient Nasopharyngeal Specimens

ABSTRACT Influenza antigen detection assays (Sofia fluorescent immunoassay [FIA] and Veritor) yield objective results, which are potentially useful for point-of-care testing. The assays were evaluated with reverse transcriptase PCR (RT-PCR) using 411 nasopharyngeal swab specimens. Sensitivity and specificity values (percentages) of 79.0/99.0 and 64.0/99.4 for influenza A and 92.9/96.7 and 78.6/98.7 for influenza B were obtained for the Sofia and Veritor assays, respectively.

Use of Continuous Human Lung Cell Culture for Adenovirus Isolation

Adenovirus sensitivity, TCD50 value and mean time to cytopathic effect (CPE) were compared in Madin-Darby canine kidney (MDCK), rhesus monkey kidney (RhMK), human lung carcinoma (A549) and in newly identified continuous human lung (CHL) cell cultures. High-titer stock and positive original specimens were inoculated in culture. Patients specimens were also placed in shell-vial cultures. A549 and CHL cells demonstrated comparable sensitivity, however the former generally produced CPE earlier. Sensitivity was respectively reduced and poor in RhMK and MDCK cultures, as expected in cells of nonhuman origin. The performance of CHL for adenoviruses and other pathogens makes them a useful addition in diagnostic virology laboratories.

Rapid Identification of 2009 H1N1 Influenza A Virus Using Fluorescent Antibody Methods

Identification of the 2009 H1N1 influenza A virus requires emergency use authorized (EUA) molecular reverse transcriptase-polymerase chain reaction. Laboratories lacking molecular capabilities outsource testing, which is costly and may delay result reporting. A fluorescent antibody (FA; D(3) Ultra 2009 H1N1 influenza A virus ID Kit, Diagnostic Hybrids, Athens, OH) recently received Food and Drug Administration EUA status for 2009 H1N1 virus identification. The performance of this FA reagent was evaluated in this study. Influenza A-positive nasopharyngeal specimens (seasonal H1, H3, and 2009 H1N1) were prepared for culture and FA testing and were stained using influenza A antibodies and the 2009 H1N1 reagent. Other respiratory viruses were also evaluated. The FA reagent demonstrated 100% sensitivity and specificity. Bright, apple-green fluorescence was effortlessly identified in culture-positive cells, particularly around cell membrane perimeters. Laboratory-prepared slides were preferred over bedside-prepared specimens because background fluorescence obscured identification in the latter. The new FA reagent provides an accurate, rapid, and inexpensive assay for identifying the 2009 H1N1 virus in nonmolecular diagnostic laboratories.

Use of monoclonal antibody blends in the identification of enteroviral aseptic meningitis

Monoclonal antibody pools directed against group B coxsackievirus and echovirus antigens (Chemicon, Temecula, California) were evaluated as tools in the identification of enteroviral aseptic meningitis. Cerebrospinal fluid (CSF) and serial dilutions of stock coxsackievirus were inoculated into tube and shell vial Rhesus monkey kidney (RhMk) cell cultures. Positive cellular fluorescence was observed only in conjunction with cytopathic effect (CPE). The time from inoculation to CPE was similar with both tube and shell vial cultures.Direct CSF testing failed to reliably identify positive specimens as fluorescent debris, and a lack of available cells hindered results.Viral components of each antibody blend demonstrated positive cellular fluorescence when appropriately stained. False-positive fluorescence was not observed when cells, infected with other CSF viruses, were stained with these reagents.The findings suggest a role for these reagents (available both as blends and type-specific reagents) in the culture confirmation and identification of many common enteroviral serotypes associated with aseptic meningitis.

A rapid microscopic method for the confirmation of rhinoviruses in cell culture.

Objective: Culture-confirmation of rhinovirus is done using acid lability testing, a laborious and time-consuming method which delays the reporting of patient results by 1–2 days. A fluorescent monoclonal antibody pool (Light Diagnostics Pan-Enterovirus Blend; Millipore Inc., Temecula, Calif., USA) developed to identify various enterovirus isolates in culture was recently reported to also cross-react with rhinoviruses. We evaluated the use of this cross-reacting antibody, used in tandem with non-cross-reacting enterovirus antibodies (D3 IFA; Diagnostic Hybrids Inc., Athens, Ohio, USA) to rapidly identify rhinoviruses in cell culture. Methods: Microscope slides were prepared from cell cultures of 11 rhinovirus clinical isolates and a variety of other respiratory viruses. Slides were stained using both enterovirus antibody pools and examined for fluorescent activity. Results: Positive fluorescence was observed in all the rhinovirus isolates tested using the pan-enterovirus antibody blend, but yielded negative results when stained using the D3 antibodies. Both antibody products produced positive fluorescence for enterovirus isolates and produced negative results when a variety of other respiratory viruses were examined. Conclusion: Staining suspected rhinovirus isolates with each antibody pool affords a rapid means of identifying rhinoviruses and distinguishing them from enteroviruses, without the need for acid lability testing.