Clinical Isolates. 168 identified and typed enterovirus clinical isolates obtained from a mid-west department of health were used to conduct this study. Super E-Mix Shell Vial Testing. Triplicate Super E-Mix monolayers were inoculated with 200ul of each isolate and centrifuged at 700xg for 1 hr then incubated at 35-370C for 17-24 hrs. One monolayer from each triplicate was scraped and dotted onto a slide. After allowing the slides to dry, they were fixed with 100% Acetone and stained with each of the 3 IFA Enterovirus MAbs; D3, Chemicon and Dako. DHI Control slides were used as positive controls. Isolates that showed a consensus IFA negative result at 17-24 hrs. were re-tested when a replicate vial showed CPE, or at day 5 post inoculation if no CPE was observed. Cell Culture Confirmation. Single tube cultures of MRC-5, Vero and PMK cells were aspirated and inoculated with 200ul of each isolate. These were placed in a 35-370C incubator for a 1 hr passive adsorption step. After 1 hour, the specimen was removed and the tube cultures were re-fed with 2ml 2% FBS refeed medium and placed back in the incubator. Tubes were examined daily for CPE. Tubes showing 2+ CPE or higher were scraped and the cell suspension was dotted onto slides. Tubes were examined for 14 days. After day 14 post inoculation, if a tube showed no CPE it was reported as negative for Enteroviruses. Slide Preparation. (shell vials and tubes) Cells from the scraped monolayers were mixed with a pipettor to break up cell clumps. These suspensions were centrifuged at 700xg for 10 min. Supernatant was carefully aspirated and 200ul of PBS was added. The cell pellet was re-suspended in the PBS and mixed until a homogenous cells suspension was achieved. DHI control slides were dotted in quadruplicate with each specimen at 12ul per well. Slides air dried and were fixed with 100% acetone for 10 min. The first 3 wells were stained with D3, Chemicon and Dako MAbs. The last well was used for a separate experiment. Slides incubated in a 5% CO2, humidified 35-370C incubator for 30 minutes. Slides were rinsed gently in a glass beaker of distilled water and stained with the appropriate secondary MAb for 30 min. They were rinsed again in the distilled water, mounting medium was added and a cover slide placed onto each slide. All slides were examined at 200x magnification.
Compare the clinical sensitivity and specificity of D3 Enterovirus IFA to Chemicon’s Pan-Enterovirus IFA and Dako’s Anti-Enterovirus IFA in Super E-Mix shell vials. Determine the performance characteristics of these IFA’s when using MRC-5, Vero and PMK tube cultures for the isolation of Enteroviruses. Compare the sensitivity of MRC-5, Vero and PMK cultures for the isolation of Enterovirus.
* Negative for Coxsackie A16 after 24hrs. Positive with all stains at day 5 **Dako was negative for 2 Coxsackie A9 isolates ***Dako was negative for the Echo 24 isolate **** Negative at day 5 on Super E-mix ***** Dako was negative for Enterovirus 71 isolates Results from the Super E-Mix cells showed 166/168 positive with D3 reagent, 161/168 positive with the Dako reagent and 166/168 positive with the Chemicon reagent. Non-specific background staining was low with both D3 and Dako reagents; however Chemicon’s Pan-Enterovirus reagent showed moderate to high non-specific staining which made reading the lower titered-positive scrapings difficult. Tube culture results showed similar staining results. What was of note was the Vero cells appeared to be superior to both MRC-5 and PMK for the isolation and quality of the staining for the enterovirus isolates Conclusion: Diagnostic HYBRIDS D3 IFA Enterovirus Identification Kit performed well, when compared to the Chemicon’s Pan-Enterovirus Blend and Dako’s Anti-Enterovirus reagent, for the ability to identify enterovirus positive cell cultures. The fluorescent intensity level for the D3 kit was equivalent or better than both of the Chemicon and Dako reagents. Non-specific background staining was minimal with D3 and Dako reagents while Chemicon showed higher background staining which made reading results difficult.
Introduction: The Diagnostic HYBRIDS, Inc. D3 IFA Enterovirus Kit uses a blend of enterovirus protein derived murine monoclonal antibodies for the rapid detection and identification of Echoviruses, Coxsackie A/B viruses, Polioviruses and Enteroviruses in pre-CPE shell vial cultures and traditional tube cultures. Objective: This study compared the performance of the DHI IFA Enterovirus MAb reagents with the Light Diagnostics Pan-Enterovirus MAb blend and the Dako MAb mouse Anti-enterovirus clone 5-D8/1 for the ability to identify enterovirus isolated in multiple cell culture systems. Design/Methods: This study utilized 168 archived enterovirus isolates obtained from clinical specimens between the years of 1985-2005. The isolates were re-cultured in Super E-Mix (DHI) shell vial cultures, as well as an MRC-5, Vero and PMK tube cultures. Cultures were processed according to the respective product insert. One shell vial was scraped and cells spots were made after 17-24 hours of incubation. The cells spots were stained with D3 IFA Enterovirus Identification reagent, Chemicon’s Pan-Enterovirus Blend and Dako’s Monoclonal Mouse Anti-Enterovirus Clone 5-D8/1 reagent. An additional vial was scraped and stained with the 3 reagents when CPE was seen, up to day 5 post infection, if the initial cell spot results were negative. Tube cultures were processed by making cell scrapes and staining with all three reagents when CPE of at least 2+ was seen. Tube cultures were incubated 14 days before reporting a negative result. Results: The isolates tested were the following: 3 Coxsackie B1, 16 Coxsackie B2, 8 Coxsackie B3, 5 Coxsackie B4, 12 Coxsackie B5, 15 Coxsackie A9, 2 Coxsackie A16, 6 Echo 4, 6 Echo 5, 7 Echo 6, 11 Echo 7, 17 Echo 9, 19 Echo 11, 3 Echo 14, 2 Echo 24, 15 Echo 30, 2 Enterovirus 70, 3 Enterovirus 71, 4 Polio 1, 5 Polio 2 and 6 Polio 3 isolates. Pre-CPE Super E-Mix Results:
Coxsackie A9: Chemicon
Coxsackie B1: Chemicon
Coxsackie A9: Dako
Coxsackie B1: Dako
Negative Control on PMK: Chemicon
Negative Control on PMK: D3
Echovirus 9 on PMK: Chemicon
Echovirus 9 on PMK: D3
Echovirus 9 on VERO: Chemicon
Echovirus 9 on MRC-5: Chemicon
Echovirus 9 on VERO: D3
Echovirus 9 on MRC-5: D3
Negative Control on PMK: Dako
Echovirus 9 on PMK: Dako
Echovirus 9 on VERO: Dako
Echovirus 9 on MRC-5: Dako
Figure 2: The pictures below show the staining patterns of MRC-5, VERO and PMK tube cultures that were scraped and processed once 2+ CPE from Echovirus 9 was observed based on standard tube culture protocol. Slides were prepared and stained with D3, Chemicon’s Pan-Enterovirus and Dako’s Enterovirus MAbs. As above, Chemicon showed the dullest staining, with Dako equivalent to D3. The un-infected PMK showed specific staining with the Chemicon IFA, while D3 and Dako showed no background staining. Un-infected VERO and MRC-5 also had a moderate to high background with the Chemicon IFA while Dako and D3 showed no background, (pictures not taken).
Staining Intensity of D3 IFA Compared to Chemicon and Dako IFA’s In MRC-5, Vero and PMK cells.
Coxsackie A9: D3
Coxsackie B1: D3
Staining Intensity of D3 IFA Compared to Chemicon and Dako IFA’s In Super E-Mix DHI x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x
Chemicon x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x
x x x
x x x x x x x x x x x x x x x x x x x x: m issed isolates x x
Dako x: m issed isolates
Figure 3: the table on the left shows combined data from two testing sites in which known Enterovirus and nonEnteroviruses were inoculated onto Super E-Mix. “x” in the table indicates a positive stained monolayer with the reagent. DHI Reagent specifically detected all of the Enteroviruses and did not cross-react with the nonEnteroviruses. Dako did not crossreact with non-Enteroviruses, however it missed CA16 and several isolates of CA9, as well as Enterovirus 68 and 71. Chemicon cross-reacted strongly with HSV, Rhinovirus, Hepatitis A and Coronavirus.
127/168 148/168 152/168 152/168
Day 2 Day 4 Day 7 Day 14
167/168 (day 5)
Super E-mix w/D3 IFA
Figure 4: The table and the accompanying values in each square above show the number of positive isolates at each time point examined. All of the CPE positive tube cultures were confirmed by IFA. Tube cultures that showed no CPE after 14 days of incubation were called negative for enterovirus. It is interesting that the same two isolates, (Both Enterovirus 70) were missed by both the Vero and PMK cultures but the MRC-5 cultures isolated both of them. This data shows that by using Super E-Mix shell vials, we can detect 166/168 isolates within 24 hours of incubation, and 167/168 by day 5. A low titer Enterovirus 71 was missed by Super E-mix and took between 7 and 14 days to culture on MRC-5 tubes.
Tube culture CPE results compared to Super E-Mix stained with D3 IFA Enterovirus reagents.
Conclusion – D3 IFA Enterovirus Identification Kit is as sensitive and more specific than both Chemicon and Dako IFA reagents. Super E-Mix is significantly more sensitive than MRC-5, Vero and PMK cultures combined for the isolation of Enterovirus.
•Overall, Vero cells isolated just as many Enteroviruses as PMK cells, and in a equal amount of time.
•Super E-Mix shell vials, with D3 IFA was positive for 166/168 isolates at 24 hours. Tube cultures at best had 102/168 at 24 hours.
•Objective #3: Comparison of Vero, MRC-5 and PMK tubes cultures to Super E-Mix shell vials for the isolation of Enterovirus.
•Chemicon strongly cross-reacted to all of the above.
•Dako missed all of the Enterovirus 71 isolates.
•D3 and Dako were negative when tested against Rhinovirus, Coronavirus, Hepatitis A and Herpes Simplex Virus.
•Objective #2: Specificity of D3 IFA compared to Chemicon and Dako IFA’s.
•Dako and D3 showed the lowest background. Chemicon showed moderate to high background staining making it difficult to confirm low level positive results; 1+ or lower.
•Overall, D3 IFA stained 1+ to 2+ brighter than Chemicon’s Pan-Enterovirus IFA and equivalent or slightly brighter than Dako’s IFA.
•Stain intensity varied among manufacturers.
•Detection sensitivity varied dependent on virus strain
•Objectives #1 and #2: Compare D3 IFA to Chemicon and Dako IFA’s in Super E-Mix and MRC-5, Vero and PMK cultures.
Summary of Results
Figure 1: The pictures on the left show the staining of Coxsackie B1 and Coxsackie A9 infected Super E-Mix monolayers which were stained with the D3 IFA Enterovirus MAbs and with Chemicon’s Pan-Entero MAb and Dako’s Enterovirus MAb after 24hrs of culture. Chemicon’s stain was very faint for both of the viruses. Dako showed good staining for CB1, and slightly duller staining on CA9 than D3. Overall, this pattern of D3 and Dako giving the brightest staining with Chemicon slightly duller or equal in intensity remained consistent with the rest of the 168 isolates tested.
Viruses Tested Coxsackie A9 Coxsackie A16 Coxsackie A24 Coxsackie B1 Coxsackie B2 Coxsackie B3 Coxsackie B4 Coxsackie B5 Coxsackie B6 ECHOVirus 3 ECHOVirus 4 ECHOVirus 5 ECHOVirus 6 ECHOVirus 7 ECHOVirus 9 ECHOVirus 11 ECHOVirus 13 ECHOVirus 14 ECHOVirus 15 ECHOVirus 18 ECHOVirus 21 ECHOVirus 24 ECHOVirus 25 ECHOVirus 30 Enterovirus 68 Enterovirus 70 Enterovirus 71 Polio 1 Polio 2 Polio 3 Parechovirus 1 (E22) Parechovirus 2 HSV Rhinovirus Coronavirus Hepatitis A
Specificity of D3 IFA Compared to Chemicon and Dako IFA’s
Joseph Jollick, Jeff Houtz, Steve Ewers. Diagnostic HYBRIDS, Athens, OH
EVALUATION OF THE DIAGNOSTIC HYBRIDS D3 IFA ENTEROVIRUS IDENTIFICATION KIT FOR CULTURE CONFIRMATINON OF ENTEROVIRUSES.