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EVALUATION OF THE DIAGNOSTIC HYBRIDS D3 DUET DFA INFLUENZA A/RESPIRATORY VIRUS AND D3 DUET DFA RSV/RESPIRATORY VIRUS SCREENING KITS FOR THE DETECTION OF RESPIRATORY VIRUSES FROM CLINICAL SPECIMENS A. Munson, D. Bennion, D. Pombo, K. Gibson and P. Magee. LDS Intermountain Laboratory, Salt Lake City, Utah Comparison of Diagnostic Hybrids D3 Respiratory Duet DFA Kits to D3 Ultra

Amended Abstract Introduction: The rapid detection of respiratory viruses (influenza A, B, parainfluenza 1, 2, 3, adenovirus and RSV) by direct immunofluorescence (DFA) has had a significant impact on patient management and, more recently, therapeutic options. The two predominant viruses identified during the winter respiratory season are influenza A and respiratory syncytial virus (RSV). The new D3 Duet DFA Influenza A/ Respiratory Virus Screening Kit and D3 Duet DFA RSV/Respiratory Virus Screening Kit (Diagnostic Hybrids , Athens, OH) allow, by the use of fluorophore Phycoerytherin, the specific identification of these two viruses, respectively, while screening for the other 6 respiratory viruses. Objective: The purpose of this study was to evaluate the performance of the D3 Duet DFA Influenza A/ Respiratory Virus Screening Kit and D3 Duet DFA RSV/Respiratory Virus Screening Kit with the D3 Ultra DFA screening reagent (DHI) and SimulFluor™ Respiratory Screen reagent (CHEMICON International, Temecula, CA). Materials and methods: 319 specimens (nasopharyngeal [NP] swabs, NP aspirates, NP washes and bronchial washes) were processed according to standard laboratory practices for the preparation of cell spots for DFA testing. Cell spots were stained with the four reagents according to manufacturer’s instructions.

3

14

0

D Duet RSV / Respy Positive

0

305

D Duet RSV / Respy Negative

Influenza A w/ Influenza A/Respiratory Duet

3

D Ultra RSV Positive

D Ultra RSV Negative

107

0

1

211

3

3

3

D Duet Flu A / Respy Negative

3

D Ultra FluA Negative

3

D Duet Flu A / Respy Positive

Results: DFA Screening Reagent

3

D Ultra Flu A Positive

Influenza A w/Influenza A/Respiratory Duet

Final Identification

Neg

Pos

RSV

Flu A

Flu A

Flu B

RSV

Adeno

Para 1

Para 2

Para 3

D3 Ultra

178

141

NA

D3 Duet FluA/ * Respy

178

129

NA

14

14

5

108

11

1

1

1

14

5

108

11

1

1

1

D3Duet *,† RSV/Respy

179

35

107

NA

14

5

107

11

1

1

1

Chemicon Simulfluor

187

30

104

NA

11

5

104

11

1

1

1

Comparison of Light Diagnostics™ Simulfluor® to D3 Ultra Respiratory 3

D Ultra FluA Negative

Simulfluor FluA Positive

11

0

Simulfluor FluA Negative

3

305

1-Flu A/RSV dual infection was detected by both D3 Duet Kits. † 1-RSV/Parainfluenza 3 dual infection was detected by the D3 Duet RSV/Respy Conclusions: The D3 Duet DFA Influenza A/ Respiratory Virus Screening Kit correlated completely with the D3 Ultra DFA screening reagent (DHI). The PE labeled antibody identified the target virus with 100% sensitivity/specificity. The remaining FITC labeled antibodies also had a 100% sensitivity/specificity. The D3 Duet DFA RSV/Respiratory Virus Screening Kit failed to detect 1 positive RSV culture. The PE labeled antibody identified the target virus with 99% sensitivity and 100% specificity. The remaining FITC labeled antibodies had a 100% sensitivity/specificity. The Chemicon SimuFluor™ Respiratory Screen reagent was somewhat less sensitive for both the detection of RSV (96%) and for detection of Flu A (79%). The ability to rapidly detect influenza A and RSV specifically within a respiratory screening pool will reduce turn around time to results, technical time by eliminating additional cell spots and typing, as well the expenses relating to additional virus identification procedures. .

Influenza A w/RSV/Respiratory Duet

Influenza A w/RSV Respiratory Duet

Study Objective Determine the performance characteristics of the Diagnostic Hybrids D3 Duet DFA Influenza A/Respiratory Virus Screening Kit and the D3 Duet DFA RSV/Respiratory Virus Screening Kit when directly staining fresh respiratory specimens as compared to that of Diagnostic Hybrids D3 Ultra DFA Screening Reagent and simultaneously to Chemicon Simulfluor™ Respiratory Screening Reagent.

Simulfluor RSV Positive

Simulfluor RSV Negative

3

D Ultra RSV Positive

D Ultra RSV Negative

104

0

4

211

Summary of Results

RSV w/RSV/Respiratory Duet

Dual infection w/RSV/Respiratory Duet

Methods Three hundred-nineteen prospective respiratory specimens were collected at a large, western United States reference laboratory using approved collection methods for nasopharyngeal aspirations, nasopharyngeal washings, nasopharyngeal swabs, bronchioalveolar lavage, tracheal aspirations and sputa, respectively. Patient sex, age and specimen source were recorded for each specimen (see table #2). Each specimen was processed and fixed to slides using the manufacturer’s product insert instructions for each of the kits used. Control slides were also included with each day’s stain batch to insure functionality of the respective stains. Staining was performed according to manufacturer’s instruction and examined using a UV microscope with a broad-band filter system that captures fluorescence from both FITC and phycoerythrin labels. Results of the respective Duet stains, Ultra screening reagent and Chemicon Simulfluor™ dual stain were recorded. Discrepant analysis was accomplished by culture and virus identification using either D3 Ultra or Chemicon respiratory virus identification systems.

3

3

D Ultra FluA Positive

One-hundred forty-one of the 319 specimens were determined to be positive by Diagnostic Hybrids D3 Ultra DFA Respiratory Virus Screening Reagent. The distribution of positive samples by virus isolated is listed below in Table #1. The D3 Duet DFA Influenza A/Respiratory Virus Screening Kit was able to detect all viruses detected by D3 Ultra for a 100% specificity and sensitivity,. The D3 Duet DFA RSV/Respiratory Virus Screening Kit failed to detect one RSV isolate detected by D3 Ultra resulting in a specificity of 100% and a sensitivity of 99.7%, respectively. The Light Diagnostics™ Simulfluor® Kit failed to detect Influenza A in three specimens and RSV in four specimens resulting in a sensitivity for the detection of influenza A and RSV of 79% and 96%, respectively. All results are listed below in Table #1. Distribution of confirmed virus detections by age group is also provided in Table #2. Two dual infections were detected by the D3 Duet DFA RSV/Respiratory Virus Screening Kit (Flu A & RSV; RSV & Parainfluenza 3) and one dual infection was detected by the D3 Duet DFA Influenza A/Respiratory Virus Screening Kit (Flu A & RSV).

Conclusion – RSV w/Influenza A/Respiratory Duet

Dual Infection w/Flu A/Respiratory Duet

The ability to rapidly detect influenza A and RSV specifically within a respiratory screening pool will reduce turn around time to results, technical time by eliminating additional cell spots and typing, as well as the expenses related to additional virus identification procedures.


Evaluation of D3 Duet DFA Influenza A/RespVirus & D3 Duet DFA RSV/Resp Virus Screening Kits