Specimen Collection and Lab Analysis Related Issues with Influenza in Nebraska Objectives: Describe the Nebraska Department of Health and Human Services State Influenza Surveillance Plan, including implementation during a pandemic. Discuss processes for safe specimen collection, test order and transport. Review testing methodologies and technical complications associated to RIDT Role of the NPHL in the influenza program Pandemic full scale exercise overview

Influenza Surveillance Overview The Nebraska influenza surveillance system is a collaborative effort between DHHS and its many partners in the state including, local health departments, public health and clinical laboratories, vital statistics offices, healthcare providers, clinics and emergency departments. Nebraska monitors influenza activity in several ways: Laboratory Surveillance Outpatient Surveillance – ILINet Emergency Department Syndromic Surveillance Inpatient Surveillance Mortality Surveillance Outbreak Surveillance Long-term care facilities School absenteeism   Helping People Live better Lives.

Influenza Laboratory Surveillance Laboratory Specimen Surveillance Submission of isolates by clinical virology laboratories and sentinel provider offices to the Nebraska Public Health Laboratory (NPHL). Rightsizing Influenza Virologic Surveillance Vaccine Strain Selection Antigenic drift – annual update Predict which strains are most likely to be spreading the following winter Antiviral Resistance Monitoring Situational Awareness Determine beginning and end of season Monitor prevalence of viruses Novel Event Detection Detect a rare event (e.g. novel flu)  Year-round surveillance is necessary to maintain these detection goals. -1 sentinel hospital laboratory in each local public health jurisdiction -16 ILINet participating sentinel providers -Statistical calculations define the desired number of specimens needed to ensure adequate confidence in virologic surveillance capacity throughout the state. Influenza Right-Size surveillance objectives and thresholds are centered on four aspects: Vaccine Strain Selection- minor change in the virus, Anti-viral Resistance Monitoring (timely detection of resistant viruses among influenza positive surveillance specimens), Situational Awareness (monitoring the prevalence and spread of influenza viruses throughout the year and to determine the beginning and end of the season), and Novel Event Detection (early identification of a novel influenza virus among influenza positive surveillance specimens for effective intervention and control measures). Year-round surveillance is necessary to maintain these detection goals. Helping People Live better Lives.

Influenza Laboratory Surveillance Laboratory Reporting Surveillance Reporting by virology laboratories of positive test results and total number of respiratory virus specimens tested. Weekly online influenza/RSV testing survey Influenza, rapid tests summary report only (laboratories only) Electronic Laboratory Reporting (ELR) Influenza, all tests positive and negative (applies only to laboratories performing electronic lab reporting as specified in 173 NAC 1-005.02C)   Helping People Live better Lives.

Influenza Specimen Collection

Influenza Specimen Collection Recommended nasopharyngeal swab or wash Although manufacturers list nasal swab as approved, the fine print states “To obtain accurate results, use Viral Transport Media (VTM)” NPHL accepts only nasopharyngeal swabs in VTM

Swab Types

NP Collection Procedure https://www.youtube.com/watch?v=hXohAo1d6tk

NP Collection Procedure Demonstration by Kate Boulter, NBU Key points: Wear gown, gloves and protective face shield Stand to side of patient to avoid aerosols Have patient close eyes tight to avoid sneeze reflex Gently guide swab to nasopharyngeal area, pointing toward ear, not up into nasal area Rotate as you pull out. Cover VTM tube with alcohol swab or gauze when bending swab, to snap off into media

NUlirt NUlirt website: https://nulirt.nebraskamed.com/login Nebraska University’s Laboratory Information Reporting Technology NUlirt website: https://nulirt.nebraskamed.com/login NUlirt new user form: NPHL NUlirt Access Request NUlirt user guides: Full Condensed NUlirt Training Webinar: Click here

NUlirt Main Page Navigation Header and the Primary Action buttons The Navigation Header is where most non-order entry actions will be initiated from, and includes: Home – return to the main page. New Lab Order - start the ordering process for a new lab test Batch List – navigates to the updated Order Batch List page Results – navigates to a Result and Order review on a site or patient level. Patients – navigates to the patient view to view or edit patient files The User Menu – includes a Logout option and access to user settings

NUlirt Main Page Navigation Header and the Primary Action buttons Change Site - In order to begin the ordering process, a site must be selected. This is similar to the way a user needed to select an Account in ELIRT to proceed. Account visibility is restricted to the location the user is currently logged in under and each user is limited based on their given rights. This will need used if a user needs to change their ordering site. Begin New Order - Quick action button to start the ordering process at the users selected site.

Patient Records Viewing and Selecting Patients The user can select a patient by using either the Navigation Header menu Patients option or by attempting to create a new order via the Primary Action buttons and Begin New Order. Either process will give the user the opportunity to review and edit an existing patient, or to create a new patient. Once the appropriate patient has been located, they are selected by using the green checkbox. If the proper patient record cannot be located, a new record can be created by using the green add a patient button near the top of the page.

Patient Records Editing a patient record Users have the ability to review saved information and make any necessary changes, saving the record once changes are complete. Several patient demographic fields are now required upon data entry for reporting purposes. If a required field is not completed, NUlirt won’t continue to the order page until it is complete. In addition, NUlirt will look up and automatically generate the City, State, FIPS code, and Health District information after a valid 5-digit ZIP code is entered.

Placing an Order New Order A new order is generated by selecting New Order on the lower left hand side of the patient view. This will redirect the user to the order entry screen. Select an Ordering Provider or enter an Alternative Provider Select the appropriate ordering Account Select the proper Lab Test Enter the appropriate Specimen Source Correct the collect date and time When ordering is complete, select Continue and NUlirt will direct the user to the Ask at Order Entry (AOE) questions page.

AOE Questions Ask at Order Entry Questions All data fields and selection fields need completed. Once complete, select Update AOE Answers to continue.

Submit an Order Reviewing and Submitting Before final submission, the user has the ability to change or update the lab testing ordered and AOE questions via the review order page by clicking on any of the Edit buttons, and then saving changes. Once finalized, click Submit Order to send the order to the lab.

Printing a Batch List Viewing and Printing Via the Navigation Header select Batch List. Select the appropriate date ranges, the ordering Site, and the Account code for the orders submitted. Click Print Reports to view and print the batch list. The resulting Batch List view includes all of the orders associated with the Account(s) selected. Selecting Print will have each Account’s Batch List appear on its own page for easy printing and submission to the lab

Viewing Results To view results, hover over Results in the Navigation Header. A drop down will generate, first will be the Site you are currently under, followed by all programs the user has access to. Once on a results page, all results for that site or program will be displayed in descending order. The user can also search for a specific patient by using the search pane. Search by patient name, account, patient record, or Site. To view more detailed results or more order information, select View to the right of an order. By default, NUlirt only displays the patients’ most recent results. To view a more in depth patient chart, go back to the matching Patient Record and select View Results on the bottom of the page.

Packaging Specimens for Transfer 4/6/2019 Packaging Specimens for Transfer Label the specimen on viral transport media tube and ensure cap on tube is tightly sealed. Complete paperwork in accordance with state health department guidelines. Include a frozen cold pack with the specimen(s). Pack specimens in accordance with U.S. Department of Transportation regulations regarding shipment of biological substances, see www.cdc.gov/flu/professionals/diagnosis/index.htm. = Category B 2019 Sentinel Biohazard & Bioterrorism Preparedness Training

4/6/2019 Influenza Testing 2019 Sentinel Biohazard & Bioterrorism Preparedness Training

4/6/2019 Influenza Testing 2019 Sentinel Biohazard & Bioterrorism Preparedness Training

Rapid Influenza Diagnostic Tests 4/6/2019 Rapid Influenza Diagnostic Tests Antigen detection Assay 2 types: ImmunoChromatographic – Read visually Fluorescent ImmunoAssay – Requires reader 50-70% Sensitivity >90% Specificity Some testing CLIA Waved Because of the lower sensitivity of the rapid influenza diagnostic tests, clinicians should consider confirming negative test results with molecular assays, especially during periods of peak community influenza activity and/or during suspected institutional influenza outbreaks  because of the possibility of false-negative RIDT results. In contrast, false-positive RIDT results are less likely, but can occur and are more common during periods of low influenza activity. Therefore, when interpreting results of a rapid influenza diagnostic test, clinicians should consider the of the test in the context of the level of influenza activity in their community ( See Algorithm to assist in the interpretation of influenza testing results and clinical decision-making during periods when influenza viruses are circulating in the community(https://www.cdc.gov/flu/professionals/diagnosis/algorithm-results-circulating.htm) and Algorithm to assist in the interpretation of influenza testing results and clinical decision-making during periods when influenza viruses are NOT circulating in the community(https://www.cdc.gov/flu/professionals/diagnosis/algorithm-results-not-circulating.htm) for more information). Package inserts and the laboratory performing the test should be consulted for more details regarding use of rapid influenza diagnostic tests. 2019 Sentinel Biohazard & Bioterrorism Preparedness Training

Rapid Influenza Diagnostic Tests 4/6/2019 Rapid Influenza Diagnostic Tests In vitro immunochromatographic assay for the qualitative detection of influenza A and B nucleoprotein antigens Mode of action of common RIDT format: (a) Dyelabelled antibody (Ab), specific for target antigen, is present on the lower end of the nitrocellulose strip or in a well provided with the strip. Antibody, also specific for the target antigen, is bound to the strip in a thin (test) line and either antibody specific for the labelled antibody, or antigen, is bound at the control line, (b) Respiratory specimen and buffer, which have been placed on the strip or in the well, are mixed with the labelled antibody and are drawn up the strip across the lines of bound antibody. (c) If antigen is present, some labelled antibody will be trapped on the test line. Other labelled antibody is trapped on the control line. https://apps.who.int/iris/bitstream/handle/10665/44304/9789241599283_eng.pdf;jsessionid=866118BB62A1CE3FE39C93BC9C92E116?sequence=1 https://apps.who.int 2019 Sentinel Biohazard & Bioterrorism Preparedness Training

Rapid Influenza Diagnostic Tests 4/6/2019 Rapid Influenza Diagnostic Tests Advantages: Rapid results <15 min May be CLIA waived Disadvantages: One swab collected, recollection required if forwarding to NPHL Clinics tend to test dry swab Extract reagents used in testing (NOT accepted by NPHL) Subtyping NOT provided Novel strains NOT detected False negative results common at peak of influenza activity False positive results less likely, more common during periods of low influenza activity Mode of action of common RIDT format: (a) Dyelabelled antibody (Ab), specific for target antigen, is present on the lower end of the nitrocellulose strip or in a well provided with the strip. Antibody, also specific for the target antigen, is bound to the strip in a thin (test) line and either antibody specific for the labelled antibody, or antigen, is bound at the control line, (b) Respiratory specimen and buffer, which have been placed on the strip or in the well, are mixed with the labelled antibody and are drawn up the strip across the lines of bound antibody. (c) If antigen is present, some labelled antibody will be trapped on the test line. Other labelled antibody is trapped on the control line. https://apps.who.int/iris/bitstream/handle/10665/44304/9789241599283_eng.pdf;jsessionid=866118BB62A1CE3FE39C93BC9C92E116?sequence=1 2019 Sentinel Biohazard & Bioterrorism Preparedness Training

Molecular Pathogen Detection 4/6/2019 Molecular Pathogen Detection https://blog.ucdmc.ucdavis.edu/labbestpractice/index.php/2017/11/16/molecular-pathogen-detection-at-the-point-of-care-next-generation-flu-testing/ Rather than using antibodies against proteins found on the influenza virus (i.e., RIDTs), molecular assays target the virus’ genetic make-up. https://blog.ucdmc.ucdavis.edu 2019 Sentinel Biohazard & Bioterrorism Preparedness Training

Influenza Testing in Nebraska *Surveillance Sites only

Influenza Testing in Nebraska DNS = Deep Nasal Swab N = Nurse NNW = NP Swab & Wash L = Laboratory NS = NP Swab RT = Respiratory Therapy D = Doctor *Surveillance Sites only

BioSafety at Testing Site 2017 Sentinel Biohazard & Bioterrorism Preparedness Training 4/6/2019 BioSafety at Testing Site Rapid Influenza Detection Testing (RIDT) Many hospital labs and physician offices have space constraints that limit implementation of CDC guidelines: “Influenza testing is at BSL-2, recommends use of Biological Safety Cabinets” or Splash Guards Disclaimer – not showing quality of kit, simply showing some kits are more prone to aerosols. Despite experienced CLS performing, non-enclosed method shows aerosols. CDC recommends protection

BioSafety at Testing Site 2017 Sentinel Biohazard & Bioterrorism Preparedness Training 4/6/2019 BioSafety at Testing Site Wear suitable protective gown/coat, gloves, and eye/face protection when testing Best practice to test behind plastic shield Gloves overlap gown/coat Doff gloves with glove-in-glove technique Wash hands thoroughly after handling Disclaimer – not showing quality of kit, simply showing some kits are more prone to aerosols. Despite experienced CLS performing, non-enclosed method shows aerosols. CDC recommends protection

Influenza Testing @ NPHL Use the CDC Influenza assay FDA cleared assay PCR (diagram) Results (diagram) Family tree (diagram)

Polymerase Chain Reaction FDA Cleared RT-PCR

Positive Influenza Results

Influenza Family Tree

2017 Sentinel Biohazard & Bioterrorism Preparedness Training 4/6/2019 Additional Resources http://www.nphl.org