# Presentation: 183544

## Influenza 2004:  Meeting the Laboratory Challenges

- December 14, 2004
- Sponsored by
- National Laboratory Training Network
- Peter Shult, Ph.D.				Carol Kirk
- Director, 				Laboratory Network Coordinator
-    Emergency Laboratory Response 		Telephone 608-262-1021
-    & Communicable Disease Division	E-mail _[cjk@mail.slh.wisc.edu](mailto:cjk@mail.slh.wisc.edu)_
- Telephone 608-262-5419
- E-mail shult@mail.slh.wisc.edu
- Wisconsin State Laboratory of Hygiene
- Madison, WI

## Program Overview 

- Resources
- Factors Affecting the 2004-5 Influenza Season
- Challenges Facing the Laboratory
- Influenza: The Basics
- What is it?
- Nomenclature
- Antigenic Characteristics
- Public Health Importance
- Vaccine Strain Selection
- Prevention and Treatment
- Laboratory Diagnosis of Influenza
- Laboratory-Based Influenza Surveillance
- Meeting the Challenges

## Resources

- CDC Influenza home page
- _[http://](http://www.cdc.gov/flu/index.htm)__[www.cdc.gov/flu/index.htm](http://www.cdc.gov/flu/index.htm)_
- National Influenza Summary–Weekly Update
- _[http://www.cdc.gov/ncidod/diseases/flu/weekly.htm](http://www.cdc.gov/ncidod/diseases/flu/weekly.htm)_
- APHL listing of Public Health Laboratories
- _[http://www.aphl.org/Public_Health_Labs/index.cfm](http://www.aphl.org/Public_Health_Labs/index.cfm)_
- WHO Influenza home page
- _[http://](http://www.who.int/topics/influenza/en/)__[www.who.int](http://www.who.int/topics/influenza/en/)__[/topics/influenza/en/](http://www.who.int/topics/influenza/en/)_

## Factors Affecting the 2004-5 Season:  Complications, Concerns & Confounders

- Vaccine Availability
- Shortage of inactivated influenza virus vaccine
- Use of live attenuated influenza virus vaccine
- Increasing reliance on anti-viral medications
- Emerging/re-emerging diseases
- Pertussis
- Avian influenza
- Increased public & media attention
- Impact
- Increased need for timely surveillance data
- Increased need for communication between clinical and public health communities.
- Increased need for rapid diagnostic testing.

## Potential Challenges Facing the Laboratory

- Increased demand for rapid, accurate laboratory test results
- Staff shortages
- Shortages of supplies and test kits
- Staying current with emerging/re-emerging diseases and new test methodologies
- Increased biosafety risks posed by emerging diseases

## The Ultimate Challenge ...Fulfilling the laboratory’s role in meeting this season’s clinical and public health needs

## INFLUENZA: THE BASICS 

## What is “Influenza”?

- _Not _stomach flu
- _Abrupt_ onset of high fever and chills
- Associated symptoms: headache, malaise, myalgia, cough, sore throat, rhinitis; gastrointestinal symptoms in children
- Duration of _intense_ symptoms 3 to 5 days with prolonged recovery common; nevertheless, usually self-limiting
- Types A and B produce similar disease; type A, particularly H3N2, tends to be more severe
- Difficult to distinguish influenza from other respiratory infections based on symptoms alone
- Note: CDC definition of *influenza-like illness:*
-    Temp > 100°F orally and either cough or sore throat

## Influenza Virus

## Influenza

- Family:	Orthomyxoviridae
- segmented (8), ssRNA genome
- lipid envelop
- Genera:	Influenzavirus Types A, B and C
- Subtypes:	Human - **H****1****N****1****, H****3****N****2**, **H****1****N****2, **H2N2
-                         Birds - H1 to H15, N1 to N9
-                                     Bird →Human -  H5N1, H9N2 ,H7N7, H7N2, H7N**3**
- Strains:	A/Fujian/411/2002 (H3N2)-like
- (2004-05)	A/New Caledonia/20/99 (H1N1)-like
- 			B/Shanghai/361/2002-like

## Influenza: Nomenclature

- A/Fujian/411/2002 (H3N2)-like

**Original Lab ID #**

**Type**

**Subtype**

**Location of Original Isolation**

**Year of Initial Isolation**

## Influenza: Subtyping & Strain Characterization

- State Public Health Laboratories
- perform subtyping of *influenza A* viruses using hemagglutination inhibition, immunofluorescence, and/or pcr; influenza B has no subtypes.
- perform *preliminary* lineage grouping of influenza B viruses using reagents provided by CDC
- CDC
- performs subtyping of unusual samples/isolates
- performs strain characterization of selected viruses
*Note: Only CDC can perform complete strain **	**	**	**characterization of influenza viruses*

## Influenza: Epidemic (Inter-Pandemic) Influenza

- Disease caused by a new _strain _of influenza virus that has evolved gradually by **point mutations within the hemagglutinin or neuraminidase or both**** **_**of influenza A or B**_ such that preexisting antibody cannot completely neutralize the new strain.
*The mechanism: ****ANTIGENIC DRIFT***

## Interpandemic Influenza Wisconsin, 1995 - 2004

## Influenza:  Annual Impact

**Seek Care**** **

**~10%-20% Infected (29-59 million)**

**~50% Symptomatic (15-30 million)**

**Treat at Home**

**Hospitalized>200,000**

**Lab Tested **

**Death**
**~36,000**

**U.S. population ~290 million**

- Influenza:  *Annual Impact*

- CDC

**Notes:**
Each year ~10-20% of the US population becomes infected with influenza virus
Most, but not all of those infected will be symptomatic.
Of those that are symptomatic, most will treat at home but some will seek healthcare.
Of those that seek healthcare, some will require hospitalization and a subset of both the outpatients and the hospitalized patients will be tested for influenza.
Some of the people with influenza virus infection will die.

## Pandemic Influenza

- Disease caused by a **reassortment of **_**Influenza A **_**subtypes** that results in the emergence of a new virus containing a novel hemagglutinin and/or neuraminidase (a novel subtype) that is _immunologically distinct _from previously circulating strains.
*The mechanism:  ****ANTIGENIC SHIFT***

## Generation of Pandemic Influenza Strain

- CDC

##     Timeline of Emergence of 

- Influenza Viruses in Humans

**1918**

**1957**

**1968**

**1977**

**1997**

**1998/9**

**2003**

**H1**

**H1**

**B**

**H2**

**H7**

**H5**

**H5**

**H9**

**Spanish**
**Influenza**

**Asian**
**Influenza**

**Russian**
**Influenza**

**Avian**
**Influenza**

**Hong Kong**
**Influenza**

**H3**

- CDC

## Direct Interspecies Transmission of Avian Viruses to Humans Exposed to Infected Poultry

**1997: 18 cases of human respiratory illness caused by HP avian H5N1; 6 deaths – Hong Kong**** **
**1998/9: 7 cases of human respiratory illness caused by H9N2; no deaths – China, HK**** **
**2003: 2 additional HP H5N1 cases in humans; 1 death – China, HK**** **
**2003: >80 cases of infection by HP H7N7 avian viruses; 1death – The Netherlands**** **

- CDC

## Influenza A(H5N1)

- December, 2003 – October 25, 2004

- CDC

**Notes:**
H5N1 enzootic of unprecedented size and complexity now established
Poultry outbreaks in 9 countries
Ongoing poultry outbreaks in Malaysia, Thailand, Vietnam an China
Additional recent human cases and deaths in Vietnam and Thailand 
Substantial economic impact

## HP Avian Influenza A(H5N1), 2004 

**Poultry outbreaks reported in Cambodia, China, Laos, Indonesia, Japan, Malaysia, S. Korea, Thailand and Vietnam**
**Isolation of H5N1 in Hong Kong in wild birds**** **
**Total of 44 confirmed human cases in Vietnam (n=27) and Thailand (n=17); 32 deaths**
**Most cases had exposure to sick/dead birds; ****family clusters raise questions about person-to-person transmission**

- CDC

## Avian Influenza (H5N1)

-  Recent events
-  How close are we to a pandemic?
-  The recipe for a human pandemic :
**   ****Emergence of a **_**novel subtype**_** of influenza **
**An immunologically naïve population**
**   ****Replication in humans ****→ ****disease**
**   ****Efficient human-to-human transmission**

## FIGURE 1. Crude death rate for infectious 

- FIGURE 1. Crude death rate for infectious
- diseases - United States, 1900 - 1996.

## An Influenza Pandemic Will be Unlike Other Public Health Emergencies or Common Disasters

- Inevitable yet unpredictable
- Will arrive with very little warning
***Widespread, not focused like a bioterrorism event***
***Will be a  global event***
***Will affect entire national infrastructure rapidly***
- Locally explosive epidemics; unusually high morbidity and mortality
- Rapid person-to-person spread
- Extraordinary strain on human and material resources
- Effect will be relatively prolonged - weeks to months
- Multiple waves of disease
- Can’t stockpile vaccine

## Estimates For Next Pandemic in U.S. Alone

- 200 million will be infected
- 40 - 100 million will be clinically ill
- 18 - 45 million will require outpatient care
- 300,000 - 800,000 will be hospitalized
- 88,000 - 300,000 will die
- Economic losses of $71 - $166 billion

## Slide 26

## Influenza:  Prevention and Treatment

- Immunization
- Inactivated vaccine
- Live attenuated influenza vaccine
- 	_[http://www.cdc.gov/flu/professionals/vaccination/](http://www.cdc.gov/flu/professionals/vaccination/)_
- Antiviral Prophylaxis and Treatment
- Adamantanes
- Neuraminidase inhibitors
- _[http://www.cdc.gov/flu/professionals/pdf/0405antiviralguide.pdf](http://www.cdc.gov/flu/professionals/pdf/0405antiviralguide.pdf)_

## Outline of the Annual Process of Development, Manufacturing, and Distribution of Influenza Vaccine [Copyright © 2004 Massachusetts Medical Society.  All rights reserved. ]

## Laboratory Diagnosis of Influenza

## Influenza:  Laboratory Diagnosis

- _Culture_
- Traditional PRMK tubes vs. MDCK shell vials
- MDCK shell vials more rapid, often more sensitive
- For optimum sensitivity, use both
- Neither method timely (1-5d) for patient management
- Highly specific; variable sensitivity
- Need to maintain virus viability
- Limited to laboratories with viral culture capability
- BUT ...
- Variable sensitivity
- Provides isolate for strain typing & vaccine development

## Influenza:  Laboratory Diagnosis

- _Antigen Detection - Immunofluorescence_
- No need for viable virus...but no virus isolate
- Rapid (20+ minutes - 2+ hours)
- Can assess quality of specimen
- Subjective reading - reader expertise required
- Dependent on reagent quality
- Limited to laboratories with IF capability
- BUT...
- Positive predictive values dependent on prevalence & expertise
- Variable sensitivity & specificity
- No virus isolate for strain typing & study

## Influenza:  Laboratory Diagnosis

- _Molecular Diagnosis – PCR_
- Rapid results (hours) with “real-time” pcr
- No need for viable virus...but no virus isolate
- CDC protocols available to State Labs
- BUT...
- Not widely used
- Limited to laboratories with pcr capability
- Expertise & expensive equipment required
- Role in routine diagnosis & surveillance unclear
- Have same predictive value issues as other tests

## Influenza:  Rapid Laboratory Diagnosis

- _Antigen Detection – Rapid Tests_
- Rapid (10-40 minutes)
- Minimal expertise required
- Non-waived  or waived complexity
- No need for viable virus...but no virus isolate
- BUT...
- Positive predictive values dependent on prevalence
- Variable sensitivity & specificity
- No isolate for strain typing & study
**Widely available**
**Amenable to point - of - care testing; results available for patient management decisions**

## Influenza: Rapid Tests

| Test | CLIA Status | Antigen Detected |
| --- | --- | --- |
| Directigen Flu A | Non-waived | A |
| Directigen Flu A & B | Non-waived | A & B |
| Flu OIA | Non-waived | A / B |
| Flu OIA A/B | Non-waived | A & B |
| NOW Flu A and NOW Flu B | Waived/Non-waived | A & B |
| NOW Influenza A & B | Non-waived | A & B |
| QuickVue Influenza | Waived | A / B |
| QuickVue Influenza A + B | Waived | A & B |
| SAS Influenza A and Influenza B | Non-Waived | A & B |
| Clearview Flu A/B | Non-Waived | A & B |
| Xpect Flu A & B | Non-waived | A & B |
| ZstatFlu | Waived | A / B |

## Rapid Tests:  Specimens & Storage

| Test | Specimen Type | Specimen Storage |
| --- | --- | --- |
| Directigen Flu A | NP wash/asp/sw,Th Sw | 2-8C/72 hr. |
| Directigen Flu A+B | NP wash/asp/sw, Nasal wash, Th Sw, BAL | 2-8C/72 hr. |
| Flu OIA | Nasal asp, NP Sw, Th Sw, Sputum | 2-8C/24 hr. |
| Flu OIA A/B | Nasal asp, NP Sw, Th Sw, Sputum  | 2-8C/24 hr. |
| NOW Flu A & NOW Flu B | Nasal wash, NP Sw | 2-8C/24 hr. (elute swabs) |
| BinaxNOW Influenza A & B | Nasal wash/asp, NP Sw | 2-8C/24 hr. (elute swabs) |
| QuickVue Influenza | Nasal Sw, wash, asp | 2-8 C/8 hr or 15-30C/8 hr.  |
| QuickVue Influenza A + B | Nasal Sw, wash, asp | 2-8 C/8 hr or 15-30C/8 hr.  |
| SAS Influenza A & SAS Influenza B | NP wash, asp | 4-8C/24 hr.  |
| Clearview Flu A/B | Nasal wash, Sw, Th Sw | 2-8C/72 hr (in transport medium) or -20C/6 mo. |
| Xpect Flu A & B | Nasal wash, Sw, Th Sw | 2-8C/72 hr (in transport medium) or -20C/6 mo. |
| ZstatFlu | Th Sw | 0-40C/24 hr. |

- 35

## Rapid Tests: Performance Characteristics(Per Manufacturer, without discrepant analysis)

| Test | Sensitivity | Specificity |
| --- | --- | --- |
| Directigen Flu A | 91% (A) | 95% (A) |
| Directigen Flu A+B | 86%(A); 81%(B)  | 91%(A); 99.5%(B)  |
| FLU OIA | 62-88% (A/B) | 52-80% (A/B) |
| FLU OIA A/B | 62-88% (A/B)  | 52-80% (A/B)  |
| NOW Flu A and Flu B | 78-82%(A);58-71%(B) | 92-94%(A); 97%(B)  |
| NOW Influenza A & B | 100%(A); 92-100%(B)
(Compared to NOW Flu A & Flu B tests) | 93-99%(A); 94-99%(B) 
(Compared to NOW Flu A & Flu B tests) |
| QuickVue Influenza | 73-81% (A/B) | 96-99% (A/B) |
| QuickVue Influenza A + B | 72-77%(A) 73-82%(B) | 96-99%(A); 96-99%(B) |
| SAS Influenza A & Influenza B | 76%(A) 91%(B) | 98%(A); 100%(B) |
| Clearview Flu A/B | 92%(A); 98%(B)  | 100%(A); 100%(B)  |
| Xpect Flu A & B | 92%(A); 98%(B)  | 100%(A); 100%(B)  |
| ZstatFlu | 58-65% (A/B)  | 98-100% (A/B)  |

- 36

## Test Performance Characteristics:  Definitions

- Sensitivity:  The probability of a positive test result given
- the presence of disease
- Specificity:  The probability of a negative test result
- given the absence of disease.
- Predictive Value:  The probability of the presence or
- absence of disease given the results of a test
- PVP is the probability of disease in a patient with a positive test result.
- PVN is the probability of not having disease when the test result is negative.
- Determined by the sensitivity and specificity of the test, ***and*** the prevalence rate of disease in the population being tested.
- Prevalence Rate:  Number of cases of illness existing at
- a given time divided by the population at risk

## Test Performance Characteristics:  Formulae

- Disease

- Test

- Sensitivity = TP/TP+FN
- Specificity = TN/TN+FP

- PVP = TP/TP+FP
- PVN =TN/TN+FN

## Impact of Prevalence on Predictive ValuesUsing A Hypothetical Influenza Test with 95.0% Sensitivity, 96.0% Specificity 

-   	 _Prevalence = 20.0%_
- 				Disease
**Test**
-    PVP = 380/444 = 85.6%
-    PVN = 1536/1556 = 98.7%

- _Prevalence = 1.0%_ 		         Disease
**Test**
-    PVP = 19/99 = 19.2%
-    PVN = 1900/1901 = 99.9%

## Predictive Value PositiveDependence on Sensitivity, Specificity & Prevalence

## Predictive Values using Laboratory Test Data as the Prevalence Indicator, 2000-2001

- PPV & NPV of Hypothetical
- Test with 78% Sensitivity &
- 91% Specificity, Using
- Wisconsin Virology
- Laboratory Positivity Rates
- as a Prevalence Indicator
- Qualitative Estimate of
- Prevalence Based on
- Number of Specimens
- Reported Positive for
- Influenza by Wisconsin
- Virology Laboratories

**Low Prevalence**

**Low Prevalence**

**High Prevalence**

## Recommendations to Improve Accuracy of Rapid Influenza Test Results

- Educate clinicians on predictive values & limitations of test results
- Culture confirm early-season and late-season positives (when prevalence is low)
- Culture confirm peak-season negatives (when prevalence is high)
- Recognize the value of negative results (negative predictive value)
- Use prevalence indicators to decide:
- When to test
- When to qualify result
- When to confirm results
- Prevalence Indicators
- Laboratory data
- “Influenza-like illness” (ILI) data

## Recommendations for this Season

- Develop recommendations for test utilization, based on influenza prevalence and need for test results for patient management decisions.
- Consider use of clinical diagnosis at peak season
- Develop laboratory contingency plans
- Identify alternate test kits
- Identify alternate sources of critical supplies
- Develop plan to prioritize testing
- Develop surge capacity protocol for staffing.
- Develop collaborative plans with other laboratories.

## More Recommendations for this Season

- Develop “knowledge pool” via a communications network with other laboratories and state laboratory.
- Consider enhanced biosafety for *select* specimens.
- Participate in state and national laboratory-based surveillance efforts.
- Develop protocols to identify & submit specimens from suspected cases of avian influenza

## Influenza Surveillance Systems

## U.S. Influenza Surveillance

**CDC**

**Laboratories**

**Sentinel Providers**

**State and Territorial **
**Epidemiologists**

**Vital Statistics**
**Registrars**

**Other**

**Public Health**
**Officials**

**Physicians**

**Media**

**Public**

**Health Departments**

**Pediatric **
**Hospitalization**

**Pediatric Mortality**

- CDC

## Objectives of Influenza Surveillance

- Determine _when_ influenza viruses are circulating
- Determine _where _influenza viruses are circulating
- Determine _how much_ influenza activity is occurring (intensity and impact)
- Identify the _types & strains_ of circulating influenza viruses
- Detect unusual events
- Infection by unusual viruses
- Unusual syndromes caused by influenza viruses
- Unusually large/severe outbreaks of influenza
- Increased mortality

## Laboratory Component of CDC National Influenza DataWeekly Updates at http://www.cdc.gov/flu/weekly.htm

## State-Level Laboratory-Based Influenza Surveillance

## Statewide Laboratory-Based Influenza Surveillance:  The Wisconsin Model

**State Lab**

**Sentinel Submitters**

**Virology Laboratories**

**Rapid Test Sites**

**Public Health**
**Epidemiologists**

**Clinicians**

**Laboratories**

- CDC

## Role of the Wisconsin State Laboratory of Hygiene in Wisconsin’s Laboratory-Based Surveillance

- Coordinate Laboratory-Based Surveillance in collaboration with State Influenza Coordinator
- Act as liaison between clinical laboratories & public health
- Perform initial culture of diagnostic specimens (limited number), specimens in support of surveillance, outbreak investigations, & enhanced surveillance.
- Perform virus typing, subtyping & referral to CDC.
- Provide sites with no-cost specimen collection supplies, shipping supplies & transport.
- Analyze & disseminate laboratory data; distribute information from CDC, state epidemiologists.
- Request additional isolates & samples in support of special investigations.

## Advantages of Wisconsin’s Three-tiered Laboratory-based Surveillance System

- Provides complementary data (verification).
- Allows focal enhanced surveillance (e.g., reassortant viruses).
- Allows public health lab to focus on public health testing
- Provides information to enhance the quality and understanding of rapid test data
- Improves understanding of the complementary roles of clinical and public health laboratories
- Provides infrastructure to apply to other agents of public health concern

## Tier One of Wisconsin’s Laboratory-Based Surveillance: Sentinel Submitters 

- Role
- Provide limited number of patient samples for testing at State Laboratory
- Submit specimens from _selected_ patients with recent onsets and appropriate symptoms with complete patient information
- Respond to requests for additional samples in special investigations
- Benefits to System
- Provide source of specimens that is independent of changing practices/guidelines.
- Can select specimens to optimize viral recovery.
- Provide a corps of clinicians for enhanced surveillance needs.

## Tier Two of Wisconsin’s Laboratory-Based Surveillance:  Virology Laboratories

- Role
- Provide weekly reports of number & types of specimens tested, number & types of viruses found
- Provide selected viral isolates for subtyping
- Benefits to System
- Provide representative sampling of state isolates.
- Provide additional capacity for testing in state (beyond public health laboratory).
- Provide capability to identify other viruses that may be circulating.

## Tier Three of Wisconsin’s Laboratory-Based Surveillance:  Rapid Test Sites

- Role
- Provide weekly reports of number of specimens tested & positive for influenza (also RSV & rotavirus)
- Provide first influenza positive specimens for culture-confirmation
- Benefits to System
- Provide “pre-screening” of large numbers of specimens
- Improve “sensitivity” of surveillance system.
- Provide _large numbers_ of test sites & samples for enhanced surveillance needs.
- Enhance the quality & understanding of rapid test results.

## Example of Surveillance Data Generated by Wisconsin Virology Laboratories & Rapid Test Sites, 2002/3 – 2003/4

## Steps to Develop State Surveillance System with Rapid Test Sites

- Identify rapid test sites
- Provide information on optimal usage of tests
- Send them surveillance “kit”
- Encourage culture confirmation of first influenza positives, others as needed
- Fee-exempt culture
- Fee-exempt transport supplies & system
- Gather their weekly testing data
- Provide weekly surveillance reports to them

## Keys to Developing a Statewide Surveillance System

- Minimize cost and effort for clinical laboratories.
- Designate laboratory outreach team at State Laboratory.
- Develop laboratory staff contacts.
- Keep it simple.
- Take the leap – it doesn’t have to be perfect immediately, but ... someone has to take the initiative..
- Create the opportunity from current surveillance needs, bioterrorism preparedness efforts, and existing laboratory networks.
- Consolidate/integrate laboratory networks.

## Meeting the Ultimate Challenge this Influenza Season

- We can fulfill the laboratory’s role in meeting this season’s clinical and public health needs through:
- Enhanced Surveillance
- Improved Communication
- Increased Networking

## Enhanced Surveillance:  Potential Partners

- Enhanced Surveillance:  Potential Partners

**State Lab**

**Sentinel Submitters**

**Virology Laboratories**

**Rapid Test Sites**

**Public Health**
**Epidemiologists**

**Clinicians**

**Laboratories**

- CDC

## A Two-Tiered Plan for Laboratory Communications

- Preparedness
- “Just In Case”

- Response
- “Just in Time”

## Laboratory Network Development

- Present?

- Future

- Past