12/2/2004 1

Modern Methods for Influenza Detection and Sub-typing

Ronald Cheshier

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Introduction Training provided by the CDC, Association of

Public Health Laboratories (APHL), and the National Laboratory Training Network (NLTN) October 25, 2004 to October 29, 2004 at the Georgia State Laboratory

This course provided up to date information on Influenza epidemiology, surveillance, and laboratory testing protocols. One of the primary goals was to encourage the State Laboratories to develop the tools necessary for rapid response to a Pandemic Influenza A scenario.

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Influenza Virus review

Family Orthomyxoviridae: three types

Influenza A Influenza B Influenza C (not considered of

critical importance) Segmented (8) ssRNA genome

with lipid envelop

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Influenza A

Further classified by Hemagglutinin (H) and Neuraminidase (N) sub-types

Current circulating strains are H1N1 and H3N2

Human subtypes include H1N1, H3N2, H1N2, and H2N2

Avian subtypes include H1 to H15 and N1 to N9

Bird human H5N1, H9N2, H7N7, H7N2, H7N3

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Influenza B

Produces less serious disease than does Influenza type A

Not categorized as by H or N type as Influenza A is

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Influenza C

First isolated in 1949 Not known to be responsible for

epidemics

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Anticipated circulating strains for 2004/05

Northern hemisphere A/Fujian/411/2002(H3N2)-like A/New Caledonia/20/99 (H1N1)-like B/Shanghai/361/2002-like

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Influenza as a public health threat Influenza Viruses are the respiratory

viruses of greatest public health importance, particularly Influenza A

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Epidemiology, Prevention, and Control of Influenza; esp. Influenza A

Why is Influenza A such a public health threat? antigen drift (variation within the HN sub-type) or antigenic

shift (variation between different HN sub-types) makes large portions of the population immunologically naïve on a regular basis

Influenza epidemics can be characterized as inter-pandemic or pandemic

Annual average US winter epidemics affect 5% to 20% of the population with approximately 200,000 influenza-related hospitalizations during the 1990’s and 36,000 influenza related deaths.

At one time it was thought that new HN variants were due to re-assortment of genetic material from an avian strain and a human strain in a third animal, like a pig. Modern evidence suggests that humans may act as this mixing vessel

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For example

A pig or person is infected with an avian influenza like an H5N4 at the same time the pig or person is infected with a human strain like H1N1;

During the infections the two genomes re-assort to an H5N1 (with the avian H and the human N)

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Global and US surveillance

Avian influenza: H5N1: HIGHLY PATHOGENIC

Present in Asia since 1996 Extent/distribution not firmly

established Threat level is high In 1997 there were 18 confirmed

cases of H5N1 with 6 deaths Other avian strains being watched

include H7N7 and H9N2

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Pandemic Influenza

Recipe for a human pandemic Emergence of a novel sub-type of

influenza to which the population is immunologically naïve

Replication in humans disease Efficient human-to-human

transmission Note: H5N1 has meet all criteria

except the third one.

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Pandemic planning

An influenza pandemic will be unlike other public health emergencies or common disasters.

Inevitable Will arrive with very little warning Locally explosive epidemics Widespread, not focused like a bio-

terrorism event Will put an extraordinary strain on human

and material resources Effect will be relatively prolonged –weeks

to months

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Laboratory issues

Laboratory safety Tissue culture techniques Rapid test kits HA/HI sub-typing Immuno-fluorescent testing Real time PCR analysis

Molecular typing and sub-typing

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Laboratory/Epidemiology Topics of Discussion

Reviewing current Influenza surveillance testing algorithm

Brief comments on alternative Influenza surveillance algorithms and the ability of this laboratory to support them.

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Current testing algorithm

Inoculation of specimens into cell culture; one diploid, one Hep-2, one Viromed Rhmk and two Diagnostic Hybrid Rhmk

In the absence of CPE, “blind” Hemadsorption (HAD) at days 7 and 14.

In the absence of CPE “blind” passage of Hep2 with “blind” FA for RSV at day 14 for all patients ≤5 years old

Identification of Influenza isolates: Immuno-fluorescent testing to identify

type (A or B) followed by Hemagglutination Inhibition (HI) testing to identify sub-type

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Alternatives to be considered

The use of shell vials for more rapid isolation and identification of Influenza Studies by Wisconsin and Iowa suggest that MDCK

shell vials are more sensitive for Influenza than Rhmk cell culture

MDCK shell vials inoculated, incubated, and “blind” stained at day 5 for Influenza A and B with supernatant saved and used for HI testing (if necessary).

Things to consider:• Cost: purchase shell vials commercially with goal

of preparing our own shell vials once the cell culture preparation method has been established.

• The CDC provides the FA reagents as part of the WHO/CDC Influenza Kits received each year. In addition, commercial sources are available.

• Time and labor requirements; Not an issue once Arbovirus season is over.

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Alternatives to be considered

The use of Immuno-fluorescence (IFA) for Influenza A sub-typing The CDC/WHO kit provides staining

reagents for H1N1 and H3N2 Cost: We would have to purchase

additional anti-mouse IgG conjugate commercially.

This procedure would decrease turn-around-time

Has the potential for allowing us to test for 2 sub-types at once by using different conjugates, I.e. FITC and Rhodamine

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Alternatives to be considered

Use of Rapid Test kits Things to consider

Cost: These kits are very expensive and have relatively short shelf-lives

Poor positive predictive values in the absence of an outbreak (high sensitivity but low specificity)

These kits are probably more effective for use at a primary care setting during influenza season

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Alternatives to be considered PCR: The CDC has provided the sequence data for the

primers and probes for Influenza A (group) and Influenza B (group). In addition, it has provided the data for H1, H3, and H5 (avian considered as possible candidate for next pandemic). Things to consider

Cost: While the cost of primers is probably manageable, probes are very expensive.

There will be a lag time as we will have to obtain all the probes and primers and do validation studies. This includes validation for H5; Note, we do not have any controls for that agent. The CDC has indicated they will provide a Proficiency set sometime next year.

The CDC primer sets are for H1, H3, and H5, not H1N1, H3N2, and H5N1 (no neuraminidases). The CDC is not overly concerned about this because it is the “H” (Hemagglutinin) that is related to pathogenicity but, if we choose to report based on PCR we will only be able to report on the Hemagglutinin result.

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Suggested algorithms It is not the intent of the CDC to replace culture with

PCR but rather to allow the States to have PCR testing available as a surveillance/rapid diagnostic tool

Isolation will still be needed for vaccine related surveillance and production efforts

Consider the purpose of this surveillance effort

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Next Steps

Laboratory Management and the State Epidemiology will have to meet to more carefully review and discuss the options available to us in terms of surveillance enhancement.

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Items to consider

It has been 20+ years since our Influenza algorithm has been changed

A number of exciting new methods and tools are available to us including The use of shell vials Immuno-fluorescent sub-typing including

multiplexing PCR

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Final algorithm

Should allow for the most rapid response possible

Cost considerations Sensitivity and specificity of the

tests Validation studies, not just to

satisfy CLIA requirements but also to ensure that the tests being performed are providing accurate information.

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Presented By:

Ron Cheshier, Virology Section Manager

Arizona Department of Health Services - Bureau of State Laboratory Services

(602) 542-6134 or cheshir@azdhs.gov