________________________________________________________________________

List of Contents

1. Influenza Epidemiology and

Surveillance………................................................4

2. Collection, Storage and Shipping Clinical Specimens ….……..................

3. Overview of Influenza
Diagnosis ................................................................... ..7

4. 2. Non Molecular diagnostic methods.................................................... 21

5. 4-2. Molecular/PCR based diagnostic methods................................................

22

6. 5

7. 7. References .................................................. 26

Annexure:
Annex 1. Personal Protective Equipment
Annex 2. Hand Hygiene Technique
Annex 3. Lab Safety and Disinfection.................................................................. 28
Annex 4. Viral transport media (VTM)
Annex 5. Forms to accompany specimens......................................................... 30
Annex 6. WHO document Guidance on regulations for the Transport of Infectious
Substances
...........................................................................................
Annex 7. Content of the WHO AI Investigation Kit............................................
Annex 8. Field data sheets...................................................................................
Annex 9. …………………………………………………………………………………

INFLUENZA EPIDEMIOLOGY AND SURVEILLANCE

Influenza viruses belong to the Orthomyxoviridae family and are divided into types A, B,
and C. Influenza types A and B are responsible for epidemics of respiratory illness that
occur almost every winter in temperate climates and are often associated with
increased rates of hospitalization and death. Influenza type C infection is a milder
infection that does not cause epidemics and does not have the severe public health
impact of influenza A and B.

Influenza type A viruses are divided into

subtypes based on surface proteins called
hemagglutinin (HA) and neuraminidase (NA).
To date, 15 HA subtypes and 9 NA subtypes
have been identified. However, in the 20th
century, influenza viruses bearing only 3 HA
and 2 NA subtypes [i.e., influenza A (H1N1),
A(H1N2), A(H2N2), and A(H3N2) viruses] have
circulated extensively in humans. Influenza A
viruses can infect a number of animal species
Electron micrograph of influenza type A virus.
including pigs, birds, horses, seals, and
whales.
 ANTIGENIC CHANGE

The hallmark of human influenza viruses

is their ability to undergo antigenic
change, which occurs in two ways:
antigenic drift and antigenic shift.

Antigenic drift is a process of gradual,

relatively continuous change in the virus
HA and NA proteins. Drift results from the
accumulation of point mutations in the
genes during viral replication. Both
influenza A and B viruses undergo
antigenic drift leading to new virus
strains. The emergence of new strains
necessitates the frequent updating of Influenza is a negative-strand RNA virus with a
segmented genome. Influenza A and B viruses have 8
influenza vaccine virus strains. genes that code for 10 proteins, including the surface
proteins hemagglutinin and neuramimidase

In addition to antigenic drift, influenza

type A viruses can undergo a more
dramatic and abrupt type of antigenic change called an antigenic shift. By definition,
shift has occurred when an influenza A virus bearing either a “novel” HA protein or a
combination of novel HA and NA proteins (i.e., HA/NA that have not been circulating
among humans in recent years) emerges among humans. While antigenic drift occurs
continuously, antigenic shift occurs infrequently and unpredictably. Since antigenic shift
results in the emergence of a new influenza virus, a large proportion, or even all, of the
world’s population has no antibody against the new virus. If the new virus is capable of
causing illness in humans and sustained person-to-person transmission, a pandemic
may occur. There are at least three possible mechanisms by which antigenic shift can
occur: 1) a virus bearing a new HA/NA can arise through genetic reassortment between
non-human and human influenza viruses; 2) an influenza virus from another species
(e.g., birds or pigs) can infect a human directly without undergoing genetic
reassortment; or 3) a non-human virus may be passed from one species (e.g. birds)
through an intermediate animal host, such as a pig, to humans.

INFLUENZA DISEASES

Influenza is an acute respiratory disease caused by influenza type A or B viruses. The

incubation period ranges from 1-4 days. Peak virus shedding usually occurs from 1 day
before onset of symptoms to 3 days after in adults but may last longer in young children.
Typical features of influenza include abrupt onset of fever and respiratory symptoms
such as cough (usually nonproductive), sore throat, and coryza, as well as systemic
symptoms such as headache, muscle aches, and fatigue. The clinical severity of
infection can range from asymptomatic infection to primary viral pneumonia and death.
Acute illness generally lasts about 1 week, although malaise and cough may continue
for 2 weeks or longer. Common complications of influenza infection include secondary
bacterial pneumonia and exacerbation of underlying chronic health conditions and, in
children, otitis media. Uncommon complications include myositis, myocarditis, toxic-
shock syndrome, and Reye’s syndrome (generally associated with the use of aspirin in
children and adolescents with influenza-like illness).

EPIDEMIOLOGY OF INFLUENZA

In temperate climate regions, epidemics of influenza occur nearly annually, although the
rates and severity of influenza illness can vary substantially from year to year depending
on several factors including the (sub)types and strains of circulating viruses, the
incidence of infections, and levels of protective antibody in the population. Although
influenza epidemics may also occur regularly in subtropical and tropical regions,
disease patterns in these regions have been
less well established.
Seasonality of influenza virus isolation
United States, cumulative number of isolates and Timing and seasonality of regional
percent of specimens testing positive for influenza,
1998 – 2001 influenza activity

The timing of influenza activity around the

7000

6000
world varies depending on the climate of the
25

region. In temperate climates, the onset and

Number of isolates

20

5000
% positive

4000
peak of influenza activity may vary
15

substantially from one influenza season to

3000
10

2000

1000 the next but generally begins to increase in

5

0
the late fall. In the Northern Hemisphere’s
0
1

1
1

temperate regions, influenza viruses

Week

isolates %positive

frequently are isolated during a several

Hong Kong, cumulative
Seasonality of Influenzanumber of isolates
In United States, 1998 and - 2001 month period in the fall, winter, and spring.
percent of specimens testing positive for influenza,
1998 – 2001*
Periods of peak activity typically occur
sometime between December and March
and last for 6-8 weeks. In temperate regions
3500 of the Southern Hemisphere, influenza
45

activity typically peaks during May through

40
3000
Number of isolates

35

August. Although temperate regions of the

2500
% positive

30

2000 25

1500 world experience a seasonal peak in

20

influenza activity, influenza viruses can be

15
1000
10

isolated year-round.
500
5

0 0
JAN FEB MAR APR MAY JUN
Month
JUL AUG SEPT OCT
The timing of seasonal peaks in influenza
NOV DEC

isolates %positive
activity in tropical and subtropical countries
varies by region, and multiple peaks of
Brazil, cumulative number of isolates, 1997 – 2001**
activity during the same year have been
seen in some areas. This variability in
60
influenza seasonal peaks among countries
in tropical and subtropical regions illustrates
Number of isolates

50

40
the importance of country-specific and
regional epidemiologic and virologic data.
30

20

10

0
JAN FEB MAR APR MAY JUNE JULY AUG SEP OCT NOV DEC
Month

isolates

*Data from Hong Kong Department of Health website,

Brazil 1997 -2001
http://www.info.gov.hk/dh/diseases/influenza.htm.
** Data from FluNet
The variability in the timing of influenza activity also has implications for persons
traveling to another part of the world and for persons traveling in large international
groups. For example, outbreaks of influenza have occurred on cruise ships during the
summer months in temperate climates, presumably after influenza was introduced by
infected persons from an area where influenza was in season. International or large
group travel may result in exposure to influenza virus outside of an expected time
period. Thus persons counseling travelers about influenza prevention should consider
the travel mode and destination when considering timing of vaccination and the
advisability of using influenza antiviral medications for treatment or prophylaxis.

Regardless of the overall seasonality of influenza in an area, influenza viruses can

circulate at low levels during any time of the year and can cause both isolated cases of
influenza in individuals as well as outbreaks during “off season” periods.
Impact of influenza

Increases in the circulation of influenza viruses have been associated with elevations in
acute respiratory illnesses, physician visits, hospitalizations, and deaths. In general,
rates of primary influenza illness are highest among school-aged children, exceeding
30% in some years, and lower among adults. During non pandemic years, influenza
infection rates among adults generally range from 1% to 15%.

Although the highest rates of illness occur among school-aged children, the highest
rates of hospitalizations from influenza-related causes occur among children <2 years of
age, among persons of any age with certain chronic medical conditions (including
chronic heart disease, lung disease such as asthma, diabetes, renal failure, or immune
compromising conditions), and among those ≥65 years of age. Among persons of the
same age group, rates of hospitalizations are higher among those with chronic medical
conditions than healthy persons of the same age group. At both extremes of the age
spectrum, however, rates of hospitalizations are elevated, even among those without
chronic medical conditions.

In the current virologic era, hospitalization rates have been higher in seasons in which
influenza A (H3N2) viruses have predominated than in years in which influenza B or
influenza A (H1N1) viruses have predominated. In a study of U.S. hospital discharge
data from 1969-1995, the average estimated rates of influenza-related hospitalization
for persons >65 years were 228/100,000 during A(H3N2)-predominant years and
125/100,000 during A(H1N1)- or B-predominant years. Among persons <65 years, the
respective hospitalization rates were 42/100,000 and 20/100,000.

Influenza-related deaths can result from pneumonia, exacerbations of existing

cardiopulmonary conditions or exacerbations of other chronic conditions.

Pregnant women also appear to be at increased risk of complications from influenza.

An increased risk of influenza-related deaths among pregnant women first was
observed during the 1918-1919 and 1957-1958 pandemics.
Spread of influenza

Influenza is spread from infectious persons to susceptible persons by large droplets and
aerosols containing influenza viruses that are produced by coughing, sneezing or
talking. Influenza may also be spread less commonly by contaminated fomites or by
direct touching. Children are important in the spread of influenza within communities
and within households. Influenza outbreaks among school children can herald the start
of influenza activity in a community. During some community outbreaks, illness rates
among school-aged children have been shown to rise and decline earlier in the
outbreak than rates among adults. Households with young children have higher
influenza illness rates overall and secondary attack rates are higher in households
where the index case is a young child.

Pandemic influenza

In contrast to the annual seasonal epidemics of influenza seen in some global regions,
pandemics of influenza have occurred infrequently and at irregular intervals. Three
pandemics occurred in the 20th Century. During 1918-19, the “Spanish Flu” (H1N1)
pandemic led to >500,000 deaths in the United States and >20 million deaths
worldwide. Nearly half of the deaths were among persons 20-40 years of age and
case-fatality rates of 30% were reported among pregnant women. In the United States,
the 1957-58 “Asian Flu” (H2N2) pandemic was associated with an estimated 70,000
deaths while the 1968-69 “Hong Kong Flu” (H3N2) pandemic was associated with an
estimated 34,000 deaths. In all three pandemics, persons <65 years accounted for a
larger proportion of deaths than is usually seen in interpandemic influenza seasons. In
all age groups, influenza infection rates generally are higher during pandemics than
annual epidemics. School-aged children also play an important role in the spread of
pandemic influenza in the community.

WORLD HEALTH ORGANIZATION INFLUENZA PROGRAM

An international network for

WHO Influenza Surveillance Network –
influenza surveillance first was
National Influenza Centers
envisioned in 1947. In 1948, the
World Health Organization
(WHO), upon its founding,
became responsible for
administration of the system.
Currently, there are 112 National
Influenza Laboratories in 83
countries that participate in the
WHO influenza surveillance
system.
The purpose of the network is to detect the emergence of new influenza virus drift
variants of known circulating types and subtypes as well as the emergence of novel
influenza A subtypes in human populations. Information collected through this system
is used to develop annual vaccine strain selection recommendations.

Four WHO Collaborating Centers for Reference and Research on Influenza in Atlanta,
London, Melbourne, and Tokyo serve as influenza reference laboratories and conduct
detailed molecular and antigenic analysis of a subset of viruses from around the world.
The WHO Collaborating Center at the Centers for Disease Control and Prevention
(CDC) in Atlanta develops and produces the antisera included in the WHO reagent kits.
These kits are sent to all WHO national laboratories to assist them in the identification of
circulating influenza A subtypes. If a national laboratory is not able to subtype an
influenza virus using available reagents, WHO recommends that the isolate be shipped
to one or more collaborating centers for further analysis.

DISEASE SURVEILLANCE – MORBIDITY REPORTING

Morbidity surveillance is used to detect and monitor patterns of illness related to circulation
of influenza viruses. Such data are particularly useful for assessing the health impact that
influenza is having in the community. One of the most common outcomes monitored in
influenza morbidity surveillance systems is influenza-like illness (ILI), but other outcomes
such as acute respiratory illness (ARI) or hospitalizations can be monitored. A variety of
surveillance methods and data sources can be used. The choice of method should take
several considerations into account, including the availability of existing data sources, the
healthcare structure, the ease of collecting and reporting the data, the potential for
sustainable reporting, and the potential for collecting data that are reasonably
representative of the groups of interest. Below are examples of systems currently in use
worldwide.

Sentinel outpatient surveillance

Information on visits for influenza-like illness or acute respiratory infections can be

obtained from several different “sentinel” healthcare sites such as physician offices,
outpatient or hospital associated outpatient clinics, university student health clinics, or
emergency departments. Persons reporting the data can be physicians, physician
assistants, nurse practitioners, or other health care staff.

Influenza activity level assessment

The WHO, EISS, and U.S. surveillance systems each report estimated levels of overall
influenza activity. In the WHO and EISS systems, estimated levels of activity are
reported for countries or regions of a country. While each of these systems use
standard definitions to report activity levels, it is important to understand that the
surveillance methods used to make the activity level determination may vary from
country to country and state to state. Currently, these assessments are not done in a
standardized way and can be made subjectively. Nonetheless, they do provide a level
of local interpretation of influenza activity and surveillance data that may be lacking
otherwise. A more unified approach to assessing local levels of activity would provide
reports that are more directly comparable.

The activity levels in the WHO system are reported as:

No activity - no influenza viral isolates or clinical signs of influenza activity

Sporadic - an isolated case of ILI or laboratory/culture confirmed cases in a
limited area
Local outbreak - ILI activity above baseline values with laboratory confirmed
cases in a limited area
Regional activity - outbreaks of ILI or laboratory confirmed influenza in one or
more regions with a population comprising less than 50% of the country’s total
population
Widespread activity - outbreaks of ILI or laboratory confirmed influenza in one
or more regions with a population comprising 50% or more of the country’s
population

Participating countries can report their influenza activity level through WHO’s internet
reporting system, FluNet.

The EISS system activity levels are similar to those used by WHO. However, EISS
incorporates a second variable to describe the intensity of influenza activity in addition
to the geographic distribution of influenza viruses. The intensity of influenza activity is
described as low, medium, high, or very high.

Hospital-based surveillance

Hospital-based surveillance for influenza can be useful in tracking levels of severe

illness related to influenza. It is also helpful to collect viruses from hospitalized patients
since they can differ from those collected from outpatients, for example, in the
proportion of viruses from one subtype.

Collection of hospital discharge diagnosis is useful in documenting the impact of

influenza. However, the availability of such data usually is delayed, and therefore,
analysis of discharge data may be more appropriate for studies rather than weekly
surveillance. As an alternative, some surveillance systems have monitored hospital
admission diagnosis or chief complaint data, which can be available sooner than
discharge data. Since admission data often may not be coded or available in
computerized files, analysis may be time consuming. Both admission data and
discharge data are prone to coding biases and errors.

Other sources of data

Other events that may reflect levels of influenza activity include school or workplace
absenteeism, sales of over-the-counter or prescription medicines used to treat
influenza, or increases in ambulance calls. Each of these systems has its own
strengths and weaknesses. In particular, outcomes such as absenteeism are highly
nonspecific, and therefore, trends in such outcomes should be interpreted with caution.
Nonetheless, these outcomes can complement other surveillance methods if the data is
readily available.

Local influenza outbreak control and case management

Local public health staff should make information on currently circulating respiratory
viruses available to health care providers to aid in their diagnostic and treatment
decisions including avoidance of antibiotics if inappropriate and use of antiviral agents if
available. Information that influenza viruses have begun to circulate also can be used
to prompt members of target groups to receive vaccine. Since the transmission of
influenza within hospitals can mimic community activity, surveillance findings can help
institutions to anticipate nosocomial influenza outbreaks, which can be devastating in
some situations such as bone marrow transplant units, and to focus hospital infection
control efforts.

Influenza vaccination programs

Since influenza vaccination is most effective when recipients are vaccinated just before
the start of influenza activity, knowledge of the seasonal pattern of influenza viruses and
disease is essential for the effective implementation of influenza disease prevention
programs.

Scientific studies

The local and regional epidemiology of influenza, including the seasonality, types of
circulating viruses, and disease impact, is essential background information for many
scientific studies of influenza. Surveillance provides most of such information.

Vaccine strain selection

Both viral and disease surveillance provide important information for selecting influenza
vaccine strains. Viral surveillance is essential for identifying new influenza virus variants
to replace vaccine strains. A vaccine strain is replaced when 1) the antigenicity of newly
identified variants is significantly different from the current vaccine strains; 2)
populations vaccinated with the current vaccine demonstrate a reduced serologic
response to the new variant; 3) there are common genetic changes among the new
variants suggesting the emergence of a new lineage; and 4) the variant appears to be
associated with disease.

Situation in Pakistan
Developing country like Pakistan has a lot other major health problems and scarcity of
resources have made this problem a burning issue like else where in the world.
Because of its high potential for traveling across the borders it has placed Pakistan in
situation where we need to look into our routine as well as outbreak surveillance for its
timely preventive and control measures.

There is no scientific based data available that shows true picture of the disease burden
caused by the Influenza in Pakistan, or if available it is underutilized and not used in key
decision making for the resource allocation.

National Influenza Lab Based Surveillance Project (NILSP) although established in

2002, became functional in 2007. The project is being implemented by the Public Health
Laboratories Division of the National Institute of Health. Main goal of this project
(NILSP) is to establish & maintain a country wide influenza surveillance system based
on epi and lab components. Its three main objectives are:

1. To monitor trends of influenza-like illness (ILI)

2. To monitor predominant circulating influenza strain, and finally
3. To detect any novel influenza variant for further R & D

There are two main components of the project, Epi and the Lab, both being equally very
important. Case Identification, Gathering Demographic factors and case follow up are
done by the Epi component and Sample processing, analysis, typing & subtyping by its
Lab Component. The data is stored in databank and analysed later for decision making
and coordination later on.

Total of five laboratories have been envisaged to be set up for influenza surveillance,
diagnostics and isolation of influenza viral isolates and sharing of influenza information
with the stakeholders. These sentinel sites will collect data and samples and initially
sent these to NIH for processing. Once their labs will be set up they can process same
samples at their centers.

NISLP Accomplishments

In order to achieve NILSP goal and objectives it is envisaged to establish one sentinel
site in each province in major cities, like Karachi, Lahore, Quetta, Peshawar and
Federal Capital, Islamabad. Sentinel sites at these provinces have been identified and
evaluated for the establishment of the laboratory.

Two sentinel sites at Federal Government Services Hospital (FGSH) Islamabad and
King Edward Medical University (KEMU), Lahore are operational. The nominated
sentinel site physicians at these sites have been trained on data collection and
sampling. These physicians screen ILI and SARI cases as per case definitions.
Specimens are collected and transported to NIH for processing. Hayatabad Medical
Complex (HMC), Peshawar and Civil Hospital, Karachi will be soon operational.
Evaluation of the remaining provincial sentinel site (Balochistan), i.e at Bolan Medical
Complex (BMC), Quetta is in streamline.

Procurement of laboratory equipment/ supplies has been partially completed for the
central (NIH) as well as provincial laboratories. These equipment/ supplies will be
moved to these nominated sentinel sites when their bio-safety enhancement is
completed. All necessary sample collection supplies have been sent to high risk districts
of Influenza.

Flu vaccine (Inj. Fluarix) procured for the staff of the NIH and provinces have been
dispatched to the provincial sentinel sites. Anti-virals (Oseltamivir, brand Tamiflu) has
been procured for the same as well.

Number of seasonal Influenza samples received from Federal Government Services

Hospital (FGSH) Islamabad (23 November 2007 to date) is 653 samples. Out of these
samples, 26 (4.1%) influenza isolates are confirmed positive by Real-time PCR/ viral
culture. Number of seasonal Influenza samples received from King Edward Medical
University (KEMU), Lahore; (30 January 2009 to date) is 70 samples. Panel of
representative seasonal influenza samples (nine) have been submitted to Global
Information Surveillance Network (GISN) in April, 2009 and data on virological
information is entered on FluNet subsequently.

TOTAL RESULTS ON IN
LOCATION PROCESSED
SAMPLES CULTURE PROCESS

+VE -VE
FGSH,
653 632 26 608 21
Islamabad
KEMU,
70 53 - 53 17
Lahore
Grand
Total 723 685 24 661 38

Staff officers of NILSP and NIH have actively participated in the international events
regarding Influenza activity. Number of trainings/ scientific workshops have been
organized by the NILSP within country. Total number of sentinel site physicians trained
at the nominated sites is more than forty (41). Rapid response training has been
imparted to public health professionals of high risk districts of Punjab and NWFP. Plan
has been worked out for sentinel site and rapid response trainings at Balochistan as
well as to organize a National seminar on Influenza.
Bio-safety enhancement (BSE) is considered important part of NILSP. BSE at the NIH
(Islamabad), KEMU (Lahore) and HMC (Peshawar) is under process. Layout of BSE at
other provincial labs (Sindh and Balochistan) is being worked out for civil works.
 N u m b e r o f s p e c im e n s p r o c e s s e d f o r in f lu e n z a
20 70

18
60
16

14 50

% of all specimens that were

Number of specimens

12

influenza_positive
40
processed

10
30
8

6 20
4
10
2

0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
W e e k n um be r, 2009
Influe nza _ p o s itive Influ e nza _ ne g a tive % o f a ll s p e c im e ns th a t w e re influ e nza _ p o s itive

2.5
Number of specimenspositive for influenza by subtypes 6
Number of specimens positive for

5
2

ILI activity level

4
1.5
influenza

1
2

0.5
1

0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Week number, 2009

A(H1) A(H3) A(Not subtyped) B(Yamagata lineage)

B(Victoria lineage) Lineage not determined Pandemic A(H1N1) A(H5)
Other subtypes ILI activity level
 Number of specimens positive for seasonal and non-seasonal [A(H1N1) and H5] influenza
3.5 1

Proportion of all influenza that were

3
1
Number of specimens

pandemic A(H1N1) 2009

2.5
1

2
1
1.5

0
1

0
0.5

0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Week number, 2009

Seasonal influenza Pandemic A(H1N1) 2009
A(H5) Other non-seasonal
Proportion of all influenza that were pandemic A(H1N1) 2009

4 COLLECTION, STORAGE AND SHIPPING OF CLINICAL

SPECIMENS

INTRODUCTION

The success of virus diagnosis largely depends on the quality of the specimen and the
conditions for transport and storage of the specimen before it is processed in the
laboratory. Specimens for isolation of respiratory viruses in cell cultures and for the
direct detection of viral antigens or nucleic acids should generally be taken during the
first 3 days after onset of clinical symptoms.

Collection of appropriate specimens from human and animal cases for rapid viral RNA
detection by any qualified laboratory, together with rapid and precise characterization of
virus isolates of the specimens at specialized reference laboratories, is essential for
early detection of cases, proper management of patients and understanding the
epidemiology of the disease. In addition, the development of resistance to antivirals can
be determined, effective vaccines produced in a timely manner and quality control
improved.

The rapid confirmation of the precise nature of isolates of the virus permits effective
surveillance and in particular the proper documentation of the spread of the infection in
human and animal populations and detection of changes in the virus that could indicate
improved transmissibility to humans.
Specimens should be collected and transported in a suitable transport medium. Some
transport systems which have proven to be satisfactory for the recovery of a wide
variety of viruses are commercially available. Hanks balanced salt solution, cell culture
medium, tryptose-phosphate broth, veal infusion broth, and sucrose-phosphate buffer
are commonly used transport media. They should be supplemented with protein, such
as bovine serum albumin (BSA) or gelatin, to a concentration of 0.5% to 1%, to stabilize
viruses. The addition of antibiotics and antimycotics helps prevent microbial growth.
Physiological saline solution, phosphate-buffered saline, Hanks balanced salt solution,
or cell culture medium without antibiotics and proteins can be used as washing or
gargling fluids.
The ongoing threat of infections with Highly pathogenic avian influenza (HPAI) caused
by the A(H5N1) influenza subtype in animal populations, particularly wild waterfowl and
domestic poultry such as chickens and ducks, poses a continuing global human public
health risk. Furthermore the world is currently in pandemic Phase 6 of Swine origin
Influenza A H1N1influenza with over 60,000 confirmed cases world wide. Each new
human case gives the virus an opportunity to evolve further towards a rapidly
transmissible pandemic strain.

This protocol is intended to:

• Describe the minimum number and types of specimens to be collected for
lab testing in cases of suspected seasonal influenza;
• The protocol also covers in brief the universal precautions that enhance the
chances of safely obtaining a positive result if the patient is infected with A
(H5N1) or Swine Influenza H1N1
 • allow the potential identification of respiratory pathogens other than
seasonal and novel influenza viruses
• contribute to work designed to increase understanding of the pathogenesis
of seasonal and novel Influenza A(H5N1) and H1N1 disease including the
potential duration of infectiousness.
This protocol covers the following aspects of sampling:
• The specimens required from humans;
• Collection of the specimens;
• Preservation before and for shipping;
• Correct packing of the specimens;
• Shipment to laboratories;
• The information that needs to accompany the shipment.

TAKING SPECIMENS

SAFETY

In general PPE should include:

1. a suitable form of respiratory protection ; surgical Mask, N95
2. non-sterile latex gloves (or equivalent if allergic)
3. goggles or a face shield
4. gown
5. head covering

The level of PPE to be employed will be determined by the exposure risk. For example
work in intensive poultry units, where virus contaminated material can accumulate and
there is a great deal of potentially contaminated dust in the air, requires greater
personal protective equipment levels than sampling from wild birds. Yuen & Wong
(2005) recommend that: “In view of the high fatality of the disease, a combination of
contact, droplet, and airborne precautions are recommended as long as resources allow
despite the fact that the relative importance of these three modes in nosocomial
transmission of avian influenza is still unknown.”

It may also be necessary to include:

− impermeable apron
− suitable rubber boots.
High risk activities such as post mortem examination of a confirmed or strongly
suspected human case, should only be conducted in a full body cover-all, with easily
cleaned waterproof boots, heavy rubber gloves and eye protection.
PPE is essential to prevent infection during sampling but is not the whole answer.
Those taking specimens should comply with all recommended infection control
precautions including specific personal hygiene measures, and the correct use of
disinfectants.
Further details of safety procedures are given in Annex 1(PPE) & 2 (Hand hygiene
technique), and Annex 3 (Disinfection).
Each sample taken should be given a unique identifier number and accompanied by
epidemiological data/investigation form

- All specimens taken from that source should be marked with that unique identifier
as well as any other numbers needed to identify the particular specimen.
- This identifier should be used on all documentation concerning the specimen
from that source.
- Specimen tubes should also be marked with information about the type of
specimen in the tube and the date when the specimen was taken.

a) What specimens to collect

A variety of specimens are suitable and easily obtainable for the diagnosis of virus
infections of the upper respiratory tract:

• Nasal swab
• Nasopharyngeal aspirate /nasal wash
• Throat swab/throat wash or gurgle
• Combined nasal and throat swabs

If clinically indicated, invasive procedures can be performed for the diagnosis of virus
infections of the lower respiratory tract, this is more applicable in case of highly
pathogenic viruses such as H5N1:

• Transtracheal aspirate
• Bronchoalveolar lavage
• Lung biopsy

Specimens for direct detection of viral antigens by immunofluorescence staining of

infected cells should be kept on ice and processed within 1 - 2 hours. Specimens for
virus isolation should be refrigerated immediately after collection and inoculated into
susceptible cell cultures as soon as possible. If the specimen cannot be processed
within 48 - 72 hours, the specimen should be kept frozen at or below -70oC.

1. Preferred samples

a. Upper respiratory tract

(take both types of specimen to allow detection of influenza viruses):

- Nasal swabs with nasal secretions (from the anterior turbinate area)
ornasopharyngeal aspirates or swabs are appropriate specimens for detecting
human influenza A and B
- Posterior-pharyngeal (throat) swabs are currently the highest yield upper
respiratory tract specimen for detecting A(H5N1) (unlike human influenza).
b. Lower respiratory tract:
- If the patient is intubated, take a tracheal aspirate or collect a sample during
bronchoalveolar lavage.

c. Blood:
- Serum (acute and convalescent if possible).This is not applicable in cases of
seasonal influenza

2. Secondary specimens

(these are not required for seasonal influenza , but can be useful in outbreaks, if
materials are available)

• Plasma in EDTA (for detection of viral RNA)

• Rectal swab —especially if the patient has diarrhoea
• Spinal fluid if meningitis is suspected and a spinal tap is to be performed for
diagnostic/therapeutic purposes.

b) When to collect the specimens

Figure1. Virus excretion and antibody response in human Influenza infections

 • A nasopharyngeal and/or throat swab should be taken within first three days of
onset of symptoms. Note that the virus is generally detectable in throat swabs
from most patients from the point of onset of symptoms (or even just before) until
towards the end of the second week, and infrequently beginning of the third
week, after onset of symptoms.

• Virus may be detectable in tracheal aspirates from onset of lower respiratory

complaints (dyspnoea, difficulty breathing, marked cough) or pneumonia until the
second or third week of illness.

• An acute phase serum sample should be taken seven days or less after
symptom onset (this will usually be done when the patient presents and begins
treatment) and a

• convalescent sample after 3 to 4 weeks. Note that the limited data available on
antibody

• kinetics indicate development of positivity (initially ELISA and not necessarily

neutralizing antibody) from day 10 onwards.

• Single serum samples. To be collected at day 14 or later after symptom onset

since the likelihood of detecting neutralizing antibodies increases over time,
certainly during the first 3 to 4 weeks after onset of symptoms.

• Blood serum or plasma for the detection of viral RNA should be taken during the
first 7 to 9 days after the development of symptoms because the patient is most
likely to be RNAaemic (have detectable RNA in the bloodstream) at that time .

• Initial specimens (respiratory and blood) should ideally be collected from

suspected patients before antiviral therapy is begun but treatment must not be
delayed in order to take specimens. (Note that standard treatment may render
throat swabs negative for virus after three or more days of treatment but probably
has no effect on the development of neutralizing antibody).

• Specimens should be collected from deceased patients as soon as possible after

death.

PROCEDURES FOR SPECIMEN COLLECTION

Materials Required

Polyester fiber-tipped applicator Tongue depressor, wood

Falcon Cat. # 2069 Citmed Corp., Cat. # NSN-6515-324-
5500
Polyester fiber-tipped applicator
with malleable aluminum shaft
(for Nasopharyngeal swabs)
Viral transport medium* (Commercial /or
sent from NIH)

Sputum/Mucus Trap with 10FG catheter

Vygon Cat. #542.10(for Nasopharyngeal
aspiration)

15 ml conical centrifuge tubes( incase of

mouth gurgles) Falcon Cat. # 2097
Clinical specimens should be collected as described below and added
to transport medium
Specimens from the respiratory tract

Sampling from the respiratory tract is hazardous as the operator is very close to the
patient and the procedure can generate aerosols and droplets. Full PPE is therefore
essential. Chose a sitting position for adults and a supine position for infants and
younger children. Children often find sampling from the respiratory tract very distressing
and need to be reassured. They may also need to be restrained during the sampling
process.

If the child’s parents or guardians are present they must be fully informed of what is to
take place, and must be made aware that the child may become distressed. The
parent(s) should not usually be in the room during the sampling procedure. The
sampling procedure can generate aerosols which could present a risk to others in the
immediate vicinity, and also the parent(s) may react to the child’s distress by attempting
to interfere with the sampling procedure and risk injury to the child.

Very young children can be restrained by being held by an assistant against the
assistant’s chest with the child’s arms restrained by the assistant’s enfolding arms and
the child's legs held between the assistant’s legs (These are general considerations and
should be adjusted accordingly to the local cultural sensitivity and social
circumstances).

When taking throat (or nasal) swabs, the swabs must be held correctly. They should be
held between the thumb and the first and second fingers with the shaft protruding
beyond the web of the thumb (like a pencil) and not between the thumb and forefinger
with the base in the palm of the hand.
• Use only sterile dacron or rayon swabs with plastic shafts. Calcium alginate or cotton
swabs, or swabs with wooden sticks may contain substances that inactivate some
viruses and inhibit PCR testing and should only be used if dacron or rayon swabs are
not available.

Figure 2a. Swab held correctly Figure 2b. Swab held incorrectly

21
 • Prepare two vials containing at least 2–3 ml of a suitable transport/preservative
medium (Viral Transport Medium - VTM) for each specimen. These should be
marked with:
- the unique identifier
- the specimen date
- the type of specimen in the tube (e.g. blood serum, throat swab etc.).

Note: Always mark the tube itself with identifying details, never the cap as this
can get switched during handling). Use an indelible and alcohol resistant marker
pen. Be aware that stick-on labels can easily come off, especially when the
specimen is chilled to very low temperatures. Relevant field data sheets (see
Annex 9) should be filled in.

Anterior nasal swab

Use the same type of rigid swab as for sampling from the throat. Advance the swab tip
past the vestibule (anterior nares) to the nasal mucosa (approximately 2–3 cm from the
nostrils in adults) and gently rotate to collect nasal secretions from the anterior portions
of the turbinate and septal mucosa. Specimens from both nostrils are obtained with the
same swab. The tip of the swab is put into a vial containing 2 - 3 ml of transport medium
and the applicator stick is broken off.

Nasopharyngeal swab
• Insert a flexible, fine-shafted polyester swab into the nostril and back to the
nasopharynx. The swab should be slid straight into the nostril with the patient’s
head held slightly back The swab is inserted following the base of the nostril
towards the auditory pit and will need to be insert at least 5–6 cm in adults to
ensure that it reaches the posterior pharynx . (Do NOT use rigid shafted swabs
for this sampling method —a flexible shafted swab is essential).
• Leave the swab in place for a few seconds
• Withdraw slowly with a rotating motion
• Put the swab into VTM
• A second swab should be used for the other nostril and put into a second tube.

This can serve as the second sample from the patient.

Note: Nasopharyngeal sampling is an invasive process that can cause

considerable distress to the patient.

Posterior pharyngeal swab (throat swab)

• Hold the tongue out of the way with a tongue depressor;
• Use a sweeping motion to swab the posterior pharyngeal wall and tonsilar pillars.
Have the subject say "aahh" to elevate the uvula. Avoid swabbing the soft palate
and do not touch the tongue with the swab tip. (N.B. This procedure can induce
the gag reflex);
• Put the swab into VTM

22
 Taking a throat swab Restraining a small child while sampling

Nasal and throat swabs

Nasal- and throat swabs taken as described above are collected into the same vial of
transport medium.

Figure 3. Taking a nasopharyngeal (a) and Throat swab (b)

After a specimen is taken, the tip of the swab should be put into the vial and the shaft
broken or cut off sufficiently short for the lid to be closed. Plastic swab handles usually
have a weak point in them to allow them to be broken off for insertion into a specimen
tube. Others have a handle made of a brittle plastic that will snap easily. If the shaft
cannot easily be broken off short enough to be put into a small tube such as a cryo-vial
it will have to be cut.

23
To do this:
- cut the shaft with scissors taking care not to touch the tip;
- allow the tip to slide into the VTM and then cap the tube. Do not let cut
- portions of the bag or wrap fall into the tube. Sterilize the cutting edge of the
scissors by the use of flame (e.g. by the use of a spirit burner, a Bunsen burner
or another suitable heat source). Allow scissors to cool before reuse. If this
procedure cannot be followed, agitate the swab tip in the medium for 30 seconds
and squeeze it against the side of the tube before removing it from the medium
and disposing of it in a safe manner (not suitable for ethanol storage).

Nasopharyngeal aspirate

• Easier and safer than swabbing in infants and young children.

• Nasopharyngeal secretions are aspirated through a catheter connected to a
mucus trap and fitted to a vacuum source. The catheter is inserted into the nostril
parallel to the palate. Then the vacuum is applied and the catheter is slowly
withdrawn with a rotating motion. Mucus from the other nostril is collected with
the same catheter in a similar manner. After mucus has been collected from both
nostrils, the catheter is flushed with 3 ml of transport medium.

Figure 4. Nasopharyngeal aspiration

Nasal wash

The patient sits in a comfortable position with the head slightly tilted backward and is
advised to keep the pharynx closed by saying "K" while washing fluid (usually
physiological saline) is applied to the nostril. With a transfer pipette, 1 to 1.5 ml of
washing fluid is applied to one nostril at a time. The patient then tilts the head forward
and lets the washing fluid flow into a beaker or a petri dish. The process is repeated
alternatively with both nostrils until a total of 10 to 15 ml of washing fluid has been used.
Dilute approximately 3 ml of washing fluid 1:2 in transport medium.

Mouth wash
The patient gargles with 10 ml of washing fluid. The fluid is collected into a beaker or
petri dish and diluted 1:2 with transport medium.

24
Blood specimens

1. Standard precautions should always be observed when taking and handling

blood specimens because the patient may be infected with a blood born
pathogen (for example HIV or Hepatitis B).
2. Use PPE — at least gloves (plus face-shields, masks and gowns if splashes are
anticipated).
3. Remove and discard PPE items immediately after completion of task.
4. Perform hand hygiene every time gloves are removed.

Serum samples

The best "all round" specimen to collect is serum. Acute and convalescent sera are
useful for detection of changes in antibody titre and serum can be used for detection of
viral RNA. An acute phase serum specimen should be taken soon after onset of clinical
symptoms and not later than seven days after onset.
EDTA-anti-coagulated plasma is also valuable for detection of viral RNA in blood and
may be better than serum for this particular purpose since EDTA inactivates RNAses
present in the specimen. Heparin is not suitable as an anticoagulant for this type of
specimen because of potential inhibition of PCR reactions. Note that specimens for the
detection of viral RNA in the blood should be collected during the first week after the
development of symptoms (Fig 1).
At least 1ml of whole blood is needed to obtain a sufficient amount of serum or plasma
for tests. This is the maximum that should be taken from infants. However larger
specimens of 3–5 ml should be taken from older children and adults as this will allow a
greater range of tests or repeat tests to be done.
A convalescent-phase serum specimen should ideally be collected 3–4 weeks after the
onset of symptoms. When a patient is critically ill, a second ante-mortem specimen
should be collected. Blood should be collected either by use of a vacuum venepuncture
system or syringes and needles. The specimens should be collected either in a serum
separator tube (SST) or a clotting tube (for serum) and in an EDTA tube (for plasma).

Taking a blood specimen

1. Label the tubes, including the unique patient identification number, using an
indelible marker pen. Always check to ensure that the correct tubes are used for
each patient.
2. Place a tourniquet above the venepuncture site, palpate and locate the vein 3.
Disinfect the venepuncture site meticulously with 70% isopropyl alcohol (an
alcohol swab) or 10% polyvidone iodine by swabbing the skin concentrically from
the centre of the venepuncture site outwards (Let the disinfectant evaporate. Do
not re-palpate the vein.

25
Figure 5 a & b
Tourniquet on, palpating the vein Disinfecting the venepuncture site

3. Perform venepuncture.
4. If withdrawing blood with conventional disposable syringes, withdraw 3–5 ml of
whole blood from adults and older children and 1ml from infants. Under asepsis,
transfer the specimen to appropriate transport tubes. Secure caps tightly.
5. If withdrawing blood with a vacuum system (e.g. Vacutainer®), withdraw the
desired amount of blood directly into each transport tube

Figure 5 c & d Venepuncture with a vacuum venepuncture system

6. Remove the tourniquet. Use a cotton swab to apply pressure to the venepuncture
site until bleeding stops (Fig 18) and apply a band-aid.
7. Never recap used sharps. Discard directly into a suitable container (a proper
sharps disposal container if available or a container such as a coffee or other
metal can which should be appropriately labelled before use).
8. Recheck that the tubes used for sampling have been correctly labelled.
9. After taking all the samples, complete the appropriate field data sheets or case
investigation forms and the required laboratory request forms using the same
identification numbers used on the tubes.
Separation of serum and plasma
Blood samples need to be centrifuged for at least five minutes at 1500g (3000 rpm).
This requires an electric centrifuge (ideally with a swing out head rather than an angle
head rotor). Hand centrifuges are not adequate for the separation of serum or plasma
from red cells.

26
Transport Blood specimen bottles and tubes should be transported upright and secured
in a screw cap container or in a rack in a transport box. They should have enough
absorbent paper around them to soak up all the liquid in case of spillage. See Table 1
below and the notes following the table for details of storage and shipment conditions.

Figure 6. Serum separator tube

Storing Specimens

• Aliquots of specimens should be taken before the specimens are frozen.

• Repeated freezing and thawing of specimens must be avoided to prevent loss of

infectivity. Note that certain types of freezer are designated "frost free" and these

27
 should not be used for specimen storage as the temperature cycling involved in
keeping them free of ice accumulation can damage specimens.

• If specimens in VTM (or blood sera/plasma) for viral isolation can be taken to the
laboratory within four days, they may be kept at +4 °C and frozen at -70 °C on
arrival if they are to be stored. Otherwise they should be frozen at or below -70
°C until they can be transported to the laboratory.

• Freezing at -20 °C is not recommended because the virus does not survive well
at this temperature, particularly in frost-free freezers.

• n the absence of freezers (or of VTM), ethanol-preserved swabs are a possible

alternative. Storage of such specimens at +4 °C (in a standard refrigerator) is
better than at room temperature.

• Blood serum samples should be frozen at -70 °C for PCR and at -20 °C or lower
for antibody determination but they can be stored at +4 °C for approximately one
week.

• Specimens for influenza virus isolation should not be stored or shipped in dry ice
(solid

• carbon dioxide) unless they are sealed in glass or sealed, taped and double
plastic-bagged. Carbon dioxide can rapidly inactivate influenza viruses if it gains
access to the specimens. (Note: Take care not to place dry ice in an hermetically
sealed containers as it could cause an explosion).

Taking aliquots of specimens

• It is better to take more than one specimen when sampling from a patient than to
subdivide specimens later. However this may not be possible and if specimens
have to be sub-divided, the smallest volume of VTM or serum that should be
stored is 0.5ml (thus a 3ml sample can be divided into six separate sub-
samples). Fresh sterile or disposable pipettes should be used for each sample
and these should be discarded safely (into 1/100 chlorine solution).

• Taking aliquots of samples should only be undertaken under appropriate levels of

laboratory safety (e.g. preferably in a certified1 a Class II biosafety cabinet).

• Great care must be taken not to contaminate or cross contaminate specimens.

This is especially so when they are intended for analysis by PCR because PCR
procedures are especially vulnerable to cross contamination after amplification
and uncapping of the tube.

• Taking aliquots should be done before the specimen is frozen as repeated

freezing and thawing of specimens can reduce the content of virus and should
therefore be avoided.

28
Packing specimens and shipping them by air
As a sentinel sites sending specimens to the central lab is going to be one of the regular
activities and therefore the staff handling samples should be aware of basic handling
and packing principles for these samples. The protective precautions must be taken at
all times, and all samples are to be treated as infectious materials.

All the samples that are to be sent by your lab are on domestic route and
shipment is handled by the nominated courier (M/S Leopards Courier services)
Dry shippers should be well marked with ownership details. The courier service
can pick up the samples from your laboratory during working hours and return
the container for future use in 1-3 days .Please ensure proper disinfection of the
sample transport boxes between trips.

The packing of specimens and their shipment to external laboratories by air is complex
and is governed by international and national regulations and operator variations. These
are summarized below. Links to other relevant documents are given in Annex 1.
Readers are also referred to the WHO document Guidance on regulations for the
Transport of Infectious Substances (Annex---).

Shipment of specimens from humans

There are two categories covering shipment of specimens by air:

• Category A covers infectious substances (included in an indicative list of
specified pathogens) that are capable of causing permanent disability, life
threatening or fatal disease in otherwise healthy humans or animals. Highly
pathogenic avian influenza is part of the indicative list with the mention "cultures
only”. Thus only cultures of HPAI (i.e. virus isolates) must be transported as
Category A.
• Category B covers all other infectious substances that are not included in
Category A.

• As far as shipment of human or animal samples suspected or confirmed to

contain A (H5N1) or H1N1 viruses is concerned, human blood and other
human samples or animal blood and other animal samples known or
suspected to contain the A(H5N1) subtype, can be transported as “diagnostic
specimens" (UN 3373) and are included in Category B. Note that individual
airlines may adopt their own policies and these may be stricter than those issued
by IATA.

Shipment of specimens in Category A requires shippers who have undergone special

training.

For the transport of Category B infectious substances there is a requirement for clear
instructions on the use of the packaging to be supplied to the user; and this is regarded
as sufficient “training” for the shipping of these substances. However, if such specimens

29
are consigned with other dangerous goods (including liquid nitrogen or dry ice), then
shippers trained in the proper procedures for the transport of those substances must be
used.

Detailed instructions for packing specimens for shipment in the two categories can be
found in the WHO document Guidance on regulations for the Transport of Infectious
Substances (Annex).

Triple packaging method for shipment of Infectious Substances

30
SECTION II: DIAGNOSTIC METHODS

31
 OVERVIEW OF INFLUENZA DIAGNOSIS

Virologic surveillance is the foundation on which international and national influenza

surveillance systems are built. Within a country, one or more WHO National Influenza
Center laboratories or a national network of laboratories reporting to a National
Influenza Center may participate. The data are compiled within the country and then
sent to WHO. Virological data can be reported by the National Influenza Center to
WHO via their internet-based reporting system, FluNet, at
http://www.who.int/GlobalAtlas/home.asp.

Each WHO National Influenza Center will send selected influenza virus isolates to one
of the 4 WHO Centers for Reference and Research on Influenza for complete
identification and antigenic characterization. Specimens for virus isolation usually are
collected as a part of routine patient care through a surveillance network of emergency
rooms, outpatient clinics, and private physicians, or from respiratory disease outbreak
investigations.

The viral data gathered through this system are summarized each week during the
influenza season and are reported to WHO, Geneva via FluNet, and these submitted
isolates are then utilized to formulate the next year vaccine for seasonal influenza for
both the southern and northern hemispheres.

The diagnostic methods used for influenza lab diagnosis can be broadly divided
in to 2 groups

1. Non Molecular diagnostic methods

2. Molecular/PCR based diagnostic methods

32
 NON MOLECULAR DETECTION METHODS FOR INFLUENZA

1
RAPID DIAGNOSTIC KITS
Directigen Flu A & B Kit
In this test, a treated specimen is applied to each of
two wells of a filter membrane. Viral antigens, if
present, are bound to the membrane. An enzyme-
labeled monoclonal antibody specific to influenza virus
is added, which binds to any virus captured on the
membrane. Activity of any bound enzyme is detected
by two substrate-chromogen solutions. In the presence
of viral antigens, a purple triangle becomes visible on
the membrane. Virus antigen disposed on a small
round area in the center of the membrane serves as a test control. The manufacturer
reported sensitivity and specificity of this test ranges from 77% to 96% and 90% to 91%,
respectively.

Flu OIA
This test involves the extraction and detection of
influenza A or B nucleoprotein. The Optical
ImmunoAssay technology enables the direct visual
detection of a physical change in the optical
thickness of molecular thin films. The change is a
result of antigen-antibody binding on an optical
surface, which is a silicon wafer. Slight changes in
optical thickness produce a distinct visible color
change. A positive result appears as a purple spot
on the predominant gold background. The
manufacturer reported sensitivity and specificity of
this test ranges from 62% to 81% and 52% to 80%,
respectively.

QuickVue Influenza Test

This test involves the extraction of influenza A and B viral antigens. The patient
specimen is placed in the extraction reagent tube,
during which time the virus particles in the specimen
are disrupted, exposing internal viral nucleoproteins.
After extraction, the test strip is placed in the
extraction reagent tube where nucleoproteins in the
specimen will react with the reagents in the test
strip. If the extracted specimen contains influenza
antigens, a pink-to-red test line along with a blue
procedural control line will appear on the test strip
indicating a positive result. If influenza type A or type
B antigens are not present, or are present at very low levels, only a blue procedural

33
control line will appear. The manufacturer reported sensitivity and specificity of this test
ranges from 73% to 81% and 96% to 99%, respectively.

ZstatFlu Test
This test is based on the detection of influenza-
specific neuraminidase activity. Neuraminidase
reacts with a chromogenic substrate that
precipitates upon reaction. The recommended
sample type for this test is a throat swab. The
specimen is added to the kit reagents and
incubated at 41oC for 20 minutes. The reaction
mixture is then transferred into a collection device
and the colored precipitate is collected on a filter.
Positive specimens are identified by a blue color,
and negative specimens are identified by a white color or any shades of color other than
blue. The manufacturer reported sensitivity of this test in 2 clinical sites compared to
culture was 62.2%, with a 95% confidence interval of 50.8%-72.7% and specificity was
98.7% with a confidence interval of 92.8%-100%. The reported sensitivity was higher
for influenza A viruses than influenza B viruses, 65.3% vs 57.6%, but specificity was
similar for both type A and B viruses.

ISOLATION OF INFLUENZA VIRUSES

Virus isolation is a highly sensitive and very useful technique when the clinical
specimens are of good quality and have been collected in a timely manner (optimally
within 3 days of the start of illness). Isolation of a virus in cell culture along with the
subsequent identification of the virus by immunologic or genetic techniques are
standard methods for virus diagnosis. One important advantage of virus isolation is that
this method amplifies the amount of virus from the original specimen making a sufficient
quantity of virus available for further antigenic and genetic characterization, and for
drug-susceptibility testing if required.
Virus isolation is considered the “gold standard” for diagnosis of influenza virus
infections.

Cytopathic effect typical of influenza

Egg Inoculation of influenza viruses
infections in tissue culture

34
However, several factors limit the use of virus isolation as a diagnostic technique. Each
of the currently available mammalian cell lines supports the replication of only a limited
number of clinically important respiratory viruses. A laboratory, therefore, must maintain
several cell lines if they wish to detect a variety of respiratory pathogens. On the basis
of the clinical information and the epidemiologic situation, the appropriate cell lines to be
inoculated have to be selected for each specimen.

Madin-Darby canine kidney (MDCK) cells are the preferred line in which to culture
influenza viruses. Primary rhesus or cynomolgus monkey kidney cells also support the
growth of influenza viruses. In recent years, the use of cell lines has surpassed the use
of embryonated eggs to culture influenza viruses. However, only viruses grown in
embryonated eggs can be used as seed viruses for vaccine production.
Standard isolation procedures require several days before results are available, making
this test less useful to the clinician. Rapid culture assays that use immunologic
methods to detect viral antigens in cell culture have become available. The results of
these assays can be obtained in 18 - 40 hours, compared to an average of 4.5 days
using standard culture techniques.

Appropriate clinical specimens for virus isolation include nasal washes, nasopharyngeal
aspirates, nasal and throat swabs, transtracheal aspirates, and bronchoalveolar lavage.
Ideally, specimens should be collected within 72 hours of onset of illness.

In the past, influenza viruses often were isolated in embryonated eggs, however, today
the majority of laboratories use cell culture. Since vaccine candidate viruses must be
isolated in eggs the trend to use cell cultures has decreased the availability of suitable
vaccine viruses. For this reason, laboratories that have the capability to isolate influenza
in eggs are encouraged to continue.

PRECAUTIONS & QUALITY CONTROL

Over a number of passages, MDCK cells might lose their susceptibility to respiratory
viruses. For this reason, the laboratory should keep a stock of frozen cells at a low
passage level, and at the beginning of each influenza season, low passage MDCK cells
should be thawed and used during the entire season. Cell lines should be free of
Mycoplasma contamination.

a. Never use -20oC for storage of isolates!

b. Be aware of contamination of clinical specimens with laboratory strains:

Since influenza viruses are continuously evolving, older strains are antigenically distinct
from currently circulating strains. Because laboratory-adapted viruses and commercial
reference viruses are prepared using older strains, complete antigenic analysis by HAI
using selected ferret anti-sera and sequencing can be performed to determine if an
isolate has been inadvertently contaminated.

GOLDEN RULE 1:
NEVER PROCESS CLINICAL SPECIMENS FOR VIRUS ISOLATION AND
LABORATORY-ADAPTED INFLUENZA STRAINS AT THE SAME TIME.

GOLDEN RULE 2:
NEVER PROCESS CLINICAL SPECIMENS FROM HUMANS AND FROM SWINE OR
BIRDS IN THE SAME LABORATORY.

35
IDENTIFICATION OF INFLUENZA ISOLATES BY HEMAGGLUTINATION INHIBITION

The HAI test was originally described by Hirst (1942) and later modified by Salk (1944).
The hemagglutinin (HA) protein of influenza viruses can bind to red blood cells causing
the cells to agglutinate. The traditional method for identifying influenza isolates takes
advantage of this property.
When antibodies against an influenza HA protein bind to the antigenic sites on the HA,
these antigenic sites become blocked and unavailable for binding with red blood cells,
thereby, inhibiting hemagglutination. This effect is the basis for the hemagglutination
inhibition (HAI) test. Currently, the HAI test is performed in micro-titer plates.A
standardized quantity of HA antigen is mixed with serially diluted antiserum, and red
blood cells are added to detect specific binding of antibody to the HA molecule.
The HAI test is extremely reliable provided that the reference antiserum contains
antibody to currently circulating viruses. The antiserum is newly prepared each year
and is based on the vaccine strains that are the prototypes for currently circulating
strains.

Disadvantages of the HAI test include the need to remove nonspecific inhibitors of
hemagglutination that occur naturally in sera, the need to standardize reference and test
antigens each time a test is performed, and the need for specialized expertise in
reading the results of the test.

Regardless, the HAI assay remains the test of choice for WHO global influenza
surveillance and for determining the antigenic characteristics of influenza virus
isolates.

36
 Influenza Hemagglutination with Different Species of RBCs

Chicken Turkey Guinea Pig Human Type O

Concentration 0.5% 0.5% 0.75% 0.75%

Microtiter plate “V” “V” “U” “U”
Incubation time, 25oC. 30min 30min 1 hour 1 hour
Appearance of control cells button* button* halo halo

* = flows when tilted

37
 1IMMUNOFLUORESCENCE STAINING OF INFECTED CELL CULTURES

Immunofluorescence antibody (IFA) staining of virus-infected cells is a rapid and sensitive

method to diagnose influenza and other virus
infections. During recent years, monoclonal
antibodies against several clinically important
respiratory viruses have become available
through commercial sources. These antibodies
have been thoroughly evaluated in many
laboratories. With the proper monoclonal
antibodies, the type and subtype of an influenza
isolate can be determined.

The sensitivity of this method is greatly

influenced by the quality of the isolate, the
specificity of the reagents used, and by the
experience of the person(s) performing,
reading and interpreting the test. Although IFA
can be used for staining smears of clinical
specimens directly, it is preferable to first MDCK cells infected with influenza A virus and
increase the amount of virus in cell cultures. stained with rhodamine
However, this procedure is often done on the
clinical specimen if rapid diagnosis is needed.

1ENZYME IMMUNOASSAY

Commercially available enzyme immunoassays,

which allow influenza type A viruses to be rapidly
detected in clinical specimens, have been on the
market for several years. In recent years, test kits
have become available that allow both influenza A
and B viruses to be rapidly detected. Some tests can
detect both influenza type A and type B infections but
do not distinguish between the two, while others can
detect either influenza A or B viruses and distinguish
between them. These tests are particularly useful in clinical settings because they can
be performed in hospital laboratories or in a doctor's office and in outbreak
investigations. All required equipment and reagents are included in the test kit. A result
can be obtained within 30 min.

Nasopharyngeal aspirates, nasal washes, and nasopharyngeal and/or throat swabs are
generally suitable specimens for these assays, but the instruction sheet for each test kit
should be consulted for specific information. The sensitivity and specificity of these tests
vary by test type and by specimen type. In outbreak situations and when used outside the
period of peak influenza activity, it is important to use these tests in combination with virus
isolation to confirm the rapid test kit results.

38
SEROLOGIC METHODS
HEMAGGLUTINATION INHIBITION

In situations where the identification of influenza viruses is not feasible or possible

(clinical specimens for virus isolation cannot be obtained, the cases are identified after
shedding of virus has stopped, or the laboratory may not have the resources or staff to
perform virus isolation), serologic methods can be very useful. Since most human sera
contain antibodies to influenza viruses, serologic diagnosis requires demonstration of a
four-fold or greater rise in antibody titer using paired acute and convalescent serum
samples. Many laboratories rely on serology for determining recent individual infections.
The HAI test is the preferred diagnostic test for determining antibody rises. In
general, the acute phase sera should be collected within 1 week of illness onset and the
convalescent sera should be collected 2 to 3 weeks later.

There are two exceptions in which the collection of single serum samples can be helpful
in the diagnosis of influenza. In investigations of outbreaks due to novel viruses, testing
of single serum samples has been done to identify antibody to the novel virus. In some
other outbreak investigations, antibody test results from single specimens collected from
individuals in the convalescent phase of illness (cases) have been compared with
results from age-matched individuals either in the acute phase of illness or from non-ill
controls. In such situations, the geometric mean titers between the 2 groups to a single
influenza virus type or subtype can be compared. In general, these approaches are not
optimal and paired sera should be collected whenever possible.

MICRONEUTRALIZATION ASSAY

Serology methods rarely yield an early diagnosis of acute influenza virus infections;
however, the demonstration of a significant increase in antibody titers (greater than or
equal to 4-fold) between acute- and convalescent-phase sera may establish the
diagnosis of a recent infection even when attempts to detect the virus are negative.
Apart from their diagnostic value, serologic techniques such as the hemagglutinin
inhibition test (HI), the virus neutralization test, and enzyme-linked immunosorbent
assay (ELISA) are the fundamental tools in epidemiologic and immunologic studies as
well as in the evaluation of vaccine immunogenicity.

The virus neutralization test is a highly sensitive and specific assay applicable to the
identification of virus-specific antibody in animals and humans. The neutralization test is
performed in two stages consisting of (1) a virus-antibody reaction step, in which the
virus is mixed and inoculated with the appropriate antibody reagents, and (2) an
inoculation step, in which the mixture is inoculated into the appropriate host system (e.g.
cell cultures, embryonated eggs, or animals). The absence of infectivity constitutes a
positive neutralization reaction and indicates the presence of virus-specific antibodies in
human or animal sera.

The virus neutralization test gives the most precise answer to the question of whether or
not an individual has antibodies that can neutralize infectivity of a given virus strain. The

39
neutralization test has several additional advantages for detecting antibody to influenza
virus. First, the assay primarily detects antibodies to the influenza virus HA, and thus
can identify functional, strain-specific antibodies in animal and human serum. Second,
since infectious virus is used, the assay can be developed quickly upon recognition of a
novel virus and is available before suitable purified viral proteins become available for
use in other assays.

The microneutralization test is a sensitive and specific assay for detecting virus-specific
antibody to avian influenza A (H5N1) virus in human serum and potentially, for detecting
antibody to other avian subtypes. The microneutralization test could detect H5-specific
antibody in human serum at the titers that could not be detected by the HI assay, the
traditional test used for the detection of antibodies to human influenza A and B viruses.
Because antibody to avian influenza subtypes is presumably low or absent in most
human populations, single serum samples may be used in screening for the prevalence
of antibody to avian viruses. However, if infection of humans with avian viruses is
suspected, the testing of paired acute and convalescent sera in the microneutralization
test would provide a more definitive answer as to whether infection has occurred.

1 . A d d S e r a ( h e a t i n a c . )
2 - f o ld
d ilu t io n s

2 . A d d V i r u s
2 H @ 3 7 ° C

3 . A d d M D C K C e l l s 1 0 0 T 5 C 0/ wI D e l l

1 . 5 x 41 0
c e lls / w e ll

1 8 H @ 3 7 ° C

+ + +
5 . E L I S A

N P s p e c if . m A b

40
 An Algorithm summarizing the Microneutralization Assay
Conventional neutralization tests for influenza viruses based on the inhibition of
cytopathogenic effect (CPE)-formation in Madin-Darby Canine Kidney (MDCK) cell
cultures are laborious and rather slow, but in combination with rapid culture assay
principles, the neutralization test can yield results within 2 days. In our influenza virus
microneutralization assay, it is expected that serum neutralizing antibodies to influenza
virus HA will inhibit the infection of MDCK cells with virus. Serially diluted sera should be
pre-incubated with a standardized amount of virus prior to the addition of MDCK cells.
After an overnight incubation, the cells are fixed and the presence of influenza A virus
NP protein in infected cells is detected by ELISA.

41
111MOLECULAR DETECTION OF INFLUENZA VIRUSES
Two influenza A subtypes, H3N2 and H1N1, have co-circulated in human populations
since 1977. Also, two antigenically and genetically distinct lineages of influenza B
viruses, represented by B/Shanghai/361/2002 (or B/Yamagata/16/88-lineage) and
B/Hong Kong/330/2001 (or B/Victoria/2/87-lineage) viruses are currently circulating
among humans. Co-circulation of multiple types and subtypes of influenza viruses
increases the difficulty of diagnosis and virus identification. Routine diagnostic methods
for influenza infections, including virus culture and antigen detection, are both highly
sensitive and specific. However, the use of molecular techniques to directly detect virus
in respiratory samples facilitates the investigation of respiratory outbreaks and may
provide rapid identification of viruses.

Polymerase Chain Reaction (PCR) is a method used for amplification of specific regions
of DNA from very low levels of starting template DNA. RT-PCR is an extension of this
technique where RNA is first reverse transcribed using reverse transcriptase (RT) into a
DNA copy (cDNA) that is complementary to the template RNA. This cDNA then
undergoes cyclic amplification by PCR. This procedure is thus termed RT-PCR. RT-
PCR reactions require a pair of oligonucleotides, or primers; four deoxyribonucleoside
triphosphates (dNTPs); RNA template; RT and a thermostable DNA polymerase that is
not inactivated by heat such as that of the microbe Thermus aquaticus (Taq
polymerase). Following reverse transcription of the RNA target to cDNA, the cDNA is
subjected to repeated thermal cycling causing template denaturation (95°C), primer
annealing (45-60°C) and product extension (72°C). Because the products of one round
of amplification serve as templates for the next, each successive cycle essentially
doubles the amount of the desired DNA product.

PCR and RT-PCR strategies have been designed for detection and characterization of
many different infectious agents including bacteria and viruses. Because the genome of
influenza viruses consist of eight single stranded RNA segments, RT-PCR is necessary
for amplification of specific influenza gene targets. RT-PCR can be used for the
detection of influenza viruses in original respiratory samples taken from patients with
influenza-like illness, or for the characterization of viruses grown in tissue culture or
embryonated eggs. The recent availability of improved “one-step” RT-PCR reaction
chemistries have decreased the number of necessary pipetting steps, making the
process technically easier and less susceptible to contamination.

Recently, a number of strategies have been designed that utilize fluorescent dyes to
detect or quantitate amplification of DNA by PCR in real time. Such methods include
the use of SYBR® green that binds to double stranded DNA and fluoresces when
excited by light of the appropriate wavelength. Although SYBR® green will detect
amplification of DNA, all amplified products, specific and non-specific, will fluoresce
making it difficult to determine if the amplified DNA is the desired product.
Subsequently, melt-curve analysis is required to determine if primer-dimer, or other non-
specific products are present. Another method of real-time detection incorporates a
dually labeled hydrolysis oligonucleotide probe (eg. Taqman® probe) that is labeled with
a fluorescent label, or fluorophore, and a quencher dye. In this case, because the
probe binds to the specific target and does not bind to non-specific products,

42
fluorescence is only observed if the probe is able to bind to amplified target DNA and
hydrolyzed. A number of other techniques, such as “minor groove binding” (MGB)
probes, FRET probes, Molecular Beacons, Scorpion probes and others have also been
designed that are useful to detect amplification of specific targets on real-time formats.

However, it is important to consider that, as with conventional RT-PCR, the

sensitivity and specificity of “real-time” systems are directly attributable to the
quality of the oligonucleotide primers used for amplification of the desired target.

Real-Time RTPCR amplification plot

Since genetic sequences differ among the various types/subtypes of influenza viruses, it
is possible to design PCR primers and probes that will specifically detect only one
influenza type or subtype. The M gene is the least variable of the influenza virus genes,
and thus, is a useful target for the detection of all type A influenza viruses. Because
influenza A subtypes are defined by their surface hemagglutinin (HA) and
neuraminidase (NA) genes, primers that specifically amplify these genes are effective
for determining the subtype of influenza A viruses. Similarly, a number of protocols have
been designed for the detection of influenza B viruses based on their NS gene. In this
course we will use real-time RT-PCR (rRT-PCR) based on dual-labeled probe
(Taqman®) chemistry for detection and characterization of influenza viruses.

The efficient extraction and purification of viral RNA are critical for downstream
molecular applications, whether it is the sensitive and specific detection of virus in
clinical samples, virus gene cloning and expression, or quantification of the influenza
virus by molecular methods from humans. Samples can generally be divided into two
types: enriched (e.g., virus stocks) and clinical. Clinical type samples, which are swab
material, the most difficult to process due to the complex sample composition and
possibly low virus titers. Viral RNA is extracted using a commercially available RNA
extraction kit (Qaigen). The protocol of viral RNA extraction for reverse amplification of
RNA by Real-Time RT-PCR is included in this course manual.

43
 RNA EXTRACTION

Influenza viral genome is constituted by negative single stranded RNA that can be
extracted from a variety of specimens such as respiratory secretions or swabs, blood,
organs, tissues, faeces of infected cases. The most important factor to consider during
RNA manipulation is its chemical fragility as compared to DNA. RNA has to be handled
very carefully in order to avoid degradation or damage by Rnase enzymes. Therefore,
the handling and storage of RNA is important.

RNA EXTRACTION FROM SWAB SPECIMENS

A. PURPOSE
To extract nucleic acid from Throat / Nasopharyngeal samples to perform molecular test
of virology.

B. PRINCIPLE:
QIAamp Viral RNA Mini Kits provide the fastest and easiest way to purify viral RNA for
reliable use in amplification technologies. Viral RNA can be purified from plasma
(treated with anticoagulants other than heparin), serum, and other cell-free body fluids.
Samples may be fresh or frozen, but if frozen, should not be thawed more than once.
Repeated freeze-thawing of plasma samples will lead to reduced viral titers and should
be avoided for optimal sensitivity.

C. SCOPE:

This procedural format is utilized by the National Influenza Lab-Based Surveillance

Project under the supervision of National Institute of Health within the Virology
Department.

D. PROTOCOL:
This protocol is for purification of viral RNA from 140µl plasma, serum, urine, cell-culture
media or cell-free body fluids using a micro-centrifuge. Larger starting volumes, up to
560µl (in multiples of 140µl), can be processed by increasing the initial volumes

44
 proportionally and loading the QIAamp Mini spin column multiple times, as described
below in protocol.
Note: All centrifugation steps are carried out at room temperature (15-25oC).
Procedure carried out on the RNA bench
• QIAamp MiniElute Spin Kit
• Wear gloves all the times.
• 70% Alcohol make up with RNase free H2O in the kit.
• Equilibrate samples to room temperature (15-25oC).
• Equilibrate buffer AVE to room temperature for elution in step 11.
• Check that buffer AW1 and buffer AW2 have been prepared according to the
instructions.
• Add carrier RNA reconstituted in buffer AVE to buffer AVL.
E. PROCEDURE:
1. Pipette 560µl of prepared Buffer AVL containing carrier RNA into a
1.5ml microcentrifuge tube.
If the sample volume is larger than 140 µl, increase the amount of Buffer
AVL-carrier RNA proportionally (e.g., a 280 µl sample will require 1120 µl
Buffer AVL-carrier RNA) and use a larger tube.
2. Add 140 µl plasma, serum, urine, cell-culture supernatant or cell-free body fluid to
the Buffer AVL-carrier RNA in the microcentrifuge tube. Mix by pulse vortexing
for 15s.
To ensure efficient lysis, it is essential that the sample is mixed thoroughly
with Buffer AVL to yield a homogeneous solution. Frozen samples that
have only been thawed once can also be used.
3. Incubate at room temperature (15-25oC) for 10 min.
Viral particle lysis is complete after lysis for 10 min at room temperature.
Longer incubation times have no effect on the yield or quality of the
purified RNA. Potentially infectious agents and RNase are inactivated in
the Buffer AVL.
4. Briefly centrifuge the tube to remove drops from the inside of the lid.
5. Add 560 µl of ethanol (96-100%) to the sample, and mix by pulse vortexing for
15s. After mixing, briefly centrifuge the tube to remove drops from inside the lid.

45
 Only ethanol should be used since other alcohols may result in reduced
RNA yield and purity. Do not use denatured alcohol, which contains
other substances such as methanol or methylethyleketone. If the
sample volume is greater than 140 µl, increase the amount of ethanol
proportionally (e.g., a 280 µl sample will require 1120 µl of ethanol). In
order to ensure efficient binding, it is essential that the sample is mixed
thoroughly with the ethanol to yield a homogeneous solution.
6. Carefully Apply 630 µl of the solution from step 5 to the QIAamp Mini spin column
(in a 2 ml collection tube) without wetting the rim. Close the cap, and centrifuge at
6000 x g (8000rpm) for 1 min. Place the QIAamp spin column into a clean 2 ml
collection tube, and discard the tube containing the filtrate.
Close each spin column in order to avoid cross-contamination during
centrifugation.
Centrifugation is performed at 6000 x g (8000 rpm) in order to limit micro-
centrifuge noise. Centrifugation at full speed will not affect the yield or
purity of the viral RNA. If the solution has not completely passed through
the membrane, centrifuge again at a higher speed until all the solution has
passed through.
7. Carefully open the QIAamp Mini spin column, and repeat step 6.
If the sample volume was greater than 140 µl, repeat this step until all of
the lysate has been loaded onto the spin column.
8. Carefully open the QIAamp Mini spin column, and add 500µl of Buffer AW1. Close
the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini
spin column in a clean 2 ml collection tube (provided), and discard the tube
containing the filtrate.
It is not necessary to increase the volume of Buffer AW1 even if the
original sample volume was larger than 140µl.
9. Carefully open the QIAamp Mini spin column, and add 500µl of Buffer AW2. Close
the cap, and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min. continue
directly with step 11, or to eliminate any chance of possible Buffer AW2
carryover, perform step 10, and the continue with step 11.

46
10. Recommended: place the QIAamp Mini spin column in a new 2 ml collection tube
(not provided), and discard the old collection tube with the filtrate. Centrifuge at
full speed for 1 min.
11. Place the QIAamp Mini spin column in a clean 1.5 ml microcentrifuge tube (not
provided). Discard the old collection tube containing the filtrate. Carefully open
the QIAamp Mini spin column and add 60µl of Buffer AVE equilibrated to room
temperature. Close the cap, and incubate at room temperature for 1 min.
Centrifuge at 6000 x g (8000 rpm) for 1 min.
A single elution with 60 µl of Buffer AVE is sufficient to elute at least 90%
of the viral RNA from the QIAamp Mini spin column. Performing a double
elution using 2 x 40 µl of Buffer AVE will increase yield by up to 10%.
Elution with volumes of less than 30 µl will lead to reduced yields and will
not increase the final concentration of RNA in the elute.
12. Viral RNA is stable for up to one year when stored at -20oC or -70oC.

APPENDIX I
Preparation of Buffer AVL and addition of carrier RNA

Add 310ul Buffer AVE to the tube containing 310ug lyophilized carrier RNA to obtain a
solution of 1ug/ul. Dissolve the carrier RNA thoroughly, divide it into conveniently sized
aliquots, and store it at -20°C (Do not freeze-thaw the aliquots of carrier RNA more than
3 times.
Check Buffer AVL for precipitate, and if necessary incubate at 80°C until the precipitate
is dissolved. Calculate the volume of Buffer AVL-carrier RNA mix needed per batch of
samples by selecting the number of samples to be sim u ltan e o u sly
processed from Table 1.
For larger numbers of samples, volumes can be calculated using the following sample
calculation:
n x 0.56 ml = y ml
y ml x 10ul/ml = z ul
Where: n = number of samples to be processed simultaneously
y = calculated volume of Buffer AVL
z = volume of carrier RNA-Buffer AVE to add to Buffer AVL
Gently mix by inverting the tube 10 times. To avoid foaming, do not vortex.

47
Table 1. Volumes of Buffer AVL and Carrier RNA-Buffer AVE Mix Required for the
QIAamp Viral RNA Procedure
No. V ol. B uffe r V ol. ca rrier No. V o l. B u ffer V o l. carrie r
sa m ples A V L(m l) R N A -A V E (u l) sam ple s A V L (m l) R N A -A V E (ul)
1 0.56 5.6 13 7.28 72.8
2 1.12 11.2 14 7.84 78.4
3 1.68 16.8 15 8.40 84.0
4 2.24 22.4 16 8.96 89.6
5 2.80 28.0 17 9.52 95.2
6 3.36 33.6 18 10.08 100.8
7 3.92 39.2 19 10.64 106.4
8 4.48 44.8 20 11.20 112.0
9 5.04 50.4 21 11.76 117.6
10 5.60 56.0 22 12.32 123.2
11 6.16 61.6 23 12.88 128.8
12 6.72 67.2 24 13.44 134.4

Note: The sample-preparation procedure is optimized for 5.6 ug of carrier RNA per
sample. If less carrier RNA has been shown to be better for your amplification system,
transfer only the required amount of dissolved carrier RNA to the tubes containing
Buffer AVL. (Use of less than 5.6 ug carrier RNA per sample must be validated for each
particular sample type and downstream assay.)
Buffer AVL-carrier RNA should be prepared fresh, and is stable at 2-8oC for up to 48
hours. This solution develops a precipitate when stored at 2-8 oC that must be redis-
solved by warming at 80oC before use. Do not warm Buffer AVL-carrier RNA solution
more than 6 times. Do not incubate at 80oC for more than 5 minutes. Frequent warming
and extended incubation will cause degradation of carrier RNA, leading to reduced
recovery of viral RNA and eventually false negative RT-PCR results. This is particularly
the case with low-titer samples.
Buffer AW 1
Buffer AWl is supplied as a concentrate. Before using for the first time, add the
appropriate amount of ethanol (96-100%) as indicated on the bottle and in Table 2.
Buffer AWl is stable for 1 year when stored closed at room temperature, but only until
the kit expiration date.

48
 No. of Preps AW1 Concentrates Ethanol Final Volume
50 19ml 25ml 44ml

Buffer AW 2
Buffer AW2 is supplied as a concentrate. Before using for the first time, add the
appropriate amount of ethanol (96-100%) to Buffer AW2 concentrate as indicated on the
bottle and in Table 3.
Buffer AW2 is stable for 1 year when stored closed at room temperature, but only until
the kit expiration date.

No. of Preps AW2 Concentrates Ethanol Final Volume

50 13ml 30ml 43ml

REAL-TIME RT-PCR (rRT-PCR) PROTOCOL

A. PURPOSE:
This procedure assumes a basic familiarity with RRT-PCR assays for detection and
characterization of influenza virus.

49
B. PRINCIPLE:
The CDC Realtime RTPCR (RRT-PCR) Protocol for Detection and Characterization
of Influenza includes a panel of oligonucleotide primers and dual-labeled hydrolysis
(Taqman®) probes to be used in real-time RT-PCR assays for the in- vitro qualitative
detection and characterization of human influenza viruses in respiratory specimens and
viral cultures. The influenza A and B primer and probe sets are designed for universal
detection of type A and type B influenza viruses. Influenza A subtyping primer and
probe sets are designed to specifically detect contemporary human A/H1, human A/H3,
and A/H5 (Asian lineage) influenza viruses.

C. SCOPE:
This procedural format is utilized by the National Influenza Lab-Based Surveillance
Project under the supervision of National Institute of Health within the Virology
Department.

Limitations: These protocols were optimized using quantitative one-step probe RT-
PCR chemistries including Invitrogen SuperScript™III Platinum® One-Step Quantitative
Kit, Biorad /iScript One-Step RT-PCR Kit, and Ambion AgPath-ID™ One-Step RT-PCR
Kit that have been shown to produce comparable results on 96-well format
thermocycler systems such as Applied BiosystemsTM real-time PCR systems (7000,
7300, 7500, etc.), BioRad real-time PCR detection systems (iQTM or iQ5TM) or
Stratagene QPCR instruments (MX4000®, MX3000® or MX3005®).

Safety Information: Specimen processing should be performed in accordance with

pertaining national biological safety regulations.

D. PROTOCOL:

SPECIMENS:
Respiratory specimens including: broncheoalveolar lavage, tracheal aspirates, sputum,
nasopharyngeal or oropharyngeal aspirates or washes, and nasopharyngeal or
oropharyngeal swabs. Swab specimens should be collected only on swabs with a
synthetic tip (such as polyester or Dacron®) and an aluminum or plastic shaft. Swabs

50
with cotton tips and wooden shafts are not recommended. Specimens collected with
swabs made of calcium alginate are not acceptable.

Rejection criteria:
- Specimens not kept at 2-4°C (≤4 days) or frozen at -70°C or below.
- Inappropriate specimens not listed above.

NUCLEIC ACID EXTRACTION:

Performance of RT-PCR amplification based assays depends on the amount and
quality of sample template RNA. RNA extraction procedures should be qualified and
validated for recovery and purity before testing specimens. Commercially available
extraction procedures including QIAamp® Viral RNA Mini Kit, QIAamp® MinElute Virus
Spin Kit or RNeasy® Mini Kit (QIAGEN), Roche MagNA Pure Compact RNA Isolation
Kit, and Invitrogen ChargeSwitch® Total RNA Cell Kit have been shown to generate
highly purified RNA when following manufacturer’s recommended procedures for
sample extraction.
MATERIALS
Reagents:
1. One-step quantitative RT-PCR probe hydolysis (e.g., Taqman®) kit
a. Invitrogen SuperScript™ III Platinum® One-Step Quantitative Kit (cat#
11732-020 or 11745-100)
b. Biorad iScript One-Step RT-PCR Kit (cat# 170-8895)
c. Ambion AgPath-ID™ One-Step RT-PCR Kit (cat# AM1005)
2. Molecular grade sterile distilled water (RNase and DNase free)
3. Forward and reverse primers (40μM)
4. Dual-labeled probes (10μM)
5. Positive control RNAs
Supplies:
1. Laboratory marking pen
2. Cooler racks for 1.5 microcentrifuge tubes and 96-well 0.2ml PCR reaction
tubes
3. 20μl and 200μl adjustable pipettes and aerosol barrier tips
4. 0.2ml PCR reaction tube strips or plates

51
 5. Optical strip caps
6. Sterile, nuclease free 1.5 ml microcentrifuge tubes
7. Disposable powder-free gloves

Equipment:
1. Microcentrifuge
2. Vortex
3. Real-time PCR detection system with a 96-well format thermocycler reaction
block such as Applied BiosystemsTM real-time PCR systems (7000, 7300, 7500,
etc.), BioRad real- time PCR detection system (iQTM or iQ5TM) or Stratagene
QPCR instruments (MX4000®, MX3000® or MX3005®).

E. PROCEDURE

Preparation:
1. Avoiding sample contamination because of the sensitivity of fluorogenic 5’
nuclease assays, special precautions must be taken to avoid false positive
amplifications. The following precautionary steps are recommended:
a. Maintain separate areas for assay setup and handling of nucleic acids.
b. Maintain separate, dedicated equipment (e.g., pipettes, micro-centrifuges) and
supplies (e.g., micro-centrifuge tubes, pipette tips) for assay setup and handling
of extracted nucleic acids.
c. Wear a clean lab coat and powder-free disposable gloves (not previously worn)
when setting up assays.
d. Change gloves between samples and whenever you suspect they may be
contaminated.
e. Keep reagent and reaction tubes capped or covered as much as possible.
2. Equipment preparation
Work surfaces, pipettes, and centrifuges should be cleaned and decontaminated with
cleaning products such as 5% bleach, “DNAzap™” or “RNase AWAY®” to minimize risk
of nucleic acid contamination.

3. Reagent preparation

NOTE: Keep all reagents on cold rack during assay set up

52
 1 (a) Primers and probes
- Thaw frozen aliquots of primer and probes (Thawed aliquots of probes may be
stored in the dark up to 3 months at 2-8°C. Do not re-freeze probes).
- Vortex all primers and probes.
- Briefly centrifuge all primers and probes and then place in cold rack.

(b) Real time RTPCR reagents

- Place Master Mix and enzyme in cold rack
- Thaw the 2X Reaction Mix vial.
- Mix the 2X Reaction Mix by inversion.
- Briefly centrifuge 2x Reaction Mix and enzyme then place in cold rack

Tests for each RT-PCR run

1. Each sample RNA extract is tested by separate primer/probe sets: InfA, InfB,
AH1, AH3, AH5a, AH5b and RNaseP. The RNaseP primer and probe set targets the
human RNase P gene and thus serves as an internal positive control for human nucleic
acid.
2. No template controls (NTC) and positive template controls (PTC) for all
primer/probe sets should be included in each run.
3. Mock extraction control (MOCK) provides a secondary negative control that
validates the nucleic extraction procedure and reagent integrity.
Reaction setup:
Reaction assay mixtures are made as a cocktail and dispensed into the 96-well reaction
plate. Water, extracted nucleic acid or positive template controls, are then added to the
appropriate test reactions and controls.
1. Label one 1.5 ml microcentrifuge tube for each primer/probe set.
2. Determine the number of reactions (N) to set up per assay. It is necessary to
make excess reaction cocktail to allow for the NTC, PTC, MOCK reactions and pipetting
error. See below:
- If number of samples (n) including controls = 1 to 14, then N = n + 1
- If number of samples (n) including controls > 15, then N = n + 2
3. Master Mix: calculate the amount of each reagent to be added for each
primer/probe set reaction master mix. The calculations are as follows:

53
 Invitrogen/BioRad Ambion AgPath
2X PCR Master Mix N x 12.5 μl N x 12.5 μl
RT Mix N x 0.5 μl N x 1.0 μl
Forward primer (0.8 μM final concentration) N x 0.5 μl N x 0.5 μl
Reverse primer (0.8 μM final concentration) N x 0.5 μl N x 0.5 μl
Probe (0.2 μM final concentration) N x 0.5 μl N x 0.5 μl
Nuclease free water N x 5.5 μl N x 5.0 μl
Total volume N x 20.0 μl N x 20.0 μl
4. In the assay set up area, dispense reagents into labeled 1.5 ml microcentrifuge
tubes. After addition of the water, mix reaction mixtures by pipetting up and down. Do
not vortex.
5. Centrifuge for 5 sec to collect contents at bottom of the tube, and then place the
tube in cold rack.
6. Set up reaction strip tubes or plates in 96-well cooler rack.
7. Dispense 20μl of each master mix into each well going across the row as below:
Example Test setup:
1 2 3 4 5 6 7 8 9 10 11 12
A InfA InfA InfA InfA InfA InfA InfA InfA InfA InfA InfA
B InfB InfB InfB InfB InfB InfB InfB InfB InfB InfB InfB
C AH1 AH1 AH1 AH1 AH1 AH1 AH1 AH1 AH1 AH1 AH1
D AH3 AH3 AH3 AH3 AH3 AH3 AH3 AH3 AH3 AH3 AH3
E RNaseP RNaseP RNaseP RNaseP RNaseP RNaseP RNaseP RNaseP RNaseP RNaseP RNaseP
F AH5a AH5a AH5a AH5a AH5a AH5a AH5a AH5a AH5a AH5a AH5a
G AH5b AH5b AH5b AH5b AH5b AH5b AH5b AH5b AH5b AH5b AH5b
H

Example Sample Setup:

1 2 3 4 5 6 7 8 9 10 11 12
A NTC S1 S2 S3 S4 S5 S6 S7 S8 S9 Mock PTC
B NTC S1 S2 S3 S4 S5 S6 S7 S8 S9 Mock PTC
C NTC S1 S2 S3 S4 S5 S6 S7 S8 S9 Mock PTC
D NTC S1 S2 S3 S4 S5 S6 S7 S8 S9 Mock PTC
E NTC S1 S2 S3 S4 S5 S6 S7 S8 S9 Mock PTC
F NTC S1 S2 S3 S4 S5 S6 S7 S8 S9 Mock PTC
G NTC S1 S2 S3 S4 S5 S6 S7 S8 S9 Mock PTC
H

54
 Note: Negative template controls (NTC) should be added first (column 1) before any of
the samples are added to check for contamination in the master mix. MOCK should be
added after the samples have been added (column 11) to check for cross-contamination
during sample preparation or addition. Positive template controls (PTC) should be
added last after all samples and NTCs are sealed.
8. Before moving the plate to the nucleic acid handling area, set up the NTC
reactions for column 1 in the assay set-up area. As shown above, samples can be
added by column.
9. Pipette 5 μl of nuclease free water into the NTC wells. Cap NTC wells.
10. Cover the reaction plate and move the reaction plate to the nucleic acid handling
area.
11. Vortex the tubes containing the samples for 5 sec. Centrifuge tubes for 5 sec.
12. Set up the extracted nucleic acid samples in the cold rack.
13. As shown above, samples can be added by column. Pipette 5 μl of the first
sample into all the wells labeled for that sample (for example, Sample “S1” as shown
above). Change tips after each addition
14. Cap the column to which the sample has been added. This will help to prevent
sample cross contamination and enable you to keep track of where you are on the
plate.
15. Change gloves when necessary to avoid contamination.
16. Repeat steps 13. through 15. for the remaining samples.
17. Add 5 μl of mock extracted sample to the MOCK wells (column 11). Cap MOCK
wells.
18. Finally, pipette 5 μl of positive template control RNA into all PTC wells. Cap PTC
wells.
19. If using 8-tube strips, label the TAB of each strip to indicate sample position (DO
NOT LABEL THE TOPS OF THE REACTION TUBES!). Briefly centrifuge tube strips
for 10-15 seconds. Return strip tubes to cold rack.
If using plates, centrifuge at 500 x g for 30 seconds at 4°C. Return to cold rack.

RT-PCR amplification conditions:

The reaction volume is 25μl. Program the thermocycler as follows:
Invitrogen/BioRad Ambion AgPath

55
 Reverse Transcription 50°C for 30 min 50°C for 30 min
Taq inhibitor inactivation 95°C for 2 min 95°C for 10 min
PCRamplification (45 cycles) 95°C for 15 sec 95°C for 15 sec
55°C for 30 sec* 55°C for 30 sec*

• Fluorescence data (FAM) should be collected during the 55°C incubation step.
INTERPRETATION OF RESULTS:

1. The NTC reactions for probe/primer sets should not exhibit fluorescence growth
curves that cross the threshold line. If a false positive occurs with one or more of the
primer and probe NTC reactions, sample contamination may have occurred. Invalidate
the run and repeat the assay with stricter adherence to the procedure guidelines.
2. All clinical samples should exhibit RP reaction curves that cross the threshold
line at or before 35 cycles, thus indicating the presence of sufficient RNA from human
RNase P gene indicating the specimen is of acceptable quality. However, it is possible
that some samples may fail to give positive reactions due to low cell numbers in the
original clinical sample. Also, samples taken from animal/avian species or cell culture
typically exhibit either no RP reaction, or a weak RP reaction. Failure to detect RNase P
in any of the clinical samples may indicate:
a. Improper extraction of nucleic acid from clinical materials resulting in loss
of RNA or carry-over of RT-PCR inhibitors from clinical specimens
b. Absence of sufficient human cellular material in sample to enable
detection
c. Improper assay set up and execution
d. Reagent or equipment malfunction

56
 Primary Growth Curve

3. The MOCK should NOT exhibit fluorescence growth curves for primer/probe sets
InfA, InfB, AH1, AH3, AH5a, AH5b or MOCK that cross the threshold line within 45
cycles. If any influenza specific primer/probes exhibit a growth curve that crosses the
threshold line, interpret as follows:
a. Contamination of RNA extraction reagents may have occurred. Invalidate
the run and confirm reagent integrity of RNA extraction reagents prior to further testing.
b. Cross contamination of samples occurred during RNA extraction
procedures or assay setup. Invalidate the run and repeat the assay with stricter
adherence to procedure guidelines.

4. PTC reactions should produce a positive result with the InfA, InfB, AH1, AH3,
H5a, H5b and RP reactions between 20 and 30 cycles. If expected positive reactivity is
not achieved, invalidate the run and repeat the assay with stricter adherence to
procedure guidelines. Determine the cause of failed PTC reactivity, implement
corrective actions, and document results of the investigation and corrective actions. Do
not use PTC reagents that do not generate expected result.

57
5. When all controls meet stated requirements, a specimen is considered
presumptive positive for influenza A virus if the InfA reaction growth curves cross the
threshold line within 40 cycles. If the reaction for influenza A is positive, it should also be
positive for one of the following subtypes: AH1, AH3, or AH5a and/or AH5b. A specimen
is considered presumptive positive for influenza A/H1, A/H3 or A/H5 (Asian lineage)
virus if BOTH the InfA and the respective subtype (H1, H3 or AH5a AND AH5b) reaction
growth curves cross the threshold line within 40 cycles. If a specimen is positive for InfA
and only one of the AH5 reactions, or positive of InfA only, contact CDC for guidance.
6. When all controls meet stated requirements, a specimen is considered
presumptive positive for influenza B virus if InfB reaction growth curves cross the
threshold line within 40 cycles.
7. When all controls meet the stated requirements, a specimen is considered
negative for influenza virus if growth curves for neither InfA nor InfB cross the threshold
within 40 cycles.
Limitations:
1. Analysts should be trained and familiar with testing procedures and interpretation
of results prior to performing the assay.
2. A false negative result may occur if inadequate numbers of organisms are
present in the specimen due to improper collection, transport or handling.
3. A false negative result may occur if an excess of DNA/RNA template is present in
the in the reaction. If inhibition of the RP control reaction is noted for a particular
sample, extracted RNA can be tested at 2 or more dilutions (e.g., 1:10 and 1:100) to
verify the result.

58
 4 SEQUENCING

What is sequencing?
“Sequencing” means determining the order of nucleotides of a piece of DNA. Historically
there were two main methods of DNA sequencing:
• Maxam & Gilbert, using chemical sequencing
• Sanger, using dideoxynucleotides.

Modern sequencing equipment uses the principles of the Sanger technique.

Principle of Capillary Electrophoresis

1Sequence Analysis of Influenza Viruses

Antigenic analysis by traditional serologic techniques continues to be the method of

choice for the comparative analysis of large numbers of influenza viruses. However,
genetic analysis of influenza virus genes coding for the HA and NA proteins helps to
monitor the evolution of influenza viruses and to determine the degree of relatedness
between viruses isolated in different geographical areas and during different times of
the year. By comparing sequence data from isolates collected during one influenza
season, the worldwide spread of a variant virus can be followed closely. Although
recommendations for the composition of the trivalent influenza virus vaccine are
primarily based on the antigenic analysis of a large number of influenza viruses isolated
in laboratories collaborating in the world-wide surveillance of influenza activity,
sequence analysis of a subset of these viruses has provided very useful supplementary
information.

59
Example of automated sequencing output

Good quality sequence

60
ANNEXURES

61
ANNEXURE I: PERSONAL PROTECTIVE EQUIPMENT
The level of respiratory protection required when sampling depends on a number of
factors including:
• the type of sample to be taken (e.g. sampling for blood is less risky than taking
a throat swab which may cause the patient to cough);
• the situation (e.g. taking a swab from a dead bird in the open air requires less
protection than sampling inside a poultry shed);
• the type of respiratory risk (droplets, aerosols and dusts require different types
of protection).
There are many types of respirators and masks available and the different types offer
very different levels of respiratory protection. However it must be accepted that in some
situations high efficiency respirators will not be available and basic gauze masks may
be all that can be used.
• Appropriate procedures should be used to select a particulate respirator that fits
well and a user seal check (fit check) should be performed each time a
disposable particulate respirator is worn.
• Disposable particulate respirators, although similar in appearance to surgical
masks, differ significantly from surgical masks because they are specifically
designed to protect the wearer from exposure to airborne infectious diseases
by sealing tightly to the face and filtering infectious particles from the air.
• If a particulate respirator is not available, a tightly fitting surgical or procedure
mask should be used.
• Surgical and procedure masks do not provide protection against small-particle
aerosols (droplet nuclei), and aerosol-generating procedures should not be
performed if a particulate respirator is not available.

Some common terms:

Fit test: evaluating the fit of a respirator on an individual.

High efficiency particulate air (HEPA) filter: means a filter that is at least 99.97%
efficient in removing particles of 0.3 micrometers in diameter.

NIOSH: The National Institute for Occupational Safety and Health is the USA Federal
agency responsible for conducting research and making recommendations for the
prevention of work related injury and illness.

62
 ANNEXURE II: HAND HYGIENE
When they are visibly dirty or contaminated with proteinaceous material, or visibly soiled
with blood or other body fluids, wash hands with soap and water. If hands are not visibly
dirty, use an alcohol-based preparation. Ethyl alcohol has greater activity against
viruses than isopropyl alcohol, therefore, ethyl alcohol-based hand disinfection products
may be preferred over isopropyl alcoholproducts in settings where transmission of HPAI
is likely.
a) Soap and water
Liquid, bar, leaflet or powdered forms of plain soap are acceptable when washing hands
with a non-antimicrobial soap and water. Wet hands with water and apply the amount of
product necessary to cover all surfaces. Vigorously perform rotational hand rubbing on
both palms and interlace fingers to cover all surfaces.

63
Rinse hands with water and dry thoroughly with a single use towel. Use the towel to turn
off the tap/faucet. Make sure the hands are dry. Use a method that does not
recontaminate hands. Make sure towels are not used multiple times or by multiple
people. Use running and clean water for hand hygiene whenever possible. Avoid using
hot water, as repeated exposure to hot water may increase the risk of dermatitis. When
bar soap is used, small bars of soap in racks that facilitate drainage should be used.

b) Hand cleansers
When using an alcohol-based formulation (or another disinfectant based hand
cleanser), apply a palmful of the product and cover all surfaces of the hands. Rub hands
until they are dry.

64
 ANNEXURE III: LABORATORY SAFETY

BIOSAFETY
Safety is the responsibility of everybody working in the laboratory and safe procedures
must be adhered to at all times. The following statements provide some basic rules for
safety in the laboratory.
• Blood and body fluid precautions are to be consistently used with all clinical
specimens of blood or other potentially infectious material (Universal
Precautions).
• Use barrier protection at all times (laboratory coats, gloves, or other appropriate
barriers).
• Good laboratory practices should be followed. Eating, drinking, or smoking is
not permitted in the laboratory.
• Mechanical pipetting devices are to be used for all liquids in the laboratory.
Mouth pipetting is dangerous.
• Biosafety Level 2 practices should be followed when handling all specimens.
Class I or II biological safety cabinets or other physical containment devices
should be used for all manipulations of agents that cause splashes or aerosols
of infectious materials.
• Adequate and conveniently located biohazard containers for disposal of
contaminated materials should be available.
• Countertops and surfaces of biological safety cabinets should be wiped with a
disinfectant (0.5 sodium hypochlorite is preferred) routinely after working with
infectious agents or clinical specimens.
Wash hands often -- Especially before leaving the laboratory and before eating.
Protective clothing should be removed before leaving the laboratory.
HAZARDOUS CHEMICALS
Acetone will be used for fixation of cell cultures in the micro-neutralization assay.
Caution: acetone is extremely flammable as liquid and vapor, and harmful if inhaled.
Eye and Skin contact: Immediately flush eyes with plenty of water; get medical
attention
Ingestion: Get medical attention
Inhalation: Remove to fresh air

Hydrogen peroxide will be used as substrate for the HRPO-conjugate in immuno-

peroxidase staining.
Caution: hydrogen peroxide may cause severe irritation of skin, eyes, and mucous
membranes and respiratory irritation.
Eye and Skin contact: Flush with plenty of water; get medical attention.
Ingestion: Give large volumes of water or milk if conscious;
DO NOT INDUCE VOMITING; get medical attention.
Inhalation: Remove to fresh air

65
Sodium azide is added as a preservative in a concentration of 0.1% to some of the
reagents included in the WHO Influenza Reagent Kit and the monoclonal antibodies.
Caution: sodium azide may be fatal if swallowed, inhaled, or absorbed through skin.
Eye and skin contact: Flush with plenty of water, remove contaminated clothing
Inhalation: Remove to fresh air, give artificial respiration or oxygen if required.
Ingestion: Give large quantities of water if conscious and not convulsive. Induce
vomiting; get medical attention.

Chloroform is used in the alternate methods for RNA extraction and the dsDNA
purification.
Caution: Chloroform is a possible human carcinogen and may be fatal if swallowed or
inhaled. Inhalation or ingestion may cause nausea, vomiting, headache, or dizziness.
Eye contact may cause corneal damage. Skin contact may cause irritation or dermatitis.
Eye and skin contact: Flush with water for at least 15 minutes.
Ingestion: If swallowed and conscious, give large amounts of water. Induce vomiting.
Call a physician.
Inhalation: Remove to fresh air. Give oxygen if breathing becomes difficult. Call a
physician.

Ethidium Bromide is an intercalating dye that will be used to visualize DNA in agarose
gels.
Caution: Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves
should be worn when working with solutions containing this dye, and a mask should be
used when weighing it out.
Eye and skin contact: Flush with water for at least 15 minutes.
Ingestion: Call physician immediately. Do not induce vomiting unless instructed by
medical personnel
Inhalation: Remove to fresh air. Give oxygen if breathing becomes difficult. Call a
physician.

Phenol is used in the alternate methods for RNA extraction and dsDNA purification.
Caution: Phenol is highly corrosive and can cause severe burns. This agent is rapidly
absorbed through the skin. Wear gloves, lab coat, and safety glasses when handling
phenol. All manipulations should be carried out in a chemical hood.
Eye and skin contact: Rinse with large volumes of water and wash with soap and
water. Do not use ethanol.
Ingestion: If conscious, give activated charcoal in water, olive oil, or margarine; then
induce vomiting. Call a physician.
Inhalation: Remove to fresh air. Give oxygen if necessary. Call a physician.

66
STERILIZATION

Sterilization means complete killing or removal of all living micro organisms from an
article. Sterilization is done by physical agents and chemical agents. Physical agents
are heat, low temperature, filtration and radiations.

Heat

Fire and boiling water have been used for sterilization and disinfection since the time
of Greeks, and heating is one of the most popular ways to destroy microorganisms.
Either moist or dry heat may be applied. Moist heat readily kills viruses, bacteria and
fungi. Exposure to boiling water for 10 minutes is sufficiently to destroy vegetative
cells and eukaryotic spores. Unfortunately the temperature of boiling water 100 c is
not high enough to destroy bacterial endospores that may survive hours of boiling
.therefore boiling can be used for disinfection of drinking water and objects not
harmed by water, but boiling does not sterilize. Because heat is so useful in
controlling microorganisms, it is essential to have precise measure of heat killing
efficiency.

Moist heat Sterilization

Moist heat sterilization must be carried out at temperature above 100oC. In order to
destroy bacterial endospores and this requires the use of saturated steam under
pressure. Steam sterilization is carried out with an autoclave.

Autoclave

Autoclave is a device somewhat like a fancy pressure cooker. The development of

the autoclave by Chamberland in 1884 tremendously stimulated the growth of
microbiology.

Principle of autoclave (How to work)

Water is boiled to produce steam, which is released through a jacket and into the
autoclave chamber. The air initially present in the chamber is forced out until the
chamber is filled with saturated steam and the outlets are closed. Hot, saturated
steam continues to until the chamber reaches the desired temperature and pressure,
usually 121o C and 15 pounds of pressure. At this temperature, saturated steam
destroys all vegetative cells and endospores in a small volume of liquid within 10 to
12 min. Treatment is continued for about 15 min. to provide a margin of safety. Moist
heat is thought to kill so effectively by degrading nucleic acid and by denaturing
enzymes and other essential proteins. It also may disrupt cell membranes.

Important Note

Autoclaving must be carried out properly or the processed material will not be
sterilized. If all air has not flushed out the chamber, it will not reach 121 oC even
though it may reach a pressure of 15 pounds. The chamber should not be packed

67
too tightly because steam needs to circulates freely and contact everything in the
autoclave.

Control Check

A biological indicator is often autoclaved along with other material. This indicator
commonly consists of a culture tube containing an ampoule of medium and paper
strip covered spores of bacillus stearothermophilus or clostridium PA3679. After
autoclaving ampoule aseptically broken and culture incubated for several days. If the
test bacterium does not grow in the medium, the sterilization run has been
successful. Sometime either special tape or paper indicator strip that change color
upon sufficient heating is autoclaved with a load of material. If the color changes
after autoclaving, the material is supposed to be sterilized.

Following items can be sterilized within an autoclave

Liquid solutions, Rubber items, Plastic material, culture media and cotton swabs etc.

Procedure of Autoclaving

1. Fill the autoclave with tap water upto the level of basket.
2. Place the material in the basket.
3. Close the autoclave door and draining valve.
4. Switch on the autoclave and open the air valve until the air is removed completely.
5. Close the air valve on releasing the steam.
6. Start the stop watch on reaching the temp at 121o C at 15 pound pressure.
7. Switch off the autoclave after 15 min and open the air valve until removal of
complete steam.
8. Open the door and remove the material.

Dry Heat Sterilization

Many objects are best sterilized in the absence of water by Dry Hot Sterilization. The
item to be sterilized is placed in an oven at 160 to 170o C for 2 to 3 hours.

Principle

Microbial death apparently results from the oxidation of the cell constituent and
denaturation of protein although dry heat is less effective than moist heat.

Following items can be sterilized with Dry heat

Glassware, powders, oils, scissors, forceps, etc.

Filtration

Filtration is an excellent way to reduce the microbial population in solutions of heat

sensitive material, and sometimes it can be used to sterilization rather than directly

68
destroying contaminating microorganisms, the filters simply removes them. There
are two types of filters. Depth Filters, Membrane Filters.

Membrane Filters

These circular filters are porous membrane, a little over 0.1 mm thick, made of
cellulose acetate, cellulose nitrate, and polycarbonate. Although a wide variety of
pour sizes are available, membranes with pores about 0.2 micron in diameter are
used to remove most vegetative cells but not viruses, from solutions ranging in
volume from 1 ml to many liters.

Radiations

Following radiations are used in sterilization

• Ultraviolet (UV) Radiation.

• Ionizing Radiation

Ultraviolet Radiation

Around 260 nm is quite lethal but does not penetrate glass, dirt films, water, and
other substances very effectively. Because of this disadvantage, UV radiation is
used as sterilization only in few specific situations. UV lamps are sometime placed
on the ceilings of rooms or in biological safety cabinets to sterilize the air and any
exposed surfaces. Because UV radiation burns the skin and damages eyes, people
working in such areas must be certain the UV lamps are off when the areas are in
use.

Ionizing Radiation

Ionizing radiation is an excellent sterilizing agent and penetrates deep into objects. It
will destroy bacterial endospores and vegetative cells, both prokaryotic and
eukaryotic. However, ionizing radiation is not always as effective against viruses.

Sterilization by Chemical Agent

On the basis of potency these chemical are divided in to two types;

• Disinfectants
• Antiseptic

Disinfectants

These chemicals are used for killing of microorganisms. these are more
concentrated, more irritant and more poisonous. These chemicals cannot be used
on living surface.

69
Antiseptics

These chemicals are used only for the prevention of microorganisms. These are
diluted, non irritant and mild. These chemicals can be used on living surface.

These chemicals act on the cytoplasmic membrane of microbes and produce

various changes which damage to these microbes. For example,

• Phenol and acid

• Alcohols
• Halogens
• Aldehydes
• Surface active agents i.e. soaps and detergents

70
 Worksheet for SOP: MOL-002
National Influenza Lab Based Surveillance Project NILSP, NIH
Developed: 14th August, 2008 Revised: 17th September, 2008

PROTOCOL#: MOL-007-01 DATE: - Procedure #:RRT-0 -09

PURPOSE: - Seasonal flu PERFORMED BY: _Nazish Badar_____

SAMPLES: Sample testing

INSTRUMENT #: ABI-7500-1
1. Materials and Methods:
1.1. Method: (Circle one)
RT-PCR N-PCR RT-Multiplex

Reagents and Supplies:

1.1.1. Invitrogen SSIII PlatinumTaq RT-PCR kit (Lot# 289834 )
1.1.2. Taq (Lot# 289834 )
1.1.3. dNTPs (Lot#: 359560 )
1.1.4. PCR Tubes (Lot # ABI )
1.1.5. NA-free water (Lot# 0703002 )
1.1.6. Primer working dilution/volume (Date prepared: . by: )
1.1.7. Probe working dilution (Date prepared: . by: )
1.2. Plate Setup
1 2 3 4 5 6 7 8 9 10 11 12

A- NTC NTC NTC NTC

B-

C-

D-

E-

F-

G-

H- PTC PTC PTC PTC

1.3. Master Mix Preparation by

1.3.1. Mix: Calculate from relevant Excel sheet
1.3.2. Reaction volume: 25 50 100 (Circle one) Other: µl

71
Influenza ASeasonal &H5 RT-PCR(CDCProtocol, 2007)
No. of Rx 4 5
Master mix Volume(ul) Volume/Tube Total

2XPCRMaster Mix, 12.5 12.5 50

Enzyme(RT) Mix 0.5 0.5 2 2.5
Forward primer (0.8 µM final conc.) 0.5 0.5 2 2.5
Reverse primer (0.8 µM final conc.) 0.5 0.5 2 2.5
Probe (0.2 µM final conc.) 0.5 0.5 2 2.5
PCRWater 5.5 5.5 22
Mix without water 20 20 80
Template volume/RNA 5
Reaction volume 25

Reverse Transcription - Hold 50Cfor 30 min

Taqinhibitor Inactivation 95°Cfor 2 min

72
 National Lab based Influenza Surveillance; Laboratory Log Book
Hospital __________ District ____________ Province ________________ Country _______________

Hospital Patient Lab Nature of Sample Tissue Real Time Other

S. No Date Remarks
ID (if any) ID ID Sample Appearance Culture PCR Tests