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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.

While antigenic drift occurs continuously. as well as systemic symptoms such as headache. and coryza. 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. By definition. Influenza A and B viruses have 8 influenza vaccine virus strains.g. ANTIGENIC CHANGE The hallmark of human influenza viruses is their ability to undergo antigenic change. birds or pigs) can infect a human directly without undergoing genetic reassortment. HA/NA that have not been circulating among humans in recent years) emerges among humans. 2) an influenza virus from another species (e. muscle aches. The emergence of new strains necessitates the frequent updating of Influenza is a negative-strand RNA virus with a segmented genome. INFLUENZA DISEASES Influenza is an acute respiratory disease caused by influenza type A or B viruses. Acute illness generally lasts about 1 week. Drift results from the accumulation of point mutations in the genes during viral replication. although malaise and cough may continue for 2 weeks or longer. of the world’s population has no antibody against the new virus. The incubation period ranges from 1-4 days. antigenic shift occurs infrequently and unpredictably. or 3) a non-human virus may be passed from one species (e. Common complications of influenza infection include secondary . Since antigenic shift results in the emergence of a new influenza virus. If the new virus is capable of causing illness in humans and sustained person-to-person transmission. shift has occurred when an influenza A virus bearing either a “novel” HA protein or a combination of novel HA and NA proteins (i. Both influenza A and B viruses undergo antigenic drift leading to new virus strains. relatively continuous change in the virus HA and NA proteins. such as a pig. or even all. a pandemic may occur. sore throat. birds) through an intermediate animal host.e. to humans. which occurs in two ways: antigenic drift and antigenic shift. and fatigue. including the surface proteins hemagglutinin and neuramimidase In addition to antigenic drift. Typical features of influenza include abrupt onset of fever and respiratory symptoms such as cough (usually nonproductive). 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.. genes that code for 10 proteins.g. a large proportion. influenza type A viruses can undergo a more dramatic and abrupt type of antigenic change called an antigenic shift. The clinical severity of infection can range from asymptomatic infection to primary viral pneumonia and death.. Antigenic drift is a process of gradual.

Although temperate regions of the 2500 % positive 30 2000 25 1500 world experience a seasonal peak in 20 influenza activity. cumulative number of isolates.bacterial pneumonia and exacerbation of underlying chronic health conditions and. and Reye’s syndrome (generally associated with the use of aspirin in children and adolescents with influenza-like illness). 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. In temperate climates. percent of specimens testing positive for influenza. 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. influenza 45 activity typically peaks during May through 40 3000 Number of isolates 35 August. the incidence of infections. Brazil 1997 -2001 http://www. Although influenza epidemics may also occur regularly in subtropical and tropical regions. ** Data from FluNet . Seasonality of influenza virus isolation United States. and spring.gov. winter. influenza viruses can be 15 1000 10 isolated year-round. and multiple peaks of Brazil. 1998 – 2001* Periods of peak activity typically occur sometime between December and March and last for 6-8 weeks. 1998 and . toxic- shock syndrome. disease patterns in these regions have been less well established. In temperate regions 3500 of the Southern Hemisphere. influenza viruses Week isolates %positive frequently are isolated during a several Hong Kong. and levels of protective antibody in the population. 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. 1998 – 2001 influenza activity The timing of influenza activity around the 7000 6000 world varies depending on the climate of the 25 region.info. cumulative number of isolates and Timing and seasonality of regional percent of specimens testing positive for influenza. cumulative Seasonality of Influenzanumber of isolates In United States.htm. In the Northern Hemisphere’s 0 1 3 5 7 9 1 3 5 7 9 1 3 5 7 9 1 3 5 7 9 1 1 3 5 7 9 1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 4 4 4 4 4 5 temperate regions. Uncommon complications include myositis. 1997 – 2001** activity during the same year have been seen in some areas.2001 month period in the fall. 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. epidemics of influenza occur nearly annually. in children. EPIDEMIOLOGY OF INFLUENZA In temperate climate regions. myocarditis. otitis media.hk/dh/diseases/influenza. 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.

At both extremes of the age spectrum. rates of hospitalizations are elevated. During non pandemic years. presumably after influenza was introduced by infected persons from an area where influenza was in season. Influenza-related deaths can result from pneumonia. Among persons <65 years. For example. 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. outbreaks of influenza have occurred on cruise ships during the summer months in temperate climates. or immune compromising conditions). and deaths. diabetes.000 during A(H1N1). hospitalizations. the highest rates of hospitalizations from influenza-related causes occur among children <2 years of age.or B-predominant years. renal failure. the average estimated rates of influenza-related hospitalization for persons >65 years were 228/100.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. physician visits. rates of hospitalizations are higher among those with chronic medical conditions than healthy persons of the same age group. In general. 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. even among those without chronic medical conditions. and lower among adults. In the current virologic era. . Although the highest rates of illness occur among school-aged children. and among those ≥65 years of age. In a study of U. influenza infection rates among adults generally range from 1% to 15%. rates of primary influenza illness are highest among school-aged children. Pregnant women also appear to be at increased risk of complications from influenza. among persons of any age with certain chronic medical conditions (including chronic heart disease. International or large group travel may result in exposure to influenza virus outside of an expected time period. Among persons of the same age group.000 during A(H3N2)-predominant years and 125/100. Regardless of the overall seasonality of influenza in an area.000 and 20/100. the respective hospitalization rates were 42/100. An increased risk of influenza-related deaths among pregnant women first was observed during the 1918-1919 and 1957-1958 pandemics. 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. exacerbations of existing cardiopulmonary conditions or exacerbations of other chronic conditions.000. exceeding 30% in some years. lung disease such as asthma. Impact of influenza Increases in the circulation of influenza viruses have been associated with elevations in acute respiratory illnesses. however.S. hospital discharge data from 1969-1995.

illness rates among school-aged children have been shown to rise and decline earlier in the outbreak than rates among adults.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. the World Health Organization (WHO). Influenza outbreaks among school children can herald the start of influenza activity in a community. Pandemic influenza In contrast to the annual seasonal epidemics of influenza seen in some global regions. In all three pandemics. In all age groups. the “Spanish Flu” (H1N1) pandemic led to >500. Currently. there are 112 National Influenza Laboratories in 83 countries that participate in the WHO influenza surveillance system. the 1957-58 “Asian Flu” (H2N2) pandemic was associated with an estimated 70. In 1948. Three pandemics occurred in the 20th Century. persons <65 years accounted for a larger proportion of deaths than is usually seen in interpandemic influenza seasons. Influenza may also be spread less commonly by contaminated fomites or by direct touching. 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.000 deaths while the 1968-69 “Hong Kong Flu” (H3N2) pandemic was associated with an estimated 34. During 1918-19. Nearly half of the deaths were among persons 20-40 years of age and case-fatality rates of 30% were reported among pregnant women. upon its founding. became responsible for administration of the system. pandemics of influenza have occurred infrequently and at irregular intervals. In the United States. WORLD HEALTH ORGANIZATION INFLUENZA PROGRAM An international network for WHO Influenza Surveillance Network – influenza surveillance first was National Influenza Centers envisioned in 1947. sneezing or talking.000 deaths in the United States and >20 million deaths worldwide. Children are important in the spread of influenza within communities and within households. During some community outbreaks. 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. .000 deaths.

surveillance systems each report estimated levels of overall influenza activity. the ease of collecting and reporting the data. Four WHO Collaborating Centers for Reference and Research on Influenza in Atlanta. physician assistants. Such data are particularly useful for assessing the health impact that influenza is having in the community. 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. EISS. WHO recommends that the isolate be shipped to one or more collaborating centers for further analysis. In the WHO and EISS systems. or other health care staff. One of the most common outcomes monitored in influenza morbidity surveillance systems is influenza-like illness (ILI). Information collected through this system is used to develop annual vaccine strain selection recommendations.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. and U. estimated levels of activity are reported for countries or regions of a country. Melbourne. 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. and the potential for collecting data that are reasonably representative of the groups of interest.S. If a national laboratory is not able to subtype an influenza virus using available reagents. These kits are sent to all WHO national laboratories to assist them in the identification of circulating influenza A subtypes. London. Influenza activity level assessment The WHO. including the availability of existing data sources. A variety of surveillance methods and data sources can be used. or emergency departments. these assessments are not done in a . 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. the healthcare structure. Persons reporting the data can be physicians. the potential for sustainable reporting. Below are examples of systems currently in use worldwide. but other outcomes such as acute respiratory illness (ARI) or hospitalizations can be monitored. outpatient or hospital associated outpatient clinics. Currently. university student health clinics. DISEASE SURVEILLANCE – MORBIDITY REPORTING Morbidity surveillance is used to detect and monitor patterns of illness related to circulation of influenza viruses. nurse practitioners. and Tokyo serve as influenza reference laboratories and conduct detailed molecular and antigenic analysis of a subset of viruses from around the world. The choice of method should take several considerations into account.

they do provide a level of local interpretation of influenza activity and surveillance data that may be lacking otherwise. for example. or very high.ILI activity above baseline values with laboratory confirmed cases in a limited area Regional activity . A more unified approach to assessing local levels of activity would provide reports that are more directly comparable. high. analysis of discharge data may be more appropriate for studies rather than weekly surveillance. Both admission data and discharge data are prone to coding biases and errors. FluNet. The EISS system activity levels are similar to those used by WHO. medium. Nonetheless. The activity levels in the WHO system are reported as: No 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 .standardized way and can be made subjectively. which can be available sooner than discharge data.no influenza viral isolates or clinical signs of influenza activity Sporadic . The intensity of influenza activity is described as low. in the proportion of viruses from one subtype.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. EISS incorporates a second variable to describe the intensity of influenza activity in addition to the geographic distribution of influenza viruses. Collection of hospital discharge diagnosis is useful in documenting the impact of influenza. Hospital-based surveillance Hospital-based surveillance for influenza can be useful in tracking levels of severe illness related to influenza. However. Other sources of data . analysis may be time consuming. As an alternative. It is also helpful to collect viruses from hospitalized patients since they can differ from those collected from outpatients. Since admission data often may not be coded or available in computerized files. some surveillance systems have monitored hospital admission diagnosis or chief complaint data.an isolated case of ILI or laboratory/culture confirmed cases in a limited area Local outbreak . the availability of such data usually is delayed. However. and therefore.

Information that influenza viruses have begun to circulate also can be used to prompt members of target groups to receive vaccine. or increases in ambulance calls. outcomes such as absenteeism are highly nonspecific. and disease impact. knowledge of the seasonal pattern of influenza viruses and disease is essential for the effective implementation of influenza disease prevention programs. Surveillance provides most of such information. including the seasonality. Viral surveillance is essential for identifying new influenza virus variants to replace vaccine strains. 3) there are common genetic changes among the new variants suggesting the emergence of a new lineage. Nonetheless. sales of over-the-counter or prescription medicines used to treat influenza. trends in such outcomes should be interpreted with caution. and therefore. 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. Each of these systems has its own strengths and weaknesses. and to focus hospital infection control efforts. A vaccine strain is replaced when 1) the antigenicity of newly identified variants is significantly different from the current vaccine strains. Situation in Pakistan . Since the transmission of influenza within hospitals can mimic community activity. In particular. and 4) the variant appears to be associated with disease. Scientific studies The local and regional epidemiology of influenza. Vaccine strain selection Both viral and disease surveillance provide important information for selecting influenza vaccine strains. surveillance findings can help institutions to anticipate nosocomial influenza outbreaks. these outcomes can complement other surveillance methods if the data is readily available.Other events that may reflect levels of influenza activity include school or workplace absenteeism. is essential background information for many scientific studies of influenza. which can be devastating in some situations such as bone marrow transplant units. Influenza vaccination programs Since influenza vaccination is most effective when recipients are vaccinated just before the start of influenza activity. types of circulating viruses. 2) populations vaccinated with the current vaccine demonstrate a reduced serologic response to the new variant.

The nominated sentinel site physicians at these sites have been trained on data collection and sampling. diagnostics and isolation of influenza viral isolates and sharing of influenza information with the stakeholders. Islamabad. became functional in 2007. These physicians screen ILI and SARI cases as per case definitions. Epi and the Lab. Sentinel sites at these provinces have been identified and evaluated for the establishment of the laboratory. To monitor trends of influenza-like illness (ILI) 2. NISLP Accomplishments In order to achieve NILSP goal and objectives it is envisaged to establish one sentinel site in each province in major cities. National Influenza Lab Based Surveillance Project (NILSP) although established in 2002. Peshawar and Federal Capital. Peshawar and Civil Hospital. Hayatabad Medical Complex (HMC). 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. or if available it is underutilized and not used in key decision making for the resource allocation. Karachi will be soon operational. analysis. Two sentinel sites at Federal Government Services Hospital (FGSH) Islamabad and King Edward Medical University (KEMU). To detect any novel influenza variant for further R & D There are two main components of the project. Its three main objectives are: 1. Specimens are collected and transported to NIH for processing. The project is being implemented by the Public Health Laboratories Division of the National Institute of Health. both being equally very important. There is no scientific based data available that shows true picture of the disease burden caused by the Influenza in Pakistan. Quetta. Lahore. like Karachi. Case Identification.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. and finally 3. To monitor predominant circulating influenza strain. Total of five laboratories have been envisaged to be set up for influenza surveillance. Once their labs will be set up they can process same samples at their centers. . typing & subtyping by its Lab Component. The data is stored in databank and analysed later for decision making and coordination later on. Gathering Demographic factors and case follow up are done by the Epi component and Sample processing. These sentinel sites will collect data and samples and initially sent these to NIH for processing. Main goal of this project (NILSP) is to establish & maintain a country wide influenza surveillance system based on epi and lab components. Lahore are operational.

Lahore. TOTAL RESULTS ON IN LOCATION PROCESSED SAMPLES CULTURE PROCESS +VE -VE FGSH. brand Tamiflu) has been procured for the same as well. 70 53 . Procurement of laboratory equipment/ supplies has been partially completed for the central (NIH) as well as provincial laboratories.e at Bolan Medical Complex (BMC). Anti-virals (Oseltamivir. Bio-safety enhancement (BSE) is considered important part of NILSP. BSE at the NIH (Islamabad). Fluarix) procured for the staff of the NIH and provinces have been dispatched to the provincial sentinel sites. 26 (4. Quetta is in streamline. KEMU (Lahore) and HMC (Peshawar) is under process.Evaluation of the remaining provincial sentinel site (Balochistan). Number of trainings/ scientific workshops have been organized by the NILSP within country. 2009 and data on virological information is entered on FluNet subsequently. Flu vaccine (Inj. Number of seasonal Influenza samples received from Federal Government Services Hospital (FGSH) Islamabad (23 November 2007 to date) is 653 samples. Panel of representative seasonal influenza samples (nine) have been submitted to Global Information Surveillance Network (GISN) in April. All necessary sample collection supplies have been sent to high risk districts of Influenza. Layout of BSE at other provincial labs (Sindh and Balochistan) is being worked out for civil works. Plan has been worked out for sentinel site and rapid response trainings at Balochistan as well as to organize a National seminar on Influenza. i. Number of seasonal Influenza samples received from King Edward Medical University (KEMU). . (30 January 2009 to date) is 70 samples. Total number of sentinel site physicians trained at the nominated sites is more than forty (41). Out of these samples. 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. These equipment/ supplies will be moved to these nominated sentinel sites when their bio-safety enhancement is completed.1%) influenza isolates are confirmed positive by Real-time PCR/ viral culture. 653 632 26 608 21 Islamabad KEMU. Rapid response training has been imparted to public health professionals of high risk districts of Punjab and NWFP.

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 .5 influenza 3 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.5 Number of specimenspositive for influenza by subtypes 6 Number of specimens positive for 5 2 ILI activity level 4 1. 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. 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.

5 1 2 1 1. . proper management of patients and understanding the epidemiology of the disease. 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. effective vaccines produced in a timely manner and quality control improved. In addition. the development of resistance to antivirals can be determined.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.5 0 1 0 0. 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. Collection of appropriate specimens from human and animal cases for rapid viral RNA detection by any qualified laboratory. is essential for early detection of cases. together with rapid and precise characterization of virus isolates of the specimens at specialized reference laboratories.5 1 Proportion of all influenza that were 3 1 Number of specimens pandemic A(H1N1) 2009 2. Number of specimens positive for seasonal and non-seasonal [A(H1N1) and H5] influenza 3. 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.

tryptose-phosphate broth. The addition of antibiotics and antimycotics helps prevent microbial growth. veal infusion broth. Furthermore the world is currently in pandemic Phase 6 of Swine origin Influenza A H1N1influenza with over 60. The ongoing threat of infections with Highly pathogenic avian influenza (HPAI) caused by the A(H5N1) influenza subtype in animal populations. phosphate-buffered saline. particularly wild waterfowl and domestic poultry such as chickens and ducks. cell culture medium. such as bovine serum albumin (BSA) or gelatin. Hanks balanced salt solution. or cell culture medium without antibiotics and proteins can be used as washing or gargling fluids. Hanks balanced salt solution. Physiological saline solution. 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. Some transport systems which have proven to be satisfactory for the recovery of a wide variety of viruses are commercially available. and sucrose-phosphate buffer are commonly used transport media. poses a continuing global human public health risk.5% to 1%. to stabilize viruses. They should be supplemented with protein. • 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 . Each new human case gives the virus an opportunity to evolve further towards a rapidly transmissible pandemic strain.Specimens should be collected and transported in a suitable transport medium.000 confirmed cases world wide. to a concentration of 0.

surgical Mask. Yuen & Wong (2005) recommend that: “In view of the high fatality of the disease. a combination of contact. N95 2. gown 5. • Preservation before and for shipping. should only be conducted in a full body cover-all. droplet. non-sterile latex gloves (or equivalent if allergic) 3. and the correct use of disinfectants. Further details of safety procedures are given in Annex 1(PPE) & 2 (Hand hygiene technique). High risk activities such as post mortem examination of a confirmed or strongly suspected human case. TAKING SPECIMENS SAFETY In general PPE should include: 1. goggles or a face shield 4. This protocol covers the following aspects of sampling: • The specimens required from humans. . requires greater personal protective equipment levels than sampling from wild birds. where virus contaminated material can accumulate and there is a great deal of potentially contaminated dust in the air. • The information that needs to accompany the shipment. heavy rubber gloves and eye protection. • Collection of the specimens. For example work in intensive poultry units. and Annex 3 (Disinfection). with easily cleaned waterproof boots. a suitable form of respiratory protection . head covering The level of PPE to be employed will be determined by the exposure risk. PPE is essential to prevent infection during sampling but is not the whole answer. • Correct packing of the specimens. • Shipment to laboratories.” It may also be necessary to include: − impermeable apron − suitable rubber boots. 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. Those taking specimens should comply with all recommended infection control precautions including specific personal hygiene measures. • 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.

invasive procedures can be performed for the diagnosis of virus infections of the lower respiratory tract. If the specimen cannot be processed within 48 .Nasal swabs with nasal secretions (from the anterior turbinate area) ornasopharyngeal aspirates or swabs are appropriate specimens for detecting human influenza A and B . . 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 . Upper respiratory tract (take both types of specimen to allow detection of influenza viruses): . Preferred samples a. 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.Posterior-pharyngeal (throat) swabs are currently the highest yield upper respiratory tract specimen for detecting A(H5N1) (unlike human influenza). 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.Each sample taken should be given a unique identifier number and accompanied by epidemiological data/investigation form . . . Specimens for virus isolation should be refrigerated immediately after collection and inoculated into susceptible cell cultures as soon as possible. This identifier should be used on all documentation concerning the specimen from that source.72 hours. the specimen should be kept frozen at or below -70oC. 1. Specimen tubes should also be marked with information about the type of specimen in the tube and the date when the specimen was taken.2 hours.

Lower respiratory tract: .If the patient is intubated. take a tracheal aspirate or collect a sample during bronchoalveolar lavage. Virus excretion and antibody response in human Influenza infections . but can be useful in outbreaks. Secondary specimens (these are not required for seasonal influenza . c.b. b) When to collect the specimens Figure1. 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.Serum (acute and convalescent if possible).This is not applicable in cases of seasonal influenza 2. Blood: .

• Single serum samples. (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. To be collected at day 14 or later after symptom onset since the likelihood of detecting neutralizing antibodies increases over time. Note that the limited data available on antibody • kinetics indicate development of positivity (initially ELISA and not necessarily neutralizing antibody) from day 10 onwards. # 2069 Citmed Corp. after 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. • Virus may be detectable in tracheal aspirates from onset of lower respiratory complaints (dyspnoea.. • 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. wood Falcon Cat. # NSN-6515-324- 5500 Polyester fiber-tipped applicator with malleable aluminum shaft (for Nasopharyngeal swabs) . marked cough) or pneumonia until the second or third week of illness. • 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 . • 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. difficulty breathing. PROCEDURES FOR SPECIMEN COLLECTION Materials Required Polyester fiber-tipped applicator Tongue depressor. certainly during the first 3 to 4 weeks after onset of symptoms. • A nasopharyngeal and/or throat swab should be taken within first three days of onset of symptoms. Cat.

# 2097 .10(for Nasopharyngeal aspiration) 15 ml conical centrifuge tubes( incase of mouth gurgles) Falcon Cat.Viral transport medium* (Commercial /or sent from NIH) Sputum/Mucus Trap with 10FG catheter Vygon Cat. #542.

Swab held correctly Figure 2b.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. 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. 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. Figure 2a. the swabs must be held correctly. and must be made aware that the child may become distressed. • Use only sterile dacron or rayon swabs with plastic shafts. 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. Chose a sitting position for adults and a supine position for infants and younger children. Full PPE is therefore essential. If the child’s parents or guardians are present they must be fully informed of what is to take place. Swab held incorrectly 21 . When taking throat (or nasal) swabs. They may also need to be restrained during the sampling process. Calcium alginate or cotton swabs. 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). 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. Children often find sampling from the respiratory tract very distressing and need to be reassured.

blood serum. Be aware that stick-on labels can easily come off. Have the subject say "aahh" to elevate the uvula. 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 . Posterior pharyngeal swab (throat swab) • Hold the tongue out of the way with a tongue depressor. • Prepare two vials containing at least 2–3 ml of a suitable transport/preservative medium (Viral Transport Medium .the unique identifier . (Do NOT use rigid shafted swabs for this sampling method —a flexible shafted swab is essential). This procedure can induce the gag reflex). fine-shafted polyester swab into the nostril and back to the nasopharynx. These should be marked with: .B.the type of specimen in the tube (e. Nasopharyngeal swab • Insert a flexible. Note: Nasopharyngeal sampling is an invasive process that can cause considerable distress to the patient. This can serve as the second sample from the patient. Anterior nasal swab Use the same type of rigid swab as for sampling from the throat. (N.). Relevant field data sheets (see Annex 9) should be filled in.3 ml of transport medium and the applicator stick is broken off.the specimen date . • Use a sweeping motion to swab the posterior pharyngeal wall and tonsilar pillars. 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. • 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. Avoid swabbing the soft palate and do not touch the tongue with the swab tip. never the cap as this can get switched during handling).g. The tip of the swab is put into a vial containing 2 . Use an indelible and alcohol resistant marker pen.VTM) for each specimen. throat swab etc. especially when the specimen is chilled to very low temperatures. Specimens from both nostrils are obtained with the same swab. • Put the swab into VTM 22 . Note: Always mark the tube itself with identifying details.

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. Taking a throat swab Restraining a small child while sampling Nasal and throat swabs Nasal. 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. Figure 3. Taking a nasopharyngeal (a) and Throat swab (b) After a specimen is taken. 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.and throat swabs taken as described above are collected into the same vial of transport medium. 23 .

Mouth wash The patient gargles with 10 ml of washing fluid. Sterilize the cutting edge of the scissors by the use of flame (e. If this procedure cannot be followed. by the use of a spirit burner. 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). Figure 4.portions of the bag or wrap fall into the tube.g. 24 .To do this: . . The catheter is inserted into the nostril parallel to the palate.allow the tip to slide into the VTM and then cap the tube. The patient then tilts the head forward and lets the washing fluid flow into a beaker or a petri dish. 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. Allow scissors to cool before reuse. Dilute approximately 3 ml of washing fluid 1:2 in transport medium.5 ml of washing fluid is applied to one nostril at a time. the catheter is flushed with 3 ml of transport medium. With a transfer pipette. 1 to 1. The process is repeated alternatively with both nostrils until a total of 10 to 15 ml of washing fluid has been used. • Nasopharyngeal secretions are aspirated through a catheter connected to a mucus trap and fitted to a vacuum source. a Bunsen burner or another suitable heat source). Do not let cut .cut the shaft with scissors taking care not to touch the tip. The fluid is collected into a beaker or petri dish and diluted 1:2 with transport medium. Then the vacuum is applied and the catheter is slowly withdrawn with a rotating motion. After mucus has been collected from both nostrils. Nasopharyngeal aspirate • Easier and safer than swabbing in infants and young children. Mucus from the other nostril is collected with the same catheter in a similar manner.

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. 3. palpate and locate the vein 3. 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). Heparin is not suitable as an anticoagulant for this type of specimen because of potential inhibition of PCR reactions. At least 1ml of whole blood is needed to obtain a sufficient amount of serum or plasma for tests. Serum samples The best "all round" specimen to collect is serum. Perform hand hygiene every time gloves are removed. Use PPE — at least gloves (plus face-shields. masks and gowns if splashes are anticipated). 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 second ante-mortem specimen should be collected. An acute phase serum specimen should be taken soon after onset of clinical symptoms and not later than seven days after onset. Place a tourniquet above the venepuncture site. 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). 2. This is the maximum that should be taken from infants. Do not re-palpate the vein. 4. A convalescent-phase serum specimen should ideally be collected 3–4 weeks after the onset of symptoms. Label the tubes. When a patient is critically ill. Always check to ensure that the correct tubes are used for each patient.Blood specimens 1. 2. including the unique patient identification number. Blood should be collected either by use of a vacuum venepuncture system or syringes and needles. Acute and convalescent sera are useful for detection of changes in antibody titre and serum can be used for detection of viral RNA. 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. using an indelible marker pen. 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). 25 . Remove and discard PPE items immediately after completion of task. Taking a blood specimen 1.

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

Shipment of specimens in Category A requires shippers who have undergone special training. • Category B covers all other infectious substances that are not included in Category A. Links to other relevant documents are given in Annex 1. Thus only cultures of HPAI (i. 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. 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 .e. and all samples are to be treated as infectious materials.Please ensure proper disinfection of the sample transport boxes between trips. virus isolates) must be transported as Category A. human blood and other human samples or animal blood and other animal samples known or suspected to contain the A(H5N1) subtype. 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. The protective precautions must be taken at all times. can be transported as “diagnostic specimens" (UN 3373) and are included in Category B. • As far as shipment of human or animal samples suspected or confirmed to contain A (H5N1) or H1N1 viruses is concerned. and this is regarded as sufficient “training” for the shipping of these substances.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. These are summarized below. 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. However. Readers are also referred to the WHO document Guidance on regulations for the Transport of Infectious Substances (Annex---). if such specimens 29 . 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. life threatening or fatal disease in otherwise healthy humans or animals. Note that individual airlines may adopt their own policies and these may be stricter than those issued by IATA. Highly pathogenic avian influenza is part of the indicative list with the mention "cultures only”.

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

SECTION II: DIAGNOSTIC METHODS 31 .

one or more WHO National Influenza Center laboratories or a national network of laboratories reporting to a National Influenza Center may participate. FluNet.asp. Non Molecular diagnostic methods 2. The viral data gathered through this system are summarized each week during the influenza season and are reported to WHO. Specimens for virus isolation usually are collected as a part of routine patient care through a surveillance network of emergency rooms. OVERVIEW OF INFLUENZA DIAGNOSIS Virologic surveillance is the foundation on which international and national influenza surveillance systems are built. and these submitted isolates are then utilized to formulate the next year vaccine for seasonal influenza for both the southern and northern hemispheres. outpatient clinics. Virological data can be reported by the National Influenza Center to WHO via their internet-based reporting system. and private physicians. Molecular/PCR based diagnostic methods 32 . Geneva via FluNet. The data are compiled within the country and then sent to WHO. Within a country. or from respiratory disease outbreak investigations.int/GlobalAtlas/home.who. at http://www. The diagnostic methods used for influenza lab diagnosis can be broadly divided in to 2 groups 1. 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.

or are present at very low levels. exposing internal viral nucleoproteins. The Optical ImmunoAssay technology enables the direct visual detection of a physical change in the optical thickness of molecular thin films. a purple triangle becomes visible on the membrane. if present. Flu OIA This test involves the extraction and detection of influenza A or B nucleoprotein. 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. 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. The patient specimen is placed in the extraction reagent tube. If influenza type A or type B antigens are not present. respectively. only a blue procedural 33 . An enzyme- labeled monoclonal antibody specific to influenza virus is added. Virus antigen disposed on a small round area in the center of the membrane serves as a test control. Slight changes in optical thickness produce a distinct visible color change. a pink-to-red test line along with a blue procedural control line will appear on the test strip indicating a positive result. respectively. Activity of any bound enzyme is detected by two substrate-chromogen solutions. Viral antigens. are bound to the membrane. which binds to any virus captured on the membrane. If the extracted specimen contains influenza antigens. The manufacturer reported sensitivity and specificity of this test ranges from 77% to 96% and 90% to 91%. The manufacturer reported sensitivity and specificity of this test ranges from 62% to 81% and 52% to 80%. during which time the virus particles in the specimen are disrupted. QuickVue Influenza Test This test involves the extraction of influenza A and B viral antigens. which is a silicon wafer. The change is a result of antigen-antibody binding on an optical surface. A positive result appears as a purple spot on the predominant gold background. In the presence of viral antigens.

Virus isolation is considered the “gold standard” for diagnosis of influenza virus infections. but specificity was similar for both type A and B viruses. The manufacturer reported sensitivity of this test in 2 clinical sites compared to culture was 62. 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).6%. ZstatFlu Test This test is based on the detection of influenza- specific neuraminidase activity. Neuraminidase reacts with a chromogenic substrate that precipitates upon reaction. and for drug-susceptibility testing if required. The recommended sample type for this test is a throat swab. The manufacturer reported sensitivity and specificity of this test ranges from 73% to 81% and 96% to 99%.7% and specificity was 98.8%-72. The specimen is added to the kit reagents and incubated at 41oC for 20 minutes. with a 95% confidence interval of 50.3% vs 57. Positive specimens are identified by a blue color.8%-100%.2%. The reported sensitivity was higher for influenza A viruses than influenza B viruses. Cytopathic effect typical of influenza Egg Inoculation of influenza viruses infections in tissue culture 34 . The reaction mixture is then transferred into a collection device and the colored precipitate is collected on a filter. 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. 65. 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. respectively.7% with a confidence interval of 92. and negative specimens are identified by a white color or any shades of color other than blue.control line will appear.

For this reason. the laboratory should keep a stock of frozen cells at a low passage level. must maintain several cell lines if they wish to detect a variety of respiratory pathogens. Madin-Darby canine kidney (MDCK) cells are the preferred line in which to culture influenza viruses. Rapid culture assays that use immunologic methods to detect viral antigens in cell culture have become available. low passage MDCK cells should be thawed and used during the entire season. however. Standard isolation procedures require several days before results are available.40 hours. MDCK cells might lose their susceptibility to respiratory viruses. therefore. nasopharyngeal aspirates. PRECAUTIONS & QUALITY CONTROL Over a number of passages. complete antigenic analysis by HAI using selected ferret anti-sera and sequencing can be performed to determine if an isolate has been inadvertently contaminated. the appropriate cell lines to be inoculated have to be selected for each specimen.5 days using standard culture techniques. Because laboratory-adapted viruses and commercial reference viruses are prepared using older strains. For this reason. and at the beginning of each influenza season. However. GOLDEN RULE 2: NEVER PROCESS CLINICAL SPECIMENS FROM HUMANS AND FROM SWINE OR BIRDS IN THE SAME LABORATORY. Be aware of contamination of clinical specimens with laboratory strains: Since influenza viruses are continuously evolving. specimens should be collected within 72 hours of onset of illness. GOLDEN RULE 1: NEVER PROCESS CLINICAL SPECIMENS FOR VIRUS ISOLATION AND LABORATORY-ADAPTED INFLUENZA STRAINS AT THE SAME TIME.However. the use of cell lines has surpassed the use of embryonated eggs to culture influenza viruses. Each of the currently available mammalian cell lines supports the replication of only a limited number of clinically important respiratory viruses. influenza viruses often were isolated in embryonated eggs. transtracheal aspirates. Primary rhesus or cynomolgus monkey kidney cells also support the growth of influenza viruses. making this test less useful to the clinician. Never use -20oC for storage of isolates! b. 35 . 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. older strains are antigenically distinct from currently circulating strains. In the past. and bronchoalveolar lavage. A laboratory. The results of these assays can be obtained in 18 . only viruses grown in embryonated eggs can be used as seed viruses for vaccine production. several factors limit the use of virus isolation as a diagnostic technique. nasal and throat swabs. In recent years. a. Cell lines should be free of Mycoplasma contamination. Appropriate clinical specimens for virus isolation include nasal washes. compared to an average of 4. On the basis of the clinical information and the epidemiologic situation. Ideally. laboratories that have the capability to isolate influenza in eggs are encouraged to continue.

Disadvantages of the HAI test include the need to remove nonspecific inhibitors of hemagglutination that occur naturally in sera. The traditional method for identifying influenza isolates takes advantage of this property. and red blood cells are added to detect specific binding of antibody to the HA molecule. Regardless. thereby. When antibodies against an influenza HA protein bind to the antigenic sites on the HA. and the need for specialized expertise in reading the results of the test.IDENTIFICATION OF INFLUENZA ISOLATES BY HEMAGGLUTINATION INHIBITION The HAI test was originally described by Hirst (1942) and later modified by Salk (1944). the need to standardize reference and test antigens each time a test is performed. these antigenic sites become blocked and unavailable for binding with red blood cells. inhibiting hemagglutination. the HAI test is performed in micro-titer plates. The hemagglutinin (HA) protein of influenza viruses can bind to red blood cells causing the cells to agglutinate. 36 . The antiserum is newly prepared each year and is based on the vaccine strains that are the prototypes for currently circulating strains. Currently.A standardized quantity of HA antigen is mixed with serially diluted antiserum. the HAI assay remains the test of choice for WHO global influenza surveillance and for determining the antigenic characteristics of influenza virus isolates. The HAI test is extremely reliable provided that the reference antiserum contains antibody to currently circulating viruses. This effect is the basis for the hemagglutination inhibition (HAI) test.

Influenza Hemagglutination with Different Species of RBCs Chicken Turkey Guinea Pig Human Type O Concentration 0.5% 0. 25oC.5% 0.75% 0.75% Microtiter plate “V” “V” “U” “U” Incubation time. 30min 30min 1 hour 1 hour Appearance of control cells button* button* halo halo * = flows when tilted 37 .

nasal washes. and by the experience of the person(s) performing. 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. A result can be obtained within 30 min. while others can detect either influenza A or B viruses and distinguish between them. 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. During recent years. 1ENZYME IMMUNOASSAY Commercially available enzyme immunoassays. monoclonal antibodies against several clinically important respiratory viruses have become available through commercial sources. the specificity of the reagents used. The sensitivity and specificity of these tests vary by test type and by specimen type. the type and subtype of an influenza isolate can be determined. In recent years. have been on the market for several years. and nasopharyngeal and/or throat swabs are generally suitable specimens for these assays. The sensitivity of this method is greatly influenced by the quality of the isolate. Nasopharyngeal aspirates. In outbreak situations and when used outside the period of peak influenza activity. Although IFA can be used for staining smears of clinical specimens directly. this procedure is often done on the clinical specimen if rapid diagnosis is needed. reading and interpreting the test. These antibodies have been thoroughly evaluated in many laboratories. which allow influenza type A viruses to be rapidly detected in clinical specimens. it is preferable to first MDCK cells infected with influenza A virus and increase the amount of virus in cell cultures. With the proper monoclonal antibodies. 38 . it is important to use these tests in combination with virus isolation to confirm the rapid test kit results. All required equipment and reagents are included in the test kit. but the instruction sheet for each test kit should be consulted for specific information. 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. stained with rhodamine However.

these approaches are not optimal and paired sera should be collected whenever possible. Many laboratories rely on serology for determining recent individual infections. The neutralization test is performed in two stages consisting of (1) a virus-antibody reaction step. and enzyme-linked immunosorbent assay (ELISA) are the fundamental tools in epidemiologic and immunologic studies as well as in the evaluation of vaccine immunogenicity. 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. In investigations of outbreaks due to novel viruses. In such situations. The virus neutralization test is a highly sensitive and specific assay applicable to the identification of virus-specific antibody in animals and humans. The HAI test is the preferred diagnostic test for determining antibody rises. serologic techniques such as the hemagglutinin inhibition test (HI). In general. testing of single serum samples has been done to identify antibody to the novel virus. 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. in which the virus is mixed and inoculated with the appropriate antibody reagents. or the laboratory may not have the resources or staff to perform virus isolation). In some other outbreak investigations. The absence of infectivity constitutes a positive neutralization reaction and indicates the presence of virus-specific antibodies in human or animal sera. the demonstration of a significant increase in antibody titers (greater than or equal to 4-fold) between acute. MICRONEUTRALIZATION ASSAY Serology methods rarely yield an early diagnosis of acute influenza virus infections.g. or animals). Apart from their diagnostic value. 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 cases are identified after shedding of virus has stopped. embryonated eggs. There are two exceptions in which the collection of single serum samples can be helpful in the diagnosis of influenza. and (2) an inoculation step. however. in which the mixture is inoculated into the appropriate host system (e. the geometric mean titers between the 2 groups to a single influenza virus type or subtype can be compared.and convalescent-phase sera may establish the diagnosis of a recent infection even when attempts to detect the virus are negative.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. 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. The 39 . serologic methods can be very useful. cell cultures. In general. the virus neutralization test.

the testing of paired acute and convalescent sera in the microneutralization test would provide a more definitive answer as to whether infection has occurred.f o ld d ilu t io n s 2 . First. strain-specific antibodies in animal and human serum. 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. and thus can identify functional. m A b 40 . ) 2 . However. Second. if infection of humans with avian viruses is suspected. 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 traditional test used for the detection of antibodies to human influenza A and B viruses. 5 x 41 0 c e lls / w e ll 1 8 H @ 3 7 ° C + + + 5 . single serum samples may be used in screening for the prevalence of antibody to avian viruses. since infectious virus is used. Because antibody to avian influenza subtypes is presumably low or absent in most human populations. A d d V i r u s 2 H @ 3 7 ° C 3 . 1 . The microneutralization test could detect H5-specific antibody in human serum at the titers that could not be detected by the HI assay.neutralization test has several additional advantages for detecting antibody to influenza virus. A d d S e r a ( h e a t i n a c . E L I S A N P s p e c if . 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 . the assay primarily detects antibodies to the influenza virus HA.

41 . the cells are fixed and the presence of influenza A virus NP protein in infected cells is detected by ELISA. it is expected that serum neutralizing antibodies to influenza virus HA will inhibit the infection of MDCK cells with virus. but in combination with rapid culture assay principles. In our influenza virus microneutralization assay. After an overnight incubation. Serially diluted sera should be pre-incubated with a standardized amount of virus prior to the addition of MDCK cells. 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. the neutralization test can yield results within 2 days.

Recently. The recent availability of improved “one-step” RT-PCR reaction chemistries have decreased the number of necessary pipetting steps. RT and a thermostable DNA polymerase that is not inactivated by heat such as that of the microbe Thermus aquaticus (Taq polymerase). Although SYBR® green will detect amplification of DNA. primer annealing (45-60°C) and product extension (72°C). or fluorophore. 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. Because the genome of influenza viruses consist of eight single stranded RNA segments. making the process technically easier and less susceptible to contamination. have co-circulated in human populations since 1977. This cDNA then undergoes cyclic amplification by PCR. Such methods include the use of SYBR® green that binds to double stranded DNA and fluoresces when excited by light of the appropriate wavelength. 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. are both highly sensitive and specific. or primers. Subsequently. Taqman® probe) that is labeled with a fluorescent label. Polymerase Chain Reaction (PCR) is a method used for amplification of specific regions of DNA from very low levels of starting template DNA. all amplified products. will fluoresce making it difficult to determine if the amplified DNA is the desired product. PCR and RT-PCR strategies have been designed for detection and characterization of many different infectious agents including bacteria and viruses. or for the characterization of viruses grown in tissue culture or embryonated eggs. Following reverse transcription of the RNA target to cDNA. Also. two antigenically and genetically distinct lineages of influenza B viruses. because the probe binds to the specific target and does not bind to non-specific products. H3N2 and H1N1. or other non- specific products are present. and a quencher dye.111MOLECULAR DETECTION OF INFLUENZA VIRUSES Two influenza A subtypes. RT-PCR can be used for the detection of influenza viruses in original respiratory samples taken from patients with influenza-like illness. Another method of real-time detection incorporates a dually labeled hydrolysis oligonucleotide probe (eg. 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. Because the products of one round of amplification serve as templates for the next. RNA template. RT- PCR reactions require a pair of oligonucleotides. RT-PCR is necessary for amplification of specific influenza gene targets. This procedure is thus termed RT-PCR. Routine diagnostic methods for influenza infections. 42 . In this case. including virus culture and antigen detection. a number of strategies have been designed that utilize fluorescent dyes to detect or quantitate amplification of DNA by PCR in real time. melt-curve analysis is required to determine if primer-dimer. Co-circulation of multiple types and subtypes of influenza viruses increases the difficulty of diagnosis and virus identification. each successive cycle essentially doubles the amount of the desired DNA product. specific and non-specific. the cDNA is subjected to repeated thermal cycling causing template denaturation (95°C). However. four deoxyribonucleoside triphosphates (dNTPs).

Clinical type samples.. 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. as with conventional RT-PCR. such as “minor groove binding” (MGB) probes. The protocol of viral RNA extraction for reverse amplification of RNA by Real-Time RT-PCR is included in this course manual. Because influenza A subtypes are defined by their surface hemagglutinin (HA) and neuraminidase (NA) genes. whether it is the sensitive and specific detection of virus in clinical samples.fluorescence is only observed if the probe is able to bind to amplified target DNA and hydrolyzed. which are swab material. Similarly. is a useful target for the detection of all type A influenza viruses. and thus. a number of protocols have been designed for the detection of influenza B viruses based on their NS gene. it is possible to design PCR primers and probes that will specifically detect only one influenza type or subtype. The efficient extraction and purification of viral RNA are critical for downstream molecular applications. The M gene is the least variable of the influenza virus genes. Viral RNA is extracted using a commercially available RNA extraction kit (Qaigen). FRET probes.g. virus stocks) and clinical. Molecular Beacons. it is important to consider that. 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. A number of other techniques. or quantification of the influenza virus by molecular methods from humans. 43 . Scorpion probes and others have also been designed that are useful to detect amplification of specific targets on real-time formats. primers that specifically amplify these genes are effective for determining the subtype of influenza A viruses. Samples can generally be divided into two types: enriched (e. However. Real-Time RTPCR amplification plot Since genetic sequences differ among the various types/subtypes of influenza viruses. the most difficult to process due to the complex sample composition and possibly low virus titers. virus gene cloning and expression.

RNA has to be handled very carefully in order to avoid degradation or damage by Rnase enzymes. C. 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. 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. RNA EXTRACTION FROM SWAB SPECIMENS A. B. can be processed by increasing the initial volumes 44 . the handling and storage of RNA is important. Larger starting volumes. serum. Repeated freeze-thawing of plasma samples will lead to reduced viral titers and should be avoided for optimal sensitivity. D. should not be thawed more than once. faeces of infected cases. Samples may be fresh or frozen. and other cell-free body fluids. PRINCIPLE: QIAamp Viral RNA Mini Kits provide the fastest and easiest way to purify viral RNA for reliable use in amplification technologies. cell-culture media or cell-free body fluids using a micro-centrifuge. Viral RNA can be purified from plasma (treated with anticoagulants other than heparin). but if frozen. up to 560µl (in multiples of 140µl). serum. tissues. organs. urine. blood. PURPOSE To extract nucleic acid from Throat / Nasopharyngeal samples to perform molecular test of virology. The most important factor to consider during RNA manipulation is its chemical fragility as compared to DNA. Therefore. PROTOCOL: This protocol is for purification of viral RNA from 140µl plasma.

cell-culture supernatant or cell-free body fluid to the Buffer AVL-carrier RNA in the microcentrifuge tube.5ml microcentrifuge tube. • Check that buffer AW1 and buffer AW2 have been prepared according to the instructions. Pipette 560µl of prepared Buffer AVL containing carrier RNA into a 1. Note: All centrifugation steps are carried out at room temperature (15-25oC). Briefly centrifuge the tube to remove drops from the inside of the lid. Incubate at room temperature (15-25oC) for 10 min. Mix by pulse vortexing for 15s. If the sample volume is larger than 140 µl. 2. 45 . proportionally and loading the QIAamp Mini spin column multiple times. Add 140 µl plasma. 4. 5.. • 70% Alcohol make up with RNase free H2O in the kit. serum. After mixing.g. increase the amount of Buffer AVL-carrier RNA proportionally (e. Frozen samples that have only been thawed once can also be used. 3. Potentially infectious agents and RNase are inactivated in the Buffer AVL. To ensure efficient lysis. PROCEDURE: 1. urine. • Equilibrate samples to room temperature (15-25oC). a 280 µl sample will require 1120 µl Buffer AVL-carrier RNA) and use a larger tube. briefly centrifuge the tube to remove drops from inside the lid. E. it is essential that the sample is mixed thoroughly with Buffer AVL to yield a homogeneous solution. • Add carrier RNA reconstituted in buffer AVE to buffer AVL. Viral particle lysis is complete after lysis for 10 min at room temperature. • Equilibrate buffer AVE to room temperature for elution in step 11. as described below in protocol. Add 560 µl of ethanol (96-100%) to the sample. Longer incubation times have no effect on the yield or quality of the purified RNA. and mix by pulse vortexing for 15s. Procedure carried out on the RNA bench • QIAamp MiniElute Spin Kit • Wear gloves all the times.

g. and discard the tube containing the filtrate. 7. Carefully open the QIAamp Mini spin column. continue directly with step 11. Place the QIAamp spin column into a clean 2 ml collection tube. and centrifuge at full speed (20. 46 .000 x g. a 280 µl sample will require 1120 µl of ethanol). 9. and repeat step 6.. 6. and the continue with step 11. 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.000 rpm) for 3 min. Centrifugation at full speed will not affect the yield or purity of the viral RNA. Only ethanol should be used since other alcohols may result in reduced RNA yield and purity. or to eliminate any chance of possible Buffer AW2 carryover. In order to ensure efficient binding. Close each spin column in order to avoid cross-contamination during centrifugation. and centrifuge at 6000 x g (8000rpm) for 1 min. 14. Carefully open the QIAamp Mini spin column. increase the amount of ethanol proportionally (e. and centrifuge at 6000 x g (8000 rpm) for 1 min. and add 500µl of Buffer AW2. it is essential that the sample is mixed thoroughly with the ethanol to yield a homogeneous solution. Close the cap. Carefully open the QIAamp Mini spin column. If the solution has not completely passed through the membrane. which contains other substances such as methanol or methylethyleketone. Do not use denatured alcohol. and add 500µl of Buffer AW1. Centrifugation is performed at 6000 x g (8000 rpm) in order to limit micro- centrifuge noise. and discard the tube containing the filtrate. Close the cap. If the sample volume is greater than 140 µl. Close the cap. centrifuge again at a higher speed until all the solution has passed through. perform step 10. repeat this step until all of the lysate has been loaded onto the spin column. 8. Place the QIAamp Mini spin column in a clean 2 ml collection tube (provided). It is not necessary to increase the volume of Buffer AW1 even if the original sample volume was larger than 140µl. If the sample volume was greater than 140 µl.

and store it at -20°C (Do not freeze-thaw the aliquots of carrier RNA more than 3 times. 12. do not vortex. Performing a double elution using 2 x 40 µl of Buffer AVE will increase yield by up to 10%. and incubate at room temperature for 1 min. Recommended: place the QIAamp Mini spin column in a new 2 ml collection tube (not provided). Centrifuge at full speed for 1 min. divide it into conveniently sized aliquots. Discard the old collection tube containing the filtrate. To avoid foaming. and discard the old collection tube with the filtrate. Close the cap. Viral RNA is stable for up to one year when stored at -20oC or -70oC. 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. 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. Place the QIAamp Mini spin column in a clean 1. Dissolve the carrier RNA thoroughly. volumes can be calculated using the following sample calculation: n x 0. For larger numbers of samples. and if necessary incubate at 80°C until the precipitate is dissolved. Centrifuge at 6000 x g (8000 rpm) for 1 min. 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 1.5 ml microcentrifuge tube (not provided).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. 47 . 11. Check Buffer AVL for precipitate. 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.10. Carefully open the QIAamp Mini spin column and add 60µl of Buffer AVE equilibrated to room temperature.

8 2 1.44 134. V o l.0 4 2.6 10 5.0 9 5.52 95.56 5. ca rrier No.84 78.6 23 12.4 3 1.4 Note: The sample-preparation procedure is optimized for 5.28 72.24 22.32 123.64 106. Buffer AW 1 Buffer AWl is supplied as a concentrate.8 12 6.2 14 7.48 44.2 11 6.8 15 8. If less carrier RNA has been shown to be better for your amplification system.0 17 9. Before using for the first time.4 8 4.Table 1.) Buffer AVL-carrier RNA should be prepared fresh.36 33. and is stable at 2-8oC for up to 48 hours. B u ffer V o l. but only until the kit expiration date.6 13 7.68 16.4 21 11.6 18 10. Do not incubate at 80oC for more than 5 minutes.92 39.04 50.2 19 10. Volumes of Buffer AVL and Carrier RNA-Buffer AVE Mix Required for the QIAamp Viral RNA Procedure No. 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. This solution develops a precipitate when stored at 2-8 oC that must be redis- solved by warming at 80oC before use. leading to reduced recovery of viral RNA and eventually false negative RT-PCR results. V ol.12 11.08 100.72 67.2 24 13. add the appropriate amount of ethanol (96-100%) as indicated on the bottle and in Table 2.8 20 11. B uffe r V ol.6 ug carrier RNA per sample must be validated for each particular sample type and downstream assay.2 6 3.60 56.96 89. This is particularly the case with low-titer samples.20 112.40 84.8 7 3.6 5 2.76 117. Frequent warming and extended incubation will cause degradation of carrier RNA. (Use of less than 5.80 28.0 22 12. 48 . transfer only the required amount of dissolved carrier RNA to the tubes containing Buffer AVL. Buffer AWl is stable for 1 year when stored closed at room temperature. Do not warm Buffer AVL-carrier RNA solution more than 6 times.16 61.4 16 8.88 128.6 ug of carrier RNA per sample.

No. of Preps AW1 Concentrates Ethanol Final Volume 50 19ml 25ml 44ml Buffer AW 2 Buffer AW2 is supplied as a concentrate. No. add the appropriate amount of ethanol (96-100%) to Buffer AW2 concentrate as indicated on the bottle and in Table 3. PURPOSE: This procedure assumes a basic familiarity with RRT-PCR assays for detection and characterization of influenza virus. but only until the kit expiration date. 49 . Before using for the first time. of Preps AW2 Concentrates Ethanol Final Volume 50 13ml 30ml 43ml REAL-TIME RT-PCR (rRT-PCR) PROTOCOL A. Buffer AW2 is stable for 1 year when stored closed at room temperature.

Limitations: These protocols were optimized using quantitative one-step probe RT- PCR chemistries including Invitrogen SuperScript™III Platinum® One-Step Quantitative Kit. PROTOCOL: SPECIMENS: Respiratory specimens including: broncheoalveolar lavage. Biorad /iScript One-Step RT-PCR Kit. Safety Information: Specimen processing should be performed in accordance with pertaining national biological safety regulations. human A/H3. 7300. BioRad real-time PCR detection systems (iQTM or iQ5TM) or Stratagene QPCR instruments (MX4000®.vitro qualitative detection and characterization of human influenza viruses in respiratory specimens and viral cultures. 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. nasopharyngeal or oropharyngeal aspirates or washes. Influenza A subtyping primer and probe sets are designed to specifically detect contemporary human A/H1. D. 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 . tracheal aspirates. and A/H5 (Asian lineage) influenza viruses. The influenza A and B primer and probe sets are designed for universal detection of type A and type B influenza viruses. 7500.B. MX3000® or MX3005®). sputum.). 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. 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. and nasopharyngeal or oropharyngeal swabs. etc.

Inappropriate specimens not listed above. Molecular grade sterile distilled water (RNase and DNase free) 3. Positive control RNAs Supplies: 1. Cooler racks for 1. RNA extraction procedures should be qualified and validated for recovery and purity before testing specimens. Ambion AgPath-ID™ One-Step RT-PCR Kit (cat# AM1005) 2. . Rejection criteria: .2ml PCR reaction tubes 3. and Invitrogen ChargeSwitch® Total RNA Cell Kit have been shown to generate highly purified RNA when following manufacturer’s recommended procedures for sample extraction.2ml PCR reaction tube strips or plates 51 .. Forward and reverse primers (40μM) 4. One-step quantitative RT-PCR probe hydolysis (e. Taqman®) kit a. Specimens not kept at 2-4°C (≤4 days) or frozen at -70°C or below. Roche MagNA Pure Compact RNA Isolation Kit. Dual-labeled probes (10μM) 5.with cotton tips and wooden shafts are not recommended. Commercially available extraction procedures including QIAamp® Viral RNA Mini Kit. NUCLEIC ACID EXTRACTION: Performance of RT-PCR amplification based assays depends on the amount and quality of sample template RNA.g. 20μl and 200μl adjustable pipettes and aerosol barrier tips 4. QIAamp® MinElute Virus Spin Kit or RNeasy® Mini Kit (QIAGEN). Biorad iScript One-Step RT-PCR Kit (cat# 170-8895) c.5 microcentrifuge tubes and 96-well 0. Laboratory marking pen 2. MATERIALS Reagents: 1. 0. Specimens collected with swabs made of calcium alginate are not acceptable. Invitrogen SuperScript™ III Platinum® One-Step Quantitative Kit (cat# 11732-020 or 11745-100) b.

Keep reagent and reaction tubes capped or covered as much as possible.). micro-centrifuge tubes. c. Optical strip caps 6. PROCEDURE Preparation: 1. e. BioRad real.5 ml microcentrifuge tubes 7. Disposable powder-free gloves Equipment: 1. Vortex 3. Reagent preparation NOTE: Keep all reagents on cold rack during assay set up 52 . 2. Maintain separate areas for assay setup and handling of nucleic acids. 3. Maintain separate. micro-centrifuges) and supplies (e. dedicated equipment (e. The following precautionary steps are recommended: a. etc. Microcentrifuge 2.g. 5.time PCR detection system (iQTM or iQ5TM) or Stratagene QPCR instruments (MX4000®. special precautions must be taken to avoid false positive amplifications. and centrifuges should be cleaned and decontaminated with cleaning products such as 5% bleach. Wear a clean lab coat and powder-free disposable gloves (not previously worn) when setting up assays. Equipment preparation Work surfaces. Sterile. b. pipettes. pipettes. 7500. 7300. Real-time PCR detection system with a 96-well format thermocycler reaction block such as Applied BiosystemsTM real-time PCR systems (7000. d. Avoiding sample contamination because of the sensitivity of fluorogenic 5’ nuclease assays.. “DNAzap™” or “RNase AWAY®” to minimize risk of nucleic acid contamination. E.. nuclease free 1. Change gloves between samples and whenever you suspect they may be contaminated.g. pipette tips) for assay setup and handling of extracted nucleic acids. MX3000® or MX3005®).

Vortex all primers and probes. MOCK reactions and pipetting error. Do not re-freeze probes). InfB. . 1. . AH3. 2. then N = n + 2 3. 1 (a) Primers and probes . If number of samples (n) including controls = 1 to 14. Briefly centrifuge all primers and probes and then place in cold rack. . (b) Real time RTPCR reagents . . Determine the number of reactions (N) to set up per assay. Water. The calculations are as follows: 53 . If number of samples (n) including controls > 15. extracted nucleic acid or positive template controls. No template controls (NTC) and positive template controls (PTC) for all primer/probe sets should be included in each run. Master Mix: calculate the amount of each reagent to be added for each primer/probe set reaction master mix. Mock extraction control (MOCK) provides a secondary negative control that validates the nucleic extraction procedure and reagent integrity. AH5a. are then added to the appropriate test reactions and controls. Label one 1. Place Master Mix and enzyme in cold rack .5 ml microcentrifuge tube for each primer/probe set. See below: . PTC. Each sample RNA extract is tested by separate primer/probe sets: InfA. Mix the 2X Reaction Mix by inversion. AH1. Briefly centrifuge 2x Reaction Mix and enzyme then place in cold rack Tests for each RT-PCR run 1. The RNaseP primer and probe set targets the human RNase P gene and thus serves as an internal positive control for human nucleic acid. then N = n + 1 . Thaw the 2X Reaction Mix vial. AH5b and RNaseP. Reaction setup: Reaction assay mixtures are made as a cocktail and dispensed into the 96-well reaction plate. 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. It is necessary to make excess reaction cocktail to allow for the NTC. 3. 2.

After addition of the water.0 μl Forward primer (0. Invitrogen/BioRad Ambion AgPath 2X PCR Master Mix N x 12.0 μl 4.5 μl N x 0. 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 .5 μl N x 5.5 ml microcentrifuge tubes.5 μl Probe (0. In the assay set up area. mix reaction mixtures by pipetting up and down. Set up reaction strip tubes or plates in 96-well cooler rack.0 μl N x 20.5 μl N x 1.2 μM final concentration) N x 0.5 μl N x 0.0 μl Total volume N x 20. Do not vortex.5 μl N x 0.5 μl N x 12.8 μM final concentration) N x 0. 6.5 μl RT Mix N x 0. dispense reagents into labeled 1. and then place the tube in cold rack.5 μl Nuclease free water N x 5. Centrifuge for 5 sec to collect contents at bottom of the tube. 7.5 μl Reverse primer (0.8 μM final concentration) N x 0. 5.

Cap MOCK wells. Cover the reaction plate and move the reaction plate to the nucleic acid handling area. Program the thermocycler as follows: Invitrogen/BioRad Ambion AgPath 55 . Centrifuge tubes for 5 sec. If using 8-tube strips. 18. for the remaining samples. This will help to prevent sample cross contamination and enable you to keep track of where you are on the plate. Pipette 5 μl of the first sample into all the wells labeled for that sample (for example. 15. Vortex the tubes containing the samples for 5 sec. 10. Change gloves when necessary to avoid contamination. Repeat steps 13. centrifuge at 500 x g for 30 seconds at 4°C. pipette 5 μl of positive template control RNA into all PTC wells. through 15. As shown above. 13. Finally. 8. Briefly centrifuge tube strips for 10-15 seconds. Sample “S1” as shown above). 11. Return to cold rack. Pipette 5 μl of nuclease free water into the NTC wells. 17. set up the NTC reactions for column 1 in the assay set-up area. Cap PTC wells. If using plates. Set up the extracted nucleic acid samples in the cold rack. Add 5 μl of mock extracted sample to the MOCK wells (column 11). samples can be added by column. Cap NTC wells. Positive template controls (PTC) should be added last after all samples and NTCs are sealed. RT-PCR amplification conditions: The reaction volume is 25μl. Cap the column to which the sample has been added. Return strip tubes to cold rack. label the TAB of each strip to indicate sample position (DO NOT LABEL THE TOPS OF THE REACTION TUBES!). 9. Before moving the plate to the nucleic acid handling area. MOCK should be added after the samples have been added (column 11) to check for cross-contamination during sample preparation or addition. 12. Change tips after each addition 14. samples can be added by column. 19. 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. 16. As shown above.

Failure to detect RNase P in any of the clinical samples may indicate: a. 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. 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. 2. Reagent or equipment malfunction 56 . However. All clinical samples should exhibit RP reaction curves that cross the threshold line at or before 35 cycles. or a weak RP reaction. thus indicating the presence of sufficient RNA from human RNase P gene indicating the specimen is of acceptable quality. Also. The NTC reactions for probe/primer sets should not exhibit fluorescence growth curves that cross the threshold line. sample contamination may have occurred. INTERPRETATION OF RESULTS: 1. it is possible that some samples may fail to give positive reactions due to low cell numbers in the original clinical sample. If a false positive occurs with one or more of the primer and probe NTC reactions. Invalidate the run and repeat the assay with stricter adherence to the procedure guidelines. Improper assay set up and execution d. samples taken from animal/avian species or cell culture typically exhibit either no RP reaction.

PTC reactions should produce a positive result with the InfA. interpret as follows: a. AH3. AH1. and document results of the investigation and corrective actions. The MOCK should NOT exhibit fluorescence growth curves for primer/probe sets InfA. 4. If expected positive reactivity is not achieved. 57 . Contamination of RNA extraction reagents may have occurred. AH5b or MOCK that cross the threshold line within 45 cycles. AH5a. Primary Growth Curve 3. implement corrective actions. Cross contamination of samples occurred during RNA extraction procedures or assay setup. Determine the cause of failed PTC reactivity. InfB. If any influenza specific primer/probes exhibit a growth curve that crosses the threshold line. Do not use PTC reagents that do not generate expected result. b. AH3. H5b and RP reactions between 20 and 30 cycles. Invalidate the run and confirm reagent integrity of RNA extraction reagents prior to further testing. InfB. H5a. Invalidate the run and repeat the assay with stricter adherence to procedure guidelines. AH1. invalidate the run and repeat the assay with stricter adherence to procedure guidelines.

If the reaction for influenza A is positive. If a specimen is positive for InfA and only one of the AH5 reactions. When all controls meet stated requirements. 6. contact CDC for guidance. 58 .g.. H3 or AH5a AND AH5b) reaction growth curves cross the threshold line within 40 cycles. 2. or positive of InfA only. a specimen is considered presumptive positive for influenza A virus if the InfA reaction growth curves cross the threshold line within 40 cycles. transport or handling. When all controls meet the stated requirements. 1:10 and 1:100) to verify the result. When all controls meet stated requirements. A/H3 or A/H5 (Asian lineage) virus if BOTH the InfA and the respective subtype (H1.5. it should also be positive for one of the following subtypes: AH1. Analysts should be trained and familiar with testing procedures and interpretation of results prior to performing the assay. A false negative result may occur if inadequate numbers of organisms are present in the specimen due to improper collection. a specimen is considered presumptive positive for influenza B virus if InfB reaction growth curves cross the threshold line within 40 cycles. AH3. Limitations: 1. A false negative result may occur if an excess of DNA/RNA template is present in the in the reaction. A specimen is considered presumptive positive for influenza A/H1. extracted RNA can be tested at 2 or more dilutions (e. 3. 7. If inhibition of the RP control reaction is noted for a particular sample. or AH5a and/or AH5b. a specimen is considered negative for influenza virus if growth curves for neither InfA nor InfB cross the threshold within 40 cycles.

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. using chemical sequencing • Sanger. using dideoxynucleotides. 4 SEQUENCING What is sequencing? “Sequencing” means determining the order of nucleotides of a piece of DNA. 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. Modern sequencing equipment uses the principles of the Sanger technique. sequence analysis of a subset of these viruses has provided very useful supplementary information. 59 . However. 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. Historically there were two main methods of DNA sequencing: • Maxam & Gilbert.

Example of automated sequencing output Good quality sequence 60 .

ANNEXURES 61 .

• the type of respiratory risk (droplets.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. taking a swab from a dead bird in the open air requires less protection than sampling inside a poultry shed). Some common terms: Fit test: evaluating the fit of a respirator on an individual. sampling for blood is less risky than taking a throat swab which may cause the patient to cough).3 micrometers in diameter. a tightly fitting surgical or procedure mask should be used. aerosols and dusts require different types of protection). 62 . although similar in appearance to surgical masks.97% efficient in removing particles of 0. • Disposable particulate respirators. • Surgical and procedure masks do not provide protection against small-particle aerosols (droplet nuclei). High efficiency particulate air (HEPA) filter: means a filter that is at least 99. 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. and aerosol-generating procedures should not be performed if a particulate respirator is not available. 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. There are many types of respirators and masks available and the different types offer very different levels of respiratory protection.g. • 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. • If a particulate respirator is not available. • the situation (e. 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.g.

or visibly soiled with blood or other body fluids. therefore. bar. 63 . If hands are not visibly dirty. Vigorously perform rotational hand rubbing on both palms and interlace fingers to cover all surfaces. ANNEXURE II: HAND HYGIENE When they are visibly dirty or contaminated with proteinaceous material. leaflet or powdered forms of plain soap are acceptable when washing hands with a non-antimicrobial soap and water. Ethyl alcohol has greater activity against viruses than isopropyl alcohol. use an alcohol-based preparation. ethyl alcohol-based hand disinfection products may be preferred over isopropyl alcoholproducts in settings where transmission of HPAI is likely. Wet hands with water and apply the amount of product necessary to cover all surfaces. a) Soap and water Liquid. wash hands with soap and water.

Make sure the hands are dry. Use a method that does not recontaminate hands. apply a palmful of the product and cover all surfaces of the hands. Make sure towels are not used multiple times or by multiple people.Rinse hands with water and dry thoroughly with a single use towel. b) Hand cleansers When using an alcohol-based formulation (or another disinfectant based hand cleanser). 64 . Use running and clean water for hand hygiene whenever possible. Use the towel to turn off the tap/faucet. Avoid using hot water. When bar soap is used. small bars of soap in racks that facilitate drainage should be used. Rub hands until they are dry. as repeated exposure to hot water may increase the risk of dermatitis.

• Mechanical pipetting devices are to be used for all liquids in the laboratory. • Good laboratory practices should be followed. Ingestion: Give large volumes of water or milk if conscious. • Countertops and surfaces of biological safety cabinets should be wiped with a disinfectant (0. or smoking is not permitted in the laboratory. get medical attention.Especially before leaving the laboratory and before eating. Caution: hydrogen peroxide may cause severe irritation of skin. or other appropriate barriers). Wash hands often -. DO NOT INDUCE VOMITING. The following statements provide some basic rules for safety in the laboratory. gloves. Mouth pipetting is dangerous. Protective clothing should be removed before leaving the laboratory. Inhalation: Remove to fresh air 65 . • Adequate and conveniently located biohazard containers for disposal of contaminated materials should be available. drinking. get medical attention. • Biosafety Level 2 practices should be followed when handling all specimens. • Use barrier protection at all times (laboratory coats. Eye and Skin contact: Flush with plenty of water. and mucous membranes and respiratory irritation. Caution: acetone is extremely flammable as liquid and vapor. • Blood and body fluid precautions are to be consistently used with all clinical specimens of blood or other potentially infectious material (Universal Precautions). 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.5 sodium hypochlorite is preferred) routinely after working with infectious agents or clinical specimens. and harmful if inhaled. eyes. 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. 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. HAZARDOUS CHEMICALS Acetone will be used for fixation of cell cultures in the micro-neutralization assay. Eating. Eye and Skin contact: Immediately flush eyes with plenty of water.

or absorbed through skin. Caution: Phenol is highly corrosive and can cause severe burns. vomiting. then induce vomiting. Chloroform is used in the alternate methods for RNA extraction and the dsDNA purification. olive oil. or dizziness. and safety glasses when handling phenol. Gloves should be worn when working with solutions containing this dye. Ingestion: Call physician immediately. Skin contact may cause irritation or dermatitis. Inhalation: Remove to fresh air. Inhalation or ingestion may cause nausea. Do not use ethanol. Caution: Ethidium bromide is a powerful mutagen and is moderately toxic. Give oxygen if breathing becomes difficult. Call a physician.Sodium azide is added as a preservative in a concentration of 0. get medical attention. lab coat. All manipulations should be carried out in a chemical hood. Call a physician.1% to some of the reagents included in the WHO Influenza Reagent Kit and the monoclonal antibodies. Ingestion: Give large quantities of water if conscious and not convulsive. Wear gloves. give large amounts of water. Give oxygen if breathing becomes difficult. Induce vomiting. Eye and skin contact: Flush with plenty of water. Ingestion: If conscious. Induce vomiting. Eye and skin contact: Rinse with large volumes of water and wash with soap and water. or margarine. Inhalation: Remove to fresh air. This agent is rapidly absorbed through the skin. Eye and skin contact: Flush with water for at least 15 minutes. Call a physician. Call a physician. Eye and skin contact: Flush with water for at least 15 minutes. Caution: sodium azide may be fatal if swallowed. give artificial respiration or oxygen if required. Ingestion: If swallowed and conscious. Do not induce vomiting unless instructed by medical personnel Inhalation: Remove to fresh air. give activated charcoal in water. and a mask should be used when weighing it out. Ethidium Bromide is an intercalating dye that will be used to visualize DNA in agarose gels. 66 . inhaled. Caution: Chloroform is a possible human carcinogen and may be fatal if swallowed or inhaled. remove contaminated clothing Inhalation: Remove to fresh air. Eye contact may cause corneal damage. Call a physician. headache. Phenol is used in the alternate methods for RNA extraction and dsDNA purification. Give oxygen if necessary.

and heating is one of the most popular ways to destroy microorganisms. The chamber should not be packed 67 . Moist heat Sterilization Moist heat sterilization must be carried out at temperature above 100oC. At this temperature. In order to destroy bacterial endospores and this requires the use of saturated steam under pressure. Heat Fire and boiling water have been used for sterilization and disinfection since the time of Greeks. Steam sterilization is carried out with an autoclave. The development of the autoclave by Chamberland in 1884 tremendously stimulated the growth of microbiology.therefore boiling can be used for disinfection of drinking water and objects not harmed by water. Moist heat is thought to kill so effectively by degrading nucleic acid and by denaturing enzymes and other essential proteins. Sterilization is done by physical agents and chemical agents. Unfortunately the temperature of boiling water 100 c is not high enough to destroy bacterial endospores that may survive hours of boiling . but boiling does not sterilize. Moist heat readily kills viruses. Because heat is so useful in controlling microorganisms. filtration and radiations. Important Note Autoclaving must be carried out properly or the processed material will not be sterilized. it will not reach 121 oC even though it may reach a pressure of 15 pounds. saturated steam destroys all vegetative cells and endospores in a small volume of liquid within 10 to 12 min. It also may disrupt cell membranes. Principle of autoclave (How to work) Water is boiled to produce steam. which is released through a jacket and into the autoclave chamber. to provide a margin of safety. low temperature. The air initially present in the chamber is forced out until the chamber is filled with saturated steam and the outlets are closed. usually 121o C and 15 pounds of pressure. bacteria and fungi. Autoclave Autoclave is a device somewhat like a fancy pressure cooker. saturated steam continues to until the chamber reaches the desired temperature and pressure.STERILIZATION Sterilization means complete killing or removal of all living micro organisms from an article. Physical agents are heat. Treatment is continued for about 15 min. it is essential to have precise measure of heat killing efficiency. If all air has not flushed out the chamber. Exposure to boiling water for 10 minutes is sufficiently to destroy vegetative cells and eukaryotic spores. Hot. Either moist or dry heat may be applied.

Close the autoclave door and draining valve. Switch off the autoclave after 15 min and open the air valve until removal of complete steam. culture media and cotton swabs etc. the material is supposed to be sterilized. Control Check A biological indicator is often autoclaved along with other material. Fill the autoclave with tap water upto the level of basket. Switch on the autoclave and open the air valve until the air is removed completely. 2. Rubber items. powders. Filtration Filtration is an excellent way to reduce the microbial population in solutions of heat sensitive material. 7. forceps.too tightly because steam needs to circulates freely and contact everything in the autoclave. Following items can be sterilized within an autoclave Liquid solutions. 5. the sterilization run has been successful. and sometimes it can be used to sterilization rather than directly 68 . Sometime either special tape or paper indicator strip that change color upon sufficient heating is autoclaved with a load of material. 3. 6. Dry Heat Sterilization Many objects are best sterilized in the absence of water by Dry Hot Sterilization. Following items can be sterilized with Dry heat Glassware. This indicator commonly consists of a culture tube containing an ampoule of medium and paper strip covered spores of bacillus stearothermophilus or clostridium PA3679. Place the material in the basket. Close the air valve on releasing the steam. 8. 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. The item to be sterilized is placed in an oven at 160 to 170o C for 2 to 3 hours. Start the stop watch on reaching the temp at 121o C at 15 pound pressure. 4. If the color changes after autoclaving. etc. If the test bacterium does not grow in the medium. Open the door and remove the material. Plastic material. Procedure of Autoclaving 1. oils. After autoclaving ampoule aseptically broken and culture incubated for several days. scissors.

It will destroy bacterial endospores and vegetative cells. These chemicals cannot be used on living surface.2 micron in diameter are used to remove most vegetative cells but not viruses. and other substances very effectively. water. However. Ionizing Radiation Ionizing radiation is an excellent sterilizing agent and penetrates deep into objects. a little over 0. Membrane Filters These circular filters are porous membrane. UV lamps are sometime placed on the ceilings of rooms or in biological safety cabinets to sterilize the air and any exposed surfaces. Depth Filters. • Ionizing Radiation Ultraviolet Radiation Around 260 nm is quite lethal but does not penetrate glass. Radiations Following radiations are used in sterilization • Ultraviolet (UV) Radiation. membranes with pores about 0. these are more concentrated. Although a wide variety of pour sizes are available. Membrane Filters. UV radiation is used as sterilization only in few specific situations. the filters simply removes them. ionizing radiation is not always as effective against viruses. and polycarbonate. people working in such areas must be certain the UV lamps are off when the areas are in use.destroying contaminating microorganisms. made of cellulose acetate.1 mm thick. cellulose nitrate. Because of this disadvantage. Because UV radiation burns the skin and damages eyes. dirt films. both prokaryotic and eukaryotic. more irritant and more poisonous. There are two types of filters. Sterilization by Chemical Agent On the basis of potency these chemical are divided in to two types. 69 . from solutions ranging in volume from 1 ml to many liters. • Disinfectants • Antiseptic Disinfectants These chemicals are used for killing of microorganisms.

Antiseptics These chemicals are used only for the prevention of microorganisms. These chemicals can be used on living surface. soaps and detergents 70 . non irritant and mild.e. For example. • Phenol and acid • Alcohols • Halogens • Aldehydes • Surface active agents i. These chemicals act on the cytoplasmic membrane of microbes and produce various changes which damage to these microbes. These are diluted.

3.2.1. NTC NTC NTC NTC B- C- D- E- F- G- H. Mix: Calculate from relevant Excel sheet 1.2.1.5.1. by: ) 1. PCR Tubes (Lot # ABI ) 1. by: ) 1.1. Procedure #:RRT-0 -09 PURPOSE: . NIH Developed: 14th August. Reaction volume: 25 50 100 (Circle one) Other: µl 71 . Taq (Lot# 289834 ) 1. NA-free water (Lot# 0703002 ) 1. PTC PTC PTC PTC 1. Master Mix Preparation by 1. Seasonal flu PERFORMED BY: _Nazish Badar_____ SAMPLES: Sample testing INSTRUMENT #: ABI-7500-1 1. Method: (Circle one) RT-PCR N-PCR RT-Multiplex Reagents and Supplies: 1. dNTPs (Lot#: 359560 ) 1.4. Plate Setup 1 2 3 4 5 6 7 8 9 10 11 12 A.1. 2008 Revised: 17th September. 2008 PROTOCOL#: MOL-007-01 DATE: .3.3. Primer working dilution/volume (Date prepared: . Materials and Methods: 1.6. Worksheet for SOP: MOL-002 National Influenza Lab Based Surveillance Project NILSP.1.1.7.1. Invitrogen SSIII PlatinumTaq RT-PCR kit (Lot# 289834 ) 1.3.2.1. Probe working dilution (Date prepared: .1.

5 2 2.8 µM final conc.Influenza ASeasonal &H5 RT-PCR(CDCProtocol.5 2 2.5 Probe (0. 12.5 Reverse primer (0.5 0.2 µM final conc.5 22 Mix without water 20 20 80 Template volume/RNA 5 Reaction volume 25 Reverse Transcription .5 5.) 0.) 0.5 50 Enzyme(RT) Mix 0.5 0. of Rx 4 5 Master mix Volume(ul) Volume/Tube Total 2XPCRMaster Mix.5 Forward primer (0.) 0.8 µM final conc. 2007) No.5 12.5 0.5 PCRWater 5.5 2 2.5 2 2.Hold 50Cfor 30 min Taqinhibitor Inactivation 95°Cfor 2 min 72 .5 0.

No Date Remarks ID (if any) ID ID Sample Appearance Culture PCR Tests . Laboratory Log Book Hospital __________ District ____________ Province ________________ Country _______________ Hospital Patient Lab Nature of Sample Tissue Real Time Other S. National Lab based Influenza Surveillance.