Respiratory syncytial and influenza viruses in children under 2 years old with severe acute respiratory infection (SARI) in Maputo, 2015

Introduction Although respiratory syncytial virus (RSV) and influenza virus (influenza) infections are one of the leading causes of Severe Acute Respiratory Infections (SARI) and death in young children worldwide, little is known about the burden of these pathogens in Mozambique. Material and methods From January 2015 to January 2016, nasopharyngeal swabs from 450 children, aged ≤2 years, who had been admitted to the Pediatric Department of the Maputo Central Hospital (HCM) in Mozambique, suffering with SARI were enrolled and tested for influenza and RSV using a real-time PCR assay. Results Influenza and RSV were detected in 2.4% (11/450) and 26.7% (113/424) of the participants. Children with influenza were slightly older than those infected with RSV (10 months in influenza-infected children compared to 3 months in RSV-infected children); male children were predominant in both groups (63.6% versus 54.9% in children with influenza and RSV, respectively). There was a trend towards a higher frequency of influenza (72.7%) and RSV (93.8%) cases in the dry season. Bronchopneumonia, bronchitis and respiratory distress were the most common diagnoses at admission. Antibiotics were administered to 27,3% and 15,9% of the children with influenza and RSV, respectively. Two children, of whom, one was positive for RSV (aged 6 months) and another was positive for Influenza (aged 3 months) died; both were children of HIV seropositive mothers and had bronchopneumonia. Conclusions Our data demonstrated that RSV, and less frequently influenza, occurs in children with SARI in urban/sub-urban settings from southern Mozambique. The occurrence of deaths in small children suspected of being HIV-infected, suggests that particular attention should be given to this vulnerable population. Our data also provide evidence of antibiotics prescription in children with respiratory viral infection, which represents an important public health problem and calls for urgent interventions.


Intended Use
The Influenza A/B Typing Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR instrument in conjunction with clinical and epidemiological information: , sputum, and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture.  To provide epidemiologic information for surveillance of circulating influenza viruses.
Performance characteristics for influenza were established during a season when seasonal influenza viruses A/H1 and A/H3 were the predominant influenza A viruses in circulation and during a season when the A/H1pdm09 influenza virus was the predominant influenza A virus in circulation. Performance characteristics may vary with other emerging influenza A viruses.
Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. Conversely, positive results do not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease.
If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.
All users, analysts, and any person reporting results from use of this device should be trained to perform and interpret the results from this procedure by a competent instructor prior to use. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed a training course provided by CDC instructors or designees.

Summary and Explanation
Influenza viruses are the causative agents of influenza infection, an acute respiratory illness that is highly contagious and results in significant morbidity each year with flu-related deaths ranging from a low of about 3,000 to a high of about 49,000 people (http://www.cdc.gov/flu/about/disease/symptoms.htm). Influenza virus types A and B primarily infect the nasopharyngeal and oropharyngeal cavities. Influenza A viruses are further categorized into subtypes based on two major surface proteins, hemagglutinin (HA) and neuraminidase (NA). Similarly, Influenza B viruses can also be separated into two major lineages. The infective potential of influenza is frequently underestimated and can result in high morbidity and mortality rates, especially in elderly persons and in high risk patients, such as the very young and immunocompromised. Typical influenza viral infections in humans have a relatively short incubation period of 1 to 2 days, with symptoms that last about a week including an abrupt onset of fever, sore throat, cough, headache, myalgia, and malaise. When a subject is infected with a highly virulent strain of influenza, these symptoms can progress rapidly to pneumonia and in some circumstances death. Pandemic outbreaks of highly virulent influenza present a serious risk to human and animal health worldwide.
Influenza viruses may be detected and characterized from clinical specimens by several methods that vary in sensitivity, speed, and capability with regard to distinguishing influenza types and subtypes. Laboratories may use the influenza A, B, A/H1, A/H1pdm09, and A/H3 and A/H5 (Asian Lineage) primer and probe sets simultaneously to type and/or subtype suspected influenza positive clinical specimens. Additionally, influenza B viruses may be further characterized as B/Victoria or B/Yamagata using the VIC and YAM primer and probe sets. The CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel assists in routine surveillance of seasonal influenza following an algorithm as follows: The Influenza A/B Typing Kit consists of oligonucleotide primers and dual-labeled hydrolysis (TaqMan ® ) probes and controls which may be used in rRT-PCR assays for the in vitro qualitative detection and characterization of the human influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness (ILI). The oligonucleotide primers and probes for detection of Influenza A and B viruses were selected from highly conserved regions of the matrix (M) and non-structural (NS) genes, respectively. Detection of viral RNA not only aids in the diagnosis of illness caused by seasonal, newly emerging, and novel influenza viruses in patients with ILI, but also provides epidemiological and surveillance information on influenza and aids in the presumptive laboratory identification of specific novel influenza A viruses. .  Use personal protective equipment such as (but not limited to) gloves and lab coats when handling kit reagents while performing this assay and handling materials including samples, reagents, pipettes, and other equipment and reagents.  Amplification technologies such as PCR are sensitive to accidental introduction of product from previous amplification reactions. Incorrect results could occur if either the clinical specimen or the real-time reagents used in the amplification step become contaminated by accidental introduction of amplification product (amplicon). Workflow in the laboratory should proceed in a unidirectional manner.
 Maintain separate areas for assay setup and handling of nucleic acids.  Always check the expiration date prior to use. Do not use expired reagent. Do not substitute or mix reagent from different kit lots or from other manufacturers.  Change aerosol barrier pipette tips between all manual liquid transfers.  During preparation of samples, compliance with good laboratory techniques is essential to minimize the risk of cross-contamination between samples, and the inadvertent introduction of nucleases into samples during and after the extraction procedure. Proper aseptic technique should always be used when working with nucleic acids.  Maintain separate, dedicated equipment (e.g., pipettes, microcentrifuges) and supplies (e.g., microcentrifuge tubes, pipette tips) for assay setup and handling of extracted nucleic acids.  Wear a clean lab coat and powder-free disposable gloves (not previously worn) when setting up assays.  Change gloves between samples and whenever contamination is suspected.  Keep reagent and reaction tubes capped or covered as much as possible.  Primers, probes (including aliquots), and enzyme master mix must be thawed and maintained on cold block at all times during preparation and use.  Work surfaces, pipettes, and centrifuges should be cleaned and decontaminated with cleaning products such as 5% bleach, "DNAzap™" or "RNase AWAY ® " to minimize risk of nucleic acid contamination. Residual bleach should be removed using 70% ethanol.  Reagents, master mix, and RNA should be maintained on cold block or on ice during preparation and use to ensure stability.  Dispose of unused kit reagents and human specimens according to local, state, and federal regulations.

Reagent Storage, Handling, and Stability
 Store all primers and probes at 2-8°C until re-hydrated for use; Store all control materials (HSC, PIPC) at ≤ -20°C.  Always check the expiration date prior to use. Do not use expired reagents.  Protect fluorogenic probes from light.  Primers, probes (including aliquots), and enzyme master mix must be thawed and kept on cold block at all times during preparation and use.  Do not refreeze probes.  Controls and aliquots of controls must be thawed and kept on ice at all times during preparation and use.

Specimen Collection, Handling, and Storage
Inadequate or inappropriate specimen collection, storage, and transport are likely to yield false negative test results. Training in specimen collection is highly recommended due to the importance of specimen quality. CLSI MM13-P may be referenced as an appropriate resource.  Collecting the Specimen  Follow specimen collection devices manufacturer instructions for proper collection methods.  Swab specimens should be collected using only swabs with a synthetic tip, such as nylon or Dacron ® , and an aluminum or plastic shaft. Calcium alginate swabs are unacceptable and cotton swabs with wooden shafts are not recommended.  Transporting Specimens  Ensure that, when transporting human respiratory specimens, all applicable regulations for the transport of etiologic agents are met. Human respiratory specimens, to be tested within 72 hours post-collection, should be transported refrigerated at 2-8°C. Alternatively, specimens may be frozen and transported for testing.  Respiratory specimens should be collected and placed into viral transport media (VTM) as described by CDC and WHO guidelines.  For more information refer to WHO "Guidelines for the collection of clinical specimens during field investigation of outbreaks", http://www.who.int/csr/resources/publications/surveillance/whocdscsredc2004.pdf, and/or www.cdc.gov/h1n1flu/specimencollection.htm.  Storing Specimens  Specimens received cold should be stored refrigerated (2-8°C) for up to 72 hours before processing. Store any residual specimens at ≤ -70°C.  Although optimal performance is met when testing fresh specimens within 72 hours of collection, performance has been demonstrated with frozen specimens. If testing of a fresh specimen is not possible within 72 hours storage at 2-8°C, the specimen may be frozen at ≤ -70°C and tested at a later time.  Specimens received frozen should be stored at ≤ -70°C until processing. Store any residual specimens at ≤ -70°C.  Storing Purified Nucleic Acid  Store purified nucleic acids at ≤ -70°C.

Specimen Referral to CDC
 Referring a specimen to the CDC  Ship all specimens and related RNA overnight to CDC.  Ship frozen specimens on dry ice and non-frozen specimens on cold packs. Ship extracted RNA on dry ice.  Refer to the International Air Transport Association (IATA -www.iata.org) for requirements for shipment of human or potentially infectious biological specimens.  Prior to shipping, notify CDC Influenza Division (see contact information below) that you are sending specimens.  Fill out the Influenza Specimen Submission Form completely -include specimen type and Ct values detected by your laboratory.

Reagent and Controls Preparation
Primer and Probe Preparation: 1. Upon receipt, store lyophilized primers and probes at 2-8°C. 2. Rehydration a. Remove primers and probes from 2-8°C. b. Allow primers and probes to sit at ambient temperature for 15 minutes. c. Quickly centrifuge dried primers and probes to collected pellet in the bottom of the tube. d. Pipette 0.5 ml (500 µL) of 10 mM Tris, pH 7.4-8.2 or PCR-grade water into each dried PCR primer or probe. e. Allow primers and probes to fully rehydrate for at least 15 minutes at room temperature. f. After primers and probes are fully rehydrated, pulse vortex to ensure a homogenous solution.

Prepare a Combined Oligonucleotide Mix for Each Marker Set
a. For each marker set (e.g. InfA), pipette the entire volume (0.5 mL) of the reconstituted forward and reverse primers and probe into a single new, nucleasefree, sterile tube (henceforth, the primer/probe mix). b. Pulse vortex the combined primer/probe mix to ensure a homogenous solution. 4. Aliquot a. Label five new, nuclease-free, sterile tubes for each combined primer/probe mix with the following information:  Primer or probe name  Kit lot #  Expiration date b. Aliquot 300 µL of each combined primer/probe mix into respective labeled tubes and store at -20°C.

Storage
a. After rehydration i. Aliquots of primers and probes are stored at -20°C or below until expiration date as long as QC requirements are met. ii. Thawed aliquots of primers and probes may be stored at 2-8°C for up to 3 months. Combined primers/probe mix aliquots should be stored in the dark.

General Preparation Equipment Preparation
Clean and decontaminate all work surfaces, pipettes, centrifuges, and other equipment prior to use. Decontamination agents should be used including 5% bleach, 70% ethanol, and DNAzap™ or RNase AWAY ® to minimize the risk of nucleic acid contamination.

Reagent Preparation NOTE: All reagents should be kept on ice or cold rack during assay preparation.
Primers and Probes Reagents  Thaw frozen aliquots of primer/probe mix. Thawed aliquots may be stored at 2-8°C in the dark for up to 3 months. Do not re-freeze.  Vortex each combined primer/probe mix for 15 seconds.  Briefly centrifuge each primer/probe mix.  Place combined primer/probe mix in cold rack during master mix preparation.

Invitrogen SuperScript™ III Platinum ® One-Step Quantitative RT-PCR System
 Place Invitrogen 2X PCR Master Mix and Superscript III RT/Platinum Taq enzyme mix in a cold rack at 2-8°C.

Nucleic Acid extraction
Performance of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel is dependent upon the amount and quality of template RNA purified from human specimens. The following commercially available RNA extraction kits and procedures have been qualified and validated for recovery and purity of RNA for use with the panel:   4. In a sterile labeled 1.5 mL tube, prepare master mix for each marker set to be tested by first calculating the amount of each reagent to be added for each primer/probe set reaction master mix required for the test being performed. 5. In the assay preparation area, dispense reagents into each respective labeled 1.5 mL microcentrifuge tube. After addition of the water, mix reaction mixtures by pipetting up and down. Do not vortex. 6. Centrifuge for 5 seconds to collect contents at the bottom of the tube, and then place the tube in a cold rack. 7. Set up reaction strip tubes or plates in a 96-well cooler rack. 8. Dispense 20 µL of each master mix into the appropriate wells going across the row as shown below (Figure 2): H empty empty empty empty empty empty empty empty empty empty empty empty 9. Prior to moving to the nucleic acid handling area, prepare the NTC reactions for column #1 in the assay preparation area. 10. Pipette 5 µL of nuclease-free water into the NTC sample wells. Securely cap NTC wells before proceeding. 11. Cover the entire reaction plate and move the reaction plate to the specimen nucleic acid handling area.

Template Addition
1. Gently vortex nucleic acid sample tubes for approximately 5 seconds. 2. After centrifugation, place extracted nucleic acid sample tubes in the cold rack.
3. Samples should be added to column 2-11 (column 1, 11 (bottom half), and 12 are for controls) to the specific assay that is being tested as illustrated in Figure 3. Carefully pipette 5.0 µL of the first sample into all the wells labeled for that sample (i.e. Sample "S1" down column #2). Keep other sample wells covered during addition. Change tips after each addition. 4. Securely cap the column to which the sample has been added to prevent cross contamination and to ensure sample tracking. 5. Change gloves often and when necessary to avoid contamination. 6. Repeat steps #3 and #4 for the remaining samples. 7. Add 5 µL of Human Specimen Control (HSC) extracted sample to the HSC wells (Figure 3, column 11). Securely cap wells after addition. This is applicable to any plate set-up. 8. Cover the entire reaction plate and move the reaction plate to the positive template control handling area.  A NTC S1 S3 S5 S7 S9 S11 S13 S15 S17 S19 PIPC B NTC S1 S3 S5 S7 S9 S11 S13 S15 S17 S19 PIPC C NTC S1 S3 S5 S7 S9 S11 S13 S15 S17 S19 PIPC D empty empty empty empty empty empty empty empty empty empty empty empty E empty S2 S4 S6 S8 S10 S12 S14 S16 S18 HSC empty F empty S2 S4 S6 S8 S10 S12 S14 S16 S18 HSC empty G empty S2 S4 S6 S8 S10 S12 S14 S16 S18 HSC empty

5.
After selecting next, the Select Detectors screen (Figure 5) will appear. 6. Click the New Detector button (see Figure 5). 7. The New Detector window will appear (Figure 6). A new detector will need to be defined for each influenza primer and probe set. Creating these detectors will enable you to analyze each primer and probe set individually at the end of the reaction.   10. After each Detector is added, the Detector Name, Description, Reporter and Quencher fields will become populated in the Select Detectors screen (Figure 7). 11. Before proceeding, the newly created detectors must be added to the document. To add the new detectors to the document, click ADD (see Figure 7). Detector names will appear on the right hand side of the Select Detectors window (Figure 7). 12. Once all detectors have been added, select (none) for Passive Reference at the top right hand drop down menu (Figure 8).

Figure 8. Select Passive Reference
Passive reference should be set to "(none)" as described above. 13. Click Next at the bottom of the Select Detectors window to proceed to the Set Up Sample Plate window (Figure 8). 14. In the Set Up Sample Plate window (Figure 9), use your mouse to select row A from the lower portion of the window, in the spreadsheet (see Figure 9). 15. In the top portion of the window, select detector InfA. A check will appear next to the detector you have selected (Figure 9). You will also notice the row in the spreadsheet will be populated with a colored "U" icon to indicate which detector you've selected. 16. Repeat step 14-15 for each detector that will be used in the assay.  18. After clicking "Finish", there will be a brief pause allowing the ABI 7500 Fast Dx to initialize. This initialization is followed by a clicking noise. Note: The machine must be turned on for initialization. 19. After initialization, the Plate tab of the Setup (Figure 11) will appear. 20. Each well of the plate should contain colored U icons that correspond with the detector labels that were previously chosen. To confirm detector assignments, select Tools from the file menu, then select Detector Manager.  h.
Step 2 of Stage 3 should be highlighted in yellow to indicate data collection (see Figure 13).

Figure 13. Instrument Window
4. After making changes to the Instrument tab, the template file is ready to be saved. To save the template, select File from the top menu, then select Save As. Since the two enzyme options have different instrument settings it is recommended that the template be saved with a name indicating the enzyme option. 5. Save the template as Influenza AB Typing Kit SuperScript or Influenza AB Typing Kit qScript as appropriate in the desktop folder labeled "ABI Run Templates" (you must create this folder). Save as type should be SDS Templates (*.sdt) (Figure 14).  Influenza AB Typing Kit qScript). 6. There will be a brief pause allowing the ABI 7500 Fast Dx Real-Time PCR Instrument to initialize. This initialization is followed by a clicking noise. Note: The machine must be turned on for initialization.

Figure 15. Plate Set-up Window
7. After the instrument initializes, a plate map will appear (Figure 15). The detectors and controls should already be labeled as they were assigned in the original template.
8. Click the Well Inspector icon from the top menu. 9. Highlight specimen wells of interest on the plate map. 10. Type sample identifiers to Sample Name box in the Well Inspector window (Figure 16). 14. The reaction conditions, volumes, and type of 7500 reaction should already be loaded. (Figure 17). 9. Click the Analyze button in the lower right corner of the window. The red threshold line will turn to green, indicating the data has been analyzed. 10. Repeat steps 5-9 to analyze results generated for each set of markers (i.e. InfA, InfB, etc). 11. Save analysis file by selecting File then Save As from the main menu. 12. After completing analysis for each of the markers, select the Report tab above the graph to display the Ct values. To filter report by sample name in ascending or descending order, simply click on Sample Name in the table.

Interpretation of Results and Reporting Extraction and Positive Control Results and Interpretation No Template Control (NTC)
 The NTC consists of using nuclease-free water in the rRT-PCR reactions instead of RNA.
The NTC reactions for all primer and probe sets should not exhibit fluorescence growth curves that cross the threshold line. If any of the NTC reactions exhibit a growth curve that crosses the cycle threshold, sample contamination may have occurred. Invalidate the run and repeat the assay with strict adherence to the guidelines.

Pooled Influenza Positive Control (PIPC)  The PIPC consists of four different influenza viruses representing influenza A/H1N1, A/H3N2, A/H1pdm09, and influenza B viruses suspended in cultured human cells (A549).
Purified RNA from the PIPC will yield a positive result with the following primer and probe sets: InfA, InfB, H1, H3, pdmInfA, pdmH1, and RP.

Human Specimen Control (HSC) (Extraction Control)
 The HSC control consists of noninfectious cultured human cell (A549) material. The HSC is used as an RNA extraction procedural control to demonstrate successful recovery of RNA as well as extraction reagent integrity. Purified RNA from the HSC should yield a positive result with the RP primer and probe set and negative results with all influenza specific markers.

Influenza A/B Typing Assay Results Interpretation Guide
The

Standards-Based Electronic Laboratory Reporting for Influenza Background
This section contains the recommendations for uniform coding and vocabulary for CDC Human Influenza Virus Real-time RT-PCR Diagnostic Panel.
The following information is provided to assist the performing laboratory in complying with new federal guidelines for the meaningful use of electronic health information systems. The implementation of adopted standards should be harmonized across all performing laboratories to ensure semantic interoperability to better support electronic data exchange.
The CDC developer of this assay through collaboration has established Standard English terminology for the test name and test results with the testing community and expert knowledge of the processes involved. It is recognized that this terminology will differ in countries outside the United States. However, through the use of national and international agreements, it is possible to establish a universal set of codes and terms to accurately characterize laboratory observations. Recommendations in this package insert apply to the reporting of results of this assay only within the United States.

Process for achieving uniformity in laboratory test results
The laboratory performing the influenza assay may utilize a Laboratory Information Management Systems (LIMS) with connections to a hospital or medical system Electronic Health Record (EHR). The coding systems include LOINC -Logical Observation Identifiers Names and Codes (LOINC ® --http://www.loinc.org) and SNOMED CT -Systematic Nomenclature of Medicine--Clinical Terms (http://www.ihtsdo.org/). These coding systems have specific capabilities that are essential for achieving uniformity. The test request and results are to be incorporated into a standard Health Level 7 (HL7) electronic format for laboratory test messaging. More information about HL7 can be found at http://www.hl7.org.
LOINC provides for a common understanding of the medical procedure or process related to the specific assay, in this case the process of detecting the presence of influenza virus and the potential sub-typing of the detected influenza virus. The LOINC codes specified here describe the important information about the methodology employed by the assay; recovery and amplification of one or more RNA targets. Multiple LOINC codes are utilized to convey that the assay is composed of multiple components, i.e. it is a panel or a battery of subtests. In the case of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, the LOINC also provides for conveying that an interpretive test summary is appropriate.
SNOMED CT codes provide for unambiguous representation of the test results and allow the application of specific concepts such as "detected" or "positive" or the identification of detected organism names. Though not further defined in this document, SNOMED CT can also be used to provide for description of the type and source/ location of the specimen being tested or for conveying information about failures of the test procedure or the lack of adequate specimen.

Specific Recommendations for Standards-Based Electronic Data Exchange for Influenza
Laboratories can find more information regarding implementation of HL7 messaging for CDC Flu rRT-PCR Dx Panel, including applicable LOINC test codes and SNOMED result codes at http://www.cdc.gov/flu/professionals/diagnosis/rtpcr-test-kits.htm

Quality Control
 Quality control requirements must be performed in conformance with local, state, and federal regulations or accreditation requirements and the user's laboratory's standard quality control procedures. It is recommended that the user refer to CLSI document C24-A2, Statistical Quality Control for Quantitative Measurements: Principles and Definitions: [Approved Guideline-Second Edition] or other published guidelines for general quality control recommendations. For further guidance on appropriate quality control practices, refer to 42CFR 493.1202(c).  Quality control procedures are intended to monitor reagent and assay performance.  Test all positive controls prior to running diagnostic samples with each new kit lot to ensure all reagents and kit components are working properly.  Good laboratory practice (cGLP) recommends including a positive extraction control in each nucleic acid isolation batch.  HSC extraction control must proceed through nucleic acid isolation per batch of specimens to be tested.  Always include a negative control (NTC), and the appropriate positive control (i.e PIPC) in each amplification and detection run.

Limitations
 All users, analysts, and any person reporting diagnostic results should be trained to perform this procedure by a competent instructor. They should demonstrate their ability to perform the test and interpret the results prior to performing the assay independently. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed training provided by CDC instructors or designees. This device is subject to a special control requiring that distribution be limited to laboratories with (i) experienced personnel who has training in standardized molecular testing procedures and expertise in viral diagnosis, and (ii) appropriate biosafety equipment and containment (21 CFR866.3332(b)(2)).  Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions.  A false negative result may occur if a specimen is improperly collected, transported or handled. False negative results may also occur if amplification inhibitors are present in the specimen or if inadequate numbers of organisms are present in the specimen. Children tend to shed virus more abundantly and for longer periods of time than adults. Therefore, testing specimens from adults will have lower sensitivity than testing specimens from children.
 Positive and negative predictive values are highly dependent on prevalence. False negative test results are more likely during peak activity when prevalence of disease is high. False positive test results are more likely during periods of low influenza activity when prevalence is moderate to low.  The performance of the assay has not been established in individuals who received nasally administered influenza vaccine. Individuals who received nasally administered influenza A vaccine may have positive test results for up to three days after vaccination. http://www.cdc.gov/mmwr/preview/mmwrhtml/rr57e717a1.htm  Do not use any reagent past the expiration date.  Optimum specimen types and timing for peak viral levels during infections caused by a novel influenza A virus have not been determined. Collection of multiple specimens from the same patient may be necessary to detect the virus.  If the virus mutates in the rRT-PCR target region, a specific novel influenza A virus may not be detected or may be detected less predictably. Inhibitors or other types of interference may produce a false negative result. An interference study evaluating the effect of common cold medications was not performed.  Test performance can be affected because the epidemiology and pathology of disease caused by a specific novel influenza A virus is not fully known. For example, clinicians and laboratories may not know the optimum types of specimens to collect, and when during the course of infection these specimens are most likely to contain levels of virus that can be readily detected.  Detection of viral RNA may not indicate the presence of infectious virus or that influenza is the causative agent for clinical symptoms.  The performance of this test has not been established for monitoring treatment of influenza A or B infection.  The performance of this test has not been established for screening of blood or blood product for the presence of influenza A or B.  This test cannot rule out diseases caused by other bacterial or viral pathogens.

Expected values
The

Performance Characteristics Clinical Performance
The performance characteristics of oligonucleotide primer and probe sets of the Influenza A/B Typing assay were evaluated in multiple clinical investigations and influenza seasons. The combined summary clinical data are presented as well as an additional clinical evaluation to demonstrate the performance of the panel with an alternate enzyme chemistry.

Analytical Reactivity (Inclusivity)
The analytical inclusivity of the InfA and InfB primer and probe sets was demonstrated by testing ten (10)

Analytical Specificity -Exclusivity
Non-Influenza Human Respiratory Pathogens Analytical specificity (cross-reactivity) was evaluated by testing InfA and InfB primer/probe sets with nucleic acids extracted from 36 organisms (16 viruses, 19 bacteria, and 1 yeast) representing common respiratory pathogens or flora commonly present in specimens collected from the nasopharynx region. Bacteria and yeast were tested at concentrations greater than or equal to10 6 cfu/ml with the exception of Chlamydia pneumoniae which was quantified by determining inclusion forming units and Mycobacterium tuberculosis where DNA was extracted from pure culture and quantified by spectrophotometry. Non-influenza respiratory viruses were tested at concentrations greater than 10 6 TCID 50 /mL with the exception of human parainfluenza type 2 (10 3.1 TCID 50 /ml due to difficulty generating a high titer virus stock in culture) and human parainfluenza type 1 where RNA was extracted from viral culture and quantified by spectrophotometry. Similarly, RNA was extracted from viral cultures of Coronaviruses CoV 229E and CoV OC43 and concentrations were determined by spectrophotometry. The study demonstrated that the primer and probe sets contained within the CDC Human Influenza Real-Time RT-PCR Diagnostic Panel did not cross-react with any of the non-influenza respiratory pathogens or commensal organisms and demonstrated 100% concordance with the expected results.

Exclusivity with Influenza Viruses with Pandemic Potential
To demonstrate the ability of the InfA primer and probe set to detect influenza A/H5 viruses with pandemic potential, 10 avian A/H5 influenza viruses that have been shown to infect humans were tested. The avian A/H5 influenza viruses were grown and harvested from allantoic/amniotic fluid from infected embryonated chicken eggs (ECE) and titered. Each virus was extracted using the Roche MagNA Pure Compact RNA Isolation kit, and the purified RNA was tested in triplicate. To demonstrate the ability of the InfA primer and probe set to detect potential pandemic influenza A viruses and exclusivity of the InfB primer and probe set, 15 non-human influenza viruses that have been shown to infect humans were tested. The non-human influenza viruses were propagated in ECE, grown to high titer, and harvested as described previously. Each virus was extracted using Roche MagNA Pure Compact RNA kit. extraction (QIAGEN Ltd.), the Roche MagNA Pure LC Automated Isolation System with Total RNA isolation method, and the QIAGEN QIAamp ® Viral RNA manual extraction method. The manufacturer's instructions for use provided in the package insert were followed.
The panels and assay controls were tested at each site by 2 operators on 5 different days within a 10-day period. Each site tested one of the four extraction methods associated with this device. Simulated samples in the panel used in the reproducibility evaluation were:  Sample #1 Influenza A/H1N1 at low viral RNA titer range  Sample #2 Influenza A/H1N1 at a 1:10 dilution of sample 1  Sample #3 Influenza A/H3N2 at low viral RNA titer range  Sample #4 Influenza A/H3N2 at a 1:10 dilution of sample 3  Sample #5 Influenza A/H5N1 WT at low viral RNA titer range  Sample #6 Influenza A/H5N1 WT at a 1:10 dilution of sample 5  Sample #7 Influenza B Yamagata at low viral RNA titer range  Sample #8 Influenza B Yamagata at a 1:10 dilution of sample 7  Sample #9 Influenza Negative (Uninfected A549 cells) LB-067 Rev 1 CDC/OID/NCIRD/Influenza Division Effective: June 13, 2014

Enzyme Equivalency
Clinical Comparison A prospective clinical study was conducted during the 2011-2012 influenza season to compare the performance of the CDC Human Influenza rRT-PCR Diagnostic Panel using Quanta BioSciences qScript™ One-Step qRT-PCR Kit, Low ROX (Quanta qScript™) and Invitrogen Superscript™ III Platinum® One-Step Quantitative RT-PCR Kit (Invitrogen Superscript™). Residual material from a total of 1,002 respiratory specimens from patients who were symptomatic for influenza-like illness (ILI) was collected and tested at 6 clinical sites. Nine hundred thirty-one specimens were included in the data analysis after exclusion of samples with inconclusive results (42), technician or instrument error (25), or unspecified specimen type (4).
The results from the prospective study are summarized in the table below showing the percent sensitivity or specificity with the two-sided 95% confidence interval. The analytical and clinical performance of the CDC Human Influenza rRT-PCR Diagnostic Panel using Quanta qScript™ is substantially equivalent when compared with the Invitrogen Superscript™. Both enzyme kits are cleared for use with the CDC Human Influenza rRT-PCR Diagnostic Panel.