key: cord-0733603-asf5c7xu
authors: Jenny, Shireen L.; Hu, Yaobi; Overduin, Pieter; Meijer, Adam
title: Evaluation of the Xpert Flu A Panel nucleic acid amplification-based point-of-care test for influenza A virus detection and pandemic H1 subtyping
date: 2010-07-31
journal: J Clin Virol
DOI: 10.1016/j.jcv.2010.07.005
sha: d2882f37917b6c8f2061da45bd1b58f467ac5cff
doc_id: 733603
cord_uid: asf5c7xu

BACKGROUND: Influenza antigenic point-of-care (POC) tests are too insensitive for individual reliable diagnosis of influenza virus infections without additional laboratory confirmation. Molecular POC tests could be a valuable alternative. OBJECTIVES: To evaluate the first influenza molecular POC test commercially available, the Cepheid Xpert Flu A Panel designed to simultaneously detect influenza A virus and subtype A(H1N1) 2009 pandemic virus, and compare it with in-house real-time RT-PCR (qRT-PCR). STUDY DESIGN: Clinical specimens positive for influenza virus and influenza virus isolates with different viral loads and of different type and subtype were used to determine the analytical reactivity and sensitivity. A panel of pathogen negative specimens and isolates of 19 different respiratory pathogens were used to determine the analytical specificity. RESULTS: Except A(H9N2) virus the Xpert Flu A Panel detected A(H1N1) seasonal and 2009 pandemic, A(H3N2), A(H5N2), A(H5N1) and A(H7N7) viruses and correctly subtyped A(H1N1) 2009 virus. Analytical sensitivity was similar to qRT-PCR in the range of 400–5000 viral particles per ml. However, of most subtypes some specimens with cycle threshold values greater than 30 in qRT-PCR and A(H1N1) 2009 specimens with inconsistent results in the qRT-PCR due to primer or probe mismatches were not detected in the Xpert Flu A Panel. Analytical specificity was 100%. CONCLUSIONS: The Xpert Flu A Panel is the first commercially available POC molecular test for detection of influenza A virus and determination of the H1 2009 subtype and is analytically reasonable sensitive compared with qRT-PCR and highly specific and therefore a welcome alternative to antigenic POC tests.

For diagnosis of influenza virus infections a variety of techniques is being used, of which the application depends on the clinical setting and purpose. 1 Time consuming virus isolation is required to determine the antigenic match with the influenza vaccine whilst point-of-care (POC) antigenic tests are used for rapid determination of infection to be able to timely implement therapy for the patient or preventive measures to limit spread in outbreak situations. [1] [2] [3] [4] A major drawback, however, of POC antigenic tests is their relative insensitivity and hence unsuitability for individual patient diagnosis without additional (confirmatory) tests. 2,3 Therefore, a need for more sensitive POC tests arose, in particular for diagnosing infections with the highly fatal A(H5N1) influenza virus. 5 Although real-time RT-PCR (qRT-PCR) is rapid it needs expensive equipment, a well suited laboratory and trained personnel for RNA purification and RT-PCR reagent preparation and still takes about 4 hours. 1, 3 The main bottle-neck in developing POC qRT-PCR has been sample preparation. 6 . They all combine sample preparation with single or multiplex qRT-PCR in a closed system and an assay can be completed within an hour. A(H1N1) 2009 pandemic virus was included in these assays as POC qRT-PCR systems could be particular useful for on-site triage and for rapid and easy diagnosis during off-office hours. The first commercial available POC qRT- 

Evaluation of the Xpert Flu A Panel POC test and comparison with in-house real-time RT-PCR (qRT-PCR) assays used for influenza A virus detection and subtyping of the A(H1N1) 2009 pandemic virus.

The primers en probes in the Xpert Flu A Panel are designed for the generic detection of the type A influenza virus matrix gene and for the specific detection of the haemagglutinin gene of the A(H1N1) 2009 pandemic virus. The panel consists of a single-use cartridge with freeze-dried reagents for RNA extraction and realtime RT-PCR to which nucleic acid binding reagent in a squeeze ampoule and the specimen using a disposable pipette provided in the kit has to be added. A 4-site GeneXpert Dx System (Cepheid, Sunnyval, CA) attached to a laptop computer with a barcode reader was used. Assays were performed according to manufacturer's instructions.

Briefly, nucleic acid was purified from specimens using a Magna-Pure LC system with the MagnaPure LC total nucleic acid isolation kit (Roche Diagnostics, Almere, The Netherlands). qRT-PCR was performed on a LightCycler 480 (Roche Diagnostics). Primary qRT-PCR diagnostics of clinical specimens was initially done using the Taqman Master kit for two-step qRT-PCR (Roche Diagnostics, Almere, The Netherlands), replaced during the 2009 A(H1N1) pandemic by the TaqMan EZ RT-PCR core reagents kit for one-step qRT-PCR (Applied Biosystems, Nieuwerkerk aan den IJssel, The Netherlands). For optimal comparison, influenza virus containing specimens were repeated using the one-step qRT-PCR in parallel with the Xpert Flu A Panel on the same day. Detailed information about the (qRT-)PCR assays for the pathogens listed in Tables 1 and 2 are available upon request. 

For determination of the analytical reactivity, clinical specimens (combined nose and throat swabs in Glucose-Lactalbumin-Yeast viral transport medium [GLY]) containing different influenza viruses with a variety of viral loads or with inconsistent qRT-PCR results, several influenza virus isolates in different dilutions in GLY and the Quality Control for Molecular Diagnostics (QCMD) 2009 influenza virus haemagglutinin typing External Quality Assessment (EQA) panel consisting of various dilutions of influenza virus isolates in virus transport medium were used (Table 1) . For determination of the analytical specificity, a respiratory panel and negative specimens were used (Table 2 ). For determination of the analytical sensitivity, qRT-PCR cycle threshold (Ct) values were used as a semi-quantitative measure whereas for determination of the exact viral load in virus particles per ml an electron microscopy (EM) counted standard of human influenza A/Puerto Rico/8/1934 (H1N1) was used. All specimens that were parallel tested with the Xpert Flu A panel and the qRT-PCR had the same freeze/thaw cycle. As Universal Transport Medium (UTM) (Copan Italia S.p.A, Italy) is the preferred specimen transport medium for the Xpert Flu A Panel and we use GLY transport medium, we compared performance of both transport media by diluting clinical specimens in UTM and GLY and analysing the diluted specimens by both assays. Table 1) 

None of the 19 specimens of the respiratory panel containing viruses other than influenza A virus and bacteria commonly detected in specimens from patients with influenza-like illness were positive in the Xpert Flu A Panel tests (Table 2 ). In addition, four virus negative specimens were also negative in the Xpert Flu A Panel tests.

Using the EM counted standard the viral load of virus isolates were determined, for A/Bilthoven/4310800347/2008 (H1N1) seasonal 5.2E + 08 ± SD 3.2E + 07 virus particles per ml, for A/Bilthoven/4310902347/2009 (H1N1) 2009 4.0E + 08 ± SD 2.7E + 07 virus particles per ml and for A/Bilthoven/ 4310801070/2008 (H3N2) 4.2E + 08 ± SD 1.8E + 07 virus particles per ml. Both qRT-PCR and Xpert Flu A Panel matrix tests performed similar for all three viruses with an analytical sensitivity of about 400-5000 viral particles per ml (Fig. 1) shown in Table 1 were specimens with lowest viral load tested for a given subtype; Ct values in the qRT-PCR matrix assay greater than 30.

Preliminary results from other users of the Xpert Flu A Panel assay suggested that freeze-thawing of nose or throat swabs collected in UTM might result in clogging of the cartridge. With none of the freeze-thawed clinical specimens in GLY analysed in this study we noticed any reduced performance due to clogging of the cartridge. No significant differences were found between the Xpert Flu A Panel and the qRT-PCR with three original clinical specimens diluted 1:10 in GLY or UTM and subsequently freeze-thawed (Fig. 2) . 

The US Food and Drug Administration (FDA) granted Emergency Use Authorization for the use of the Cepheid Xpert Flu A Panel (granted December 2009) and the Iquum Liat Influenza A/2009 H1N1 Assay (granted May 2010) tests for detection of influenza A virus and identification of the H1N1 2009 subtype in laboratories certified under the Clinical Laboratory Improvement Amendments (CLIA) to perform "moderate complexity" testing, which enables the tests to be performed in hospital near-patient, aka POC, settings. For POC use the GeneXpert system is available as a 1-site apparatus linked to a laptop and barcode reader. The Iquum system is an all-in-one system and does not need additional equipment. However, compared to the Iquum system the GeneXpert system offers already other useful rapid diagnostics, e.g. methicillin-resistant Staphylococcus aureus and enterovirus in cerebrospinal fluid. 7, 8 Nevertheless, most promising for rapid diagnosis of respiratory illness is the FilmArray Respiratory Panel which combines sample preparation with real-time (RT-)PCR detection of 18 viruses, including the A(H1N1) 2009 variant, and three bacteria commonly found in acute respiratory infections within one hour (http://www.idahotech.com/FilmArray). 9 However, this system is currently still in its clinical trial phase for FDA clearance.

The Xpert Flu Panel A test was easy to perform and took only one hour from start to result with less than one minute hands-on time. Results were easy to interpret with clear positive/negative information, for e.g. clinicians and information in Ct values and amplification curves for users with qRT-PCR experience (Fig. 3) . Validity of the test was clearly indicated by the results of the sample processing control and the probe-checks.

Our analytical evaluation shows the potential of the Xpert Flu A Panel to become a real POC molecular test, however, some limitations were determined. The Xpert Flu A Panel does detect human influenza A viruses including the A(H1N1) 2009 variant with an analytical sensitivity similar to our qRT-PCR, however, some low viral load specimens were not detected. In addition, viral load limitation was particularly noticed for avian influenza viruses A(H5N1) and A(H5N2), whilst A(H9N2) was not detected at all. This is an important limitation as there is an urgent need for molecular POC tests detecting avian influenza viruses. 5 The Xpert Flu A Panel H1 2009 test was found specific for the H gene of the A(H1N1) 2009 virus and was able to subtype all A(H1N1) 2009 virus containing specimens that were found positive in the matrix test of the Xpert Flu A Panel. However, all specimens with repeatable inconsistent A(H1N1) 2009 results in the qRT-PCR were not detected in the Xpert Flu A Panel. Sequencing of the primer and probe targets of the qRT-PCR of these viruses revealed that there were some mismatches explaining the incon- sistent results (not shown). As the primer and probe binding targets included in the Xpert Flu A Panel were not revealed by Cepheid it was impossible to determine whether lack of performance with some specimens was caused by mismatching of primers and/or probes. Drift of influenza viruses necessitates regular updating of primers and probes and especially quick updating is needed when a virus with mutated primer or probe binding site becomes the dominant virus in a season. Emergency use authorisation of commercial influenza molecular tests might overcome this problem, however, even emergency development and validation might take longer than a season lasts. Therefore, molecular POC can never fully replace in-house qRT-PCR for detection and identification of influenza viruses.

In contrast to findings of others we could not confirm clogging problems due to the use of UTM and freeze/thawing of clinical specimens. All our historical specimens have been vigorously vortex on arrival disrupting large particles and mucus flocks which might play a role in clogging when omitted in POC settings.

Although the analytical sensitivity of the Xpert Flu A Panel exceeds the published analytical sensitivity of antigenic A(H1N1) 2009 tests 10 by at least 1000-fold, the purchase of a 1-site Gen-eXpert machine and the costs per test (approximately 45 euros per cartridge) will be still a bottle-neck compared to the antigenic tests (approximately 15 euros per test) for real POC use. However, the Xpert Flu A Panel is a welcome alternative to antigenic rapid tests in large practices and to qRT-PCR in laboratories for emergency diagnostics during off-office hours. To become really useful as POC test, especially in areas where avian influenza viruses infecting humans are endemic, the Xpert Flu A Panel should be improved for detection of all influenza A viruses and for determination of relevant subtypes.

In conclusion, the Xpert Flu A Panel is the first commercially available POC molecular test for detection of influenza A virus and determination of the H1 2009 subtype, is analytically reasonable sensitive compared with qRT-PCR and highly specific and therefore a welcome alternative to antigenic POC tests.

None.

None declared.

Not required.

Laboratory diagnosis of human seasonal and pandemic influenza virus infection

World Health Organization. WHO recommendations on the use of rapid testing for influenza diagnosis

Rapid tests for the diagnosis of influenza

Importance of rapid testing to combat the global threat of bird flu

Expert consultation on diagnosis of H5N1 avian influenza infections in humans

Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings

Evaluation of the Xpert MRSA assay for rapid detection of methicillin-resistant Staphylococcus aureus from nares swabs of geriatric hospitalized patients and failure to detect a specific SCCmec type IV variant

Multicenter beta trial of the GeneXpert enterovirus assay

Comparison of two multiplex respiratory pathogen PCR panels: The FilmArray TM 20-Pathogen Panel versus Luminex xTAG ® 12-Pathogen Panel. Poster session presented at: annual meeting Pan American Society for Clinical Virology

Analytical sensitivity of rapid influenza antigen detection tests for swine-origin influenza virus (H1N1)

The authors thank Ngoc Hoa Chung