Title

Evaluation of NxTAG Respiratory Pathogen Panel andComparison with xTAG Respiratory Viral Panel Fast v2 and FilmArray Respiratory Panel for Detecting Respiratory Pathogens inNasopharyngeal Aspirates and SwineAvian-Origin Influenza ASubtypes in Culture Isolates

Author(s) Chan KH To KKW Li TW Wong TL Zhang R Chik KKHChan G Yip CY Chen H Hung FNI Chan JFW Yuen KY

Citation Advances in Virology 2017 v 2017 p 13242761-13242768

Issued Date 2017

URL httphdlhandlenet10722259397

Rights This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 40 International License

Research ArticleEvaluation of NxTAG Respiratory Pathogen Paneland Comparison with xTAG Respiratory Viral Panel Fast v2and Film Array Respiratory Panel for Detecting RespiratoryPathogens in Nasopharyngeal Aspirates and SwineAvian-OriginInfluenza A Subtypes in Culture Isolates

K H Chan1 K K W To123 P T W Li1 T L Wong1 R Zhang1 K K H Chik1

G Chan1 C C Y Yip4 H L Chen123 I F N Hung5 J F W Chan123 and K Y Yuen1236

1Department of Microbiology The University of Hong Kong Pokfulam Hong Kong2State Key Laboratory for Emerging Infectious Diseases The University of Hong Kong Pokfulam Hong Kong3Carol Yu Centre for Infection The University of Hong Kong Pokfulam Hong Kong4Department of Microbiology Queen Mary Hospital Pokfulam Hong Kong5Department of Medicine The University of Hong Kong Pokfulam Hong Kong6The Collaborative Innovation Center for Diagnosis and Treatment of Infectious DiseasesThe University of Hong Kong Pokfulam Hong Kong

Correspondence should be addressed to K H Chan chankh2hkucchkuhk and K Y Yuen kyyuenhkucchkuhk

Received 16 May 2017 Accepted 26 July 2017 Published 29 August 2017

Academic Editor Subhash C Verma

Copyright copy 2017 K H Chan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This study evaluated a newmultiplex kit Luminex NxTAG Respiratory Pathogen Panel for respiratory pathogens and compared itwith xTAG RVP Fast v2 and FilmArray Respiratory Panel using nasopharyngeal aspirate specimens and culture isolates of differentswineavian-origin influenzaA subtypes (H2N2H5N1H7N9H5N6 andH9N2) NxTAGRPP gave sensitivity of 952 specificityof 996 PPV of 935 andNPVof 997NxTAGRPP xTAGRVP and FilmArray RP had highly concordant performance amongeach other for the detection of respiratory pathogens The mean analytic sensitivity (TCID50ml) of NxTAG RPP xTAG RVPand FilmArray RP for detection of swineavian-origin influenza A subtype isolates was 07 418 and 08 respectively All threemultiplex assays correctly typed and genotyped the influenza viruses except for NxTAG RRP that could not distinguish H3N2from H3N2v Further investigation should be performed if H3N2v is suspected to be the cause of disease Sensitive and specificlaboratory diagnosis of all influenza A viruses subtypes is especially essential in certain epidemic regions such as Southeast AsiaThe results of this study should help clinical laboratory professionals to be aware of the different performances of commerciallyavailable molecular multiplex RT-PCR assays that are commonly adopted in many clinical diagnostic laboratories

1 Introduction

Respiratory tract infections (RTIs) are caused by variouspathogens that are often indistinguishable from one anotherby clinical diagnosis RTIs are a major cause of mortalitymorbidity and hospitalization especially in children agedbelow five years and pose significant economic burden [1ndash6]

Most RTIs are caused by viruses Common respiratory virusesinclude influenza virus adenovirus parainfluenza virus res-piratory syncytial virus (RSV) human coronaviruses humanmetapneumovirus (hMPV) and rhinovirus [7 8]

Rapid and accurate identification of the causativepathogens of RTIs can help guide treatment decisionspossibly reducing the length of hospital stays and associated

HindawiAdvances in VirologyVolume 2017 Article ID 1324276 8 pageshttpsdoiorg10115520171324276

Research ArticleEvaluation of NxTAG Respiratory Pathogen Paneland Comparison with xTAG Respiratory Viral Panel Fast v2and Film Array Respiratory Panel for Detecting RespiratoryPathogens in Nasopharyngeal Aspirates and SwineAvian-OriginInfluenza A Subtypes in Culture Isolates

K H Chan1 K K W To123 P T W Li1 T L Wong1 R Zhang1 K K H Chik1

G Chan1 C C Y Yip4 H L Chen123 I F N Hung5 J F W Chan123 and K Y Yuen1236

1Department of Microbiology The University of Hong Kong Pokfulam Hong Kong2State Key Laboratory for Emerging Infectious Diseases The University of Hong Kong Pokfulam Hong Kong3Carol Yu Centre for Infection The University of Hong Kong Pokfulam Hong Kong4Department of Microbiology Queen Mary Hospital Pokfulam Hong Kong5Department of Medicine The University of Hong Kong Pokfulam Hong Kong6The Collaborative Innovation Center for Diagnosis and Treatment of Infectious DiseasesThe University of Hong Kong Pokfulam Hong Kong

Correspondence should be addressed to K H Chan chankh2hkucchkuhk and K Y Yuen kyyuenhkucchkuhk

Received 16 May 2017 Accepted 26 July 2017 Published 29 August 2017

Academic Editor Subhash C Verma

Copyright copy 2017 K H Chan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This study evaluated a newmultiplex kit Luminex NxTAG Respiratory Pathogen Panel for respiratory pathogens and compared itwith xTAG RVP Fast v2 and FilmArray Respiratory Panel using nasopharyngeal aspirate specimens and culture isolates of differentswineavian-origin influenzaA subtypes (H2N2H5N1H7N9H5N6 andH9N2) NxTAGRPP gave sensitivity of 952 specificityof 996 PPV of 935 andNPVof 997NxTAGRPP xTAGRVP and FilmArray RP had highly concordant performance amongeach other for the detection of respiratory pathogens The mean analytic sensitivity (TCID50ml) of NxTAG RPP xTAG RVPand FilmArray RP for detection of swineavian-origin influenza A subtype isolates was 07 418 and 08 respectively All threemultiplex assays correctly typed and genotyped the influenza viruses except for NxTAG RRP that could not distinguish H3N2from H3N2v Further investigation should be performed if H3N2v is suspected to be the cause of disease Sensitive and specificlaboratory diagnosis of all influenza A viruses subtypes is especially essential in certain epidemic regions such as Southeast AsiaThe results of this study should help clinical laboratory professionals to be aware of the different performances of commerciallyavailable molecular multiplex RT-PCR assays that are commonly adopted in many clinical diagnostic laboratories

1 Introduction

Respiratory tract infections (RTIs) are caused by variouspathogens that are often indistinguishable from one anotherby clinical diagnosis RTIs are a major cause of mortalitymorbidity and hospitalization especially in children agedbelow five years and pose significant economic burden [1ndash6]

Most RTIs are caused by viruses Common respiratory virusesinclude influenza virus adenovirus parainfluenza virus res-piratory syncytial virus (RSV) human coronaviruses humanmetapneumovirus (hMPV) and rhinovirus [7 8]

Rapid and accurate identification of the causativepathogens of RTIs can help guide treatment decisionspossibly reducing the length of hospital stays and associated

HindawiAdvances in VirologyVolume 2017 Article ID 1324276 8 pageshttpsdoiorg10115520171324276

2 Advances in Virology

healthcare costs Moreover it aids in epidemiologic trackingof local outbreaks or epidemics by implementing properinfection control measures and administering effectiveantimicrobial therapy Rapid laboratory identification of RTIpathogens has been associated with up to 50 reductionin hospital stays 30 reduction in the inappropriate use ofantibiotics and 20 reduction in unnecessary diagnostictests and procedures [9] Multiplexed molecular assays thatcan concurrently detect multiple pathogens from the samesample in a single reaction have been increasingly adoptedin clinical microbiology laboratories in the recent years[10] Several studies have shown that these molecular testshave superior diagnostic capacity and cost-effectiveness ofmolecular tests when compared to conventional and otherdiagnostic methods [11 12]

For influenza viruses rapid molecular tests based onpolymerase chain reaction (PCR) using primers that targetthe relatively conserved matrix (M) gene are frequentlyused for achieving rapid and accurate laboratory diagnosisHowever variations in the gene and amino acid sequences indifferent influenza virus subtypes due to rapid mutations ofthe viral RNA polymerase may affect the results of these testsWith the emergence of human infections due to the swine-origin influenza A H3N2 variant (H3N2v) virus in NorthAmerica in 2010 andmore recently the avian-origin influenzaA H7N9 and H5N6 in China [13ndash15] it would be importantto ascertain the analytical sensitivities of these rapid tests forthe novel influenza viruses [16 17] In a previous study wehave demonstrated that xTAG RVP has lower sensitivity fordetection of H7N9 in patient samples and culture isolates[16 17]

NxTAG Respiratory Pathogen Panel (RPP) CE-IVD(Luminex Molecular Diagnostics Toronto Canada) is arecent new qualitative multiplex molecular test for thedetection of nucleic acids from multiple respiratory virusesand bacteria extracted from respiratory specimens It detects22 viral and bacterial targets including influenza A virusinfluenza A virus subtype seasonal H1 influenza A virussubtype seasonal H3 influenza A virus subtype swine-originH1N1pdm09 influenza B virus RSV A and B parainfluenzaviruses types 1 to 4 adenovirus hMPV rhinovirusentero-virus human coronavirus- (HCoV-) 229E HCoV-OC43HCoV-NL63 HCoV-HKU1 human bocavirusChlamydoph-ila pneumoniae Mycoplasma pneumoniae and Legionellapneumophila (Table 1) Although there were several stud-ies about the performance of NxTAG RPP using RUO(research use only) assay the performance of the NxTAGRPP CV-IVD for detection of respiratory viruses partic-ularly the sensitivity and specificity of influenza A sub-types remains unclear [18ndash20] The purpose of this studyis to evaluate the NxTAG RPP CE-IVD and compare itwith Luminex xTAG Respiratory Viral Panel (RVP) Fast v2(Luminex Molecular Diagnostics Toronto Canada) and Fil-mArray Respiratory Panel (RP) (bioMerieux Marcy lEtoileFrance) using nasopharyngeal aspirate (NPA) samples ob-tained frompatientswithRTIs and culture isolates of differentswine and avian-origin influenza A subtypes (H2N2 H3N2vH5N1 H5N6 H7N9 and H9N2)

2 Materials and Methods

21 Clinical Specimens The 133 NPA specimens selectivelycollected from patients with symptoms and signs of RTIswho were managed in Queen Mary Hospital Hong Kongand with clinical PCR results were used in this studyThese NPA samples were collected into a viral transportmediumas described previously [21] andwere tested by directimmunofluorescence (DFA) (D3 Ultra 8 DFA RespiratoryVirus Screening and Identification Kit Diagnostic HybridsInc (Quidel) USA) for influenza A virus influenza B virusparainfluenza viruses types 1 to 4 respiratory syncytial virushuman metapneumovirus and adenovirus and an aliquotof each NPA was sent to Hong Kong Government VirusLaboratory the Department of Health for further testing byRT-PCR for detection of influenza A virus influenza B virusparainfluenza viruses types 1 to 4 respiratory syncytial virusadenovirus and rhinovirusenterovirus as part of the routineclinical diagnostic protocol Single RT-PCR for detectionof human metapneumovirus human coronavirus- (HCoV-)229E HCoV-OC43 HCoV-NL63 HCoV-HKU1 humanbocavirus Chlamydophila pneumoniae Mycoplasma pneu-moniae and Legionella pneumophila was used as describedpreviously [22ndash27] All these PCR results were used as posi-tive referenceThe remaining samples were then recruited forNxTAG RVP evaluation A sufficient quantity of the sampleswas used for further testing with xTAG RVP and FilmArrayRP

22 Influenza Isolates Two swine-origin and four avian-origin influenza A human isolates namely H1N1pdm09 (A41574209H1N1) H3N2v (AWisconsin122010) H2N2 (AAsia573) H5N1 (AVietnam302804) H7N9 (AAnhui12013) and H9N2 (AHK107399) were used in thisstudy Additionally an H5N6 avian isolate [H5N6 (AGreatEgretH5N6HK2016)] was also included The viruses werecultured for 2 days inMadin-Darby Canine Kidney (MDCK)cells with tosyl sulfonyl phenylalanyl chloromethyl ketone-(TPCK-) treated trypsin (2 120583gml) (Sigma St Louis MO)as previously described [28] Aliquots of culture supernatantwere frozen at minus80∘C until use Serial tenfold dilutions ofthe virus stock with serum-free minimal essential medium(MEM) (Gibco BRL Grand Island NY) were tested inNxTAG RPP xTAG RVP and FilmArray RP The viral Mgene genome copy numbers per ml of each virus dilutionwere determined by real-time quantitative RT-PCR (q-PCR)as previously described with modification [29 30] Briefly5 120583L purified RNA was amplified in a 25120583L reaction con-taining 05120583L Superscript III Reverse TranscriptasePlatinumTaq DNA polymerase (Invitrogen Carlsbad CaliforniaUSA) 005 120583L ROX reference dye (25mM) 125 120583L of 2xreaction buffer 800 nmolL forward primer (51015840-GACCRA-TCCTGTCACCTCTGAC-31015840) 800 nmolL reverse primer(51015840-AGGGCATTYTGGACAAAKCGTCTA-31015840) and probe200 nmolL (FAM-51015840-TGCAGTCCTCGCTCACTGGGC-ACG-31015840-BHQ1) The thermal cycling conditions were 30minutes at 50∘C for reverse transcription and then 2minat 95∘C for RT inactivationinitial denaturation followed by50 cycles of 15 s at 95∘C and 30 s at 55∘C All reactions

Advances in Virology 3

Table 1 Detectable pathogens and targets

Viruses NxTAG RPP xTAG RVP Fast v2 FilmArray RPInfluenza A virus radic radic radic

Subtype (H1 H1pdm09 H3) radic (H1 H3) radic

Influenza B virus radic radic radic

Respiratory syncytial virus (A B) (A B) radic

Human coronavirus (229E OC43 NL63 HKU1) radic radic radic

Parainfluenza viruses 1ndash4 radic radic radic

Human metapneumovirus radic radic radic

Adenovirus radic radic radic

Human bocavirus radic radic

Human rhinovirusEnterovirus radic radic radic

Chlamydophila pneumoniae radic radic

Legionella pneumophila radic

Mycoplasma pneumoniae radic radic

Bordetella pertussis radic

Total targets 22 18 20

were performed using StepOnePlus Real-Time PCR System(Applied Biosystems Foster City) For quantitative assaya reference standard plasmid was prepared by cloning thetarget M gene amplicon into pCRII-TOPO vector (TOPOTA Cloning Kit Invitrogen San Diego CA) according tothemanufacturerrsquos instructionsThe copy number of the stan-dard plasmid was determined based on molar concentrationof the plasmid (1 molar is equivalent to about 60221415 times 1023copies numbers) A series of 6 log10 dilutions equivalent to 1times 101 to 1 times 106 copies per reaction were then prepared fromthe reference standard plasmid to generate calibration curvesand run in parallel with the test samples The limit ofdetection of the M gene qRT-PCR is 10 copies per reactionat 95 confidence level The same virus dilutions wereinoculated onto MDCK cells to determine the 50 tissueculture infective dose (TCID50) using the Reed-Muenchmethod as previously described [28]

23 Nucleic Acid Extraction Nucleic acid was detected usingeasyMAG extraction platform (bioMerieux Marcy lEtoileFrance) and extracted for NxTAG RPP and xTAG RVP fromthe specimens as previously described [31] Total nucleicacid extraction was isolated by using the NucliSENS easy-MAG instrument (bioMerieux) Briefly 10 120583l of MS2 internalcontrol was first added to 200120583l of sample The mixturewas added to 2ml of lysis buffer and was incubated for 10minutes at room temperature The lysed sample was thentransferred to the well of a plastic vessel with 100120583l ofsilica This was followed by automatic magnetic separationNucleic acid was recovered in 110 120583l elution buffer By usinga parallel specimen (300 120583l) nucleic acid extraction PCRand detection were performed in FilmArray according to themanufacturerrsquos instructions The NxTAG RPP incorporatesmultiplex Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR)with the Luminex proprietary universal tag sortingsystem on the Luminex platform for detecting respiratorypathogen targets according to the manufacturerrsquos instruc-tions Briefly the extracted total nucleic acid is added to

preplated Lyophilized Bead Reagents (LBRs) and mixed toresuspend the reaction reagentsThe reaction is amplified viaRT-PCR and the reaction product undergoes bead hybridiza-tion within the sealed reaction well The hybridized taggedbeads are then sorted and read on the MAGPIX instrumentand the signals are analyzed using the NxTAG RespiratoryPathogen Panel Assay File for SYNCT Software (LuminexAustin Texas USA)

24 Statistical Analysis Diagnostic sensitivity specificityPPV and NPV of the assays against the positive refer-ences and agreement between the assays were calculatedby VassarStats Website for Statistical Computation (httpvassarstatsnet)

3 Results

31 Clinical Samples and Performance of NxTAG RPP Atotal of 133 NPA specimens were tested in this study Thespecimens were collected from 75 males and 58 females witha mean age of 325 years (range 2 months to 99 years ofage) Of 133 specimens 75 positives were detected by DFAand 151 positives were detected by PCR [influenza A virus =16 (H3 = 12 H1pdm09 = 3 H7 = 1) influenza B virus =10 parainfluenza virus 1 = 10 parainfluenza virus 2 = 10parainfluenza virus 3 = 10 parainfluenza virus 4 = 11 RSV =11 hMPV = 11 adenovirus = 4 rhinovirusenterovirus = 28human coronavirus- (HCoV-) 229E = 3 HCoV-OC43 = 6HCoV-NL63 = 1 HCoV-HKU1 = 5 human bocavirus = 9Chlamydophila pneumoniae = 1 Mycoplasma pneumoniae =4 and Legionella pneumophila = 1] All 133 specimens wereevaluated by NxTAG RPP Overall 153 pathogens were iden-tified byNxTAGRPP Ten positives (H1pdm09times 1 RSVAtimes 1P1F1 times 1 adenovirus times 1 EVrhinovirus times 4 and bocavirus times2) were considered as false positives when compared withpositive reference The sensitivity specificity positive predic-tive value and negative predictive value of NxTAG RPP for

Advances in Virology 3

Table 1 Detectable pathogens and targets

Viruses NxTAG RPP xTAG RVP Fast v2 FilmArray RPInfluenza A virus radic radic radic

Subtype (H1 H1pdm09 H3) radic (H1 H3) radic

Influenza B virus radic radic radic

Respiratory syncytial virus (A B) (A B) radic

Human coronavirus (229E OC43 NL63 HKU1) radic radic radic

Parainfluenza viruses 1ndash4 radic radic radic

Human metapneumovirus radic radic radic

Adenovirus radic radic radic

Human bocavirus radic radic

Human rhinovirusEnterovirus radic radic radic

Chlamydophila pneumoniae radic radic

Legionella pneumophila radic

Mycoplasma pneumoniae radic radic

Bordetella pertussis radic

Total targets 22 18 20

were performed using StepOnePlus Real-Time PCR System(Applied Biosystems Foster City) For quantitative assaya reference standard plasmid was prepared by cloning thetarget M gene amplicon into pCRII-TOPO vector (TOPOTA Cloning Kit Invitrogen San Diego CA) according tothemanufacturerrsquos instructionsThe copy number of the stan-dard plasmid was determined based on molar concentrationof the plasmid (1 molar is equivalent to about 60221415 times 1023copies numbers) A series of 6 log10 dilutions equivalent to 1times 101 to 1 times 106 copies per reaction were then prepared fromthe reference standard plasmid to generate calibration curvesand run in parallel with the test samples The limit ofdetection of the M gene qRT-PCR is 10 copies per reactionat 95 confidence level The same virus dilutions wereinoculated onto MDCK cells to determine the 50 tissueculture infective dose (TCID50) using the Reed-Muenchmethod as previously described [28]

23 Nucleic Acid Extraction Nucleic acid was detected usingeasyMAG extraction platform (bioMerieux Marcy lEtoileFrance) and extracted for NxTAG RPP and xTAG RVP fromthe specimens as previously described [31] Total nucleicacid extraction was isolated by using the NucliSENS easy-MAG instrument (bioMerieux) Briefly 10 120583l of MS2 internalcontrol was first added to 200120583l of sample The mixturewas added to 2ml of lysis buffer and was incubated for 10minutes at room temperature The lysed sample was thentransferred to the well of a plastic vessel with 100120583l ofsilica This was followed by automatic magnetic separationNucleic acid was recovered in 110 120583l elution buffer By usinga parallel specimen (300 120583l) nucleic acid extraction PCRand detection were performed in FilmArray according to themanufacturerrsquos instructions The NxTAG RPP incorporatesmultiplex Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR)with the Luminex proprietary universal tag sortingsystem on the Luminex platform for detecting respiratorypathogen targets according to the manufacturerrsquos instruc-tions Briefly the extracted total nucleic acid is added to

preplated Lyophilized Bead Reagents (LBRs) and mixed toresuspend the reaction reagentsThe reaction is amplified viaRT-PCR and the reaction product undergoes bead hybridiza-tion within the sealed reaction well The hybridized taggedbeads are then sorted and read on the MAGPIX instrumentand the signals are analyzed using the NxTAG RespiratoryPathogen Panel Assay File for SYNCT Software (LuminexAustin Texas USA)

24 Statistical Analysis Diagnostic sensitivity specificityPPV and NPV of the assays against the positive refer-ences and agreement between the assays were calculatedby VassarStats Website for Statistical Computation (httpvassarstatsnet)

3 Results

31 Clinical Samples and Performance of NxTAG RPP Atotal of 133 NPA specimens were tested in this study Thespecimens were collected from 75 males and 58 females witha mean age of 325 years (range 2 months to 99 years ofage) Of 133 specimens 75 positives were detected by DFAand 151 positives were detected by PCR [influenza A virus =16 (H3 = 12 H1pdm09 = 3 H7 = 1) influenza B virus =10 parainfluenza virus 1 = 10 parainfluenza virus 2 = 10parainfluenza virus 3 = 10 parainfluenza virus 4 = 11 RSV =11 hMPV = 11 adenovirus = 4 rhinovirusenterovirus = 28human coronavirus- (HCoV-) 229E = 3 HCoV-OC43 = 6HCoV-NL63 = 1 HCoV-HKU1 = 5 human bocavirus = 9Chlamydophila pneumoniae = 1 Mycoplasma pneumoniae =4 and Legionella pneumophila = 1] All 133 specimens wereevaluated by NxTAG RPP Overall 153 pathogens were iden-tified byNxTAGRPP Ten positives (H1pdm09times 1 RSVAtimes 1P1F1 times 1 adenovirus times 1 EVrhinovirus times 4 and bocavirus times2) were considered as false positives when compared withpositive reference The sensitivity specificity positive predic-tive value and negative predictive value of NxTAG RPP for

4 Advances in Virology

detection of respiratory viruses were 952 [95 confidencelevel (Cl) 904ndash977] 996 (95 Cl 993ndash998) 935 (95Cl 884ndash965) and 997 (95 Cl 994ndash999) respectively(Table 2)

32 Performance and Concordance of xTAG RVP and FilmAr-ray RP Of 133 tested samples 71 had enough quantity tobe used for further testing with xTAG RVP and FilmArrayRP The results of these 71 samples tested by the three assayswere then compared Out of these 71 specimens 66 werepositive and 5 were negative by NxTAG RPP Both the xTAGRVP and FilmArray RP detected respiratory pathogens in 63(887) specimens with 96 and 83 pathogens respectivelyThe sensitivity specificity positive predictive value and neg-ative predictive value of xTAG RVP and FilmArray RP were969 999 990 and 997 and 853 999 988and 989 respectively When NxTAG RPP was comparedwith xTAG RVP and FilmArray RP the Kappa value was 097with 95 CI of 095ndash099 and 093 with 95 CI of 089ndash097respectivelyWhen xTAGRVPwas comparedwith FilmArrayRP the Kappa value was 095 with 95 CI of 091ndash098Theseresults suggested that the three assays were highly concordantas a Kappa value higher than 080 indicated concordance

We have previously shown that xTAG RVP has lowsensitivity for detecting H7N9 [16 17] In this study oneof the patient specimens with confirmed H7N9 influenza Ainfection was also not detected by xTAGRVP influenza A butwas detected by NxTAG RPP and FilmArray RP influenza AxTAG RVP has a 1000 and 10 times higher limit of detectionfor H7N9 than NxTAG RPP and FilmArray RP respectively(Table 3) These findings show that NxTAG RRP has greatlyimproved the sensitivity for detecting H7N9 over the xTAGRVP Besides NxTAG RPP has also improved sensitivity fordetecting other respiratory viruses over xTAG RVP (1 aden-ovirus and 2 bocavirus) These findings corroborate anotherrecent report on the partial comparison between the NxTAGRespiratory Pathogen Panel Assay and the Luminex xTAGRespiratory Panel Fast Assay v2 [32] NxTAG RPP detected12more pathogens than FilmArray RP in our study with 50(612) being enterovirusrhinovirus The other additionalpositives detected by NxTAG RPP were adenovirus (2)parainfluenza virus 4b (1) hMPV (1) H1N1pdm (1) and Mpneumoniae (1) Overall 8 positives hMPV (1) P2 (1) P4 (3)enterovirusrhinovirus (2) andM pneumoniae (1) were notdetected by NxTAG RPP All these positives indeed had highCt value (ge35) thatmay be lower than the limit of detection byNxTAG RPP Further study should be required if the primersthat are suspected do not detect epidemic strains

Furthermore the results of 5 samples (4 H1N1pdm 2009and 1 H7N9) were reported to be ldquoequivocalrdquo by FilmArrayRP (Flu A-pan-1 positive Flu A-pan-2 negative) These 5specimens were in fact true positive as confirmed by othertesting methods These findings indicated that M gene (FluA-pan-1) was more sensitive than NS1 gene (Flu A-pan-2) fordetection of H1N1pdm 2009

33 Analytical Sensitivity of Influenza A Culture IsolatesWhen influenza A virus subtype isolates were tested themean (range) analytic sensitivities (viral load log10 copiesml

and TCID50ml) of NxTAG RPP xTAG RVP and FilmArray

RP were 30 (18ndash42) and 07 (00056ndash32) 43 (30ndash64)and 418 (056ndash178) and 34 (27ndash44) and 08 (01ndash178)respectively (Table 3) All avian- and swine-origin influenzaA strains were detected by M gene in NxTAG RPP xTAGRVP and FilmArray RP The swine-origin H1N1pdm09 wascorrectly genotyped byNxTAGRPP and FilmArray RPHow-ever H3N2v and seasonal H3N2 could not be differentiatedby NxTAG RPP

4 Discussion

In the recent years the availability of molecular diagnos-tic tests has revolutionized the diagnosis and surveillanceof infectious diseases and treatment of RTIs Multiplexedmolecular assays simultaneously detect multiple pathogensfrom the same sample in a single reaction vessel providinga more comprehensive picture of infection In this study theclinical performance of the Luminex NxTAG RPP (CV-IVD)in NPA specimens was found to have high sensitivity (952)and specificity (996) for detection of respiratory virusesThese findings are similar to those reported in recent eval-uation studies using NxTAG RPP (RUO) kit by other groups[18ndash20] The performances of the NxTAG RPP xTAG RVPand FilmArray RP were also compared Their results werehighly concordant with the highest concordance reportedbetween NxTAG RRP and xTAG RVP [Kappa value = 097(95 Cl 095ndash099)]

According to the manufacturer M gene (Flu A-pan-1)and NS1 gene (Flu A-pan-2) are used for influenza A typingby FilmArray RP The result is only interpreted as positivewhen both genes are positive The result will be interpretedas ldquoequivocalrdquo when only Flu A-pan-1 is positive and thesystem will suggest the users to repeat testing by the sameassay or other assays In this study the results showed that anldquoequivocalrdquo result by FilmArray RP is most likely to be a truepositive The correct interpretation of these results shouldbe considered to avoid delay in diagnosis and treatmentparticularly in patients with severe influenza A virus infec-tion

In general the analytical sensitivities of the NxTAGRPP and FilmArray RP for the swine- and avian-origininfluenza viruses were comparable but xTAG RVP had morethan one log lower analytical sensitivity than NxTAG RPPand FilmArray RP (Table 3) NxTAG RPP also had higheranalytical sensitivity for detecting H5N6 than the xTAGRVP but the same sensitivity as FilmArray RP and higheranalytical sensitivity for detecting H3N2v than xTAG RVPand FilmArray RP All threemultiplex assays accurately typedand genotyped the influenza viruses except for NxTAG RRPthat does not distinguish subtyped H3N2 from H3N2v Thisfinding was further confirmed by testing another H3N2vstrain AIndiana082011 with 500TCID

50 A recent study

also showed that NxTAG RPP (RUO) only correctly geno-typed 955 of influenza A virus H1N1pdm09 strains [19]

Rapidmultiplex RT-PCR assays for respiratory pathogensdetection are important for establishing diagnosis to facilitatethe implementation of appropriate treatment and infectioncontrol measures Sensitive and specific laboratory diagnosis

Advances in Virology 5

Table2Perfo

rmance

ofNxT

AGRR

Pford

etectin

grespira

tory

virusesinnasoph

aryngealaspirates

Viruses

NxT

AGPo

sRe

fPos

NxT

AGNeg

RefP

osNxT

AGPo

sRe

fNeg

NxT

AGNeg

RefN

egSensitivity

(95

Cl)

Specificity

(95

Cl)

PPV

(95

Cl)

NPV

(95

Cl)

Influ

enza

A16

01

116

100(759ndash

100)

991(946ndash

100)

941(692

ndash997

)100(960ndash

100)

H3

120

0121

100(699

ndash100)

100(962ndash100)

100(699

ndash100)

100(962ndash100)

H1pdm

093

01

129

100(310

ndash100)

992(951ndash100)

75(219

ndash987)

100(964ndash

100)

H1

00

0133

NA

NA

NA

NA

Influ

enza

B10

00

123

100(655ndash100)

100(962ndash100)

100(655ndash100)

100(955ndash100)

RSVA

90

1123

100(629ndash

100)

992(949ndash

100)

90(541ndash995)

100(962ndash100)

RSVB

20

0131

100(19

8ndash100)

100(964ndash

100)

100(198

ndash100)

100(964ndash

100)

PIF1

100

1122

100(655ndash100)

992(949ndash

100)

999(571ndash9

95)

100(962ndash100)

PIF2

91

0123

900(541ndash994)

100(962ndash100)

100(629ndash

100)

992(949ndash

100)

PIF3

100

0123

100(655ndash100)

100(962ndash100)

100(655ndash100)

100(962ndash100)

PIF4

83

0122

727(390

ndash926)

100(962ndash100)

100(598

ndash100)

976(926ndash

99)

Adenoviru

s4

01

128

100(396

ndash100)

992(951ndash100)

800(299

ndash989)

100(964ndash

100)

hMPV

101

0122

909(571ndash9

95)

100(962ndash100)

100(665ndash100)

992(949ndash

100)

HCoV

-229E

30

0130

100(310

ndash100)

100(964ndash

100)

100(310

ndash100)

100(964ndash

100)

HCoV

-OC4

36

00

127

100(517

ndash100)

100(963ndash100)

100(517

ndash100)

100(963ndash100)

HCoV

-NL6

31

00

132

100(55ndash100)

100(965ndash100)

100(55ndash100)

100(965ndash100)

HCoV

-HKU

15

00

128

100(463ndash100)

100(964ndash

100)

100(463ndash100)

100(964ndash

100)

EVrhino

virus

262

4101

929(750ndash

988)

962(900ndash

988)

867(684ndash

956)

981(924ndash

100)

Bocavirus

90

2122

100(629ndash

100)

984(937ndash997)

818(478

ndash968)

100(962ndash100)

Cpn

eumoniae

10

0132

100(55ndash100)

100(965ndash100)

100(55ndash100)

100(965ndash100)

Mpneum

oniae

31

0129

750(219

ndash987)

100(963ndash100)

100(310

ndash100)

992(952ndash100)

Lpn

eumophila

10

0132

100(55ndash100)

100(965ndash100)

100(55ndash100)

100(965ndash100)

Bpertussis

00

0133

NA

NA

NA

NA

Total

158

811

2882

952(904ndash

977)

996(993

ndash998

)935(884ndash

965)

997(994

ndash999

)EV

enteroviru

sHCoV

hum

ancoronavirushM

PVh

uman

metapneum

oviru

sPIFparainflu

enza

virusPP

Vpo

sitivepredictiv

evalueNPV

negativepredictiv

evalueRS

Vrespira

tory

syncytialv

irusNAn

otapplicableR

efreferencePospositiveN

egn

egative

Overalltotaln

umbero

ffalse

positives

is10

becauseinfl

uenzaA

andsubtypingweretestedfora

samples

imultaneou

sly

6 Advances in Virology

Table3Analytic

alsensitivityof

NxT

AGRP

PxT

AGRV

PandFilm

Array

RPford

etectin

ginflu

enza

Aavian-o

rswine-origin

subtypes

Influ

enza

ALimitof

detection[vira

lload(lo

g10c

opiesml)

log10TC

ID50m

l]of

influ

enza

Asubtype

NxT

AGRP

PxT

AGRV

PFilm

Array

RPSubtype

Strains

Viralload

TCID50

Viralload

TCID50

Viralload

TCID50

H1N

1pdm

09AH

K41574209H1N

130

01

30

01

30

01

H2N

2AA

sia573

37

32

37

32

27

032

H3N

2vAW

iscon

sin12

2010

20

01

40

1030

1H5N

1AVietnam

302804

42

056

42

056

42

056

H5N

6AH

5N6HK

2016

30

150

100

30

1H7N

9AA

nhui12013

34

0178

64

178

44

178

H9N

2AH

K107399

1800056

38

056

38

056

Mean

30

07

43

418

34

08

Advances in Virology 7

of all influenza A virus subtypes is especially essentialin certain epidemic regions such as Southeast Asia Thecorrect interpretation of these results is also important andwould avoid delay in patient management and infectioncontrol especially in patients with severe seasonal influenzaA or avianswine-origin influenza A virus subtype infectionClinical microbiologists infectious disease specialists andlaboratory managers should be aware of and understand thedifferent clinical performances of these commercially avail-able molecular multiplex RT-PCR assays that are commonlyadopted in many clinical microbiology laboratories Thesensitivity and specificity of these molecular multiplex assaysmust be predetermined and be able to confidently detectmostof the important avian or swine influenza A subtype infectionincluding H5N1 and H7N9 and this capability is particularlyimportant for use in Southeast Asia

A limitation of this study is that some target numbers aresmall This is due to low prevalent disease and may require alonger period in order to collect a higher number of positivesHigh cost for these multiplex assays is another limitation forus to study more samples

5 Conclusion

In this study the results showed thatNxTAGRPP xTAGRVPand FilmArray RP assays had high sensitivity and specificityfor diagnosis of respiratory diseases They are suitable for usein clinical diagnostic laboratories for detection of respiratorypathogens in patients with RTIs However awareness shouldbe raised that H3N2 could not be distinguished from H3N2vby NxTAGRPP Further investigation should be performed ifH3N2v is suspected to be the cause of disease

Ethical Approval

This study was approved by the Institutional Review Board ofthe University of Hong KongHospital Authority Hong KongWest Cluster

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors would like to thank the staff of the Departmentof Microbiology Queen Mary Hospital for the facilitation ofthis projectThis work was partly supported by the donationsof Larry Chi-Kin Yung and Hui Hoy and Chow Sin LanCharity Fund Limited the authors also obtained fundingfrom the Consultancy Service for Enhancing LaboratorySurveillance of Emerging Infectious Diseases the Depart-ment of Health Hong Kong Special Administrative Regionthe University Development Fund and the Committee forResearch and Conference Grant the University of HongKong the Collaborative Innovation Center for Diagnosis andTreatment of Infectious Diseases the Ministry of Educationof China and the Commissioned Research on Control ofInfectious Diseases (Phase III) of the Health and Medical

Research Fund (HKM-15-M-04) of the Food and HealthBureau of the HKSAR Government

References

[1] W Olszewska M Zambon and P J M Openshaw ldquoDevel-opment of vaccines against common coldsrdquo British MedicalBulletin vol 62 no 1 pp 99ndash111 2002

[2] World Health Organization Burden of Disease Project WorldHealth Organization Geneva Switzerland 2005

[3] B Ehlken G Ihorst B Lippert et al ldquoEconomic impact ofcommunity-acquired and nosocomial lower respiratory tractinfections in young children in Germanyrdquo European Journal ofPediatrics vol 164 no 10 pp 607ndash615 2005

[4] M M Massin J Montesanti P Gerard and P Lepage ldquoSpec-trum and frequency of illness presenting to a pediatric emer-gency departmentrdquo Acta Clinica Belgica vol 61 no 4 pp 161ndash165 2006

[5] K G Nicholson T McNally M Silverman P Simons J DStockton and M C Zambon ldquoRates of hospitalisation forinfluenza respiratory syncytial virus and human metapneu-movirus among infants and young childrenrdquo Vaccine vol 24no 1 pp 102ndash108 2006

[6] A Sauro F Barone G Blasio L Russo and L Santillo ldquoDoinfluenza and acute respiratory infective diseases weigh heavilyon general practitionersrsquo daily practicerdquo European Journal ofGeneral Practice vol 12 no 1 pp 34ndash36 2006

[7] J S Tregoning and J Schwarze ldquoRespiratory viral infections ininfants causes clinical symptoms virology and immunologyrdquoClinical Microbiology Reviews vol 23 no 1 pp 74ndash98 2010

[8] S S Chiu P-L Ho M J S Peiris K H Chan and E L YChan ldquoPopulation-based hospitalization incidence of respira-tory viruses in community-acquired pneumonia in childrenyounger than 5 years of agerdquo Influenza and other RespiratoryViruses vol 8 no 6 pp 626-627 2014

[9] K J Henrickson ldquoCost-effective use of rapid diagnostic tech-niques in the treatment and prevention of viral respiratoryinfectionsrdquo Pediatric Annals vol 34 no 1 pp 24ndash31 2005

[10] E B Popowitch S S OrsquoNeill andM B Miller ldquoComparison ofthe biofire filmarray RP Genmark eSensor RVP Luminex xTAGRVPv1 and Luminex xTAGRVP fastmultiplex assays for detec-tion of respiratory virusesrdquo Journal of Clinical Microbiology vol51 no 5 pp 1528ndash1533 2013

[11] J B Mahony ldquoDetection of respiratory viruses by molecularmethodsrdquo Clinical Microbiology Reviews vol 21 no 4 pp 716ndash747 2008

[12] J B Mahony A Petrich and M Smieja ldquoMolecular diagnosisof respiratory virus infectionsrdquo Critical Reviews in ClinicalLaboratory Sciences vol 48 no 5-6 pp 217ndash249 2011

[13] Centers for Disease Control and Prevention Swine-OriginInfluenza A (H3N2) Virus Infection in Two Children mdash Indianaand Pennsylvania JulyndashAugust MMWR 2011

[14] Y Chen W Liang S Yang et al ldquoHuman infections withthe emerging avian influenza A H7N9 virus from wet marketpoultry clinical analysis and characterisation of viral genomerdquoLancet vol 381 pp 1916ndash1925 2013

[15] Z-F Yang C K P Mok J S M Peiris and N-S ZhongldquoHuman infection with a novel avian influenza A(H5N6) virusrdquoNew England Journal of Medicine vol 373 no 5 pp 487ndash4892015

8 Advances in Virology

[16] K-H Chan K K W To J F W Chan C P Y Li H Chenand K-Y Yuen ldquoAnalytical sensitivity of seven point-of-careinfluenza virus detection tests and two molecular tests fordetection of avian origin H7N9 and swine origin H3N2 variantinfluenza a virusesrdquo Journal of Clinical Microbiology vol 51 no9 pp 3160-3161 2013

[17] K-H Chan K K W To J F W Chan et al ldquoAssessmentof antigen and molecular tests with serial specimens from apatient with influenza A(H7N9) infectionrdquo Journal of ClinicalMicrobiology vol 52 no 6 pp 2272ndash2274 2014

[18] J H K Chen H-Y Lam C C Y Yip et al ldquoClinical evalua-tion of the new high-throughput luminex nxtag respiratorypathogen panel assay for multiplex respiratory pathogen detec-tionrdquo Journal of Clinical Microbiology vol 54 no 7 pp 1820ndash1825 2016

[19] Y-W Tang S Gonsalves J Y Sun et al ldquoClinical evaluationof the luminex nxtag respiratory pathogen panelrdquo Journal ofClinical Microbiology vol 54 no 7 pp 1912ndash1914 2016

[20] C Beckmann andHHHirsch ldquoComparing LuminexNxTAG-Respiratory Pathogen Panel and RespiFinder-22 for multiplexdetection of respiratory pathogensrdquo Journal ofMedical Virologyvol 88 no 8 pp 1319ndash1324 2016

[21] I Hung V Cheng A Wu et al ldquoViral Loads in Clinical Spec-imens and SARS Manifestationsrdquo Emerging Infectious Diseasesvol 10 no 9 pp 1550ndash1557 2004

[22] MWelti K Jaton M Altwegg R Sahli A Wenger and J BilleldquoDevelopment of a multiplex real-time quantitative PCR assayto detect Chlamydia pneumoniae Legionella pneumophilaand Mycoplasma pneumoniae in respiratory tract secretionsrdquoDiagnosticMicrobiology and Infectious Disease vol 45 no 2 pp85ndash95 2003

[23] L J R Van Elden A M Van Loon F Van Alphen et al ldquoFre-quent Detection of Human Coronaviruses in Clinical Speci-mens from Patients with Respiratory Tract Infection by Useof a Novel Real-Time Reverse-Transciptase Polymerase ChainReactionrdquo Journal of Infectious Diseases vol 189 no 4 pp 652ndash657 2004

[24] P C YWoo S K P Lau C-MChu et al ldquoCharacterization andcomplete genome sequence of a novel coronavirus coronavirusHKU1 from patients with pneumoniardquo Journal of Virology vol79 no 2 pp 884ndash895 2005

[25] S S Chiu K H Chan K W Chu et al ldquoHuman coronavirusNL63 infection and other coronavirus infections in childrenhospitalized with acute respiratory disease in Hong KongChinardquoClinical Infectious Diseases vol 40 no 12 pp 1721ndash17292005

[26] J Kuypers N Wright L Corey and R Morrow ldquoDetectionand quantification of human metapneumovirus in pediatricspecimens by real-time RT-PCRrdquo Journal of Clinical Virologyvol 33 no 4 pp 299ndash305 2005

[27] S K P Lau C C Y Yip T-L Que et al ldquoClinical and molecu-lar epidemiology of human bocavirus in respiratory and fecalsamples from children in Hong Kongrdquo Journal of InfectiousDiseases vol 196 no 7 pp 986ndash993 2007

[28] K H Chan S Y Lam P Puthavathana et al ldquoComparativeanalytical sensitivities of six rapid influenza A antigen detectiontest kits for detection of influenza A subtypes H1N1 H3N2 andH5N1rdquo Journal of Clinical Virology vol 38 no 2 pp 169ndash1712007

[29] I F Hung K K To J F Chan et al ldquoEfficacy of Clarithromycin-Naproxen-Oseltamivir Combination in the Treatment of

Patients Hospitalized for Influenza A(H3N2) Infectionrdquo Chestvol 151 no 5 pp 1069ndash1080 2017

[30] Centers for Disease Control and Prevention CDC protocol ofrealtime RTPCR for influenza A (H1N1) Revision 2 Centers forDisease Control and Prevention Atlanta GA 2009

[31] K H Chan W C Yam C M Pang et al ldquoComparison of theNucliSens easyMAG and Qiagen BioRobot 9604 nucleic acidextraction systems for detection of RNA and DNA respiratoryviruses in nasopharyngeal aspirate samplesrdquo Journal of ClinicalMicrobiology vol 46 no 7 pp 2195ndash2199 2008

[32] S Esposito A Scala S Bianchini et al ldquoPartial comparisonof the NxTAG Respiratory Pathogen Panel Assay with theLuminex xTAG Respiratory Panel Fast Assay V2 and singleplexreal-time polymerase chain reaction for detection of respiratorypathogensrdquo Diagnostic Microbiology and Infectious Disease vol86 no 1 pp 53ndash57 2016

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Advances in Virology 5

Table2Perfo

rmance

ofNxT

AGRR

Pford

etectin

grespira

tory

virusesinnasoph

aryngealaspirates

Viruses

NxT

AGPo

sRe

fPos

NxT

AGNeg

RefP

osNxT

AGPo

sRe

fNeg

NxT

AGNeg

RefN

egSensitivity

(95

Cl)

Specificity

(95

Cl)

PPV

(95

Cl)

NPV

(95

Cl)

Influ

enza

A16

01

116

100(759ndash

100)

991(946ndash

100)

941(692

ndash997

)100(960ndash

100)

H3

120

0121

100(699

ndash100)

100(962ndash100)

100(699

ndash100)

100(962ndash100)

H1pdm

093

01

129

100(310

ndash100)

992(951ndash100)

75(219

ndash987)

100(964ndash

100)

H1

00

0133

NA

NA

NA

NA

Influ

enza

B10

00

123

100(655ndash100)

100(962ndash100)

100(655ndash100)

100(955ndash100)

RSVA

90

1123

100(629ndash

100)

992(949ndash

100)

90(541ndash995)

100(962ndash100)

RSVB

20

0131

100(19

8ndash100)

100(964ndash

100)

100(198

ndash100)

100(964ndash

100)

PIF1

100

1122

100(655ndash100)

992(949ndash

100)

999(571ndash9

95)

100(962ndash100)

PIF2

91

0123

900(541ndash994)

100(962ndash100)

100(629ndash

100)

992(949ndash

100)

PIF3

100

0123

100(655ndash100)

100(962ndash100)

100(655ndash100)

100(962ndash100)

PIF4

83

0122

727(390

ndash926)

100(962ndash100)

100(598

ndash100)

976(926ndash

99)

Adenoviru

s4

01

128

100(396

ndash100)

992(951ndash100)

800(299

ndash989)

100(964ndash

100)

hMPV

101

0122

909(571ndash9

95)

100(962ndash100)

100(665ndash100)

992(949ndash

100)

HCoV

-229E

30

0130

100(310

ndash100)

100(964ndash

100)

100(310

ndash100)

100(964ndash

100)

HCoV

-OC4

36

00

127

100(517

ndash100)

100(963ndash100)

100(517

ndash100)

100(963ndash100)

HCoV

-NL6

31

00

132

100(55ndash100)

100(965ndash100)

100(55ndash100)

100(965ndash100)

HCoV

-HKU

15

00

128

100(463ndash100)

100(964ndash

100)

100(463ndash100)

100(964ndash

100)

EVrhino

virus

262

4101

929(750ndash

988)

962(900ndash

988)

867(684ndash

956)

981(924ndash

100)

Bocavirus

90

2122

100(629ndash

100)

984(937ndash997)

818(478

ndash968)

100(962ndash100)

Cpn

eumoniae

10

0132

100(55ndash100)

100(965ndash100)

100(55ndash100)

100(965ndash100)

Mpneum

oniae

31

0129

750(219

ndash987)

100(963ndash100)

100(310

ndash100)

992(952ndash100)

Lpn

eumophila

10

0132

100(55ndash100)

100(965ndash100)

100(55ndash100)

100(965ndash100)

Bpertussis

00

0133

NA

NA

NA

NA

Total

158

811

2882

952(904ndash

977)

996(993

ndash998

)935(884ndash

965)

997(994

ndash999

)EV

enteroviru

sHCoV

hum

ancoronavirushM

PVh

uman

metapneum

oviru

sPIFparainflu

enza

virusPP

Vpo

sitivepredictiv

evalueNPV

negativepredictiv

evalueRS

Vrespira

tory

syncytialv

irusNAn

otapplicableR

efreferencePospositiveN

egn

egative

Overalltotaln

umbero

ffalse

positives

is10

becauseinfl

uenzaA

andsubtypingweretestedfora

samples

imultaneou

sly

6 Advances in Virology

Table3Analytic

alsensitivityof

NxT

AGRP

PxT

AGRV

PandFilm

Array

RPford

etectin

ginflu

enza

Aavian-o

rswine-origin

subtypes

Influ

enza

ALimitof

detection[vira

lload(lo

g10c

opiesml)

log10TC

ID50m

l]of

influ

enza

Asubtype

NxT

AGRP

PxT

AGRV

PFilm

Array

RPSubtype

Strains

Viralload

TCID50

Viralload

TCID50

Viralload

TCID50

H1N

1pdm

09AH

K41574209H1N

130

01

30

01

30

01

H2N

2AA

sia573

37

32

37

32

27

032

H3N

2vAW

iscon

sin12

2010

20

01

40

1030

1H5N

1AVietnam

302804

42

056

42

056

42

056

H5N

6AH

5N6HK

2016

30

150

100

30

1H7N

9AA

nhui12013

34

0178

64

178

44

178

H9N

2AH

K107399

1800056

38

056

38

056

Mean

30

07

43

418

34

08

Advances in Virology 7

of all influenza A virus subtypes is especially essentialin certain epidemic regions such as Southeast Asia Thecorrect interpretation of these results is also important andwould avoid delay in patient management and infectioncontrol especially in patients with severe seasonal influenzaA or avianswine-origin influenza A virus subtype infectionClinical microbiologists infectious disease specialists andlaboratory managers should be aware of and understand thedifferent clinical performances of these commercially avail-able molecular multiplex RT-PCR assays that are commonlyadopted in many clinical microbiology laboratories Thesensitivity and specificity of these molecular multiplex assaysmust be predetermined and be able to confidently detectmostof the important avian or swine influenza A subtype infectionincluding H5N1 and H7N9 and this capability is particularlyimportant for use in Southeast Asia

A limitation of this study is that some target numbers aresmall This is due to low prevalent disease and may require alonger period in order to collect a higher number of positivesHigh cost for these multiplex assays is another limitation forus to study more samples

5 Conclusion

In this study the results showed thatNxTAGRPP xTAGRVPand FilmArray RP assays had high sensitivity and specificityfor diagnosis of respiratory diseases They are suitable for usein clinical diagnostic laboratories for detection of respiratorypathogens in patients with RTIs However awareness shouldbe raised that H3N2 could not be distinguished from H3N2vby NxTAGRPP Further investigation should be performed ifH3N2v is suspected to be the cause of disease

Ethical Approval

This study was approved by the Institutional Review Board ofthe University of Hong KongHospital Authority Hong KongWest Cluster

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors would like to thank the staff of the Departmentof Microbiology Queen Mary Hospital for the facilitation ofthis projectThis work was partly supported by the donationsof Larry Chi-Kin Yung and Hui Hoy and Chow Sin LanCharity Fund Limited the authors also obtained fundingfrom the Consultancy Service for Enhancing LaboratorySurveillance of Emerging Infectious Diseases the Depart-ment of Health Hong Kong Special Administrative Regionthe University Development Fund and the Committee forResearch and Conference Grant the University of HongKong the Collaborative Innovation Center for Diagnosis andTreatment of Infectious Diseases the Ministry of Educationof China and the Commissioned Research on Control ofInfectious Diseases (Phase III) of the Health and Medical

Research Fund (HKM-15-M-04) of the Food and HealthBureau of the HKSAR Government

References

[1] W Olszewska M Zambon and P J M Openshaw ldquoDevel-opment of vaccines against common coldsrdquo British MedicalBulletin vol 62 no 1 pp 99ndash111 2002

[2] World Health Organization Burden of Disease Project WorldHealth Organization Geneva Switzerland 2005

[3] B Ehlken G Ihorst B Lippert et al ldquoEconomic impact ofcommunity-acquired and nosocomial lower respiratory tractinfections in young children in Germanyrdquo European Journal ofPediatrics vol 164 no 10 pp 607ndash615 2005

[4] M M Massin J Montesanti P Gerard and P Lepage ldquoSpec-trum and frequency of illness presenting to a pediatric emer-gency departmentrdquo Acta Clinica Belgica vol 61 no 4 pp 161ndash165 2006

[5] K G Nicholson T McNally M Silverman P Simons J DStockton and M C Zambon ldquoRates of hospitalisation forinfluenza respiratory syncytial virus and human metapneu-movirus among infants and young childrenrdquo Vaccine vol 24no 1 pp 102ndash108 2006

[6] A Sauro F Barone G Blasio L Russo and L Santillo ldquoDoinfluenza and acute respiratory infective diseases weigh heavilyon general practitionersrsquo daily practicerdquo European Journal ofGeneral Practice vol 12 no 1 pp 34ndash36 2006

[7] J S Tregoning and J Schwarze ldquoRespiratory viral infections ininfants causes clinical symptoms virology and immunologyrdquoClinical Microbiology Reviews vol 23 no 1 pp 74ndash98 2010

[8] S S Chiu P-L Ho M J S Peiris K H Chan and E L YChan ldquoPopulation-based hospitalization incidence of respira-tory viruses in community-acquired pneumonia in childrenyounger than 5 years of agerdquo Influenza and other RespiratoryViruses vol 8 no 6 pp 626-627 2014

[9] K J Henrickson ldquoCost-effective use of rapid diagnostic tech-niques in the treatment and prevention of viral respiratoryinfectionsrdquo Pediatric Annals vol 34 no 1 pp 24ndash31 2005

[10] E B Popowitch S S OrsquoNeill andM B Miller ldquoComparison ofthe biofire filmarray RP Genmark eSensor RVP Luminex xTAGRVPv1 and Luminex xTAGRVP fastmultiplex assays for detec-tion of respiratory virusesrdquo Journal of Clinical Microbiology vol51 no 5 pp 1528ndash1533 2013

[11] J B Mahony ldquoDetection of respiratory viruses by molecularmethodsrdquo Clinical Microbiology Reviews vol 21 no 4 pp 716ndash747 2008

[12] J B Mahony A Petrich and M Smieja ldquoMolecular diagnosisof respiratory virus infectionsrdquo Critical Reviews in ClinicalLaboratory Sciences vol 48 no 5-6 pp 217ndash249 2011

[13] Centers for Disease Control and Prevention Swine-OriginInfluenza A (H3N2) Virus Infection in Two Children mdash Indianaand Pennsylvania JulyndashAugust MMWR 2011

[14] Y Chen W Liang S Yang et al ldquoHuman infections withthe emerging avian influenza A H7N9 virus from wet marketpoultry clinical analysis and characterisation of viral genomerdquoLancet vol 381 pp 1916ndash1925 2013

[15] Z-F Yang C K P Mok J S M Peiris and N-S ZhongldquoHuman infection with a novel avian influenza A(H5N6) virusrdquoNew England Journal of Medicine vol 373 no 5 pp 487ndash4892015

8 Advances in Virology

[16] K-H Chan K K W To J F W Chan C P Y Li H Chenand K-Y Yuen ldquoAnalytical sensitivity of seven point-of-careinfluenza virus detection tests and two molecular tests fordetection of avian origin H7N9 and swine origin H3N2 variantinfluenza a virusesrdquo Journal of Clinical Microbiology vol 51 no9 pp 3160-3161 2013

[17] K-H Chan K K W To J F W Chan et al ldquoAssessmentof antigen and molecular tests with serial specimens from apatient with influenza A(H7N9) infectionrdquo Journal of ClinicalMicrobiology vol 52 no 6 pp 2272ndash2274 2014

[18] J H K Chen H-Y Lam C C Y Yip et al ldquoClinical evalua-tion of the new high-throughput luminex nxtag respiratorypathogen panel assay for multiplex respiratory pathogen detec-tionrdquo Journal of Clinical Microbiology vol 54 no 7 pp 1820ndash1825 2016

[19] Y-W Tang S Gonsalves J Y Sun et al ldquoClinical evaluationof the luminex nxtag respiratory pathogen panelrdquo Journal ofClinical Microbiology vol 54 no 7 pp 1912ndash1914 2016

[20] C Beckmann andHHHirsch ldquoComparing LuminexNxTAG-Respiratory Pathogen Panel and RespiFinder-22 for multiplexdetection of respiratory pathogensrdquo Journal ofMedical Virologyvol 88 no 8 pp 1319ndash1324 2016

[21] I Hung V Cheng A Wu et al ldquoViral Loads in Clinical Spec-imens and SARS Manifestationsrdquo Emerging Infectious Diseasesvol 10 no 9 pp 1550ndash1557 2004

[22] MWelti K Jaton M Altwegg R Sahli A Wenger and J BilleldquoDevelopment of a multiplex real-time quantitative PCR assayto detect Chlamydia pneumoniae Legionella pneumophilaand Mycoplasma pneumoniae in respiratory tract secretionsrdquoDiagnosticMicrobiology and Infectious Disease vol 45 no 2 pp85ndash95 2003

[23] L J R Van Elden A M Van Loon F Van Alphen et al ldquoFre-quent Detection of Human Coronaviruses in Clinical Speci-mens from Patients with Respiratory Tract Infection by Useof a Novel Real-Time Reverse-Transciptase Polymerase ChainReactionrdquo Journal of Infectious Diseases vol 189 no 4 pp 652ndash657 2004

[24] P C YWoo S K P Lau C-MChu et al ldquoCharacterization andcomplete genome sequence of a novel coronavirus coronavirusHKU1 from patients with pneumoniardquo Journal of Virology vol79 no 2 pp 884ndash895 2005

[25] S S Chiu K H Chan K W Chu et al ldquoHuman coronavirusNL63 infection and other coronavirus infections in childrenhospitalized with acute respiratory disease in Hong KongChinardquoClinical Infectious Diseases vol 40 no 12 pp 1721ndash17292005

[26] J Kuypers N Wright L Corey and R Morrow ldquoDetectionand quantification of human metapneumovirus in pediatricspecimens by real-time RT-PCRrdquo Journal of Clinical Virologyvol 33 no 4 pp 299ndash305 2005

[27] S K P Lau C C Y Yip T-L Que et al ldquoClinical and molecu-lar epidemiology of human bocavirus in respiratory and fecalsamples from children in Hong Kongrdquo Journal of InfectiousDiseases vol 196 no 7 pp 986ndash993 2007

[28] K H Chan S Y Lam P Puthavathana et al ldquoComparativeanalytical sensitivities of six rapid influenza A antigen detectiontest kits for detection of influenza A subtypes H1N1 H3N2 andH5N1rdquo Journal of Clinical Virology vol 38 no 2 pp 169ndash1712007

[29] I F Hung K K To J F Chan et al ldquoEfficacy of Clarithromycin-Naproxen-Oseltamivir Combination in the Treatment of

Patients Hospitalized for Influenza A(H3N2) Infectionrdquo Chestvol 151 no 5 pp 1069ndash1080 2017

[30] Centers for Disease Control and Prevention CDC protocol ofrealtime RTPCR for influenza A (H1N1) Revision 2 Centers forDisease Control and Prevention Atlanta GA 2009

[31] K H Chan W C Yam C M Pang et al ldquoComparison of theNucliSens easyMAG and Qiagen BioRobot 9604 nucleic acidextraction systems for detection of RNA and DNA respiratoryviruses in nasopharyngeal aspirate samplesrdquo Journal of ClinicalMicrobiology vol 46 no 7 pp 2195ndash2199 2008

[32] S Esposito A Scala S Bianchini et al ldquoPartial comparisonof the NxTAG Respiratory Pathogen Panel Assay with theLuminex xTAG Respiratory Panel Fast Assay V2 and singleplexreal-time polymerase chain reaction for detection of respiratorypathogensrdquo Diagnostic Microbiology and Infectious Disease vol86 no 1 pp 53ndash57 2016

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 Advances in Virology

Table3Analytic

alsensitivityof

NxT

AGRP

PxT

AGRV

PandFilm

Array

RPford

etectin

ginflu

enza

Aavian-o

rswine-origin

subtypes

Influ

enza

ALimitof

detection[vira

lload(lo

g10c

opiesml)

log10TC

ID50m

l]of

influ

enza

Asubtype

NxT

AGRP

PxT

AGRV

PFilm

Array

RPSubtype

Strains

Viralload

TCID50

Viralload

TCID50

Viralload

TCID50

H1N

1pdm

09AH

K41574209H1N

130

01

30

01

30

01

H2N

2AA

sia573

37

32

37

32

27

032

H3N

2vAW

iscon

sin12

2010

20

01

40

1030

1H5N

1AVietnam

302804

42

056

42

056

42

056

H5N

6AH

5N6HK

2016

30

150

100

30

1H7N

9AA

nhui12013

34

0178

64

178

44

178

H9N

2AH

K107399

1800056

38

056

38

056

Mean

30

07

43

418

34

08

Advances in Virology 7

of all influenza A virus subtypes is especially essentialin certain epidemic regions such as Southeast Asia Thecorrect interpretation of these results is also important andwould avoid delay in patient management and infectioncontrol especially in patients with severe seasonal influenzaA or avianswine-origin influenza A virus subtype infectionClinical microbiologists infectious disease specialists andlaboratory managers should be aware of and understand thedifferent clinical performances of these commercially avail-able molecular multiplex RT-PCR assays that are commonlyadopted in many clinical microbiology laboratories Thesensitivity and specificity of these molecular multiplex assaysmust be predetermined and be able to confidently detectmostof the important avian or swine influenza A subtype infectionincluding H5N1 and H7N9 and this capability is particularlyimportant for use in Southeast Asia

A limitation of this study is that some target numbers aresmall This is due to low prevalent disease and may require alonger period in order to collect a higher number of positivesHigh cost for these multiplex assays is another limitation forus to study more samples

5 Conclusion

In this study the results showed thatNxTAGRPP xTAGRVPand FilmArray RP assays had high sensitivity and specificityfor diagnosis of respiratory diseases They are suitable for usein clinical diagnostic laboratories for detection of respiratorypathogens in patients with RTIs However awareness shouldbe raised that H3N2 could not be distinguished from H3N2vby NxTAGRPP Further investigation should be performed ifH3N2v is suspected to be the cause of disease

Ethical Approval

This study was approved by the Institutional Review Board ofthe University of Hong KongHospital Authority Hong KongWest Cluster

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors would like to thank the staff of the Departmentof Microbiology Queen Mary Hospital for the facilitation ofthis projectThis work was partly supported by the donationsof Larry Chi-Kin Yung and Hui Hoy and Chow Sin LanCharity Fund Limited the authors also obtained fundingfrom the Consultancy Service for Enhancing LaboratorySurveillance of Emerging Infectious Diseases the Depart-ment of Health Hong Kong Special Administrative Regionthe University Development Fund and the Committee forResearch and Conference Grant the University of HongKong the Collaborative Innovation Center for Diagnosis andTreatment of Infectious Diseases the Ministry of Educationof China and the Commissioned Research on Control ofInfectious Diseases (Phase III) of the Health and Medical

Research Fund (HKM-15-M-04) of the Food and HealthBureau of the HKSAR Government

References

[1] W Olszewska M Zambon and P J M Openshaw ldquoDevel-opment of vaccines against common coldsrdquo British MedicalBulletin vol 62 no 1 pp 99ndash111 2002

[2] World Health Organization Burden of Disease Project WorldHealth Organization Geneva Switzerland 2005

[3] B Ehlken G Ihorst B Lippert et al ldquoEconomic impact ofcommunity-acquired and nosocomial lower respiratory tractinfections in young children in Germanyrdquo European Journal ofPediatrics vol 164 no 10 pp 607ndash615 2005

[4] M M Massin J Montesanti P Gerard and P Lepage ldquoSpec-trum and frequency of illness presenting to a pediatric emer-gency departmentrdquo Acta Clinica Belgica vol 61 no 4 pp 161ndash165 2006

[5] K G Nicholson T McNally M Silverman P Simons J DStockton and M C Zambon ldquoRates of hospitalisation forinfluenza respiratory syncytial virus and human metapneu-movirus among infants and young childrenrdquo Vaccine vol 24no 1 pp 102ndash108 2006

[6] A Sauro F Barone G Blasio L Russo and L Santillo ldquoDoinfluenza and acute respiratory infective diseases weigh heavilyon general practitionersrsquo daily practicerdquo European Journal ofGeneral Practice vol 12 no 1 pp 34ndash36 2006

[7] J S Tregoning and J Schwarze ldquoRespiratory viral infections ininfants causes clinical symptoms virology and immunologyrdquoClinical Microbiology Reviews vol 23 no 1 pp 74ndash98 2010

[8] S S Chiu P-L Ho M J S Peiris K H Chan and E L YChan ldquoPopulation-based hospitalization incidence of respira-tory viruses in community-acquired pneumonia in childrenyounger than 5 years of agerdquo Influenza and other RespiratoryViruses vol 8 no 6 pp 626-627 2014

[9] K J Henrickson ldquoCost-effective use of rapid diagnostic tech-niques in the treatment and prevention of viral respiratoryinfectionsrdquo Pediatric Annals vol 34 no 1 pp 24ndash31 2005

[10] E B Popowitch S S OrsquoNeill andM B Miller ldquoComparison ofthe biofire filmarray RP Genmark eSensor RVP Luminex xTAGRVPv1 and Luminex xTAGRVP fastmultiplex assays for detec-tion of respiratory virusesrdquo Journal of Clinical Microbiology vol51 no 5 pp 1528ndash1533 2013

[11] J B Mahony ldquoDetection of respiratory viruses by molecularmethodsrdquo Clinical Microbiology Reviews vol 21 no 4 pp 716ndash747 2008

[12] J B Mahony A Petrich and M Smieja ldquoMolecular diagnosisof respiratory virus infectionsrdquo Critical Reviews in ClinicalLaboratory Sciences vol 48 no 5-6 pp 217ndash249 2011

[13] Centers for Disease Control and Prevention Swine-OriginInfluenza A (H3N2) Virus Infection in Two Children mdash Indianaand Pennsylvania JulyndashAugust MMWR 2011

[14] Y Chen W Liang S Yang et al ldquoHuman infections withthe emerging avian influenza A H7N9 virus from wet marketpoultry clinical analysis and characterisation of viral genomerdquoLancet vol 381 pp 1916ndash1925 2013

[15] Z-F Yang C K P Mok J S M Peiris and N-S ZhongldquoHuman infection with a novel avian influenza A(H5N6) virusrdquoNew England Journal of Medicine vol 373 no 5 pp 487ndash4892015

8 Advances in Virology

[16] K-H Chan K K W To J F W Chan C P Y Li H Chenand K-Y Yuen ldquoAnalytical sensitivity of seven point-of-careinfluenza virus detection tests and two molecular tests fordetection of avian origin H7N9 and swine origin H3N2 variantinfluenza a virusesrdquo Journal of Clinical Microbiology vol 51 no9 pp 3160-3161 2013

[17] K-H Chan K K W To J F W Chan et al ldquoAssessmentof antigen and molecular tests with serial specimens from apatient with influenza A(H7N9) infectionrdquo Journal of ClinicalMicrobiology vol 52 no 6 pp 2272ndash2274 2014

[18] J H K Chen H-Y Lam C C Y Yip et al ldquoClinical evalua-tion of the new high-throughput luminex nxtag respiratorypathogen panel assay for multiplex respiratory pathogen detec-tionrdquo Journal of Clinical Microbiology vol 54 no 7 pp 1820ndash1825 2016

[19] Y-W Tang S Gonsalves J Y Sun et al ldquoClinical evaluationof the luminex nxtag respiratory pathogen panelrdquo Journal ofClinical Microbiology vol 54 no 7 pp 1912ndash1914 2016

[20] C Beckmann andHHHirsch ldquoComparing LuminexNxTAG-Respiratory Pathogen Panel and RespiFinder-22 for multiplexdetection of respiratory pathogensrdquo Journal ofMedical Virologyvol 88 no 8 pp 1319ndash1324 2016

[21] I Hung V Cheng A Wu et al ldquoViral Loads in Clinical Spec-imens and SARS Manifestationsrdquo Emerging Infectious Diseasesvol 10 no 9 pp 1550ndash1557 2004

[22] MWelti K Jaton M Altwegg R Sahli A Wenger and J BilleldquoDevelopment of a multiplex real-time quantitative PCR assayto detect Chlamydia pneumoniae Legionella pneumophilaand Mycoplasma pneumoniae in respiratory tract secretionsrdquoDiagnosticMicrobiology and Infectious Disease vol 45 no 2 pp85ndash95 2003

[23] L J R Van Elden A M Van Loon F Van Alphen et al ldquoFre-quent Detection of Human Coronaviruses in Clinical Speci-mens from Patients with Respiratory Tract Infection by Useof a Novel Real-Time Reverse-Transciptase Polymerase ChainReactionrdquo Journal of Infectious Diseases vol 189 no 4 pp 652ndash657 2004

[24] P C YWoo S K P Lau C-MChu et al ldquoCharacterization andcomplete genome sequence of a novel coronavirus coronavirusHKU1 from patients with pneumoniardquo Journal of Virology vol79 no 2 pp 884ndash895 2005

[25] S S Chiu K H Chan K W Chu et al ldquoHuman coronavirusNL63 infection and other coronavirus infections in childrenhospitalized with acute respiratory disease in Hong KongChinardquoClinical Infectious Diseases vol 40 no 12 pp 1721ndash17292005

[26] J Kuypers N Wright L Corey and R Morrow ldquoDetectionand quantification of human metapneumovirus in pediatricspecimens by real-time RT-PCRrdquo Journal of Clinical Virologyvol 33 no 4 pp 299ndash305 2005

[27] S K P Lau C C Y Yip T-L Que et al ldquoClinical and molecu-lar epidemiology of human bocavirus in respiratory and fecalsamples from children in Hong Kongrdquo Journal of InfectiousDiseases vol 196 no 7 pp 986ndash993 2007

[28] K H Chan S Y Lam P Puthavathana et al ldquoComparativeanalytical sensitivities of six rapid influenza A antigen detectiontest kits for detection of influenza A subtypes H1N1 H3N2 andH5N1rdquo Journal of Clinical Virology vol 38 no 2 pp 169ndash1712007

[29] I F Hung K K To J F Chan et al ldquoEfficacy of Clarithromycin-Naproxen-Oseltamivir Combination in the Treatment of

Patients Hospitalized for Influenza A(H3N2) Infectionrdquo Chestvol 151 no 5 pp 1069ndash1080 2017

[30] Centers for Disease Control and Prevention CDC protocol ofrealtime RTPCR for influenza A (H1N1) Revision 2 Centers forDisease Control and Prevention Atlanta GA 2009

[31] K H Chan W C Yam C M Pang et al ldquoComparison of theNucliSens easyMAG and Qiagen BioRobot 9604 nucleic acidextraction systems for detection of RNA and DNA respiratoryviruses in nasopharyngeal aspirate samplesrdquo Journal of ClinicalMicrobiology vol 46 no 7 pp 2195ndash2199 2008

[32] S Esposito A Scala S Bianchini et al ldquoPartial comparisonof the NxTAG Respiratory Pathogen Panel Assay with theLuminex xTAG Respiratory Panel Fast Assay V2 and singleplexreal-time polymerase chain reaction for detection of respiratorypathogensrdquo Diagnostic Microbiology and Infectious Disease vol86 no 1 pp 53ndash57 2016

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Advances in Virology 7

of all influenza A virus subtypes is especially essentialin certain epidemic regions such as Southeast Asia Thecorrect interpretation of these results is also important andwould avoid delay in patient management and infectioncontrol especially in patients with severe seasonal influenzaA or avianswine-origin influenza A virus subtype infectionClinical microbiologists infectious disease specialists andlaboratory managers should be aware of and understand thedifferent clinical performances of these commercially avail-able molecular multiplex RT-PCR assays that are commonlyadopted in many clinical microbiology laboratories Thesensitivity and specificity of these molecular multiplex assaysmust be predetermined and be able to confidently detectmostof the important avian or swine influenza A subtype infectionincluding H5N1 and H7N9 and this capability is particularlyimportant for use in Southeast Asia

A limitation of this study is that some target numbers aresmall This is due to low prevalent disease and may require alonger period in order to collect a higher number of positivesHigh cost for these multiplex assays is another limitation forus to study more samples

5 Conclusion

In this study the results showed thatNxTAGRPP xTAGRVPand FilmArray RP assays had high sensitivity and specificityfor diagnosis of respiratory diseases They are suitable for usein clinical diagnostic laboratories for detection of respiratorypathogens in patients with RTIs However awareness shouldbe raised that H3N2 could not be distinguished from H3N2vby NxTAGRPP Further investigation should be performed ifH3N2v is suspected to be the cause of disease

Ethical Approval

This study was approved by the Institutional Review Board ofthe University of Hong KongHospital Authority Hong KongWest Cluster

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors would like to thank the staff of the Departmentof Microbiology Queen Mary Hospital for the facilitation ofthis projectThis work was partly supported by the donationsof Larry Chi-Kin Yung and Hui Hoy and Chow Sin LanCharity Fund Limited the authors also obtained fundingfrom the Consultancy Service for Enhancing LaboratorySurveillance of Emerging Infectious Diseases the Depart-ment of Health Hong Kong Special Administrative Regionthe University Development Fund and the Committee forResearch and Conference Grant the University of HongKong the Collaborative Innovation Center for Diagnosis andTreatment of Infectious Diseases the Ministry of Educationof China and the Commissioned Research on Control ofInfectious Diseases (Phase III) of the Health and Medical

Research Fund (HKM-15-M-04) of the Food and HealthBureau of the HKSAR Government

References

[1] W Olszewska M Zambon and P J M Openshaw ldquoDevel-opment of vaccines against common coldsrdquo British MedicalBulletin vol 62 no 1 pp 99ndash111 2002

[2] World Health Organization Burden of Disease Project WorldHealth Organization Geneva Switzerland 2005

[3] B Ehlken G Ihorst B Lippert et al ldquoEconomic impact ofcommunity-acquired and nosocomial lower respiratory tractinfections in young children in Germanyrdquo European Journal ofPediatrics vol 164 no 10 pp 607ndash615 2005

[4] M M Massin J Montesanti P Gerard and P Lepage ldquoSpec-trum and frequency of illness presenting to a pediatric emer-gency departmentrdquo Acta Clinica Belgica vol 61 no 4 pp 161ndash165 2006

[5] K G Nicholson T McNally M Silverman P Simons J DStockton and M C Zambon ldquoRates of hospitalisation forinfluenza respiratory syncytial virus and human metapneu-movirus among infants and young childrenrdquo Vaccine vol 24no 1 pp 102ndash108 2006

[6] A Sauro F Barone G Blasio L Russo and L Santillo ldquoDoinfluenza and acute respiratory infective diseases weigh heavilyon general practitionersrsquo daily practicerdquo European Journal ofGeneral Practice vol 12 no 1 pp 34ndash36 2006

[7] J S Tregoning and J Schwarze ldquoRespiratory viral infections ininfants causes clinical symptoms virology and immunologyrdquoClinical Microbiology Reviews vol 23 no 1 pp 74ndash98 2010

[8] S S Chiu P-L Ho M J S Peiris K H Chan and E L YChan ldquoPopulation-based hospitalization incidence of respira-tory viruses in community-acquired pneumonia in childrenyounger than 5 years of agerdquo Influenza and other RespiratoryViruses vol 8 no 6 pp 626-627 2014

[9] K J Henrickson ldquoCost-effective use of rapid diagnostic tech-niques in the treatment and prevention of viral respiratoryinfectionsrdquo Pediatric Annals vol 34 no 1 pp 24ndash31 2005

[10] E B Popowitch S S OrsquoNeill andM B Miller ldquoComparison ofthe biofire filmarray RP Genmark eSensor RVP Luminex xTAGRVPv1 and Luminex xTAGRVP fastmultiplex assays for detec-tion of respiratory virusesrdquo Journal of Clinical Microbiology vol51 no 5 pp 1528ndash1533 2013

[11] J B Mahony ldquoDetection of respiratory viruses by molecularmethodsrdquo Clinical Microbiology Reviews vol 21 no 4 pp 716ndash747 2008

[12] J B Mahony A Petrich and M Smieja ldquoMolecular diagnosisof respiratory virus infectionsrdquo Critical Reviews in ClinicalLaboratory Sciences vol 48 no 5-6 pp 217ndash249 2011

[13] Centers for Disease Control and Prevention Swine-OriginInfluenza A (H3N2) Virus Infection in Two Children mdash Indianaand Pennsylvania JulyndashAugust MMWR 2011

[14] Y Chen W Liang S Yang et al ldquoHuman infections withthe emerging avian influenza A H7N9 virus from wet marketpoultry clinical analysis and characterisation of viral genomerdquoLancet vol 381 pp 1916ndash1925 2013

[15] Z-F Yang C K P Mok J S M Peiris and N-S ZhongldquoHuman infection with a novel avian influenza A(H5N6) virusrdquoNew England Journal of Medicine vol 373 no 5 pp 487ndash4892015

8 Advances in Virology

[16] K-H Chan K K W To J F W Chan C P Y Li H Chenand K-Y Yuen ldquoAnalytical sensitivity of seven point-of-careinfluenza virus detection tests and two molecular tests fordetection of avian origin H7N9 and swine origin H3N2 variantinfluenza a virusesrdquo Journal of Clinical Microbiology vol 51 no9 pp 3160-3161 2013

[17] K-H Chan K K W To J F W Chan et al ldquoAssessmentof antigen and molecular tests with serial specimens from apatient with influenza A(H7N9) infectionrdquo Journal of ClinicalMicrobiology vol 52 no 6 pp 2272ndash2274 2014

[18] J H K Chen H-Y Lam C C Y Yip et al ldquoClinical evalua-tion of the new high-throughput luminex nxtag respiratorypathogen panel assay for multiplex respiratory pathogen detec-tionrdquo Journal of Clinical Microbiology vol 54 no 7 pp 1820ndash1825 2016

[19] Y-W Tang S Gonsalves J Y Sun et al ldquoClinical evaluationof the luminex nxtag respiratory pathogen panelrdquo Journal ofClinical Microbiology vol 54 no 7 pp 1912ndash1914 2016

[20] C Beckmann andHHHirsch ldquoComparing LuminexNxTAG-Respiratory Pathogen Panel and RespiFinder-22 for multiplexdetection of respiratory pathogensrdquo Journal ofMedical Virologyvol 88 no 8 pp 1319ndash1324 2016

[21] I Hung V Cheng A Wu et al ldquoViral Loads in Clinical Spec-imens and SARS Manifestationsrdquo Emerging Infectious Diseasesvol 10 no 9 pp 1550ndash1557 2004

[22] MWelti K Jaton M Altwegg R Sahli A Wenger and J BilleldquoDevelopment of a multiplex real-time quantitative PCR assayto detect Chlamydia pneumoniae Legionella pneumophilaand Mycoplasma pneumoniae in respiratory tract secretionsrdquoDiagnosticMicrobiology and Infectious Disease vol 45 no 2 pp85ndash95 2003

[23] L J R Van Elden A M Van Loon F Van Alphen et al ldquoFre-quent Detection of Human Coronaviruses in Clinical Speci-mens from Patients with Respiratory Tract Infection by Useof a Novel Real-Time Reverse-Transciptase Polymerase ChainReactionrdquo Journal of Infectious Diseases vol 189 no 4 pp 652ndash657 2004

[24] P C YWoo S K P Lau C-MChu et al ldquoCharacterization andcomplete genome sequence of a novel coronavirus coronavirusHKU1 from patients with pneumoniardquo Journal of Virology vol79 no 2 pp 884ndash895 2005

[25] S S Chiu K H Chan K W Chu et al ldquoHuman coronavirusNL63 infection and other coronavirus infections in childrenhospitalized with acute respiratory disease in Hong KongChinardquoClinical Infectious Diseases vol 40 no 12 pp 1721ndash17292005

[26] J Kuypers N Wright L Corey and R Morrow ldquoDetectionand quantification of human metapneumovirus in pediatricspecimens by real-time RT-PCRrdquo Journal of Clinical Virologyvol 33 no 4 pp 299ndash305 2005

[27] S K P Lau C C Y Yip T-L Que et al ldquoClinical and molecu-lar epidemiology of human bocavirus in respiratory and fecalsamples from children in Hong Kongrdquo Journal of InfectiousDiseases vol 196 no 7 pp 986ndash993 2007

[28] K H Chan S Y Lam P Puthavathana et al ldquoComparativeanalytical sensitivities of six rapid influenza A antigen detectiontest kits for detection of influenza A subtypes H1N1 H3N2 andH5N1rdquo Journal of Clinical Virology vol 38 no 2 pp 169ndash1712007

[29] I F Hung K K To J F Chan et al ldquoEfficacy of Clarithromycin-Naproxen-Oseltamivir Combination in the Treatment of

Patients Hospitalized for Influenza A(H3N2) Infectionrdquo Chestvol 151 no 5 pp 1069ndash1080 2017

[30] Centers for Disease Control and Prevention CDC protocol ofrealtime RTPCR for influenza A (H1N1) Revision 2 Centers forDisease Control and Prevention Atlanta GA 2009

[31] K H Chan W C Yam C M Pang et al ldquoComparison of theNucliSens easyMAG and Qiagen BioRobot 9604 nucleic acidextraction systems for detection of RNA and DNA respiratoryviruses in nasopharyngeal aspirate samplesrdquo Journal of ClinicalMicrobiology vol 46 no 7 pp 2195ndash2199 2008

[32] S Esposito A Scala S Bianchini et al ldquoPartial comparisonof the NxTAG Respiratory Pathogen Panel Assay with theLuminex xTAG Respiratory Panel Fast Assay V2 and singleplexreal-time polymerase chain reaction for detection of respiratorypathogensrdquo Diagnostic Microbiology and Infectious Disease vol86 no 1 pp 53ndash57 2016

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 Advances in Virology

[16] K-H Chan K K W To J F W Chan C P Y Li H Chenand K-Y Yuen ldquoAnalytical sensitivity of seven point-of-careinfluenza virus detection tests and two molecular tests fordetection of avian origin H7N9 and swine origin H3N2 variantinfluenza a virusesrdquo Journal of Clinical Microbiology vol 51 no9 pp 3160-3161 2013

[17] K-H Chan K K W To J F W Chan et al ldquoAssessmentof antigen and molecular tests with serial specimens from apatient with influenza A(H7N9) infectionrdquo Journal of ClinicalMicrobiology vol 52 no 6 pp 2272ndash2274 2014

[18] J H K Chen H-Y Lam C C Y Yip et al ldquoClinical evalua-tion of the new high-throughput luminex nxtag respiratorypathogen panel assay for multiplex respiratory pathogen detec-tionrdquo Journal of Clinical Microbiology vol 54 no 7 pp 1820ndash1825 2016

[19] Y-W Tang S Gonsalves J Y Sun et al ldquoClinical evaluationof the luminex nxtag respiratory pathogen panelrdquo Journal ofClinical Microbiology vol 54 no 7 pp 1912ndash1914 2016

[20] C Beckmann andHHHirsch ldquoComparing LuminexNxTAG-Respiratory Pathogen Panel and RespiFinder-22 for multiplexdetection of respiratory pathogensrdquo Journal ofMedical Virologyvol 88 no 8 pp 1319ndash1324 2016

[21] I Hung V Cheng A Wu et al ldquoViral Loads in Clinical Spec-imens and SARS Manifestationsrdquo Emerging Infectious Diseasesvol 10 no 9 pp 1550ndash1557 2004

[22] MWelti K Jaton M Altwegg R Sahli A Wenger and J BilleldquoDevelopment of a multiplex real-time quantitative PCR assayto detect Chlamydia pneumoniae Legionella pneumophilaand Mycoplasma pneumoniae in respiratory tract secretionsrdquoDiagnosticMicrobiology and Infectious Disease vol 45 no 2 pp85ndash95 2003

[23] L J R Van Elden A M Van Loon F Van Alphen et al ldquoFre-quent Detection of Human Coronaviruses in Clinical Speci-mens from Patients with Respiratory Tract Infection by Useof a Novel Real-Time Reverse-Transciptase Polymerase ChainReactionrdquo Journal of Infectious Diseases vol 189 no 4 pp 652ndash657 2004

[24] P C YWoo S K P Lau C-MChu et al ldquoCharacterization andcomplete genome sequence of a novel coronavirus coronavirusHKU1 from patients with pneumoniardquo Journal of Virology vol79 no 2 pp 884ndash895 2005

[25] S S Chiu K H Chan K W Chu et al ldquoHuman coronavirusNL63 infection and other coronavirus infections in childrenhospitalized with acute respiratory disease in Hong KongChinardquoClinical Infectious Diseases vol 40 no 12 pp 1721ndash17292005

[26] J Kuypers N Wright L Corey and R Morrow ldquoDetectionand quantification of human metapneumovirus in pediatricspecimens by real-time RT-PCRrdquo Journal of Clinical Virologyvol 33 no 4 pp 299ndash305 2005

[27] S K P Lau C C Y Yip T-L Que et al ldquoClinical and molecu-lar epidemiology of human bocavirus in respiratory and fecalsamples from children in Hong Kongrdquo Journal of InfectiousDiseases vol 196 no 7 pp 986ndash993 2007

[28] K H Chan S Y Lam P Puthavathana et al ldquoComparativeanalytical sensitivities of six rapid influenza A antigen detectiontest kits for detection of influenza A subtypes H1N1 H3N2 andH5N1rdquo Journal of Clinical Virology vol 38 no 2 pp 169ndash1712007

[29] I F Hung K K To J F Chan et al ldquoEfficacy of Clarithromycin-Naproxen-Oseltamivir Combination in the Treatment of

Patients Hospitalized for Influenza A(H3N2) Infectionrdquo Chestvol 151 no 5 pp 1069ndash1080 2017

[30] Centers for Disease Control and Prevention CDC protocol ofrealtime RTPCR for influenza A (H1N1) Revision 2 Centers forDisease Control and Prevention Atlanta GA 2009

[31] K H Chan W C Yam C M Pang et al ldquoComparison of theNucliSens easyMAG and Qiagen BioRobot 9604 nucleic acidextraction systems for detection of RNA and DNA respiratoryviruses in nasopharyngeal aspirate samplesrdquo Journal of ClinicalMicrobiology vol 46 no 7 pp 2195ndash2199 2008

[32] S Esposito A Scala S Bianchini et al ldquoPartial comparisonof the NxTAG Respiratory Pathogen Panel Assay with theLuminex xTAG Respiratory Panel Fast Assay V2 and singleplexreal-time polymerase chain reaction for detection of respiratorypathogensrdquo Diagnostic Microbiology and Infectious Disease vol86 no 1 pp 53ndash57 2016

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology