Respiratory pathogens 33 february 2011 version 1 - Mikrogen

Validation 

FTD Respiratory pathogens 33 

The following data demonstrate that the FTD respiratory pathogens 33 test (FTD 

Resp 33) is suitable for its intended purpose and performs according to predetermined 

criteria. 

Principle of the test: 

The FTD Resp 33 test is an in vitro test with eight multiplex RT-PCR reactions for 

the detection of the following viruses, bacteria and fungi: influenza A (FluA), B 

(FluB) and C (FluC); parainfluenza viruses 1, 2, 3 and 4 (para1, para2, para3, 

para4); coronaviruses NL63 (Cor63), 229E (Cor229), OC43 (Cor43) and HKU1 

(HKU1); human metapneumoviruses A and B (HMPVA and B), rhinovirus (rhino), 

respiratory syncytial viruses A and B (RSVA and B), adenovirus (AV), enterovirus 

(EV), parechovirus (PV), bocavirus (HboV), cytomegalovirus (CMV); 

pneumocystis jirovecii (PCP); mycoplasma pneumoniae (Mpneu), chlamydia 

pneumoniae (Cpneu); streptococcus pneumoniae (Spneu), haemophilus 

influenzae typ B (HIB); staphylococcus aureus (Saur); moraxella catarrhalis 

(Morax); bordetella pertussis (Bord); klebsiella pneumoniae (Kpneu); legionella 

species (Legio); salmonella species (Salm); haemophilus influenzae species 

(Haeinf) as in aid in the evaluation of infections by respiratory pathogens. 

Eight tube multiplex PCR for detection of respiratory pathogens 

1. Multiplex: Influenza A, Influenza B,Influenza and Rhinovirus 

2. Multiplex: Parainfluenza 2, 3, 4 and internal control 

3. Multiplex: Coronavirus NL 63, 229E, OC43 and HKU1 

4. Multiplex: Parainfluenza 1, Human metapneumovirus A and B, Bocavirus 

and mycoplasma pneumoniae 

5. Multiplex: Respiratory syncytial viruses A and B, adenovirus, 

enterovirus, parechovirus and cytomegalovirus 

6. Multiplex: chlamydia pneumoniae, streptococcus pneumoniae, 

haemophilus influenza type B and staphylococcus aureus 

7. Multiplex: legionella species, klebsiella pneumoniae, pneumocystis 

jirovecii and salmonella species 

8. Multiplex: Influenza C, haemophilus influenzae species, moraxella 

catarrhalis and bordetella pertussis 

Fast-track Diagnostics Luxembourg S.a.r.l. 

Z. I. Langwies 

L-6131 Junglinster, Luxembourg, 

Tel. +352 780290 332/329 Fax: +352 780290 514 

http://www.fast-trackdiagnostics.com/ 

Page 1 

March 2011

Table of contents 

Page 

1. Specificity 3 

2. Sensitivity 10 

3. Clinical evaluation 13 






Page 2 


1. Specificity 

The BLAST (www.ncbi.nlm.nih.gov/blast) search shows that the selected 

primers and probes only detect the correspondent respiratory viruses, 

bacteria and fungi. The primer and probes of different pathogens are not 

cross-reacting. The targeted sequences for each pathogen are listed in the 

table below (table1). There are 4 exceptional cases. 

Table 1: Overview of targeted gene sequences of the pathogens within the FTD Resp 33 kit 


FTD Resp 33 

Pathogen Target 

Adenovirus hexon gene 

Bocavirus NP1 gene 

Bordetella pertussis IS481 

Chlamydia pneumoniae RNA polymerase beta chain gene 

Cytomegalovirus US7 +US8 genes 

Coronavirus 43 nucleocapsid protein (N) gene 



Coronavirus HKU1 nucleocapsid protein (N) gene 

Enterovirus parts of domain IV and V 

Haemophilus influenzae ompP6 gene 

Haemophilus influenzae TypB BexA gene 

Human metapneumovirus A fusion glycoprotein (F) gene 

Human metapneumovirus B fusion glycoprotein (F) gene 

Influenza A Matrix gene (pos1) 

Influenza B segment 8 NS1/NEP 

Influenza C Matrix gene 

Klebsiella pneumoniae khe hemolysin gene 

Legionella species 16S rRNA 

Moraxella catarrhalis copB gene 

Mycoplasma pneumoniae Adhesin P1 

Parainfluenza 1 hemagglutinin-neuraminidase (HN) mRNA 



Parainfluenza 4 fusion protein gene 

Parechovirus 5`untranslated Region (UTR) 

Pneumocystis jiroveci (mtLSU)rRNA gene 

Respiratory syncytial virus A nucleocapsid protein gene 

Respiratory syncytial virus B nucleoprotein mRNA 

Rhinovirus 5`untranslated Region (UTR) 

Salmonella species tetrathionate subunit B (ttrB) 

Streptococcus pneumoniae LytA gene 

Staphylococcus aureus sensor histidin kinase vick gene 





Page 3 


1. The primers and probe for the detection of Rhino also detect EV 

(RsEPAcmv PPmix), but not the other way around. If you receive a 

positive signal for Rhino and EV, the patient is EV positive. If you 

detect Rhino only, the patient is Rhino positive. 

2. The primers and probe for the detection of HIB (Respbac2 PPmix) 

also give a positive signal for haemophilus parahaemolyticus which 

is associated with paryngitis; however, it has to be negative for the 

detection of Haeinf (MoBoCH PPmix). If there is a positive signal for 

HIB (Respbac2 PPmix) and for Haeinf (MoBoCH PPmix) the sample 

is positive for Haemophilus influenzae TypB. 

3. The primers and probe for the Kpneu assay also detect Klebsiella 

variicola (98%) which is not important as a human pathogen. 

4. The primers and probes detecting the IS481-Element in Bordetella 

pertussis (100%) are also target other Bordetella species: 

B. holmesii (99-98%) and some strains of B. bronchiseptica (99- 

96%) and B. parapertussis (90-88%) which are also associated 

with respiratory infections in humans. 

� The specificity was validated with different respiratory negative samples. 

These did not generate any positive signal with primers and probes 

included in the FTD Resp 33 test. 

� For the specificity of the FTD Resp 33 assay we analyzed a broad range of 

positive material containing bacteria, viruses and fungi. No other than the 

expected bacteria, viruses or fungi were detected by the eight PPmixes, 

except for the above mentioned cases (table 2). 






Page 4 


Table 2: Evaluation of the specificity of the FTD Resp 33 test. A huge range of different viruses, 

bacteria and fungi got tested for cross-reactivity. 

Detected = positive 

Not detected= neg 

* 1 = the primers and probe of rhinovirus also detect enterovirus, but not the other way 

around 

* 2 = HIB positive samples have to be also positive for Haeinf (MoBoCH PPmix) 

* 3 = if you detect a HIB positive, it is also possible that it is haemophilus 

parahaemolyticus, which is associated with pharyngitis; it has to be negative for Haeinf 

(MoBoCH PPmix) 

pathogens amount 






Page 5 

FTD Resp 33 ppmixes 

FluAB_Rhino ParaEAV Cor Bo-mp-Pf1 RsEPAcmv RespBac2 KLePSa MoBoCH 

Adenovirus 31 neg neg neg neg positive neg neg neg 

Bocavirus 7 neg neg neg positive neg neg neg neg 

Bordetella pertussis 6 neg neg neg neg neg neg neg positive 

Chlamydia pneumoniae 6 neg neg neg neg neg positive neg neg 

Coronavirus 229 13 neg neg positive neg neg neg neg neg 



Coronavirus HKU1 6 neg neg positive neg neg neg neg neg 

Cytomegalievirus 20 neg neg neg neg positive neg neg neg 

Enterovirus 16 positive* 1 neg neg neg positive neg neg neg 

Haemophilus influenzae 38 neg neg neg neg neg neg neg positive 

Haemophilus influenzae Type B 18 neg neg neg neg neg positive neg positive* 2 

Haemophilus parahaemolyticus 4 neg neg neg neg neg positive* 3 neg neg 

HMPV A & B 19 neg neg neg positive neg neg neg neg 

Influenza-A-virus 23 positive neg neg neg neg neg neg neg 

Influenza-B-virus 20 positive neg neg neg neg neg neg neg 

Influenza-C-virus 25 positive neg neg neg neg neg neg neg 

Klebsiella pneumoniae 21 neg neg neg neg neg neg positive neg 

Legionella anisa 1 neg neg neg neg neg neg positive neg 

Legionella lonbeachae 7 neg neg neg neg neg neg positive neg 

Legionella pneumoniae 10 neg neg neg neg neg neg positive neg 

Moraxella catarrhalis 29 neg neg neg neg neg neg neg positive 

Mycoplasma pneumoniae 16 neg neg neg positive neg neg neg neg 

Parainfluenza-virus 1 18 neg neg neg positive neg neg neg neg 

Parainfluenza-virus 2 21 neg positive neg neg neg neg neg neg 



Parechovirus 14 neg neg neg neg positive neg neg neg 

Pneumocystis jirovecii 16 neg neg neg neg neg neg positive neg 

RSV A & B 22 neg neg neg neg positive neg neg neg 

Rhinovirus 25 positive neg neg neg neg neg neg neg 

Salmonella anatum 1 neg neg neg neg neg neg positive neg 

Salmonella virchow 1 neg neg neg neg neg neg positive neg 


pathogens amount 


FTD Resp 33 ppmixes 





FluAB_Rhino ParaEAV Cor Bo-mp-Pf1 RsEPAcmv RespBac2 KLePSa MoBoCH 

Salmonella schwartzengrund 1 neg neg neg neg neg neg positive neg 

Salmonella hadar 1 neg neg neg neg neg neg positive neg 

Salmonella java 1 neg neg neg neg neg neg positive neg 

Salmonella kentucky 1 neg neg neg neg neg neg positive neg 

Salmonella braenderup 1 neg neg neg neg neg neg positive neg 

Salmonella enteritidis 3 neg neg neg neg neg neg positive neg 

Salmonella typhimurium 2 neg neg neg neg neg neg positive neg 

Salmonella species 16 neg neg neg neg neg neg positive neg 

Staphylococcus aureus 40 neg neg neg neg neg positive neg neg 

Streptococcus pneumoniae 33 neg neg neg neg neg positive neg neg 

Astrovirus 1 neg neg neg neg neg neg neg neg 

Chlamydia trachomatis 1 neg neg neg neg neg neg neg neg 

Epstein-Barr virus 1 neg neg neg neg neg neg neg neg 

Haemophilus parainfluenzae 2 neg neg neg neg neg neg neg neg 

Herpes-simplex virus 1 1 neg neg neg neg neg neg neg neg 

Herpes-simplex virus 2 1 neg neg neg neg neg neg neg neg 

Human-herpesvirus 6 1 neg neg neg neg neg neg neg neg 

Human-herpesvirus 7 1 neg neg neg neg neg neg neg neg 

Klebsiella oxytoca 2 neg neg neg neg neg neg neg neg 

Measles virus 1 neg neg neg neg neg neg neg neg 

Mumps virus 1 neg neg neg neg neg neg neg neg 

Mycoplasma genitalium 1 neg neg neg neg neg neg neg neg 

Neisseria Gonorrhoeae 1 neg neg neg neg neg neg neg neg 

Norovirus G1 1 neg neg neg neg neg neg neg neg 

Norovirus G2 1 neg neg neg neg neg neg neg neg 

Parvovirus B19 1 neg neg neg neg neg neg neg neg 

Rotavirus 1 neg neg neg neg neg neg neg neg 

Treponema pallidum 1 neg neg neg neg neg neg neg neg 

Varizella-zoster virus 1 neg neg neg neg neg neg neg neg 

� The specificity of the FluA, FluB, EV, AV, Cpneu and Bord primers and 

probes were evaluated with different panels 

(Ringversuche: http://www.instandev.de/ringversuche/). 

For the FluA and FluB distribution the “Instand Ringversuch for virus 

confirmation of Influenza A- and B-viruses including Influenza A(H1N1)virus 

(swine lineage) and avian influenza A(H5N1)-virus 2009 and 2010” 

was analyzed with the FluAB_Rhino PPmix of the FTD Resp 33 test (table 

3). 

Page 6 


Table 3: “instand Ringversuch FluA, FluB 2009/2010” All samples were dissolved 

in accordance to the instand instruction and analyzed with the FluAB_Rhino PPmix of the 

FTD Resp 33 test. 

samples 

2009 

sample content 






Page 7 

FTD Resp 33/ 

FluAB_Rhino PPmix 

20088 FluB (saisonal) Detected (FluB) 

20089 FluA (H1N1) (swine lineage) Detected (FluA) 

20090 FluA (saisonal) Detected (FluA) 

20091 negative media Negative 

20092 FluA (avian) Detected (FluA) 




samples 

2010 







20107 FluB (saisonal) Detected (FluB) 



20110 FluA (saisonal) Detected (FluA) 

20111 FluA (H1N1) (swine lineage) Detected (FluA) 

The “Instand Ringversuch for confirmation of enteroviruses and 

adenoviruses 2010” was evaluated with the new RsEPAcmv PPmix of the 

FTD Resp 33 test. (table 4) 

Table 4: “instand Ringversuch EV and AV 2010” All samples got dissolved in accordance 

to the instand instruction and analyzed with the RsEPAcmv PPmix of the FTD Resp 33 

test. 

samples 

2010 


371001 Adenovirus B Type 11 Detected (AV) 

371002 Adenovirus D Type 37 Detected (AV) 


371004 Adenovirus C Type 2 Detected (AV) 

372001 Echovirus 30 

372002 Coxsackievirus B4 

372003 Echovirus 7 


RsEPAcmv PPmix 

Detected (EV) 

Detected (EV) 

Detected (EV) 



For Cpneu the “Instand Ringversuch for confirmation of chlamydia 

pneumoniae 2010” was analyzed with the new Respbac2 PPmix of the 

FTD Resp 33 test. (table 5) 

Table 5: “instand Ringversuch chlamydia pneumoniae 2010” All samples got 

dissolved accordant to the instand instruction and analyzed with the RespBac2 PPmix of 

the FTD Resp 33 test. 

samples 

2010 

1015401 


Chlamydia pneumoniae 

1E+04 IFU/ml 

1015402 Haemophilus influenzae spp. Negative 

1015403 Escherichia coli Negative 

1015404 

Chlamydia pneumoniae 

1E+03 IFU/ml 






Page 8 

FTD Resp 33/ RespBac2 

PPmix 

Detected (Cpneu) 

Detected (Cpneu) 

The “Instand Ringversuch for confirmation of bordetella pertussis 2010” 

was evaluated with the new MoBoCH PPmix of the FTD Resp 33 test. 

(table 6) 

Table 6: “instand Ringversuch bordetella pertussis 2010” All samples got 

dissolved accordant to the instand instruction and analyzed with the MoBoCH PPmix of 

the FTD Resp 33 test. 

samples 

2010 


1015321 Escherichia coli Negative 

1015322 

1015323 

1015324 

Bordetella pertussis 

10 5 organisms/ml 






MoBoCH PPmix 

Detected (bord) 



� The specificity of salm was evaluated using a panel of 10 strains of 

Salmonella enterica. These 10 different serotypes represent the “top-ten” 

most frequently isolated from humans in Scotland (Scottish, Salmonella, 

Shigella and Clostridium difficile Reference Laboratory). All different 

serotypes got detected by the FTD Resp 33 test, with an equal efficiency 

(table7). 


Table 7: Evaluation of the salm panel with known serotypes. 

number identification material pathogen 






Page 9 


KLePSa-PPmix (CT) 

1 1 (090057) culture McF 0.5 Salmonella enteridis 15.6 

2 2 (090763) culture McF 0.5 Salmonella typhimurium 15.9 

3 3 (090696) culture McF 0.5 Salmonella anatum 15.3 

4 4 (090005) culture McF 0.5 Salmonella virchow 15.9 

5 5 (091259) culture McF 0.5 Salmonella newport 15.2 

6 6 (091119) culture McF 0.5 Salmonella schwartzengrund 15.5 

7 7 (090918) culture McF 0.5 Salmonella hadar 15.3 

8 8 (090200) culture McF 0.5 Salmonella java 15.8 

9 9 (090871) culture McF 0.5 Salmonella kentucky 15.3 

10 10 (090097) culture McF 0.5 Salmonella braenderup 15.2 

For further specificity tests of salm we used a panel of 13 unknown 

Salmonella enterica serotypes (Scottish, Salmonella, Shigella and 

Clostridium difficile Reference Laboratory). The FTD Resp 33 test 

identified all 13 serotypes as salm species (table8). 

Table 8: Evaluation of a salm panel with unknown serotypes. 

number identification material pathogen 


KLePSa-PPmix (CT ) 

1 185323P/24F stool Salmonella enterica 28.8 

2 18576/N stool Salmonella enterica 23.5 

3 185863H stool Salmonella enterica 31.6 

4 185493S stool Salmonella enterica 24.4 

5 1855012 cell culture Salmonella enterica 14.3 

6 1860392 stool Salmonella enterica 15.0 

7 184837M stool Salmonella enterica 14.0 

8 186012Y stool Salmonella enterica 29.0 

9 185083M stool Salmonella enterica 26.8 

10 18532/E stool Salmonella enterica 23.2 

11 186142R stool Salmonella enterica 29.7 

12 185867/C stool Salmonella enterica 30.3 

13 185511K stool Salmonella enterica 26.3 


2. Sensitivity 

� The analytical detection limit was tested by using a tenfold dilution row of 

plasmid DNA of known concentration (1E+09-1E+00 copies/ ml) and the 

FTD Resp 33 test. The linear range of quantitation is derived from the 

detection limit until 1E+09 copies/ml (table 9). Furthermore, it was 

analyzed by using diluted positive patient samples, calculated with 

quantitation standards containing plasmid DNA of known concentration 

(1E+09-1E+06 copies/ml) and the FTD Resp 33 test (table 9). 

Table 9: Detection limit of plasmid DNA and positive respiratory samples with plasmid 

DNA as quantitation standard. 

PPmix Pathogen 

FluAB_Rhino 

ParaEAV 

Cor 

RsEPAcmv 

Bo-mp-Pf1 

RespBac2 

KLePSa 

MoBoCH 






positive sample plasmid 

Detection 

limit 

(copies/ml) 

Detection 

limit 

(copies/10µl 

PCR) 

Page 10 

Detection 

limit 

(copies/ml) 

Detection 

limit 

(copies/10µl 

PCR) 

FluA 2,05E+03 2,05E+01 1,00E+02 1,00E+00 

FluB 6,98E+02 6,98E+00 1,00E+03 1,00E+01 

Rhino 2,78E+02 2,78E+00 1,00E+04 1,00E+02 

PF2 1,68E+02 1,68E+00 1,00E+02 1,00E+00 

PF3 5,04E+02 5,04E+00 1,00E+03 1,00E+01 

PF4 1,12E+04 1,14E+02 1,00E+04 1,00E+02 

Cor63 3,66E+01 3,66E-01 1,00E+03 1,00E+01 

Cor229 1,98E+03 1,98E+01 1,00E+04 1,00E+02 

Cor43 4,47E+02 4,47E+00 1,00E+02 1,00E+00 

HKU 5,17E+02 5,17E+00 1,00E+03 1,00E+01 

EV 2,70E+02 2,70E+00 1,00E+04 1,00E+02 

PV 7,35E+03 7,35E+01 1,00E+03 1,00E+01 

RSV 1,18E+04 1,18E+02 1,00E+03 1,00E+01 

CMV 2,80E+02 2,80E+00 1,00E+04 1,00E+02 

AV 2,17E+02 2,17E+00 1,00E+04 1,00E+02 

PF1 2,37E+03 2,37E+01 1,00E+03 1,00E+01 

HMPV 5,75E+02 5,75E+00 1,00E+01 1,00E-01 

HboV 1,03E+03 1,03E+01 1,00E+03 1,00E+01 

Mpneu 2,32E+02 2,32E+00 1,00E+02 1,00E+00 

Cpneu 2,24E+02 2,24E+00 1,00E+03 1,00E+01 

Spneu 5,90E+01 5,90E-01 1,00E+03 1,00E+01 

HIB 6,25E+02 6,25E+00 1,00E+02 1,00E+00 

Saur 3,47E+02 3,47E+00 1,00E+02 1,00E+00 

Legio 6,89E+02 6,89E+00 1,00E+03 1,00E+01 

Kpneu 2,46E+02 2,46E+00 1,00E+03 1,00E+01 

PCP 9,56E+02 9,56E+00 1,00E+02 1,00E+00 

Salm 1,96E+02 1,96E+00 1,00E+03 1,00E+01 

Morax 4,39E+01 4,39E-01 1,00E+03 1,00E+01 

Bord 1,35E+03 1,35E+01 1,00E+04 1,00E+02 

FluC 3,02E+03 3,02E+01 1,00E+04 1,00E+02 

Haeinf 3,05E+03 3,05E+01 1,00E+03 1,00E+01 


� The analytical detection limit was retested in measuring tenfold dilution 

series of plasmid DNA of known concentration (1E+06-1E+02 copies/ ml) 

and the FTD Resp 33 multiplexes. (table 10). 

Table 10: Detection limit of plasmid DNA in tenfold measuring. It is given the amount of positive 

detected rating (x/10) and the accordant detection percentage (%). 

neg = not detected 

n.t. = not tested 

pathogen 

plasmid 10 6 cop/ml plasmid 10 5 cop/ml plasmid 10 4 cop/ml plasmid 10 3 cop/ml plasmid 10 2 x/10 % x/10 % x/10 % x/10 % 

cop/ml 

x/10 % 

FluA n.t. n.t. 10/10 100% 10/10 100% 9/10 90% 5/10 50% 

FluB n.t. n.t. 10/10 100% 10/10 100% 7/10 70% neg neg 

Rhino 10/10 100% 10/10 100% 10/10 100% 10/10 100% n.t. n.t. 

Para2 n.t. n.t. 10/10 100% 10/10 100% 10/10 100% 5/10 50% 

Para3 10/10 100% 10/10 100% 10/10 100% 10/10 100% n.t. n.t. 

Para4 10/10 100% 10/10 100% neg neg n.t. n.t. n.t. n.t. 

Cor63 10/10 100% 10/10 100% 10/10 100% 10/10 100% n.t. n.t. 

Cor229 10/10 100% 10/10 100% 10/10 100% 9/10 90% n.t. n.t. 

Cor43 n.t. n.t. 10/10 100% 10/10 100% 10/10 100% 7/10 70% 

HKU1 10/10 100% 10/10 100% 10/10 100% neg neg n.t. n.t. 

Para1 10/10 100% 10/10 100% 10/10 100% 10/10 100% n.t. n.t. 

Hump n.t. n.t. n.t. n.t. 10/10 100% 10/10 100% 10/10 100% 

Boca 10/10 100% 10/10 100% 10/10 100% 10/10 100% n.t. n.t. 

Mpneu n.t. n.t. 10/10 100% 10/10 100% 10/10 100% 7/10 70% 

Adeno 10/10 100% 10/10 100% 10/10 100% 9/10 90% 2/10 20% 

RSV A/B 10/10 100% 10/10 100% 10/10 100% 5/10 50% n.t. n.t. 

PV 10/10 100% 2/10 20% neg neg n.t. n.t. n.t. n.t. 

CMV 10/10 100% 10/10 100% 10/10 100% 10/10 100% n.t. n.t. 

EV 10/10 100% 10/10 100% 10/10 100% 9/10 90% n.t. n.t. 

Spneu 

Hib 10/10 100% 10/10 100% 10/10 100% 6/10 60% 

n.t. 

Saur 10/10 100% 10/10 100% 10/10 100% 3/10 30% 


10/10 100% 10/10 100% 10/10 100% 4/10 40% 

Cpneu 10/10 100% 10/10 100% 10/10 100% 5/10 50% 

Legio 

10/10 100% 10/10 100% 10/10 100% 4/10 40% 

Kpneu 10/10 100% 10/10 100% 10/10 100% 1/10 10% 

n.t. 

PCP 10/10 100% 10/10 100% 9/10 90% 2/10 20% 

Salm 10/10 100% 10/10 100% 9/10 90% 3/10 30% 

Morax n.t. n.t. 10/10 100% 10/10 100% 10/10 100% 3/10 30% 

Bord 10/10 100% 10/10 100% 10/10 100% 9/10 90% n.t. n.t. 

FluC 10/10 100% 10/10 100% 10/10 100% 7/10 70% n.t. n.t. 

Haeinf 10/10 100% 10/10 100% 10/10 100% 9/10 90% n.t. n.t. 

� The endpoint detection limit of the multiplex PCR`s were compared to the 

singleplex PCR`s. Fivefold and tenfold diluted positive patient material was 

tested by singleplex and multiplex. All multiplex PCR`s show pretty similar 

detection limits than the singleplexes (table 11) 





Page 11 


Table 11: Comparison of multiplex (mp) to singleplex (sp) PCR`s. Dilution row (5 fold and 

10 fold) for each pathogen until endpoint detection limit. 


Sample FluA Sample FluB Sample Rhino 

mp sp mp sp mp sp 

CT CT CT CT CT CT 

none 27,69 23,66 none 31,26 28,44 none 31,14 33,15 

Dil1 (1:5) 29,31 27,31 Dil1 (1:5) 32,25 30,74 Dil1 (1:5) 34,51 36,53 

Dil2 (1:5) 31,18 29,83 Dil2 (1:5) 35,65 36,07 Dil2 (1:5) 36,24 36,5 

Dil3 (1:5) 35,15 34,39 Dil3 (1:5) 37,15 36,34 Dil3 (1:5) neg neg 

Dil4 (1:5) neg 36,67 Dil4 (1:5) neg neg Dil4 (1:5) neg neg 

ParaEAV PPmix 

Sample PF2 Sample PF3 Sample PF4 

mp sp mp sp mp sp 

CT CT CT CT CT CT 

none 29,24 31,1 none 30,97 29,79 none 31,26 26,83 

Dil1 (1:10) 30,73 31,9 Dil1 (1:10) 33,84 33,43 Dil1 (1:5) 33,39 29,62 

Dil2 (1:5) 33,87 34,8 Dil2 (1:5) 36,18 36,25 Dil2 (1:5) 36,64 32,68 

Dil3 (1:5) 37,02 38,28 Dil3 (1:5) 38,07 38,28 Dil3 (1:5) 38,73 36,92 

Dil4 (1:5) 38,75 39,27 Dil4 (1:5) neg neg Dil4 (1:5) neg 37,88 

Cor PPmix 

Sample Cor63 Sample Cor229 Sample Cor43 Sample HKU 

mp sp mp sp mp sp mp sp 

CT CT CT CT CT CT CT CT 

none 27,42 26,76 none 28,17 26,49 none 24,17 22,95 none 28,13 28,15 

Dil1 (1:10) 31,39 31,4 Dil1 (1:10) 31,39 30,64 Dil1 (1:10) 28,25 28,23 Dil1 (1:10) 32,13 31,69 

Dil2 (1:10) 36,26 35,94 Dil2 (1:10) 36,99 35,16 Dil2 (1:10) 31,55 31,44 Dil2 (1:10) 36,48 35,99 

Dil3 (1:10) neg 38,02 Dil3 (1:10) neg 38,41 Dil3 (1:10) 35,32 neg Dil3 (1:10) neg 37,79 

Dil4 (1:10) neg neg Dil4 (1:10) neg neg Dil4 (1:10) neg neg Dil4 (1:10) neg neg 

RsEPAcmv PPmix 

Sample EV Sample RSV Sample AV Sample PV Sample CMV 

mp sp mp sp mp sp mp sp mp sp 

CT CT CT CT CT CT CT CT CT CT 

none 27,1 24,3 none 26,9 25,9 none 32,4 32,4 none 23,4 21,4 none 31,1 31,4 

Dil1 (1:5) 29,2 27,3 Dil1 (1:5) 29,8 30 Dil1 (1:5) 36,2 34,9 Dil1 (1:5) 24,8 23,6 Dil1 (1:5) 34,4 33,8 

Dil2 (1:5) 32,1 30 Dil2 (1:5) 33,2 38 Dil2 (1:5) neg 34,5 Dil2 (1:5) 27,8 26,9 Dil2 (1:5) neg neg 

Dil3 (1:5) 34,2 33,4 Dil3 (1:5) neg neg Dil3 (1:5) neg neg Dil3 (1:5) 33 28,9 Dil3 (1:5) neg neg 

Dil4 (1:5) neg neg Dil4 (1:5) neg neg Dil4 (1:5) neg neg Dil4 (1:5) neg neg Dil4 (1:5) neg neg 

Bo-mp-pf1 PPmix 

Sample PF1 Sample HMPV Sample HboV Sample Mpneu 




Dil1 (1:5) 32,34 30,49 Dil1 (1:5) 33,31 34,15 Dil1 (1:5) 38,43 36,42 Dil1 (1:5) 32,78 31,73 

Dil2 (1:5) 34,79 32,44 Dil2 (1:5) 36,96 36,39 Dil2 (1:5) neg neg Dil2 (1:5) 36,23 33,99 

Dil3 (1:5) neg 36,27 Dil3 (1:5) 38,42 neg Dil3 (1:5) neg neg Dil3 (1:5) 38,76 36,99 


RespBac PPmix 

Sample HIB Sample Spneu Sample Cpneu Sample Saureus 




Dil1 (1:5) 33,8 31,06 Dil1 (1:5) 29,22 29,32 Dil1 (1:5) 30,89 31,3 Dil1 (1:5) 29,35 29,35 

Dil2 (1:5) 36,96 34,55 Dil2 (1:5) 31,92 31,68 Dil2 (1:5) 34,18 33,95 Dil2 (1:5) 31,44 31,51 

Dil3 (1:5) neg neg Dil3 (1:5) 35,6 34,04 Dil3 (1:5) 36,94 38,13 Dil3 (1:5) 33,33 33,76 

Dil4 (1:5) neg neg Dil4 (1:5) 37,66 36,48 Dil4 (1:5) neg neg Dil4 (1:5) 35,42 37,56 


KLePSa PPmix 

Sample Legio Sample Kpneu Sample PCP Sample Salm 




Dil1 (1:10) 31,5 28,8 Dil1 (1:10) 31,2 31,2 Dil1 (1:10) 34,3 33,3 Dil1 (1:10) 31,2 31,5 

Dil2 (1:10) 34,1 31,9 Dil2 (1:10) 34,4 33,9 Dil2 (1:5) 36,3 38,5 Dil2 (1:10) 34,8 35,2 

Dil3 (1:5) neg 37,4 Dil3 (1:5) 37,8 36,7 Dil3 (1:5) neg 38,9 Dil3 (1:10) 36,8 neg 

Dil4 (1:5) neg neg Dil4 (1:5) neg neg Dil4 (1:5) neg neg Dil4 (1:10) 37,2 neg 






Page 12 


MoBoCH PPmix 

Sample Morax Sample Bord Sample FluC Sample Haeinf 




Dil1 (1:10) 30,4 31,7 Dil1 (1:10) 30,3 30,5 Dil1 (1:10) 35 34,9 Dil1 (1:10) 29,9 30,3 

Dil2 (1:5) 36,6 33,6 Dil2 (1:10) neg 35,3 Dil2 (1:5) neg 38,5 Dil2 (1:5) 32,7 32,5 

Dil3 (1:5) neg 36 Dil3 (1:5) neg 34,4 Dil3 (1:5) neg neg Dil3 (1:5) neg 34,4 

3. Clinical evaluation 

� All clinical samples were tested with existing single/multiplex PCRs or 

culture and the FTD Respiratory pathogens 33 test. Pharyngeal, pleural 

effusion and other samples, collected in different sites were analyzed. The 

following statistic shows how many positives of each pathogen had been 

detected (table12). The results for the reference method and the new FTD 

Resp 33 test were comparable. 

� 

Table 10: Statistic of clinical evaluation data for the new FTD Resp 33 kit. 

pathogen 

FluA 

FluB 

Rhino 

PF2 

PF3 

PF4 

Cor43 

Cor63 

pathogen 

... method 

Resp 33 

pos neg 

pos a c 

neg b d 


pos neg 

pos 23 / 

FTD resp21 plus neg / 221 

pos 20 


pos 25 / 


pos 19 / 


pos 18 / 


pos 16 / 


pos 23 / 


pos 12 / 







Page 13 

total sensitivity (%) specificity (%) 

... 

a/(a+c) x 100 d/(d+b) x 100 

total sensitivity (%) specificity (%) 

244 100 100 

244 100 100 

244 100 100 

245 100 100 

245 100 100 

245 100 100 

243 100 100 

243 100 100 


Pathogen 


pos neg total Sensitivity (%) Specificity (%) 

Cor229 

pos 

FTD resp21 plus neg 

13 

/ 

/ 

230 243 100 100 

HKU 

pos 


6 

/ 

/ 

237 243 100 100 

HMPV 

pos 


19 

/ 

/ 

223 242 100 100 

HboV 

pos 


7 

/ 

/ 

235 242 100 100 

PF1 

pos 


18 

/ 

/ 

224 242 100 100 

Mpneu 

pos 


16 

/ 

/ 

226 242 100 100 

EV 

pos 

FTD resp21 plus 

16* 2 neg / 

/ 

237 253 100 100 

PV 

pos 

FTD resp21 plus 

14* 2 neg / 

/ 

239 253 100 100 

RSV 

pos 


22 

/ 

/ 

231 253 100 100 

AV 

pos 


31 

/ 

/ 

222 253 100 100 

pos 20 / 

CMV FTD CMV neg / 233 253 100 100 

Cpneu 

pos 


6 

/ 

/ 

260 266 100 100 

Spneu 

pos 


63 

/ 

/ 

203 266 100 100 

HIB 

pos 


18 

/ 248 266 100 100 

Saur 

pos 


40 

/ 

/ 

226 266 100 100 

Legio 

pos 

culture neg 

18 

/ 

/ 

68 86 100 100 

Kpneu 

pos 

culture neg 

20 

/ 

/ 

66 86 100 100 

PCP 

PCR Glasgow 

pos 

neg 

14 

/ 72 86 100 100 

Salm 

pos 

FTD bacgas neg 

29 

/ 

/ 

57 86 100 100 

Morax 

pos 

culture neg 

29 

/ 

/ 

55 84 100 100 

Bord 

pos 

culture neg 

6 

/ 

/ 

78 84 100 100 

FluC 

PCR Glasgow 

pos 

neg 

24 

/ 

/ 

60 84 100 100 

Haeinf 

pos 

culture neg 

40 

/ 

/ 

44 

84 100 100 






Page 14

Respiratory pathogens 33 february 2011 version 1 - Mikrogen

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?