OPT M T ON ND PRO PECT VE 2 YE R EV LU T ON OF THE DETECT ON OF CHL MYD R CHOM ND NE ER GONORRHOE EOPTIMISATION AND PROSPECTIVE 2 YEAR EVALUATION OF THE DETECTION OF CHLAMYDIA TRACHOMATIS AND NEISSERIA GONORRHOEAEOPTIMISATION AND PROSPECTIVE 2-YEAR EVALUATION OF THE DETECTION OF CHLAMYDIA TRACHOMATIS AND NEISSERIA GONORRHOEAEOPTIMISATION AND PROSPECTIVE 2-YEAR EVALUATION OF THE DETECTION OF CHLAMYDIA TRACHOMATIS AND NEISSERIA GONORRHOEAEOPTIMISATION AND PROSPECTIVE 2 YEAR EVALUATION OF THE DETECTION OF CHLAMYDIA TRACHOMATIS AND NEISSERIA GONORRHOEAEIN SEMEN SPECIMENS USING THE ABBOTT REALTIME CT/NG ASSAYIN SEMEN SPECIMENS USING THE ABBOTT REALTIME CT/NG ASSAYIN SEMEN SPECIMENS USING THE ABBOTT REALTIME CT/NG ASSAYIN SEMEN SPECIMENS USING THE ABBOTT REALTIME CT/NG ASSAYIN SEMEN SPECIMENS USING THE ABBOTT REALTIME CT/NG ASSAY

Fl G tt d P l O V h A C i j N th li F l d B P ttFlorence Grattard Paul O Verhoeven Anne Carricajo Nathalie Fonsale and Bruno PozzettoFlorence Grattard Paul O.Verhoeven Anne Carricajo Nathalie Fonsale and Bruno PozzettoFlorence Grattard, Paul O.Verhoeven, Anne Carricajo, Nathalie Fonsale and Bruno PozzettojL b f I f i A d H iè U i i H i l f S i E i FLaboratory of Infectious Agents and Hygiène University Hospital of Saint Etienne FranceLaboratory of Infectious Agents and Hygiène University Hospital of Saint-Etienne FranceLaboratory of Infectious Agents and Hygiène, University Hospital of Saint Etienne, Francey g yg , y p ,

24rd ECCMID BARCELONA 10 13 MAY 201424rd ECCMID • BARCELONA • 10-13 MAY 201424 ECCMID BARCELONA 10 13 MAY 2014

PERF R N E F HE /NG N E EN PE ENI F th d t ti f Chl di t h ti (CT) d N i i QUANTIFICATION OF THE DNA LOAD ON SEMEN SPECIMEN PERFORMANCES OF THE CT/NG ASSAY ON SEMEN SPECIMENIn France the detection of Chlamydia trachomatis (CT) and Neisseria QUANTIFICATION OF THE DNA LOAD ON SEMEN SPECIMEN PERFORMANCES OF THE CT/NG ASSAY ON SEMEN SPECIMENIn France, the detection of Chlamydia trachomatis (CT) and Neisseria QUANTIFICATION OF THE DNA LOAD ON SEMEN SPECIMEN PERFORMANCES OF THE CT/NG ASSAY ON SEMEN SPECIMEN, y ( )h (NG) d d d

Qgonorrhoeae (NG) in semen is mandatory prior assisted reproductivegonorrhoeae (NG) in semen is mandatory prior assisted reproductivegonorrhoeae (NG) in semen is mandatory prior assisted reproductivet chniqu s justif in th n d t h v v lid t d m th ds f r t stin D i i f h l i l i i i f h Abb CT/NG D i i f h d ibili f htechniques justifying the need to have validated methods for testing Determination of the amount of extracted cells from different volumes (50 µL 100 µL 150 µL Determination of the analytical sensitivity of the Abbott CT/NG assay on semen Determination of the reproducibility of thetechniques, justifying the need to have validated methods for testing Determination of the amount of extracted cells from different volumes (50 µL 100 µL 150 µL Determination of the analytical sensitivity of the Abbott CT/NG assay on semen Determination of the reproducibility of the q j y g g

h i k t t i PCR i hibit Determination of the amount of extracted cells from different volumes (50 µL, 100 µL, 150 µL, Determ nat on of the analyt cal sens t v ty of the Abbott C /NG assay on semen Determ nat on of the reproduc b l ty of the

such specimens known to contain PCR inhibitors 200 L 250 L) f d l d d i t t l l f 600 L f th specimens using semen spiked with known amounts of CT cells from the QCMD panels assay on different semen samples spiked with thesuch specimens known to contain PCR inhibitors. 200 µL or 250 µL) of crude semen samples suspended in a total volume of 600 µL of the specimens using semen spiked with known amounts of CT cells from the QCMD panels assay on different semen samples spiked with theph f h d d l l l

200 µL or 250 µL) of crude semen samples suspended in a total volume of 600 µL of the specimens using semen spiked with known amounts of CT cells from the QCMD panels assay on different semen samples spiked with the The aim of this study was to propose and evaluate a simple protocol on

µ µ ) p p µM l i ll b ff b i h C l C ll

p g p p y p pt f CT iti lThe aim of this study was to propose and evaluate a simple protocol on Multicollect buffer by using the r gene Control Cell assay same amount of a CT-positive sampleThe aim of this study was to propose and evaluate a simple protocol on Multicollect buffer, by using the r-gene Control Cell assay. same amount of a CT-positive sample

m n f pid nd n iti d t ti n f CT nd NG in thMult collect buffer, by us ng the r gene Control Cell assay. p p

semen for a rapid and sensitive detection of CT and NG using the Semen (200µL) Initial Amounts of CT CT crossingsemen for a rapid and sensitive detection of CT and NG using the Semen (200µL) Initial Amounts of CT CT crossing p gAbb tt R lTi CT/NG PCR th M2000 (Abb tt) i t t

( µ )i d ith f

gAbbott RealTime CT/NG PCR assay on the M2000 (Abbott) instrument S l l f ll t l l f ll t l l f ll t l l f ll t l l f ll t l mixed with freeze t fAbbott RealTime CT/NG PCR assay on the M2000 (Abbott) instrument. Samples volume of cell control volume of cell control volume of cell control volume of cell control volume of cell control S i (200 L) CT i IC iSemen (200 µL) Amounts of CT crossing IC crossing

mixed with freeze component of m y ( ) m pi i i i i Semen specimens (200 µL) CT crossing IC crossingSemen (200 µL) Amounts of CT crossing IC crossing dried samples of the

co po e t osemen crossing semen crossing semen crossing semen crossing semen crossing Semen specimens (200 µL) CT crossing IC crossing S ( 00 µ ) C g C gdried samples of the

th l ll i i i tsemen #

crossing i

semen #

crossing i

semen #

crossing i

semen #

crossing i

semen #

crossing i spiked with 200 µL of a CTmixed with serial2010 QCMD panel the panel cells in copies points

tested# point tested# point tested# point tested# point tested# point spiked with 200 µL of a CT-mixed with serial CT cells in2010 QCMD panel the panel cells in copies pointstested point tested point tested point tested point tested point p µ

iti l points pointsdilutions of theCT cells in

positive sample points pointsdilutions of the positive sample points pointsdilutions of the i /t t i t i tQCMD 10-07 copies/test points pointsQCMD 10-07 copies/test points points

CTB10 01 urine 280 0Sample A 50 µL 27 63 100µL 27 45 150µL 25 72 200µL 27 03 250µL CTB10-01 urine 280 0Sample A 50 µL 27.63 100µL 27.45 150µL 25.72 200µL 27.03 250µLCTB10 02 i 57 0Sample B 50 µL 28 27 100µL 26 94 150µL 27 21 200µL 28 86 250µL CTB10-02 urine 57 0Sample B 50 µL 28.27 100µL 26.94 150µL 27.21 200µL 28.86 250µL Sample A 36 63 36 91SCTB10 02 urine 57 0CTB10 03 i 5700 37 3Sample C 50 µL 30 83 100µL 30 10 150µL 29 65 200µL 28 39 250µL 28 69 Sample A 36.63 36.91Sample 1 2850 31 63 36 02CTB10-03 urine 5700 37 3Sample C 50 µL 30.83 100µL 30.10 150µL 29.65 200µL 28.39 250µL 28.69 p

S l B 36 64 37 30Sample 1 2850 31.63 36.02CTB10 03 urine 5700 37,3µ

Sample D 50 µL 28 90 100µL 26 90 150µL 26 50 200µL 26 90 250µL 26 20 Sample B 36 64 37 30Sample 2 1425 32 43 36 05CTB10-04 urine 0 0Sample D 50 µL 28.90 100µL 26.90 150µL 26.50 200µL 26.90 250µL 26.20 Sample B 36.64 37.30Sample 2 1425 32.43 36.05CTB10-04 urine 0 0p µ µ µ µ µSample C 36 65 36 75

pS l 3 712 33 76 36 07CTB10 05 urine swedish variant 31 7 Sample C 36.65 36.75Sample 3 712 33 76 36 07CTB10-05 urine swedish variant 31,7

t t d i t t l ti l f 600 lp

S l D 36 66 37 71Sample 3 712 33.76 36.07

CTB10 06 TE buffer 280 41 6tested in a total reaction volume of 600 µl Sample D 36 66 37 71Sample 4 356 34 87 36 44CTB10-06 TE buffer 280 41,6

DNA EXTRACTION ON SEMEN SPECIMENSµ Sample D 36.66 37.71Sample 4 356 34.87 36.44,

CTB10 07 TE b ffer 5700 31 6 DNA EXTRACTION ON SEMEN SPECIMENS I i th l f t 200 L d i th t ti t i th Sample E 36 67 36 40p

S l 5 178 35 83 36 26CTB10-07 TE buffer 5700 31,6 DNA EXTRACTION ON SEMEN SPECIMENS Increasing the volume of semen up to 200 µL during the extraction step increases the Sample E 36.67 36.40Sample 5 178 35.83 36.26,CTB10 08 TE b ff 0 0Increasing the volume of semen up to 200 µL during the extraction step increases the p

S l F 36 68 37 14Sample 5 178 35.83 36.26CTB10-08 TE buffer 0 0

For optimisation of the method different volumes (from 50 µL to 250 µL)g p µ g p

f d ll ( h b l i i l i l i PCR) Sample F 36 68 37 14Sample 6 89 35 83 36 20CTB10 08 TE buffer 0 0C ffFor optimisation of the method different volumes (from 50 µL to 250 µL) amounts of extracted cells (as shown by lower crossing points values in real time PCR) Sample F 36.68 37.14Sample 6 89 35.83 36.20CTB10-09 TE buffer 57 37 7For optimisation of the method, different volumes (from 50 µL to 250 µL) amounts of extracted cells (as shown by lower crossing points values in real-time PCR). Sample G 36 69 37 28Sample 7 44 36 98 36 22CTB10 09 TE buffer 57 37,7p

f ith d ik d ith CT NG iti lamounts of extracted cells (as shown by lower crossing points values in real time PCR). Sample G 36.69 37.28Sample 7 44 36.98 36.22CTB10-10 TE buffer 28 40 6of either crude semen or semen spiked with CT- or NG-positive samples This allows to detect low bacterial charges in semen specimens

ppCTB10-10 TE buffer 28 40,6of either crude semen or semen spiked with CT or NG positive samples This allows to detect low bacterial charges in semen specimensp p pi d i h h b ff id d i h h M l i ll i

This allows to detect low bacterial charges in semen specimens. were mixed with the buffer provided with the Multicollect specimen

g pwere mixed with the buffer provided with the Multicollect specimen w r m w th th uff r pro w th th Mu t co ct sp c m n

Th n l tic l s nsitivit n s m n s simil r t th t n t d b th m nuf ctur r f r urin r s b s mpl s ( t l st 320 c pi s)collection tube (Abbott) in a final volume of 600 µL The mixture was The analytical sensitivity on semen was similar to that noted by the manufacturer for urine or swab samples (at least 320 copies)collection tube (Abbott) in a final volume of 600 µL The mixture was QUANTIFICATION OF THE BACTERIAL LOAD ON SPIKED SEMEN SPECIMENThe analytical sensitivity on semen was similar to that noted by the manufacturer for urine or swab samples (at least 320 copies) collection tube (Abbott), in a final volume of 600 µL. The mixture was QUANTIFICATION OF THE BACTERIAL LOAD ON SPIKED SEMEN SPECIMEN

y y y p ( p )lth h i hibiti ld b t d h i d ith QCMD f d i d l i i t d f it d t 4°C 20°C b f t ti th M2000 t QUANTIFICATION OF THE BACTERIAL LOAD ON SPIKED SEMEN SPECIMEN although some inhibition could be noted when semen were mixed with QCMD freeze dried sample originated from urinesstored at 4°C or -20°C before extraction on the M2000 apparatus QUANTIFICATION OF THE BACTERIAL LOAD ON SPIKED SEMEN SPECIMEN although some inhibition could be noted when semen were mixed with QCMD freeze dried sample originated from urines.stored at 4 C or 20 C before extraction on the M2000 apparatus. g Q f p g f h d d d bl l h l f d f d G l

ppThe assay provided reproducible results on semen specimens with CV values of 0 041 and 0 036 for CT and NG respectivelyThe assay provided reproducible results on semen specimens, with CV values of 0.041 and 0.036 for CT and NG, respectively.The assay provided reproducible results on semen specimens, with CV values of 0.041 and 0.036 for CT and NG, respectively.

REAL TIME AMPLIFICATION PROCEDURES D t i ti f th b t i l l ds b si th CT/NG ss f diff t l s (100 REAL-TIME AMPLIFICATION PROCEDURES Determination of the bacterial loads by using the CT/NG assay from different volumes (100 REAL-TIME AMPLIFICATION PROCEDURES Determination of the bacterial loads by using the CT/NG assay from different volumes (100 h f ll b d f h d ff l f d

y g y (L 150 L 200 L 250 L) f ik d ith th l f iti lThe amount of cells obtained from the different volumes of semen tested PROSPECTIVE EVALUATION IN ROUTINE ON A 2 YEAR PERIODµL 150 µL 200 µL or 250 µL) of semen spiked with the same volume of a positive sampleThe amount of cells obtained from the different volumes of semen tested PROSPECTIVE EVALUATION IN ROUTINE ON A 2 YEAR PERIODµL, 150 µL 200 µL or 250 µL) of semen spiked with the same volume of a positive sample The amount of cells obtained from the different volumes of semen tested PROSPECTIVE EVALUATION IN ROUTINE ON A 2-YEAR PERIODµL, µL µL µL) f m p m m f p mp

s qu ntifi d b l tim PCR n n Abi7500 pp tus usin 10 µL fPROSPECTIVE EVALUATION IN ROUTINE ON A 2 YEAR PERIOD

suspended in a total volume of 600 µL of the Multicollect buffer after extraction andwas quantified by real-time PCR on an Abi7500 apparatus using 10 µL of suspended in a total volume of 600 µL of the Multicollect buffer after extraction andwas quantified by real time PCR on an Abi7500 apparatus using 10 µL of suspended in a total volume of 600 µL of the Multicollect buffer, after extraction and q y pp g µDNA hi h lifi d ith th C ll C t l (Bi M i )

plifi ti th M2000 tDNA which was amplified with the Cell Control r-gene assay (BioMerieux) amplification on the M2000 apparatusDNA which was amplified with the Cell Control r-gene assay (BioMerieux). amplification on the M2000 apparatus. p f g y ( )

h d f G f d b l Pp pp

Th i l d d i d i h 4°C 20°C Th lid t d p t c l usin 200 µL f s m n mix d ith 400 µL lum f Multic ll ct buff s lu t d in c ns cuti s mpl sThe detection of CT or NG DNA was performed by real time PCR using The test included semen mixtures stored either at 4°C or at 20°C The validated protocol using 200 µL of semen mixed with a 400 µL-volume of Multicollect buffer was evaluated in consecutive samplesThe detection of CT or NG DNA was performed by real-time PCR using The test included semen mixtures stored either at 4 C or at -20 C. The validated protocol using 200 µL of semen mixed with a 400 µL volume of Multicollect buffer was evaluated in consecutive samples The detection of CT or NG DNA was performed by real time PCR using h t st nc u s m n m tur s stor th r at or at . p g µ µ pi d f ti t tt di th R d ti D t t f th U i it H it l f S i t Eti d i 2 i dth CT/NG PCR ss f ll i th mm d ti s f th received from patients attending the Reproduction Department of the University Hospital of Saint-Etienne during a 2-year period:the CT/NG PCR assay following the recommendations of the received from patients attending the Reproduction Department of the University Hospital of Saint-Etienne during a 2-year period:the CT/NG PCR assay, following the recommendations of the

S l ik d T t V l f CT NG IC V l f CT NG IC V l f CT NG IC V l f CT NG ICf p g p p f y p f g y p

d d h ( %) d ( %) d d f dy g

fSamples spiked Temperature Volume of CT NG IC Volume of CT NG IC Volume of CT NG IC Volume of CT NG IC between 2012 and 2013 1660 consecutive semen specimens were tested with 7 (0 42%) and 3 (0 18%) detected positive for CT andmanufacturer

p p psemen crossing crossing crossing semen crossing crossing crossing semen crossing crossing crossing semen crossing crossing crossing between 2012 and 2013, 1660 consecutive semen specimens were tested, with 7 (0.42%) and 3 (0.18%) detected positive for CT andmanufacturer. semen crossing crossing crossing semen crossing crossing crossing semen crossing crossing crossing semen crossing crossing crossing between 2012 and 2013, 1660 consecutive semen specimens were tested, with 7 (0.42%) and 3 (0.18%) detected positive for CT and manufacturer. with CT or NG* of storage tested# points points points tested# points points points tested# points points points tested# points points points NG ti l N i f ti s d t t d i th s s m l sTh d t ti f th IC (i t l t l) f d i th

with CT or NG of storage tested points points points tested points points points tested points points points tested points points points NG respectively No co-infection was detected in those samplesThe detection of the IC (internal control) was performed in the same NG, respectively. No co infection was detected in those samples. The detection of the IC (internal control) was performed in the same p y p( ) pb lid h h l PCR d Th l lid d htube to validate the whole PCR procedure The latter was validated when Sample A at 4°C 100 µL 32 58 36 69 150 µL 32 69 37 68 200µL 32 63 38 17 250µL 31 96 37 49tube to validate the whole PCR procedure. The latter was validated when Sample A at 4 C 100 µL 32.58 36.69 150 µL 32.69 37.68 200µL 32.63 38.17 250µL 31.96 37.49

/ %tube to val date the whole PCR procedure. he latter was val dated when

Sample A at -20°C 100 µL 31 85 36 96 150 µL 33 97 36 86 200µL 31 71 37 21 250µL 31 50 37 25 Interestingly using the Abbott CT/NG assay less than 3% samples were initially found to contain PCR inhibitors as detected by thethe IC specific signal was detected for semen samples with a crossingSample A 100 µL 31.85 36.96 150 µL 33.97 36.86 200µL 31.71 37.21 250µL 31.50 37.25 Interestingly using the Abbott CT/NG assay less than 3% samples were initially found to contain PCR inhibitors as detected by thethe IC specific signal was detected for semen samples with a crossing Interestingly, using the Abbott CT/NG assay, less than 3% samples were initially found to contain PCR inhibitors, as detected by the the IC specific signal was detected, for semen samples, with a crossing S °C

y y p y ybs f d t ti f th i t l t l si l d t ti b l 41 l s Aft i ht t t t b t i s K th fi l

p g p gi t f 38 ( 3) l Sample B at 4°C 100 µL 33.47 37.68 150 µL 33.32 38.45 200µL 32.53 37.51 250µL 32.50 37.94 absence of detection of the internal control signal or a detection below 41 cycles After an overnight treatment by proteinase K the finalpoint of 38 ( 3) cycles p µ µ µ µ

S l B at 20°C 100 L 32 39 36 72 150 L 32 53 37 62 200 L 32 70 37 79 250 L 31 96 37 65absence of detection of the internal control signal or a detection below 41 cycles. After an overnight treatment by proteinase K, the final point of 38 ( 3) cycles. Sample B at -20 C 100 µL 32.39 36.72 150 µL 32.53 37.62 200µL 32.70 37.79 250µL 31.96 37.65

g y g y p ,i hibiti t l f 1 4% (24 i f 1660 t t d)

p ( ) yl i h i hibi ( i i 41 l ) d i

p µ µ µ µinhibition rate was only of 1 4% (24 semen specimens from 1660 tested)Samples with inhibitors (crossing point >41 cycles) were tested again inhibition rate was only of 1.4% (24 semen specimens from 1660 tested). Samples with inhibitors (crossing point >41 cycles) were tested again Sample C at 4°C 100 µL 32 42 36 82 150 µL 32 28 36 98 200µL 32 04 37 24 250µL 31 78 37 27n t n rat wa n y f . ( m n p m n fr m 66 t t ).Samples with inhibitors (crossing point 41 cycles) were tested again Sample C at 4 C 100 µL 32.42 36.82 150 µL 32.28 36.98 200µL 32.04 37.24 250µL 31.78 37.27

after a new extraction procedure performed after an overnight Sample C at -20°C 100 µL 32 49 37 08 150 µL 32 08 37 20 200µL 31 57 37 14 250µL 31 83 37 08after a new extraction procedure performed after an overnight Sample C at 20 C 100 µL 32.49 37.08 150 µL 32.08 37.20 200µL 31.57 37.14 250µL 31.83 37.08after a new extraction procedure performed after an overnight Th t f iti it th l b t i il t th t d ib d i th F h t di H d d D di t l i 2004 f d 7 CT

p p gt t t ith t i K S l h i i hibiti f th IC iti b i l t d i M lti ll t b ff The rate of positivity was rather low but similar to that described in other French studies: Hamdad-Daoudi et al in 2004 found 7 CT-treatment with proteinase K Samples showing an inhibition of the IC positive swab or urine sample stored in Multicollect buffer The rate of positivity was rather low, but similar to that described in other French studies: Hamdad Daoudi et al. in 2004 found 7 CTtreatment with proteinase K. Samples showing an inhibition of the IC m tested in a total reaction volume of 600 µl

p y ,i i l 111 d i b h R h C b A li R d l i 2006 f d 1 CT i i f 100

p p gl f d d h b f h

m tested in a total reaction volume of 600 µl positive samples amongst 111 tested specimens by the Roche Cobas Amplicor ; Rosemond et al in 2006 found 1 CT positive sperm from 100signal after repetitive experiments were considered as an inhibitor of the I i h l f f i 200 L i h l l fpositive samples amongst 111 tested specimens by the Roche Cobas Amplicor ; Rosemond et al. in 2006 found 1 CT- positive sperm from 100 signal after repetitive experiments were considered as an inhibitor of the Increasing the volume of semen for extraction up to 200 µL increases the level ofpos t ve samples amongst tested spec mens by the Roche obas Ampl cor ; Rosemond et al. n 006 found pos t ve sperm from 00signal after repetitive experiments were considered as an inhibitor of the Increasing the volume of semen for extraction up to 200 µL increases the level of infertile patients whereas Le Roy et al in 2013 could not detect any positive sample from 100 semen specimens tested at lower volumes (25PCR test

Increasing the volume of semen for extraction up to 200 µL increases the level of infertile patients whereas Le Roy et al in 2013 could not detect any positive sample from 100 semen specimens tested at lower volumes (25PCR test detection of bacterial DNA (illustrated by lower crossing points values in real time PCR)infertile patients whereas Le Roy et al. in 2013 could not detect any positive sample from 100 semen specimens tested at lower volumes (25 PCR test. detection of bacterial DNA (illustrated by lower crossing points values in real-time PCR)

p y y p p pL) ith th C b 4800 CT/NGdetection of bacterial DNA (illustrated by lower crossing points values in real time PCR) µL) with the Cobas 4800 CT/NG assayy g p

ith t i i th t f i hibitiµL) with the Cobas 4800 CT/NG assay.

without increasing the rate of inhibitionµ ) y

without increasing the rate of inhibition. SENSITIVITY AND REPRODUCIBILITY OF THE ASSAY

g Cl l l f b l DN d d k h 4°C SENSITIVITY AND REPRODUCIBILITY OF THE ASSAY Close levels of bacterial DNA were detected on semen mixtures kept either at 4°C or at SENSITIVITY AND REPRODUCIBILITY OF THE ASSAY Close levels of bacterial DNA were detected on semen mixtures kept either at 4 C or at Finally the protocol was found suitable to test semen specimens on a routine basis in combination with urine or swab samples The wholeTh iti it d d ibilit f th d fi d i Close levels of bacterial DNA were detected on semen mixtures kept either at 4 C or at Finally the protocol was found suitable to test semen specimens on a routine basis in combination with urine or swab samples The wholeThe sensitivity and reproducibility of the assay were defined using semen 20°C

Finally, the protocol was found suitable to test semen specimens on a routine basis in combination with urine or swab samples. The whole The sensitivity and reproducibility of the assay were defined using semen -20°Cy p p p

d ti f th i l di t ti d l ti PCR 5 h f t l t 45 ly p u y f y w f u g m 20 C. duration of the process including extraction and real time PCR was 5 hours for at least 45 samplesspiked either with positive CT or NG samples or with quantified duration of the process including extraction and real time PCR was 5 hours for at least 45 samples. spiked either with positive CT or NG samples or with quantified p g pspiked either with positive CT or NG samples, or with quantified p p p ql hili d DNA f th QCMD llyophilized DNA from the QCMD panelslyophilized DNA from the QCMD panels. y p Q p

CLINICAL PROSPECTIVE STUDY CLINICAL PROSPECTIVE STUDY CLINICAL PROSPECTIVE STUDYTh l d d l l d l dThe validated protocol was evaluated in consecutive samples received A i l d li bl l i d f d d i f Chl di h i d N i i h i i f 200 L l f i b i h Abb R lTiThe validated protocol was evaluated in consecutive samples received A simple and reliable protocol is proposed for an automated detection of Chlamydia trachomatis and Neisseria gonorrhoeae in routine from a 200 µL volume of semen specimen by using the Abbott RealTimeThe validated protocol was evaluated in consecutive samples received A simple and reliable protocol is proposed for an automated detection of Chlamydia trachomatis and Neisseria gonorrhoeae in routine from a 200 µL-volume of semen specimen, by using the Abbott RealTimefrom patients attendin the Reproduction Department of Saint Etienne

A simple and reliable protocol is proposed for an automated detection of Chlamydia trachomatis and Neisseria gonorrhoeae in routine from a 200 µL volume of semen specimen, by using the Abbott RealTime from patients attending the Reproduction Department of Saint-Etienne

p p p p y g p y gfrom patients attending the Reproduction Department of Saint Etienne p g p pU i it H it l d i 2 i d CT/NG th M2000 t Thi t l bl iti d t ti f th b t i l t i f ti t i i i t d d ti dUniversity Hospital during a 2-year period CT/NG assay on the M2000 apparatus This protocol enables a sensitive detection of these bacterial agents in semen for patients requiring assisted reproductive proceduresUniversity Hospital during a 2-year period. CT/NG assay on the M2000 apparatus. This protocol enables a sensitive detection of these bacterial agents in semen for patients requiring assisted reproductive procedures.y p g y p y pp p f g f p q g p p

Ackn l d m nts: This stud c i d th fin nci l supp t f Abb tt Th uth s th nk I èn B ssu Is b ll C n Ch istin P ch nd A nès Thi f th i skilful t chnic l ssist nc References: Hamdad Daoudi et al J Med Microbiol 2004 53:985 990; Rosemond et al Path Biol 2006 54: 125 129; Le Roy et al J Med Microbiol 2013 62: 217 222Acknowledgements: This study received the financial support of Abbott The authors thank Irène Bossu Isabelle Coron Christine Peyrache and Agnès Thizy for their skilful technical assistance References: Hamdad-Daoudi et al., J. Med. Microbiol. 2004, 53:985-990; Rosemond et al. Path Biol. 2006, 54: 125-129; Le Roy et al. J. Med. Microbiol. 2013, 62: 217-222.Acknowledgements: This study received the financial support of Abbott. The authors thank Irène Bossu, Isabelle Coron, Christine Peyrache and Agnès Thizy for their skilful technical assistance. References Hamdad Daoudi et al., J. Med. Microbiol. 2004, 53 985 990; Rosemond et al. Path Biol. 2006, 54 125 129; Le Roy et al. J. Med. Microbiol. 2013, 62 217 222.