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Journal of Biology, Agriculture and Healthcare ISSN 2224-32 ! "#a$er% ISSN 222&- '3( ")nline% *ol.3, No.+!

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Genetic Study of TORCH Infections in Women with Bad Obstetric History: Multi le! "olymerase Chain Reaction for #etection of Common "atho$ens and %$ents of Con$enital Infections
+. 2. 3. Sa,reen A. A. -a.al+, /u0aya 1. J. Awadh2, Ali H. 1. Al-1ar2o0i33"4orres$onding author% 4ollege of Science for wo.en, Ba,ylon 5ni6ersity 4ollege of Science for wo.en, Ba,ylon 5ni6ersity 4ollege of Science for wo.en, Ba,ylon 5ni6ersity, #) ,o7 43&, Al-Hillah city, Ba,ylon, Ira0. 8el9 ':4;;+ 33:+2+ <-.ail9 ali=al.ar2o0i>yahoo.co.u? 3 <-.ail of the corres$onding author9 ali=al.ar2o0i>yahoo.co.u?

OB&'CTI('S: 8o re6ealed the incidence of 8)/4H infections a.ong $regnancy wastage in wo.en which had ,ad o,stetric history "B)H%. M'THO#S: 8he study included +32 wo.en with ,ad o,stetric history. @enetic e6aluation for 8)/4H infections was carried out ,y s$ecific $ri.ers designed for that $ur$ose using #4/ .ethod. R'S)*T: 8o7o$las.a was 3:.3:A, ru,ella 2 .4&A, cyto.egalo6irus 2'.&&A and her$es si.$le7 6irus +3.:4A. 1a7i.u. nu.,er of cases of a,ortion &2 "+,-+,A%, $reter. la,or 2' "./-,0A% was associated with to7o$las.a infection, early neonatal deaths +' "/1-+,A% were .a7i.ally associated with to7o$las.a and 41* infections. while congenital .alfor.ations +4 "/2-0A% were e6ident .a7i.ally with to7o$las.a infection and intrauterine death ! "0-20A%. CO3C*)SIO3S: Bo.en with B)H are significantly higher in infection co.$ared with that in control. A $re6ious history of $regnancy wastage, genetic infestation using s$ecific $ri.ers for 8)/4H agentCs detection and the serological reaction for 8)/4H infections during current $regnancy .ust ,e considered while .anaging B)H cases so as to reduce the ad6erse fetal outco.e. Introduction Bad o,stetric history "B)H% i.$lies $re6ious unfa6ora,le foetal outco.e in ter.s of two or .ore consecuti6e s$ontaneous a,ortion, history of intrauterine foetal death, intrauterine growth retardation, still ,irths, early neonatal death andDor congenital ano.alies. 4ause of B)H .ay ,e genetic, hor.onal, a,nor.al .aternal i..une res$onse and .aternal infection. /ecurrent $regnancy wastage due to .aternal infections trans.issi,le in utero at 6arious stage of gestation can ,e caused ,y a wide array of organis.s which include the 8)/4H co.$le7 "Toxoplasma gondii, Rubella virus, Cytomegalovirus, Herpes simplex virus% and other agents li?e Chlamydia trachomatis, Treponema pallidum, Niesseria gonorrhoeae, HI* etc. 8o7o$las.osis ac0uired during $regnancy .ay cause da.age to the fetus 4/5 . Infection with /u,ella during $regnancy .ay lead to congenital .alfor.ation in + -&4 $ercent of cases.6.7 8he infection caused ,y 41* in adult is usually asy.$to.atic ,ut its significance is .any ti.es increased when it occurs during $regnancy. Howe6er, the rate of $ri.ary 41* infection is significantly higher for $regnant wo.en fro. low socioecono.ic grou$. 8he .other is the usual source of trans.ission of HS* to the fetus or new,orn. #ri.ary HS* infection during first half of $regnancy is associated with increased fre0uency of s$ontaneous a,ortion, still ,irth, and congenital .alfor.ation.6+7Infections ,y 8)/4H agents in wo.en are usually asy.$to.atic and chronic. 8he social and re$roducti6e .aladEust.ent ,ecause of re$eated $regnancy wastages, cost of treat.ent, and .or,idity caused to the infant .a?e the 8)/4H grou$ of infections a .aEor cause of concern. 8he $re6alence of these infections 6aries fro. one geogra$hical area to another 617. 1any sensiti6e and s$ecific tests are a6aila,le for serological diagnosis of 8)/4H co.$le7. <FISA for Ig1 anti,odies against these infections is highly sensiti6e and s$ecific 687. Nucleic acid testing has allowed .ore sensiti6e and s$ecific detection of infectious agents and is ,eing increasingly ado$ted ,y diagnostic la,oratories 607. 8he technology is $articularly useful in 6irology as it can re$lace con6entional culture .ethods that are often e7$ensi6e and la,or intensi6e, detect fastidious organis.s such as he$atitis 4 6irus "H4*%, detect low co$y nu.,er agents such as her$es si.$le7 6irus "HS*% in cere,ros$inal fluid, and i.$ro6e turn-around ti.es for detection of treata,le agents such as her$es 6iruses 697.

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Journal of Biology, Agriculture and Healthcare ISSN 2224-32 ! "#a$er% ISSN 222&- '3( ")nline% *ol.3, No.+!, 2 +3

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To!o lasmosis caused ,y 8o7o$las.a gondii, a $arasite which can $urchase fro. handling infected do.estic ani.als "cats%, .il? or eating conta.inated .eat. 8he infection can conceded to fetus 6ia the $lacenta during $regnancy $eriod, it cause infections of the eyes or central ner6ous syste. ":%. Rubella is a 6irus that can cause e$ide.ics .ost li?ely in the s$ring. Between .+-2A of new,orns will ,e infected with ru,ella. 8he rate of fetal infection 6aries according to the ti.ing of the .otherGs infection during $regnancy. Birth defects, howe6er, are .ost li?ely "!&A% in infants infected during the first eight wee?s of $regnancy ",%. Cytome$alo;irus "41*% ,elongs to the her$es 6irus grou$ of infections. It can ,e trans.itted through ,ody secretions, as well as ,y se7ual contactH so.e new,orns which ac0uire 41* through the .otherGs ,reast .il? Infected infants .ay ha6e se6ere $ro,le.s, such as hearing loss, .ental retardation, $neu.onia, he$atitis, or ,lood disorders"/2%. Her es sim le! ;irus II the 6irus enters the infant through his eyes, s?in, .outh, and u$$er res$iratory tract. Infants ,orn with HS* infection, a,out 2 A will ha6e locali2ed infections of the eyes, .outh, or s?in, a,out & A of infected infants will de6elo$ disease s$read throughout the ,ody "disse.inated% within "'-++% days after ,irth "//%. HS*-2 is se7ually trans.itted. Sy.$to.s include genital ulcers or sores. In addition to oral and genital sores, the 6irus can also lead to co.$lications such as infection of the lining of the ,rain and the ,rain itself ".eningoence$halitis% or infection of the eye es$ecially the conEuncti6a and cornea "/.%. M%T'RI%*S %3# M'THO#S Study desi$n 1icro,ial infection during $regnancy can ,e trans.itted to the fetus, resulting in a congenital infection. 8his study was $ro$osed to analy2eDdetect the acti6e infection of H41* ,y #4/ in $regnant wo.en, which are the .aEor contri,utor in congenital H41* s$read. Source of sam les A total of +32 ,lood sa.$les were collected fro. the gynecology wards in Hos$ital and out clinic fro. Ba,ylon city, -ar,ala city and Baghdad city, the $ro6ince large hos$itals where $atients 6isit fro. different areas of Ba,ylon $ro6ince. & .l of 6enous ,lood was ta?en in a sterile tu,e with <I8AH for Nucleic acid e7traction $rocess. 8he sa.$les were di6ided into two ali0uots and were stored at -! J4. A total of +32 wo.en were in6estigated. 4ases were included in the study grou$ de$ending on $re6ious history of ha6ing 3 or .ore $regnancy wastages, intrauterine deaths, $reter. deli6eries, intrauterine growth retardation and une7$lained early neonatal deaths. 4o.$rehensi6e e7a.inations and con6entional la,oratory in6estigations were carried out. Kro. each wo.an & .l of 6enous ,lood was collected in <I8A container with strict ase$tic $recautions. INA was e7tracted fro. these sa.$les using "@<N)1I4 INA 1INI -I8 K)/ BF))IDBA48</IAFD45F85</ 4<FF-@<NAII%. 8he INA was used for genetic e6aluation for 8)/4H infections. #3% e!traction INA was e7tracted in ,iosafety hood ty$e II fro. all ,lood sa.$les with the hel$ of 5ltra scri$t INA e7traction ?it "@<NAII% according to the .anufacturer $rocedure. + Ll ,lood was used for INA e7traction and the $urified e7tracted INA was sus$ended in + Ll of elution ,uffer and stored at -! J4 in the refrigerator. 4.5 Re$ular "CR #4/ reactions were carried out in a ther.al cycler "6iriti 5SA% with 8a0 INA $oly.erase ",ioneer and $ro.ega 5SA%. 8he first round of a.$lification was $erfor.ed with & Ll of e7tracted INA ,y using a forward $ri.er and a re6erse $ri.er. 8he reaction .i7ture for a single reaction consisted of + ( #4/ Buffer- 2. Ll, 1g4l2 "2& .1%- 2.4 Ll, dN8#s "& L1%- +. Ll, forward $ri.er -+. Ll, re6erse $ri.er- +. Ll, dH2)- ++.: Ll, 8a0. INA #oly.Mrase "2 5DLl%- +. L l and e7tracted INA- &. Ll. 8he cycling conditions for regular #4/ were done for get a$$ro$riate descent results. 'lectro horesis #4/ $roducts were electro$horeses in 2.&A agarose gel $re$ared in +( 8B< ,uffer ",oiled for 2 .in in .icrowa6e o6en and cooled to &&J4%, adding ethidiu. ,ro.ide "+ LgD.l% stain and e6aluated under ultra 6iolet light. 8he s$ecific INA $roduct for 8)/4H agents of each sa.$le was deter.ined ,y identifying the s$ecific ,ase $air ",$% a.$lified INA ,ands in co.$arison with the + -,$ INA ladder "#ro.ega, 5SA%, used as INA si2e .ar?er. Results and discussion In this study, wo.en that ha6e no .edical history of high ,lood $ressure "hy$ertension%, dia,etes and ?idney disease were included. A total of +32 wo.en were screened as ,ad o,stetric history "B)H% while 3 as control. A.ongst B)H, INA $ositi6e results ,y #4/H To!o lasma "3:.3:A%, Rubella were also detected in wo.en "2 .4&A%, while "2'.&&A% and "+3.:4A% wo.en were CM( and HS( 6irus $ositi6e, res$ecti6ely, as showed in

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Journal of Biology, Agriculture and Healthcare ISSN 2224-32 ! "#a$er% ISSN 222&- '3( ")nline% *ol.3, No.+!, 2 +3

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table /. Table /: re;alence BOH causati;e a$ents BOH Control Causati;e a$ents No. A No. A 4! 3:.3: ! 2:.:; To!o lasma 2; 2 .4& 4 +3.33 Rubella 3' 2'.&& ; 23.33 Cytome$alo;irus +! +3.:4 ++ 3:.:; Her es sim le! +32 + . 3 + . Total 8he history of the +32 ,ad o,stetric history "B)H% cases consisted of a,ortion in &2 "3'.3'A%, $re.ature la,or in 2' "2+.':A%, early neonatal death in +' "+4.3'A%, congenital .alfor.ation in +4 "+ .:A% and intrauterine death in ! ":. :A%, as table . re6ealed. Table .: Causati;e a$ents of TORCH with different resentation of Obstetric history To!o lasma Rubella Cytome$alo;irus Her es sim le! #atients Total 3o< 3o< 3o< 3o< A,ortion "nN&2% 23 &3.4' : +3.'& ++ 2&.&! 3 :.'! 43 #reter. la,or "nN2'% + &2.:3 2 + .&3 : 3+.&! + &.2: +' <arly neonatal death "nN+'% 4 & . + +2.& 3 3;.& . ! 4ongenital .alfor.ation "nN+4% 2 + . . . . 2 intrauterine death "nN!% 3 & . + +:.:; + +:.:; + +:.:; : High $re6alence was recorded in the age grou$ of 2 to 3' years which was 42. &A of to7o$las.a infections, followed ,y the age grou$ of 3' years which was 3+.!;A with 41* infections, while the age grou$ of 4 to 4' years this for.ed the lowest ratio. Table +: Causati;e a$ents of TORCH with a$e $rou of women with Obstetric history To!o lasma Rubella CM( HS( %$e $rou < O+' years +!.: 2+.4 32. ; +'.4 2 - 2' 42. & 34 ++. + 23.2 3 - 3' 2+.' 3 .3 3+.!; +:.' 4 -4' ++. ; ++.: +&.+4 3:.+ P4' year :.3! 2.; '.'+ 4.4 8he results showed that + $ercent $ositi6e rates were found in all three grou$s with the 8)/4H in first, second and third tri.esters of gestation, res$ecti6ely. 8here was significant difference in 8)/4H infection a.ong the wo.en in the first and second tri.esters of gestation, as $re6iewed in ta,le 4. Table 1: "re;alence of TORCH a$ents amon$ re$nant women accordin$ trimester a$es To!o lasma Rubella Cytome$alo;irus Her es sim le! Trimester a$e No. A No. A No. A No. A /st Trimester .nd Trimester +=d Trimester Total 2& +: ; 4! &2.+ 33.3 +4.: + . ' ++ ; 2; 33.3 4 .; 2&.' + . +3 +; ' 3' 33.3 43.: 23.+ + . +! + ; ++ 3!.' :+.+ . .

"rimers >orward

Re;erse Si?e 6b -7

Table 8: "rimers used to detect TORCH causati;e a$ent To!o lasma Rubella Cytome$alo;irus 8@4 888 @44 4A@ A8@ 8@4 @A@ 448 884 @A@ 44A 8@@ @A4 @A@ A8@ AA 8AA A@@ 4@8 4A 484 @A@ @@4 @AA 4A4 A88 @44 @4A @@4 8@A @88 488 @48 4A8 4A4 4@A 8A4 8A@ @8 @@8 AAA @A @@8 44& 32+ 22'

Her es sim le! A84 4@A A4@ 4A@ 444 4@4 8@ 844 @@4 @@4 A@4 A@@ @8@ 48 3!2

Although .ono$le7 #4/ and real-ti.e assays ha6e considera,le ,enefits in targeting detection of s$ecific

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Journal of Biology, Agriculture and Healthcare ISSN 2224-32 ! "#a$er% ISSN 222&- '3( ")nline% *ol.3, No.+!, 2 +3

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organis.s, they do not necessarily allow detection of the causati6e agent, due to the s$ecificity of the $ri.er sets used. Increasingly, detection of the causati6e agent using .ulti$le7es in res$iratory s$eci.ens "/+@ /1%, gastrointestinal s$eci.ens "/8%, the eye "/0%, conditions causing ly.$hadeno$athy, cere,ros$inal fluid are re$lacing $athogen s$ecific "6ersus clinical conditions s$ecific% detection "/9%. It is o,6ious that .aternal infections show a life-threatening role in $regnancy wastage and their occurrence in $atients with B)H is a significant factor. #ersistence of encysted for.s of to7o$las.a in chronically infected uteri, and their su,se0uent ru$ture during $lacentation lead to infection of the ,a,y in the first tri.ester and often to recurrent .iscarriages. In the $resent study 8. gondii, which is a ?nown etiological agent in recurrent $regnancy wastage was found in +4.::A $regnant wo.en with B)H. 8his is si.ilar to what has ,een re$orted earlier is."/:% 8he high rate of resistance against 8o7o$las.a .ay de$end u$on the en6iron.ent and life style of the $eo$le here. 8o7o$las.a anti,odies are found to ,e higher in a,orters fro. the rural areas, where contact with soil is co..on regardless of whether cats are ?e$t $ets or notC, can ,e true here also."/,% /u,ella is a .ild 6iral infection a.ong children, ne6ertheless can occasionally infect adults. #ri.ary 6iral infection during wo.en $regnancy $ossi,ly caused fetal har.. In our study ru,ella infection was 2 .4&A while other wor?ers re$ort ranging fro. 4 to +;.;;A.".2% 8he incidence of first tri.ester .iscarriage a.ong the teenagers under this study was higher than the &.&A .iscarriage rate o,ser6ed a.ong the teenagers in other $arts of India "Bhalerao et al., +'' %. 8he teenage $regnancy rates re$orted fro. 6arious $arts of the world ranged fro. ! - +4A."./% As 8. gondii, Her$es Si.$le7 *irus infections also ha6e a statistically significant correlation with the incidence of .iscarriages. Both 41* and HS* are well-?nown to ha6e an intrauterine route of trans.ission with significant .ortality and .or,idity. 8he $resent study shows rate of 2'.&&A for 41* in wo.en with B)H. In other studies ranges fro. 3 to +2.'A. "..% It was suggested that $regnancy .ay reacti6ate the latent 6irus leading to further re$roducti6e wastages. Sero$ositi6ity rate for HS* Ig1 a.ong B)H $atients inour study was !.::A, si.ilar to what has ,een re$orted $re6iously. ".+%. )n considering the effect of the $athogens on different grou$s of a,ortions, 8o7o$las.a and 41* were found to ha6e high influence on the Qco.$lete a,ortionC category. References +. Shar.a #, @u$ta 8, @anguly N-, 1ahaEan /4, 1alla N. Increasing 8o7o$las.a sero$ositi6ity in wo.en with ,ad o,stetric history and in new ,orns. Natl 1ed J +'';H+ 9:&-:: 2. 8ur,ad?ar I, 1athur 1, /ele 1. Sero$re6alence of torch infection in ,ad o,stetric history. Indian Journal of 1edical 1icro,iology. 2 3 .*olu.e 2+. Issue 9 2. #age 9 + !-++ 3. Rashodhara #, /a.la7.i BA, Naidu AN, /a.an F. #re6alence of s$ecific Ig1 due to 8o7o$las.a, /u,ella, 4yto.egalo6irus and 4.tracho.oatis infection during $regnancy. Indian J 1ed 1icro,iol 2 +H+'9;'-!2. 4. Newton <. Iiagnosis of $erinatal 8)/4H infections. 4lin ),stet @ynecol +'''H429&'-; . &. /aEendra B Sur$a., 5sha # -a.la?ar, /- -hadse, 1S Sa2i, Suresh * Jalgaon?ar. Serological study for 8)/4H infections in wo.en with ,ad o,stetric history. 2 :. J ),stet @ynecol India *ol. &:, No. + 9 #g 4+-43. :. Syr.is, 1. B., I. 1. Bhiley, 1. 8ho.as, I. 1. 1ac?ay, J. Billia.son, I. J. Sie,ert, 1. I. Nissen, and 8. #. Sloots. A sensiti6e, s$ecific, and cost-effecti6e .ulti$le7 re6erse transcri$tase-#4/ assay for the detection of se6en co..on res$iratory 6iruses in res$iratory sa.$les. 2 4. J. 1ol. Iiagn. :9+2&T+3+. ;. 8e.$leton, -. <., S. A. Scheltinga, 1. K. Beers.a, A. 4. -roes, and <. 4. 4laas. /a$id and sensiti6e .ethod using .ulti$le7 real-ti.e #4/ for diagnosis of infections ,y influen2a A and influen2a B 6iruses, res$iratory syncytial 6irus, and $arainfluen2a 6iruses +, 2, 3, and 4. 2 4. J. 4lin. 1icro,iol. 429+&:4T+&:'. !. 4ruse, J. 1., and /o,ert <. F. Illustrated Iictionary of I..unology. New Ror?9 4/4 #ress"2 3%. '. Newton, < Iiagnosis of $erinatal 8)/4H infections. 4lin ),stet @ynecolH ."+'''% 429&'-; . + . Fewis, /. A. 8orch screen , 4olu.,ia 5ni6ersity #ediatric Kaculty #ractice, NR. /e6iew $ro6ided ,y *eri 1ed Healthcare Networ? "2 ;% . ++. An.ar Ah.ed Iawood AF T 8aie. Serological Study Kor 8)/4H Infections In Bo.en Bith High Ieli6ery /is? Kactors In 1osul. 8i?rit Journal of #ure Science *ol. +& No."+% 2 + +2. Sur$a., /.B., -.la?ar, 5.#., -hadse, /.-., Sa2i, 1.S., Jalgaon?ar, S.*. Serological study for 8)/4H infection in wo.en with ,ad o,stetric history. J ),stet @ynaecol IndiaH "2 :% &:94+-3. +3. 8e.$leton, -. <., S. A. Scheltinga, 1. K. Beers.a, A. 4. -roes, and <. 4. 4laas. 2 4. /a$id and sensiti6e .ethod using .ulti$le7 real-ti.e #4/ for diagnosis of infections ,y influen2a A and influen2a B 6iruses, res$iratory syncytial 6irus, and $arainfluen2a 6iruses +, 2, 3, and 4. J. 4lin. 1icro,iol. 429+&:4T+&:'. +4. 4oiras, 1. 8., J. 4. Aguilar, 1. F. @arcia, I. 4asas, and #. #ere2-Brena. 2 4. Si.ultaneous detection of fourteen res$iratory 6iruses in clinical s$eci.ens ,y two .ulti$le7 re6erse transcri$tion neSted-#4/ assays.

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J. 1ed. *irol. ;294!4T4'&. +&. Beuret, 4. 2 4. Si.ultaneous detection of enteric 6iruses ,y .ulti$le7 real-ti.e /8-#4/. J. *irol. 1ethods ++&9+T!. +:. 4hichili, @. /., S. Ath.anathan, S. Karhatullah, N. @ango$adhyay, S. Jalali, @. #asricha, and S. Shar.a. 2 3. 1ulti$le7 $oly.erase chain reaction for the detection of her$es si.$le7 6irus, 6aricella-2oster 6irus and cyto.egalo6irus in ocular s$eci.ens. 4urr. <ye /es. 2;9!&T' . +;. 1ac?ay, I. 1., 8. @arda., -. <. Arden, S. 1cHardy, I. 1. Bhiley, <. 4risante, and 8. #. Sloots. 2 3. 4o-detection and discri.ination of si7 hu.an her$es6iruses ,y .ulti$le7 #4/-<FAHA. J. 4lin. *irol. 2!92'+T3 2. +!. Uagar A, Bani A, 1asoodi S. Sero$re6alence of to7o$las.osis in wo.en with recurrent a,ortion and neonatal deaths, and its treat.ent outco.e. Ind J #athol 1icro,iol +'''H4294!2-3. +'. 1ladina N, 1ehi?ic @, #asic "2 2%. 8)/4H infections in .others as a cause of neonatal .or,idity. A. 1ed Arh. &4"&-:%9 2;3-2;: 2 . Sood S, #illai #, /aghunath 4 et al. Infection as a cause of s$ontaneous a,ortion with s$ecial reference to 8o7o$las.a gondii, ru,ella 6irus, 41* and 8re$one.a $allidu.. Ind J 1ed 1icro,iol +''4H +292 4- ;. 2+. Bhalerao A/, Iesai S*, Iastur Na and Iaftray SN "+'' %. )utco.e of teenage $regnancy. J. #ostgrad. 1ed. 3:"3%9 +3:-3'. 22. Sen, 1./. Shu?la, B.N. 8uhina BanerEee. #re6alence of Seru. Anti,odies to 8)/4H Infection in and Around *aranasi, Northern India. Journal of 4linical and Iiagnostic /esearch. 2 +2 No6e.,er, *ol-:"'%9 +4!3-+4!&. 23. #ad.a6athy 1, 31angala @owri, 1alini J, 5.a$athy BF, Na6aneeth B*,1ohit Bhatia, Shruthi Harle. Sero$re6alence of 8)/4H Infections and Ad6erse /e$roducti6e )utco.e in 4urrent #regnancy with Bad ),stetric History. J 4lin Bio.ed Sci 2 +3 H 3 "2%H $ :2-;+.

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