{"id":1557,"date":"2018-11-11T13:33:08","date_gmt":"2018-11-11T12:33:08","guid":{"rendered":"http:\/\/www.newslab.sk\/2018\/11\/11\/moznosti-laboratornej-diagnostiky-infekcii-vyvolanych-clostridium-difficile\/"},"modified":"2018-11-11T13:35:27","modified_gmt":"2018-11-11T12:35:27","slug":"moznosti-laboratornej-diagnostiky-infekcii-vyvolanych-clostridium-difficile","status":"publish","type":"post","link":"https:\/\/www.newslab.sk\/en\/moznosti-laboratornej-diagnostiky-infekcii-vyvolanych-clostridium-difficile\/","title":{"rendered":"Laboratory diagnostic possibilities for Clostridium difficile infections"},"content":{"rendered":"

*All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf attachment at the end of the paper.<\/span> <\/strong><\/p>\n

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\u00davod<\/strong><\/p>\n

Clostridium difficile <\/em>je grampozit\u00edvna anaer\u00f3bna bakt\u00e9ria (obr\u00e1zky <\/em><\/strong>1, 2) <\/em><\/strong>be\u017ene sa vyskytuj\u00faca v pr\u00edrode, povrchov\u00fdch a odpadov\u00fdch vod\u00e1ch a s r\u00f4znou frekvenciou tie\u017e v tr\u00e1viacom trakte \u013eud\u00ed a zvierat. Za nepriazniv\u00fdch podmienok tvor\u00ed metabolicky inakt\u00edvne formy \u2013 sp\u00f3ry, ktor\u00e9 s\u00fa v\u00fdrazne odoln\u00e9 proti vysok\u00fdm teplot\u00e1m a \u010fal\u0161\u00edm fyzik\u00e1lnym vplyvom,\u00a0 taktie\u017e proti v\u00e4\u010d\u0161ine dezinfek\u010dn\u00fdch prostriedkov (vr\u00e1tane alkoholov\u00fdch prepar\u00e1tov pou\u017e\u00edvan\u00fdch na dezinfekciu pl\u00f4ch, povrchov a r\u00fak). To zvy\u0161uje riziko ich dlhodob\u00e9ho pretrv\u00e1vania v prostred\u00ed, \u010fal\u0161ieho \u0161\u00edrenia a pravdepodobnos\u0165 koloniz\u00e1cie nemocni\u010dn\u00fdch pacientov, ktor\u00e1 m\u00f4\u017ee by\u0165 nasledovan\u00e1 rozvojom infekcie. C. difficile <\/em>nepatr\u00ed medzi invaz\u00edvne patog\u00e9ny, na za\u010diatku ochorenia iba adheruje na sliznicu hrub\u00e9ho \u010dreva. Jeho patogenita vypl\u00fdva zo schopnosti tvori\u0165 ve\u013emi \u00fa\u010dinn\u00e9 exotox\u00edny pova\u017eovan\u00e9 za hlavn\u00e9 faktory virulencie. Toxig\u00e9nne kmene produkuj\u00fa vo vegetat\u00edvnej f\u00e1ze termolabiln\u00e9 prote\u00ednov\u00e9 tox\u00edny A a B, ktor\u00e9 sa do prostredia uvo\u013e\u0148uj\u00fa rozpadom buniek. Obidva tox\u00edny p\u00f4sobia synergicky a ich produkcia je spojen\u00e1 s po\u0161koden\u00edm epitelu \u010dreva i hlb\u0161\u00edch \u0161trukt\u00far \u010drevnej steny. G\u00e9ny k\u00f3duj\u00face tox\u00edn A (g\u00e9n tcdA<\/em>) a tox\u00edn B (g\u00e9n tcdB<\/em>) s\u00fa umiestnen\u00e9 na tzv. lokuse patogenity (pathogenity locus \u2013 PaLoc<\/em>) s ve\u013ekos\u0165ou 19,6 kb, a to spolu s troma \u010fal\u0161\u00edmi g\u00e9nmi reguluj\u00facimi expresiu tox\u00ednov. Tox\u00edn A je enterotox\u00edn, vyvol\u00e1va apopt\u00f3zu enterocytov a aktivuje z\u00e1palov\u00fa reakciu. Tox\u00edn B je cytotox\u00edn, sp\u00f4sobuje de\u0161trukciu buniek po\u0161koden\u00fdch tox\u00ednom A. Okrem tox\u00ednov A a B produkuj\u00fa niektor\u00e9 kmene aj tret\u00ed, tzv. bin\u00e1rny tox\u00edn. G\u00e9ny k\u00f3duj\u00face bin\u00e1rny tox\u00edn (g\u00e9ny cdtA <\/em>a cdtB<\/em>) s\u00fa umiestnen\u00e9 mimo PaLoc <\/em>lokus(8). Jeho \u00faloha v patogen\u00e9ze ochorenia nie je zatia\u013e celkom objasnen\u00e1, ale pr\u00edtomnos\u0165 tox\u00ednov A a B s\u00fa\u010dasne s bin\u00e1rnym tox\u00ednom zhor\u0161uje priebeh ochorenia a s\u0165a\u017euje jeho lie\u010dbu (v\u00e4\u010d\u0161ina epidemick\u00fdch PCR ribotypov produkuje aj bin\u00e1rny tox\u00edn). Netoxig\u00e9nne kmene C. difficile <\/em>(neprodukuj\u00face tox\u00edn A a B) s\u00fa nepatog\u00e9nne a nepredstavuj\u00fa ohrozenie ani pre vn\u00edmav\u00e9ho jedinca(9).<\/p>\n

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Patogen\u00e9za ochorenia<\/h3>\n

Infekcie vyvolan\u00e9 C. difficile <\/em>(Clostridium difficile <\/em>infections \u2013 CDI) vykazuj\u00fa \u0161irok\u00e9 spektrum klinick\u00fdch prejavov od miernej hna\u010dky cez pseudomembran\u00f3znu kolit\u00eddu a\u017e po toxick\u00fd megakolon s \u00famrtnos\u0165ou 30 \u2013 50 %(1). Ochorenie naj\u010dastej\u0161ie vznik\u00e1 v s\u00favislosti s lie\u010dbou \u0161irokospektr\u00e1lnymi antibiotikami (ATB), ktor\u00e9 eliminuj\u00fa norm\u00e1lnu \u010drevn\u00fa fl\u00f3ru, pr\u00edp. aj ako nozokomi\u00e1lna infekcia. Na prepuknutie CDI s\u00fa potrebn\u00e9 dve podmienky: strata ochrannej \u010drevnej fl\u00f3ry (tzv. koloniza\u010dn\u00e1 rezistencia) a infekcia toxig\u00e9nnymi kme\u0148mi C. difficile<\/em>. Mechanizmus \u00fa\u010dinku oboch tox\u00ednov na bunky \u010drevn\u00e9ho epitelu je odli\u0161n\u00fd. Tox\u00edn A (TcdA) je typick\u00fd enterotox\u00edn, ktor\u00fd po\u0161kodzuje bunky \u010drevn\u00e9ho epitelu a sp\u00f4sobuje kumul\u00e1ciu tekut\u00edn v \u010dreve, \u010do m\u00e1 za n\u00e1sledok vznik vodnat\u00fdch, niekedy hemoragick\u00fdch hna\u010diek. Tox\u00edn B (TcdB) svoj\u00edm cytotoxick\u00fdm \u00fa\u010dinkom vedie k nekr\u00f3ze napadnut\u00fdch buniek. Na \u010drevnej sliznici vznikaj\u00fa po\u010detn\u00e9, typick\u00e9 ostrov\u010dekovit\u00e9 (mapovit\u00e9) ulcer\u00e1cie pokryt\u00e9 pablanami, dobre vidite\u013en\u00e9 pri endoskopickom vy\u0161etren\u00ed(17). P\u00f4soben\u00edm tox\u00ednu B na hladk\u00fa svalovinu a vegetat\u00edvne nervstvo v stene hrub\u00e9ho \u010dreva doch\u00e1dza k spomaleniu a\u017e zastaveniu peristaltiky a rozvoju ilea, \u010d\u00edm z\u00e1rove\u0148 vznik\u00e1 ide\u00e1lne prostredie na mno\u017eenie mikroorganizmov. Tak\u00e9to po\u0161kodenie m\u00f4\u017ee vy\u00fasti\u0165 a\u017e do perfor\u00e1cie \u010dreva. Termin\u00e1lne \u0161t\u00e1dium choroby sa vyzna\u010duje enormnou distenziou hrub\u00e9ho \u010dreva (megakolon) a postupnou stratou bari\u00e9rovej funkcie \u010drevnej sliznice s n\u00e1sledn\u00fdm prienikom \u010drevn\u00fdch bakt\u00e9ri\u00ed do hlb\u0161\u00edch tkan\u00edv a potom do krvi. Vznik\u00e1 sepsa, naj\u010dastej\u0161ie gramnegat\u00edvna, s r\u00fdchlym rozvojom septick\u00e9ho \u0161oku s vysokou \u00famrtnos\u0165ou(1). Diagn\u00f3za tohto ochorenia sa stanovuje na z\u00e1klade anamn\u00e9zy, klinick\u00e9ho obrazu, endoskopick\u00e9ho n\u00e1lezu pseudomembran\u00f3znej kolit\u00eddy a laborat\u00f3rneho d\u00f4kazu tox\u00ednov C. difficile <\/em>v stolici.<\/p>\n

Epidemiol\u00f3gia ochorenia<\/h3>\n

CDI maj\u00fa celosvetovo a rovnako na Slovensku narastaj\u00facu tendenciu v\u00fdskytu. Asi 90 % v\u0161etk\u00fdch foriem CDI vznik\u00e1 v nadv\u00e4znosti na predch\u00e1dzaj\u00facu alebo s\u00fabe\u017en\u00fa antimikrobi\u00e1lnu terapiu, hlavne syst\u00e9movo pod\u00e1van\u00fdmi antibiotikami. Ich n\u00e1rast s\u00favis\u00ed aj s vy\u0161\u0161\u00edm vekom pacientov (nad 65 rokov), polymorbiditou, dlhodobou hospitaliz\u00e1ciou, \u010dast\u00fdm u\u017e\u00edvan\u00edm antisekre\u010dn\u00fdch liekov a inhib\u00edtorov prot\u00f3novej pumpy, so z\u00e1krokmi na hrubom \u010dreve, s hypomobilitou \u010dreva a mnoh\u00fdmi \u010fal\u0161\u00edmi faktormi. O narastaj\u00facej incidencii sved\u010dia aj \u00fadaje poskytnut\u00e9 \u00daradom verejn\u00e9ho zdravotn\u00edctva SR (graf 1)<\/em><\/strong>. K\u00fdm v roku 2011 bolo v SR hl\u00e1sen\u00fdch 136 pr\u00edpadov ochoren\u00ed vyvolan\u00fdch C. difficile<\/em>, v roku 2017 ich po\u010det st\u00fapol na 2 604 pr\u00edpadov.\u00a0 Re\u00e1lny v\u00fdskyt infekci\u00ed je v\u0161ak vy\u0161\u0161\u00ed. Mnoho pr\u00edpadov\u00a0\u00a0 je nezachyten\u00fdch alebo poddiagnostikovan\u00fdch, ch\u00fdba \u0161tandardiz\u00e1cia poskytovan\u00fdch \u00fadajov. Z uveden\u00e9ho vypl\u00fdva d\u00f4le\u017eitos\u0165 venova\u0165 patri\u010dn\u00fa pozornos\u0165 diagnostike CDI. C. difficile <\/em>je be\u017enou s\u00fa\u010das\u0165ou \u010drevn\u00e9ho mikrobi\u00f3mu u 50 % det\u00ed do 2 rokov a u 2 \u2013 5 % be\u017enej popul\u00e1cie. Je naj\u010dastej\u0161\u00edm vyvol\u00e1vate\u013eom nozokomi\u00e1lnej hna\u010dky. Ve\u013ek\u00e1 \u010das\u0165 infikovan\u00fdch pacientov zost\u00e1va asymptomatick\u00fdch a st\u00e1va sa najv\u00e4\u010d\u0161\u00edm rezervo\u00e1rom mikr\u00f3ba(4). Odhaduje sa, \u017ee 7 \u2013 11 % hospitalizovan\u00fdch pacientov,\u00a0 5 \u2013 7 % pacientov soci\u00e1lnych zariaden\u00ed\u00a0\u00a0 a menej ako 2 % ambulantn\u00fdch pacientov s\u00fa nosi\u010dmi kme\u0148ov\u00a0difficile <\/em>produkuj\u00facich tox\u00edny. Na zvy\u0161uj\u00facej sa incidencii ochoren\u00ed sp\u00f4soben\u00fdch C. difficile <\/em>sa podie\u013ea vysok\u00e1 preskripcia antibiot\u00edk, ako aj objavenie sa hypervirulentn\u00fdch kme\u0148ov(5). Na prelome rokov 2002 \u2013 2003 boli v Severnej Amerike op\u00edsan\u00e9 epid\u00e9mie CDI vyvolan\u00e9 kme\u0148om 027\/NAP1\/B1. Tento hypervirulentn\u00fd kme\u0148 je charakteristick\u00fd vysokou produkciou tox\u00ednov (A aj B), schopnos\u0165ou tvori\u0165 viac sp\u00f3r ako kontroln\u00e9 kmene (\u013eah\u0161ie \u0161\u00edrenie v nemocni\u010dnom prostred\u00ed) a rezistenciou na fluorochinol\u00f3ny(11). Spom\u00ednan\u00fd ribotyp (RT) 027 sa n\u00e1sledne roz\u0161\u00edril aj do z\u00e1padnej Eur\u00f3py. V Spojenom kr\u00e1\u013eovstve v rokoch 2008 \u2013 2009 sp\u00f4sobil a\u017e polovicu v\u0161et-k\u00fdch nozokomi\u00e1lnych CDI. N\u00e1sledne boli op\u00edsan\u00e9 \u010fal\u0161ie epidemick\u00e9 RT, napr. RT 001, 014, 017, 078, 176. Pod\u013ea v\u00fdsledkov celogen\u00f3mov\u00e9ho sekvenovania je RT 176 (ozna\u010dovan\u00fd ako 027-like) geneticky bl\u00edzky pr\u00edbuzn\u00fd hypervirulentn\u00e9mu RT 027 a vykazuje aj podobn\u00e9 fenotypov\u00e9 vlastnosti(16). Produkuje bin\u00e1rny tox\u00edn pova\u017eovan\u00fd za prognostick\u00fd marker pre vznik\u00a0rekurentn\u00fdch epiz\u00f3d CDI(15) a klinick\u00fd priebeh ochorenia vyvolan\u00fd t\u00fdmto RT je rovnako z\u00e1va\u017en\u00fd ako pri RT 027(10). Identifik\u00e1cia izol\u00e1tov C. difficile <\/em>a znalos\u0165 lok\u00e1lnej epidemiologickej situ\u00e1cie je d\u00f4le\u017eit\u00e1 pre zavedenie opatren\u00ed zamedzuj\u00facich \u0161\u00edreniu epidemiologicky v\u00fdznamn\u00fdch kme\u0148ov v r\u00e1mci nemocn\u00edc, resp. oddelen\u00ed.<\/p>\n

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Terapia<\/h3>\n

Lie\u010debn\u00e9 postupy CDI s\u00fa zakotven\u00e9 v slovensk\u00fdch odpor\u00fa\u010daniach na diagnostiku a lie\u010dbu kolit\u00eddy sp\u00f4sobenej C. difficile<\/em>(7). V\u0161eobecn\u00e9 odpor\u00fa\u010dania v lie\u010dbe \u013eahk\u00fdch foriem CDI s\u00fa: ukon\u010denie ATB lie\u010dby, ktor\u00e1 infekciu vyvolala (ak je to mo\u017en\u00e9), rehydrat\u00e1cia, bezzvy\u0161kov\u00e1 di\u00e9ta. Pri n\u00edzkej hladine album\u00ednu u pacienta je potrebn\u00e1 jeho parenter\u00e1lna substit\u00facia. Lieky tlmiace \u010drevn\u00fa peristaltiku (spazmolytik\u00e1, opi\u00e1ty) s\u00fa kontraindikovan\u00e9. Na lie\u010dbu stredne \u0165a\u017ek\u00fdch, \u0165a\u017ek\u00fdch foriem a rekurentn\u00fdch foriem CDI sa odpor\u00fa\u010da medikament\u00f3zna antibiotick\u00e1 lie\u010dba: metronidazol, vankomyc\u00edn a nov\u00e9 ma- krocyklick\u00e9 antibiotikum fidaxomic\u00edn. Ako \u010fal\u0161ie mo\u017enosti podpornej lie\u010dby sa vyu\u017e\u00edvaj\u00fa probiotik\u00e1 a v poslednom obdob\u00ed aj tzv. fek\u00e1lna bakterioterapia. Transplant\u00e1cia stolice je ofici\u00e1lne odpor\u00fa\u010danou met\u00f3dou pri lie\u010dbe viacpo\u010detn\u00fdch rekurenci\u00ed CDI s vysok\u00fdm percentom \u00faspe\u0161nosti, ktor\u00fdm prekon\u00e1va aj existuj\u00face antibiotick\u00e9 re\u017eimy(13).<\/p>\n

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Laborat\u00f3rna diagnostika CDI<\/h3>\n

Na mikrobiologick\u00fa diagnostiku CDI sa dnes vyu\u017e\u00edva viacero met\u00f3d. Ka\u017ed\u00e1 z nich m\u00e1 in\u00fa mieru citlivosti a \u0161pecifickosti, preto sa odpor\u00fa\u010da ich kombin\u00e1cia. Cielen\u00e9 mikrobiologick\u00e9 vy\u0161etrenie stolice je indikovan\u00e9 u pacientov s klinick\u00fdm podozren\u00edm na CDI. Naopak, nie je indikovan\u00e9 u pacientov s formovanou stolicou a be\u017ene sa nerob\u00ed ani u det\u00ed do 1 \u2013 2 rokov veku.<\/p>\n

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D\u00f4kaz antig\u00e9nu <\/strong>C. difficile<\/em><\/strong><\/p>\n

Glutam\u00e1tdehydrogen\u00e1za (GDH) je \u0161pecifick\u00fd antig\u00e9n \u2013 exoenz\u00fdm, produkovan\u00fd v\u0161etk\u00fdmi kme\u0148mi C. difficile <\/em>(toxig\u00e9nnymi aj netoxig\u00e9nnymi). <\/em>D\u00f4kaz antig\u00e9nu je mo\u017en\u00fd priamo zo stolice, \u0161pecifickos\u0165 testu je 80 \u2013 100 %. M\u00e1 vysok\u00fa negat\u00edvnu predikt\u00edvnu hodnotu (NPV), t. j. negat\u00edvny v\u00fdsledok s ve\u013ekou pravdepodobnos\u0165ou vylu\u010duje mo\u017enos\u0165 klostr\u00eddiovej infekcie. Test v\u0161ak neodl\u00ed\u0161i toxig\u00e9nne kmene od netoxig\u00e9nnych. Jeho nev\u00fdhodou je aj mo\u017enos\u0165 skr\u00ed\u017eenej reakcie s in\u00fdmi druhmi Clostridium spp. <\/em>pr\u00edtomn\u00fdmi v stolici (falo\u0161ne pozit\u00edvny v\u00fdsledok v 20 %). GDH m\u00f4\u017ee by\u0165 stanoven\u00e1 samostatne alebo je s\u00fa\u010das\u0165ou testov dokazuj\u00facich z\u00e1rove\u0148 tox\u00edny C. difficile<\/em>.<\/p>\n

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D\u00f4kaz tox\u00ednov <\/strong>C. difficile <\/em><\/strong>(A\/B)<\/strong><\/p>\n

Na d\u00f4kaz tox\u00ednov A\/B sa pou\u017e\u00edvaj\u00fa diagnostick\u00e9 s\u00fapravy zalo\u017een\u00e9 na princ\u00edpe enz\u00fdmovej imunoanal\u00fdzy alebo imunochromatografie. Na trhu je v s\u00fa\u010dasnosti k dispoz\u00edcii cel\u00fd rad komer\u010dn\u00fdch s\u00faprav s r\u00f4znou citlivos\u0165ou a \u0161pecifickos\u0165ou. Ich v\u00fdhodou je cenov\u00e1 dostupnos\u0165, r\u00fdchlos\u0165, vysok\u00e1 \u0161pecifickos\u0165, jednoduch\u00e9 vyhotovenie s jednozna\u010dnou interpret\u00e1ciou d\u00f4kazu toxigenity kme\u0148a. Test m\u00e1 vysok\u00fa pozit\u00edvnu predikt\u00edvnu hodnotu (PPV), t. j. pozit\u00edvny v\u00fdsledok s ve\u013ekou pravdepodobnos\u0165ou dokazuje klostr\u00eddiov\u00fa infekciu. Nev\u00fdhodou je relat\u00edvne n\u00edzka citlivos\u0165 (90 %), \u010do v niektor\u00fdch pr\u00edpadoch vedie k falo\u0161ne negat\u00edvnym v\u00fdsledkom.<\/p>\n

Anaer\u00f3bna kultiv\u00e1cia<\/h3>\n

Anaer\u00f3bna kultiv\u00e1cia C. difficile <\/em>je pomerne n\u00e1ro\u010dn\u00e1 a zd\u013ahav\u00e1 (2-3 dni). Na kultiv\u00e1ciu sa pou\u017e\u00edvaj\u00fa selekt\u00edvne p\u00f4dy\u00a0\u00a0 s obsahom cefoxit\u00ednu a cykloser\u00ednu, ktor\u00e9 potl\u00e1\u010daj\u00fa sprievodn\u00fa fl\u00f3ru. Konfirm\u00e1cia kultiva\u010dn\u00e9ho n\u00e1lezu je mo\u017en\u00e1 mikroskopicky (orienta\u010dne), biochemicky (komer\u010dn\u00fdmi anaerotestami), latexov\u00fdm aglutina\u010dn\u00fdm testom, pr\u00edp. pou\u017eit\u00edm nov\u0161\u00edch techn\u00edk (PCR \u2013 polymerase chain reaction, MALDI TOF MS \u2013 matrix assisted laser desorption ionization-time of flight mass spectrometry). Kultiva\u010dn\u00e9 vy\u0161etrenie sa pova\u017euje za nevyhnutn\u00e9 pri v\u0161etk\u00fdch vzork\u00e1ch stolice, pri ktor\u00fdch bola potvrden\u00e1 pozitivita GDH vr\u00e1tane tox\u00edn-negat\u00edvnych stol\u00edc. Na d\u00f4kaz tox\u00ednov z narastenej kult\u00fary mo\u017eno pou\u017ei\u0165 imunochemick\u00e9 testy alebo PCR. V\u00fdhodou kultiva\u010dn\u00e9ho vy\u0161etrenia je jeho vysok\u00e1 v\u00fd\u0165a\u017enos\u0165, mo\u017enos\u0165 ulo\u017eenia izolovan\u00fdch kul- t\u00far, stanovenia citlivosti na antibiotik\u00e1 (ATB), pr\u00edp. molekul\u00e1rna typiz\u00e1cia (epidemiologick\u00e9 \u00fa\u010dely).<\/p>\n

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D\u00f4kaz cytotoxicity na tkanivov\u00fdch kult\u00farach<\/h3>\n

D\u00f4kaz cytotoxicity na tkanivov\u00fdch kult\u00farach m\u00e1 sk\u00f4r historick\u00fd v\u00fdznam. T\u00e1to met\u00f3da je n\u00e1ro\u010dn\u00e1 na \u010das, laborat\u00f3rne vybavenie a interpreta\u010dn\u00e9 sk\u00fasenosti, jej nev\u00fdhodou je aj riziko falo\u0161nej pozitivity. Senzitivita sa uv\u00e1dza 94 \u2013 100 %. Cytotox\u00edn mo\u017eno dok\u00e1za\u0165 v stolici alebo z izolovan\u00e9ho kme\u0148a\u00a0difficile <\/em>na tkanivov\u00fdch kult\u00farach fibroblastov, kde doch\u00e1dza k cytopatick\u00e9mu efektu.<\/p>\n

 <\/p>\n

PCR \u2013 d\u00f4kaz g\u00e9nov toxicity<\/h3>\n

Zavedenie PCR met\u00f3d, \u010di u\u017e ako skr\u00edningov\u00fdch alebo konfirma\u010dn\u00fdch met\u00f3d, do diagnostiky CDI je eur\u00f3psky trend. Vyzna\u010duj\u00fa sa vysokou citlivos\u0165ou (99 \u2013 100 %) a \u0161pecifickos\u0165ou, v\u00fdznamne skracuj\u00fa \u010das z\u00edskania v\u00fdsledku. Hodnota PPV je tu paradoxne zn\u00ed\u017een\u00e1, preto\u017ee PCR je tak\u00e1 citliv\u00e1, \u017ee vo vzorke dok\u00e1\u017ee i mal\u00e9 mno\u017estvo toxig\u00e9nnych C. difficile <\/em>pr\u00edtomn\u00fdch pri be\u017enej koloniz\u00e1cii, nerozl\u00ed\u0161i teda koloniz\u00e1ciu od infekcie(14). Dnes m\u00e1me k dispoz\u00edcii komer\u010dn\u00e9 s\u00fapravy (na princ\u00edpe realtime PCR) na d\u00f4kaz g\u00e9nu pre tvorbu tox\u00ednu B, g\u00e9nu pre bin\u00e1rny tox\u00edn, s\u00fapravy na detekciu del\u00e9ci\u00ed typick\u00fdch pre niektor\u00e9 epidemick\u00e9 PCR ribotypy (napr. 027, 176). PCR m\u00e1 v\u00fdznam najm\u00e4 pri verifik\u00e1cii \u0165a\u017ek\u00fdch foriem CDI a pri potvrden\u00ed nejasn\u00fdch v\u00fdsledkov predch\u00e1dzaj\u00facich testov.<\/p>\n

 <\/p>\n

Typiz\u00e1cia <\/strong>C. difficile <\/em><\/strong>na epidemiologick\u00e9 \u00fa\u010dely<\/strong><\/p>\n

Medzi met\u00f3dy nadstavbovej diagnostiky patr\u00ed ribotypiz\u00e1cia a toxinotypiz\u00e1cia, ktor\u00fdch z\u00e1kladom je PCR a makrore\u0161trik\u010dn\u00e1 anal\u00fdza pulznou g\u00e9lovou elektrofor\u00e9zou(2). Pri ribotypiz\u00e1cii C. difficile <\/em>doch\u00e1dza k amplifik\u00e1cii \u00faseku medzi g\u00e9nmi pre 16S a 23S rRNA (tzv. Intergenic Spacer Region). <\/em>Medzi jednotliv\u00fdmi ribotypmi je rozdiel v po\u010dte pr\u00edtomn\u00fdch alel t\u00fdchto g\u00e9nov aj v d\u013a\u017ekach \u00fasekov medzi alelami(6). Ka\u017ed\u00fd RT tvor\u00ed \u0161pecifick\u00fd elektroforetick\u00fd profil, pod\u013ea ktor\u00e9ho je n\u00e1sledne identifikovan\u00fd. Na ur\u010denie konkr\u00e9tneho RT s\u00fa z\u00edskan\u00e9 elektroforeogramy porovn\u00e1van\u00e9 s rak\u00faskou webovou datab\u00e1zou https:\/\/webribo.ages.at\/. <\/a>Celkovo bolo objaven\u00fdch viac ne\u017e 800 PCR ribotypov a 24 toxinotypov.<\/p>\n

 <\/p>\n

Odpor\u00fa\u010dan\u00fd diagnostick\u00fd postup<\/h3>\n

Z\u00e1kladn\u00fd diagnostick\u00fd algoritmus vych\u00e1dza z d\u00f4kazu GDH alebo d\u00f4kazu g\u00e9nu\/g\u00e9nov pre produkciu tox\u00ednov pomocou PCR a v pr\u00edpade pozitivity n\u00e1slednej detekcie tox\u00ednov A\/B imunoenzymatickou met\u00f3dou (EIA)(3). Hovor\u00edme o tzv. 2-stup\u0148ovom diagnostickom algoritme. GDH negat\u00edvne vzorky mo\u017eno interpretova\u0165 ako definit\u00edvne negat\u00edvne na pr\u00edtomnos\u0165 C. difficile <\/em>a nie je potrebn\u00e9 ich \u010falej testova\u0165. Ak je test GDH pozit\u00edvny, je nutn\u00e9 potvrdi\u0165 alebo vyl\u00fa\u010di\u0165 toxigenitu kme\u0148a C. difficile <\/em>(d\u00f4kaz tox\u00ednov). V pr\u00edpade pozitivity GDH a negativity tox\u00ednov A\/B je interpret\u00e1cia nejednozna\u010dn\u00e1. M\u00f4\u017ee \u00eds\u0165 o pr\u00edtomnos\u0165 netoxig\u00e9nneho kme\u0148a alebo falo\u0161ne negat\u00edvny v\u00fdsledok podmienen\u00fd obmedzenou senzitivitou met\u00f3dy na d\u00f4kaz tox\u00ednov. V tomto pr\u00edpade s\u00fa potrebn\u00e9 \u010fal\u0161ie laborat\u00f3rne vy\u0161etrenia ako PCR alebo kultiv\u00e1cia s n\u00e1sledn\u00fdm overen\u00edm toxigenity izolovan\u00e9ho kme\u0148a. Hovor\u00edme o tzv. 3-stup\u0148ovom diagnostickom algoritme. Vzh\u013eadom na rozdielnu citlivos\u0165 r\u00f4znych pou\u017e\u00edvan\u00fdch met\u00f3d je pod\u013ea s\u00fa\u010dasn\u00fdch odpor\u00fa\u010dan\u00ed preferovan\u00e1 kombin\u00e1cia minim\u00e1lne dvoch alebo viacer\u00fdch testov. Hlavn\u00fdm cie\u013eom pou\u017eitia dopl\u0148uj\u00facich met\u00f3d je z\u00edskanie \u010do najspo\u013eahlivej\u0161ieho v\u00fdsledku, teda vyl\u00fa\u010denie alebo potvrdenie pr\u00edtomnosti toxikog\u00e9nneho kme\u0148a C. difficile<\/em>. Pr\u00edklad algoritmu kombinuj\u00faceho r\u00f4zne metodiky je uveden\u00fd v sch\u00e9me 1<\/em><\/strong>(7). Pri z\u00e1va\u017en\u00fdch form\u00e1ch CDI a z epidemiologick\u00fdch d\u00f4vodov je vhodn\u00e1 kultiv\u00e1cia stolice a n\u00e1sledn\u00e1 molekul\u00e1rna typiz\u00e1cia izolovan\u00e9ho kme\u0148a. Interpret\u00e1ciu laborat\u00f3rnych n\u00e1lezov treba v\u017edy hodnoti\u0165 v s\u00favislosti s klinick\u00fdm n\u00e1lezom u konkr\u00e9tneho pacienta, s aktu\u00e1lnou epidemiologickou situ\u00e1ciou a ostatn\u00fdmi laborat\u00f3rnymi parametrami.<\/p>\n

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Materi\u00e1l a metodika<\/h3>\n

Na\u0161a \u0161t\u00fadia prebiehala od m\u00e1ja do septembra 2017. Celkovo 481 hna\u010dkovit\u00fdch stol\u00edc bolo vy\u0161etren\u00fdch 3-stup\u0148ov\u00fdm diagnostick\u00fdm algoritmom. V prvom kroku boli v\u0161etky vzorky testovan\u00e9 na pr\u00edtomnos\u0165 GDH kvalitat\u00edvnym imunochromatografick\u00fdm testom Rapid-Viditest C. difficile <\/em>Ag (Vidia). V druhom kroku boli v\u0161etky GDH pozit\u00edvne vzorky analyzovan\u00e9 na pr\u00edtomnos\u0165 tox\u00ednov A\/B pomocou imunochromatografick\u00e9ho testu RIDA\u00aeQUICK Clostridium difficile <\/em>Toxin A\/B (R-Biopharm). Vo v\u0161etk\u00fdch GDH pozit\u00edvnych vzork\u00e1ch stolice bola z\u00e1rove\u0148 zalo\u017een\u00e1 anaer\u00f3bna kultiv\u00e1cia po predch\u00e1dzaj\u00facom \u201ealkoholovom \u0161oku\u201c: vzorka stolice zmie\u0161an\u00e1 s 96 % etanolom v pomere 1 : 1, po d\u00f4kladnom premie\u0161an\u00ed na vortexe je inkubovan\u00e1 pri laborat\u00f3rnej teplote asi 30 min\u00fat a\u017e\u00a01 hodinu. N\u00e1sledne sa 1-2 kvapky suspenzie inokuluj\u00fa na selekt\u00edvny agar pre C. difficile <\/em>(Brazier, Oxoid) a v anaer\u00f3bnej atmosf\u00e9re kultivuj\u00fa 48 \u2013 72 hod\u00edn pri teplote 35 \u2013 37 \u00b0C. Vykultivovan\u00e9 suspektn\u00e9 kol\u00f3nie C. difficile <\/em>boli identifikovan\u00e9 pomocou hmotnostnej spektrometrie MALDI-TOF MS. Izol\u00e1- ty C. difficile <\/em>vykultivovan\u00e9 z GDH pozit\u00edvnych a tox\u00edn-negat\u00edvnych vzoriek boli znovu testovan\u00e9 na pr\u00edtomnos\u0165 tox\u00ednov A\/B (toxig\u00e9nna kultiv\u00e1cia \u2013 TC) rovnak\u00fdm imunochromatografick\u00fdm testom ako v pr\u00edpade stanovenia tox\u00ednov v stoli- ci (3. diagnostick\u00fd stupe\u0148). Molekul\u00e1rna anal\u00fdza kme\u0148ov identifikovan\u00fdch ako C. difficile <\/em>bola vykonan\u00e1 v spolupr\u00e1ci s \u00dastavom lek\u00e1rskej mikrobiol\u00f3gie FN v Motole v Prahe, \u010cesk\u00e1 Republika. Zah\u0155\u0148ala stanovenie g\u00e9nov pre produkciu tox\u00ednov A\/B (tcdA\/tcdB<\/em>), bin\u00e1rneho tox\u00ednu (cdtA, cdtB<\/em>) a ribotypiz\u00e1ciu izol\u00e1tov C. difficile<\/em>. Pr\u00edtomnos\u0165 g\u00e9nov pre produkciu tox\u00ednov bola stanoven\u00e1 pomocou multiplex PCR(12).<\/p>\n

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Odber<\/strong> biologick\u00e9ho<\/strong> materi\u00e1lu<\/strong> a transport do laborat\u00f3ria<\/strong> Na mikrobiologick\u00e9 vy\u0161etrenie stolice na pr\u00edtomnos\u0165 C. di<\/em>fficile <\/em>je potrebn\u00e9 odobra\u0165 minim\u00e1lne 2 ml stolice do sterilnej n\u00e1dobky. Stolica m\u00e1 by\u0165 spracovan\u00e1 a vy\u0161etren\u00e1 do dvoch hod\u00edn, preto\u017ee tox\u00edny s\u00fa pomerne nestabiln\u00e9, r\u00fdchlo sa rozpad\u00e1vaj\u00fa a v\u00fdsledkom m\u00f4\u017ee by\u0165 falo\u0161n\u00e1 negativita. Ak nemo\u017eno vy\u0161etri\u0165 stolicu hne\u010f po odbere, je vhodn\u00e9 vzorku uchova\u0165 pri chladni\u010dkovej teplote (asi 5 \u00b0C), vtedy je stabilita tox\u00ednu zabezpe\u010den\u00e1 na 48 hod\u00edn. Na dlhodob\u00e9 uchov\u00e1vanie vzoriek treba vzorku stolice hlboko zmrazi\u0165 na \u201370 \u00b0C.<\/p>\n

 <\/p>\n

V\u00fdsledky<\/h3>\n

V priebehu 5 mesiacov bolo vy\u0161etren\u00fdch celkovo 481 hna\u010dkovit\u00fdch stol\u00edc z 3 nemocn\u00edc. Preh\u013ead jednotliv\u00fdch oddelen\u00ed, z ktor\u00fdch poch\u00e1dzali vy\u0161etrovan\u00e9 vzorky, je uveden\u00fd v tabu\u013eke 1<\/em><\/strong>. Z celkov\u00e9ho po\u010dtu vy\u0161etren\u00fdch stol\u00edc bolo 104 vzoriek GDH pozit\u00edvnych (21,6 %). Po odstr\u00e1nen\u00ed ne\u00faspe\u0161ne kultivovan\u00fdch, duplicitn\u00fdch, resp. triplicitn\u00fdch vzoriek ostalo\u00a0 v s\u00fabore 66 vzoriek (66 pacientov). Izol\u00e1ty C. difficile <\/em>z t\u00fdchto stol\u00edc boli podroben\u00e9 molekul\u00e1rnej typiz\u00e1cii. Z po\u010dtu 66 vzoriek bolo 58 (87,9 %) toxig\u00e9nnych (pr\u00edtomn\u00e9 g\u00e9ny tcdA <\/em>a tcdB<\/em>), z nich 6 izol\u00e1tov malo navy\u0161e g\u00e9ny pre bin\u00e1rny tox\u00edn (cdtA, cdtB<\/em>). Zvy\u0161n\u00fdch 8 vzoriek zo s\u00faboru (12,1 %) bolo netoxig\u00e9nnych, t. j. nebola v nich preuk\u00e1zan\u00e1 pr\u00edtomnos\u0165 \u017eiadneho z g\u00e9nov pre produkciu tox\u00ednov. Pomocou 3- stup\u0148ov\u00e9ho diagnostick\u00e9ho algoritmu a kombin\u00e1ciou \u010fal\u0161\u00edch met\u00f3d (imunochromatografick\u00e9 stanovenie tox\u00ednov a detekcia toxigenity met\u00f3dou PCR) bolo zisten\u00e9, \u017ee z po\u010dtu 58 tox\u00ednovo pozit\u00edvnych stol\u00edc malo 35 vzoriek pozit\u00edvny tox\u00edn priamo zo stolice a v 23 vzork\u00e1ch (tox\u00edn-negat\u00edvnych zo stolice) a\u017e toxig\u00e9nna kultiv\u00e1cia potvrdila pozitivitu tox\u00ednov (tabu\u013eka 2)<\/em><\/strong>.<\/p>\n

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Z\u00e1ver<\/h3>\n

R\u00fdchla a v\u010dasn\u00e1 diagnostika C. difficile <\/em>je d\u00f4le\u017eit\u00e1 nielen pre lie\u010dbu samotn\u00e9ho pacienta, ale aj z d\u00f4vodu prevencie nozokomi\u00e1lnych n\u00e1kaz. Aj ke\u010f je mikrobiologick\u00e9 vy\u0161etrenie pre konfirm\u00e1ciu CDI k\u013e\u00fa\u010dov\u00e9, nesl\u00fa\u017ei na kontrolu \u00fa\u010din- nosti lie\u010dby ani ako argument pre izola\u010dn\u00e9 opatrenia, ak u\u017e u pacienta nie je hna\u010dka pr\u00edtomn\u00e1. Hlavn\u00fdm krit\u00e9riom vylie\u010denia CDI je \u00fastup klinick\u00fdch pr\u00edznakov, nie negativita mikrobiologick\u00e9ho vy\u0161etrenia. Pozit\u00edvne v\u00fdsledky klasick\u00fdch a molekul\u00e1rnych vy\u0161etren\u00ed m\u00f4\u017eu pretrv\u00e1va\u0165 dlhodobo po ukon\u010den\u00ed lie\u010dby (koloniz\u00e1cia C. difficile<\/em>) a nie s\u00fa d\u00f4vodom na \u010fal\u0161iu ATB terapiu. Aj v tomto pr\u00edpade je d\u00f4le\u017eit\u00fd klinick\u00fd stav pacienta. Neodpor\u00fa\u010da sa ani opakovan\u00e9 vy\u0161etrenie v intervale krat\u0161om ako 7 dn\u00ed u pacientov, u ktor\u00fdch predch\u00e1dzaj\u00face vy\u0161etrenie na C. difficile <\/em>bolo negat\u00edvne, s v\u00fdnimkou situ\u00e1ci\u00ed, ak d\u00f4jde k progresii ochorenia a boli vyl\u00fa\u010den\u00e9 aj in\u00e9 pr\u00ed\u010diny(3). Na\u0161e v\u00fdsledky ukazuj\u00fa, \u017ee ani 2-stup\u0148ov\u00fd diagnostick\u00fd algoritmus nie je v mnoh\u00fdch pr\u00edpadoch posta\u010duj\u00faci. Takmer 40 % GDH pozit\u00edvnych vzoriek v na\u0161om s\u00fabore bolo ur\u010den\u00fdch ako toxig\u00e9nne a\u017e pou\u017eit\u00edm 3-stup\u0148ov\u00e9ho diagnostick\u00e9ho algoritmu. Preto je potrebn\u00e9 zv\u00e1\u017ei\u0165 jeho zavedenie a\/alebo kombin\u00e1ciu viacer\u00fdch met\u00f3d aj do rutinnej laborat\u00f3rnej praxe. Rie\u0161enie problematiky CDI v nemocni\u010dn\u00fdch zariadeniach si vy\u017eaduje komplexn\u00fd pr\u00edstup vr\u00e1tane adekv\u00e1tnej \u0161pecifickej ATB terapie zameranej na redukciu najrizikovej\u0161\u00edch skup\u00edn ATB (chinol\u00f3ny, \u0161irokospektr\u00e1lne cefalospor\u00edny, potenciovan\u00e9 aminopenicil\u00edny), spr\u00e1vnu indik\u00e1ciu laborat\u00f3rneho vy\u0161etrenia, dodr\u017eiavanie hygienicko-epidemiologick\u00fdch opatren\u00ed na zamedzenie \u0161\u00edrenia sp\u00f3r, d\u00f4sledn\u00fa izol\u00e1ciu v\u0161etk\u00fdch infikovan\u00fdch pacientov s CDI, ale hlavne d\u00f4sledn\u00e9 dodr\u017eiavanie bari\u00e9rovej o\u0161etrovate\u013eskej techniky v\u0161etk\u00fdmi zainteresovan\u00fdmi zdravotn\u00edckymi pracovn\u00edkmi.<\/p>\n

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LITERAT\u00daRA<\/strong><\/p>\n

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      6. Voth ED, Ballard DJ, Jimmy Clostridium difficile <\/em>Toxins: Mechanism of Action and Role in Disase. Clinical Microbiology Reviews 2005; 18(2): 247-263.<\/li>\n<\/ol>\n

         <\/p>\n","protected":false},"excerpt":{"rendered":"

        *All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf attachment at the end of the paper.   \u00davod Clostridium difficile je grampozit\u00edvna anaer\u00f3bna bakt\u00e9ria (obr\u00e1zky 1, 2) be\u017ene sa vyskytuj\u00faca v pr\u00edrode, povrchov\u00fdch a odpadov\u00fdch vod\u00e1ch a s r\u00f4znou frekvenciou tie\u017e v tr\u00e1viacom trakte \u013eud\u00ed a<\/p>\n","protected":false},"author":7,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_mi_skip_tracking":false,"footnotes":""},"categories":[292],"tags":[993,995,989,990,404,563,994,991,996,992],"class_list":["post-1557","post","type-post","status-publish","format-standard","hentry","category-microbiology","tag-clostridium-difficile-infections","tag-glutamatdehydrogenase","tag-glutamatdehydrogenaza-en","tag-infekcie-vyvolane-clostridium-difficile-en","tag-laboratorna-diagnostika-en","tag-laboratory-diagnostics","tag-pseudomembranous-colitis","tag-pseudomembranozna-kolitida-en","tag-toxigenic-strain","tag-toxigenny-kmen-en","typ_clanku-original-work"],"acf":{"abstrakt":"

        Clostridium difficile with toxin production is the most common cause of nosocomial gastrointestinal infections. Clostridium difficile infection can manifest as mild diarrhea, pseudomembranous colitis but also as a life-threatening illness accompanied by paralytic ileus developing into secondary sepsis. The disease is generated in relation with a broad-spectrum antibiotic therapy which eliminates normal gut mikrobiota. Transmission of Clostridium difficile is done by spores following the faecal-oral route. Early diagnosis of Clostridium difficile infections
        \nis essential for proper initiation of an antibiotics therapy and also for anti-epidemic measures which prevent spore spreading in hospitals.<\/p>\n

        Keywords:<\/strong> Clostridium difficile infections, pseudomembranous colitis, laboratory diagnostics, glutamatdehydrogenase,
        \ntoxigenic strain<\/p>\n","casopis":[{"ID":1513,"post_author":"7","post_date":"2018-11-05 11:53:53","post_date_gmt":"2018-11-05 10:53:53","post_content":"

          \r\n \t
        • Genetically important aberrations in patients with mye-lodysplastic syndrome and laboratory methods of their detection<\/li>\r\n \t
        • Molecular \u2013 genetic diagnostics of Human Papillomavirus (HPV) and monitoring of HPV patients<\/li>\r\n \t
        • Laboratory diagnostic possibilities for Clostridium difficile infections<\/li>\r\n \t
        • Chorangiosis of Placenta - Disorder of Unclear Etiology (Case Report and Overview of Current Knowledge)<\/li>\r\n \t
        • Circulating tumor DNA and its utilization as marker with prognostic, predictive and diagnostic value in patients with oncological diseases<\/li>\r\n<\/ul>","post_title":"newsLab","post_excerpt":"","post_status":"publish","comment_status":"closed","ping_status":"closed","post_password":"","post_name":"newslab-2","to_ping":"","pinged":"","post_modified":"2018-11-05 11:57:18","post_modified_gmt":"2018-11-05 10:57:18","post_content_filtered":"","post_parent":0,"guid":"http:\/\/www.newslab.sk\/casopis\/newslab-2\/","menu_order":0,"post_type":"casopis","post_mime_type":"","comment_count":"0","filter":"raw"}],"strana":"86","upload_clanok":{"ID":1555,"id":1555,"title":"Mo\u017enosti laborat\u00f3rnej diagnostiky infekci\u00ed vyvolan\u00fdch Clostridium difficile","filename":"Mo\u017enosti-laborat\u00f3rnej-diagnostiky-infekci\u00ed-vyvolan\u00fdch-Clostridium-difficile.pdf","filesize":317058,"url":"https:\/\/www.newslab.sk\/wp-content\/uploads\/2018\/11\/Mo\u017enosti-laborat\u00f3rnej-diagnostiky-infekci\u00ed-vyvolan\u00fdch-Clostridium-difficile.pdf","link":"https:\/\/www.newslab.sk\/en\/moznosti-laboratornej-diagnostiky-infekcii-vyvolanych-clostridium-difficile\/moznosti-laboratornej-diagnostiky-infekcii-vyvolanych-clostridium-difficile-2\/","alt":"","author":"7","description":"","caption":"","name":"moznosti-laboratornej-diagnostiky-infekcii-vyvolanych-clostridium-difficile-2","status":"inherit","uploaded_to":1557,"date":"2018-11-11 12:18:51","modified":"2018-11-11 12:18:51","menu_order":0,"mime_type":"application\/pdf","type":"application","subtype":"pdf","icon":"https:\/\/www.newslab.sk\/wp-includes\/images\/media\/document.png"}},"_links":{"self":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts\/1557","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/users\/7"}],"replies":[{"embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/comments?post=1557"}],"version-history":[{"count":0,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts\/1557\/revisions"}],"wp:attachment":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/media?parent=1557"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/categories?post=1557"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/tags?post=1557"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}