Vorremmo ringraziare i tecnici di laboratorio della Clinica Mayo micobatteriologia Laboratorio per il loro aiuto durante lo studio.

Ogni laboratorio deve eseguire una valutazione dei rischi in collaborazione con il loro Comandante istituzionali di sicurezza biologica per determinare il livello appropriato di biosicurezza per la preparazione dell&#39;inoculo e per pipettaggio dell&#39;inoculo nella piastra. Nel nostro laboratorio, utilizziamo livello di biosicurezza 3 laboratorio (BSL3) pratiche fino alla micropiastre vengono inoculati, e sigillato con una guarnizione di plastica adesiva. Queste lastre vengono poi poste all&#39;interno di un sacchetto di plastica, che è caldo anche sigillato. Lettura di incubazione e l&#39;interpretazione dei micropiastra è condotto in un laboratorio di livello di biosicurezza 2 (BSL2). 1. Etichettatura delle materie <ol><li> Etichettare una piastra di agar sangue, tre Middlebrook 7H10 piatti, brodo Middlebrook 7H9, una soluzione salina tra tubo di vetro e perle micropiastra AST con gli identificatori appropriati (per esempio, il nome del paziente, il numero di cartella clinica) nel BSL2. </li><li> Etichetta l&#39;agar sangue e 7H10 placcato come il &quot;controllo della purezza&quot;. Conta delle colonie periodica degli inoculo sono raccomandati per garantire che un&#39;adeguata concentrazione di organismi viene utilizzato nel test. Conta delle colonie, quando eseguite dovranno avere due dei piatti 7H10 etichettati con &quot;conta 1 / 1 colonia&quot; e &quot;contare 1 / 50 colonia&quot;. </li></ol> 2. Tecnologo preparati per andare in BSL3 <ol><li> Appropriati dispositivi di protezione individuale è necessario lavorare in BSL3 e comprende solid-front camice, copricapo, guanti, copriscarpe, e un respiratore. Ciascuna istituzione dovrebbe stabilire il proprio piano di protezione delle vie respiratorie in collaborazione con l&#39;Ufficio Igiene Industriale loro istituzione. Nel nostro laboratorio, abbiamo respiratori diversi disponibili, tra cui respiratori alimentati ad aria purificata (PAPRs) e montato con filtri HEPA, N95 mezza faccia respiratori. </li></ol> 3. Preparazione della cappa di sicurezza biologica <ol><li> Nel BSL3, preparare la cappa di sicurezza biologica (BSC) per la disinfezione della superficie e mettendo un tovagliolo di carta imbevuto di disinfettante circa 6 centimetri dal pannello di sfiato aria. </li><li> Posizionare un contenitore per rifiuti a sinistra, e nephlometer piccole e vortex sulla destra. </li><li> Loop inoculo, pipetors e suggerimenti sono posti accanto al tovagliolo di carta. </li><li> Mettere un rack con l&#39;organismo (in brodo o su terreno solido) e il 0,5 di serie McFarland sul tovagliolo di carta. </li></ol> 4. Calibrazione del nefelometro <ol><li> Posizionare un tubo di vetro salina perla tra nel nefelometro e impostare lo strumento a zero usando il vuoto salina interpolazione. </li><li> Posizionare il 0,5 standard di McFarland nel nefelometro e calibrare secondo le istruzioni del produttore. </li></ol> 5. Preparazione dell&#39;inoculo <ol><li> Lavorare nel colonie raschiare BSC dal terreno solido utilizzando un&#39;ansa sterile nel adeguatamente etichettati salina tubo tra perle di vetro fino a ≥ 0,5 McFarland standard si ottiene. </li><li> Ispezionare visivamente il campione, e confrontare allo standard McFarland. </li><li> Esempio Vortex per 30-60 secondi. </li><li> Lasciare l&#39;inoculo di stabilirsi per 15 minuti (impostare un timer). </li></ol> 6. Inoculazione del micropiastra <ol><li> Regolare l&#39;inoculo con soluzione salina tra brodo usando il nefelometro calibrato al McFarland 0,5 in passi 4.1 e 4.2. Questo deve essere completato entro 30 minuti (secondo le specifiche del produttore). </li><li> Utilizzare un 200 pipetter microlitri e un lungo puntale resistente aerosol per rimuovere 100 l di inoculo dal rd TOP 1 / 3 della soluzione salina tubo tallone interpolazione. Inoculare il brodo Middlebrook 7H9. Lentamente vortice il brodo inoculato 7H9 per 20 secondi. </li><li> Rimuovere la piastra microtiter AST dalla confezione del produttore. NON USARLO se l&#39;essiccante è blu o se la confezione di alluminio è danneggiato. </li><li> Preparare una diluizione 1:50 tubo per l&#39;uso più tardi nella preparazione della diluizione 1:50 pipettando 50 ml di acqua sterile in una provetta sterile. </li><li> Versare cautamente l&#39;inoculo 7H9 brodo in un trogolo. Evitare di generare aerosol. </li><li> Posizionare la micropiastra AST con l&#39;etichetta rivolto verso l&#39;alto. </li><li> Usare un 12-canale pipetter per aggiungere 100 microlitri della inoculo a ciascun pozzetto. </li><li> Preparare le diluizioni per la conta delle colonie: <ol type="a"><li> L&#39;1 / 1 diluizione è preparato dal controllo positivo (H12) con striature 1 loop microlitri sul piatto 7H10 denominata &quot;1 / 1&quot;. </li><li> L&#39;1 / 50 di diluizione è preparati inoculando un loop 1 microlitri dal pozzetto del controllo positivo (H12) e vorticoso nel tubo con 50 ml di tubo di acqua preparata in precedenza. Streak 1 ml di questa diluizione alla piastra etichetta &quot;1 / 50&quot;. </li></ol></li><li> Inoculare le piastre purezza dal trogolo pipettando 50 microlitri su un agar sangue senza anticoagulante e 7H10 piastra e strisciare per l&#39;isolamento. </li><li> Postoun sigillo adesivo sul micropiastra AST. Premere con decisione su ogni bene per assicurare un&#39;adeguata tenuta utilizzando il retro bianco della tenuta. Evitare pieghe. </li><li> Disinfettare la micropiastra strofinando con un tovagliolo di carta imbevuto di un disinfettante tubercolicida e microstrips posto in un sacchetto di plastica e chiudere con una saldatura a caldo o nastro. <ol type="a"><li> Il sacchetto di plastica funge da contenitore secondario per garantire che tutti brodo è contenuto dovrebbe esserci una perdita o fuoriuscita del piatto principale. </li></ol></li></ol> 7. Disinfezione dei materiali e cabina di sicurezza biologica <ol><li> Per evitare di produrre aerosol, attraverso un altro posto sulla parte superiore della vasca inoculato prima di inserirlo delicatamente nel contenitore degli scarti. </li><li> Disinfettare tutti i materiali tra cui nephlometer, vortex, lastre, tubi, ecc strofinando giù con un disinfettante tubercolicida e aspettare il momento opportuno per assicurare la disinfezione secondo le istruzioni del produttore prima di togliere dal BSC. Per esempio, Prospray, il disinfettante tubercolicida usiamo, richiede 15 minuti per uccidere micobatteri. </li><li> Mettere il recipiente scartare in un sacchetto di rischio biologico e sigillare prima di sterilizzazione in autoclave. </li></ol> 8. Incubazione <ol><li> Piastre devono essere incubate con i media verso il basso e si deve prestare attenzione per evitare fuoriuscite o spruzzi liquidi. </li><li> Incubare la micropiastra AST piatto, 7H10 e piastre di agar sangue in un incubatore 35-37 ° C con il 5-10% di CO 2 con 5-10% di CO 2 nel laboratorio BSL2. <ol class="lalpha"><li> Nel nostro laboratorio, questo è accettabile perché la piastra è ben sigillato e dentro un sacchetto di plastica sigillato. </li></ol></li><li> Non più di 2 piastre di microtitolazione AST dovrebbe essere impilati in incubatrice secondo le istruzioni del produttore. </li></ol> 9. Rappresentante risultati Interpretazione dei risultati: Sigillato AST micropiastre possono essere letti manualmente usando uno specchio come un aiuto o con un lettore di piastre automatizzata, ad esempio il Vizion (Diagnostica TREK). Le lastre possono essere esaminata non appena 7 giorni dopo l&#39;inoculazione. Esaminare i pozzetti di controllo della crescita in posizioni H11 e H12 per determinare se sono positivi (cioè, hanno un deposito di cellule sul fondo del pozzo). Se i pozzi di controllo della crescita non sono positivi, reincubare la piastra e riesaminare periodicamente (es., giorno 10, giorno 14) fino a quando i pozzi di controllo della crescita sono positivi. <ol type="a"><li> Specchio leggere: <ol type="i"><li> Luogo sigillato AST micropiastra sulla piattaforma; </li><li> Leggi pozzetti di controllo della crescita dopo 7 giorni di incubazione, se invece è negativo, reincubare AST micropiastra; </li><li> Se i controlli di crescita sono accettabili, quindi leggere il farmaco contenente pozzi. Per per ogni farmaco, registrare la prima diluizione che non ha una crescita confrontando i pozzi della droga con il controllo della crescita. </li><li> La crescita appare come torbidità o come un deposito di cellule in fondo a un pozzo. </li></ol></li><li> Strumento di lettura: seguire le istruzioni del produttore, le istruzioni che seguono sono un esempio di come la lettura sarebbe stato fatto con la piattaforma Vizion: <ol type="i"><li> Log in </li><li> Utilizzare una piastra &quot;MYCOTB&quot; AST software. </li><li> Luogo piatto sigillato con il codice a barre rivolto verso l&#39;utente. La crescita appare come torbidità o come un deposito di cellule in fondo a un pozzo. </li><li> Leggi controlla la crescita, se questo è negativo, reincubare AST micropiastra, se positiva leggere la piastra toccando lo schermo con il primo pozzo di ogni farmaco che non mostra crescita. </li></ol></li></ol> Leggi le piastre purezza. La piastra di agar sangue dovrebbe mostrare alcuna crescita dopo 48 ore e la piastra di purezza 7H10 dovrebbe avere crescita confluente di un tipo di organismo. Se contaminati, il test AST microtiter deve essere ripetuta da una coltura pura. Calcolare la conta delle colonie dal controllo positivo utilizzando la tabella sottostante: Colony Conte chiave <table border="1"><tbody><tr><td> # Colonie contate sulla piastra denominata &quot;1 / 1&quot; </td><td> # Colonie contate sulla piastra denominata &quot;1 / 50&quot; </td><td> Inoculo conteggio delle colonie (ufc / ml) </td></tr><tr><td> &lt;50 </td><td> 0 </td><td> &lt;5 X 10 4 </td></tr><tr><td> 50-100 </td><td> 0-2 </td><td> 5 x 10 4 a 1 x 10 5 </td></tr><tr><td> &gt; 100 </td><td> ≤ 10 </td><td> 1 x 5-5 Ottobre X 10 5 </td></tr><tr><td> &gt; 100 </td><td> &gt; 10 </td><td> &gt; 5 X 10 5 </td></tr></tbody></table> Ci possono essere uscita con l&#39;acido para-aminosalicilico in alcuni ceppi di Mycobacterium tubercolosis. Questo può essere interpretato, cercando in tutti i pozzetti che sono pellet e con il primo pozzo che ha la stessa dimensione del pellet come la diluizione successiva, come il punto finale. La resistenza è rara con questo farmaco. <img src="/files/ftp_upload/3094/3094fig1.jpg" alt="Figura 1"/> Figura 1. Risultati rappresentativi utilizzando la piastra MYCOTB. <table border="1"><tbody><tr><td> Droga abbreviazione </td><td> Droga </td><td> MIC μ / ml </td></tr><tr><td> OFL </td><td> ofloxacina </td><td> 16 </td></tr><tr><td> MXF </td><td> moxifloxacina </td><td> 8 </td></tr><tr><td> RIF </td><td> rifampin </td><td> &gt; 16 </td></tr><tr><td> AMI </td><td> amikacina </td><td> 0,5 </td></tr><tr><td> STR </td><td> streptomicina </td><td> 16 </td></tr><tr><td> RFB </td><td> rifabutina </td><td> 8 </td></tr><tr><td> PAS </td><td> acido p-aminosalicilico </td><td> ≤ 0,5 </td></tr><tr><td> ETH </td><td> etionamide </td><td> 5 </td></tr><tr><td> CYC </td><td> cicloserina </td><td> 4 </td></tr><tr><td> INH </td><td> isoniazide </td><td> 2 </td></tr><tr><td> KAN </td><td> kanamicina </td><td> 1,2 </td></tr><tr><td> EMB </td><td> etambutolo </td><td> 4.0 </td></tr></tbody></table>

<ol>
	<li>Wengenack, N., Hall, L., Labombardi, V., Parrish, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+New+Sensititre+(MYCOTB)+MIC+Plate+for+the+Susceptibility+Testing+of+Mycobacterium+tuberculosis+(Mtb)+to+First+and+Second+Line+Drugs.">Evaluation of the New Sensititre (MYCOTB) MIC Plate for the Susceptibility Testing of Mycobacterium tuberculosis (Mtb) to First and Second Line Drugs.</a> , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HHS+Publication+No.+(CDC)+21-112:+Biosafety+in+Microbiological+and+Biomedical+Laboratories+(BMBL).">HHS Publication No. (CDC) 21-112: Biosafety in Microbiological and Biomedical Laboratories (BMBL).</a> U.S. Department of Health and Human Services Centers for Disease and Preventions. , (2009).</li><li>Morcillo, N., Imperiale, B., Digiulio, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+MGIT+960+and+the+colorimetric-based+method+for+tuberculosis+drug+susceptibility+testing.">Evaluation of MGIT 960 and the colorimetric-based method for tuberculosis drug susceptibility testing.</a> Int J Tuberc Lung Dis. 14, 1169-1175 (2010).</li><li>Bemer, P., Bodmer, T., Munzinger, J., Perrin, M., Vincent, V., Drugeon, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+Evaluation+of+the+MB/BACT+System+for+Susceptibility+Testing+of+Mycobacterium+tuberculosis.">Multicenter Evaluation of the MB/BACT System for Susceptibility Testing of Mycobacterium tuberculosis.</a> J Clin Microbiol. 42, 1030-1034 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SENSITITRE+MYCOTB+Susceptibility+Plate:+For+Mycobacterium+Susceptibility+Testing.">SENSITITRE MYCOTB Susceptibility Plate: For Mycobacterium Susceptibility Testing.</a> Product Insert. , (2010).</li><li>CLSI. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Susceptiblity Testing of Mycobacteria, Nocardia, and Other Aerobic Actinomycetes; Approved Standard. Document M24-A. , (2003).</li><li>Sullivan, N., Sotos, J., Anhalt, K., Allen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+results+from+a+new+TREK+Sensititre+Mycobacterium+tuberculosis+MIC+plate+(MYCOTB).">Preliminary results from a new TREK Sensititre Mycobacterium tuberculosis MIC plate (MYCOTB).</a> , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VersaTREK+MYCO+SUSCEPTIBLITY+KIT.">VersaTREK MYCO SUSCEPTIBLITY KIT.</a> Product insert. , 7115-7160 (2008).</li><li>Parrish, N. M., Carroll, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+the+Clinical+Mycobacteriology+Laboratory+in+the+Diagnosis+and+Management+of+Tuberculosis+in+Low+Prevalance+Settings.">The Role of the Clinical Mycobacteriology Laboratory in the Diagnosis and Management of Tuberculosis in Low Prevalance Settings.</a> J Clin Microbiol. , (2010).</li><li>Kam, K. M., Sloutsky, A., Yip, C. W., Bulled, N., Zingol, M., Espinal, M., Kim, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+critical+concentrations+of+second-line+anti-tuberculosis+drugs+with+clinical+and+microbiological+relevance.">Determination of critical concentrations of second-line anti-tuberculosis drugs with clinical and microbiological relevance.</a> Int J Tubercl Lung Dis. 14, 282-288 (2010).</li><li>Woods, G. L., Warren, N. G., Inderlied, C. B., Murray, P. R., Barron, E. J., Jorgensen, J. H., Landry, M. L., Pfaller, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+Test+Methods:+Mycobacteria,+Nocardia,+and+Other+Actinomycetes.">Susceptibility Test Methods: Mycobacteria, Nocardia, and Other Actinomycetes.</a> Manual of Clinical Microbiology. , 1223-1247 (2007).</li><li>Bergmann, J. S., Woods, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+ESP+Culture+System+II+for+Testing+Susceptibilities+of+Mycobacterium+tuberculosis+Isolates+to+Four+Primary+Antituberculosis+Drugs.">Evaluation of the ESP Culture System II for Testing Susceptibilities of Mycobacterium tuberculosis Isolates to Four Primary Antituberculosis Drugs.</a> J Clin Microbiol. 36, 2940-2943 (1998).</li></ol>

Un esempio della micropiastra AST che controlla la crescita positiva è illustrato nella figura 1 e il punto finale MIC (mg / ml) per ogni farmaco è cerchiato. Uno studio condotto nel nostro laboratorio hanno indicato che il metodo microtiter AST piastra è equivalente al metodo gold standard proporzione agar per testare la sensibilità del Mycobacterium tuberculosis. Più veloce comunicazione dei risultati è stato ottenuto con micropiastra AST microtiter rispetto al metodo tradizionale proporzione agar con l&#39;interpretazione finale per piastra AST microtitolazione e proporzione metodo agar a 14 e 21 giorni, respectively.The AST micropiastra ha il vantaggio di test prima e farmaci di seconda linea contemporaneamente che può aiutare a prevenire ritardi eccessivi con ceppi resistenti. La piastra microtiter AST è anche molto più facile da impostare e leggere che l&#39;APM e sia uno specchio manuale e un sistema semi-automatico come il Vizion può essere utilizzato per leggere le piastre. La disponibilità di un valore di MIC per testare la resistenza ai farmaci TB può fornire preziose informazioni ai medici nuove rispetto al valore tradizionale concentrazione critica, soprattutto per quelli isolati con suscettibilità &quot;borderline&quot;.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> AST micropiastre e sigillo adesivo </td><td> Diagnostica TREK, Cleveland Ohio </td><td> MYCOTB </td></tr><tr><td> McFarland 0,5 di serie </td><td> Diagnostica TREK, Cleveland, Ohio </td><td> E1041 </td></tr><tr><td> Middlebrook 7H10 piastre </td><td> Pescatore </td><td></td></tr><tr><td> Piastre di agar sangue </td><td> Pescatore </td><td></td></tr><tr><td> Sterile loop microlitri 1 </td><td> Pescatore </td><td></td></tr><tr><td> Anse sterili 10 L </td><td> Pescatore </td><td></td></tr><tr><td> Sensititre Saline-Tween perline di vetro brodo </td><td> Diagnostica TREK, Cleveland, Ohio </td><td> 27143 </td></tr><tr><td> 7H9 OADC brodo </td><td> Diagnostica TREK, Cleveland, Ohio </td><td> T3440 </td></tr><tr><td> Puntali lungo </td><td> Pescatore </td><td></td></tr><tr><td> Trogoli sterile </td><td> Diagnostica TREK, Cleveland, Ohio </td><td></td></tr><tr><td> M. tuberculosis </td><td> ATCC </td><td> 27294 </td></tr><tr><td> Pipeters: 1000, 200, 20μL </td><td> Pescatore </td><td></td></tr><tr><td> Consigli 1000, 20, 20 l </td><td> Pescatore </td><td></td></tr><tr><td> Multicanale Pipetter </td><td> Pescatore </td><td></td></tr><tr><td> Incubatore di CO% 05-10 febbraio </td><td> Pescatore </td><td></td></tr><tr><td> Sacchetti di plastica, piccoli, CO 2 permeabile </td><td> Poly plastica, Delano, PA </td><td></td></tr><tr><td> TB disinfettante (Come ad esempio ProSpray C-60) </td><td> Certol internazionale, Commerce City, CO </td><td> PSC60/32 </td></tr><tr><td> Specchio Box Reader </td><td> Pescatore </td><td></td></tr></tbody></table>

antimicrobial susceptibility testing of mycobacterium tuberculosis complex for first and second line drugs by broth dilution in a microtiter plate format

La rilevazione rapida della resistenza antimicrobica è importante nel tentativo di controllare l&#39;aumento della resistenza Mycobacterium tuberculosis (Mtb). Test di suscettibilità antimicrobica (AST) di Mtb è stata tradizionalmente eseguita con il metodo di agar proporzione o testando macrobroth su uno strumento come il BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (Diagnostica TREK, Cleveland, OH) o BacT / ALERT (bioMérieux, Hazelwood, MO). Il metodo delle proporzioni agar, mentre considerato lo standard &quot;oro&quot; di AST, è laborioso e richiede il calcolo della resistenza effettuando la conta delle colonie su agar contenenti droga rispetto al drug-free agar. Se vi è una crescita ≥ 1% sul farmaco contenente medio rispetto al drug-free medio, l&#39;organismo è considerato resistente a quel farmaco. I metodi macrobroth richiedono strumentazione e rompere punto di prova (&quot;critical&quot;) concentrazioni del farmaco per i farmaci di prima linea (isoniazide, etambutolo, rifampicina e pirazinamide). Il metodo qui descritto è disponibile in commercio in un formato da 96 pozzetti micropiastra [MYCOTB (Diagnostica TREK)] e contiene concentrazioni crescenti di 12 antimicrobici usati per il trattamento della tubercolosi inclusi sia prima (isoniazide, rifampicina, etambutolo) e farmaci di seconda linea (amikacina, cicloserina, etionamide, kanamicina, moxifloxacina, ofloxacina, acido para-aminosalicilico, rifabutina, e streptomicina). Pirazinamide, un farmaco di prima linea, non è incluso nella piastra microtiter a causa della sua necessità di condizioni di prova acida. Vantaggi del sistema micropiastra includono sia la facilità di configurazione e veloce girare intorno a tempo (14 giorni) rispetto al rapporto tradizionale agar (21 giorni). Inoltre, la piastra può essere impostato da inoculo preparato utilizzando brodo o terreno solido. Poiché il formato micropiastra è nuova e da Mtb presenta sfide di sicurezza unici nel laboratorio, questo protocollo si descrive come impostare in modo sicuro, incubare e leggere la micropiastra.

Watch this Scientific Journal Video about Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format at J

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

Test di suscettibilità antimicrobica di Mycobacterium tuberculosis è impegnativo, ma fondamentale per la cura del paziente. Questo formato micropiastra offre test di 12 farmaci antimicobatterici e fornisce minima concentrazione inibente (MIC) valori, che sarà di aiuto nel trattamento appropriato.

Test di suscettibilità antimicrobica del complesso Mycobacterium tuberculosis delle droghe prima e seconda linea per diluizione brodo in un formato micropiastra

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

antimicrobial-susceptibility-testing-mycobacterium-tuberculosis

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Immunology and Infection

38.1K Views.  Mayo Clinic. Test di suscettibilità antimicrobica di Mycobacterium tuberculosis è impegnativo, ma fondamentale per la cura del paziente. Questo formato micropiastra offre test di 12 farmaci antimicobatterici e fornisce minima concentrazione inibente (MIC) valori, che sarà di aiuto nel trattamento appropriato.

Video: Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

A 96 Beh microtest piastra a base di Metodo per la Formazione di monitoraggio e test di suscettibilità antifungini Candida albicans Biofilm

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

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Immunologia e infezioni

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Preparazione del campione di<em&gt; Mycobacterium tuberculosis</em&gt; Estratti di studi di risonanza magnetica nucleare metabolomica

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Crescita Mycobacterium tuberculosis I biofilm

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Dimostrazione di un multi-resistente ai farmaci<em&gt; Mycobacterium tuberculosis</em&gt; Amplificazione Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Un riferimento brodo di microdiluizione per Dalbavancina<em&gt; In Vitro</em&gt; Test della Sensibilità di batteri che crescono aerobico

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Un 3D Polmone umano tessuto modello per studi funzionali su<em&gt; Mycobacterium tuberculosis</em&gt; Infezione

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Sistema per l&#39;efficacia e la citotossicità Proiezione di inibitori di targeting intracellulare<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Analisi di 18FDG PET/CT Imaging come strumento per studiare l'infezione di tubercolosi del micobatterio e trattamento in primati Non umani

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Tubercolosi del micobatterio in topi con fluorescenza enzima Reporter di imaging

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Modellazione di tubercolosi in Zebrafish adulto infettato di Mycobacterium marinum

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...