Bu çalışma hibe RC3 AI089106 altında Ulusal Sağlık Enstitüleri (NIH) tarafından desteklenmiştir. MDR-TB nükleik asitler Tropikal Hastalıklar (TDR), Cenevre, İsviçre Araştırma ve Eğitim için Birleşmiş Milletler Çocuk Fonu / Birleşmiş Milletler Kalkınma Programı / Dünya Bankası / Dünya Sağlık Örgütü Özel Programı tarafından sağlanmıştır. Biz prototip tahlilde dahil spesifik genlerin ve mutasyonların rehberlik için Hastalık Kontrol ve Önleme Merkezleri ABD Dr Tom Shinnick teşekkür ederim.

Evrensel PCR önlemleri takip laboratuarları için, birkaç amplifikasyon mikrodizinleri ve substrat başına contalar bulunmaktadır ve burada anlatıldığı gibi, bir toplu kapta aynı anda tüm büyütme mikrodizinleri yıkamak için operasyonel daha etkilidir. Başka yerde 30,33 bildirildiği gibi sarf biçimleri, tamamen kapalı, kapalı Amplikon testinde post-amplifikasyon mikroarray yıkama adımları gerçekleştirmek için kullanılabilir. 1.. Kurulum <ol><li> Özü ve tercih edilen bir yöntem ile, uygun koşullar altında biyo-güvenlik numuneden nükleik asitleri saflaştırmak. Gereksinimi nükleik asitler asimetrik, multipleks PCR amplifikasyonu için yeterli saflıkta olmasıdır. </li><li> Dekontaminasyon çözümleri ile yüzeyleri ve ekipmanı silerek çalışma alanını hazırlayın. </li><li> Buz veya soğuk blokta güçlendirme reaktifleri yerleştirin. </li><li> Amplifikasyon ana karışımı için bir steril 1.5 ml mikrofuge&#39;de tüp etiketleyin ve bir steril 0.2 ml mikrofuge&#39;de tu etiketHer bir numune için olabilir. </li><li> Reaktifler, kısa bir süre girdap çözülmüş ve mini santrifüjünde bir darbe spin tüpün dibine içeriğini toplamak sonra. Taq polimeraz vorteks etmeyin. Hafifçe karıştırın ve mini-santrifüj bir darbe spin ile takip tüp fiske. </li><li> Tablo 1&#39;de gösterilen başına örnek reaksiyon hacimleri kullanılarak amplifikasyon master karışımı hazırlayın. Olası pipetleme yanlışlıklar için hesap için, işlenen örneklerin toplam sayısından en az bir fazla reaksiyon hacmini hazırlamak. Ana karışım ilave Taq polimeraz, üzerinde ve muliplex PCR tampon maddesi ile sağlanmaktadır ne yukarıda içerdiğini unutmayın. Sadece diğer tüm maddeleri birleştirildikten sonra ana karıştırmak için amplifikasyon / inhibisyon denetimi ekleyin ve stok reaktif tüpler depolama döndü. </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Reaktif </td><td> Per-Numune Hacmi (ul) </td><td> &lt;strong&gt; Final [] </td></tr><tr><td> HotStar Taq Plus ile Mulitplex PCR Tampon </td><td> 25 </td><td> 1x </td></tr><tr><td> Sığır serum albümini (BSA) </td><td> 0.55 </td><td> 0.6 mg / ml </td></tr><tr><td> Formamide </td><td> 3.8 </td><td> 7.6% </td></tr><tr><td> Ek Taq polimeraz </td><td> 0.8 </td><td> birim / ul (4 adet toplam) </td></tr><tr><td> MDR-TB primer karışımı </td><td> 15.75 </td><td> - </td></tr><tr><td> RNase içermeyen H2O </td><td> 2.1 </td><td> - </td></tr><tr><td> Amplifikasyon / önlenmesi, kontrol </td><td> 1 </td><td> 5,0 fg / ul </td></tr><tr><td> Toplam </td><td> 49 </td><td></td></tr></tbody></table> Tablo 1. ÇİD-TB amplifikasyon mikroarray master miks bileşimi. <ol start="7"><li> Vortex ana karışımı ve mini santrifüjünde bir darbe spin tüpün dibine içeriğini toplamak. </li><li> Ana karışımı 49 ul ve M. 1 ul birleştirin yukarıdaki aşama 1.4 &#39;den örnek tüplerinin her birine tüberküloz DNA. Bir dış, hiç şablon kontrol (NTC), M. yerine moleküler biyoloji dereceli su 1 ul kullanımı tüberküloz DNA örneği. Vortex ve darbe-spin bitmiş tüp (ler). </li></ol> 2.. Amplifikasyon mikrodizileri yükleyin <ol><li> Mikrotertibi kendisi dokunmayın dikkatli olmak, kendi microarrays merkezi üzerine her ana mix / numunenin 48 ul ekleyin. </li><li> Mikroarray odasında herhangi bir hava kabarcığı değil tuzak için dikkatli olmak, mikroarray conta ve mühür üstüne bir kapak kayma yerleştirin. Hava kabarcıkları olumsuz amplifikasyon verimliliğini etkileyebilirve eserler veya sonraki mikroarray görüntüde gürültü yaratabilir. </li></ol> 3.. Termal Bisiklete binme <ol><li> Bir kez tüm mikroarray&#39;ler düz blok termal döngü örnek, yer yüzeylerde yüklenir. Isıtmalı kapak seçeneği &quot;Kapalı&quot; açık olduğundan emin olun. Akonni Biyosistem elde Ekipmanları zaten kullanıma ölçütlere olacaktır. Aksi takdirde, düz blok termal döngü (ler) tüm mikrotertip yüzeyler üzerinde (ler) üniforma, tutarlı ısıtma sağlamak doğrulamak için çok önemlidir. </li><li> , Termal döngü yazılımı açın uygun programı seçin, reaksiyon hacmi için &quot;50 ul&quot; girin ve Çalışmayı başlatmak. Burada anlatılan Termal döngü protokol oluşur: <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Termal Bisiklet Adımlar </td></tr><tr><td> 1 </td><td> 88 ° C </td><td> 5 dakika </td></tr><tr><td> 2 </td><td> 88 ° C </td><td> 30 sn </td></tr><tr><td> 3 </td><td> 55 ° C </td><td> 1 dakika </td></tr><tr><td> 4 </td><td> 65 ° C </td><td> 30 sn </td></tr><tr><td> 5 </td><td> 50 devir için adımlar (2-4) tekrarlayın </td></tr><tr><td> 6 </td><td> 65 ° C </td><td> 3 dakika </td></tr><tr><td> 7 </td><td> 55 ° C </td><td> 3 saat </td></tr></tbody></table></li><li> Toplam termal döngü sayısı ve 55 ° C amplifikasyon sonrası bekleme süresi gerekli ya da belirli bir deney için uygun kullanıcı, değiştirebilir ki bağımsız değişkenler. MDR-TB ana karışım, asimetrik (ağırlıklı olarak tek şeritli) amplikonları oluşturduğu için, başka türlü bir geleneksel, üslü PCR protokolü için kullanılan olabilir daha fazla ısı döngüsü dahil etmek genellikle yararlıdır. </li><li> Termal bisiklet ve melezleme programı tamamlandığında, programını sona amplifikasyon mikrodizinleri kaldırmak, yazılımı kapatın ve termal döngü (ler) kapatın.</li></ol> 4.. Yıkama ve Kuru <ol><li> % 0.1 Triton X-100 yıkama tamponu, ve en az 250 ml deiyonize veya Milli-Q dereceli su her iki konteyner - aynı anda 24 alt tabakalara kadar yıkanması için, en az 250 ml 1x SSPE hazırlamak. Yıkama tamponu önceden hazırlanabilir. </li><li> Dikkatle düz uç forseps kullanarak her amplifikasyon mikroarray odasından kapak kayma ve contayı çıkarın. Bu adım, fiziksel olarak zarar jel eleman dizilerin büyük risk var olduğu noktadır. Çoğaltılan nükleik asitlerin yayılmasını önlemek için uygun PCR kontaminasyonu denetimlerini kullanın çalışma ortamında. </li><li> Bir histoloji slayt tutucu mikroküme yüzeylerde yerleştirin ve yıkama tamponu içeren yıkama kutusu içine tüm slaytlar yerleştirin. Bir kapak ile konteyner örtün. </li><li> Yavaşça sallanarak, oda sıcaklığında 10 dakika boyunca mikrodizileri yıkayın. </li><li> Microarrays üç deiyonize iki ardışık yıkar kez her veya DipMilli-Q kaliteli su ve hava kuru. </li></ol> 5.. Görüntüleme Asimetrik MDR-TB primer karışımı Cy3-etiketli amplikonları oluşturur. Jel eleman mikroarrayler görüntüleme Cy3 yeteneğine sahip herhangi bir standart mikroarray görüntüleyicisi ile görüntülü olabilir. Aşağıdaki görüntüleme yöntemidir Akonni tarafından sağlanan ÇİD-TB ana karışımı, Dx2100 alan taşınabilir görüntüleme cihazı ve otomatik analiz yazılımı için özeldir. <ol><li> Görüntüleyici gelen ethernet kablosunun bilgisayara bağlı olduğundan emin olun. </li><li> Görüntüleyici açın. Güç anahtarı cihazın arka panelinde yer almaktadır. Kamera açıkken görüntüleyici önündeki mavi LED yanacaktır. </li><li> Bilgisayarı açın. </li><li> Bilgisayar masaüstünde, çift otomatik görüntü analiz yazılımı başlatmak için yazılım simgesini tıklatın. </li><li> Kamera kamera ile bağlantı kurmak için yazılım için bekleyin. Bağlantı kurulduktan sonra, analiz düğmesi kullanılabilir hale gelir, vemesajı &quot;Başarılı Connection&quot; ekranın sol alt köşesinde görünecektir. </li><li> Mikrodizi kullanıcıdan uzağa bakan ve objektif lens doğru ile kurutuldu, amplifikasyon mikrodizisinin yerleştirin. Mikrodizi yanlış lens ve kamera ile ilgili yönde ise, otomatik analiz yazılımı floresan görüntü üzerinde bir ızgara yer başarısız olur. </li><li> Erişim kapağını kapatın. Bir önizleme görüntüsü bilgisayar ekranında mevcut olacaktır. </li><li> Açılır menüden ÇİD-TB analiz komut dosyası seçin ve (milisaniye) istenilen pozlama süresini girin. Jel eleman güçlendirme mikrodizilerde tipik bir başlangıç ​​noktası 100-500 msn. </li><li> Doymuş piksel kırmızı Preview Image görüntülenir. Bireysel noktalar içinde doymuş piksel sayısını en aza indirmek için pozlama süresini ayarlayın. </li><li> Floresan görüntü elde etmek ve analiz etmek için analiz tıklayın. Yazılım otomatik olarak yerleştirecektirGörüntü üzerine ÇİD-TB tahlil özgü ızgara, entegre yoğunluğu ve arka plan değerleri ayıklamak ve ilaç direnci ve genotip sonuçları rapor. Otomatik analizi, genellikle birkaç saniye sürer. Çizik, toz, floresan eserler veya mikroarray özellikleri fiziksel zarar içeren zorlu görüntülerde, yazılım analizi tamamlamak için 1 dakika kadar gerekebilir. </li><li> Analiz tamamlandıktan sonra, Kaydet üzerine tıklayın bir dosya adı hedef klasör, türünü seçin ve Tamam &#39;ı tıklatın. İsterseniz yatan mikroarray veri bir. Xls dosyası olarak kaydedilir ve off-line için ihraç edilebilir, analiz eder. </li><li> Analiz tamamlandıktan sonra, görüntüleyici gelen büyütme mikrotertibi kaldırmak ve sonraki analizi başlatmak için Yeni tıklayın. </li><li> Veri toplama bittiğinde, yazılımı kapatın bilgisayarı kapatın ve görüntüleyici kapatın. </li></ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diagnostic+performance+of+the+GenoType+MTBDRplus+Version+2+line+probe+assay+is+equivalent+to+that+of+the+Xpert+MTB/RIF+assay.">The diagnostic performance of the GenoType MTBDRplus Version 2 line probe assay is equivalent to that of the Xpert MTB/RIF assay.</a> J. Clin. Microbiol. 50, 3712-3716 (2012).</li><li>Sekiguchi, J. -. I., Nakamura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+a+line+probe+assay+for+rapid+identification+of+pncA+mutations+in+pyrazinamide-resistant+Mycobacterium+tuberculosis+strains.">Development and evaluation of a line probe assay for rapid identification of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis strains.</a> J. Clin. Microbiol. 45, 2802-2807 (2007).</li><li>Chandler, D. P., Bryant, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+amplification+microarrays+for+infectious+disease+diagnostics.">Integrated amplification microarrays for infectious disease diagnostics.</a> Microarrays. 1, 107-124 (2012).</li><li>Vincent, V., Rigouts, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TDR+Tuberculosis+Strain+Bank:+a+resource+for+basic+science,+tool+development+and+diagnostic+services.">The TDR Tuberculosis Strain Bank: a resource for basic science, tool development and diagnostic services.</a> Int. J. Tuberc. Lung Dis. 16, 24-31 (2012).</li><li>Golova, J. B., Chernov, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-volatile+copolymer+compositions+for+fabricating+gel+element+microarrays.">Non-volatile copolymer compositions for fabricating gel element microarrays.</a> Anal. Biochem. 421, 526-533 (2012).</li><li>Cooney, C. G., Sipes, D., Thakore, N., Holmberg, R., Belgrader, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plastic,+disposable+microfluidic+flow+cell+for+coupled+on-chip+PCR+and+microarray+detection+of+infectious+agents.">A plastic, disposable microfluidic flow cell for coupled on-chip PCR and microarray detection of infectious agents.</a> Biomed. Microdevices. 14, 45-53 (2012).</li><li>Lane, S., Evermann, J., Loge, F., Call, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplicon+secondary+structure+prevents+target+hybridization+to+oligonucleotide+microarrays.">Amplicon secondary structure prevents target hybridization to oligonucleotide microarrays.</a> Biosens. Bioelectron. 20, 728-735 (2004).</li><li>El-Hajj, H. H., Marras, S. A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+sloppy+molecular+beacon+probes+for+identification+of+Mycobacterial+species.">Use of sloppy molecular beacon probes for identification of Mycobacterial species.</a> J. Clin. Microbiol. 47, 1190-1198 (2009).</li><li>Boehme, C. C., Nabeta, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+molecular+detection+of+tuberculosis+and+rifampin+resistance.">Rapid molecular detection of tuberculosis and rifampin resistance.</a> N. Engl. J. Med. 363, 1005-1015 (2010).</li></ol>

Yazarlar herhangi bir mali çıkarlarını beyan.

Bir amplifikasyon mikroarray ile çoğullama ölçüde sonuçta katlı asimetrik PCR verimi değil, mikrodizi tarafından belirlenir. Bizim tecrübelerimize göre, 10-12 benzersiz primer çiftleri kolaylıkla bir amplifikasyon mikroarray formatında çoğaltılmış olabilir. Geleneksel primer ve prob tasarım kriterleri, bu nedenle, bu çözelti, bir de-faz nükleik asitler ve hareketsiz mikrodizi prob, bir PCR tamponu içinde termal termal döngü verimliliği ve prob hibridizasyon davranış arasındaki olası etkile...

Erken vaka tespiti ve hızlı tedavi Mycobacterium tuberculosis (MTB) iletim 1 azaltmak için en etkin kontrol stratejileri kabul ve bakım (POC) veya POC testinde yakın bir noktadan aynı anda TB teşhis TB toplumda geniş bir mutabakat bulunmaktadır vardır ve ilaç direnci (DR) gereklidir. Böyle Sefeid en GeneXpert ve diğer nükleik asit amplifikasyon testleri gibi teknolojiler birçok TBC hastaları için tanı süresini azaltmak, ve rifampin veya diğer birinci veya ikinci basamak ilaçlara 2 direnç kazandıran seçilen mutasyonlara karşı direnç gösteren bir hızlı okuma-çıkış sağlamak. Gerçek zamanlı ve izotermal nükleik asit amplifikasyon testleri MDR-TB yol açan ilaç direnç mutasyonları tanımlamak için tasarlanmış olsa da, bunlar tespit mutasyonların spektrumu, hastanın ilaç direnci profiline karşılık gelen kişiselleştirilmiş bir ilaç rejimi tasarımı genellikle yetersizdir ve ilgili teknik kısıtlarıoptik çapraz konuşma veya amplifikasyon ve raporlama kimyaları karmaşıklığına 3-7 loci veya tespit edildiği mutasyonların sayısını sınırlandırabilir. Böylece, yüksek çoklayıcı kapasiteli algılama teknolojileri ÇİD-TB POC teşhis bilinen boşlukları gidermek için gereklidir. Mikroarray&#39;ler ve WHO onaylı Hain hat prob deneyleri ÇİD-TB 8-29 teşhis &quot;birden fazla gen, multipl mutasyonlar&quot; aşabilirsiniz. Ne yazık ki, bu hibridizasyon temelli, mültipleksli algılama platformları çok adımlı, karmaşık ve moleküler teknikler önemli eğitim ve uzmanlık gerektiren açık Amplikon protokollerini kullanır. Amplifikasyon Mikrodizi 30 bu mikroarray iş akışı ve operasyonel bazı endişelerini gidermek için tasarlanmıştır. Basitleştirilmesi akışkan ilkeleri, yükseltmek hibridize olan ve tek bir mikroakışkan odası içinde nükleik asit hedefleri tespit etmek için bulunmaktadır. Kullanıcı, nükleik asit amplifikasyonu ve mas getirmektedirter bir pipet ile bir akışkan odasına karıştırın ve termal döngü protokolü başlar. Burada gösterilen toplu işlem yöntemi için, mikro sıraları, daha sonra, yığın solüsyonu ile yıkanır, kurutulur ve görüntülü. Bu çalışma rpoB için bir ÇİD-TB mikroarray test (30 mutasyon), katG (2 mutasyonları), Inha (4 mutasyonlar), rpsL (2 mutasyonları), embB (1 mutasyon), IS1245, IS6110 kullanarak bir amplifikasyon mikrotertibin işlevselliğini gösteriyor ve bir iç amplifikasyon ve hibridizasyon kontrolü. Mikrodizi sondalar (vahşi tip (WT) ve tek nükleotid mutant (MU)) en az bir çift eşleşen ilgi her mutasyon için dahil edilir. Çoklu ilaç dirençli M. saflaştırılmış nükleik asitleri tüberküloz TDR Verem Zorlanma Bankası 31 vardır. Başka bir yerde tarif edildiği gibi 32 Gel elemanı mikroarrayleri biz polymeriz her sonda% 4 monomer ve 0.05 mM kullanmak dışında, esas olarak kopolimerizasyonu ile, cam alt-tabakalar üzerinde üretilmektediration karışımı ile gerçekleştirilmektedir. Diziler, kullanımdan önce, 50 ml conta ile çevrilidir. Termal bisiklet, melezleme ve yıkama aşamalarından sonra, amplifikasyon mikroarray&#39;ler bir Akonni taşınabilir analizörü ile görüntülenmiş. Background-düzeltilmiş, entegre sinyal yoğunlukları ham elde edilir. Sabit bir daire algoritma kullanarak tif görüntüler. Her jel elemanı için Gürültü yerel nokta kökenden üç kat standart sapma olarak hesaplanmıştır. Gürültü oranı (SNR) sinyal için ≥ 3 değerleri gen hedefleri tipik bir şekilde saptanabilir kabul edilir. Her bir gen ya da kodon belirgin bir mutasyonun varlığını veya yokluğunu belirlemek için, ayırt edici bir oranı (WT-MU) / (WT + MU) halinde SNR değerlerinden hesaplanır. Discriminant oranları 0, vahşi tip dizisinin göstergesidir &lt;oranları ise 0, lokusuna bir ilaç-dirençli mutasyon göstergesidir&gt;.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;">MDR-TB amplification microarrays, with applied gasket and pre-cut cover slips</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Multiplex PCR kit, containing 2X PCR buffer with HotStar Taq plus</td>
			<td>Qiagen</td>
			<td>#206143</td>
			<td></td>
		</tr>
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			<td height="22" style="height:22px;">Taq polymerase</td>
			<td>Qiagen</td>
			<td>#201207</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">RNAse-free water</td>
			<td>Qiagen</td>
			<td>#206143&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Formamide</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP227-500</td>
			<td></td>
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		<tr height="26">
			<td height="26" style="height:26px;">20 mg mL-1 non-acetlyated bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>#3B6917</td>
			<td></td>
		</tr>
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			<td height="22" style="height:22px;">5X concentrated MDR-TB primer mix</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">500 fg uL-1 amplification and inhibition control</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">20X SSPE</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP1328-4</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Triton X-100</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP151-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Disinfecting Spray&#160;</td>
			<td>Current Technologies, Inc.</td>
			<td>#BRSPRAY128</td>
			<td></td>
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			<td height="22" style="height:22px;">70% Isopropyl Alcohol</td>
			<td>Decon Labs, Inc.</td>
			<td>#8416</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Equipment</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Microarray imager, with automated image and data analysis software</td>
			<td>Akonni Biosystems</td>
			<td>100-20011</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Thermal cycler with flat block insert</td>
			<td>Akonni Biosystems</td>
			<td>100-10021</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">High-throughput wash station and slide holder</td>
			<td>ArrayIt</td>
			<td>HTW</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dissecting forceps</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#10-300</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Mini Vortexer</td>
			<td>VWR</td>
			<td>#3365040</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;">Mini-centrifuge</td>
			<td>VWR</td>
			<td>#93000-196</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Airbrush Compressor Kit</td>
			<td>Central Pneumatic</td>
			<td>#95630</td>
			<td></td>
		</tr>
	</tbody>
</table>

demonstrating a multi-drug resistant mycobacterium tuberculosis amplification microarray

Mikrodizi iş akışı basitleştirilmesi rutin alt-kaynak ortamlarda kullanılabilir ÇİD-TB mikroarray tabanlı tanı oluşturmak için gerekli bir ilk adımdır. Bir amplifikasyon mikroarray tek bir çözüm içinde ve tek bir mikroakışkan odasına asimetrik PCR, hedef boyutu seçimi, hedef etiketleme ve mikroarray hibridizasyon birleştirir. Bir toplu işleme yöntemi çoklu ilaca dirençli Mycobacterium tuberculosis (MDR-TB) genotiplemeye 9-karmaşık asimetrik ana karışımı ve düşük yoğunluklu jel elemanı mikrotertipte ile gösterilmiştir. Burada açıklanan protokol 6 saat içinde tamamlanmış ve genomik DNA&#39;sının en az 1000 hücre eşdeğer doğru genotipleme temin edilebilir. Çip üzerinde yıkama adımları birleştirilerek tamamen kapalı bir amplikon, yöntem ve sistem ile sonuçlanır ki, mümkündür. Bir amplifikasyon mikroarray ile çoğullama ölçüde sonuçta tek bir ana m birleştirilebilir primer çiftlerinin sayısı ile kısıtlanırix ve hala oldukça dizide hareketsiz olan probların sayısından daha istenen duyarlılık ve özgüllük performans ölçümleri, başarmak. Aynı şekilde, toplam analiz süresi belirli bir kullanım amacı, araştırma sorusu ve tespiti istenen sınırları bağlı olarak kısaltılabilir veya uzatılabilir. Bununla birlikte, genel bir yaklaşım önemli ölçüde elle yoğun ve zaman alıcı bir işlem adımlarının sayısını azaltarak son kullanıcı için mikroarray iş akışını düzenler, ve rutin klinik uygulamaya mikroarray tabanlı tanı çeviri için basitleştirilmiş bir biyokimyasal ve mikroakışkan yolu sağlar.

Nitel görüntü analizi otomatik görüntü analiz yazılımı tarafından oluşturulan veri tabloları tanımlamak için zorlu deneysel gürültü veya değişkenlik kaynaklarını içgörü sağlayabilir. Böylece, 1) Tüm jel elemanları sağlam ve hasarsız olduğu, 2) Küresel plan gürültü oranı (SNR) değerleri tek bir sinyalin etkileyebilecek floresan eserler ücretsiz olduğunu tespit görsel olarak yararlı olabilir, 3) hiçbir kanıt yoktur kabarcık oluşumu ya da kuralsız amplifikasyon / melezleme dizi...

Watch this Scientific Journal Video about Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray at JoVE.com

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Bir amplifikasyon mikrodizi önemli ölçüde son kullanıcı için mikro-dizi iş akışını tek bir oda içine asimetrik PCR amplifikasyonu ve mikrodizi hibridizasyon birleştirir. Mikrodizi iş akışı basitleştirilmesi rutin alt-kaynak ortamlarda kullanılabilir mikroarray tabanlı tanı oluşturmak için gerekli bir ilk adımdır.

Çok ilaca dirençli Demonstrating<em&gt; Mycobacterium tuberculosis</em&gt; Amplifikasyon Microarray

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

demonstrating-multi-drug-resistant-mycobacterium-tuberculosis

Research

JoVE Journal

Immunology and Infection

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

12.5K Görüntüleme.  Akonni Biosystems, Inc..  Bir amplifikasyon mikrodizi önemli ölçüde son kullanıcı için mikro-dizi iş akışını tek bir oda içine asimetrik PCR amplifikasyonu ve mikrodizi hibridizasyon birleştirir. Mikrodizi iş akışı basitleştirilmesi rutin alt-kaynak ortamlarda kullanılabilir mikroarray tabanlı tanı oluşturmak için gerekli bir ilk adımdır. 

Video: Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

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

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

Microtiter Plate Format Broth Seyreltme Birinci ve İkinci Line İlaçları Mycobacterium tuberculosis kompleksi Antimikrobiyal Duyarlılık Testi

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

İmmünoloji ve Enfeksiyon

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.

Numune Hazırlama<em&gt; Mycobacterium tuberculosis</emNükleer Manyetik Rezonans metabolomik Araştırmaları&gt; özler

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.

Büyüme Mycobacterium tuberculosis Biyofilmler

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

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Prionlar protein misfolding Döngüsel Amplifikasyon

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Doğal İletim üzerine Tüberküloz-Sıtma Koenfeksiyonu Çalışması için Deneysel Bir Model Mycobacterium tuberculosis Ve Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

Kurtarma Bir Roman Mikrodiseksiyon Yaklaşımı<em&gt; Mycobacterium tuberculosis</emAkciğer granülomlara Gömülü Formalinle Sabit Parafin dan&gt; Özel Transkript

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

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.

Fonksiyonel Araştırmaları 3D İnsan Akciğer Doku Modeli<em&gt; Mycobacterium tuberculosis</em&gt; Enfeksiyon

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.

Hücre içi Hedefleme İnhibitörleri Etkinliği ve Sitotoksisite Taraması için sistem<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 ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

Bir Yüksek verim Uyumlu Deneyi Makrofaj, pasajlamaya karşı ilaç etkinliğini değerlendirmektir<em&gt; Mycobacterium tuberculosis</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

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.

Analiz 18FDG PET/BT uzmanı Mycobacterium tüberküloz enfeksiyon ve insan olmayan primatlarda tedavisi için bir araç olarak görüntüleme

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