Questo lavoro è stato parzialmente sostenuto dal governo spagnolo &quot;Ministerio de Educación y Ciencia&quot; (progetto AGL2008-05275-C01/ALI), dall&#39;Unione Europea Quadro di Ricerca 7 progetti finanziati VIROBATHE (contratto n ° 513648), VIROCLIME (contratto n ° 243923 ) e dall&#39;Agenzia catalana di acqua, Agencia Catalana de l&#39;Aigua (ACA), Departament de controllo i Millora dels Ecosistemes Aquatics. Durante lo studio ha sviluppato Marta Rusiñol era un collega del governo catalano &quot;AGAUR&quot; (FI-DGR).

1. Concentrazione delle particelle virali presenti nei campioni di acqua <ol><li> Raccolta e condizionamento dei campioni di acqua <ol><li> Raccogliere un minimo di 2 repliche di 10 litri per campione in contenitori di plastica con fondo piatto e un campione in più come un controllo di processo. Questo ultimo esempio sarà arricchito con una quantità nota di particelle virali e utilizzato come controllo. Nota: Si raccomanda di avere particolari materiali utilizzati (bottiglie, tubi, ecc) per i campioni a spillo. </li><li> Controllare la taratura del conduttimetro e ricalibrare se necessario. Preparare un controllo negativo utilizzando l&#39;acqua del rubinetto in precedenza regolato la conducibilità adeguato (vedi sotto 1.6) in un ulteriore contenitore in plastica 10 L. </li><li> Per i campioni a spillo: Aggiungere il volume standard del virus di controllo (circa 10 5 copie del genoma per 10 L di acqua) al campione. Mescolare agitando evitando schizzi e aerosol. I controlli positivi potrebbe consistere in un raro ceppo di adenovirus such come HAdV-35 o un batteriofago come MS2. </li><li> Se il campione presenta elevate quantità di materiale in sospensione (sabbia o altri materiali), si lascia sedimentare per 15 minuti. Trasferire l&#39;acqua in un contenitore nuovo. </li><li> Regolare il pH del campione di acqua di 3,5 (± 0,1) con l&#39;aggiunta di 1 N HCl. Questo passaggio è importante per la concentrazione del virus, in modo da assicurarsi che il pH è stato regolato correttamente. Mescolare accuratamente l&#39;acqua agitando vigorosamente, mentre l&#39;aggiunta del HCl. (Nota: se il pH è inferiore a 3,5 aggiungere 1 M di NaOH). </li><li> Regolare la conduttività. Se il campione ha una conducibilità di 1500 mS / cm o superiore, questo passaggio non è necessario. Se la conducibilità è inferiore a 1500 mS / cm la formazione di materiale flocculato (fiocchi) non è garantita in modo regolare la conducibilità a 1500 mS / cm con l&#39;aggiunta di sali marini artificiali (Sigma). Mescolare agitando vigorosamente, mentre l&#39;aggiunta del sale marino. </li><li> Registra il pH dei campioni prima e dopo il condizionamento anche unas il volume di HCl utilizzato. La conducibilità dovrebbero anch&#39;essi essere registrati dopo la regolazione del pH. Disinfettare sempre il pH-metro e conduttimetro elettrodi con una soluzione fresca HCl, dechlorinate con una soluzione di sodio al 10% tiosulphate ed infine risciacquare con acqua distillata. </li></ol></li><li> Preparazione di pre-flocculato 1% di latte scremato (PSM) <ol><li> Controllare la taratura del pH-metro e conduttimetro e ricalibrare se necessario. </li><li> Preparare pre-flocculato soluzione latte scremato (1 PSM%, w / v), sciogliendo 10 g di latte scremato in polvere (Difco) in 1 L di acqua di mare artificiale (sciogliere 33,3 g di sale marino artificiale in 1 L di acqua del rubinetto declorurata e autoclave) e con attenzione la regolazione del pH a 3,5 con HCl 1N. I fiocchi dovrebbe essere visibile. Preparare la soluzione poco prima da utilizzare o conservare a 4 ° C per 24 h. Per declorazione usare 1 ml di soluzione di tiosulphate 10% per ogni 100 ml di acqua. </li></ol></li><li> Flocculazione di particelle virali presenti nei campioni di acqua<ol><li> Aggiungere 100 ml di 1% PSM al 10-L campione di acqua. </li><li> Mescolare i campioni per 8-10 ore per permettere al virus di adsorbire le fiocchi. Utilizzare un timer per lo spegnimento l&#39;agitazione dopo 8-10 ore </li><li> Fermare l&#39;agitazione e lasciare il sedimento fiocchi per gravità per 8-10 h. </li></ol></li><li> Raccolta e ri-sciogliendo i fiocchi. Centrifugazione <ol><li> Rimuovere il surnatante utilizzando una pompa peristaltica e una pipetta di plastica collegato ad un tubo di plastica. Per i campioni addizionati il ​​surnatante devono essere raccolti in una bottiglia e disinfettati secondo le procedure interne. In tutti i casi ATTENZIONE a non riscuotere il pellet. </li><li> Raccogliere il sedimento con i fiocchi (circa 500 ml) in una bottiglia di centrifuga. </li><li> Equilibrio i piatti con l&#39;aggiunta di pH PSM 3.5. </li><li> Centrifugare i vasi in una centrifuga ad alta velocità a 8.000 xg per 30 min a 4 ° C. Non appena la centrifuga si ferma, rimuovere con cura i vasi centrifuga dalla centrifuga. </li><li> Molto gdipendentemente versare fuori e scartare il surnatante. Seguire le misure appropriate per materiale infetto. </li><li> Aggiungere 7 ml di tampone fosfato a sciogliere il precipitato in ogni bottiglia centrifuga. </li><li> Una volta che i fiocchi sono stati sciolti, misura e aggiungere tampone fosfato a raggiungere un volume totale di 10 ml. </li><li> Omogeneizzare la concentrazione virale vortex e distribuire i 10 ml in microtubi pulite che devono essere congelati a -80 ° C fino al momento in ulteriori analisi. </li></ol></li></ol> 2. Estrazione acido nucleico <ol><li> Effettuare una estrazione degli acidi nucleici con il QIAamp Viral RNA Mini Kit seguenti istruzioni del produttore. Questo kit consente l&#39;utilizzo di una piattaforma automatizzata (come Qiacube, Qiagen). </li></ol> 3. PCR quantitativa di adenovirus umani (HAdV), poliomavirus JC (JCPyV), adenovirus suina (PAdV) e poliomavirus bovina (BPyV) <ol><li> Quantificazione delle copie del genoma nei campioni <ol><li> Preparareil mix qPCR in una zona pulita separata utilizzando TaqMan Ambientale 2x PCR Master Mix (Applied Biosystems). La reazione avviene in una piastra di reazione 96-ottico ben coperti con coperture adesive ottica. Le concentrazioni del Master Mix, primer e le sonde sono descritte nella tabella 1. </li><li> Quando l&#39;impasto è stato preparato, 15μl aliquota in ciascun pozzetto inclusi i controlli (vedi 3.2). Il volume totale per una reazione dopo l&#39;aggiunta dei target sarà 25μl (mix 15μl campione + 10μl o standard). </li><li> Aggiungere le estrazioni acidi nucleici dai campioni (10μl) in una zona separata. Eseguire diretta e diluizione decimale in acqua purificata di ogni campione in duplicato. Coprire i pozzetti contenenti i campioni con una parte di una cover adesiva, mantenere l&#39;altra parte della copertura per il passo successivo. </li><li> Aggiungi diluizioni di sospensioni standard del DNA (10μl) da 10 0 a 10 6 GC/10μl dal triplice copia e l&#39;utilizzo di una micropipetta utilizzati esclusivamente per le norme sianostandard del DNA. Si consiglia di aggiungere gli standard in una zona attrezzata con radiazioni UV per distruggere il DNA plasmidico e per pulire la micropipetta dopo ogni utilizzo. Coprire i pozzetti contenenti gli standard con il pre-taglio copertina adesiva. Nota: Per preparare sospensioni standard da utilizzare nella quantificazione delle copie del genoma, la regione di DNA target dovrebbe essere clonato in un plasmide e linearizzato. Nella seguente indirizzo troverete una procedura dettagliata su come creare curve standard con i modelli di DNA plasmidico da utilizzare in qPCR: <a href="http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf">http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf</a> </li><li> Eseguire la qPCR in un adeguato sistema selezionando i parametri appropriati (considerando l&#39;uso di copertura adesiva e il volume totale in ogni pozzetto, ecc.) Dopo l&#39;attivazione del AmpliTaq Gold per 10 min a 95 ° C, 40 cicli di amplificazione sono effettuate come segue: 15 s a 95 ° Ce 1 min a 60 ° C per HAdV, JCPyV e BPyV, e 15 s a 95 ° C, 20s a 55 ° C e 20s a 60 ° C per PAdV. </li><li> Una volta che le reazioni sono completate, memorizzare i dati ed i risultati come descritto nel manuale d&#39;uso delle apparecchiature utilizzate. La quantità di DNA verrà definito come la media dei dati ottenuti dopo la correzione del fattore di diluizione in caso di necessità. </li></ol></li><li> Controlli <ol><li> Utilizzare i controlli positivi e negativi. Il dosaggio deve includere più di un non-modello di controllo (NTC) per dimostrare mix non produce fluorescenza. Si consiglia di eseguire il controllo positivo di processo al fine di valutare il potenziale inibizione enzimatica a causa di inibitori presenti nei campioni studiati. </li><li> Risultato record di saggi qPCR di due diverse diluizioni del DNA standard e dal controllo di processo. Utilizzare i risultati per preparare carte di controllo per il controllo qualità (QC) i programmi relativi alla sensibilità e l&#39;efficienza del test. </li></ol></li><li> Conferma dellarisultati <ol><li> Risultati positivi possono essere ulteriormente confermata mediante sequenziamento nested-PCR e nucleotidi degli amplificati, producendo ulteriori dati sulle sequenze nucleotidiche dei ceppi individuati 1,5,6,7,9,12. </li></ol></li></ol> Seguendo la procedura descritta, i virus umani e animali sono stati individuati e quantificati in acque di balneazione, l&#39;acqua di mare e di acqua di fiume 2,3. Come un esempio rappresentativo, campioni di terra di acqua dai locali che presentano alti livelli di nitrati sono stati valutati per definire le fonti di contaminazione. Dieci litri sono stati raccolti campioni di acqua da 4 pozzi diverse nelle zone rurali di una regione nord-est della Spagna. Cinque replica sono stati raccolti in ciascun pozzetto essere uno replicare seminato con adenovirus umano 2 utilizzati come controllo di processo. I campioni sono stati trattati secondo il protocollo rappresentato nella Figura 1. Le quattro repliche analizzati in uno dei quattro siti studiati hanno mostrato risultati positivi per PAdV (valore medio 7,74x10 2 GC / L) che sarebbe legato alla presenza di fanghi di maiale nelle zone circostanti il sito di campionamento e sosterrebbe la contaminazione fecale suina come fonte di nitrati nelle acque sotterranee (Tabella 2). 4. Rappresentante dei risultati: <img src="/files/ftp_upload/2820/2820fig1.jpg" alt="Figura 1" /> Figura 1. Procedura per l&#39;individuazione e la quantificazione dei virus in acqua. <img src="/files/ftp_upload/2820/2820table1.jpg" alt="Tabella 1" /> Tabella 1. Concentrazioni dei primer e sonde per saggi qPCR. <img src="/files/ftp_upload/2820/2820table3.jpg" alt="Tabella 3" /> Tabella 2. Rilevazione e quantificazione di adenovirus animale e umana e poliomavirus in campioni di acque sotterranee. n numero di repliche analizzato % Percentuale di positivi replica (-) Non rilevati

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterococcus+species+distribution+among+human+and+animal+hosts+using+multiplex+PCR.">Enterococcus species distribution among human and animal hosts using multiplex PCR.</a> J. Appl. Microbiol. 109, 539-547 (2010).</li><li>de Motes, M. a. l. u. q. u. e. r., Clemente-Casares, C., Hundesa, P., Mart&#237;n, M., Girones, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+bovine+and+porcine+adenoviruses+for+tracing+the+source+of+fecal+contamination.">Detection of bovine and porcine adenoviruses for tracing the source of fecal contamination.</a> Appl. Environ. Microbiol. 70 (3), 1448-1454 (2004).</li><li>Pal, A., Sirota, L., Maudru, T., Peden, K., Lewis, A. 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Due domande di brevetto sono state depositate nel 2009 per proteggere la proprietà intellettuale dei protocolli per la quantificazione della PAdV e BPyV.

<html> La procedura descritta potrebbe soddisfare le condizioni per un metodo adatto per routine laboratori ambientali e sanitarie: riproducibile, affidabile, semplice e conveniente. Il protocollo è semplice, ma che devono essere seguite attentamente. Bassa conducibilità nei campioni senza aggiungere la concentrazione richiesta di sali di acqua di mare artificiale ridurrebbe drasticamente il recupero di virus come sarebbe il caso se il tempo di agitazione per la flocculazione è significativamente ridotto (meno di 5 or...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Centrifuga ad alta velocità (8.000 xg) </td><td> Berckman Coulter </td><td> Avanti J-20XP </td><td></td></tr><tr><td> pH-metro, termometro e conduttimetro </td><td> Afora </td><td> LPPC3003 </td><td></td></tr><tr><td> Tubi di plastica 100-200 cm di lunghezza </td><td> DeltaLab </td><td> 350059 </td><td></td></tr><tr><td> Pipette graduate sterili monouso </td><td> Labclinics </td><td> PN10E1 </td><td></td></tr><tr><td> Sterili tubi di plastica di 1,5 e 10-15 ml (Eppendorf, Falchi, ecc) </td><td> Afora </td><td> KA298/00 </td><td></td></tr><tr><td> Pentole centrifuga (500 ml) </td><td> Fisher Scientific </td><td> SE5753512 </td><td></td></tr><tr><td> Agitatori magnetici e magneti (uno percampione) </td><td> Fisher Scientific </td><td> 10510 </td><td></td></tr><tr><td> Contenitori di vetro o plastica, che ha fondo piatto per consentire l&#39;uso di agitatori magnetici </td><td> DeltaLab </td><td> 191642 </td><td></td></tr><tr><td> Una pompa peristaltica per la rimozione del surnatante (o un getto d&#39;acqua pompa del vuoto) </td><td> Watson-Marlow </td><td> 323E / D </td><td></td></tr><tr><td> Timer per lo spegnimento l&#39;agitazione dopo 8-10 ore </td><td> DeltaLab </td><td> 900400 </td><td></td></tr><tr><td> Acido cloridrico (1N e 0,1 N) </td><td> Panreac </td><td> 141020.1611 </td><td></td></tr><tr><td> Idrossido di sodio (1N) </td><td> Panreac </td><td> 131687.1211 </td><td></td></tr><tr><td> Artificiale con acqua di mare mare sali </td><td> Sigma </td><td> S9883 </td><td></td></tr><tr><td> Latte scremato (SM) </td><td> Difco </td><td> 232100 </td><td></td></tr><tr><td> Tampone fosfato a pH 7, 5 </td><td></td><td></td><td> 01:02 v / v di sterile Na 2 HPO 4 0,2 M e NaH 2 PO 4 0,2 M a pH 7,5 </td></tr><tr><td> Tiosolfato </td><td> Panreac </td><td> 121879.1209 </td><td> Fai una soluzione al 10% in acqua </td></tr><tr><td> QIAamp Viral RNA Mini Kit </td><td> Qiagen </td><td> 52904 </td><td></td></tr><tr><td> 96-pozzetti di reazione ottico (500 unità) </td><td> Applied Biosystems </td><td> 43426659 </td><td></td></tr><tr><td> Ottico copre adesivo (100 unità) </td><td> Applied Biosystems </td><td> 4311971 </td><td></td></tr><tr><td> TaqMan Ambientale PCR Master Mix 2x </td><td> Applied Biosystems </td><td> 4396838 </td><td></td></tr></tbody></table>

cost-effective method for microbial source tracking using specific human and animal viruses

La contaminazione microbica dell&#39;ambiente rappresenta un rischio significativo per la salute. Classica indicatori batterici fecali hanno dimostrato di avere notevoli limitazioni, i virus sono più resistenti ai processi di inattivazione molti indicatori e standard fecale non informare sulla fonte di contaminazione. Lo sviluppo di metodi economici per la concentrazione di virus da acqua e saggi molecolari facilita l&#39;applicabilità di virus come indicatori di contaminazione fecale e come fonte microbica tracking (MST) strumenti. Adenovirus e poliomavirus sono virus a DNA infettare specie di vertebrati specifici compresi gli esseri umani e sono costantemente escreto nelle feci e / o urine in tutte le aree geografiche studiate. In studi precedenti, abbiamo suggerito la quantificazione di adenovirus umani (HAdV) e poliomavirus JC (JCPyV) mediante PCR quantitativa (qPCR) come indice di contaminazione fecale umana. Recentemente, abbiamo sviluppato saggi qPCR per la quantificazione specifica di poradenovirus cine (PAdV) e poliomavirus bovina (BPyV) come animali indicatori di contaminazione fecale con sensibilità di copie del genoma 1-10 per provetta. In questo studio, presentiamo la procedura da seguire per identificare la fonte di contaminazione nei campioni di acqua di questi strumenti. Come esempio dei risultati rappresentativi, l&#39;analisi dei virus nelle acque sotterranee presenta alti livelli di nitrati è mostrato. Individuazione di virus in acque inquinate o moderatamente bassa richiede la concentrazione del virus da almeno diversi litri di acqua in un volume molto più piccolo, una procedura che di solito comprende due fasi concentrazione in serie. Questa procedura alquanto complesso e la variabilità osservata in recuperi virale ostacolare in modo significativo l&#39;elaborazione simultanea di un gran numero di campioni di acqua. Al fine di eliminare il collo di bottiglia causato dai due procedure dettagliate abbiamo applicato un passo protocollo sviluppato in Studie precedentes ed applicabile ad una varietà di matrici acqua. La procedura prevede: l&#39;acidificazione di dieci litri di campioni di acqua, flocculazione da latte scremato, sedimentazione dei materiali flocculato, la raccolta del precipitato e centrifugazione, risospensione del precipitato in 10 ml di tampone fosfato. Il concentrato virale è utilizzato per l&#39;estrazione di acidi nucleici virali e l&#39;adenovirus specifico e poliomavirus di interesse sono quantificati da qPCR. Elevato numero di campioni può essere contemporaneamente analizzate con questo metodo a basso costo concentrazione. La procedura è stata applicata per l&#39;analisi delle acque di balneazione, l&#39;acqua di mare e di acqua di fiume e in questo studio, presentiamo i risultati analizzando campioni di acque sotterranee. Questo high-throughput metodo quantitativo è affidabile, semplice e conveniente.

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Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Lo studio descrive un metodo conveniente per l&#39;identificazione della fonte di contaminazione fecale / urina o la contaminazione da nitrati in acqua utilizzando qPCR per la quantificazione specifica di virus DNA umano / suini / bovini, adenovirus e poliomavirus, proposti come strumenti di MST.

Costo-efficace metodo per il rilevamento di origine microbica Utilizzando specifici virus umani e animali

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

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12.1K Views.  University of Barcelona. Lo studio descrive un metodo conveniente per l'identificazione della fonte di contaminazione fecale / urina o la contaminazione da nitrati in acqua utilizzando qPCR per la quantificazione specifica di virus DNA umano / suini / bovini, adenovirus e poliomavirus, proposti come strumenti di MST.

Video: Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinazione di nastro adesivo a base di campionamento e di fluorescenza In situ</emIbridazione> per il rilevamento rapido di Salmonella Su prodotti freschi

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Ricerca

Immunologia e infezioni

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

Completa dei costi e monitoraggio di laboratorio efficace di HIV / AIDS: un modello di ruolo africano

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

Staphylococcus aureus crescita con emoglobina umana come fonte di ferro

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Creazione di un liquido coperto Cultura polarizzata Airway epiteliali umane Calu-3 celle per studio cellulare risposta dell&#39;ospite agli agenti patogeni delle vie respiratorie<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

Pairwise crescita Concorso test per la determinazione della Fitness replica del virus dell&#39;immunodeficienza umana

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Intranasale somministrazione di ricombinante Influenza vaccini nei modelli chimerico topo per studiare immunità delle mucose

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

Bacterial Artificial Chromosomes: uno strumento Genomica Funzionale per lo studio di positivo-strand virus RNA

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizzando le risposte dell'anticorpo al virus A(H3N2) in sieri umani di Microneutralization l'Influenza di misurazione analisi utilizzando cellule MDCK-SIAT1

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

VirWaTest, un metodo point-of-use per il rilevamento di virus nei campioni d'acqua

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Quantificazione delle cellule CD4 specifiche dell'antigene umano che proliferano utilizzando Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...