In our Country, the centralization of diagnostic procedures is evolving, even for microbiology diagnostic laboratories and this has a direct effect on the contamination rates. NAC-PAC RED, in this study, has demonstrated to be a valid means of overcoming this problem, by reducing the contamination rates from 13% to 3%. This reduction of contamination has led to a significant reduction of sample re-processing. These improvements directly affect the quality of the care and treatment received by the patient. This reduction moreover has afforded our labouratory a reduction in the labour commitment and cost.

Between November 2010 and February 2011 our laboratory processed 446 biological samples using the NAC-PACTM RED (Alpha-Tec, USA) (Fig. 1). We compared the percentage of contaminated samples by using NAC-PAC RED and the percentage of contaminated samples obtained in the previous years (2008-2009) using MycoPrepTM

(Becton, Dickinson and Company - BD). We evaluated only the results obtained using the liquid culture medium MGITTM 960 (BD) rather than the solid media, as the non-radiometric liquid media has a very rich composition and therefore is the most contaminated of the liquid media. Both methods use N-acetyl-L-cysteine (NALC) together with sodium hydroxide (NaOH) and sodium citrate as the digestant / decontaminating reagent. NAC-PAC RED also contains a pH indicator. A pH indicator is incorporated into the digestion and decontamination reagent to monitor the pH throughout the decontamination and buffering procedure, allowing the laboratory technologist to visually see when neutralization has been achieved. pH neutralization is indicated by a color change from red/pink to colorless.

1 Do not add water, PBS, or M/15 phosphate buffer. 2 If using individual patient test volumes, discard any remaining buffer to prevent cross contamination. 3 This volume can be altered depending on the order in which your laboratory performs diagnostic protocols and the relative importance ofsensitivities. See Directions For Use for details.

MycobacteriaAFB Specimen Processing Protocol

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Place one drop of the mixed pellet from thepipette onto a clean microscope slide(s) to stainfor AFB. Do not touch the slide with the pipette.Return unused sample in pipette to thespecimen container.

Concentration: Centrifuge the samples at 3000 xg for 15-20 minutes. (Use a nomographchart to convert rpm to xg.)

Following the centrifugation step, decant all ofthe supernatant, being careful to retain thepellet (sediment). Use an approved biohazardcontainer.

Pellet Resuspension: Using a transfer pipetteaspirate 1 ml of PRB™ and add to thespecimen pellet.1, 3

Using the same pipette, carefully mix thespecimen through gentle aspiration anddischarge. Draw up a small amount of themixed pellet (~0.2 ml) into the pipette.

Break off the top of the N-acetyl-L-cysteine(NALC) ampule and add the powdered NALCto the NAC-PAC™ RED base solution. Re-capand mix the solution well.

Digestion and Decontamination: For specimens1-5 ml add an equal volume of NAC-PAC RED,for specimens 6-7 ml add 5 ml of NAC-PAC RED,for specimens 8 ml or greater add an equal vol-ume of NAC-PAC RED and split the specimen intotwo 50 ml centrifuge tubes after decontamination.These volumes must be measured precisely.

Recap the centrifuge tube containing thespecimen and NALC/Base solution. Vortex thespecimen until it is liquified (~30 seconds).

Place the specimen upright in a test tube rackto decontaminate for 15-20 minutes. Vortexseveral times during this period.

Neutralizing: Add the NPC-67™ NeutralizingBuffer1 to the specimen tube only until effectiveneutralization is indicated by a color changefrom red / pink to colorless. Do not add morebuffer after this change occurs.2

Inoculate solid media, liquid media and a TSA or 5% SBA plate, streak and incubate at 35o-37o Cto monitor for breakthrough contamination.

Inoculate automated detection broth asappropriate for your laboratory’s equipment,per the manufacturers’ instructions.

A portion of the remaining specimen pellet can be used for molecular diagnostics.

Store the remaining specimen (in a smaller tube if necessary) at 2o-6o C so that it is available forreprocessing if required.

Add the balance of the PRB to the remainderof the specimen pellet. Cap the specimen tube.

NAC-PAC™, NPC-67™ and PRB™ are trademarks of Alpha-Tec Systems Inc., Vancouver, WA, USA. ©2014 Alpha-Tec Systems Inc. All rights reserved.

Alpha-Tec Systems, Inc. Vancouver, WA 98668 USA P [+1] 800.221.6058 INT [+1] 360.260.2779 Sales@AlphaTecSystems.com www.AlphaTecSystems.comCC14-142, Effective Date: 06 May 2014, M-IN-AFB.A

On 446 analyzed samples (Nov. 2010 - Feb. 2011), only 13 (3.00%) were contaminated. Over the same period in 2009, on 449 analyzed samples, 49 (11.42%) were contaminated. In 2008 we analyzed 465 samples and 61 (13.00%) were contaminated (Fig. 2, Fig. 3).

We also considered the number of positive samples for Mycobacteria in the same periods in order to detect if the decontamination methods had an impact on the viability of Mycobacteria.

The results of both methods were comparable; in fact, in the period between November - February, the positive MTB samples were 24 (5.0%) in 2008/2009, 15 (3.3%) in 2009/2010, 20 (4.5%) in 2010/2011 (Fig. 4).

Positive samples for NTM were 12 (2.6%) in 2008/2009, 23 (5.1%) in 2009/2010, 16 (3.6%) in 2010/2011 (Fig. 5).

CC14-239, Effective Date: 28 Aug. 2014, M-PO-ITA.ANAC-PAC™ is a trademark of Alpha-Tec Systems, Inc., Vancouver, WA, USA. ©2014, Alpha-Tec Systems, Inc. All rights reserved.

Fig. 4

Fig. 5

Fig. 3

Fig. 2

Fig. 1

// Introduction

// Materials and Methods

// Results

// Conclusion

Evaluation of the Alpha-Tec® NAC-PACTM RED System for the Decontamination/Digestion of Clinical Samples for Mycobacteria Detection Using The BACTECTM MGITTM 960 System

Camaggi Anna, Molinari Gian L., Mantovani Monia, Brunelli Maria G., Fortina Giacomo Department of Microbiology and Virology, “Maggiore della Carità” University Hospital, C.so Mazzini 18, 28100 Novara, Italy Contact: annacamaggi@libero.it

The majority of biological samples intended for mycobacteria detection are usually contaminated with oral flora resident bacteria. The mycobacteria growth, if compared to the other bacteria is considerably slower; therefore, a decontamination procedure is required of the sample before streaking on suitable culture media. Decontamination is intended to reduce the microbial contamination at the lowest possible levels so as not to interfere with the growth of mycobacteria. The decontamination procedure may affect the viability of the mycobacteria, so this should not be too aggressive in the method and timing.

EVALUATION OF THE ALPHA TEC® NAC-PAC REDTM SYSTEM FOR THE DECONTAMINATION/DIGESTION OF CLINICAL SAMPLES FOR

MYCOBACTERIA DETECTION USINGTHE BACTECTM MGITTM 960 SYSTEM

Camaggi Anna, Molinari Gian L., Mantovani Monia, Brunelli Maria G., Fortina Giacomo Department of Microbiology and Virology, “Maggiore della Carità” University Hospital,

C.so Mazzini 18, 28100 Novara, ItalyContact: annacamaggi@libero.it

CONCLUSION

In our Country, the centralization of diagnostic procedures is evolving, even for microbiology diagnostic laboratories and this has a direct effecton the contamination rates. NAC-PACTM Red, in this study, has demonstrated to be a valid means of overcoming this problem, by reducing thecontamination rates from 13% to 3%. This reduction of contamination has led to a significant reduction of sample re-processing. Theseimprovements directly affect the quality of the care and treatment received by the patient. This reduction moreover has afforded our labouratorya reduction in the labour commitment and cost.

On 446 analyzed samples (Nov. 2010 - Feb.2011), only 13 (3.00%) were contaminated.Over the same period in 2009, on 449 analyzedsamples, 49 (11.42%) were contaminated.In 2008 we analyzed 465 samples and 61(13.00%) were contaminated (Fig. 2, Fig. 3).

We also considered the number of positivesamples for Mycobacteria in the same periodsin order to detect if the decontaminationmethods had an impact on the viability ofMycobacteria.The results of both methods were comparable;in fact, in the period between November-February, the positive MTB samples were 24(5.0%) in 2008/2009, 15 (3.3%) in 2009/2010, 20(4.5%) in 2010/2011 (Fig. 4).Positive samples for NTM were 12 (2.6%) in2008/2009, 23 (5.1%) in 2009/2010, 16 (3.6%) in2010/2011 (Fig. 5).

RESULTS

Between November 2010 and February 2011 our laboratory processed 446biological samples using the NAC-PACTM Red (Alpha Tec, USA) (fig. 1).We compared the percentage of contaminated samples by using NAC-PACTM Red and the percentage of contaminated samples obtained in theprevious years (2008-2009) using MycoPrepTM (Becton, Dickinson andCompany - BD). We evaluated only the results obtained using the liquidculture medium MGITTM 960 (BD) rather than the solid media, as the non-radiometric liquid media has a very rich composition and therefore is themost contaminated of the liquid media. Both methods use N-acetyl-L-cysteine (NALC) together with sodium hydroxide (NaOH) and sodiumcitrate as the digestant / decontaminating reagent. NAC-PACTM Red alsocontains a pH indicator. A pH indicator is incorporated into the digestionand decontamination reagent to monitor the pH throughout thedecontamination and buffering procedure, allowing the laboratorytechnologist to visually see when neutralization has been achieved. pHneutralization is indicated by a color change from red/pink to colorless.

The majority of biological samples intended for mycobacteria detection are usually contaminated with oral flora resident bacteria. Themycobacteria growth, if compared to the other bacteria is considerably slower; therefore, a decontamination procedure is required of thesample before streaking on suitable culture media. Decontamination is intended to reduce the microbial contamination at the lowestpossible levels so as not to interfere with the growth of mycobacteria. The decontamination procedure may affect the viability of themycobacteria, so this should not be too aggressive in the method and timing.

INTRODUCTION

MATERIALS AND METHODS

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