The MycoDDR Product Line is Essential for Mycobacteria Survival and Recovery

Transcription

1 The MycoDDR Product Line is Essential for Mycobacteria Survival and Recovery Crider, B.J., Neary, B.P., Bauman, S.K. Abstract Tuberculosis (TB) is a serious disease that affects a significant portion of the world s population and is one of the leading causes of death in developing countries. Diagnosis of TB is important not only for correct treatment of the disease but also for containment of the highly contagious infected individual. Currently, the most widely used diagnostics for pulmonary TB benefit from pre-processing of the patient sputum sample for concentration of the sample, removal of any contaminating organisms that may interfere with the test results, and survival of any mycobacterium for culturing. Consequently, processing of the patient sample is a critical first step in facilitating the diagnosis of TB. The importance of this sample processing step necessitates that the method be effective and dependable. The MycoDDR product line was developed as an exceptionally reliable specimen processing system that facilitates the digestion and decontamination of patient specimens in a controlled process that is essential for mycobacterium survival and recovery, which will facilitate diagnosis of TB and may ultimately reduce TB-associated mortality. Introduction TB is primarily caused by Mycobacterium tuberculosis, a member of the Mycobacterium tuberculosis complex, a set of genetically similar Mycobacterium spp. that can cause respiratory disease in humans. In humans, M. tuberculosis infection is typically caused by the inhalation of infected aerosols that are expelled during coughing by people with active pulmonary disease 2. Only HIV/AIDS surpasses TB as the greatest killer worldwide due to a single infectious agent; in 2011 alone, 8.7 million people fell ill with TB and 1.4 million people died from the disease 3. The scientific and medical communities in collaboration with the World Health Organization are currently in a global health initiative to reverse the incidence of TB by 2015; however, incidence is only declining slowly - especially among underserved and vulnerable populations, notably in Africa 4. It is currently recommended that all persons with symptoms of tuberculosis, a positive tuberculin skin test, or an interferongamma release assay, which are indicative of M. tuberculosis infection, should have sputum samples collected for diagnostic evaluation 5. The digestion and decontamination method used to process these patient specimens can have a direct effect on TB diagnosis and patient outcome. Importance of ph in the Digestion/Decontamination of Patient Specimens Currently, there are several commonly used TB diagnostic techniques that benefit from specimen pre-processing. These methods include culture examination, acid-fast (AFB) microscopy, and nucleic acid amplification (NAA). Regardless of the downstream diagnostic method, the digestion/decontamination procedure is absolutely critical for liberation and concentration of any mycobacteria present within the sample and elimination of contaminating normal flora organisms that could otherwise outgrow/outcompete the mycobacterial colonies when cultured, or make visualization of mycobacteria difficult with AFB microscopy 6-8. Kubica et al. described a sputum processing technique in which 0.5% N-acetyl-L-cysteine and 2% sodium hydroxide (NALC-NaOH) in citrate buffer was used to effectively digest and decontaminate sputum sample specimens 7, 9. This processing step is accomplished by liquefying the sample with the use of the NALC reagent and then decontaminating the sample by generating alkali conditions, through the use of an agent that modifies the ph of the sample, such as sodium hydroxide (NaOH) 7, 10. Unfortunately, mycobacteria cells are also negatively impacted by prolonged exposure to elevated ph 1, 6, Approximately 30% of mycobacteria are killed during NaOH processing following the methods of Kubica et al. 6, 7. Additionally, the overkill of mycobacteria can continue during centrifugation if the NaOH decontamination step is 6, 14 not neutralized efficiently (Figure 1).

2 Figure 1 MycoDDR, unlike traditional methods, maintains Mycobacteria viability through specimen pre-processing 1. Figure 2 - ph levels and its effect during the different stages of sample pre-processing. The blue MycoDDR NaOH Reagent A is added to patient samples to elevate the ph and kill contaminating normal flora. Obtaining a neutral ph is critical for the survival of mycobacteria, so unlike the traditional method of specimen pre-processing, the MycoDDR NaOH Reagent A contains a ph indicator that turns the solution from blue to colorless, at near neutral ph, with the addition of Buffer B. This solution is then subjected to centrifugation and decanted. Failure to adequately neutralize the solution will result in additional death of mycobacteria during the centrifugation and resuspension steps.

3 Consequently, the solution ph should be tightly regulated and monitored during this pre-processing procedure to prevent excessive loss of mycobacteria viability due to chemical processing (Figure 1). In order to address this concern, the MycoDDR reagents were developed to facilitate the recovery of viable mycobacteria throughout the preprocessing procedure by providing greater neutralization efficiency and a visual indication that the NALC-NaOH solution has been completely neutralized by changing the color of the solution from blue to colorless (Figure 2 and 3). MycoDDR : Design, Technology, and Advantages The MycoDDR product line is designed based on the NALC- NaOH procedure published by Kubica et al. 7. The mucolytic compound, NALC, is combined with a NaOH in a sodium citrate solution to digest the mucus while the high ph of the NaOH kills any contaminating bacteria. As previously mentioned, the high ph of this solution can also kill Mycobacterium spp. after minutes, which makes the timing of the digestion/decontamination process critical and equally critical that the solution be brought back to a neutral ph as quickly as possible in order to facilitate the recovery of viable mycobacteria. The MycoDDR NaOH Reagent A includes a ph indicating reagent that changes from blue at basic ph, to colorless at near neutral ph (Figure 3). This allows the laboratory technologist to visually titrate the solution using the included Buffer B. This visual indicator of neutralization, in addition to the increased buffering capacity of the Buffer B, facilitates the critical timing needed for survival of the mycobacteria present in the sample. This solution is then subjected to centrifugation and decanted. The resulting uniform specimen pellet is then re-suspended in and is a ph of between 6.8 and 7.2; making it optimal for automated growth detection systems. Supports Samples up to 10 ml The MycoDDR is able to support specimens up to 10 ml due to the advanced buffering capacity of the neutralization buffer. This is a substantial advantage compared to other products on the market that have a maximum sample size limit of 6-7 milliliters. When a sample is above this lower sample size limit, labs must split the sample and digest/decontaminate both of the resulting samples. Splitting samples doubles the amount of reagents used, and forces labs to perform an extra step in the treatment process this drastically increases the material costs and labor costs associated with the digestion/decontamination process. Therefore, a higher maximum sample size limit is extremely advantageous to laboratories, as it will limit the number of samples that need to be split. Easy to Process Specimens Containing Blood Treating bloody specimens with the MycoDDR is easy because of the unique, blue color of the NALC-NaOH solution. When the NALC-NaOH is added to a sample specimen containing blood, the solution turns purple (Figure 4). Then as the neutralization buffer is added, it is easy to see the color change from purple to light pink. At this point, the lab technician knows the solution has been properly neutralized. Figure 4 - MycoDDR facilitates processing of bloody specimen samples. (A) Addition of the blue MycoDDR NaOH Reagent A to bloody sample specimens results in a purple solution that turns a light pink with the addition of Buffer B. (B) The bright pink color of other products on the market does not provide an adequate color change with the addition of neutralization buffer. Figure 3- MycoDDR Buffer B allows visualization of accurate neutralization. The MycoDDR reagent set allows the NaOH neutralization step to be visualized as (A) addition of Buffer B turns the solution from blue to (B) colorless. Other digestion/decontamination products on the market that utilize an integrated ph indicator have a pinkish- red NALC-NaOH reagent. This is problematic when dealing with bloody specimens as the NALC-NaOH reagent turns the color of the solution to pink/red, which is the same color that is achieved when the neutralization buffer is added. A video demonstrating this comparison can be found at: Color Comparison of NaOH on Videos tab

4 Table 1: Comparison of digestion/decontamination products currently on the market. Features and Benefits Competitor #1 Competitor #2 ph Indicator Pink to Red ne Blue Indicates Bloody Samples Max Sample Size Compatible with Molecular Assays Gamma Irradiation of Packaged Buffers Conclusions ---- Potential Interference ---- Interference 6-7 ml 10 ml 10 ml Tuberculosis (TB) is a serious disease that affects a significant portion of the world s population and is one of the leading causes of death in developing countries. Diagnosis of TB is important not only for correct treatment of the disease but also for containment of the highly contagious infected individual. Processing of the patient sample is a critical step in facilitating the diagnosis of TB. The MycoDDR product line was developed as an exceptionally reliable specimen processing system that can be used to digest and decontaminate patient specimens in an easy and affordable manner. It provides a number of benefits over other products on the market including improved visualization of sample ph, ability to process higher volume samples, and decreased time to positive positive culture 15. PrepBLUE vs. Trident To give all types of labs the opportunity to use the highest quality digestion/decontamination reagents, IMMY has developed two packaging options for MycoDDR : MycoDDR Trident - Convenient packaging with the least amount of work on the lab MycoDDR PrepBLUE The economical choice, but preparation work is required Table 2: PrepBLUE vs. Trident. Features PrepBLUE Trident Capable of rigid ph Control Individually Packaged Buffers Dried chemical packets for Buffer B and Preparation Work Moderate ne Digestant/Decontaminate NaOH Reagent A and NALC Buffer B NaOH Reagent A and NALC Buffer B Additional Materials MycoDDR Training Video

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