Abstract
M. Tech.
Internationally, 9.2 million new cases and 1.7 million deaths occurred from tuberculosis (TB) in 2006. The vast majority of TB deaths occurred in the developing world, that is, Asia and Africa (WHO, 2008). The risk of infection is worsened by overcrowding of healthcare facilities which share re-circulated air without high efficiency particulate arrestance (HEPA) filtration or effective decontamination devices. With emerging and re-emerging infectious agents, the importance of the microbial influence on indoor air quality is gaining momentum around the world. The transmission of Mycobacterium tuberculosis (MTB) is a recognized occupational hazard and the mode of airborne transmission in risk settings needs to be investigated.
The current study examined the efficacy of Polymerase Chain Reaction (PCR) for the early detection of airborne MTB using three types of filters: Polytetrafluoroethylene (PTFE), Polycarbonate (PC) and Gelatine, and a sedimentation gel (impaction method). A total of 520 samples, 68 internal positive controls and 68 internal negative controls were tested using two different PCR detection methods. The four different sampling types were each exposed to samples containing an avirulent strain of MTB (H37Ra) and negative controls exposed to aerosolized distilled water in an uncontrolled environment. The air was filtered at a flow rate of 2.5 L/min for a specified time. The filter membranes and sedimentation gel were removed from their respective holders, washed and analysed using conventional and real time (RT) PCR. An additional step using magnetic bead separation was used to assess its performance in overcoming inhibition.
The sampling methods used included an in - house preparation of sedimentation gel. This sampling method was used in a study by Vadrot and colleagues in 2004 for detection of airborne MTB using the PCR method. Commercially available filters that were used as sampling
methods included PTFE, PC and Gelatine. The detection methods included conventional PCR which detected the MTB complex in three hours, RT-PCR which detected MTB species in 70 minutes and RT-PCR coupled to magnetic bead separation (RT-PCR M) which detected MTB species in 90 minutes. The magnetic bead separation method purified the nucleic acids in the sample by eliminating the inhibitors that were present.
The current study showed that by using the magnetic bead capture assay in conjunction with RT-PCR, gave excellent results of 100% sensitivity and 100% specificity when using PTFE, PC and Gelatine sampling methods. The sedimentation gel showed results of 90% sensitivity and 90% specificity. The Gelatine sampling method results showed 100% inhibition with conventional PCR. In conclusion, the use of conventional PCR is limiting for the detection of airborne MTB, possibly due to inhibition factors.
In addition, the PTFE filter demonstrated excellent results for all detection methods used. The sedimentation gel did not perform well with PCR and RT-PCR, however gave excellent results with RT-PCR M. The PC filter can be considered the second sampling method of choice after PTFE filter, showing superior results for all diagnostic parameters using RT-PCR M, followed by RT-PCR and then PCR.
The greatest application of using this validated method will be in the area of infection control. Environmental practitioners and occupational hygienists would be able to use this method to evaluate environmental control measures and monitor the air quality in healthcare facilities and other workplaces.