| Staffs engaged in the process of diagnosis and researching of infectious pathogenic microorganisms are easy to get infected due to occupational exposure. In human infectious diseases, tuberculosis causes serious harm to human health, public health and socio-economic. Mycobacterium tuberculosis is Class2Biological Hazards pathogen and is the infectious agent of tuberculosis spread by aerosol. Tuberculosis (TB) diagnosis and treatment mainly rely on laboratory isolation and culture of mycobacterium tuberculosis. In TB diagnosis and research process, laboratorians suffer from occupational exposure due to direct contaction with M.tb,which results in Laboratory-Acquired Infection(LAI).The incidence of infection is3to9times than in other operations. Figuring out in which part of the M.tb laboratory could the pollution take place and what operations could lead to more serious pollution is of great significance to prevent LAI.This thesis is divided into three parts. First of all, through the establishment of detection technology with high specificity and strong sensitivity we could detect and identify the key parts and key links of the BSL-3laboratory micro-environmental surface contamination in the M.tb experimental activities; we found out the key parts of safety protection and disinfection, and then decreased pollutions on micro-environment caused by M.tb experimental activities. Thereby reduced the laboratorians’ risk of getting LAI. Furthermore, we screened out the suitable disinfectants for M.tb experimental activities and terminal clearance in M.tb laboratory, and then effectively removed of the M.tb residues in experimental activities in order to ensure the biological safety of laboratorians. Finally, through the connection of cycle disinfection fan with HEPA exhaust-air filter in the BSL-3laboratory’s core area, and the confirmation of the steam sterilization program in the same time, we realized in situ disinfection of the BSL-3laboratory’s HEPA exhaust-air filter. From starting with both pollution reduction and decontamination, we could effectively control the Laboratory-Acquired M.tb Infection. Thereby the BSL-3laboratory biosafety could be ensured.The first part of the study starts from monitoring the key parts and key links of BSL-3laboratory’s micro-environmental surface contamination during M.tb experimental activities. Firstly, we verified the specificity and sensitivity of target genes, which were commonly used in clinical tuberculosis test and diagnosis, in the detection of M.tb DNA. These target genes include16SrDNA, rpsL, IS6110, sigA, rpoB and katG. They can be used to detect the genomic DNA of M.tb, BCG and M.S, but they may not be used to monitor M.tb specifically. So these genes are not suitable for detecting pollutions on micro-environment surface caused by M.tb. Secondly, by comparing the genome sequences of M.tb, BCG and MS by Synteny MAP software, we could find the RD District, the unique region of M.tb complex. Through designing primers, we tested the specificity and sensitivity of RD District, which was used as a target gene in detecting M.tb. We screened out that Rv1980c (mpt64), Rv3875(ESAT-6) could be used to detect M.tb. And they were more sensitive than other RD District genes. The minimum amount of M.tb DNA that could be tested was5.0×101-102copies. So, we established detection technologies of Loop-mediated isothermal amplification (LAMP) based on nucleic acid amplification to detect micro-environment surface pollution. The minimum amount of M.tb DNA that could be tested was5copies. Furthermore, we used real-time quantitative PCR to detect micro-environment surface pollution of M.tb, so we could conduct relative quantitative analysis of the pollution status. The minimum amount of M.tb DNA that could be tested was5copies by this method.After that, we utilized M.tb viable bacterial culture to verify the sampling process of BSL-3laboratory microenvironment surface. The efficiency was up to70～90%.The sampling method was of high credibility. We applied this sampling method to take samples of the surface that could possibly be polluted during the M.tb experimental activities in the BSL-3laboratory. These include the operation surface of the biological safety cabinets and surface of personal protective equipment. Experimental activities include the conventional operation with M.tb bacteria, such as opening the cover of the centrifuge tube, bacteria grinding and centrifugation, turbidimetry of bacterial concentrations and bacteria preservation. The experimental operations with infected animals include animal anatomy, bedding replacement and gavage, organ grinding, vaccinations, etc. After extracting genomic DNA of these samples, we used PCR, LAMP, real-time PCR and other methods we had mentioned above to do the detection. The contaminated areas mainly include operating surface of biological safety cabinet, grid tray of the front ventilation, gloves and cuff for personal protection and feeding cage of infected animals. Therefore, these prompt that when carrying out the M.tb associated experimental procedure, we need to strengthen the protection of such areas or parts, and disinfect them promptly.In addition, we used BCG strains to imitate centrifuge tube overflow accident in the biological safety cabinet to detect micro-environment pollution status. The results show that the overflow point, surrounding area, gloves, anti-dress forearm for personal protection and the operator’s chest were BCG positive. Moreover, through bacterial cultures we studied the survival of M.tb in biological safety cabinet:2.26×104CFU M.tb was cultured in the biological safety cabinet, after7days’ arefaction and ventilation7.5×102CFU M.tb were still alive. This suggests that M.tb has high survival ability in laboratory micro-environment. Therefore, the laboratory environment should be completely disinfected after experiment activities.The second part of the study tested the efficiency of disinfectant commonly used to kill M.tb in the pathogenic microbiology laboratory. Using disinfectants is an important mean of eliminating contamination in M.tb BSL-3laboratory. Membrane (0.45μm) filtration quantitative bactericidal tests as well as direct effects were utilized to study the bactericidal effect of disinfectants (ethanol, sodium hypochlorite, hydrogen peroxide, Medicom SafeSpore disinfectant) on M.tb. The results showed that:the M.tb killing log value of75%ethanol, sodium hypochlorite containing1000mg/L available chlorine and SafeSpore disinfectant was up to5.0within five minutes. They all could be used to clear M.tb pollution in BSL-3laboratory. Ethanol is easy to volatilize, the concentration will decrease during use. The results showed that the killing log value of37.5%ethanol was3.49in5minutes, and was up to5.68in10minutes. So it is recommended to use freshly prepared disinfectant. The M.tb killing log value of7%hydrogen peroxide in10minutes was up to5.38. But due to its strong oxidizing reaction and strong stimulating effects on human mucosa, it is not recommended to be used as a routine BSL-3laboratory disinfectant. Available chlorine solutions possess oxidative corrosion, and they could lead to laesio enormis on the equipment. So it is also inadaptable to be used as a conventional disinfectant in BSL-3laboratory. The M.tb killing log value of Medicom SafeSpore disinfectant was up to4.86in1minute, and5.77in5minutes. The bactericidal effect was obvious. But it may not be largely use due to its high price. For these reasons, we used75%ethanol as the routine disinfectant to kill mycobacterium tuberculosis in BSL-3laboratory. Chlorine solutions containing1000mg/L available chlorine is mainly used in the disinfection of the BSL-3laboratory emergencies.The third part of the study verified the in situ disinfection of HEPA exhaust-air filter, an important defensive line to ensure the biosafety of BSL-3laboratory facilities. HEPA exhaust-air filter in the core area of BSL-3laboratory is the main line of defense to ensure the non-leakage of infectious aerosols generated in the experimental procedure. In situ disinfection of the HEPA filter is needed in order to protect the safety of personnels. This study designed and produced a cycle disinfection prototype (with the function of formalin volatilization, gas humidification and air circulation supply). We connected the prototype with the disinfection interface of HEPA filter’s exhaust box. And then the steam cycle of formalin was driven through the HEPA filter by air circulation supply system. Thus the in situ disinfection was realized. The results showed that: Formalin vapor concentration at room temperature was16mg/L, and the relative humidity was70%. After2hours continuous fumigation cycle running through, Staphylococcus aureus and Mycobacterium smegmatis, which was arranged in exhaust cabinets of the HEPA filter and used to indicate the disinfection, could be killed. But the Bacillus subtilis and Bacillus stearothermophilus couldn’t be eradicated. After12hours continuous fumigation cycle running through, Staphylococcus aureus, Mycobacterium smegmatis, Bacillus subtilis and Bacillus stearothermophilus were completely disinfested. As a consequence, we achieved the in situ disinfection of HEPA exhaust-air filter in both equipment and methodology.This thesis defined the areas susceptible to be contaminated by infectious aerosol, thus provided a basis for personal protection and effective site clearing. Suitable disinfectants for mycobacterium tuberculosis BSL-3laboratory were screened out simultaneously as disinfections for laboratory decontamination and biosecurity ensurance. In addition, we also designed and manufactured a disinfection prototype for in situ disinfection of the HEPA exhaust-air filter, and verified the effectiveness of disinfection program as well, thus provided device support for environmental biosafety insurance. |
PDF Full Text Request