High-precision automatic identification method for dicentric chromosome images using two-stage convolutional neural network.

Dicentric chromosome analysis is the gold standard for biological dose assessment. To enhance the efficiency of biological dose assessment in large-scale radiation catastrophes, automatic identification of dicentric chromosome images is a promising and objective method. In this paper, an automatic identification method for dicentric chromosome images using two-stage convolutional neural network is proposed based on Giemsa-stained automatic microscopic imaging. To automatically segment the adhesive chromosome masses, a k-means based adaptive image segmentation and watershed segmentation algorithm is applied. The first-stage CNN is used to identify the dicentric chromosome images from all the images and the second-stage CNN works to specifically identify the dicentric chromosome images. This two-stage CNN identification method can effectively detects chromosome images with concealed centromeres, poorly expanded and long-armed entangled chromosomes, and tricentric chromosomes. The novel two-stage CNN method has a chromosome identification accuracy of 99.4%, a sensitivity of 85.8% sensitivity, and a specificity of 99.6%, effectively reducing the false positive rate of dicentric chromosome. The analysis speed of this automatic identification method can be 20 times quicker than manual detection, providing a valuable reference for other image identification situations with small target rates.

Rapid and automatic detection of micronuclei in binucleated lymphocytes image.

Cytokinesis block micronucleus (CBMN) assay is a widely used radiation biological dose estimation method. However, the subjectivity and the time-consuming nature of manual detection limits CBMN for rapid standard assay. The CBMN analysis is combined with a convolutional neural network to create a software for rapid standard automated detection of micronuclei in Giemsa stained binucleated lymphocytes images in this study. Cell acquisition, adhesive cell mass segmentation, cell type identification, and micronucleus counting are the four steps of the software's analysis workflow. Even when the cytoplasm is hazy, several micronuclei are joined to each other, or micronuclei are attached to the nucleus, this algorithm can swiftly and efficiently detect binucleated cells and micronuclei in a verification of 2000 images. In a test of 20 slides, the software reached a detection rate of 99.4% of manual detection in terms of binucleated cells, with a false positive rate of 14.7%. In terms of micronuclei detection, the software reached a detection rate of 115.1% of manual detection, with a 26.2% false positive rate. Each image analysis takes roughly 0.3 s, which is an order of magnitude faster than manual detection.

Influence of fine particulate matter and its pure particulate fractions on pulmonary immune cells and cytokines in mice.

Particulate matter with a diameter ≤2.5 µm (PM2.5) has a complex composition and has been associated with the incidence of cardiopulmonary disease and premature death in humans. However, whether pure particulate fractions of PM2.5 (PPP2.5), which are composed primarily of carbon, are responsible for the toxicity caused by ambient particulate matter (original PM2.5 particles, OPP2.5) is currently unclear. The present study assessed the acute toxic effects of OPP2.5 sampled in Beijing, China and of its PPP2.5 fraction in male BALB/c mice. The mice were intratracheally instilled with a single dose of aerosolized OPP2.5 or PPP2.5. Blood, lungs and bronchoalveolar lavage fluid were collected after 24 h for histopathology, flow cytometry and the measurement of pro-inflammatory cytokines/chemokines and other biochemical factors. Both OPP2.5 and PPP2.5 caused acute toxicity, particularly inflammatory responses, including an increase in the levels of pro-inflammatory cytokines and an accumulation of numerous immune cells in the lungs. OPP2.5 induced a stronger inflammatory response than PPP2.5. The complex components adsorbed into the solid core granules of OPP2.5 and the granules themselves contributed to the toxic effects.

Inhalation Study of Mycobacteriophage D29 Aerosol for Mice by Endotracheal Route and Nose-Only Exposure.

BACKGROUND: Lytic mycobacteriophage D29 has the potential for tuberculosis treatment including multidrug-resistant strains. The aims of this study are to investigate deposition and distribution of aerosolized phage D29 particles in naive Balb/C mice, together with pharmacokinetics and evaluation of acute lung injury. METHODS: Pharmacokinetics and BALF (bronchoalveolar lavage fluids) were analyzed after administration of phage D29 aerosols by endotracheal route using Penn-century aerosolizer; Collison 6-jet and Spinning top aerosol nebulizers (STAG) were used to generate phage aerosols with different particle size distributions in nose-only inhalation experiments. After exposure, deposited amounts of phage D29 particles in respiratory tracts were measured, and deposition efficiencies were calculated. A typical path deposition model for mice was developed, and then comparisons were made between predictions and experimentally measured results. RESULTS: Approximately 10% of aerosolized phages D29 reached lung of mouse for pulmonary delivery, and were completely eliminated until 72 h after administration. In contrast, about 0.1% of intraperitoneal injected phages reached the lung, and were almost eliminated at 12 h time point. The inflammation was hardly observed in lung according to the results of BALF analysis. The CMADs (count median aerodynamic diameters) of generated aerosol by Collison and STAG nebulizer were 0.8 µm and 1.5 µm, respectively. After nose-only exposure, measured deposition efficiencies in whole respiratory tract for 0.8 and 1.5 µm phage particles were below 1% and 10%, respectively. Predictions of the computer deposition model compared fairly well with experimentally measured results. CONCLUSIONS: This is the first systematic study of phage D29 aerosol respiratory challenge in laboratory animals. It provides evidence that aerosol delivery of phage D29 is an effective way for treating pulmonary infections caused by Mycobacterium tuberculosis. This research will also provide important data for future inhalation experiments.

[Concentration and Particle Size Distribution of Microbiological Aerosol During Haze Days in Beijing].

In this study, we evaluated the bacterial, fungal aerosol concentration, and particle size distribution using microbiological aerosol sampler, and analyzed the particles count concentration of PM1.0, PM2.5, PM5.0 and PM10.0 using aerodynamic particle sizer during clear and haze days in Beijing during Jan 8th, 2013 to Feb 4th, 2013. The concentration of bacterial, fungal aerosol, air particulate matter and aerosol distribution were compared between haze days and clear days. Our results indicated that the proportion of fungal particles smaller than 5 micron, which could deposit in lungs or deeper regions, was much higher than bacterial particles. The biological concentration of bacteria and fungi were higher in clear days than in haze days, and there was no statistic difference of the microbiological aerosol distribution. The concentration of air particulate matter were higher in haze days than in clear days, PM10 was the main particulate matters both in clear days and haze days.

Assessment of the risk of infectious aerosols leaking to the environment from BSL-3 laboratory HEPA air filtration systems using model bacterial aerosols.

To assess the risk of infectious bacterial aerosols leaking to the environment, the filtration efficiency of a biosafety level 3 (BSL-3) laboratory high-efficiency particulate (HEPA) filter was investigated using the aerosolized bacteria Serratia marcescens. The aerosol size was measured using an Andersen sampler. Eight first stage HEPA filters (numbered 1-8) were distributed in contaminated labs and exhausts from each of the first stage HEPA filters were aggregated and filtered through one second stage HEPA filter before being released to the environment. In total, 8 first-stage and 1 second-stage HEPA filters from the BSL-3 air purification system were analyzed. No S. marcescens was detected in first stage filters 1, 2, 4, 5, 7 and 8 and the second stage HEPA filter. The filtration efficiencies against aerosolized S. marcescens were >99.9999%. First stage filter numbers 3 and 6 had filtration efficiencies of 99.9825% and 99.9906%, respectively. When filter number 3 was replaced by a new filter and the bracket for filter number 6 was sealed, no aerosolized S. marcescens was detected in the filtered air. Our work suggests that the BSL-3 laboratory HEPA filter air purification system is effective against bacterial aerosols, with little to no bacterial leakage into the environment.

Assessment the protection performance of different level personal respiratory protection masks against viral aerosol.

New viral disease such as SARS and H1N1 highlighted the vulnerability of healthcare workers to aerosol-transmitted viral infections. This paper was to assess the protection performance of different level personal respiratory protection equipments against viral aerosol. Surgical masks, N95 masks and N99 masks were purchased from the market. The masks were sealed onto the manikin in the aerosol testing chamber. Viral aerosol was generated and then sampled simultaneously before and after the tested mask using biosamplers. This allows a percentage efficiency value to be calculated against test phage SM702 aerosols which surrogates of viral pathogens aerosol. At the same time, the masks face fit factor was determined by TSI8020. The viral aerosol particles aerodynamic diameter was 0.744 µm, and GSD was 1.29. The protection performance of the material of all the tested masks against viral aerosol was all >95 %. All the five surgical masks face fit factor were <8. F model N95 mask and H model N99 mask face fit factor were all >160. G model N95 mask face fit factor was 8.2. The protection performances of N95 or N99 masks were many times higher than surgical mask when considering the face fit factor. Surgical masks cannot offer sufficient protection against the inhalation of viral aerosol because they cannot provide a close face seal.

Purification and concentration of mycobacteriophage D29 using monolithic chromatographic columns.

Bacteriophages are used widely in many fields, and phages with high purity and infectivity are required. Convective interaction media (CIM) methacrylate monoliths were used for the purification of mycobacteriophage D29. The lytic phages D29 from bacterial lysate were purified primarily by polyethylene glycol 8000 or ammonium sulphate, and then the resulting phages were passed through the CIM monolithic columns for further purification. After the whole purification process, more than 99% of the total proteins were removed irrespective which primary purification method was used. The total recovery rates of viable phages were around 10-30%. Comparable results were obtained when the purification method was scaled-up from a 0.34 mL CIM DEAE (diethylamine) monolithic disk to an 8 mL CIM DEAE monolithic column.

[Evaluation of the protective performance of a positive pressure bio-protective clothing against viral aerosol].

OBJECTIVE: To evaluate the protective performance of a positive pressure bio-protective clothing against viral aerosol. METHODS: The suspension of indicating virus phage Phi-X174 was made for viral aerosol generating in a hermetic cabin. The diameter of viral aerosol particles were measured with a aerodynamics size analyzer. By adjusting the inner humidity of the cabin, the protective efficiency of the positive pressure bio-protective clothing against viral aerosol in high and low windshield conditions was determined with Andersen six-stage air sampler sampling and plage forming unit (PFU) counting, respectively. RESULTS: The mass median diameter of Phage Phi-X174 aerosol particles was about 0.922 µm and the background concentration is beyond 2 × 104 particles/m³. The protective efficiency of the clothing against phage Phi-X174 aerosol particles was above 99.9% under different test conditions with the range of viral aerosol concentration between 0 - 23 PFU/m³. Airflow (P = 0.84), environment humidity conditions (P = 0.33) and sampling time (P = 0.07) did not affect the protective efficiency statistically. CONCLUSION: The positive pressure bio-protective clothing provided a relatively high efficiency against phage Phi-X174 aerosol regardless of airflow rate, environment humidity and sampling time.

Impact of relative humidity and collection media on mycobacteriophage D29 aerosol.

This study was conducted to evaluate the effect of aerosol generation, methods of sampling, storage conditions, and relative humidity on the culturability of the mycobacteriophage D29. The lytic phage D29 can kill Mycobacterium tuberculosis, and the phage aerosol can be treated as a potential tool for tuberculosis treatment. The culturability of D29 was tested using a test chamber designed for the bioaerosols research against three spray liquids (deionized water, phosphate-buffered saline [PBS], and normal saline), four collection media (suspension medium [SM], nutrient broth, PBS, and deionized water), two sampling systems (the all-glass impinger AGI-30 and the Biosampler) and across a range of humidities (20 to 90%). The effect of storage conditions on the culturability of collected sample was also evaluated for the AGI-30 impinger. The results proved that viable phage D29 particles generated by deionized water were approximately 30- and 300-fold higher than PBS and normal saline, respectively. As collection media, SM buffer and nutrient broth were observed to yield a higher number of plaques compared to PBS and deionized water. No difference was observed in collection efficiency between AGI-30 and Biosampler with two detection methods (culture-based technique and real-time PCR). The culturability of collected D29 in SM buffer or nutrient broth can be maintained up to 12 h irrespective of storage temperature. Relative humidity was found to strongly influence airborne D29 culturability which is 2- to 20-fold higher in low humidity (25%) than medium (55%) or high (85%) humidity. This research will help identify the optimal means for the application of D29 aerosol in animal inhalation experiments.

Effects of different sampling solutions on the survival of bacteriophages in bubbling aeration.

The present study focuses on the effects of three different sampling solutions, namely distilled water, phosphatic buffer solution, and suspension medium (SM), adding antifoam or not, on the survival probability of several different bacteriophages (EcP1,PhiX174,SM702, and F2) as surrogates for the mammalian viruses in the bubbling process. AGI-10 impinger was used as the representative for all the impingers which would bubble during operation. It was found that the survival probability of the same bacteriophage bubbling with different sampling solutions was different except that there was no significant difference observed for the bacteriophage F2. The use of SM as the collection fluid was relative to a high survival probability for the four bacteriophages. And the endurance or resistance of different kinds of bacteriophages in the same sampling solution was different. We conclude that SM is a promising sampling solution for liquid impingers in the process of sampling phages.

Determining the filtration efficiency of half-face medical protection mask (N99) against viral aerosol.

Hospital-based outbreaks of severe acute respiratory syndrome (SARS) have once again highlighted the vulnerability of healthcare workers (HCWs). Use of personal respiratory protective equipment was the main method used by HCWs to avoid nosocomial transmission. This paper describes the technology used to evaluate the filtration efficiency of the half-face medical protection mask (N99), manufactured by Firmshield Biotechnology, against viral aerosol. Viral aerosol was generated and then sampled simultaneously with and without the test mask. This enables a percentage efficiency value to be calculated against test phage f2 aerosols (surrogates of viral pathogen aerosols). At the same time the mask filtration efficiency against NaCl particle aerosol was determined by use of TSI8130 equipment and face-fit factor was tested by use of TSI8020 equipment. The half-face medical protection mask (N99) evaluated by use of the viral aerosol had a filtration efficiency >99%. The mask filtration efficiency against NaCl particle aerosol was 99.634 ± 0.024% and it had a good face-fit factor. This half-face medical protection mask (N99) can protect the wearer from viral aerosol disease transmission. The test method can be used to assess filtration efficacy against viral aerosol of masks used for respiratory protection.

[A methodological study on testing and evaluating of filtration efficiency of canister against microbial aerosol].

OBJECTIVE: To establish a testing and evaluating method for filtration efficiency of the canister against microbial aerosol. METHODS: Serratia marcescens aerosol served as model of bacterial aerosol, Bacillus subtilis var niger aerosol as model of spores aerosol, bacteriophage f(2) aerosol as model of viral aerosol. Employing the microbial aerosol testing platform was established in lab, models of microbial aerosol generated artificially were sampled quantitatively by air samplers before and after filtrating by canisters, respectively. Filtration efficiency was determined by the concentration of microbial aerosol in the air sample before and after filtrating. The four canisters of 1-1, 1-2, 1-3, 1-4 were tested for the filtration efficiency against Serratia marcescens, Bacillus subtilis var niger and phage f(2) aerosol. The two canisters of 543 and 544 canisters equipped with active carbon were tested for the filtration efficiencies against Serratia marcescens aerosol. RESULTS: The filtration efficiency of 1-1, 1-2, 1-3 canisters against Serratia marcescens, Bacillus subtilis var niger and phage f(2) aerosol was 100.000%. The filtration efficiency of 1-4 canister filtration efficiency against Bacillus subtilis var niger spores aerosol was 99.997% and efficiency of the other two aerosol was 100.000%. The filtration efficiency of the two canisters of 543 and 544 to those attached with active carbon against Serratia marcescens aerosol was 100.000%. CONCLUSION: The testing method might be used to evaluate the protective performance of the canister against microbiological aerosol. The effect of the canisters (including those equipped with active carbon) against microbiological aerosol should be reliable.

Detection of viral aerosols by use of real-time quantitative PCR.

PCR quantification is regarded as one of the most promising techniques for real-time identification of bio-aerosols. We have, therefore, validated a QPCR assay for quantification of a viral aerosol sample using the double-stranded DNA-binding dye SYBR green I, an economical alternative for quantification of target microorganisms. To achieve this objective we used mycobacteriophage D29 as model organism. Phage D29 aerosol was produced in an aerosol cabinet and then collected by use of an AGI liquid sampler. A standard curve was created by use of purified genomic DNA from the phage in liquid culture of known concentration measured by titration. To prevent false-positive results caused by formation of primer-dimers, an additional data-acquisition step was added to the three-step QPCR procedure; the new technique was called four-step QPCR. The standard curve was then used to quantify the total amount of phage D29 in liquid culture and aerosol samples. For liquid culture samples there was no significant difference (P > 0.05) between results from quantification of the virus using double-agar culture and QPCR. For aerosol samples, however, the result determined by the QPCR method was significantly (P < 0.05) higher than that from the double-agar culture method. The four-step SYBR green I QPCR method is a quick quantitative method for mycobacteriophage D29 aerosol. We believe that QPCR using SYBR green I dye will be an economical method for detection of airborne bio-aerosols.

[Isolation and characterization of Serratia marcescens phage].

OBJECTIVE: To isolate the bacteriophage of Serratia marcescens (8039) from sewage, and to study on its biological characteristics. METHODS: We used Serratia marcescens (8039) as the host to isolate phage from raw sewage from the Sewage Treatment Center of Hospital. Phage SM701 was examined in electron microscope. The genome of SM701 was extracted and the size of its nucleic acid was identified with restriction enzyme analysis. Phage isolated was subjected to plaque forming unit (PFU) assay using double layer agar medium plate method and morphological properties of plaque was observed, and finally optimal MOI multiplicity of infection and one-step growth experiments were carried out. RESULTS: Phage SM701 specific to Serratia marcescens (8039) was isolated successfully from the raw sewage. SM701 had an isometric polyhedral head (about 64 nm in diameter) and a long noncontractile tail (about 143 nm long). The nucleic acid could be cut off by dsDNA restriction enzyme BamH I or HindIII and its complete size was about 57kb. The plaque of SM701 was transparent about 1mm in diameter at the 12th hour. When MOI equaled 10, the number of phage offspring was higher. One-stepgrowth kinetics was determined according to the results of one-step growth experiment, which showed that the latent period was about 30 min, the rise period was about 100 min, and the average bust size was about 63 pfu/cell. CONCLUSION: Phage SM701 belonged to tailed family: siphoviridae and lytic bacteriophage. It was quite easy to observe and count the plaques due to existing color differences between lytic and nonlytic zone.