key: cord-008917-ek8rp4kh authors: caul, e.o.; ashley, c.r.; clarke, s.k.r.; egglestone, s.i. title: coronavirus-like particles in diarrhoea stools date: 1980-12-06 journal: lancet doi: 10.1016/s0140-6736(80)92509-x sha: doc_id: 8917 cord_uid: ek8rp4kh nan s:r,&mdash;dr dourmashkin and colleagues (nov. i, p. 971) report that they have seen pleomorphic coronavirus-like particles in a specimen of human faeces and postulate that these may have derived from an intestinal yeast-like organism and suggest blastocystis (now believed to be a protozoon 1). [2] [3] [4] [5] we have described coronavirus-like particles in human faeces. these are quite different from the cellular material with ill-defined fringes3 which is commonly present in human faecal material. to date one strain of the coronavirus-like particle has been shown to replicate in cell and organ culture systems 5 producing ultrastructural changes which are indistinguishable from those produced by a bovine coronavirus in a similar intestinal organ culture system. 6,7the sectioned particles seen inside the cells were typical of coronaviruses. we concluded that the particles were enteric coronaviruses. a common feature of these enteric coronaviruses is their pleomorphism, a finding which is not unusual with enveloped rna viruses, especially before they are adapted to in vitro culture. the above evidence of the viral nature of these particles must be considered against the findings of dourmashkin et al., who concluded that the particles they saw were probably not viruses. this conclusion was based on an interpretation of structural relationships obtained from a sectioned deposit of ultracentrifuged crude faecal suspension. we believe that interpretation of findings from these pilot experiments is not only difficult but also inappropriate to the question posed. sir,&mdash;dr berkowitz and colleagues (oct. 4, p. 752) conclude that oral contraceptives do not increase the risk of proiiferative trophoblastic sequelae when taken after the evacuation of hydatidiform mole and before gonadotrophin values have fallen to normal. it is not clear how this conclusion can be drawn from a total of 50 patients "selected at random" from their files. its statistical significance would seem highly questionable. the relevance of their data might also be questioned on the grounds that this group report elsewhere 8 that they give cytotoxic drugs to all patients judged to be at risk of malignant sequelae even before the uterus is evacuated. such prophylactic therapy affects the issue in two ways. first, it ensures that a high proportion of mole patients receive potentially mutagenic cytotoxic agents during their childbearing period, whereas others think it desirable to avoid such exposure. secondly, it renders their series unsuitable for comparison with series of patients not given prophylactic therapy. the analysis of 611 consecutively registered cases of hydatidiform mole on the registry of the royal college of obstetricians and gynaecologists suggested that taking oral contraceptives before gonadotrophin remission had been achieved increased the risk of invasive mole or choriocarcinoma almost 3-fbld compared with those not taking oestrogens and progestagens.9 these data are not conclusive since patients were not randomised to "pill" and "no pill" groups but they would seem to be a better basis for mole follow-up policy than the new england data. sir,-dr gorchein (july 19, p. 152) comments on our letter.l perhaps we should have pointed out more clearly that we knew that induction of 6-aminolaevulinic acid synthetase is a dose related phenomenon. moreover, in general, there exists a large species variation in therapeutic or toxic effects of chemical substances, and additionally an interspecies difference exists in drug metabolism and pharmacological response, especially to liposoluble drugs 2-all of which points should be considered when extrapolating our findings in rats to man. nevertheless, we feel very strongly that those drugs which evoke a positive response in our rat model are potentially harmful in the hereditary porphyrias and should be avoided. we take exception to gorchein's suggestion that the safety of new drugs in the hereditary porphyrias should be evaluated by clinical trial. it would be irresponsible to subject a population at risk to potentially harmful agents. we have had a great deal of success, as evidenced by the remarkable decrease in the number of acute attacks,3 by education of affected subjects and their medical attendants about the dire consequences of exposure to potentially dangerous drugs. furthermore, good correlation exists between our clinical findings and those in our rat model with respect to the dangerous porphyrogenic drugs. sir,-dr hawkins and his colleagues in their study of thyroid microsomal antibodies (tma) (nov. 15, p. 1057) face a dilemma which bedevils much epidemiological research. this is the problem of studying individual subjects. 89 out of 160 subjects with tma had subclinical hypothyroidism (premyxoedema), according to hawkins and colleagues' criterion of a raised basal thyroid stimulating hormone (tsh) level. in our experience, 25% of patients with premyxoedema have a normal basal tsh but exaggerated response to thyrotrophin-releasing hormone (trh). 4 blastocystis hominis, an intestinal protozoan parasite of man coronavirus particles in faeces from patients with gastroenteritis recognition of human enteric coronaviruses by electron microscopy coronavirus propagated from patients with non-bacterial gastroenteritis further studies on human enteric coronaviruses replication of an enteric bovine coronavirus in intestinal organ cultures the human enteric coronaviruses 28% of euthyroid subjects with an exaggerated response to trh have neither thyroid antibodies nor a history of partial thyroid ablation (unpublished) charing cross hospital relationship of oral contraception to development of trophoblastic tumour after evacuation of a hydatidiform mole drug safety in porphyria the value of determining the plasma concentration of drugs in animals and man. fundamentals of drug metabolism and drug disposition porphyria and the dangerous life-threatening drugs association between exaggerated responsiveness to thyrotrophin-releasing hormone and hypercholesterolaemia key: cord-007131-q0cy9dej authors: shimazaki, yasuhiro; okubo, masaaki; yamamoto, toshiaki title: three‐dimensional numerical simulation of gas‐particulate flow around breathing human and particulate inhalation date: 2006-05-05 journal: aip conf proc doi: 10.1063/1.2204538 sha: doc_id: 7131 cord_uid: q0cy9dej it is important to predict the environment around the breathing human because inhalation of virus (avian influenza, sars) is recently severe worldwide problem, and air pollution caused by diesel emission particle (dep) and asbestos attract a great deal of attention. in the present study, three‐dimensional numerical simulation was carried out to predict unsteady flows around a breathing human and how suspended particulate matter (spm, diameter∼1 μm) reaches the human nose in inhalation and exhalation. in the calculation, we find out smaller breathing angle and the closer distance between the human nose and pollutant region are effective in the inhalation of spm. in recent years, decline of air quality becomes one of significant problems and people from all over the world are concerned as human safety [1] . there are two main types of air pollutant: particulate pollutant and gas pollutant. damages by sars [2] , avian influenza [3] , diesel emission particles [4] , and asbestos [5] are the good examples of particulate pollutant. many researches have been performed to deal with the increasing problems of such air pollutants [6] , [7] . however, few studies have been performed to address the theme of human with unsteady breathing [8] , [9] . two-dimensional numerical simulation was carried out, and the time-dependent pollutant concentrations were determined until this point [10] . in this paper, calculating area was advanced to three-dimension, real particle with size and mass. in the simulation, the breathing process alternated between inhalation and exhalation as the unsteady breathing at nose. the governing equations for flow are the mass conservation and the navier-stokes equations. in these equations, ρ is the density and stays constant in the simulations, p is the static pressure, and µ is the viscosity. u r is the velocity of the surrounding air. u, v, w are the velocity of each component. as a turbulence model, standard k-ε model was adopted. it involves solutions of transport equations for turbulent kinetic energy and its rate of dissipation. the model adopted in the simulation is based on launder and spalding [11] and is expressed as the transport equations for k are and the transport equations for ε are the motions of particulates were analyzed by solving the lagrangian equations for each particle. the equation of particle motion is ( ) where m p is the mass of particle and u r =(u, v, w) is its velocity vector, c d is the drag coefficient, ρ is the density of surrounding air, a p is the projected area of a particle, and g r is the gravity vector, respectively. for a spherical particle, a p =πd 2 /4 where d is the particle diameter. each of the force considered in the analysis is stokes drag with cunningham correction [5] , gravity, saffman lifting force, pressure gradient force, and brownian diffusion [12] . the following correlations are used to calculate the drag coefficient, for very small particles, when the size of particles is of the same magnitude as distance between gas molecules, slip occurs. cunningham correction factor to fluid drag on particles should be applied for rarefied fluid-particle flows. cunningham correction factor is the knudsen number, kn, may be evaluated based on molecular mean free path of the fluid and particle diameter as d kn where λ is the molecular mean free path. the saffman lifting force arises due to the surface pressure distribution on a particle in the presence of a velocity gradient in the flow field and plays an important role in determining particle deposition patterns in simulations. generalized saffman lifting force can be expressed as . 1 (11) the pressure gradient force on a particle is given by where p ∇ is the local pressure gradient. the brownian diffusion experienced by particles results from the impact of carrier fluid molecules on the particles and is significant for sub-micron particles. brownian diffusion can be evaluated as where k b is the boltzmann constant, ∆t is the time step, and r r is a gaussian random number. in three-dimensional numerical simulations, general-purpose software, cfd-ace+2004 (cfd research corp.) was used. the grid configuration is shown in figure 2. linear orthogonal grid is mainly used in the region distant from the human body. boundary-fitted grid is used near the human body. the maximum cell dimensions ranges from 1.1 mm to 325 mm. the total numbers of cell are 110077. the grids were generated by mesh generator cfd-viscart (cfd research corp.). the finite volume approach is adopted, and then governing equations are numerically solved over each of control volumes. a co-located cell-centered variable arrangement is employed. the checkerboard instability is circumvented by rhie and chow [13] . simplec scheme has been adopted for flow phase analysis, and is an enhancement to the well known simple algorithm [14] . where α is the breathing angle between the vertical lines and breathing velocity vector. velocity is a time function f (t) and breathing volume is a product of multiplying f (t) by area of nostrils as figure 3. as a pollutant source, a thousand particles are randomly placed in the box region (0.1 m 0.6 m 1.0 m). the particle region stay away l cm from the human nose, and we describe l as source distance. particles are set for initial condition as where u p , v p , w p are particle velocities. particles are totally absorbed at the nose, on walls, and human surface. initially, the fluid is considered as stationary fluid. we first examined the dependence between breathing angles and flow field by a way of comparison of the two dimensional model. breathing angles were set at 0, 30, 60, and 90 degree. l was 0.1 m at this time. the relationship among breathing angles and time to reach the nose and particle captured efficiency is shown in table 1. the particle captured efficiency tends to become higher and particles tend to reach the human nose faster as the breathing angle becomes smaller. particles reach the human in 7 s at the earliest, and the particle captured efficiency is 2.9% at the maximum, when breathing angle is 0 degree. no particles are captured with breathing angle of 60 degree or larger. when we look at the flow field, exhalation has an impact on the field as shown in figure 6. (a) (d). in the early period of exhalation, the respiration region is small and the flow is complicated. the respiration area becomes larger and the velocity becomes higher as the time goes on. the downward stream caused by exhalation remains during inhalation, which is to say the downward stream is small in scale, but always stays and particles go down. note that each breathing cycle seems self-dependence. we focus on the particulate pollutant in the air and three-dimensional numerical simulation was carried out. the effects of breathing angle and source distance ware clarified around human with unsteady breathing. the main results are summarized as follows: (1) exhalation has an impact on the flow field more than inhalation. (2) the particle captured efficiency tends to become higher and particles tend to reach the human nose faster as source distance becomes near from human body. particles reach the human nose when source distance is less than 0.2 m. no particles are reached beyond 0.3 m. the maximum 2.6% of particles are captured when the source distance is 0.05 m. (3) the particle captured efficiency tends to become higher and particles tend to reach the human nose faster as the breathing angle becomes smaller. the human can inhale particles only breathing angle of 30 degree or smaller. (4) throughout all simulations, particles are drawn from upper region of the human. when particles are captured, motions of particles away from the human body with right-left unsymmetrical vortexes are observed. otherwise, particles just move away and no vortexes are induced. sources and measures for indoor air pollution (shitsunai kukiosen no genin to taisaku), tokyo: the nikkan kogyo shimbun ltd proceedings of jsae symposium multiphase flows with droplets and particles key: cord-010022-e19vm7vp authors: riepenhoff‐talty, m.; barrett, h. j.; spada, b. a.; ogra, p. l. title: negative staining and immune electron microscopy as techniques for rapid diagnosis of viral agents date: 2006-12-16 journal: ann n y acad sci doi: 10.1111/j.1749-6632.1983.tb22228.x sha: doc_id: 10022 cord_uid: e19vm7vp nan in 1973, bishop and her colleagues visualized reovirus-like particles in thin sections of duodenum biopsied from a young child with diarrhea. shortly afterward, in england' and canada," similar particles were seen in feces using direct visualization with negative-stain electron microscopy. now, nearly ten years later, the impact of rotaviruses on human disease is becoming apparent. in 1972, an outbreak of gastroenteritis affected 50% of students and teachers in an elementary school in norwalk, ohio. particles 27 nm in diameter were visualized in the infectious stool filtrate. this filtrate was mixed with convalescent serum from a patient who had had the disease. the resultant particles were coated with antibody and clumped together. this technique was termed immune electron microscopy and the virus-like particles were called norwalk agent.* it is estimated that these two agents account for 50% of hitherto undiagnosed gastroenteritis around the world. it is amazing that in this era of tissue culture, virology pathogens were diagnosed initially by electron microscopic techniques and that these techniques are still vital. even in 1982 cell culture isolation is not an adequate diagnostic tool for rotaviruses. norwalk agent and other antigenically related virus-like particles'.20 are still not grown in cell culture systems. even more recently electron microscopy and immune electron microscopy have been shown to be critical techniques for detecting new virus-like particles associated with gastroenteritis in humans. these particles have two things in common. they are smaller than rotavirus ( i 5-40 nm) and are round. they have logically been referred to by some investigators as small round virus-like particles (srv). they are not antigenically related to norwalk agent. because these srv have been detected by electron microscopy, their morphology is a distinguishing feature for some particles. astrovirus, a 28 nm particle with a star-like surface was described by madely and cosgrove in 1975. astrovirus has been detected in outbreaks of diarrhea in scotland, england, and canada.2.' 1~1 3~1 6 caliciviruses, also approximately 28 to 30 nm in size, have been associated with diarrhea in human infants according to several additional small round viruses (27 to 40 nm) have been distinguished by electron microscopy in fecal specimens from infants with nonbacterial gastroenteritis. twentyfive to 28 nm particles, termed picorna-parvo have been reported to be associated with diarrhea.16 finally a group of slightly larger, 30-40 nm calici-like particles associated repor~s~s.12.14.17.1* with diarrhea in several areas of the world may actually represent a single group. while they have various names: srvl," minireo,i6 fuzzy-wuzzy's,6 minirota," otofuke agent," and sapporo agent,' they appear to have a similar size range and some common morphological features, like a spiky outer surface. definitive etiological role and antigenic relatedness await good convalescent sera and serological reagents and appropriate immune electron microscopic testing. the virology laboratory of children's hospital began diagnosis by electron microscopy near the end of 1978. since then, rotavirus has been detected in approximately 500 children hospitalized with diarrhea. we have also found adenovirus, coronavirus-like particles, and representative samples of most of the s r v described above. we have begun using immune electron microscopy as a corollary diagnostic tool in an attempt to make a definitive diagnosis particularly in the case of the s r v . centrifuge serum a t 15,000 rpm for 1 hour dilute serum w i t h pbs i n 4 fold serial dilutions 0.9 ml serum + 0.1 ml undiluted virus,mix well fecal samples were obtained either in a specimen bottle, soiled diaper, or rectal swab from children suffering from gastroenteritis and diarrhea or from age-matched control infants without diarrhea. the period of study under greatest concentration (especially for s r v ) was october, 1979 to november, 1981. during that time fecal samples were obtained from 1260 infants. one thousand one hundred and sixty samples were taken from children with diarrhea. at least 90% of these were hospitalized. fifty (50%) of the control infants were housed in the neonatal nursery during the winter season when rotavirus and s r v were prevalent. the remaining 50 were non-diarrheic infants either well, living at home, or attending a clinic with another complaint. w two techniques were utilized in immune electron microscopy (iem). the first described in figure 2 was adapted from the method described by brandt and colleagues4 and was best suited for fecal samples. the second technique seen in figure 3 was developed by kapikian and is most useful for tissue culture grown and probably purified fecal virus.' finally figure 4 shows the result of iem. calicivirus-like agent (cvla) was mixed with hyperimmune serum. note the connecting bridges between the particles that have been produced by the virus-specific antibody. using the techniques just described we examined specimens from over 1,200 children during a two-year period. we found a variety of viruses and virus-like particles. figures 5 through 7 are representative examples of the particles seen. in all cases the particles were seen in feces from children who had diarrhea at the time the samples were taken. some of the agents, like rotavirus, are known pathogens and others are still awaiting a definite etiological role. in figure 8b convalescent serum containing rotavirus-specific antibody had clumped particles in a fecal sample. figure 8a is the control without the addition of virus-specific antiserum. when acute and convalescent serum samples are available, iem can be used to document the cause of viral enteritis. and were large enough to be placed in the "minireo" or "minirota" category. the remaining srv did not have a distinctive surface. they were negative in the radioimmunoassay for norwalk virus as determined by dr. h. greenberg at the national institute of health. therefore they may be left in the category described by middleton and his associates'6 as picorna-parvo. the final 29 (9%) were identified as adenovirus and four particles appeared to be coronavirus-like. we found that the mean age of children who were rotavirus positive was 11.5 months. this was significantly older than the 4.5 months, which was the mean for the infants who were positive for srv. negative-stain contrast electron microscopy can be held at least partially responsible for diagnosing 50% of the hitherto undiagnosed nonbacterial gastroenteritis in hospitalized children. in addition, iem helped establish rotavirus as a major etiological agent of gastroenteritis in infants and young children. finally, both techniques are being used to detect and to define the role of new virus-like particles associated with diarrhea. using these techniques, we were able to detect distinctive viruses or virus-like particles associated with diarrhea in 27% of all specimens examined. while the vast majority of these were rotavirus, the finding of astrovirus and other small round viruses winter vomiting disease astrovirus-associated gastroenteritis in children virus particles in epithelial cells of duodenal mucosa from children with acute nonbacterial gastroenteritis comparison of direct electron microscopy, immune electron microscopy, and rotavirus enzyme-linked immunosorbent assay for detection of gastroenteritis fecal shedding of virus in relation to the days of illness in gastroenteritis due to calicivirus electron microscopy in the diagnosis of infectious diarrhea flewett visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis the 33 to 39 nm virus-like particles tentatively designated as sapporo agent associated with an outbreak of acute gastroenteritis small round virus-like particles associated with acute gastroenteritis in japanese children astrovirus associated gastroenteritis in a children's ward viruses in infantile gastroenteritis calicivirus associated with winter vomiting disease orbivirus acute gastroenteritis of infancy viruses associated with acute gastroenteritis in young children nosocomial infantile gastroenteritis associated with minirotavirus and calicivirus the occurrence of calicivirus in infants with acute gastroenteritis virus-like particle, 35 to 40 nm associated with an institutional outbreak of acute gastroenteritis in adults detection by immune electron microscopy of 26-27 nm virus-like particles associated with two family outbreaks of gastroenteritis key: cord-265164-ybh5yljw authors: zhao, bin; zhang, zhao; li, xianting title: numerical study of the transport of droplets or particles generated by respiratory system indoors date: 2004-11-24 journal: build environ doi: 10.1016/j.buildenv.2004.09.018 sha: doc_id: 265164 cord_uid: ybh5yljw the outbreak of atypical pneumonia, referred to as severe acute respiratory syndrome (sars), has spread to many countries in the world. sars may infect human bodies by the tiny droplets or particles carrying various virus and bacteria, which are generated by the respiratory system of infected patients. this paper presents the numerical analysis of the influence of generating ways of the droplets or particles on the transport and distribution of the droplets or particles indoors. the drift flux model, which considers the settling of particles or droplets under the effect of gravitational sedimentation, is adopted to simulate the droplets transport and distribution indoors during respiration and sneezing or coughing process, while the simplified model for solving the continuous fluid flow is combined. two different cases considering the normal respiration and coughing or sneezing are studied, respectively, and two different outlet velocities from the mouth for the sneezing or coughing process are considered. the results show that droplets or particles generated by normal breathing process transport a relatively short distance, while droplets or particles generated during coughing or sneezing may travel much longer distances, which may pose adverse effect on human bodies for defending the sars or other infectious diseases. severe acute respiratory syndrome (sars) is a recently described illness of human with a high casefatality rate that has spread widely since november 2002. by late april 2003, over 4300 sars cases and 250 sars-related deaths from over 25 countries around the world were reported by the world health organization (who). most of these cases occurred after exposure to sars patients in household or healthcare settings [1] . as a kind of epidemic disease, sars may infect human bodies by the tiny droplets or particles carrying various virus and bacteria, which are generated by the respiratory system of infected individuals. some other infectious diseases such as flue, measles and mumps are also transmitted from person to person by inhaled airborne particles [2] . people staying indoors may be infected easily by the virus carried through the droplets or particles since there are limited air exchange rates indoors. normal respiration, sneezing or coughing generates the tiny droplets composed of water and air or fine particles. these generating ways have different generating rates and duration, resulting in different effects on indoor environment and human bodies. the main purpose of this paper is to study the transport of droplets or particles generated by the respiratory system indoors during different generating processes. because it is difficult to examine the transport and diffusion of droplets or particles from respiratory system by experiments, a three-dimensional and time-dependent numerical method is adopted in this study. for simplification, ''particles'' or ''droplets'' is termed uniformly as ''particle'' in this paper, which is a general rule in aerosol science [3] . to calculate the three-dimensional and non-isothermal airflow inside ventilated rooms, a well validated simplified methodology combined with n-point air supply opening model [4] , a zero equation turbulence model [5] is applied. for indoor environment, the settling under the effect of gravitational sedimentation may play an important role on the particles diffusion. to take the gravitational sedimentation into account, the drift flux model, which has been applied and validated successfully for indoor particles simulation [6, 7] , is adopted. the governing equations for airflow and particles diffusion in vector form are as follows: where r; v and p are the air density (kg/m 3 ), velocity vector (m/s) and pressure (pa), respectively. v s is the settling velocity of particles (m/s). c is the mass concentration of particle. here, relative concentration is used with an assumed unit concentration (1.0) from the mouth. the effective viscosity m eff is the sum of molecular and turbulent viscosity (kg/m s). turbulence is modeled by the zero equation turbulence model developed by chen and xu [5] . f is the scalar quantity of air. the non-dimensional numbers s f and s c represent the turbulent diffusivity of f and c, respectively. here the values are set as 1.0. f is the body force due to air density (temperature) difference, which is modeled by using the boussinesq approximation (pa). s c is the droplets generated rate indoors (mg/s). the main assumptions used for the simulation of particle transport include: (1) the airflow is isothermal. (2) the effect of particles on turbulence is not considered, as it is believed that the low particle loadings and comparatively small particle settling velocities have a very small effect compared to the high inflow turbulence levels [8] . (3) the particle size distribution will not be altered by coagulation due to low particle loadings. (4) the evaporation of particles is neglected for simplification. strictly speaking, assumption (4) above only applies to solid particles. many droplets in the 0.5-1.0 mm range can evaporate quickly in rooms with normal relative humidity levels. evaporation could lead to loss of particle momentum and increase of particle buoyancy. in this research, evaporation is neglected because only short-term behavior (in the order of seconds or less) of particles is considered. for longer time domain, the droplet evaporation could be a major factor that deserves more research. the settling velocity of a particle derived by equaling the fluid drag force on the particle with the gravitational force can be expressed as where c d is the drag coefficient, d p is the diameter of the particle (mm), r p and r a are the density of the particle and the ambient air (kg/m 3 ), respectively, and g is the gravitational acceleration (m/s 2 ). the settling velocity always has the same direction as gravitation, namely, perpendicularly downward. the drag coefficient is either derived by stokes equation (reo1) or a revised equation (1oreo1000) [3] : re is the reynolds number according to the relative velocity of particle and air. the settling velocity is a velocity relative to air under steady condition. when reo1, the settling velocity equals the product of the acceleration due to gravity and the relaxation time of the particle [3] . and when re41, the settling velocity is derived by iterating the drag balance equation based on eq. (6). the supply inlet is described by the n-point supply opening model to consider the complex geometry of actual diffusers [4] . and all variables are defined at the supply inlet. outlet boundary conditions are set as the neumann boundary condition, that is, mass flow boundaries are specified to ensure that the mass flow rate out of the domain is the same as the mass flow rate into the flow domain. for the zero equation turbulence model, wall functions are not needed for the region near the walls, where the algebraic equations of turbulent viscosity are applied directly [5] . the wall boundary condition of particle is simplified as zero normal gradients. that is, qc=qn ¼ 0; where n is the direction normal to the wall. as the number of deposited particles is usually much smaller than that of exhausted particles by ventilation [9] , the deposition process cannot exert a strong influence on the flow field in indoor environment. the equations above are discretized into algebraic equations by finite volume method and the coupling between velocity and pressure is solved by the simple algorithm [10] . in the course of discretization, the power law scheme and the second-order central difference are, respectively, implemented for the convection and diffusion terms. the resulting set of discretized equations for each variable are solved by a line-by-line procedure, combining the tri-diagonal matrix algorithm (tdma) and the linear under-relaxation iteration. non-uniform staggered grid system is employed with denser grids clustering near the bounded walls so as to resolve the boundary layer properly. a well-validated cfd program developed by the authors, stach-3 [4] , which utilized the simplified methodology mentioned above, is used as the simulation tool. to study the different transport and distribution characteristics of particles from human respiratory system indoors, a full-scale room is selected. the geometry of the room is x and the grille size is 0.5 m(z) â 0.125 m(y) and an exhaust of the same size is located at the bottom of the same wall. both the inlet and outlet are symmetrical about the middle line in the xy plane (z ¼ 1:5 m). one person is assumed to stay at the opposite side of the inlet and outlet. fig. 1 shows the schematic of the ventilated room and the person. two different cases considering the normal respiration and coughing/sneezing process are studied, respectively. the outlet velocity of normal respiration process is periodic [11] and that of coughing or sneezing process is pulse [2] . the assumed velocity as boundary conditions in simulation is depicted in fig. 2 . for respiration process, we assume breathing from the nose and the direction of inhaling/exhaling is 301 downward (fig. 2(a) ). two different outlet velocities from mouth for sneezing or coughing process are considered (v em =20 and 100 m/s, fig. 2(b) ). it should be noted that many people sneeze more than once in a cycle of sneezing. however, we only consider sneezing or coughing once for simplification. in fact, the results in the following will show that sneezing or coughing only once may cause long transport distance and high concentration of particles, which implies that sneezing or coughing more than once may cause higher concentration. the outlet particle concentration by respiration, sneezing or coughing is assumed as 1.0, which may be used as a relative value. table 1 presents the air supply parameters and other boundary conditions. as larger particles (d p 41 mm) generated from humans can only be transported within 1.0-1.5 m and these larger particles constitute less than 5% of all the particles or droplets of one human [12] , only fine particles are considered in the present study. two different sizes of particles, 1.0 and 0.5 mm, are considered. because results for the two sizes have little difference, only the results of 1.0 mm are presented next. the particle density is assumed as 600 kg/m 3 , which is the approximate density of ash particles or droplets of mixture composed of water and air. in fact, because the particles are fine ones, the density of particles does not remarkably influence the transport and distribution of the particles. the airflow and particle transport are calculated in two respiratory periods (8 s) since the respiratory process begins in the ventilated room. fig. 3 shows the streamline at the symmetrical plane (z=1.5 m) during the first respiration period (see fig. 2(a) ). because of the relatively smaller air flow volume when breathing, the inhaling and exhaling processes have little influence on the room air flow pattern. fig. 4 gives the particle concentration at different times when the human is exhaling or inhaling. it is seen that the particle concentration reduces to below 0.001% of the outlet particle concentration within 1.0 m away from the human. the air exchange rate per hour (ach) for this case is 0.5. thus, we can imagine that a larger air exchange rate may cause better indoor environment with lower particle concentration and shorter spreading distance of particles. this implies that the transport distance of particles or droplets generated by normal respiration of patients is limited. unlike normal respiration, sneezing or coughing may produce a large amount of particles of droplets, with a diameter of about 10-100 mm. however, they will split into smaller particles of 1-4 mm in a very short time [2] . therefore, 1.0 mm is again adopted as the particle diameter in the simulation. fig. 3(c) shows the streamline at the symmetrical plane (z=1.5 m) for an initial velocity v em =100.0 m/s (see fig. 2(b) ). it is seen that the large outlet velocity will maintain a much longer distance of particle transport due to the large momentum. fig. 5 compares the simulated flow pattern with the measured one by [2] . the results seem reasonable. fig. 6 further gives the particle concentration distribution at different times since the sneezing or coughing begins, which indicates that sneezing or coughing may cause long transport distance of particles, although the ventilation air exchange rate is rather large (5 ach). comparison of the two different outlet velocities of the sneezing or coughing case indicates that larger sneezing or coughing velocity will cause further transport and higher concentration of the exhaled particles or droplets. fig. 7 shows the three-dimensional view of the particle concentration distribution at several different times. the contour zone stands for the particle concentration where the value is larger than 1% of the outlet concentration. if we define ''affecting range'' as the distance from the patient to where the concentration reduces to the ''safe threshold'', and define ''safe threshold'' as the percentage of outlet concentration that will not cause adverse effect on people, fig. 7 depicts the ''affecting range'' of the sneezing or coughing if we consider 1% as the ''safe threshold'' (in fact, the exact threshold needs careful study). the figure also proves that particles generated during coughing or sneezing may travel a long distance, which may pose adverse effect on human bodies for defending the sars or other infectious diseases. from the results, good personal habits, that is, covering the nose and mouth when sneezing or coughing, is very important for preventing the transmission of the diseases from human to human. it should be noted that the above results are based on oversimplified and ideal scenarios without considering many influencing factors such as the evaporation of the droplets, supply and return air system orientation, blockage of air and particle movement, etc. thus, the results should be used with caution. this study, however, may be considered as a direction in understanding the complex phenomena of particle transport indoors and ultimately, preventing the transmission of infectious diseases such as sars. numerical studies on the transport and distribution of particles or droplets generated by normal respiration and sneezing or coughing indoors result in the following conclusions: normal respiration process produces fewer particles or droplets, which have small influence on indoor environment. and the particles or droplets may only transport a short distance by normal respiration. sneezing or coughing may produce more particles or droplets, which will transport a long distance. a normal sneezing or coughing with outlet velocity of 20 m/s will cause the particles or droplets to transport a distance longer than 3 m, and larger outlet velocity will have even longer distance in shorter time. for the purpose of defending humans against the diseases, good personal habits are very important to prevent the virus spreading via sneezing or coughing. characterization of a novel coronavirus associated with severe acute respiratory syndrome institute of cell and molecular biology, and biology teaching organisation aerosol technology: properties, behavior, and measurement of airborne particles a simplified system for indoor airflow simulation a zero-equation turbulence model for indoor air flow simulation diffusion characteristics of airborne particles with gravitational settling in a convection-dominant indoor flow field modelling of the indoor environment-particle dispersion an depositoin on predicting particle-laden turbulent flows mathematical modelling of indoor aerosol dynamics numerical heat transfer and fluid flow medical physics and biomedical engineering. bristol and philadelphia: institue of physics publishing preliminary prediction of flow and particulate concentration produced from normal human cough dispersion. proceedings of the second joint embs/bmes conference this study is financially supported by the national nature science foundation of china under grant 50346007. key: cord-233294-jnic4o2j authors: ravazi, maryam; butt, zahid; lin, mark h.e.; chen, helen; tan, zhongchao title: in situ measurement of airborne particle concentration in a real dental office: implications for disease transmission date: 2020-08-19 journal: nan doi: nan sha: doc_id: 233294 cord_uid: jnic4o2j recent guidelines by who recommend delaying non-essential oral health care amid covid-19 pandemic and call for research on aerosol generated during dental procedures. thus, this study aims to assess the mechanisms of dental aerosol dispersion in dental offices and to provide recommendations based on a quantitative study to minimize infection transmission in dental offices. the spread and removal of aerosol particles generated from dental procedures in a dental office are measured near the source and at the corner of the office. we studied the effects of air purification (on/off), door condition (open/close), and particle sizes on the temporal concentration distribution of particles. the results show that in the worst-scenario scenario it takes 95 min for 0.5 um particles to settle, and that it takes a shorter time for the larger particles. the indoor air purifier tested expedited the removal time at least 6.3 times faster than the scenario air purifier off. airborne particles may be transported from the source to the rest of the room, even when the particle concentrations in the generation zone return to the background level. these results are expected to be valuable to related policy making and technology development for infection disease control in dental offices and similar built environments. in august 2020, the world health organization (who) guidelines recommend delaying routine nonessential oral health care amid covid-19 pandemic and call for more research on indoor aerosol generated by dental procedures. the reason is that dental professionals, staff, and patients in dental offices are exposed to aerosol droplets, particles, and pathogenic microorganisms in the saliva and blood of the infected patients. the infectious microorganisms transmitted from saliva and nasopharyngeal secretions include pneumonic plague, legionella pneumophila, tuberculosis, influenza viruses, herpes viruses, sars virus (a form of coronavirus), pathogenic streptococci and staphylococci, hiv, and hepatitis viruses [1, 2] . airborne and spread to the room. extraoral high volume evacuators (ehve) can also be used to remove the aerosol particles near to the area of particle generation [1] ; however, its performance depends on the volumetric rate of evacuation and particle generation rate. in addition, using extra devices around the dental unit causes a restricted environment and inconvenience to the dentists. recent covid-19 outbreak has resulted in increased use of portable air purifiers in dental offices, despite the scarcity of published research on their performances in dental offices [26, 27] . further research on the protective effectiveness of air purifiers in dental clinics was recommended [28] . the portable air purifiers can be located at the corners in the dental offices, and they cause much less inconvenience during dental operations than extra-oral high evacuators do. in addition, these portable air purifiers do not require modification to existing ventilation systems. despite earlier research on number concentrations for micron [29, 30] and nano-size particles [31] [32] [33] related to the dental processes, to the best of our knowledge, no research has been done on the dispersion or transport of airborne particles lingering in different parts of the office. the nature of the extensive surface area in dental offices may enhance the losses of particles onto various surfaces. furthermore, research on the effects of air purifiers is needed to develop guidelines and protocols to reduce waiting time between patients and ensure the safe operation of dental offices. the objective of this study is to understand the spatial and temporal concentration-distribution of airborne particles generated from dental procedures in dental offices. the remainder of this paper is presented as follows. section 2 presents the experimental design of concentration measurements in the dental office. section 3.1 reports the number concentration distribution of particle under the effects of operating conditions during the generation; section 3.2, the spatial and temporal change of particle concentrations distribution under the effects of operating conditions at the generation zone; section 3.3, at the corner of the office. the results reveal the effective removal mechanisms that depend on particle size. finally, section 5 summarizes the entire work. results in this paper are deemed valuable to the best practices for particle removal from dental offices. the concentrations of micron and submicron particles were measured on may 15, 2020 in a dental operation room on the second floor of the dental clinic in toronto, ontario, canada. figure 1 shows the schematic of the operatory and layout of the instruments. this typical dental operatory room is 3 m wide, 3 m long, and 4 m high; it has one central dental unit. the mechanical ventilation system was turned off and the window was closed throughout the test. the temperature and relative humidity of the room air were 13.4 0 c and 88%, respectively. range of 0.5-20 µm in diameter and those smaller than 0.5 µm. the aps was located on the left-hand side of the doctor, to prevent any inconvenience for the doctor during dental operations. a stainless-steel sampling tube, which is 1/4-inch of inner diameter and 0.3 m long, was connected to the inlet of the aps for sampling air 10 cm away from the operation area (i.e., the patient's mouth). both opcs were running continuously. one opc was located beside the aps, and another opc was 1.8 m away from the source. both opcs report particles with diameters of 0.3, 0.5, 1, 2, 5, 10 µm. the first opc is calibrated against the aps. before the operation, the room was unoccupied for 15 hours before the background concentrations were measured at the source without air purification. as seen in figure 2 , all particles in the background air were less than 10 #/cm 3 and those larger than 1 m in diameter were less than 1 #/cm 3 . airborne particles were generated over 5 min of continuous drilling operation (high-speed handpiece) using a pig jaw. pig teeth are commonly used for dental studies because of similarities between the structure of human and pig enamel and dentin [34, 35] . the particle number concentrations were measured during 5 min of continuous dental operation and afterward until the number concentrations reached the background. then we measured the airborne particle concentrations under six scenarios. conditions. for all scenarios, the smaller the particle size, the higher concentration is. in closed-door scenarios, by comparing the scenario that no air purifier is running (figure 3 .a) with the scenario that the air purifier is running at the beginning of operation (figure 3 .b), it can be observed that particles have a wider distribution in figure 3 .b, which means particles are growing to the larger sizes. for instance, the concentration of higher than 200 #/cm 3 is observed for 0.5-1.3 m particles in figure 3 .a, while, this range of concentration is observed for 0.5-1.5 m particles in figure 3 from this observation, it can be inferred that running the air purifier from the beginning causes air circulation in the room. the air circulation can enhance the interaction between airborne particles leading to agglomeration in the area that particles are generated [36] . thus, the particles may grow to the larger ones when the air purifier was on at the beginning of the operation. growing to larger sizes is preferable in terms of particle removal. removal by hepa filter is size dependant; the larger sizes, the more probable filtration is. the filtration of micron particles is due to interception and impaction [37]. growing particles to larger sizes during the first 5 min while the air purifier was running from the beginning of the operation. the concentration of higher than 200 #/cm 3 is observed for 0.5-1 m particles in figure 3 .a (air purifier off), while, this range of concentration is observed for 0.5-1.4 m particles in figure 3 .d. moreover, the concentration of 200-70 #/cm 3 is detected for 1-2.2 m particles in figure 3 .c, however 1.4-2.5 m particles have this concentration range in figure 3 .d. 9 the particles generated in the 5-min long operation gradually spread in the room, and their concentrations were decreased by different mechanisms. they are introduced in the next sections. table 3 summarizes the times it takes for the number concentrations to reach their background levels (removal times) for all six scenarios. in the worst-scenario scenario, when the door is closed and no air purifier is running in the room, it takes 95 min for 0.5 m particles to return to the background level. air purifier. figure 4a shows the lowest particle concentrations in the room when the high-speed air purifier is running from the beginning of the operation. however, the removal time is almost the same for all these 3 scenarios: low-speed air purification after the dental operation, high-speed air purification after the dental operation, and high-speed air purification from the beginning of the operation. it can be inferred that particles were captured with the hepa filter and activated carbon filter installed in the air purifier. in addition to filtration, enhancing air circulation in the room by the air purifier leads to faster particle settlement on the surface areas. these results suggest that air purifier has a crucial role in removing airborne contamination of dental offices in the generation zone. is recommended as a short term solution for the dental offices without air filtration systems. the particle removal time varies with particle size although the air purifier and open door help reduce the concentration of all-size particles in the generation zone. the next section elaborates on the size dependency of particle spread and removal because smaller particles probably carry more infectious microorganisms because the concentration of smaller particles is higher than the larger ones. table 3 . at the beginning of the dental operation 8 there are several mechanisms of particle removal from the air including settling, air circulation, and air filtration. first, all particles in a closed-door room without air circulation or filtration settle down because of gravity. it is well-known that the larger particles have higher gravitational settling velocity and that their removal times are shorter than the smaller particles. figure 5a further confirms this mechanism. for example, 2.5-m particles disappeared faster than those that were smaller. second, air circulation leads to the dispersion of particles and their subsequent removal by settling on the surface areas or exiting the room or both. the drag force on a particle is also size-dependent. it usually takes a longer time for a larger particle to disperse than the smaller ones do. figure 5e indicates that air circulation through the open door expedited the particle removal, although the air purifier was off. in addition, figure 5e shows expedited removal of smaller particles and confirms that air circulation is the dominant mechanism in this scenario. third, the filtration efficiency is also size dependant and it increased with the particle size for micron particles [37] . moreover, figure 5 .b, 5.c, and 5.d show that the removal times do not vary with particle size. therefore, a combination of settling, air circulation, and air filtration all play roles in particle removal for these scenarios. comparing these scenarios with that in figure 5f demonstrates the strong effects of air circulation due to the open door. in summary, an air purifier running at high fan speed may ensure the removal of 0.5 to 3 m particles, while air circulation is more effective for smaller particles. since the door of dental offices might be open frequently, an air purifier with a strong fan may help prevent cross-contamination from one room to the other through the door. nonetheless, our study herein does not undermine the effectiveness of external highvolume evacuation (ehve) and suction, which are often used near to the generation zone. however, it does not mean that the room is completely cleaned even when the particle concentrations in the generation zone dropped back to the background. the particles may be transported from the source to the rest of the room. dental staff walks around in the same room, and they often remove their masks for a short break at the corner, where there is little air circulation. it is necessary to investigate the spread of particles by analyzing the concentration at the corner of the room, and the results are presented in the next section. these results indicate the effectiveness of high-speed high-efficiency air purification. generally, it can be inferred that the peak is observed in the corner when the rate of particle settlement and removal from the air is lower than particle transport to the corner. table 4 indicates that it took 6 min for the concentration peak to reach the corner when the travel time of the concentration peak and peak concentration ratios are close to each other for the three closed-door scenarios including air purifier off (figure 6a ), low-speed air purifier running after the operation (figure 6b) , and high-speed air purifier running after the operation (figure 6c) . thus, the same fraction of particles reaches the corner at the same time for these scenarios. this is surprising because these results imply that the air circulation result from the air purifier has little impact on the air movement to the corner of the room (see figure 1 ). removal mechanisms. second, the peak is observed in the corner when the rate of particle settlement and removal from the air is lower than particle transport to the corner. thus, a fraction of 1, 2.5 m particles, which is not removed from the air, traveled to the corner. the following conclusions can be drawn from the results of this study: • in the worst-scenario scenario with no protection system in the closed-door office and continuous high-speed drilling, it takes 95 min for 0.5 m particles to return to background level and that it takes a shorter time for particles larger than 0.5 m to be removed from the air. in the real operations with the patient, which usually is less than five minutes, air may be cleaner because of other measures like suction from the source (i.e., the mouth). • there are three size-dependent mechanisms for particle removal: gravity settling, air circulation, and air filtration. technologies that combine all of them are the most effective in air cleaning. the air purifier expedited the removal time at least 6.3 times faster than the scenario with no air purifier in the generation zone. running high-speed air purifier at the beginning of the operation is the most effective scenario in reducing airborne particle concentrations. the air purifier at one corner could not eliminate the concentration peak in the other corner of the room except for the scenario when the door was closed and the air purifier was running at the highest speed from the beginning of the operation. • in the second part of this study, the number concentrations were measured for three dental operations with real patients. the air ventilation system was blocked, and the door was closed, however, it was opened several times during the operations. the first operation was conducted in 2 parts, shown in by patterned area. the higher concentrations entered the room from outside. after closing the door, the number concentration was reduced by the air purifier. moreover, the concentration peaks were observed, in the moments that the door was open. the major fraction of particles was generated in the second part of the operation. during this time, the air purifier was running at low speed in 7 min and turbo speed in 7 min. in the first 7 min, the removal rate was 0.28 (#/cm 3 min) and the second 7 min was 1.14 (#/cm 3 min), 4 times faster than the time with low speed. the second operation was conducted in a single part, and no considerable particles were measured. similar to the fist operation, the number concentration of outside was higher than inside. the number concentration in the third operation was higher than the first two operations. the third operation was conducted in 2 parts. higher values of concentration coming from outside are observed in this operation comparing to the first two because aps was closer to the door in 3 rd operation. characterization of aerosols and fine particles produced in dentistry and their health risk assessments aerosols and splatter in dentistry: a brief review of the literature and 7 recognition of aerosol transmission of infectious agents: a commentary it is time to address airborne transmission of covid-19 modes of transmission of virus causing covid-19: implications for ipc precaution recommendations: scientific brief lung cancer, cardiopulmonary mortality, and long-term exposure to fine particulate air pollution safe handling of nanotechnology global air quality and climate high secondary aerosol contribution to particulate pollution during haze events in china the contribution of outdoor air 29 aerosol and bioaerosol particles in a dental office measurement of particle concentrations in a dental office. environmental monitoring and assessment d'arcy, assessing potential nanoparticle release during nanocomposite shredding using direct-reading instruments nanoparticle release from dental composites nanoparticle concentrations and composition in a dental office and dental laboratory: a pilot study on the influence of working procedures enamel microstructure and microstrain in the fracture the authors would like to acknowledge the financial and technical support from the natural sciences and engineering research council of canada (rgpin-2020-04687), the gci ventures capital, surgically clean air, and waterloo filtration institute. key: cord-355883-uyrn281a authors: zimmerman, alyssa; petters, markus d.; meskhidze, nicholas title: observations of new particle formation, modal growth rates, and direct emissions of sub-10 nm particles in an urban environment date: 2020-08-07 journal: atmos environ (1994) doi: 10.1016/j.atmosenv.2020.117835 sha: doc_id: 355883 cord_uid: uyrn281a ultrafine particles with diameters less than 100 nm suspended in the air are a topic of interest in air quality and climate sciences. sub-10 nm particles are of additional interest due to their health effects and contribution to particle growth processes. ambient measurements were carried out at north carolina state university in raleigh, nc between april to june 2019 and november 2019 to may 2020 to investigate the temporal variability of size distribution and number concentration of ultrafine particles. a mobile lab was deployed between march and may 2020 to characterize the spatial distribution of sub-10 nm particle number concentration. new particle formation and growth events were observed regularly. also observed were direct emissions of sub-10 nm particles. analysis against meteorological variables, gas-phase species, and particle concentrations show that the sub-10nm particles dominated number concentration during periods of low planetary boundary layer height, low solar radiation, and northeast winds. the spatial patterns observed during mobile deployments suggest that multiple temporally stable and spatially confined point sources of sub-10 nm particles are present within the city. these sources likely include the campus utility plants and the raleigh-durham international airport. additionally, the timing of data collection allowed for investigation of variations in the urban aerosol number size distribution due to reduced economic activity during the covid-19 pandemic. to investigate the spatial and temporal variability of aerosols in raleigh, three different 159 instrumentation configurations, detailed in table 1 , were used during measurements taken from 160 april 19 to june 4, 2019 and again from november 15, 2019 to may 15, 2020. instrumentation 161 spatial distribution of sub-10 nm particles throughout raleigh. this system consisted of 170 instrumentation for aerosol number concentration measurements and continually sampled from 171 the jordan hall laboratory, until it was transported to a vehicle for mobile deployments. 172 processing was used for cpc-3. the sample flow rate was continuously measured and 193 coincidence was corrected through live-time counting performed by the manufacturer's 194 algorithm. this method divides the number of counted particles by the time between electrical 195 pulses and the flow rate to provide an accurately determined true counting rate. due to 196 instrumentation failure, cpc-4 was replaced with cpc-5 (tsi 3772) in may 2020. the 197 difference in the counts between cpc-3 and cpc-4/5 provided a particle number concentration 198 for diameters between 2.5-11 nm (n 2.5-11nm , 10 nm upper limit when cpc-5 was used). was used during mobile deployments 5 and 6. 200 all raw 10 hz data from the smps system and corresponding cpc were inverted using 202 the method described in petters (2018) . the inversion method includes mapping the time varying 203 electric field to a corresponding mobility diameter (s. c. wang & flagan, 1990) . particle counts 204 are binned and inverted to the total concentration by accounting for the smps transfer function, 205 multiple charges, and diffusional broadening of the transfer function. the resulting concentration 206 and particle diameter data from the smps were binned into 83 logarithmically spaced bins 207 spanning a size range of 5-55 nm. the binned smps data was then averaged onto fixed 5 min. 208 and 30 min time grids to facilitate time-series analysis. 209 figure 1 presents the temporal variability in the 5-55 nm spectral number density during 210 had an initial mode between 10 to 20 nm and the particles also grew to larger sizes. finally, if 227 there were nucleation mode particles present, resulting in an increase in the ultrafine particle 228 number concentration, but growth was not continuous or sustained, this event was classified as 229 class c. more specifically class c events were characterized by modal growth lasting < 4 hr., or 230 significant modal shrinkage of particles. examples of class a, b, and c npf events are shown 231 in figures s5, s6 , and s7. the dates and times of all class a and b npf events are detailed in 232 tables s1 and s2. a slide deck analyzing the normalized spectral number density of all observed 233 nucleation events is included in the archived dataset. 234 visual analysis of the size distribution data also revealed the presence of pb events. 235 these events were characterized by continuous bursts of nucleation mode particles that persisted event, if multiple bursts of nucleation mode particles continued for more than 6 hr and no modal 239 growth was observed. the 30-minute average of 5-10 nm particles was visually analyzed for 240 each pb event to estimate a threshold concentration that was exceeded by each burst in an event. 241 due to variations in strength of events and background number concentration, each event was 242 assigned a separate threshold concentration (shown in table s3 ). the threshold concentrations 243 were determined by the maximum 30-minute average number concentration of the weakest 244 particle burst during each event. the threshold values were then used to estimate the pb event 245 duration. the start and end times of each event were estimated as when the 30-minute average 5-246 10 nm number concertation surpassed and receded the threshold concertation. 247 the growth rates were calculated for class a and b npf events using the maximum 249 concentration method of kulmala et al. (2012) . the detection time of each event was initially 250 estimated from visual inspection of normalized number spectral density plots. for each scan 251 during the event, the modal diameter was calculated from the maximum number concentration 252 and then plotted as a function of time. the growth rates of each event were derived using a linear 253 regression of the modal diameters across three size ranges of 5-10 nm, 10-20 nm, and 20-30 254 nm. the first size range was selected based on the lower limit of the smps instrument. 255 considering that the initial mode diameter of class b events was approximately 10 nm, the 256 second size range captures the initial growth of nucleated particles during these events. the 257 second and the third size ranges allow for comparison of growth rates between the class a and b 258 events. due to the 5 nm lower limit in the size distribution data, the growth rates calculated 259 between 5-10 nm were used to estimate the approximate start time of each class a event. average growth rates between 1.5 and 3 nm are nearly half of the growth rates measured above 3 262 nm. considering this variability in growth rates below 10 nm, the class a event start times are 263 reported within a possible range based on one standard deviation of the 5-10 nm growth rates. 264 class b start times are reported as a single time, rather than a range, because the initial mode 265 detected was greater than the lower limit of the smps and the initial appearance of these 266 particles can be accurately estimated. 267 to investigate potential sub-10 nm particle sources, the mobile system was deployed 6 269 times between march and may 2020. during mobile sampling, the inlet for the particle counters 270 was placed out the rear passenger side window. a 1000-watt lithium battery power station was 271 used to operate the cpcs, which provided approximately 3.5 hr of sample time. time series of 272 the driving route coordinates were recorded using a mobile phone application. the particle 273 counts were then averaged on a time grid, ranging between 6 to 7.5 sec depending on the mobile 274 deployment, to match the coordinate dataset. details of the individual deployments are shown in 275 table 2 . two different routes were driven during the various deployments. the first, shown in 276 figures s8 and s9 was designed to maximize distance traveled around raleigh and highlight any 277 concentration gradient present between downtown and the suburbs. this route was used in 278 mobile deployments 1-3. the second route (figs. s10 and s11) was designed locally around the 279 ncsu campus and was used in mobile deployments 4-6. 280 one-minute data for temperature, pressure, wind speed and direction, precipitation, and 282 solar radiation were acquired from the north carolina climate office weather station located on mesoscale forecast system (nam) at a 12 km spatial resolution. hourly data for co, no 2 , o 3 , 285 pm 2.5 , and pm 10 were acquired from the environmental protection agency's (epa) aqs api 286 system for the millbrook school site (aqs id: 37-183-0014). the millbrook school site is in the 287 northeast suburbs of downtown raleigh and is 12.4 km ne of the sampling site. hourly gaseous 288 pollutant data for so 2 was unavailable at any nearby monitors, and therefore was not analyzed. instruments. the average integrated smps number concentration between 5-10 nm (n 5-10nm ), 296 and 10-40 nm (n 10-40nm ) were 1.6×10 3 cm -3 and 4.3×10 3 cm -3 . these values show that on average, 297 n 5-10nm accounts for ~27% of less than 40 nm sized particle number concentration. for periods 298 where the smps and dual cpc system was available, there are concurrent time series number 299 concentrations. for example, n > 11 nm and n 10-40nm correlate. the daily average number 300 concentrations > 2.5, and >11/10 nm from cpcs 3 and 4/5 were 5.3×10 3 cm -3 and 1.1×10 4 cm -3 , 301 respectively. the average n 2.5-11nm computed from the difference in the two cpcs was 5. carolina was under a state of emergency and all k-12 public schools, restaurants, and bars were 316 mandated to close. after these executive orders, vehicular traffic on western blvd. decreased to 317 approximately 1.4×10 4 vehicles per day (i.e., by ~ 67% of the average daily traffic) (fig. s12) . 318 on march 23, gatherings of 50 or more people were banned, and service sector businesses were 319 mandated to close. on march 30 the statewide stay at home order began. during the stay at home 320 order, local traffic counts were reduced by ~75% of the average daily traffic. human movement 321 to retail and recreation, grocery and pharmacy, transit stations, and the workplace were down 322 approximately 50%, 28%, 77%, and 45%, respectively according to the wake county 323 community mobility report. as the stay at home order persisted into late april and early may, 324 movement to parks increased from 5 to 47%. as shown in figure 3 wang et al., 2020). here, the pm 2.5 concentration also decreased during this period (fig. s14) , 348 but the concentration generally remained within the statistical fluctuation of the last 10 years. 349 new particle formation events occurred during nearly all months of measurement. figure 351 the start time of npf events varies greatly based on the class of event. the average start 361 and detection times presented in table 3 show that class a npf events were generally detected 362 during the morning hours, while class b events were detected during or after midday. the 363 estimated average start time of all class a events was 9:30 ± 01:00 and the average detection 364 time was 11:00 ± 1:15. the average minimum and maximum start times reported in table 3 365 represent the starting time range estimated based on one standard deviation of the 5-10 nm grs. 366 all class a event times are detailed in table s1 . the general start time of all class a events was 367 also pronounced in the diurnal variation of particle number concentration plots shown in figure 368 5a. the average n 5-10nm and n 10-40nm increases first between 06:00 and 09:00 likely due to rush-369 hour traffic, and again between 09:00 and 12:00 correlating with the average detection time of 370 the nucleated particles. the onset of a class a npf event increases the n 5-10nm by almost a factor 371 of two compared to the concentration on a non-npf day (see fig. 5b ). the morning traffic 372 signature was also present in the n 10-40nm diurnal variation. figure 5a shows that the 373 concentration increase due to npf was delayed on average by ~60 min. from the onset of the table s2 and fig. s15) . 381 the particle grs were broken down into three different size ranges (5-10 nm, 10-20 nm, 382 and 20-30 nm) to analyze how particle growth varies with modal size. little variation was 383 observed between size ranges in the class a grs (5-10 nm: 2.3 ± 1.2 nm hr -1 ; 10-20 nm: 3.1 ± 384 2.2 nm hr -1 ; 20-30 nm: 2.6 ± 1.2 nm hr -1 ). particle growth rates during class b events were 385 slightly greater than class a events. the average grs were 3.7 ± 2.9 and 3.3 ± 2.3 nm hr -1 for 386 10-20 nm and 20-30 nm, respectively. the class b gr values measured during this study were 387 comparable to rates previously measured on the ncsu campus (10-25 nm gr: 1.6-3.9 nm hr -1 ; to non-event days. o 3 concentrations were also elevated during class b event days; however, 405 pm 2.5 levels were comparable to non-event days. 406 particle burst events play a significant role in shaping the near-surface particle 408 concentration and size distribution in raleigh. these bursts of sub-10 nm particles were observed 409 during all hours of the day and their duration lasted anywhere between 7 to 93 hr. figure 4 the pb events observed were characterized by a factor of two increase in n 5-10nm due to 416 continuous bursts of sub-10 nm particles (example event shown in fig. s18 ). however, when 417 compared to a regular day, there was no change in the n 10-40nm (figure 5c ). the average n 5-10nm 418 during all events ranged between 7.7×10 3 -6.9×10 4 cm -3 . no modal growth of the particles was 419 measured during pb events, suggesting that the particles were produced by an isolated source. 420 the average mode diameter of all pbs was between 5-7.5 nm suggesting that particles had a the mode diameter would drop below 5 nm causing the n 5-10nm to peak over 5×10 4 cm on three measurement days (jan. 17 and 21; and feb. 21, 2020) a class b npf event and 431 a pb event were detected simultaneously (fig. s19) . as newly nucleated particles grew 432 characteristically of an npf event, the pre-existing pb sub-10 nm particles persisted throughout 433 the growth event maintaining a constant mode diameter. the decoupled nature of these two 434 events also suggests that the pb particles likely originated from a local source and had similar 435 sizes when reached the detection instrument. figure s19 also shows that there was little 436 coagulation between the particles produced by two distinct sources, i.e., ones produced by a 437 mesoscale npf event and by a local source. the large spatial extent of the npf event allowed 438 for particles to grow as they advected toward the measurement instrument. providing that the 439 direct measurement of the local pollutant persisted, detection of the pb event particles continued 440 throughout the growth event. the pb event particles likely contributed to the growth event, albeit 441 without measurable modal growth. 442 wind analysis on pb days shown in figure 6 reveals that the wind was blowing from the 443 nne and ne 40% of the of time. whereas, the winds out of the ssw were most frequent on 444 exceeded 2×10 4 cm -3 . subtle peaks in s and e winds also correlated with average n 5-10nm 446 concentrations greater than 1×10 4 cm -3 . some pb events occurred during overcast weather and 447 periods of rainfall, which had no impact on removal of the sub-10 nm particles (table s3) . 448 additionally, both the pblh and solar radiation remained lower on pb days compared to regular 449 days (fig. s16 ). low pblh, solar radiation, and the strong correlation with ne winds on pb 450 days suggests that aerosol dispersion processes were weak, allowing for direct measurements of 451 a pollution plume created by a local source. studies that also observed pb events suggest weak *the average number concentration reported for mobile deployments 5 and 6 is between 2.5-10 823 nm as cpc-5 was used during this deployment. 824 observation of new particle formation 587 in subtropical urban environment effects of grades and other loads on on-road emissions of hydrocarbons and carbon monoxide a new coincidence correction method for atmospheric new particle formation at the research station melpitz, germany: connection 609 with gaseous precursors and meteorological parameters observations of new particle production in the atmosphere of a moderately polluted site 613 in eastern england emissions from an 616 international airport increase particle number concentrations 4-fold at 10 km characterization of modeling and measurements of urban aerosol processes on the neighborhood scale in atmospheric new particle formation and growth: review of field observations dispersion 642 of particle numbers and elemental carbon from road traffic, a harbour and an airstrip in 643 the netherlands characteristics of the atmospheric particle formation events observed at a borel 655 forest site in southern finland exposure 657 to sub-10 nm particles emitted from a biodiesel-fueled diesel engine: in vitro toxicity and 658 inflammatory potential impact 661 of nucleation on global ccn possible wintertime sources of fine particles in an emission of ultrafine particles from natural gas domestic burners anthropogenic and natural radiative forcing. cambridge, united kingdom and new comparison of atmospheric new particle formation events in three central european 682 cities global analysis of continental boundary layer new particle formation based on long-term 686 measurements effects of 689 continental boundary layer evolution, convection, turbulence and entrainment, on aerosol 690 formation variations of ultrafine particle concentration in urban gwangju, korea: observation of 699 ultrafine particle events respiratory effects are 702 associated with the number of ultrafine particles a language to simplify computation of differential mobility analyzer 706 response functions observation and analysis of particle 709 nucleation at a forest site in southeastern us an observational case study on the influence of atmospheric boundary-layer dynamics on new particle formation. boundary-layer meteorology spatial and vertical extent of nucleation events in the midwestern usa: insights from the a study of ambient fine 727 particles at tianjin international airport, china. science of the total environment size distribution and hygroscopic properties of aerosol particles from dry-season biomass 731 burning in amazonia frequent nucleation events at the high altitude station of chacaltaya (5240 m a effect of restricted 745 emissions during covid-19 on air quality in india sources and concentration of 748 nanoparticles (<10nm diameter) in the urban atmosphere atmospheric sulphuric acid and aerosol formation: implications from atmospheric 752 measurements for nucleation and early growth mechanisms air quality study: description and relation to key meteorological, gas phase aerosol parameters special issue of aerosol science and technology on findings from 757 the fine particulate matter supersites program particle number and size distribution from a diesel and new particle formation events at an urban site in hong kong severe air pollution events not 769 avoided by reduced anthropogenic activities during covid-19 outbreak. resources, 770 conservation and recycling scanning electrical mobility spectrometer characteristics 775 of regional new particle formation in urban and regional background environments in the horizontal homogeneity and vertical extent of new particle formation events. tellus b: 780 chemical and physical meteorology the los angeles international airport 783 as a source of ultrafine particles and other pollutants to nearby communities growth of nucleation mode particles in the summertime arctic: a case study strong 794 atmospheric new particle formation in winter in urban shanghai growth rates of nucleation mode particles in hyytiälä during 2003&minus;2009: 798 variation with particle size, season, data analysis method and ambient conditions characteristics of aerosol 802 size distributions and new particle formation in the summer in beijing a hypothesis for growth of fresh atmospheric nuclei aircraft emissions and local 816 air quality impacts from takeoff activities at a large international airport daily average integrated smps concentration for 5-10 nm (red) and 10-40 nm (blue) 832 size range vertical ticks are spaced one week apart. horizontal lines show monthly averages for 5-10 and 834 10-40 nm particles diurnal profiles of median planetary boundary layer height (pblh, left) and number 837 concentration of 10-40 nm particles (right). colors correspond to time period before and after shaded areas represent the 25 th and 75 th 839 quartiles of the data. pre-covid-19 encompasses days from spring 2019 and winter/spring 840 2019/2020 prior to march 14, 2020. covid-19 days include days between march 14 key: cord-272248-d0ybu9o3 authors: bostanci ceran, basak; karakoç, alp; taciroğlu, ertuğrul title: airborne pathogen projection during ophthalmic examination date: 2020-06-25 journal: graefes arch clin exp ophthalmol doi: 10.1007/s00417-020-04815-4 sha: doc_id: 272248 cord_uid: d0ybu9o3 purpose: microscale droplets act as coronaviruses (cov) carriers in the air when released from an infected person and may infect others during close contact such as ophthalmic examination. the main objective of the present work is to demonstrate how cov deposited droplets are projected during biomicroscopy and to discuss what kind of precautions should be taken in ophthalmic practice. methods: a coupled fluid-structure system comprising smoothed particle hydrodynamics and the finite element method has been built to assess the projection of droplets spreading from an infected person. different conditions based on the maximum exit flow velocity from the infector’s mouth during the ophthalmic examination were modeled. results: during exhalation, for which the exit flow is ~ 1000 mm/s, the average horizontal distance of the flow front was ~ 200 mm while individual particles can reach up to ~ 500 mm. in case of coughing or sneezing (corresponding to an exit flow of ~ 12,000 mm/s), the average horizontal distance of the flow front was ~ 1300 mm. conclusion: during the ophthalmic examination, the proximity to the patient’s nose and mouth was observed to be less than the horizontal distance of flow front particles. even though mounted breath shields are used, particles flew beyond the shield and contaminate the ophthalmologist. compared with the current protective breath shields, the use of a larger shield with a minimum radius of 18 cm is needed to decrease viral transmission. electronic supplementary material: the online version of this article (10.1007/s00417-020-04815-4) contains supplementary material, which is available to authorized users. in december 2019, china reported a pneumonia outbreak in wuhan, a city with more than 11 million people [1] . the causative organism was identified as a new coronavirus-namely, novel coronavirus: ncov-severe acute respiratory syndrome coronavirus 2 (sars-cov-2)-and the disease as the coronavirus disease 2019 (covid-19) [2] . after its introduction, the world health organization declared the situation as a public health emergency of international concern [3] and published suggestions for protection and prevention of transmission [4] . respiratory droplets, aerosols, and direct contact are confirmed transmission routes for covid-19 infection [5, 6] . droplets and aerosols might carry the virus in the air when they are spread from an infected person by breathing, sneezing, or coughing [7] . individuals who were infected by subclinical patients by droplets or by contact with secretions have also been reported [8] . in addition to that, some anecdotal reports suggest the possibility of transmission by aerosols through the conjunctival route if no eye protections are used [9, 10] . the diameter of the droplets spreading from an infected individual through breathing, coughing, and sneezing may range from 0.5 to 200 μm and transmission may be possible over a short distance (up to 90 cm) in the air [11] . droplets this article is part of the topical collection on perspectives on covid-19 electronic supplementary material the online version of this article (https://doi.org/10.1007/s00417-020-04815-4) contains supplementary material, which is available to authorized users. which are smaller than 100 μm have a high potential to be accumulated in the pharynx and larynx of the infected person and cause further infection [12] . ophthalmologists work in close proximity with patients during slit-lamp examinations (fig. 1a) . although protective measures like wearing masks, goggles, and breath shields (fig. 1b) are taken in large epidemics like covid-19, in daily practice, biomicroscopes are typically used without any prevention. in consideration of these occasions, the present study aims to simulate airborne pathogen projection through breathing, coughing, and sneezing during the ophthalmic examination and suggest preventive measures for diminishing transmission. in the present study, smoothed particle hydrodynamics (sph), which is a mesh-free lagrangian particle method that allows functions to be expressed in terms of their values at a set of disordered particles [13] , was implemented to study the airborne pathogens spreading under different circumstances. sph is a versatile and stabile method that address modeling needs where traditional numerical models such as the finite element method (fem) and finite difference method (fdm) are inefficient [14] . especially for the interactive applications for the highly deformable bodies and fluid flows, sph is proven to be a convenient method, which has been validated with various experiments and benchmark problems in the literature [15] [16] [17] [18] . in the sph framework, continuous field quantities a, their gradients ∇a, and laplacian ∇ 2 a at i th particle position x i are interpolated as a weighted sum of contributions from the neighboring particles as here, j is the particle index, m and ρ are the particle mass and density, respectively, h is the radius of support (or smoothing length bounding the neighboring particles used in the calculations), and w(x-x j , h) is the smoothing kernel that is chosen as a quintic function [19] . since sph is a lagrangian-based method, particles move with the domain. thus, the number of particles, each of which has constant masses m, is kept constant during the computations. this inherently satisfies the conservation of mass, where the conservation of momentum in terms of navier-stoke's formulation is expressed as where dv/dt is the substantial derivative, v is the velocity, g is the gravitational acceleration, and μ is the viscosity of the fluid. in the sph framework, for the ith particle, the pressure and viscous terms on the right-hand side of eq. (6) are expressed by means of the eqs. (1)-(3) as [20] . for the stable clusters of particles, the velocity is modified with xsph (x means unknown) introduced by monaghan where ε ∈ [0, 1]. in this framework, the relationship between the pressure and density for saliva, which is assumed to possess the mechanical characteristics of water, can be expressed with the linear us-up hugoniot form of the mie-grüneisen equation of state, here, c s is the speed of sound, γ 0 and s are material constants, ρ 0 is the reference density, e m is the internal energy per unit mass, and η is the nominal volumetric strain [22] . on the other hand, the air is assumed to be ideal gas with the equation of state given as follows where p a is the ambient pressure, r is the gas constant, t is the temperature of the gas, and t zero is the temperature of the absolute zero [14] . based on the abovementioned expressions, dv/dt of eq. (6) is solved in the scheme of explicit central-difference time integration algorithm with abaqus/explicit to obtain the time histories of field variables for all the particles. in the simulations, abaqus built-in surface behavior formulation is used to prevent the particles on opposite sides of a surface from interacting with each other, and free-slip condition is used throughout the mouth structure in consideration to the low viscosity and high flow velocity [14] . transmission of airborne pathogens eventuates by aerosol and droplets [23, 24] . airborne transmission of aerosols (≤ 5 μm) may occur over distances greater than 1 m, whereas droplet transmission is the spread of droplets (> 5 μm) over shorter distances [24] . in the present study, the transmission of all airborne pathogens, both aerosol and droplet, is included because the distance between two individuals during biomicroscopic examination is less than 50 cm. droplets and aerosols projecting from the patient's mouth and nose spread to air and contaminate the recipient. droplet size distribution does not differ between acts with expulsive pressure, such as coughing-sneezing and normal exhalation; however, the number of respiratory droplets may differ [25, 26] . the pathogen-loaded droplets that are inhaled may than be deposited in the recipient's respiratory tract, although there are reports showing the possibility of mucous membranes to be contaminated [27] . following this deposition, the pathogen gains the ability to be amplified in the respiratory tract and peripheral tissues of the recipient and the recipient become an infector [28] . the outflow from the mouth or nose during breathing, coughing, or sneezing can be treated as jet flow with several meters per second [7] . since each human has a unique mouth structure, way of breathing, coughing, or sneezing mechanisms, the models can only provide a likelihood for the pathogen spreading mechanisms. the characteristics of the saliva (e.g., density, viscosity) can simply vary for each human under different seasonal and ambient conditions. in order to standardize these effects, the flow characteristics were adapted from the experimental investigations in the literature, which have been carried out for various indoor scenarios [29] [30] [31] [32] . based on the indoor investigations, the drag force of the ambient environment was assumed to be negligible. moreover, due to the short period of analyses, which is t = 1.0 s, evaporation physics was also neglected, whereas gravity was taken into. accordingly, the maximum exit flow velocity from the infector's mouth, which was modeled as an ellipse with semiminor axis length of 2 mm and semi-major axis length of 9 mm, was regulated as~1000 mm/s (exhalation) and1 2,000 mm/s (coughing/sneezing). the solution domain was generated based on the oral volume capacity studies by nascimento et al., and which composed of a~70 ml reservoir of air (~90% volume fraction) and saliva particle (~10% volume fraction) mixture, where the air was assumed an ideal gas and saliva inherited the mechanical characteristics of water [33] as listed in table 1 . the particles were assumed to be spherical with radii of 50 μm in accordance with the previous parametric studies [11, 12, 34] . in order to understand the social distancing phenomenon and airborne pathogen spread during the ophthalmic examination, two scenarios with two different conditions based on exit flow velocities were considered: the configurations for these cases are depicted in fig. 2 . in these cases, the upper torso of the manikins was modeled as rigid bodies with three-dimensional 4-node, bilinear quadrilateral r3d4 elements provided in abaqus/explicit. the same elements were valid for the protective breath shields. the outer skin of the manikins and protective shield surfaces were modeled by using the rough friction model in conjunction with the no-separation contact pressure-overclosure relationship [14] . therefore, the particles hitting the surfaces were assumed to stick rather than bouncing back. as seen in the exhalation case of the first scenario (fig. 3a) , for which the exit flow was~1000 mm/s, the average horizontal distance of the flow front was computed as~200 mm, while the individual particles can reach up to a horizontal distance of5 00 mm. hence, it can be deduced that if the social distancing is not followed, there is a risk of airborne pathogen spreading even for the act of exhalation. the case for coughing or sneezing (fig. 3b) is unquestionably more hazardous. for instance, in case of an exit flow of~12,000 mm/s without any preventive action, fig. 4 airborne particle projection during the ophthalmic examination with the protective breath shield: a exhalation (the maximum flow velocity at the exit from the patient's mouth is~1000 mm/s), b coughing/sneezing (the maximum flow velocity at the exit from the patient's mouth is~12,000 mm/s) the average horizontal distance of the flow front was obtained to be~1300 mm. even more critical than this, some of the particles flew a horizontal distance of nearly 3000 mm in the time span of t = 1.0 s, which is greater than the social distancing recommendations, with the potent risk of carrying pathogens further than the flow front [4] . the results for the abovementioned cases show the necessity of preventive measures. especially, in case of medical examinations such as ophthalmic examination, for which the ophthalmologist and the patient have a distance around 200-250 mm. for this reason, protective breath shields that are mounted to the biomicroscopes have been in use (fig. 1b) . these shields approximately have the size of an a4 paper (210 mm × 297 mm) and are placed very close to the ophthal mologist. however, t here is no explicit recommendation/guideline regarding the size for these sheets and very little is known about the projection of airborne pathogens during the close proximity medical examinations even there exists protection. in consideration of this issue, a case study on such examination was carried out, the configuration of which is depicted in fig. 4 . in the case of exhalation (fig. 4a, clip 1) , it was deduced from the simulations that the protective breath shield configuration works well and most of the particles at the flow front stick to the shield that was assumed to have a rough surface. however, in case of coughing/sneezing (fig. 4b, clip 2) , it was observed that particles may flow beyond the protective shield and may get in contact with the ophthalmologist clothing, indicating a tangible risk. in order to minimize the risks, it appears that larger shields or more strict preventive measures are needed. based on the simulations, the minimum radius for a new shield design should be 180 mm as depicted in fig. 4b . the shield should undoubtedly cover the examiner's head and chest. a conceptual design is proposed based on the minimum size requirements deduced from the simulation data, which can be seen in fig. 5 . the new concept aims at covering the entire risk-zone perimeter and providing protection for the examinee and examiner against viral transmission if no other preventive measures such as wearing masks, goggles, or gowns are taken. a standard ophthalmologic examination relies strongly on physical evaluation to make a diagnosis. centers for disease control defines close contact for transmission risk as being 2 m from a patient for examinations that last 1 to 2 min [35] . the time we spend to complete a detailed ophthalmic examination is far beyond this period. it is therefore important to examine the projection behavior of droplets to determine effective protective measures during our usual practices. to that end, a three-dimensional model was used in the present study to predict the transmission of droplets during the ophthalmic examination. normal exhalation, coughing, and sneezing mechanisms with or without protective shields were simulated using different particle injection velocities. although protective measures like wearing masks, gowns, and goggles are recommended during covid-19 pandemic, in our study, none of these measures was taken into account for evaluating the sole effect of the breath shield to prevent transmission of airborne pathogens since these measures are difficult to standardize and might be discarded by the patients and also clinicians after the acute influence of the pandemic passes. the results indicated that the average horizontal distance of the flow even for normal breathing scenarios is enough for viral transmission during the biomicroscopic examination. although protective breath shields mounted to slit lamps offer a physical barrier between the ophthalmologist and patient, it appears that the dimensions of the currently used shields are inadequate, especially in case of coughing/sneezing. based on our simulations carried out in the present study, the minimum radius for a shield should be 180 mm, and the shield should cover the examiner's head and chest sections. having said that, one must admit that approaching the eyelids for a complete view of the ocular structures or fundus examination with a hand-held lens would be challenging with a breath shield of provided dimensions. as a result, a protective shield that allows access for fundoscopy or the ability to approach the eye will not be adequate for the protection of the ophthalmologist on its own and other measures including masks and goggles are necessary in order to perform a routine eye examination. additionally, thorough periodic cleaning of the protective breath shield and use of disposable isolation gowns, gloves, caps, eye protection, surgical masks, or thermal/chemical disinfection for the reusable laboratory clothing is needed since contaminant particles/droplets can stay in still air for several hours [4] . numerous measures-such as rapid diagnosis, tracing, and quarantine-have been adopted to counter the sars-cov-2 pandemic. however, due to delays in the initial detection of asymptomatic cases and infected individuals in the incubation period, the infection is still exhibiting an uprising trend [28] . as the occurrence and need for awareness of novel epidemic agents have been increasing from year to year, it appears paramount to accumulate knowledge for future outbreaks. stepping up infection control measures and updating current practices for preventing transmission in an evidence-based process necessitates interdisciplinary work, which requires joint efforts of healthcare professionals, engineers, and civil servants [36] . the present study aimed to serve as a starting platform for research into technical developments to reduce viral transmission during patient examination. availability of data and material (data transparency) all data and software applications support our published claims and comply with field standards. authors' contributions all authors have participated sufficiently in the preparation of this work to take public responsibility for it. conflicts of interests the authors declare that they have no conflict of interest. ethics approval this article does not contain any studies with human participants or animals performed by any of the authors. informed consent consent has been granted by the individuals for use of the fig. 1a in the submission to the journal. a novel coronavirus outbreak of global health concern china coronavirus: six questions scientists are asking detail/30-01-2020-statement-on-thesecond-meeting-of-the-international-health-regulations-(2005)-emergency-committee-regarding-the-outbreak-of-novelcoronavirus-(2019-ncov) infection-prevention-andcontrol-during-health-care-when-novel-coronavirus-(ncov)-infection-is-suspected-20200125 sars-associated coronavirus transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination study of sars transmission via liquid droplets in air protecting health-care workers from subclinical coronavirus infection ophthalmologic evidence against the interpersonal transmission of 2019 novel coronavirus through conjunctiva 2019-ncov transmission through the ocular surface must not be ignored human head model as an aerosol sampler: calculation of aspiration efficiencies for coarse particles using an idealized human head model facing the wind intercomparison of experimental regional aerosol deposition data smoothed particle hydrodynamics abaqus user's manual. abaqus standard version:6 particle-based fluid simulation for interactive applications experimental validation of smoothed particle hydrodynamics on generation and propagation of water waves numerical simulation for the air entrainment of aerated flow with an improved multiphase sph model smoothed particle hydrodynamics (sph) simulation of a high-pressure homogenization process particle-based non-newtonian fluid animation for melting objects lagrangian fluid dynamics using smoothed particle hydrodynamics on the problem of penetration in particle methods numerical simulation of sphere water entry problem using eulerian-lagrangian method respiratory and facial protection: a critical review of recent literature aerobiology and its role in the transmission of infectious diseases droplet expulsion from the respiratory tract the size distribution of droplets in the exhaled breath of healthy human subjects novel coronavirus disease 2019 (covid-19): the importance of recognising possible early ocular manifestation and using protective eyewear drivers of airborne human-to-human pathogen transmission human cough as a two-stage jet and its role in particle transport fsi analysis of the coughing mechanism in a human trachea experimental measurement of breath exit velocity and expirated bloodstain patterns produced under different exhalation mechanisms gender effect on oral volume capacity design and characterization of a cough simulator interim us guidance for risk assessment and public health management of persons with potential coronavirus disease 2019 (covid-19) exposures: geographic risk and contacts of laboratory-confirmed cases towards a conceptual framework to support one-health research for policy on emerging zoonoses this article contains video clips as additional online-only material. the following should appear online-only: clip 1 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments this work used computational and storage services associated with the hoffman2 shared cluster provided by ucla institute for digital research and education's research technology group. in this work, a.k. conducted the numerical analyses, a.k. and b.c.c. conceptualized and wrote the manuscript. e.t. provided technical guidance and editorial supervision. key: cord-255314-mqexsqzj authors: hersen, guillaume; moularat, stéphane; robine, enric; géhin, evelyne; corbet, sandrine; vabret, astrid; freymuth, françois title: impact of health on particle size of exhaled respiratory aerosols: case‐control study date: 2008-04-29 journal: clean (weinh) doi: 10.1002/clen.200700189 sha: doc_id: 255314 cord_uid: mqexsqzj individuals with viral infection could possibly emit an infectious aerosol. the distinction between exhaled breaths of infected and healthy individuals should facilitate an understanding of the airborne transmission of infections. in this context, the present study is aimed at distinguishing healthy individuals from symptomatic ones by the study of their exhaled breath. a setup composed of a modified hood connected to an electrical low pressure impactor, which allows for the study of a wide range of particle sizes (from 7 nm to 10 μm), has been developed in order to collect exhaled breaths. this setup has been used with seventy eight volunteers. the results obtained using principal component analysis (pca) showed that exhaled breaths of individuals without symptoms have statistical similarities and are different from those of individuals with symptoms. this separation was made by the greater proportional emission by individuals with symptoms of particles collected on stages 3 (d (50) = 0.09 μm), 6 (d (50 )= 0.38 μm), 8 (d (50) = 0.95 μm), 10 (d (50 )= 2.40 μm), and 12 (d (50 )= 4.02 μm) of the impactor. there was not a specific size distribution obtained for the individuals with symptoms. as a consequence, further research on the exhaled breath should be undertaken with symptomatic volunteers and would require the analysis of this wide range of particle sizes. respiratory viruses represent a major public health concern. outbreaks of bronchiolitis, influenza or cold have a major impact on the smooth running of society, e. g., the annual outbreak of bronchiolitis affects more than 460,000 infants in france [1] . airborne transmission of respiratory viruses has been well established for humans [2 -5] and for animals [6 -12] . therefore, it has been shown that infected individuals can emit particles in the air while talking, sneezing or coughing. these droplets may carry microorganisms and are likely to contaminate other people. the size of these droplets determines how long they will stay in the environment before settling and also the pulmonary site of deposition [13 -16] . until recently, only a few studies have explored the size and number of particles emitted during coughing, sneezing or talking. they present various results with the diameters of the droplets ranging between 0.3 and 2000 lm. the first experiments were performed in the early 1940's using high-speed photography [17] . duguid [18] studied these particles by microscopic measurement of stain marks found on slides exposed directly to air exhaled from the mouth. the study reported that the diameters of the droplets ranged between 1 and 2000 lm. it was also stated that 95% of these particles were less than 100 lm and that most droplets were between 4 and 8 lm in diameter. the number of test subjects and their health status were not described. loudon and roberts [19] used a chamber with bond paper placed inside to study droplet size. three subjects with a dye in their mouths coughed into this chamber. after 30 min, the air was withdrawn from inside the chamber through a filter. following this, stain marks on the bond paper and the filter were measured using microscopy. in this work, the average number of particles emitted per cough was 470. particles with a diameter of less than 10 lm represented 53.6 % of the sample. the health status of the test subjects was not described. gerone et al. [20] used a 127 l stainless steel chamber shaped as a truncated cone to minimize the impaction of particles on its sides. it was then associated with different systems, e. g., impinger, impactors and a particle-size analyzer, to collect coughs and sneezes. in the study, the volunteers were experimentally infected with coxsackievirus a-21. the authors reported that most of the particles were less than 1 lm. papineni and rosenthal [21] used an optical particle counter (opc) connected to a funnel into which five healthy subjects coughed. size spectra were reported to be weighted towards the smallest particles detectable by the opc (the opc was reported to have a lower diameter limit of 0.3 lm). approximately 85% of the particles detected had diameters of less than 1 lm and the total droplet concentrations ranged from 1 to 218 per l. finally, fennelly correspondence: e. robine, centre scientifique et technique du bâtiment (cstb), laboratoire de microbiologie des environnements intørieurs, 84, av. jean jaurs, champs-sur-marne, 77447 marne-la-valløe, france. e-mail: enric.robine@cstb.fr et al. [22] , using a chamber containing microbial air samplers, showed that most particles produced by individuals with pulmonary tuberculosis were in the respirable size range. since methods and test subjects are different from one study to another, it is difficult to compare these results. consequently, detailed information is not available. in particular, it is not possible at present to determine the impact of volunteer health on the particle size of exhaled respiratory aerosols. in this context, the present study is aimed at distinguishing healthy individuals from symptomatic ones by the study of their exhaled breath. the experimental approach consisted of firstly developing a system that allows measurement of fine particles exhaled from a greater numbers of volunteers (78 individuals), with and without, symptoms. following this, the size differences between aerosols emitted by symptomatic volunteers and controls were determined. finally, confounding factors, i. e., factors that could distinguish between the two groups of volunteers without one noticing were researched. seventy eight human volunteers, ranging in age from 6 to 66 years, participated in this study. this panel was composed of 43 patients selected by a general practitioner, called "symptomatics", with objective symptoms, i. e., cough, cold, etc. and 35 healthy persons without symptoms called "controls". among the population, 54% of the symptomatic volunteers and 50% of the controls were women. information was collected using a face to face interview with volunteers. eight questions were asked: three questions concerned volunteers themselves (sex, age, and smoking habits) and five questions described the symptoms (headache, fever, running nose, loose cough and dry cough). biological analyzes were performed for each volunteer on a nasal swab using the polymerase chain reaction (pcr) method. the viruses searched were influenza viruses a, b, and c, respiratory syncytial virus (rsv), human metapneumovirus (hmpv), rhinovirus, enterovirus and coronavirus [23] . a specific apparatus was designed in order to collect exhaled breaths [24] . this experimental system is outlined in fig. 1 and is comprised of a modified cylindrical polyurethane hood and hose (protector tornado t-9 hood, scott health and safety, lancashire, england) ventilated with powered air (protector tornado t-power, scott health and safety, lancashire, england) connected to a poly-methyl methacrylate (pmma) cone shaped part (bfp-cindar, champigny-sur-marne, france). the initial flow rate inside the hood of 140 l/min was decreased to 40 l/min in order to limit the particle loss. in order to prevent the particle content in the room obscuring the particles produced by coughing, the air in the hood was filtered with a p3 particulate filter (psl filter pf251/2, scott health and safety, lancashire, england). this modified ventilated hood was linked to an electrical low pressure impactor (elpi, dekati, tampere, finland). the elpi is a real-time particle size spectrometer. it measures airborne particles in the concentration size range of 7 n 10 -3 to 10 lm (see tab. 1). its nominal airflow is 10 l/min. a membrane filter (durapore membrane filter, millipore, ma, usa) was placed on each stage of the elpi in order to recover collected particles. no grease was added. in order to determine the particle loss in the system, an aerosol was compared before and after its passage through the ventilated hood. in this way an aerosolization setup, was positioned either directly connected to the elpi (to represent the aerosol before its passage through the hood) or inside the hood connected to the elpi. the aerosolized suspension was a ten-fold dilution of a solution that simulates saliva (artisial, laboratoire chemineau). the aerosol was generated using a collison nebulizer at 20 psi (bgi, ma, usa). then it was mixed with dry filtered air (10 l/min) in a homogenization sphere (belleville, france). each aerosolization lasted for 2 min. experiments were the same for each volunteer. after answering the questionnaire, individuals were placed inside the ventilated hood, which was connected to the elpi. volunteers were asked not to cough during the first 10 s of the experiment in order to obtain the background noise. they were then invited to cough as much as they could and for as long as possible. experiments were stopped when volunteers requested it. the ventilated hood was cleaned using ethanol following each experiment. particulate concentration obtained for one stage of the elpi throughout the size distribution for each volunteer. the dimensions of this data matrix are such that it is impossible to directly detect any similarities in statistical behavior between the volunteers (individuals) or sizes (variables). principal component analysis (pca) [25] was chosen to analyze these results. this type of analysis was employed in other studies processing the results obtained using data from microorganisms (mould or bacterium) [26, 27] . it enables a statistical representation of all data without emitting any hypothesis. pca generates an optimum and similar graphical representations of the scatterplot representing the data matrix. each pca factor is a linear combination of variables representing the maximum variance in the scatterplot. factors have an orthogonal relationship, so that they only take independent sources of variance into account. therefore, two spaces are constructed with the same factors, i. e., a vectorial space for variables and an observation space. two factors define a plane on which points (observations) or vectors are projected. the proximity of two vectors (variables) indicates a strong linear correlation between these two variables, as does the proximity of two points. therefore, groups of observations and variables are defined to provide a view of the data structure that is little evident at first sight. spad version 3.5 (døcisia) data analysis software was used for these analyzes. the answers of the volunteers to the questionnaire were transformed into a 79 n 9-point matrix. in this matrix, lines represent the volunteers and columns represent the questions. the intersection of a line and a column represents the answer to each question. the answers are binary, i. e., yes or no. a multiple correspondence analysis (mca) was applied to this matrix in order to identify the association between the selected factors. mca is an extension of correspondence analysis (ca) to the case of more than two variables. it is a method that allows the study of the association between two or more qualitative variables. mca represents for qualitative variables what principal component analysis does for quantitative variables. one can obtain maps where it is possible to visually observe the distances between the categories of the qualitative variables and between the observations [28] . in doing so, it was estimated that the lod was 140 particles cm -3 . only emissions with total concentrations greater than this value were considered. moreover, the background noise, i. e., the concentration measured before the volunteer starts to cough, was removed from the measured concentration during the emission for each stage of the elpi. in order to estimate the particle loss in the system, aerosol mimicking saliva was compared before and after its passage in the modified hood. these experiments (n = 5) permitted the assignment of a correction factor for each channel of the elpi to the emissions of each volunteer. table 1 lists the correction factors obtained for each stage of the elpi. the results of the biological analysis highlighted eight symptomatic volunteers infected by influenza virus and one by corona virus. no virus was found among the controls. due to the fact that the size of the influenza virus and corona virus are greater than 80 nm [1] the first and second channels of the elpi (see tab. 1) were not considered afterwards. the averaged size distributions of emissions are presented for each group in fig. 2 . this representation reveals that symptomatic volunteers emit more particles than controls, especially considering particles of 0.5 lm and micronics particles. these particles, considering their size, could carry some microorganisms such as virus or bacteria. these results are in accordance with the study of fennelly et al. [22] . however, the important dispersions of concentrations inside each group (210% for each group) make it difficult to draw conclusions just by the global observation of the two groups. moreover, analysis of the data collected consists of comparing two series of more than 40 size distributions, where each represents a significant quantity of information. therefore, pca, which enables a simple representation of data, was employed in order to study variances in detail without emitting hypothesis, see fig. 3 . data were standardized before applying the pca due to their important dispersion. therefore, the concentration of each particle size was divided by the total concentration for each of the 78 volunteers. the diagrammatic representation obtained using the spad program reveals two groups of individuals, which are outlined by drawing two ellipses. these groups are composed of individuals with and without symptoms, respectively. a close comparison between the two groups reveals that symptomatics are more dispersed than controls. indeed, symptomatics scatter along the two axes while "controls" only scatter along the first axis. as expected, individuals with viral infections are among the symptomatics. however, the presence of a virus had no impact on the exhaled breath, and therefore, infected people did not form a third group. nevertheless, it is difficult to conclude on the impact of virus considering the low number of infected volunteers involved. this method facilitated the separation for 80% of individuals. it should be noted that these results were obtained using the size distribution of the particles emitted in the exhaled breaths. as a result, the partition observed above is the consequence of the differences in these size distributions. as can be seen in fig. 3 , it is essentially the second factor that distinguishes the two groups. this factor corresponds to several stages of the impactor (variables). in order to identify these variables, they were projected onto a correlation circle, see fig. 4 . the length of the vectors obtained provides information on their representativeness in the circle defined by the principal plane. thus, the greater the importance of the vector norm, the more a variable is well represented in the chosen plane. by comparing the position of the volunteers on the pca representation, fig. 3 , with the directions of vectors onto the correlation circle, see fig. 4 , it is possible to determine which variables are responsible of the separation. in doing so, it should be noted that five stages of the impactor are involved. their cutpoints (d50) are 0.09, 0.38, 0.95, 2.40, and 4.02 lm, respectively. these results indicate that, in this study, symptomatics emitted more of these particles than controls. the fact that symptomatics are more dispersed than controls, fig. 3 , shows that they did not present a single type of exhaled breath. they can differ from the controls because of one or more of the particle sizes determined above. in 1997, papineni and rosenthal [21] supposed that, owing to their small size, viruses were susceptible to being among the fine particles, but until now there was no device that could measure fine particles. using the elpi, it appears that exhaled breaths contain fine particles that are possibly infectious. in addition, the data obtained with the pca permitted highlighted the necessity to take into account both the fine particles and also super-micronic particles. in this part of the study, the presence of possible confounding factors, which are other factors that could cause the separation between the two groups without one noticing, was checked. sixteen factors were compared with symptomatics factor and controls using the mca method. in the diagrammatic representation, a confounding factor is revealed by the superposition of points. the findings are summarized in fig. 5 . there is no superposition between symptomatics or controls and others factors. hence, there is no confounding factor concerning the two factors studied here, i. e., symptomatics and controls. in the same way, "sex" and "smoker" characters (near the origin in this graph) are halfway between symptomatics and controls. thus, they have no effect on the separation observed in the previous graph. finally, the symptomatics factor is halfway between the "loose cough" factor and the "dry cough" factor. subsequently, the size distributions of the symptomatics are independent of the type of cough. the purpose of this work was to distinguish healthy individuals from symptomatics by the study of their exhaled breath. from this preliminary study, it can be concluded that, on the one hand, exhaled breaths of individuals without symptoms have statistical similarities and are different from those of individuals with symptoms. on the other hand, a specific size distribution is not obtained for the individuals with symptoms. it was shown that a proportionally greater emission by symptomatic individuals of particles collected onto the impactor stages with cutpoint (d50) of 0.09, 0.38, 0.95, 2.40, and 4.02 lm make this separation. to the current authors' knowledge, differences between aerosols produced by symptomatic volunteers and controls have not been studied previously. moreover, this is the first time that such a large panel was used (78 volunteers) to study the exhaled breath [18 -21] . the knowledge of the exhaled breath of symptomatic individuals should also permit a better understanding of airborne transmission mechanisms, in particular, considering annual outbreaks and pandemic threats. i 2008 wiley-vch verlag gmbh & co. kgaa, weinheim www.clean-journal.com figure 5 . diagrammatic representation of a mca made by using the administered questionnaire in order to find out potential confounding factors. a confounding factor is revealed by the superposition of points. human influenza resulting from aerosol inhalation production of tracheobronchitis in volunteers with rhinovirus in a small-particle aerosol effect of route of inoculation on experimental respiratory viral disease in volunteers and evidence for airborne transmission an outbreak of influenza aboard a commercial airliner experimental infection of pigs with the porcine respiratory coronavirus (prcv): measure of viral excretion, veterinary microbiol experimental airborne transmission of porcine reproductive and respiratory syndrome virus and bordetella bronchiseptica, veterinary microbiol experimental infection of calves with bovine respiratory syncytial virus (quebec strain) experimental airborne transmission of prrs virus, veterinary microbiol airborne transmission of bhv1, brsv, and bvdv among cattle is possible under experimental conditions, veterinary microbiol airborne transmission of bovine herpes virus 1 infections in calves under field conditions a model for respiratory syncytial virus (rsv) infection based on experimental aerosol exposure with bovine rsv in calves airborne transmission and pulmonary deposition of respiratory viruses particle inhalability curves for humans and small laboratory animals physics of airborne particles and their deposition in the lung public health applications of high-speed photography the size and duration of air carriage of respiratory droplets and droplet nuclei droplet expulsion from the respiratory tract assessment of experimental and natural viral aerosols the size distribution of droplets in the exhaled breath of healthy human subjects cough-generated aerosols of mycobacterium tuberculosis: a new method to study infectiousness development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses etude de l'exposition aux aørosols viraux dans les environnements intørieurs, congrs français sur les aørosols statistique exploratoire multidimensionnelle etude de la contamination fongique des environnements intørieurs par la døtermination et la mesure de traceurs chimiques spøcifiques: application à l'hygine de l'habitat principal component analysis and discriminant analysis (pca-da) for discriminating profiles of terminal restriction fragment lenght polymorphism (t-rflp) in soil bacterial communities multiple correspondance analysis and related methods the authors thank marie-nol grattepanche for the selection of the volunteers with symptoms. olivier ramalho and jean-paul lucas provided excellent advice in pca and mca. key: cord-327507-ic3tdx59 authors: kirschbaum, stephanie; hommel, hagen; strache, peggy; horn, roland; falk, roman; perka, carsten title: laminar air flow reduces particle load in tka—even outside the laf panel: a prospective, randomized cohort study date: 2020-11-09 journal: knee surg sports traumatol arthrosc doi: 10.1007/s00167-020-06344-3 sha: doc_id: 327507 cord_uid: ic3tdx59 purpose: released particles are a major risk of airborne contamination during surgery. the present prospective study investigated the quantitative and qualitative particle load in the operating room (or) depending on location, time of surgery and use of laminar air flow (laf) system. methods: the particle load/m(3) was measured during the implantation of 12 total knee arthroplasties (6 × laf, 6 × non-laf) by using the met one hhpc 6 + device (beckmann coulter gmbh, germany). measurement was based on the absorption and scattering of (laser) light by particles and was performed at three different time-points [empty or, setting up, ongoing operation) at 3 fixed measurement points [or table (central laf area), anaesthesia tower (marginal laf area), surgical image amplifier (outside laf area)]. results: independent of time and location, all measurements showed a significantly higher particle load in the non-laf group (p < 0.01). with ongoing surgical procedure both groups showed increasing particle load. while there was a major increase of fine particles (size < 1 µm) with advancing activity in the laf group, the non-laf group showed higher particle gain with increasing particle size. the lowest particle load in the laf group was measured at the operating column, increasing with greater distance from the operating table. the non-laf group presented a significantly higher particle load than the laf group at all locations. conclusion: the use of a laf system significantly reduces the particle load and therefore potential bacterial contamination regardless of the time or place of measurement and therefore seems to be a useful tool for infection prevention. as laf leads to a significant decrease of respirable particles, it appears to be a protective factor for the health of the surgical team regardless of its use in infection prevention. level of evidence: i. causes of periprosthetic infections are mostly either a haematogenic spread or an intraoperative contamination [1] . intraoperative contamination can occur per continuitatem as h.hommel and s.kirschbaum are sharing first authorship. well as airborne [2] [3] [4] [5] [6] . airborne contamination is linked to the presence of suspended particles [7, 8] . approximately 5-10% of such particles carry bacteria, allowing bacteria sedimentation and contamination of the operating area or instrument table [7] . as a consequence, particle load can be used as a parameter for risk of infection [7] [8] [9] . various authors demonstrated that the use of laminar air flow (laf) systems resulted in a reduced intraoperative bacteria sedimentation [2, 5, [10] [11] [12] . it is therefore surprising that current literature didn't find a reduced infection rate when using laf [13] [14] [15] [16] . however, recommendations of existing reviews or meta-analyses examining the use of laf systems in reduction of surgical site infection (ssi) are usually based on inhomogeneous studies with different types and sizes of laf systems [13, 14, 17, 18] , where there may also be a lack of standardization of possible cofounders (antibiotic prophylaxis, patient related risk factors). another factor which is often discussed but has not yet been thoroughly examined, is the turbulent air flow occurring at the margin of the laf panel or even inside the laf area due to obstacles. only few studies compare concentration of suspended particles depending on position inside the laf area or examines particles as potential bacteria carriers in the operative arthroplasty setting [11, 12, 19] . no study currently examines the quantitative and qualitative particle load or its distribution during total knee arthroplasty (tka) in comparison with or without working of laf system. the aim of the present study is therefore to evaluate the quantitative and qualitative particle load in the operating room depending on the measurement location (inside laf area, margin of laf area and outside laf panel), the time of surgery and the use of a laf system. it was hypothesized that the laf system is able to reduce particle load at any time of surgical procedure. it was furthermore hypothesized that outside the working laf area particle load increases due to turbulent air flow. this prospective cohort study was approved by the local ethics committee (as58(bb)/2017). all the patients provided informed consent to be involved in the study. the air particle concentration was measured during the implantation of 12 tka. all patients were informed about the aims and the design of the study and agreed to participate. the patients were randomly allocated to laf or non-laf groups. only the study nurse performing the measurements was informed about the randomization. the surgical team was blinded concerning the laf function. six tka were implanted while using a laf system (laf group), 6 tka without the use of laf system (non-laf group). one measurement failed due to technical problems and therefore was not included in the data analysis. a new case was therefore included. in order to avoid cofounders due to architecture or previous operations, recommendations of edmiston et al. were followed [20] : every tka evaluated in this study was performed as first position in the same operation room (or). the measuring method of the met one hhpc 6 + device (beckmann coulter gmbh, germany) is based on the absorption and scattering of (laser) light by particles. photodiodes detect these effects and convert them into electrical signals, which are counted accordingly by the device. the measuring device can detect particles with a diameter of 0.3-10 μm. each measurement took 120 s and examined an air volume of 5.66 l. measurement was performed at three different time points during the ongoing operation. first, the particle load was referenced before the beginning of the operation day without any persons in the or. the second measurement was performed after preparing the surgical setting but before the patient entered the or. the third measurement was performed after exposure of the knee joint using the electrocautery ("safeair smoke evacuator", stryker) but before saw cuts were made. the particle load each time was measured at three fixed positions within the operating theatre ( fig. 1 ). position 1 was located centrally under the laminar flow system, directly next to the operating column. position 2 was located at a defined point of the anaesthesia device. this is located marginal in the laf area. position 3 was defined as a control point outside the laf area near the surgical image amplifier. to test for airborne contamination due to particle load, a single swab (fa. copan -floq swabs) of the electrocauteries "safeair smoke evacuator" filter was taken after each operation (n = 14). all microbiological samples were cultured on blood agar plates (37 °c, 48 h) in a standard fashion and interpreted by a consultant microbiologist. the panel of the laf system (admeco, hochdorf, switzerland) measured 3.2 × 3.2 m. air volume flow was 11,400m 3 /h, the vertical flow velocity reached 0.22 m/s. the exhaust air extraction was 50% near the floor and 50% by the ceiling device. the complete surgical team as well as all instrument tables were placed beneath the laf area. in order to minimize cofounders such as increased particle load due to door opening and number of persons in the or, those risk factors were standardized [19, 21] . door opening was reduced to a minimum: study nurse entered an empty or, 2 scrub nurses entered or to prepare surgical setting, patient and anaesthesiology team (2 persons) entered or and surgical team entered or. once the patient entered the room, there were seven persons in the or. all employees were dressed in cottonblended clothing in accordance with the applicable hygiene regulations and were equipped with surgical hoods and masks. the core team (2 surgeons, 1 instrumental surgical assistant) also wore protective goggles, sterile gloves and disposable gowns from berendsen chirutex. based on the results of sossai et al. [12] , the sample size estimation of the current study was performed using the program g*power (version 3.1, 2014) with an effect strength of 2.33, α = 0.05 and a power of 95%. first, a general comparison of qualitative and quantitative particle load between laf and non-laf groups, independent from measurement time and location, was performed. furthermore, particle load was analysed depending of measurement time and measurement location. descriptive statistics including means, standard deviation, and minimum and maximum values of continuous variables within the groups were calculated using the friedman test. the mean values between laf and non-laf were compared using the mann-whitney test (mwu). a p-value below 0.05 was considered significant. table 1 shows a comparison of all particle load measurements between activated and non-activated laf systems, independent of time and location. with and without laf system, a significant increase in particle load was observed with increasing activity in the operating room ( table 2 ). the dedicated comparison showed at each time-point a significant difference in favour of the laf group (mwu, p < 0.01 each). there was an increase of the particle concentration in the laf group with increasing distance to the operating column (table 3) . a comparison of the laf group and the non-laf group showed a significantly reduced particle load, independent of particle size and measurement location, when using laf (mwu, each p < 0.001). none of the microbiological cultures showed bacteria growth after 48 h of incubation. up to now there has been a controversial discussion concerning the effect of laf on the infection rate in arthroplasty surgery due to inhomogeneous data as well as the lack of prospective well-designed cohort studies. this is the first prospective, randomized cohort study evaluating the influence of laf on quantitative and qualitative particle load during surgical procedure at several locations of measurement. although former studies already demonstrated efficacy of the laf system in reducing overall particle load [11, 12] , the current study is the first one able to show that a laf system using a laf ceiling device significantly reduces the particle load during the entire surgical procedure at any location-even the one lying outside laf panel. those findings were independent from the particle size, confirming our hypothesis. the current study demonstrated that, independent from the use of laf device, the particle load increases with ongoing activity and number of persons in the or. the latter confirmed the finding of rezapoor et al. the authors demonstrated a decrease in particle density per person from 211.19 to 18.19 particles/ft 3 (p < 0.001) when using a laf system but failed to evaluate the influence of people activity during surgical procedure [19] . however, the current study showed significant reduction of particle load at any time when using a laf system. interestingly, while the laf group showed the highest increase in small particles during the operating procedure, the non-laf group showed the highest increase in bigger particles. so, the laf system seems more effective in reducing the quantity of bigger particles. as ongoing activity in the or is proven to increase particle load and therefore increase the number of potential bacteria carriers, it seems reasonable to outsource the patient's preparation (intubation, shaving, pre-cleaning, positioning) as far as possible from the or itself in order to reduce particle load and potential bacterial contamination. in the present study, the laf group showed an increasing particle load with increasing distance from the operation column by a factor 1.8-1900 depending on particle size. this is not surprising as with increasing distance, decreasing efficacy of the laf ceiling device can be assumed. unlike nilsson et al. suggested [11] , the current study was not able to find an increased particle load at the marginal laf area due to turbulent air flow. on the contrary, this study is the first able to show that laf system reduces particle load even outside the laf panel itself. the non-laf group showed also a significant increase in particle load (≤ 2 µm) at the operation column compared to the outer or area, but only by the factor 1.1-1.6. surprisingly there was no increase within the deactivated laf area. it remains to be assumed that the laf ceiling field influences the airflow, and thus the distribution of particles in the operating theatre, even when the laf is switched off. however, the highest particle load in the laf group was always less than the lowest particle load in the non-laf group at any measurement location, confirming the use of the laf system in reducing the particle load and therefore the risk of acrogenic bacterial contamination [9] . by now, the possible health-damaging effect of the particle load on the surgical team has received little attention. the effect and damage mechanism depends on surface charge and particle size [22] . the present study showed that the main particle load is made up from alveolar particles < 1 µm, and increases significantly with the beginning of the operation. here, too, the use of the laf system achieves a reduction of the load by a factor of 2.1-2.9. particle sizes up to 2.5 µm are considered respirable while ultrafine particles < 0.1 µm can even enter vessels [23] . unfortunately, particles smaller than 5 µm cannot be filtered by common surgical masks [24] . a positive surface charge of those ultrafine particles induces activation of the complement system and can trigger thromboembolic events after pulmonary exposure [25, 26] . furthermore, it was demonstrated that particles are able to carry bacteria as well as viral fragments [27, 28] . especially during the corona pandemic, the safety of the or team by reduction of potential risk factors should be of major interest. especially as tka surgery releases high levels of particles and aerosols by the use of saws, drills, electrocautery and jet lavage system [29] . therefore reducing particle load by means of a laf system might represent an additional safety factor for the surgical team. on the other hand, there are several studies discussing that in the case of covid-19, operations should take place within negative instead of positive pressure to reduce the risk of disseminating the virus beyond the or [30] [31] [32] . however, reducing particle and therefore virus load requires a high frequency of air changes (25 per h) and the use of a high-efficiency particulate air filter within the or [31] . besides the technical setting, correct protective equipment as well as or team discipline is needed to avoid the spread of covid-19 beyond the or [29, 30] . there are some limitations of the current study. first, it must be mentioned that only a smear test, and no evaluation of the cfu/m 3 or incubation of sedimentation plates, was carried out. furthermore, there were only 48 h of incubation of the microbiological findings. slowly growing bacteria such as cutibacterium acnes cannot be detected [33] . nevertheless, various authors demonstrated that the use of laf systems resulted in a reduced intraoperative bacteria sedimentation [2, 5, [10] [11] [12] . erichsen et al. showed that laf systems resulted in a reduction of 89% of colony forming units in comparison with the displacement system [34] . taken together, laf reduces the risk of bacterial sedimentation due to effective reduction of particles as bacteria carriers. a third limitation is that here was no examination concerning fungal findings. the lack of microbiological findings in the current study should therefore been viewed critically. another limitation is the lack measurements carried out at specific time-intervals (rather than at given surgical steps). although following highly standardized procedures, resulting in similar times of preparation and surgery, a certain bias due to time range cannot be excluded. on the other hand, measuring particle load after a fixed time might result in a bias too, as this results in the comparing of different stages of activity. furthermore, the current study showed a much higher particle load/m 3 compared to those mentioned above. due to a lack of specification regarding model and function of the particle counters used, only a different function can be assumed [2, 5, 10, 11] . a clear distinction must be made between particle load, bacterial contamination and later surgical site infection, as a reduced infection rate using laf systems could not be confirmed in recent studies [13, 14, 18] . however, as the parameter "infection rate" is strongly dependant on chosen criteria and follow-up time, particle load (as potential bacteria carrier) like used in the current study seems the more reliable parameter in evaluating aerogenic infection risk [7, 9] . regardless of the infection rate, the laf system appears to be a protective factor regarding the health burden of the surgical team, as a significant reduction of respirable particles can be achieved. the use of a laf system significantly reduces the particle load and therefore the risk of bacterial contamination regardless of the time or place of measurement and therefore seems to be useful tool for infection prevention. the current study is the first one demonstrating that the laf system is not only able to reduce general particle load during tka surgery but is also able to reduce particle load outside the laf panel. furthermore, this study was the first one demonstrating that especially respirable particles (< 2.5 µm), which cannot be filtered by the surgical mask, show a major increase in concentration with the beginning of surgery. as the use of laf leads to a significant decrease of those respirable particles it appears to be a protective factor for the health of the surgical team, regardless of use in infection prevention. periprosthetic joint infection the addition of a 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current covid-19 pandemic what we do when a covid-19 patient needs an operation: operating room preparation and guidance preparing for a covid-19 pandemic: a review of operating room outbreak response measures in a large tertiary hospital in singapore prolonged bacterial culture to identify late periprosthetic joint infection: a promising strategy comparison between mixed and laminar airflow systems in operating rooms and the influence of human factors: experiences from a swedish orthopedic center conflict of interest the authors declares that they have no conflict of interest.ethical approval approved as58(bb)/2017).open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-273382-7w8fli6w authors: guderian, daniela b.; loth, andreas g.; weiß, roxanne; diensthuber, marc; stöver, timo; leinung, martin title: in vitro comparison of surgical techniques in times of the sars-cov-2 pandemic: electrocautery generates more droplets and aerosol than laser surgery or drilling date: 2020-09-07 journal: eur arch otorhinolaryngol doi: 10.1007/s00405-020-06330-y sha: doc_id: 273382 cord_uid: 7w8fli6w introduction: based on current knowledge, the sars-cov-2 is transmitted via droplet, aerosols and smear infection. due to a confirmed high virus load in the upper respiratory tract of covid-19 patients, there is a potential risk of infection for health care professionals when performing surgical procedures in this area. the aim of this study was the semi-quantitative comparison of ent-typical interventions in the head and neck area with regard to particle and aerosol generation. these data can potentially contribute to a better risk assessment of aerogenic sars-cov-2-transmission caused by medical procedures. materials and methods: as a model, a test chamber was created to examine various typical surgical interventions on porcine soft and hard tissues. simultaneously, particle and aerosol release were recorded and semi-quantitatively evaluated time-dependently. five typical surgical intervention techniques (mechanical stress with a passive instrument with and without suction, co(2) laser treatment, drilling and bipolar electrocoagulation) were examined and compared regarding resulting particle release. results: neither aerosols nor particles could be detected during mechanical manipulation with and without suction. the use of laser technique showed considerable formation of aerosol. during drilling, mainly solid tissue particles were scattered into the environment (18.2 ± 15.7 particles/cm(2)/min). the strongest particle release was determined during electrocoagulation (77.2 ± 30.4 particles/cm(2)/min). the difference in particle release between electrocoagulation and drilling was significant (p < 0.05), while particle diameter was comparable. in addition, relevant amounts of aerosol were released during electrocoagulation (79.6% of the maximum flue gas emission during laser treatment). discussion: our results demonstrated clear differences comparing surgical model interventions. in contrast to sole mechanical stress with passive instruments, all active instruments (laser, drilling and electrocoagulation) released particles and aerosols. assuming that particle and aerosol exposure is clinically correlated to the risk of sars-cov-2-transmission from the patient to the physician, a potential risk for health care professionals for infection cannot be excluded. especially electrocautery is frequently used for emergency treatment, e.g., nose bleeding. the use of this technique may, therefore, be considered particularly critical in potentially infectious patients. alternative methods may be given preference and personal protective equipment should be used consequently. the sars-cov-2 pandemic is affecting people's lives and working environments worldwide and poses major challenges for health systems in particular [1] . the course of the disease is not yet fully understood, but varies from asymptomatic courses to severe pneumonia with life-threatening organ failure [2, 3] . in addition to the containment of the pandemic and the treatment of patients suffering from covid-19, protection of health care professionals is a priority to ensure the operational readiness of the medical system. at present, droplet and smear infection through coughing and sneezing, as well as transmission through aerosols (droplets smaller than 5 µm [4] ), is considered to be the main transmission route for this disease. sars-cov-2-positive patients present high virus densities in the upper and lower respiratory tract [5, 6] . at expiration [7] and speaking [8] virus containing aerosols are released [9, 10] . ent physicians are exposed to sars-cov-2 due to their direct proximity to the upper airways during examination and treatment. in addition, medical interventions in the area of the mucous membranes of the upper airways could promote the formation of aerosols and thus facilitate the transmission of pathogens. for otorhinolaryngology, this applies to interventions with passive instruments as well as active instruments such as electrocoagulation, laser or drills. up today, only few studies have examined the aerosol generation and distribution during specific ent medical interventions. a previously described model used fluorescein dye for detection of particle distribution in mastoidectomy and sinus surgery [11] [12] [13] . however, this method was mainly used to detect the spread of droplets and to a lesser extent the formation of aerosols, which are particularly relevant in the context of the covid-19 pandemic. the aim of the presented study was therefore to develop an experimental setup for the simultaneous assessment of aerosol and particle formation in various typical ent interventions. furthermore, a semi-quantitative comparison of these interventions was performed. these data are the basis for a risk assessment for potential aerogenic sars-cov-2 transmission and lead to recommendations for typical ent interventions. the setup developed to carry out the experiments is shown in fig. 1 . the interventions were performed in a test chamber (1) . an opening at the front wall of the chamber (2) allowed experimental interventions on the specimen (3). an acrylic glass plate on the rear wall of the chamber with a distance of 4 cm to the tissue sample was used for particle detection (4) . splashes of liquid and tissue particles released by the surgical intervention technique adhered to the plate. these splashes were recorded with a digital optical microscope (vhx5000, keyence, japan) for semi-quantitative evaluation. the acrylic glass plate was replaced for each test run. to detect the experimental aerosol formation, a black and white test picture (6) was documented by a full-hd video camera (eos 500, canon, japan) through a window (5) in the upper part of the chamber. aerosol release was quantitatively assessed by the turbidity of the sight on the text picture (see below for details on the methodology of particle and aerosol measurement). experiments were performed on fresh porcine hard and soft tissue specimens (bone and muscle). to facilitate the evaluation of particle formation, the tissue samples were immersed in royal blue ink and 1% methylene blue for 12 h and hereby stained intensely blue. five different interventions were evaluated: gmbh, germany, 60 w at power level 6, bipolar mode). the tissue specimens were treated for 3 min in each test condition. the experimental intervention was interrupted after 1, 2 and 3 min to record the particle formation. then the intervention was resumed. the video documentation to record the aerosol formation was done continuously. to eliminate inter-and intraobserver variability, measurements were performed fully automatic with different computerbased algorithms described below. the method of particle measurement via counting have been widely used to assess aerosol creation [11] [12] [13] . turbidity measurements are more commonly used in larger scale pollution measurements but were adapted to show aerosol generation in another way [14] . the acrylic glass plate on the rear wall of the sample chamber was photodocumented with a digital microscope (vhx 5000, keyence, japan) at 4 predefined positions (20 × magnification). these images were subsequently post-processed using proprietary software based on lab-view 2011 (national instruments, usa) (see fig. 2 ). a grey threshold-based segmentation was performed on a color plane extraction of the original image with good contrast of the originally blue stained tissue particles. the detected particles were evaluated with regard to their number and size (max. diameter). during manipulation of the specimen, a video file of the target object (no. 6 in fig. 1 ) was recorded with a full-hd camera at 25 frames per second. the resulting video was then post-processed frame by frame, also using proprietary software. indicator points were randomly distributed over the surface of the target object (see fig. 2 ). after averaging, the gray values were determined per image at these positions, resulting in a data graph with a time resolution of 40 ms. the measured turbidity of the camera view is an indirect measure for the aerosol density in the sample chamber. statistical evaluation was carried out using microsoft excel (excel for mac, version 16.16.20, microsoft, usa). non-parametric test procedures (wilcoxon rank sum test for comparisons within a test condition, mann whitney u test for comparisons between test conditions) were used due to the lack of detectability of a normal distribution. our results demonstrated clear differences comparing surgical intervention techniques. with the described experimental setup, all five test conditions could be assessed regarding their application-specific aerosol and particle generation. in the case of mechanical stress with a passive instrument, no particle or aerosol formation was detected even after several minutes of intensive manipulation of the tissue (see fig. 3 , first line). similarly, no particle or aerosol formation was detected during mechanical impact by use of a passive instrument in direct tissue contact with additional suction (cf. fig. 3 , second line). evaluation of particle formation: during mechanical manipulation, suction in tissue contact or laser application, no detectable particle formation occurred even after several minutes of treatment. during drilling, the slide is covered with a lot of rinsing liquid, but only a small amount of tissue particles are thrown along. there is a slight increase over time. with electrocoagulation the particle formation is much more pronounced. the mean particle size during drilling and coagulation shows no significant difference with a large variance in particle diameter. the black dots in the microscopic images are markings for finding the respective positions again and are not included in the particle evaluation the laser treatment of the tissue did not lead to a detectable particle formation at any of the three points in time of the analysis (see fig. 3 , third line). under the surgical microscope, however, a highly directed ejection of very fine droplets was observed. these droplets were ejected away from the tissue almost exclusively in the direction of the incoming laser beam. the co 2 laser application led to a strong aerosol formation (lower series of images and blue curve in fig. 4 ): within only 3 s the view of the test picture was obfuscated by 60%. after a further 9 s the maximum value was reached. additionally, rising plumes of smoke at the beginning of the intervention were created by the laser (fig. 4) . over the course of the treatment, the test chamber was filled completely with smoke gas and homogeneous fog of increasing density. this caused the scattering of the measured values to decrease over time. processing the specimens with a cutting drill resulted in clearly detectable particles. the released particles increased with additional processing time up to a density of 47.2 ± 31.8 particles/cm 2 after 3 min of treatment (mean value ± standard deviation). this corresponded to a rate of 18.2 ± 15.7 particles/cm 2 /min at a distance of 4 cm from the tissue. the average size of the particles remained constant over the experimental time (208.6 ± 17.1 µm). the particles consisted of solid tissue. in addition, a light precipitation of clear irrigation fluid appeared on the acrylic glass plate. with increasing processing time these droplets confluated. the detection of the aerosol formation showed a slightly clouded view of the target object, which, however, quickly cleared up during a short intervention pause (red arrows in fig. 4 at t = 31 s) . this effect represented a spray mist rather than a gaseous aerosol. after electrocoagulation, 189.1 ± 76.8 particles/cm 2 were detected after 3 min of intervention. the particles were mostly intensive and homogeneous blue stained droplets. solid tissue parts were found only sporadically. with 77.2 ± 30.4 particles/cm 2 /min electrocoagulation created 4.2 times more particles than drilling intervention. this difference was significant (p < 0.05). the average particle size for electrocoagulation was 266.2 ± 25.3 µm. a significant difference was neither found between the different measuring times, nor in comparison to drilling intervention. relevant amount of aerosol was also produced during electrocoagulation (orange curve in fig. 4 ). the maximum turbidity was 79.6 ± 1.2% and thus significantly lower than the aerosol formation by the laser treatment (p < 0.01). high sars-cov-2 densities were previously described in the upper respiratory area, exposing medical staff working in the head and neck area, as ent physicians, to a particular infectious risk [15] . surgical interventions such as the insertion of nasal tamponades, the aspiration of secretions from the nose, electrocoagulation of the nasal mucosa during nose bleeding or the use of laser interventions on the nasal mucosa are common ent procedures. these frequently performed procedure require special consideration and risk analysis. five typical ent intervention techniques were examined: mechanical intervention without and with simultaneous suction, laser intervention, the use of a drilling system and bipolar electrocoagulation. increased formation of particles and/or aerosols was considered as an indication of tissue or tissue components spreading and thus as an indicator of the potential risk of intervention-related infection. no droplet or aerosol formation could be detected during mechanical stress, which was simulated by scratching with a sharp dissector. this observation is consistent with the results of other authors [11] . our data indicate a comparatively low risk of aerosol formation, thus presumably low virus distribution and potentially low infection risk associated with this procedure. a typical clinical situation may be seen during insertion or removal of nasal tamponades. however, as we only used a model setup, the real live clinical situation may be different: in an awake patient, nasal interventions may result in additional effects as aerosol formation due to reflective sneezing or coughing [16] . no formation of aerosols or droplets was observed during mechanical stress with suction. postoperative suctioning of the main nasal cavities is an example of a very common ent procedure in postoperative nasal care or in the treatment of nasal bleeding. the results obtained from our model suggest that this intervention is not associated with relevant aerosol formation. no addition aerosol or particles were detected with simultaneous suction. furthermore, sharma et al. were able to demonstrate a protective effect of suction in their study on cadaver heads, as the aerosol formation was significantly reduced [12] . even with excessive tissue destruction using a microdebrider, the integrated suction effectively prevented droplet formation [11] . although our data did not show a positive effect of the application of a suction device due to the experimental design, it seems obvious that additional suction may be considered in mechanical nasal interventions. no particle formation was detected in tissue treatment with a co 2 laser. however, an emission of very fine droplets was observed under the surgical microscope. since the plate for particles was mounted in the opposite direction in our experimental setup, this minimal particle formation could not be detected. the laser-induced aerosol formation, however, was considerable and surpassed all other surgical intervention techniques. in ent medicine, lasers are widely used for various clinical applications. the co 2 laser in our study is a commonly used laser system. depending on the applied power density, thermal tissue effects range from coagulation, carbonization (charring of the tissue) to vaporization [17] . all these effects could also be shown in our experiment. it is known from laser treatment in hpv-associated papilloma that hpv-dna can be detected in the resulting flue gas [18, 19] . thus, increased rates of hpv infections among surgical staff [20, 21] have been discussed [22] . our data clearly confirm an excessive aerosol release during laser application. in the interest of employee protection adequate suction and ffp3 protective masks should be recommended when applying laser technology in sars-cov-2 patients. although the use of a drilling system showed less particle formation compared to electrocoagulation, complete pieces of tissue were released and distributed in our study. the dispersion of tissue particles during drilling was also observed previously in cadaver studies at the frontal and lateral skull base [11, 13] . depending on the viral load in these tissues, these particles have to be considered as potentially infectious (see table 1 ). as far as microscopically assessable, in our study the aerosol created by drilling consisted of clear droplets of the irrigation fluid without visible solid tissue components. other studies indicated, that even spray mist created by a drilling system and transferred over a distance of several meters was contaminated with blood particles of the patient [23] . thus, we cannot exclude that the apparently "clean" spray mist detected in our study could also contain biological material and could be infectious for sars-cov-2. intervention with electrocoagulation revealed a considerable aerosol formation, even though the density of the flue gas did not reach the same level as with laser treatment. with regard to particle formation, electrocoagulation showed the strongest effect in comparison to all other intervention techniques. in contrast to the application of the drilling system; however, these particles did not consist of solid pieces of tissue, but of liquid drops. in addition, with electrocoagulation these liquid drops were colored deep blue (from prepared blue-dyed tissue). this is an indication for intra-and/or extracellular fluid, that could be associated with a potentially higher risk of pathogen transmission. similar to the use of a drilling system, the extent of tissue disruption in electrocoagulation is time-dependent: the transfer of tissue and tissue components is linearly related to the duration of application. our data indicate, that particle formation, thus the risk of infection, may be increased with time exposed to electrocoagulation intervention. for everyday clinical practice, this should result in short time application of electrocoagulation if clinically possible in addition to individual protective measures. our data suggest that simple interventions without the use of active instruments carry a comparably low risk of infection, whereas technically supported interventions are associated with exposure to potentially infectious particles and aerosols. laser applications and drilling are techniques that are more likely to be used in elective procedures. hence, infectious status of a patient can be determined before intervention. if sars-cov2-infection is detected, these intervention techniques should be avoided. in addition, protective measures should be used if applicable [24] . emergency treatment in cases of uncertain infectious status represents a particular challenge. bipolar or monopolar coagulation has been frequently used here up to now. the clinical results of electrocautery are favorable [25] . however, against the background of the results obtained in this study, treatment alternatives may be discussed. this includes the use of nasal tamponade alone, if clinically possible. alternatively, local etching of the mucosa with silver nitrate sticks could also be considered [26] . primary conservative treatment with hemostyptics and nasal tamponades may also be an alternative treatment. if electrocautery is unavoidable, additional suction most likely reduces particle and aerosol exposure [27] . regardless of the chosen treatment option, wearing protective glasses is recommended to reduce the risk of conjunctival blood and pathogen transmission [28] . the present study presents a pre-clinical model, which differs from the clinical setting regarding several aspects. firstly, the particle measurement in our setup was carried out in a test chamber that does not correspond to the spatial dimensions of an oral or nasal cavity. future studies may look at an anatomic model with naturally shaped nasal and oral cavity, which may differ the spreading of particles and aerosols. secondly, breathing, which would be airflow directed towards the surgeon, was not simulated in our study. nevertheless, our setup allowed a reliable detection of differences for typical ent intervention techniques. in our study, aerosol formation was detected indirect via the optical turbidity. methodically, a maximum value could not be exceeded. thus, an increase of aerosol formation beyond the maximum value cannot be measured. despite these limitations, the methodology of our experimental setup was suitable to rank the intervention methods with respect to the amount of particle and aerosol formation. the extent to which aerosols and particles are generated during laser treatment and cautery depends largely on the basic technical setting chosen. the settings chosen in this study represent the standard used in our hospital. however, these technical settings may vary between institutions and can therefore influence aerosol and particle creation. we used aerosol and particle detection as a model for potential distribution. our approach allowed a semi-quantitative classification of particle and aerosol formation. although we did not use real virus particles our setup is a direct model for aerosols and particles that are clearly related to sars-cov-2 infections. clearly real virus exposure depends on various factors, e.g., individual virus load, not considered in our model. however, our model provides good indirect evidence for aerosol and particle exposure as a risk factor for potential sars-cov-2 infection. using an experimental setup, typical ent interventions on hard and soft tissue were compared semi-quantitatively with regard to particle and aerosol formation. simple mechanical stress with a passive surgical instrument did not generate any detectable aerosols or particles, whereas the application of a laser and a drill system is associated with a strong material release. electrocoagulation leads to the highest droplet and aerosol formation. in the light of the current pandemic, we suggest careful use of the above procedures to minimize the possible exposure of medical staff to potentially infectious material. especially common procedures as electrocoagulation in the emergency treatment of nose bleeding should be viewed critically and adequate measures to reduce aerosol exposure such as ppe and consideration of conservative treatment seem to be appropriate until further data regarding transmission of sars-cov-2 is available. conflict of interest there are no conflicts of interest to disclose by the authors. human and animal rights the submitted manuscript does not report data derived from experimental or clinical observations in human or animal subjects. informed consent for the same reason informed consent was neither necessary nor applicable. a novel coronavirus from patients with pneumonia in china covid-19: a novel zoonotic disease caused by a coronavirus from china: what we know and what we don't covid-19): update für anästhesisten und intensivmediziner märz 2020 airborne transmission and precautions: facts and myths sars-cov-2 viral load in upper respiratory specimens of infected patients viral load of sars-cov-2 in clinical samples detection of air and surface contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in hospital rooms of infected patients effect of voicing and articulation manner on aerosol particle emission during human speech transmission potential of sars-cov-2 in viral shedding observed at the university of nebraska medical center aerodynamic analysis of sars-cov-2 in two wuhan 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papillomavirus in the vapor of warts treated with carbon dioxide laser or electrocoagulation: detection and protection risk of acquiring human papillomavirus from the plume produced by the carbon dioxide laser in the treatment of warts does exposure to laser plume place the surgeon at high risk for acquiring clinical human papillomavirus infection? larynxpapillomatose -erstmalige anerkennung als berufskrankheit bei einer op-schwester [laryngeal papillomatosis-first recognition in germany as an occupational disease in an operating room nurse floating aerial blood mists in the operating room endoscopic skull base and transoral surgery during covid-19 pandemic: minimizing droplet spread with negative-pressure otolaryngology viral isolation drape comparison of outcomes between endoscopic surgery and conventional nasal packing for epistaxis in the posterior fornix of the inferior nasal meatus outcomes analysis in epistaxis management: development of a therapeutic algorithm surgical smoke and ultrafine particles epistaxis and conjunctival contamination-are our ent trainees at risk? key: cord-312236-8eaqn8eu authors: lai, alvin c.k.; cheng, y.c. title: study of expiratory droplet dispersion and transport using a new eulerian modeling approach date: 2007-06-02 journal: atmos environ (1994) doi: 10.1016/j.atmosenv.2007.05.045 sha: doc_id: 312236 cord_uid: 8eaqn8eu understanding of droplet nuclei dispersion and transport characteristics can provide more engineering strategies to control transmission of airborne diseases. droplet dispersion in a room under the conventional well-mixed and displacement ventilation is simulated. two droplet nuclei sizes, 0.01 and 10 μm, are selected as they represent very fine and coarse droplets. the flow field is modeled using k–ε rng model. a new eulerian drift-flux methodology is employed to model droplet phase. under the conventional ventilation scheme, both fine and coarse droplets are homogeneously dispersed within approximately 50 s. droplet nuclei exhibit distinctive dispersion behavior, particularly for low airflow microenvironment. after 270 s of droplet emission, gravitational settling influences the dispersion for 10 μm droplets, and concentration gradient can still be observed for displacement ventilation. after the outbreak of severe acute respiratory syndrome (sars) and avian influenza in east and southeast asia, there has been increasing research interest in studying the transport and control of airborne bacteria and viruses indoors (beggs et al., 2006; chao and wan, 2006; li et al., 2007; nicas et al., 2005) and in confined environments such as aircraft cabin (mangili and gendreau, 2005) . nosocomial transmission is also receiving significant attention in the medical literature (tellier, 2006) . in a review, eickhoff (1994) estimated that as high as 10% of all hospital-acquired infections occurred through airborne transmission. transmission of airborne disease is a function of the concentration of respirable infectious particles in air and the contact time. when a contagious individual coughs or sneezes, droplets containing infectious particles (bacteria, viruses) are released. the larger ones fall to the floor within a few meters. smaller droplets remain airborne long enough that the moist coating of saliva and mucus evaporate, leaving a residual dry nucleus of the droplet, that may include one or more bacteria or viruses (referred to droplet nuclei). by applying a simple water vapor balance calculation and using major components of mucus, nicas et al. (2005) the equilibrium diameter and the evaporating time required to reach the equilibrium state. due to the presence of the nonvolatile compounds in mucus, the droplet nuclei equilibrium size is roughly half of the original droplet size and the evaporating time is in the order of 0.5 s. similar finding is also reported by another review article (morawska, 2006) . depending on the original (and final) size, droplet nuclei can remain suspended in air for several hours, hence they can travel over long distances, distribute widely throughout indoors, and lead to airborne transmitted infections. aerosol droplet dispersion and transport in a ventilated enclosure depend on the ventilation scheme, particle size, density, concentration, source location, etc. among all the parameters, the ventilation scheme is the most important parameter influencing the droplet transport and dispersion indoors. the global airflow pattern affects significantly on the overall distribution of pollutants. high-level supply and high-level return (hereafter referred to well-mixed) scheme is the most popular ventilation arrangement for commercial buildings. high velocity, cooled air discharges through supply grills and warm air exhausts through return grills. for displacement ventilation systems low momentum, cooled air is supplied to lower part of the room and is exhausted through high ceiling return grills. previous studies using passive gaseous as contaminant sources concluded that with heat sources, displacement ventilation is more favorable to remove the pollutants without mixing to the whole indoor environments (brohus and nielsen, 1996; he et al., 2005) . droplet nuclei are aerosols, and some of their physical characteristics are very distinct from those of the gaseous counterparts. gravitational deposition and inertia are among the most important characteristics that distinguish aerosols from gaseous, and the importance of both features increases with size. nevertheless, some previous studies used passive gaseous as surrogates to investigate the transport and dispersion of aerosols (beggs and sleigh, 2002; qian et al., 2006; zhao et al., 2005) or applied well-mixed assumption to estimate the risk of droplet exposure (nazaroff et al., 1998; rudnick and milton, 2003) . plausible reason of using gas surrogate is that generation of gaseous is relatively straightforward, and the detection is fairly simple with very high accuracy. on the other hand, generation and real-time detection of aerosol droplets concentration are more complicated. due to the complexity of the indoor airflow, the temporal and spatial distributions of droplet transport must be solved by computational fluid dynamics (cfd). either eulerian or lagrangian frameworks can be employed to resolve the particulate phase. the author has developed a new eulerian methodology (drift-flux model), and the model has been validated experimentally for a scaled chamber. detailed descriptions of the model can be found elsewhere lai and chen, 2006; wang and lai, 2006) . the key objectives of the present work are (i) to apply the new eulerian approach to study droplet dispersion and transport in a ventilated room, and (ii) to highlight the influence of droplet sizes and ventilation scheme on mixing characteristics. to investigate droplet nuclei dispersion and transport using the new drift-flux approach, an enclosure with two identical model occupants with heat energy dissipated is selected (fig. 1 ). there is one occupant emitting droplets (source) and faces directly to another occupant (receptor). the only differences between the well-mixed and displacement ventilation configurations are the inlet boundary conditions, location and geometry. for the wellmixed ventilation, a high-level supply grill is used while for displacement ventilation, a floor supply is employed. table 1 shows the details of the room geometry and the boundary conditions. the geometry of a human occupant was originally suggested by brohus and nielsen (1996) . an opening, 0.01 â 0.02 m, measured at 1.54 m above floor, locating at the centerline of the head is added to simulate the mouth of the occupant. two planes are defined in the geometry; a breathing plane and a mid-plane. the breathing plane is 1.54 m above and parallel to the x-y plane. the mid-plane is symmetrical about the middle line in the x-z plane. table 2 shows the details of the geometry of the model occupant used for this study. the wall temperature is set to 298 k while the surface of the model is specified by body temperature 309.5 k. it has been reported that the initial emission velocity can be up to 100 m s à1 (wells, 1955) . recent study recording the coughed airflow of healthy males shows the velocity ranges from 6 to 22 m s à1 (zhu et al., 2006) . in the present study, the source emits unit density spherical droplet nuclei lasting for 0.5 s with initial velocity 50 m s à1 . two recent review articles demonstrate the time scale of evaporation. nicas et al. (2005) estimated that the shrinkage time from the original droplets to droplet nuclei is rapid and is in the order of 0.5 s. morawska (2006) also draws similar conclusion showing a very rapid evaporation rate. she modeled three pure water droplet sizes, 1, 10 and 100 mm. for the two smaller droplet sizes, the time required to evaporate to the equilibrium size ranges from 0.001 to 0.4 s. this time scale is at least an order of magnitude shorter than the residence time of the droplet nuclei suspended in the room, hence the approach adopted here is to ignore the ''evaporation period'' and model the droplet nuclei directly. nicas et al. (2005) also summarized the droplet size data available from the literature, and the size ranges from submicron to over 1000 mm. it is understood that the expiratory droplets follows a certain size distribution, however, in this work, two representative droplet nuclei sizes, 0.01 and 10 mm, are chosen. the smaller size represents those fine droplets of which inertia and gravitational settling can be ignored, and their motions follow the air streamline. on the other hand, the coarse size represents the upper size limit that can be inhaled, deposited into lungs, and cause health problems. to simplify the model, the droplets are assumed trapped once they touch any surfaces and do not resuspend or break-up. these assumptions are valid for the present low air velocity environment. coagulation effect has been examined by applying a simple estimation (hinds, 1999) . the particle table 1 room configuration wall b breathing plane source z x y nicas et al. (2005) . the result reveals that the coagulation effect can be neglected. renormalization group (rng) k-e turbulent model is adopted here to simulate the airflow. the rng k-e model is more appropriate for indoor airflow simulation, and better agreement between simulated results and measured data has been achieved compared to the standard k-e and other turbulence or laminar models (chen, 1995; posner et al., 2003) . a generic commercial cfd code fluent (fluent, 2005) was used to simulate the airflow. the piso algorithm was employed to couple the pressure and velocity fields. grid independent tests were performed and the optimal grid densities for the well-mixed and displacement ventilation geometries are 404,000 and 375,000 cells, respectively. since two dummy heat sources (occupants) are involved, there are buoyancy flows around the occupants. here, air density is defined as a function of temperature by a piecewise-linear function. the simulation was performed on an sgi onyx 3800 shared server. a simplified eulerian drift-flux model has been developed to take full advantage of the extremely low volume fraction of indoor particles lai and chen, 2006) . the term ''drift-flux'' (or drift velocity) stands for particle flux (or velocity) caused by effects other than convection, i.e. gravitational settling and diffusion for the current work. the advantage of this approach is the feasibility of incorporating other external forces i.e. electrostatic (wang and lai, 2006) into the model. as the convective velocity of the particle phase is the same as the air phase, the complexity of the two-phase flow system is greatly reduced. the governing equation for particle transport in turbulent flow field is given as where u is the air phase velocity vector, c i is the particle mass concentration, kg m à3 (or number concentration, m à3 ) of particle size group i (here-after the subscription i denotes particle size group), v s;i is the particle settling velocity, p is the particle eddy diffusivity, and d i is the brownian diffusion coefficient and s c i is the mass concentration source term. the drift-flux methodology is incorporated into fluent by a user-written sub-program. the performance of the new eulerian model is compared with a lagrangian approach, as discrete phase tracking has been employed to solve many types of two-phase engineering problems for more than a few decades. zhao et al. (2005) conducted an order analysis and concluded that for indoor aerosol particles, only drag force, brownian force and gravity, were important. the lagrangian particle tracking is carried out by fluent builtin features. the equation of motion of a small aerosol particle can be written as where u p;i and u i are the velocity of the particle and fluid, respectively, t is the particle relaxation time (lai and nazaroff, 2000) , n i ðtþ is the brownian force per unit mass, r p and r are the particle and air density, respectively, and g i is the gravitational acceleration. in the following section, some eulerian and lagrangian predictions will be presented sideby-side. for the lagrangian approach, a sampling plane must be defined prior to counting the particles. here, the droplets within 1.535-1.545 m (for breathing plane) and 1.745-1.755 m (for midplane) are selected. the purpose of including the lagrangian approach is to compare the results qualitatively only. it must be emphasized here that direct quantitative comparison of these two approaches is impossible as the variables and governing equations solved are different. fig . 2 shows the velocity field at the mid-plane for the two ventilation schemes at time ''0'' (momentarily before injection). for the well-mixed ventilation, the inlet jet flow causes air recirculation in the room, and the jet velocity is significantly higher than the neighboring region. in this figure, elapsed times which represent the arrival of the droplet puff to some specific locations are labeled. for instances, under the well-mixed ventilation, the expiratory droplets takes approximately 0.5 s to reach the receptor's face, 2 s to reach the wall a, 5 s to the receptor's legs, 10 s to the wall b. all the above times mentioned refer to the elapsed times from the commencing of the droplet emitting process. as expected, the displacement ventilation exhibits a very different airflow pattern. cooled, low velocity air flows at near floor level absorbing heat from the two occupants, and a vertical thermal plume dominates the airflow field in the boundary layer around each occupant. droplet cloud takes approximately 7 s to reach the exhaust. except for the mid-plane, the airflow velocity is fairly weak in all other regions. although it is not the prime objective to study the statistical properties of the lagrangian approach, a brief justification for the selection of the injection number seems necessary prior to discussing the results. three injection numbers were tested; 3000, 10,000 and 90,000. particles escaped through the outlet and deposited to the receptor's head region were recorded and counted. inferring from fig. 3 , there is no statistical difference between the results of 10,000 and 90,000 injections. hence, 10,000 injections were selected for all lagrangian simulations. fig. 4 shows the combined results modeled by the drift-flux and lagrangian approaches at the breathing plane and the mid-plane for 0.75 and 2 s elapsing from the commencing of the droplet emitting process. the high-speed droplet nuclei emitted from the mouth opening carry high momentum. they impact to the face of receptor directly resulting in significant particle loss. inferring from the results, it can be observed that the two approaches predict fairly similar profiles, and the essential feature of the droplet dispersion is captured by the current eulerian model. fig. 5 depicts the mid-plane results for 0.01 mm and 10 mm droplets under both ventilation schemes at 5 and 10 s. it is found out that for the same ventilation scheme, the difference between the two droplet sizes is not significant. the two modeling approaches give close matches, but this time the number of particles tracked are much less than those in fig. 3. as mentioned, the droplet dispersion depends on the bulk air movement and the physical properties of the droplet itself. the insignificant difference between two droplet sizes is attributed to the relatively short elapsed time presented here. even for 10 mm droplets with unit density, the settling velocity is just 3 â 10 à3 m s à1 , hence it needs very long time to distinguish the dispersion characteristics (cf. fig. 7) . at those elapsed times reported, the droplet trajectories follow closely to the airflow pattern (cf. fig. 2) . for instances at 5 s for well-mixed ventilation, the puff just arrives at the receptor's legs, and it matches very closely to the bulk airflow pattern. once the droplets are emitted, due to the much lower surrounding airflow speed they encountered, the droplets start to decelerate to attain the same velocity as the surrounding air, and then thereafter considered to be airborne. particle relaxation time (t) is used to characterize the time required for the particle to ''relax'' to become airborne where c c is cunningham slip correction factor, d p is the droplet diameter, and m is the kinematics viscosity of air. for the present system, the relaxation time ranges from 10 à4 to 10 à10 s. with these negligible relaxation times, the droplets decelerate almost instantaneously, and hence the droplets follow closely the airflow. as expected the concentration profile is significantly different between the two ventilation schemes. for the well-mixed scheme, due to the much higher airflow velocity (in the x-direction) compared to the displacement scheme (2 m à1 s vs. 0.2 m s à1 ), the droplets reach the vertical wall a in approximately 2 s. after the impact, many droplets are deposited to the wall, and the rest follows the large recirculation eddy. the predictions are very different for the displacement ventilation. in contrast to the well-mixed scheme where the airflow in x-direction dominates, the velocity in x-direction is very slow for the displacement ventilation. due to this characteristic, the transport of droplets in that direction is fairly weak, and the droplets move slowly to the exhaust outlet. fig. 6 shows the dispersion at 50 s for the two ventilation schemes at the mid-plane; under the well-mixed scheme, 0.01 mm droplets are well-mixed, whereas large concentration gradient can still be observed under the displacement ventilation. in fact, the droplets do not disperse to most regions of the room until 270 s (refers to fig. 7 ). under such a single emission event studied here, droplets under displacement ventilation take approximately 14 times longer than that for the well-mixed ventilation to achieve moderate room dispersion. some salient features regarding the two approaches are worth discussion. first, under the lagrangian methodology each particle has a unique id, and hence the position for each particle can be tracked throughout the entire simulation domain and time. in risk exposure applications, this feature seems attractive only for some cases such as to investigate the individual ''contribution'' of multiple sources. for a single source, particle tracking feature is not important most of the time. instead, for many exposure assessments, the spatial and temporal concentration levels are the vital pieces of information needed. due to its discrete phase nature, the post-processing of the lagrangian simulation for the results shown in figs. 4, 5 and 7 is not trivial. in these figures, a 1-cm thick ''slice'' is chosen and particles enclosed in the slice are counted. if a thicker slice is chosen, it cannot represent the correct spatial concentration; on the other hand, if the slice is too thin, there may be no particle contained. the selection is, however, quite arbitrary. there is one transformation methodology, called particle-source-in-cell (psic), which can convert the discrete particle trajectories into concentration (crowe et al., 1977) . by using the residence time the particle stays in a pre-determined cell volume, the particle mass flow rate can be transformed to concentration. however, particle trajectories are still required prior to applying the transformation. this means that the accuracy of the transformation depends critically on the number of the injections. in contrast, since the droplet phase is treated as a continuum, the drift-flux approach can directly present the concentration magnitude over the entire spatial domain. this feature is very attractive for exposure assessment. secondly, due to the nature of the governing equations resolved, direct comparison between lagrangian and eulerian results is impossible. here a qualitative comparison is performed by counting the ''discrete particles'' in fig. 5 and comparing to the scalar magnitude. thirty three, 7, 17 and 10 are counted in the 1-cm slices of figs. 5(a)-(d), respectively, and the trend is consistent with the scalar legend. in the future, more comprehensive comparison can be made by performing volume integration to get the spatial-average concentration. fig. 7 depicts the mid-plane results by the lagrangian and drift-flux approaches at 210 and 270 s. since the droplets are already homogeneously mixed at 50 s for the well-mixed system which is presented in fig. 5 , only the results for displacement ventilation are shown. due to the relatively few suspended droplets, for those lagrangian simulation results, the slice thickness is increased to 5 cm. apparent difference between the 0.01 and 10 mm is observed at these two longer elapsed times. sedimentation is observed for 10 mm particles as more droplets are found at the lower region, while for 0.01 mm particles no apparent settling is seen. in fact, a particle-free region is found for the 0.01 mm droplets. the same observation is found for the lagrangian simulation for 10 mm: more discrete droplets can be found near ground level. literature results have shown similar conclusions. a recent study also shows that 3 mm particle concentration in a chamber exhibits inhomogeneity, and can be attributed by the turbulent diffusion and gravity (richmond-bryant et al., 2006) . chang et al. (2006) performed a detailed large eddy simulation on indoor particles under natural ventilation. he found out that, due to the larger inertia and gravitational settling, coarse particles (pm 10 ) can move easily from one circulation region to other ones while fine particles (pm 2.5 ) are easily influenced by the surrounding complex indoor air pattern. based on the literature and the current results, it reveals that droplet nuclei may exhibit distinctive dispersion behavior, particularly for low airflow microenvironment. approximately about 40% of droplets are less than 10 mm in terms of number (nicas et al., 2005) . these droplets can still be inhaled and deposited deep to lungs. hence, the present result has important implication; using passive gaseous as surrogates of droplet nuclei or applying the well-mixed assumption may cause incorrect exposure risk assessment results. in spite of the low relaxation time, deposition and gravitation effects may influence the accuracy if they are not properly taken into accounted. an alternative eulerian drift-flux model is adopted to simulate dispersion of droplet nuclei in a ventilated room. two particle sizes (0.01 and 10 mm) are chosen to mimic very fine and coarse droplet nuclei. results show that both lagrangian and drift-flux approaches give similar concentration profiles. however, there are a few inherent drawbacks for the lagrangian approach including the difficulty in generating particle density plots at a certain plane, and more importantly the uncertainty in the number of droplets injected may lead to inaccurate conclusion, particularly if the number of droplet suspension is low. on the other hand, using the drift-flux approach with proper account on sedimentation gives straightforward concentration profile. in addition, the computational time and resources required for the drift-flux are much less than those required for the traditional lagrangian particle tracking methodology (typically less than 50%). this advantage becomes more significant for complex geometries with substantial grid elements. inferring from the results presented, it can be observed that for the well-mixed ventilation scheme, the dispersion pattern is dominated by the high velocity airflow, and the different between droplet sizes is not obvious. the droplets are homogeneously mixed within 1 min. when the global airflow speed is lower, the distinctive characteristics of coarse size start to appear. ten micrometer droplets begin to settle at the lower region of the room under displacement ventilation. a quantitative method for evaluating the germicidal effect of upper room uv fields methodology for determining the susceptibility of airborne microorganisms to irradiation by an upper-room uvgi system personal exposure in displacement ventilated rooms numerical investigation of airflow pattern and particulate matter transport in naturally ventilated multi-room buildings a study of the dispersion of expiratory aerosols in unidirectional downward and ceilingreturn type airflows using a multiphase approach comparison of different k-e models for indoor airflow computations modeling particle distribution and deposition in indoor environments with a new drift-flux model the particlesource in cell method for gas droplet flow airborne nosocomial infection: a contemporary perspective removal of contaminants released from room surfaces by displacement and mixing ventilation: modeling and validation aerosol technology, second ed comparison of a new eulerian model with a modified lagrangian approach for particle distribution indoors modeling indoor particle deposition from turbulent flow onto smooth surfaces role of ventilation in airborne transmission of infectious agents in the built environment-a multidisciplinary systematic review transmission of infectious diseases during commercial air travel droplet fate in indoor environments, or can we prevent the spread of infection? framework for evaluating measures to control nosocomial tuberculosis transmission toward understanding the risk of secondary airborne infection: emission of respirable pathogens measurement and prediction of indoor air flow in a model room dispersion of exhaled droplet nuclei in a two-bed hospital ward with three different ventilation systems transport of airborne particles within a room risk of indoor infection transmission estimated from carbon dioxide concentration review of aerosol transmission of influenza a virus a new drift-flux model for particle transport and deposition in human airways airborne contagion and air hygiene numerical study of the transport of droplets or particles generated by respiratory system indoors study on transport characteristics of saliva droplets produced by coughing in a calm indoor environment key: cord-287751-52e0tlcu authors: dai, qili; ding, jing; song, congbo; liu, baoshuang; bi, xiaohui; wu, jianhui; zhang, yufen; feng, yinchang; hopke, philip k. title: changes in source contributions to particle number concentrations after the covid-19 outbreak: insights from a dispersion normalized pmf date: 2020-11-06 journal: sci total environ doi: 10.1016/j.scitotenv.2020.143548 sha: doc_id: 287751 cord_uid: 52e0tlcu factor analysis models use the covariance of measured variables to identify and apportion sources. these models, particularly positive matrix factorization (pmf), have been extensively used for analyzing particle number concentrations (pncs) datasets. however, the variation of observed pncs and particle size distribution are driven by both the source emission rates and atmospheric dispersion as well as chemical and physical transformation processes. this variation in the observation data caused by meteorologically induced dilution reduces the ability to obtain accurate source apportionment results. to reduce the influence of dilution on quantitative source estimates, a methodology for improving the accuracy of source apportionment results by incorporating a measure of dispersion, the ventilation coefficient, into the pmf analysis (called dispersion normalized pmf, dn-pmf) was applied to a pnc dataset measured from a field campaign that includes the spring festival event and the start of the covid-19 lockdown in tianjin, china. the data also included pollutant gases and hourly pm2.5 compositional data. eight factors were resolved and interpreted as municipal incinerator, traffic nucleation, secondary inorganic aerosol (sia), traffic emission, photonucleation, coal combustion, residential heating and festival emission. the dn-pmf enhanced the diel patterns of photonucleation and the two traffic factors by enlarging the differences between daytime peak values and nighttime concentrations. the municipal incinerator plant, traffic emissions, and coal combustion have cleaner and more clearly defined directionalities after dispersion normalization. thus, dispersion normalized pmf is capable of enhancing the source emission patterns. after the covid-19 lockdown began, pnc of traffic nucleation and traffic emission decreased by 41% and 44%, respectively, while photonucleation produced more particles likely due to the reduction in the condensation sink. the significant changes in source emissions indicate a substantially reduced traffic volume after the implement of lockdown measures. positive associations between ambient particulate matter (pm) pollution and subsequent detrimental health outcomes have been well documented in many epidemiologic studies (song et al., 2017; yin et al., 2019) . given the severe fine pm (pm 2.5 , with aerodynamic diameter less than 2.5 µm) pollution in china, pm 2.5 was ranked as the first leading mortality risk factor in 2015 (cohen et al., 2018) . compared with larger sized particles with higher mass concentrations, smaller particles have lower mass concentrations but higher particle number concentrations (pncs) and larger surface areas that are more likely resulting in increased adverse health effects (meng et al., 2013; yin et al., 2019) . a recent time-series analysis in northern china (shenyang) reported that short-term exposure to increased pncs for particles with size less than 0.5 µm were significantly associated with total and cardiovascular mortality (meng et al., 2013) . they found that these associations between mortality and pncs tended to be independent of particle mass concentrations and simultaneous exposures to gaseous pollutants. previous studies also highlighted that the limited availability of size-resolved pnc measurements prevented obtaining adequate epidemiologic evidence regarding associations with pncs (ibald-mulli et al., globally (isaifan, 2020; venter et al., 2020) . for example, air quality in china has improved with the reduction of non-essential industrial and motor vehicle usage as reported by liu et al. (2020) , who has investigated the spatial and temporal characteristics of nighttime light radiance and air quality index values before and during the pandemic in mainland china. the covid-19 outbreak provides an opportunity to evaluate the public health benefits of air quality improvement. a recent study found that covid-19 forced-industrial and anthropogenic activities lockdown are likely have saved more lives by preventing ambient air pollution than by preventing infection . their cross-sectional study conducted in the united states indicated that an increase of only 1 μg m -3 in pm 2.5 was associated with an 8% increase in the covid-19 death rate. thus, an emphasis is needed on the importance of continuing to enforce existing air pollution regulations to protect human health both during and after the covid-19 crisis. the environmental impact of the pandemic is of particular interest to governments and the public since it is crucial for developing post-pandemic pollution control strategies. however, there are no reports to date on changes in source contributions to health risk relevant pncs during the outbreak of covid-19. to identify particle sources and calculate their contributions, source apportionment studies have been conducted to support control strategies that can further reduce the health burden of pm. positive matrix factorization (pmf) has been widely applied to 6 et al., 2011; lorelei de jesus et al., 2020) . dilution is recognized as a crucial process that induces other processes to alter the particle number and size distributions (gidhagen et al., 2005; jacobson and seinfeld, 2004; ketzel and berkowicz, 2004) . kumar et al. (2011) reviewed the dynamics and dispersion modelling of nanoparticles from road traffic in the urban atmospheric environment and concluded that dilution is the most important process that needed to be considered with highest priority in dispersion models irrespective of any spatial scales. freshly emitted particles and new particles formed via nucleation process are diluted in the air and undergone transformation processes. the conventional use of pmf to apportion pncs is to extract size distribution profile from observed particle size distributional data on the basis of the internal covariance of particles from different detected sizes. due to the variation in dispersion, some of the information content in the observation data will be certainly lost. to investigate the changes in source contributions to pncs after the outbreak of covid-19, a newly proposed dispersion normalized pmf (dn-pmf) that incorporated the ventilation coefficient (vc) into the pmf analysis aimed of reducing meteorological influence on the analysis was applied to a pncs dataset measured from january 15, 2020 to february 13, 2020, included the start of the covid-19 lockdown in tianjin, china. the database also included pollutant gases and hourly pm 2.5 speciated composition data. suburban site surrounded by several universities, and distant from major highways and high traffic zones. a suite of instruments was operated in the supersite building with sampling inlets on the roof terrace. particle number concentrations with sizes ranging from 7.2 nm to 778 nm were measured using a universal scanning mobility particle sizer spectrometer (u-smps) (palas, germany). other air pollutants, including pm 1 and pm 2.5 , sulfur dioxide (so 2 ), nitrogen oxides (no 2 ), carbon monoxide (co) and ozone (o 3 ) were also recorded hourly. organic carbon (oc) and element carbon (ec) were measured hourly using a semicontinuous thermal-optical carbon analyzer (focused photonics inc., china) and water-soluble ions (so 4 where is dispersion normalized concentrations, is the concentration observed during period i, and is the average value of vc over the entire measurement campaign. in this case, is 582 m 2 s -1 . the concentrations and uncertainties of pnc dataset were normalized on a sample by sample basis. the dispersion normalized concentrations and uncertainties were used as the input matrix to the pmf analysis. us epa pmf 5.0 was used for pmf analyses. details of pmf are available elsewhere (hopke, 2016; paatero, 1999) . in addition to measured pnc data, auxiliary variables include gaseous pollutants (so 2 , no 2 , co and o 3 ), pm 1 , pm 1-2.5 (pm 2.5 -pm 1 ), radiation and selected potential source tracer species (oc, ec, no 3 -analyses to support factor interpretation. adding additional variables increases the numbers of edge points and thereby reduce rotational ambiguity (emami and hopke, 2017) . all missing data values were replaced by the median value for the given size bin or species and the uncertainty was set to three times that value. measurement errors for pnc were estimated as follows (ogulei et al., 2006b; squizzato et al., 2019) : where is the estimated measurement error for size bin j of i th sample, nij is the measured concentration for size bin j of i th sample, ̅ is the arithmetic mean of the measured concentration for size bin j. was commonly used as a constant throughout all size bins and pollutants ( =0.01). given that the lowest and highest bins of pnc were reported with elevated measurement error (liu et al., 2014; wiedensohler et al., 2018) , we set higher uncertainties to these size bins. the first two lowest size bins and highest size bins were assigned with a multiplier of , and the second two lowest size bins and highest size bins were assigned with multiplier of . for subsequent lower and higher size bins, the multiplier was set with a decrement of 0.5 until it approaches unit. the overall uncertainty matrix was computed as (ogulei et al., 2006a) : where was estimated from eq. 3 and is a constant. c 3 values from 0.01 to 0.5 were tested. a value of 0.1 was assigned to c 3 for all size bins and 0.15 to so 2 , no 2 , co, o 3 , pm 1 , pm 1-2.5 and radiation, while c 3 was set as 0.2 for the total pnc to obtain an optimal solution in mathematical sense. for selected chemical species, these species concentrations below method detection limit (mdl) values were replaced by j o u r n a l p r e -p r o o f journal pre-proof half of the mdl value. the corresponding uncertainties of these values were set at five sixths of the mdl values (polissar et al., 1998) . missing chemical species values were estimated by linearly interpolating the closet non-missing values and the corresponding uncertainties were increased by a factor of 3. all auxiliary variables were assigned as "weak" and the total pnc was set as the "total variable" with uncertainty tripled. the f-value for total pnc was used to normalize the size bin data and factor contributions. the f-value for pm 2.5 was used to normalize the chemical the time series of pnc, pm 1 mass concentration and vc values are shown in fig. 1 . the hourly average total pnc from 7.2 to 778 nm over the measurement campaign was 14397 ±5695 pt cm -3 , ranged from 5490 to 48372 pt cm -3 . the average mass concentration of pm 1 was 52.2 ± 42.6 µg m -3 , ranged from 1.0 to 224.1 µg m -3 . in solutions using pmf with four to nine factors were explored for the original data and dispersion normalized data. the best solution with the optimal number of factors was evaluated with selection criteria of appropriately narrow distributions of scaled residuals of pncs and the physical interpretability of factors in terms of (a) examination of size factor profiles and its association with external variables, (b) source directionality from cbpf plots, and (c) diel patterns. eight-factor models were finally selected as the best solutions for both the s4 and 3, respectively. the factors are interpreted and discussed individually in the following section. this factor presented three major size modes: a nucleation mode peaked at ~8-20 nm, a small sized mode peaked around 30-50 nm and an accumulation mode peaked at 100-200 nm (fig. 3 ). on-line measurements of the size distribution of particles in flue j o u r n a l p r e -p r o o f and stack gas of a municipal waste incineration plant in europe showed a bimodal size distribution with peak mode at 90-140 nm and a smaller mode at approximately 40 nm (maguhn et al., 2003) . it was also found that particles tend to growth by absorption and coagulation after the boiler. the chemical species profile had high contributions of cr and cu with narrow disp intervals (fig. 3) , which are tracers of municipal waste incineration. lu et al. (2018) reported abundant cr in bottom ash from plastic materials incineration. the cbpf plot of incinerator also shows a very clear nw wind direction pointing to the municipal incineration plant. thus, this factor was assigned to municipal incinerator. on average, municipal incinerator had the lowest contribution to total pnc during the campaign (3.2%, fig. s5 ). traffic has been recognized as one of the major sources of pnc. two traffic factors were identified as traffic nucleation and traffic emission with particle major size modes about 10-30 nm and 30-150 nm, respectively. as been previously reported in literature (morawska et al., 2008; vu et al., 2015) , diel pattern of the traffic nucleation factor has a sharp morning rush hour peak (6:00-8:00 am) and had enhanced contributions more broadly distributed beginning in the afternoon. while the traffic emission factor showed a strong peak in the morning after the rush hour (8:00-10:00 am) and a minor peak in the evening rush hour. both traffic factors had high concentrations of no 2 and explained 5-10% of the ec variation, indicating its traffic exhaust nature including diesel emissions . the presence of some o 3 and radiation in the traffic emission factor, suggests that particles exhaust from vehicle emitted in the morning were possibly subjected to photochemistry processes. the cbpf plot of traffic nucleation indicates its directionality corresponded to the roads located ~1.5 km and 3.0 km southwest of the measurement site (fig. s1 ). the traffic emission factor has a stronger association with southeasterly winds and a predominant occurrence for wind speeds between 3-5 m s -1 . there are two major highways situated ~18 km east of the measurement site and a highway extending to the southeast. both traffic factors are heavily affected by local traffic emissions. the sia (ammonium nitrate and sulfate) had three modes in the particle number size profiles with the major size mode between 180-600 nm, an aitken mode and an ultrafine mode size ranges, which is similar with the observations such as in rochester, ny (squizzato et al., 2019) . the explained variations of particles in the accumulated mode increased with increasing size. the presence of high nitrate, sulfate and ammonium in the species profile support its assignment as secondary inorganic material. the wide particle size range of sia factor suggests that it originated from both gas-to-particle conversion of local no x and regional transportation of sulfate. some ec present in the species factor may be caused by the condensation of secondary material on freshly emitted ec particles. the morning peak of sia at 9:00-10:00 am likely resulted from downmixing of transported secondary particles from aloft after the breakup of overnight inversions. the broad afternoon minimum was likely due to the mixing layer height dynamics. the cbpf plot of sia shows higher probability values with moderate speed winds (~2 m s -1 ) come from downtown tianjin (nw) more than other directions, highlighting the regional transport nature of sia during heavy particulate pollution episodes. it was the largest contributor to pm mass concentration but accounted for only 6.2% of total pnc over the measurement campaign. in addition to new particle formation from traffic emission with a dominant particle size ranged from ~10-30 nm as typically observed during rush hour as discussed above, a factor with particle size peaking in the nucleation mode size range (<20 nm), and characterized by high explained variations of o 3 and solar radiation with narrow disp bands was identified as new particle formation (npf) through photochemistry. it shows a strong sharp increase around midday (12:00) concurrent with the highest solar intensities that drive photochemical processes. photonucleation occurred primarily during clean days with low pm mass concentrations (rivas et al., 2020) . the low pm concentrations result in low condensation sinks and allow for npf to occur (mcmurry and friedlander, 1979) . as suggested by the cbpf plot, this factor is associated primarily with relatively higher wind velocities for east-south-easterly winds and southerly winds. the cbpf plot of photonucleation overlaps with traffic nucleation in south-south-west direction, suggesting air masses from this direction j o u r n a l p r e -p r o o f likely facilitate npf. easterly winds often associated with relatively clean bohai bay air possibly favors the nucleation process because of its low condensation sink. interestingly, the non-local feature of photonucleation tends to indicate that the measurement site is likely well separated from the emission sources. on average, this factor contributed 7.8% of total pnc. coal combustion was reported as an important source of ambient particles with size ranged from nanoparticles to coarse particles, depending upon various factors such as coal type, combustion condition, dilution ratio and residence time, and pollution control device (vu et al., 2015) . the formation of particles emission from coal combustion involved in complex chemistry as it will undergo vaporizationnucleation/condensation-growth processes (carbone et al., 2010) . lipsky et al. (2002) reported a nucleation peak mode around 10 nm in the stack plume of coal combustion. particles tend to shift to large sizes after release into ambient air experiencing dilution, cooling, and condensation/coagulation processes. an experimental measurement of pulverized coal combustion suggested that ultrafine particles exhibited a unimodal distribution under 1500 k with size peak shifting from 10.76 nm to 38.46 nm as time evolved (gao et al., 2017) . there is a paper mill with hot steam supplied by pulverized coal boilers situated ~6.5 km se from the measurement site (fig. s1 ). the cbpf plot presents a clear se direction linking this factor to the paper mill. thus, this factor with particle sizes peaking around 30 nm was attributed to coal combustion. the gaseous and species profiles corroborate the assignment of this factor to coal combustion since it had relatively high concentrations of no 2 , ec, so 4 2and no 3 -, and explained some of the so 2 , co, and as variations. the diel pattern presents a j o u r n a l p r e -p r o o f journal pre-proof middle day peak as the particles can be transported by the afternoon sea breeze from bohai bay. the factor has a size mode ranging from ~50 nm to 300 nm and peaked in the accumulation mode (~120 nm). it was identified as residential heating. its size distribution is similar with particles emission from residential wood pellet boilers (chandrasekaran et al., 2011; wang et al., 2019) . it is associated with some so 2 , no 2 , and co with tight disp bands, and moderate loading of pm mass concentration. the chemical species profile also supports coal as it explained ~15% of cland ~10% oc with small intervals in mass fractions. cl is a common marker of coals in northern china (yu et al., 2013; li et al. 2019 ). the diel pattern for this factor presents a clear morning peak (7:00-8:00 am) during stable atmosphere conditions with low wind speeds. pnc of residential heating during nighttime appears to be higher than that during daytime. the cbpf plot of residential heating factor has an association with all wind directions and low wind speeds (<2 m s -1 ). the festival emission factor is attributed largely on the basis of strong chemical association with oc, ec, no 3 -, so 4 2-locations (joshi et al., 2019; kong et al., 2015; moreno et al., 2010; tian et al., 2014) . these species have relatively narrow disp intervals in the species profiles supporting the inclusion of firework emissions in this factor. oc, ec and sulfate in the profile were also likely emitted from coal/wood burning in large bond fires to celebrate the festival as reported in dai et al. (2020) . fireworks were generally banned in urban areas and tianjin implemented a no-fireworks policy this year. however, people still likely utilize fireworks during these holiday periods. based on the cbpf plot, the origins of these particles were located in residential areas where coal/biomass was burned for heating/cooking during the spring festival. people were required to stay at home after the implement of lockdown measures to prevent the spread of covid-19 and thus, more people were in residential areas throughout the day requiring more heating and cooking than under normal circumstances. fireworks emissions were correlated with residential coal/biomass burning. it should be noted that this factor contributed significantly to pm mass concentration, particularly pm 1 in the period after the start of the spring festival (january 24, 2020). since the dn-pmf reduced the influence of dispersion, the cbpf plots derived from dn-pmf results tend to have better defined directionality than the regular pmf based plots. this measurement campaign included the unusual event of the chinese spring festival (sf) and lockdown measures enforced to prevent the spread of covid-19. unlike the common sources under the "business as usual" scenario prior to january 24, there was a change in the number of sources due to the fireworks displayed during the sf. source emission strengths also changed after the covid-19 outbreak as emergency responses were implemented beginning on january 25 in tianjin. to j o u r n a l p r e -p r o o f to reduce the influence of meteorology on quantitative source estimates, dispersion normalized pmf incorporating data normalized with the ventilation coefficient into pmf analyses, was applied to a pncs dataset measured from a field campaign that includes the spring festival event and the start of the covid-19 outbreak in tianjin, china. in addition to pncs data, other variables include solar radiation, gaseous pollutants, particle mass concentrations and chemical compositional data were measured and included to facilitate the interpretation of factors. the pncs dataset combined with these additional variables were normalized by vc values and then subjected to pmf analysis. eight factors were resolved and labelled as municipal incinerator, traffic nucleation, secondary inorganic aerosol (sia), traffic emissions, photonucleation, coal combustion, residential heating and festival emission. to examine the effects of reducing meteorological influence on estimated source patterns, the diel pattern and source directionality for each source derived from conventional pmf (unnormalized) and dn-pmf were compared. the dn-pmf enhanced the diel patterns of photonucleation and the two traffic factors by sharpening the differences between daytime peak values and nighttime concentrations. after dispersion normalization, pmf yielded better defined directionality of sources. the directionalities of municipal incinerator plant, traffic emission and 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covid-19 lockdowns cause global air pollution declines with implications for public health risk. medrxiv review: particle number size distributions from seven major sources and implications for source apportionment studies emissions from in-use residential wood pellet boilers and potential emissions savings using thermal storage exposure to air pollution and covid-19 mortality in the united states: a nationwide cross-sectional study. medrxiv associations between size-fractionated particle number concentrations and copd mortality in characterization and source apportionment of pm 2.5 in an urban environment in beijing particle size and mixing state of freshly emitted black carbon from different combustion sources in china methodology, writing-original draft preparation jing ding: assisting in sample collection, data curation congbo song: result interpretation, writing -review & editing baoshuang liu: writing -review & editing xiaohui bi: resources, investigation jianhui wu: sample collection and analysis yufen zhang: writing -review & editing yinchang feng: resources result interpretation, writing -review & editing this work was financially supported by the national key r&d program of china key: cord-007363-h8xa83nw authors: yan, yihuan; li, xiangdong; ito, kazuhide title: numerical investigation of indoor particulate contaminant transport using the eulerian-eulerian and eulerian-lagrangian two-phase flow models date: 2019-05-09 journal: exp comput multiph flow doi: 10.1007/s42757-019-0016-z sha: doc_id: 7363 cord_uid: h8xa83nw transport of micron particles in a displacement ventilated room was simulated using both the eulerian-eulerian model and the eulerian-lagrangian model. the same inter-phase action mechanisms were included in both models. the models were compared against each other in the aspects of air velocity, particle concentration, and particle-wall interactions. it was found that the two models have similar accuracy in predicting the airflow field while each of them has its own advantage and drawback in modelling particle concentration and particle-wall interactions. the e-e model is capable of providing a mechanistic description of the inter-phase interactions, whilst the e-l model has obvious advantage in modelling particle-wall interactions. advices were given for choosing an appropriate model for modelling particulate contaminant transport in indoor environments. particulate pollutants in indoor environments have drawn increasing attentions in recent years since a number of diseases including asthma, neurogenic diseases, and lung cancer have been found to be related to exposure to inhalable airborne particles (mølhave et al., 2000; fucic et al., 2011) . in addition, virus could be carried by the particles or droplets and widely spread due to particle dispersion. recalling the global outbreaks of sars in 2003 and h1n1 flu in 2009, a great concern of infectious disease transmission through airborne particles in building environments and in small enclosed spaces, such as vehicle and airliner cabins, has been raised (rothman et al., 2006) . epidemiologic studies (buonanno et al., 2013) have proven that health risk associated with particulate matters is subject to the exposure dose which could be represented by the pollutant concentrations. thus, knowledge about the concentration and distribution of particulate matters in indoor environments is crucial to risk assessment and disease prevention associated with particle exposure. the computational fluid dynamics (cfd) technique has been proven to be an efficient approach to analyse transport of particulate matters in indoor environments as cfd is not only able to provide full-scale simulations and visualisation of the transport processes in a cost-efficient way, but also capable of leading to an in-depth understanding of the complicated physical phenomena. basically, two distinct approaches, namely the lagrangian approach and the eulerian approach, each having its own advantages and drawbacks, have been employed in cfd simulations of particulate transport in indoor air. both the lagrangian approach and the eulerian approach simulate the airflow using the same set of conservation equations, but use different methods to model particle movement through the air. the lagrangian approach, which tracks a number of representative particles separately through the air, has its unique advantage in whole-process description of particle movement from the injection point to the final destination. however, this approach cannot give direct prediction to the vol. 2, no. 1, 2020, 31-40 experimental and computational multiphase flow https://doi.org/10.1007/s42757-019-0016-z y. yan, x. li, k. ito 32 particle concentration as only particle dynamic equations are solved. therefore, additional post-process is required to calculate particle concentrations through the statistics of a large number of particle trajectories yielded from cfd computations. during the past years, the so-called sampling volume method (zhang and chen, 2007; salmanzadeh et al., 2012) and the kernel method (chang et al., 2012a (chang et al., , 2012b have been developed to estimate particle concentration based on the lagrangian cfd results. however, the stability and accuracy of these post-process procedures are still not satisfactory as reported by chang et al. (2012a chang et al. ( , 2012b . on the other hand, the eulerian approach, which treats the dispersed particles as a continuous phase, has gained relatively higher reputation in saving computational cost and simulating pollutant concentration, whilst it cannot predict particle motions or provide particle transport tracks. the eulerian approach comes with different models depending on the integrality of the description of the gas-particle interactions. during the past years, some simplified eulerian models have been utilized to model gas-particle flows in indoor environments, including the single fluid model by zhang and chen (2007) , the mixture model by zhao et al. (2008) , and the drift-flux model by chen et al. (2006) and zhao et al. (2008) . zhao et al. (2008) found that when compared with the mixture model, the drift-flux model has better accuracy since more mechanisms such as gravitational settling are included in the latter model. in fact, all of the aforementioned eulerian models are simplified by assuming the gas-particle mixture as a pseudo fluid whose physical properties are calculated based on the local volume or mass fractions of the two phases. despite a transport equation is solved for a dimensionless transportable scalar representing the particle concentration in some models, it should be noted that only one set of transport equations are solved for the balance of mass, momentum, and energy of the two phases. this drawback actually makes the inter-phase actions between the phases could not be fully described. therefore, a more comprehensive eulerian model which is capable of describing the transport of each phase as well as the inter-phase actions is in demand. in this study, another type of eulerian approach, known as the eulerian-eulerian two-fluid model (tu and fletcher, 1995; mohanarangam and tu, 2007) , is employed to simulate the transport and concentration distribution of particulate pollutants in an indoor environment. being different to the aforementioned eulerian models, the eulerian-eulerian model solves two sets of conservation equations governing the balance of mass, momentum, and energy for each phase. since the macroscopic fields of one phase are not independent of the other phase, the interaction terms which couple the transport of mass, momentum, and energy across the interfaces are solved in the field equations. the eulerian-eulerian model is believed to be capable of leading to a more mechanistic modelling of two-phase flows than the simplified eulerian models. although the eulerian-eulerian model has rarely been utilized to analyse contaminant transport in indoor environments, it has been widely employed in modelling aerosol (armand et al., 1998) and other gas-particle flows (chen and wang, 2014) . it was found that the eulerian-eulerian model is not only more cost-efficient, but also more accurate when the particle concentration is relatively high (chen and wang, 2014) . for the purpose of comparison, the lagrangian approach, termed as the eulerian-lagrangian model in this study, was also employed with the same inter-phase action terms included. the eulerian-eulerian model and the eulerian-lagrangian model are abbreviated as the e-e model and the e-l model in the following sections, respectively. a displacement ventilated room with dimensions of 5.5 m (length) × 4.5 m (width) × 2.7 m (height) was built for the purpose of model test, as illustrated in fig. 1 . all the room walls were assumed to be adiabatic. air with constant temperature of 18 °c was supplied from a square inlet (0.53 m × 0.53 m) located near the floor and exhausted through a circular outlet (diameter 0.25 m) located near the ceiling. this ventilation layout created a displacing airflow pattern in the room. the air exchange rate was carefully selected to be 3 h −1 in terms of the ashrae standard (2013b), which yielded an air supply rate of 0.066 kg/s at the inlet. for the dual purposes of a realistic simulation and achieving a low-momentum airflow condition at the inlet, a separate computation was firstly conducted to simulate air flowing through a displacement diffuser with the same dimensions as those of the inlet (0.53 m × 0.53 m) and containing 3200 small holes (each with 3 mm in diameter) on its discharging plate. the predicted velocity profile at the perforated plate was extracted and then set as the inlet velocity boundary condition at the room inlet. the numerical procedure of obtaining the inlet boundary condition is illustrated in fig. 1 as well. as demonstrated in our previous study (li et al., 2013) , when an occupant's thermal plume overlaps with its breathing zone, the thermal plume has a significant effect on the characteristics of particle inhalation. therefore, in order to create a breathing zone affected the thermal plume in this study, a seated female manikin model with detailed body features (available at www.ie.dtu.dk/manikin) was place in the middle of the room with its back towards the inlet. detailed information about the manikin geometry could be found in sørensen and voigt (2003) . the original manikin was slightly modified in this study to achieve a surface area of 1.50 m 2 . in terms of the ashrae handbook (2013a), the heat release rate from a sedentary human body (by both radiation and convection) with that skin area magnitude is around 85 w. as heat transfer by radiation is not taken into account in this study, a convective heat release rate of 45 w was equally applied at the manikin surface, as recommended by rim and novoselac (2009) . particles with a density of 1000 kg/m 3 and different sizes were released from a circular area with diameter of 0.5 m and located 0.5 m upstream of the manikin so that the particle trajectories could be effectively affected by the thermal plume of the manikin, as illustrated in fig. 1 . it should be noted that the shape and location of the particle injection area are for computational purpose only and they donot stand for any scenario. the selected particle sizes were 0.2, 0.77, and 2.5 μm, representing ultrafine, fine, and coarse aerosol particles, respectively. the rate of particle release was 0.1 g/s and the injection velocity was 0.15 m/s. in fact, it was found that the particle velocity dropped immediately after being released so that the particle injection velocity just had an invisible effect on the overall particle trajectories or particle concentration distribution. this is because that for particles with such small sizes, their movement is mainly controlled by the airflow due to the low inertial and gravitational effects (longest et al., 2004) . furthermore, comparative computations also demonstrated that even the particle injection rate is as high as 0.1 g/s, the normalized particle concentration pattern is free from the effects of particle injection concentration. this means that for cfd simulations of particulate transport in most indoor environments, it is safe to ignore the effects of particle injection concentration on the overall concentration pattern. in an e-e model, the particle phase is treated as an additional continuous phase inter-penetrating with the continuous air phase and two sets of conservations governing the balance of mass, momentum, and energy of each phase are solved. as inter-phase heat and mass transfers are not considered in this study, the conservation equations take the following form (ansys, 2011) : the continuity equation where i and j are the phase denotations (i, j = a for the air phase and i, j = p for the particle phase). , α , ρ , u  p, h, t, and λ represent the volume fraction, density, velocity, pressure, enthalpy, temperature, and thermal conductivity, respectively. it should be noted that the energy equation (eq. (3)) was solved only for the air phase while heat transfer within the particle phase was ignored. is the momentum source due to buoyancy, which is defined in terms of a reference density ref ρ which takes value of air density at the inlet: when calculating the thermal buoyancy force induced by the thermal plume, the buossinesq approximation is employed in the momentum equation to take into account thermal expansion of the air. where β is the coefficient of thermal expansion and ref t is the reference temperature which takes value of air temperature at the inlet. (2)) represents the interfacial forces, which is formulated based on the assumption of spherical particles and includes the drag force d f  , the turbulent dispersion force td f  , and the virtual mass force where d c is the drag coefficient correlated to the particle reynolds number, td c is the turbulent dispersion coefficient, and vm c is the virtual mass coefficient. when an e-l model is employed for a gas-particle flow, the air phase is still governed by the eulerian equations (eqs. (1)-(3) with a 1 α = , which means the volume fraction occupied by the particles is negligible), while the particles are tracked using the lagrangian method separately through the airflow field. being different to the e-e model, the effect of turbulent dispersion on particle transport is modelled by adding an eddy fluctuating component onto the mean air velocity. it is the fluctuating component of the air velocity which causes the dispersion of particles in turbulent flow. therefore, the local air velocity is redefined by where φ is a normally distributed random number which accounts for the randomness of turbulence about a mean value. in the e-l model, the particles are tracked using the equation of motion. for a spherical particle with a diameter of p d immersed in continuous air, the drag force d f  , the buoyancy force buoy f  , and the virtual mass force vm f  are considered here in order to keep the same inter-phase momentum transfer mechanisms as those considered in the e-e model. the room model containing the manikin (fig. 1) was discretized using unstructured tetrahedral and prism meshes. fine meshes were used around the manikin surface in order to capture the geometric features and the human thermal plume. the grid sensitivity test proved that mesh independence was achieved at 3.4 million cells for the both models. the re-normalisation group (rng) k-ε model was chosen for the airflow turbulence because of its successful utilization in the simulations dilute gas-particle flows (tu and fletcher, 1995) . to resolve the boundary layer in the near wall regions, the scalable wall function (ansys, 2011) was used for the air phase in all the models. particle deposition was not considered in this study. in the e-l model, the particles are assumed to bounce back with the same momentum magnitude after they collide with the solid walls. however, as the particles are treated as a continuous phase in the e-e model, their bouncing behaviour is hard to be modelled (tu and fletcher, 1995) . as an approximation, a free-slip boundary condition was applied at the solid walls for the particle phase in the e-e model. the models were solved by the commercial cfd code cfx 14.5 (ansys inc.) and convergence was achieved when the rms residual of the continuity equation dropped down to 1.0 × 10 −6 . a typical airflow field yielded from the computations is illustrated in fig. 2(a) . it was found that due to the effect of manikin body heat, a significant thermal buoyancy flow was observed above the manikin head. the thermal buoyancy flow was so strong that it was the major airflow in the room, besides the airflow near the floor which was induced by the ventilating jet. salmanzadeh et al. (2012) the airflow in a displacement ventilated room containing a seated thermal manikin, and the predicted thermal plume is illustrated in fig. 2(b) for the purpose of comparison. it was found that this study predicted a very close pattern of the human thermal plume to that by salmanzadeh et al. (2012) ; especially, the low-velocity region immediately above the head was successfully predicted. as shown in fig. 2 , as the uprising airflow detaches the manikin head, it keeps accelerating until it reaches its maximum velocity. therefore, the highest speed of thermal plume exists at somewhere above the manikin head. as the thermal plume hits the ceiling, it then changes its direction to horizontally spread. this actually contributes to the temperature stratification in a displacement ventilated room. however, due to the difference in the geometry and boundary conditions such as the ventilating jet direction and velocity, as well as the heat flux at the manikin surface, this study predicted a slightly lower buoyancy flow velocity than that by salmanzadeh et al. (2012) . the numerical results demonstrated that the both models yielded very similar airflow fields. for the purpose of quantitative comparison of these models, velocity distributions along a horizontal line (line 1, fig. 2(a) ) crossing the whole computation domain at a height of 2.0 m and a vertical line (line 2, fig. 2(a) ) which has a length of 200 mm and is located 20 mm in front of the manikin nose tip were plotted in figs. 3(a) and 3(b), respectively. air velocity distribution along line 1 could represent the overall airflow field while the air velocity distribution along the short vertical line could represent the local airflow pattern. it was found that the two models gave very close predictions for the air velocity profile. especially in the breathing zone ( fig. 3(b) ), the value difference between the e-e model and e-l model was less than 1%. rim and novoselac (2009) once investigated experimentally the airflow field and human thermal plume in a displacement ventilated room containing a seated manikin, which had a very similar setup with the model of this study. the measurements revealed that the air velocity in the bulk region was negligibly small (less than 0.05 m/s), which agreed well with the predictions of this study. as shown in fig. 3(a) , the models predicted almost quiescent air in the bulk region with air velocity less than 0.02 m/s. rim and novoselac (2009) also measured the average thermal plume velocity in a circular plane with 0.25 m diameter above the manikin head using 8 velocity sensors. the experimental data was analysed in this study and an average air velocity of 0.215 m/s was obtained and compared to the numerical results, as shown in fig. 4 . the area-averaged air velocities in the circular region were found to be 0.201 and 0.203 m/s for the e-e and e-l model, respectively. this small difference (less than 5%) is within the uncertainty of the experimental measurement and computational model rim and novoselac (2009) and velocity in the circular region (250 mm in diameter) 250 mm above the manikin head. prediction. the predicted air velocities in the thermal plume region also agreed well with most experimental measurements (craven and settles, 2006) and numerical simulations (sørensen and voigt, 2003) . again, the e-e model and e-l model yielded very close predictions. this indicates that a complete description of inter-phase actions would help further improve the model reliability. typical particulate contaminant distributions in the room predicted by the aforementioned models are illustrated in fig. 5 . it is clear that the e-e model (fig. 5(a) ) gives a direct prediction to the particle concentration while the e-l model (fig. 5(b) ) predicts the particle trajectories (0.77 μm). despite this, the overall transport or distribution patterns of the particles predicted by the both models are very close. as the particles approach the manikin, they bend their way upwards due to the buoyancy effect of the thermal plume, then after the particles hit the ceiling, they bend their way again into the horizontal direction, which causes the particles spreading all over the room. locally, it is important to notice that some of the particles which are released at lower height are entrained into the breathing zone by the thermal plume ( fig. 5(a) ). the particle concentration in the breathing zone is therefore larger than the ambient concentration. this is consistent with a number of experimental observations that the human thermal plume is capable of increasing the exposure risk of the occupants to particulate contaminants by entraining particles from a lower level into the breathing zone (bjorn and nielsen, 2002; rim and novoselac, 2009 ). typical particulate contaminant distributions yielded by the models (0.77 μm). cn presents the normalized particle concentration, which was normalized based on the average particle concentration in the circular region of particle injection. in order to keep the comparability of the models in predicting particle transport, the so-called particle source in cell (psi-c) method developed by zhang and chen (2007) was utilized to calculate the particle concentration based on the particle trajectories yielded from the e-l model. where c j is the local particle concentration in the jth cell and v j is the volume of that cell, m is the mass flow rate represented by a particle trajectories, and dt(i, j) is the residence time of the ith particle in the jth cell. it should be noted that the control volume here for concentration calculation is different from the computational mesh for model solution. in terms of the psi-c method, the computational domain needs firstly to be divided into a number of small control volumes (or cells, which are for post-processing purpose only and are different to the computational meshes), then a number of particle trajectories are selected for concentration calculation from the numerical results yielded from the e-l model. it was found that the resultant particle concentration is highly sensitive to both the cell size and the particle trajectory number. at first, the cell size needs to be carefully determined so that an appropriate number of particle trajectories are contained in the each cell. then, for a given cell division scheme, the number of particle trajectories needs to be tested to obtain an acceptable concentration calculation. for the issue of this study, when the computational domain was divided into 100(x) × 80(y) × 50(z) cells, sensitivity test proved that 50,000 particle trajectories were sufficient for a stable particle concentration since a further increase to 60,000 trajectories just caused a negligible change in the concentration profile along a randomly selected vertical line, as shown in fig. 6 . in fact, since the stability of the psi-c fig. 6 sensitivity test of particle trajectory number on particle concentration calculation. method and its resultant particle concentration are not only affected by the trajectory number, but also highly impacted by the cell size, when a quantitative index is absent for the assessment of optimal cell size, it is anything but an easy job to obtain a stable solution. therefore, more advanced postprocessing procedures for particle concentration calculation based on the e-l model are in urgent demand. the predicted particle concentration distributions in a vertical plane cutting through the manikin (y = 2.25 m) and a horizontal plane close to the ceiling (z = 2.50 m) are compared in fig. 7 , which clearly illustrates the process of particle transport. after the particles are released from the injection area, they follow the airflow and move forward. when approaching the manikin, most of the particles bend their way upwards before reaching the manikin while minority of them go around the manikin and then are brought up by the thermal plume. after the particles approach the ceiling, they bend their way and move horizontally and finally to the whole room. this is consistent with the airflow velocity pattern as illustrated in fig. 2 . a comparison of fig. 2 and fig. 7 reveals that the size of the particle plume, which is the region in which the particle concentration is obviously higher than the ambient concentration, is significantly smaller than that of the thermal plume (fig. 2) in which the air velocity is higher than the ambient air velocity. furthermore, the particle plume exists mainly upstream of head-top point where it detaches the manikin. this is physically reasonable since the solid manikin behaves as an obstacle against particle movement, most of the particles have to change their way upward and then were brought further by the uprising airflow and finally detach the manikin before reaching the head-top point. on the other hand, the heat-releasing manikin works as a driving force of the thermal plume, which causes uprising airflow exists mainly on the downstream side. therefore, in the plane cutting through the manikin (y = 2.25 m), the particle plume was observed to exist mainly upstream of the head top while the thermal plume exists in a wider area. in the plane of z = 2.50 m, it was found that the particle transport in horizontal directions was successfully predicted by the both models, although the patterns and the local values of particle concentration yielded from different models were slightly different. figure 7 also demonstrates that the e-e model and the e-l model yield similar overall particle concentration profiles in the room, and they give significantly different particle concentration prediction in some local areas close to the walls. as shown in fig. 7(b) , several distinct local regions with high particle concentration were predicted by the e-l model, while the e-e model just predicted a smoothly changing concentration distribution near the walls. it is supposed that this is caused by the different methods employed by the models to describe the particle-wall interactions. in the e-l model, the particles are assumed to bounce back after they hit the wall. this actually enables a near-mechanistic description of particle behaviours in the wall boundary layer. it is physically reasonable that due to uprising airflow caused by the thermal plume, particles close to the ceiling would be pushed back again to the ceiling after they bounce back from it, which would cause particle gathering and lead to higher local particle concentration in some local regions. on the other hand, since particles are treated either as a continuous phase in the e-e model, it is hard to realize an accurate modelling of their actual behaviours such as bouncing-back in the boundary layers. in this study, the e-e model actually failed to achieve a mechanistic modelling of particle-wall interactions and the particle movement in the boundary layer. for the purpose of a mechanistic modelling of gas-particle flows using the eulerian method, fletcher (1994, 1995) derived a wall boundary condition for the particulate phase, through which the particle-wall interactions such as the momentum exchange between the particles and the solid walls could be included in the particle momentum equation. this mechanistic method is believed to be promising in improving the eulerian model for gas-particle flows. for the purpose of quantitative comparison, particle concentration profiles along line 1 (fig. 2(a) ) and line 3 which is located 20 mm in front of the nose tip ( fig. 7(a) ) are shown in fig. 8 . line 1 and line 3 penetrate through the whole computational domain horizontally and vertically respectively, and line 3 is also located in the thermal plume region; therefore, they could be used to verify both the overall and local particle concentration predictions. it seems that the agreement between the two models increases with increasing particle size up to 2.5 μm. for the overall particle concentration (figs. 8(a) , 8(c), and 8(e)), the e-e models predicted obviously higher particle concentration in the bulk region, except the e-l model predicted a higher particle concentration in the area above the manikin head. with increasing particle size, the predicting error of particle concentration in the bulk region decreases. however, the e-l model still gave a higher prediction than the e-e model. it is supposed that the peak distribution of particle concentration is caused by the psi-c algorithm which actually deals with the particle resident time. comparatively, the particle concentration profiles predicted by the models along line 3 agree better with each other and the agreement for coarse and fine particles is better than that for ultra-fine particles. this is especially true for regions close to the walls. however, as experimental data is unavailable at this moment to validate the models, it's hard to tell which model behaves better for ultra-fine particles. further research is still needed. recalling fig. 7 and fig. 8 , the particle concentration patterns predicted by the e-l model seem discontinuously distributed while the e-e model yields smoothly changing particle concentration fields. it is suspected that the discontinuous concentration pattern is induced by the psi-c method for converting particle trajectories into particle concentration. it was found the psi-c method is not stable enough as its results are highly sensitive to both the domain discretization and the number of particle trajectories. the complicated geometry of the manikin of this study further increased the instability. even a large amount of efforts have been devoted; however, the final results of the particle concentration field were still not satisfactory. therefore, a more robust model or algorithm for converting particle trajectories into particle concentration is in urgent demand. both the eulerian-eulerian model and the eulerian-lagrangian model were employed in this study to simulate particulate contaminant transport in a displacement ventilated room containing a thermal manikin. the two models were compared against each other in terms of airflow velocity and particle concentration. it was found the both models give very close prediction to the airflow field; however, each of the models have its own advantages and drawbacks in modelling particle transport. this study not only highlighted the advantages of each model, but also gave detailed recommendations for improving the both models. conclusions arising from this study mainly include: (1) the e-e model, which treats the dispersed particles as a continuous phase, not only gives a direct prediction to the particle concentration, but also is capable of providing a mechanistic description of the inter-phase interactions. however, since the particles are treated as a continuous phase, their interactions with the solid walls are hard to be modelled accurately. further research is needed to develop a model to describe particle behaviours in the boundary layer. (2) the e-l model, which tracks the particles through the air separately, not only can give a whole-process tracking of particle movement, but also has obvious advantage in modelling particle-wall interactions. however, further study is still need to develop a reliable post-processing procedure for converting particle tracks into particle concentration. ansys cfx-solver theory guide two-fluid modeling of aerosol transport in laminar and turbulent flows ashrae. 2013a. 2013 ashrae handbook-fundamentals. atlanta. ashrae. 2013b. standard 62.1-2013-ventilation for acceptable indoor air quality (ansi approved) dispersal of exhaled air and personal exposure in displacement ventilated rooms health effects of daily airborne particle dose in children: direct association between personal dose and respiratory health effects lagrangian modeling of particle concentration distribution in indoor environment with different kernel functions and particle search algorithms comparison of a new kernel method and a sampling volume method for estimating indoor particulate matter concentration with lagrangian modeling modeling particle distribution and deposition in indoor environments with a new drift-flux model a comparison of two-fluid model, dense discrete particle model and cfd-dem method for modeling impinging gas-solid flows a computational and experimental investigation of the human thermal plume radiochemical indoor environment and possible health risks in current building technology numerical investigation of particle transport and inhalation using standing thermal manikins efficient computation of micro-particle dynamics including wall effects two-fluid model for particleturbulence interaction in a backward-facing step sensory and other neurogenic effects of exposures to airborne office dust transport of particulate and gaseous pollutants in the vicinity of a human body communicable respiratory threats in the ed: tuberculosis, influenza, sars, and other aerosolized infections computational modeling of effects of thermal plume adjacent to the body on the indoor airflow and particle transport modelling flow and heat transfer around a seated human body by computational fluid dynamics an improved model for particulate turbulence modulation in confined two-phase flows numerical computation of turbulent gas-solid particle flow in a 90° bend comparison of the eulerian and lagrangian methods for predicting particle transport in enclosed spaces particle dispersion and deposition in ventilated rooms: testing and evaluation of different eulerian and lagrangian models the financial supports provided by australian research council (project id: dp160101953) and jsps (japan society for the promotion of science) fund for the promotion of joint international research (grant no. 15kk0211) are gratefully acknowledged. key: cord-253092-igic5pxc authors: lee, byung hee; yee, su whan; kang, dong hwa; yeo, myoung souk; kim, kwang woo title: multi-zone simulation of outdoor particle penetration and transport in a multi-story building date: 2016-12-20 journal: build simul doi: 10.1007/s12273-016-0340-1 sha: doc_id: 253092 cord_uid: igic5pxc in areas with poor ambient air quality, indoor particle concentrations can be significantly affected by particulate matter originating outdoors. the indoor environments of multi-zone and multi-story buildings are affected differently by outdoor particles compared with single-family houses, because of the buildings’ more complicated airflow characteristics. the objective of this study is to analyze outdoor particle penetration and transport, and their impact on indoor air, in a multi-zone and multi-story building using a contamw simulation. for the airflow and particle transport analysis, the building leakage, penetration coefficients, and deposition rates were determined by on-site experiments. the results of airflow simulations for cold winters show that outdoor air infiltrates through the lower part of building and exfiltrates from the upper part. the results of the particle simulation also indicated that the airflow characteristics, combined with deposition rates, cause the lower floors of a multi-story building to be exposed to higher fine particle concentrations compared with the upper floors of the building. the study demonstrated that the contamw simulation can be useful in analyzing the impact of outdoor particles on indoor environments through the identification of key particle transport parameters and validated airflow simulations. rapid development over the last two decades has caused a progressive deterioration in outdoor air quality in many developing countries (abdul rahim 2000) . in particular, there has been an exponential increase in urbanization and industrialization over the last few decades in several east asian countries, including china and korea, leading to increases in air pollutant emissions (lee et al. 1999) . during asian dust storm episodes, the outdoor pm 10 concentrations in korea can increase more than four-fold compared with the concentrations before the storm episodes (chun et al. 2001) . according to the seoul air quality information, the outdoor daily peak in pm 10 concentrations in 2015 reached above 700 μg/m 3 . especially in cold winters, high daily average pm 10 concentrations of above 560 μg/m 3 were observed in seoul; these high concentrations are attributed to the use of combustion devices for heating. governments and research institutes have made various efforts to reduce the risk of outdoor pm pollution in recent years. since many people spend a considerable amount of time indoors, indoor air quality, particularly in relation to particle pollution, has received significant attention from numerous researchers (ligocki et al. 1993; pope et al. 1995; abt et al. 2000; long et al. 2001; wallace et al. 2003; stephens 2015) . indoor particle concentrations are influenced by particles originating both indoors and outdoors. in areas with poor ambient air quality, the indoor particle concentration can be significantly affected by particulate matter originating outdoors (chen and li 2014) . particles infiltrate from outdoors through gaps in the building envelope, and through window and door frames, even when they are closed (chao et al. 2003) . therefore, outdoor particles may have a lasting and significant impact on indoor environments. a number of researchers have investigated the contribution of outdoor particles to indoor air quality, predominantly by measuring ratios of indoor to outdoor particle concentrations. (alzona et al. 1979; ligocki et al. 1993; ozlaynnak et al. 1996; abt et al. 2000; lee and chang 2000; long et al. 2001; liu et al. 2003; wallace et al. 2003; cao et al. 2005; kuo and shen 2010; chatoutsidou et al. 2015) . these studies mainly focused on single family houses, and the investigations have suggested a strong relationship between outdoor and indoor particle concentrations. unlike single family houses where a single zone assumption is usually made, multi-zone and multi-story buildings involve more complicated airflow characteristics, meaning that outdoor particles have a different effect on the indoor air quality. for example, in winter, when the outdoor particle concentration is generally high, outdoor air infiltrates through the building envelop at the lower floors of the building, and exfiltrates from the upper floors owing to the stack effect. air pollutants can also spread from lower floors to upper floors due to the inter-floor airflow caused by the buoyancy force (zhou and deng 2014) . this phenomenon results in a distinctive indoor particle concentration distribution within a building. moreover, deposition loss in multi-zone buildings may lead to different outdoor particle effects on the indoor air depending on the building zone. a few studies (rim et al. 2013; oh et al. 2015) have involved field measurements, and results postulated the relationships between outdoor and indoor particles in multizone and multi-story buildings. however, a full understanding of the impact of outdoor particles within the multi-zone and multi-story building is still lacking because of the limited number of air sampling locations. in practice, using a large number of particle sampling devices simultaneously is expensive and time-consuming (ma et al. 2012) . a careful computer simulation study with reliable inputs for key simulation parameters, such as the particle penetration coefficient and deposition rate, may be capable of providing comprehensive insights for understanding particle transport in multi-story buildings. the objective of this study is to analyze outdoor particle penetration and transport in a multizone and multi-story building, and their impact on indoor air. to this end, contamw, a multi-zone airflow and contaminant transport analysis program, was used to predict size-resolved particle concentrations in each zone and on each floor. two key simulation parameters, the penetration coefficient and the deposition rate of size-resolved particles, were determined by field measurements for the reference building, and used for the simulation. 2 multi-zone building simulation to investigate the impact of outdoor particle penetration and transport in a multi-zone and multi-story building, the contamw, which is a multi-zone airflow and contaminant transport analysis software package, was used. as particle transport is affected by a building's airflow, both the airflow and particle transport analyses were simultaneously considered in the simulation for a reference building. for the airflow simulation, the leakage data for the building components were measured using the typical blower door pressurization and depressurization method in compliance with iso 9972 (iso 2015a). to validate the airflow simulation, the pressure prediction results were compared with the measured pressure distribution in the reference building. for the particle transport analysis, the two key parameters, the penetration coefficients and deposition rates, were determined by field experiments. contamw is capable of considering particle deposition loss in the inbuilt model, but does not provide a model for particle penetration. a modification to consider particle penetration, which is a function of airflow rates and penetration coefficients, was made in the contamw model. a conceptual diagram of the simulation approach is illustrated in fig. 1 . contamw, a multi-zone airflow and contaminant transport analysis software package, was used for this study. the program has been widely used in research focusing on various contaminant transport issues. recently, several researchers (lai 2004; sohn et al. 2007; demetriou and khalifa 2009; liu and zhai 2009; lim et al. 2011; miller 2011; dols et al. 2016 ) used contamw to analyze aerosol transport issues, including not only airborne contaminants but also infectious bio-aerosols such as severe acute respiratory syndrome (sars) and influenza a (h1n1). detailed governing equations for contamw can be found in (walton 1989) and (walton and dols 2005) . for this study, contamw provides the airflow prediction results depending on the zone leakage data, and also the indoor particle transport load in the interzonal airflow. however, particle penetration through a building envelope and deposition indoors could not be directly considered in contamw. to incorporate these two key parameters when predicting particle concentrations, the application of several input values in contamw was modified. contamw calculates contaminant concentrations using the following equation: where α i m is the mass of contaminant α in control volume i, j i f  is the rate of air mass flow from control volume j to control volume i, α j η is the filter efficiency in the airflow path, α j c is the mass concentration of contaminant α in control volume j, α i g is contaminant α generation rate in control volume i, , α β k is the kinetic reaction coefficient in control volume i between contaminant α and contaminant β, β i c is the mass concentration of contaminant β in control volume i, i j f  is the rate of air mass flow from control volume i to control volume j, α i c is the mass concentration of contaminant α in control volume i, and α i r is removal rate of contaminant α in control volume i. in contrast to gaseous contaminants, losses from particleladen flows may be caused by penetration through the building envelope and deposition on surfaces in the indoor environment. in this study, to employ the particle penetration concept, a filter efficiency term α j η was used as a substitute for the penetration coefficient in each envelope. in other words, each of the building's walls was assumed to act as a filter by using a penetration coefficient as the value for the filter efficiency: (1 ) α j η -. the removal rate term α i r was also employed to model particle deposition indoors. particle deposition was also represented as a sink element by choosing the deposition rate sink model in contamw. in general, it is difficult to initiate chemical reactions between particles and other contaminants indoors without the presence of a catalyst, so this study neglected the "chemical reactions" term. a 5-story office building, located in seoul, korea, was chosen as a reference building for this study. the building has multiple zones including office spaces, corridors, bathrooms, an elevator, and stairwell shafts. the typical floor plan and a section of the reference building are shown in fig. 2 . each office space in the building consists of similar sets of the building envelope, interior finishing materials, and furnishings. a glass curtain wall system is installed over the entire exterior wall of the building. as the reference building was recently built (in 2013), the leakage characteristics for the entire wall are expected to be relatively uniform. the reference building is a relatively small office building with natural ventilation features throughout the office spaces above ground level. mechanical ventilation systems are only equipped in the basement. the building's vertical spaces, such as elevators and stairwell shafts, are located in the center and on the west side of this 5-story (with a 2-story basement) building. we assumed that infiltration of airflow and ambient particles can occur in the lower part of the building and be transported through the vertical spaces, especially in winter when the outdoor air quality is usually poor. the simulation was conducted for a typical winter day between january and february, when the outdoor particle concentrations were notably high and the outdoor temperature was low, resulting in increased airflow between indoorsoutdoors as well as inter-zonal airflows. the outdoor temperature was assumed to be −6.7 °c, the relative humidity was set at 60%, and the external wind velocity was assumed to be 2 m/s. measured data for the reference building during a cold season were used as input variables, such as temperature and relative humidity, for the indoor environment. the simulation assumed that all openings, including windows and doors, were closed as people usually close the openings during winter. particle transport through buildings is a complex matter that depends on the nature of air movements. particle penetration and transport are also affected by characteristics relating to particle diameter. therefore, particles, which were defined as having a diameter of less than or equal to 10 μm, were sorted into six groups (0.3 μm, 0.5 μm, 1 μm, 3 μm, 5 μm, and 10 μm) in line with particle sizes based on iso 14644-1 (iso 2015b). in contamw, the six differentsized outdoor particles were regarded as different contaminant species, having different input variables for penetration and deposition. particle sources induced by human activities, office equipment, or other potential internal sources were disregarded in the indoor environment, in order to limit the investigation to the effect of outdoor particles on the indoor environment. to acquire the building leakage area data necessary for the airflow and particle transport simulation, a typical blower door pressurization and depressurization test was utilized. the tested building components included exterior and interior walls, elevator doors, stairwell doors, lobby doors, and interior doors. the blower door equipment used in the test was the retrotec 3101 blower door system (retrotec, usa) with one fan. the maximum airflow rate is 4 m 3 /s at 50 pa and the measurement accuracy is ±5% for airflow rate. for validation of the airflow simulation, absolute outdoor minus indoor pressure difference profiles were measured in the reference building, and the pressure distribution predicted by the contamw airflow simulation was compared with the field measurement results. two parameters are considered essential particle simulation input values: the penetration coefficient and deposition rate. the penetration coefficient is a dimensionless parameter defined as the fraction of particles that can penetrate indoors (alzona et al. 1979; tung et al. 1999; chao et al. 2003) . the deposition rate is the particle loss rate due to deposition, which is generated throughout the process whereby particles are transported and attached to surfaces (elimelech et al. 1995) . in this study, a field test on the reference building was conducted using the natural decay method to identify the particle size-resolved penetration coefficient and deposition rate; the obtained values were used as simulation parameters. the determination of the penetration coefficient and deposition rate can be conducted using natural decay methods (ozkaynak et al. 1996; tung et al. 1999; chao et al. 2003) . the natural decay method is based on an experimental test where the natural decay of particle concentrations should be measured. the penetration coefficient and deposition rate were determined using the following equations. the indoor particle concentration level can be expressed by the following mass balance equation (chao et al. 2003) : where v is the effective volume of the room, in c is the indoor particle concentration, p is the penetration coefficient, q is the air change rate, out c is the outdoor particle concentration, k is the deposition rate on the wall, floor, and other surfaces, and e(t) is the source generation rate inside the control volume. assuming that the measured room was unoccupied during the experiments and no specific indoor particle generation sources existed in the room, the term e(t) can be neglected. all the parameters in eq. (2) are known values obtained from field measurements except for the penetration coefficient p, and the deposition rate k. assuming that the outdoor particle concentration and the air change rate were constant during the experiments, two terms, out / pqc ( ) q k + and ( ) q k + , can be regarded as constants. as a result, eq. (3) describes the particle decay profile, and the two key parameters can be estimated by fitting the measured particle concentrations with the following equation. where int c is the initial indoor particle concentration. four repetitions of the field measurements to acquire the key parameters were carried out in a typical room in the reference building in january and february, as shown in fig. 3 . before the experiment, the test room was vacuumed, and the particle concentration level was increased to a relatively high level by opening windows to allow the inflow of outdoor particles. to achieve well-mixed conditions in the room, mixing fans were operated for an hour. after turning off the fans and closing the windows, the natural decay of the indoor particle number concentration, as well as the outdoor particle concentrations, were monitored by fig. 3 test room and sampling point for the key parameter identification two identical optical particle counters (tsi 9306-v2, tsi, usa). the optical particle counter is capable of counting the 6 sizes of particles (0.3 μm, 0.5 μm, 1.0 μm, 3.0 μm, 5.0 μm and 10 μm) simultaneously at a flow rate of 2.83 l/min with ±5% accuracy. samples of outdoor pm were taken in the center of the roof, while the indoor pm samples were taken 1 m away from the window and 1.2 m above the floor. particle number concentrations were recorded every 10 minutes during the experiment periods. at the same time, the tracer decay method, which uses a co 2 monitor with a real time data logger (mch-383sd, lutron, usa), was used to estimate airflow exchange rates in the test room during the natural decay period. when all openings were closed, co 2 gas was injected into the test room with a concentration above 1500 ppm. indoor and outdoor co 2 concentrations during the test period were recorded by identical co 2 monitors, and were analyzed to compute the air exchange rates. the estimated penetration coefficient and deposition rates are shown in fig. 4 . the average values of the four repeated measurements are displayed. as shown in fig. 4 (a) , the highest penetration coefficient was 0.85 at 0.3 μm and the lowest penetration coefficient was 0.04 at 10 μm. it was observed that particles with larger diameters (>1.0 μm) tended to have lower penetration coefficients. figure 4 (b) presents the deposition rate of size-resolved particles. deposition rates ranged from 0.05 h −1 to 0.55 h −1 , and the highest deposition rate was found to be 0.55 h −1 at 10 μm. the coarse particles (3.0 μm, 5.0 μm, 10 μm) generally showed higher deposition rates than the fine particles. these penetration coefficients and deposition rates fall within similar ranges to values suggested by other researchers (long et al. 2001; williams et al. 2003; chao et al. 2003) . in their studies, penetration coefficients ranged from 0.72 to 0.79 for fine particles and from 0.58 to 0.48 for coarse particles, while deposition rates ranged from 0.12 h −1 to 0.42 h −1 for fine particles and from 0.37 h −1 to 0.55 h −1 for coarse particles. figure 5 shows the predicted vertical pressure distribution of the reference building, and its validation results. the simulation results for the pressure distribution were compared with the measured pressure difference between the inside fig. 4 size-resolved penetration coefficients and deposition rates of particles, estimated in the test room: (a) penetration coefficients; (b) deposition rates and outside of the building. generally, good agreement was found between the simulated and measured values. the pressure difference between the outside and inside was 10 pa on the first floor. the pressure difference gradually decreased on the upper floors, as demonstrated by a pressure difference on the top floor of −5 pa. in the simulated building, the neutral pressure level (δp = 0) was approximately located at the 3rd floor. these pressure simulation results indicate that the outdoor air infiltrates into the lower level of building and exfiltrates from the upper level of the building, which is commonly known as the stack effect. generally, it is known that the pressure difference between indoors and outdoors is ranged from 0 to 5 pa normally applied to single family houses. the higher pressure difference of 10 pa predicted on the first floor suggests that particle penetration, which is dependent on the pressure figure 6 shows the airflow rate of three representative zones ("exterior," "corridor," and "interior") in the reference building. the three zones were set to examine the differences in airflow and particle transport in the multi-zone building. the exact representative locations can be found in fig. 2(a) . the airflow rate for "exterior (z1)" indicates air exchange rates with the outdoors, while the airflow rates for "corridor (z2)" and "interior (z3)" indicate the airflow rates between adjacent zones. the "+" and "−" sign in fig. 6 represent the airflow direction, in that "+" indicates that the airflow travels from the outside to the inside and "−" indicates the opposite direction of travel. at the building exterior, the outdoor the results clearly suggest that the infiltration of air on each floor is significantly different in terms of the amount of air as well as the airflow direction. also, they indicate that the outdoor particles' impact on the indoor environment differs significantly from floor to floor. figure 7 illustrates the amount of size-resolved particle penetration (penetration coefficient × airflow rate) through the building envelope zone of the reference building. the "+" and "−" signs in fig. 7 represent the direction of particle penetration: "+" indicates particle penetration from the outside to inside, and "−" indicates the opposite direction. the amounts of particle penetration ranged from 0.12 h −1 to 2.17 h −1 on the lower floors and from −0.49 h −1 to −0.06 h −1 on the upper floors. the particle transport trends through the building envelope are generally similar to the airflow results. the results show that the outdoor particles penetrate into the lower floors of the building. this particle penetration was prominent for fine particles (0.3 μm, 0.5 μm, 1.0 μm), while the amount of penetration from coarse particles (3 μm, 5 μm, 10 μm) not significant owing to the low penetration coefficients. the result does not indicate that the outdoor particles only affect the lower floors of multi-story buildings, because the outdoor particles that penetrate into the building can be transported through vertical spaces such as elevator shafts and stairwells, resulting in the influence of outdoor particles on the upper floors of the building. figure 8 shows the size-resolved particle i/o ratio in each zone and for each floor of the reference building. as no indoor particle generation or resuspension was assumed, the simulated i/o ratio indicates the influence of particles originating outdoors on the indoor particle concentrations, considering the particle penetration, deposition, and transport within a building. the results show that the i/o ratios of coarse particles were generally lower than those of fine particles. this is attributed to the low penetration coefficients and high deposition rates of coarse particles. as indicated above, it was found that the i/o ratios for the lower floors were higher than for the upper floors. for fine particles, the maximum i/o ratio for the lower floors was 0.71, while that for the upper floors was 0.13. these findings were true for both fine and coarse particles. the simulated i/o ratio strongly suggests that the impact of outdoor particles would be significant for the lower floors of a multi-story building. from the results, the upper floors were marginally affected by coarse particles owing to the higher deposition loss rate (0.11 h −1 -0.52 h −1 ), but significantly affected by the fine outdoor particles. in terms of the zone location, the exterior zone is more affected by outdoor particles than other zones. the average i/o ratio of exterior zone was 0.15, and those of corridor and interior zones were 0.08 and 0.06, respectively. for fine particles, the i/o ratios of the exterior zone ranged from 0.08 to 0.71 and those of the interior zone ranged from 0 to 0.45. however, there was almost no difference between zones for the coarse particles: for coarse particles, the maximum i/o ratio of the exterior zone was 0.1 and that of the interior zone was approximately 0. the results from the airflow simulation suggest that the outdoor air infiltrates through the lower floors of the building and exfiltrates from the upper floors. the particle simulation also confirms that the amount of penetrating particles differs from floor to floor, indicating that the impact of external particles on indoor air can differ according to the floor in a multi-story building. south korea and some northern chinese regions have cold winters when the outdoor air quality is very poor owing to industrial and domestic heating. in cold winters, the stack effect occurs frequently, affecting the building airflow characteristics. the results of this study suggest that the lower part of a multi-story building will experience higher particle concentrations than the higher part of the building. this phenomenon can explained by the deposition loss of the penetrating particles. according to the results of the predicted i/o ratio distributions within the reference building, the outdoor particle loss increases as the particle flow passes through multiple zones. in terms of the influence of particle sizes on indoor air, the simulation results suggest that the fine particles (0.3 μm, 0.5 μm, 1.0 μm) showed higher i/o ratios than the coarse particles (3.0 μm, 5.0 μm, 10.0 μm). this result can be confirmed by several previous experimental researches which reported that the i/o ratios for fine particles were higher than those for coarse particles (thatcher and layton 1995; rojas-bracho et al. 2002; wang et al. 2006; colbeck et al. 2010) . our simulation results indicate that the fine ambient particles significantly affect the indoor environment. fine particles are known to pose more serious health hazards to occupants. this study suggests that appropriate particle control measures should be implemented especially in the lower part of multi-story buildings. a potential criticism of this study is that the impact of fine outdoor particles on indoor air could be exaggerated because the simulation results only suggest the particles' i/o ratios without consideration of the actual mass fraction of outdoor particles. a very low mass fraction of fine particles would result in a low impact on the indoor environment despite the higher i/o ratios of fine particles. to examine the actual impact of fine outdoor particles on the indoor environment, an additional analysis was conducted that considered the mass fraction of outdoor particles, using a winter smog episode as an example. figure 9 shows the size-resolved particle mass concentrations outdoors and indoors during a winter smog episode. the outdoor mass concentration shows that the amount of fine fig. 9 size-resolved particle mass concentrations outdoors and indoors particles is similar to that of coarse particles, and that the indoor fine particle mass concentration is much higher than that for the coarse particles. the analysis confirms the significant impact of fine outdoor particles on indoor environments, especially on the lower floors of a multistory building. it should be noted that the actual impact of fine particles will vary with the outdoor mass fraction of fine particles. this study demonstrates that the contamw simulation can be useful in analyses of the impact of outdoor particles on indoor environments through the identification of key particle transport parameters and a validated airflow simulation. the study also suggests that the outdoor particle impact on the indoor environment can differ from floor to floor, indicating that lower levels of a multi-story building in particular can be exposed to higher fine particle concentrations. some limitations of this study should be addressed. the airflow and particle transport simulation was only conducted for the cold winter season. in many asian cities, the frequency of outdoor particle pollution events has increased in other seasons. the airflow and particle transport characteristics will significantly alter with a change in the outdoor climate conditions. in this study, the airflow simulation was validated and the key particle parameters were determined by field tests. however, a direct validation was not performed for the particle transport simulation owing to the complexity and high cost of multiple point particle measurements. for future studies, a field test measuring particle concentrations at multiple points in a multi-story and multi-zone building is necessary to confirm our conclusions. also, it should be noted that this simulation has not considered indoor generation and resuspension of particles in the building, as the study aimed at exploring the impact of particle originated outdoors on indoor air. in real indoor environment, the indoor particle concentration is affected not only by outdoor originated particles, but also by indoor originated particles and resuspended particles. a comprehensive analysis considering various scenarios of particle infiltration, indoor-generation, and resuspension would contribute to fully understanding the actual particle distribution in the multi-zone and multi-story building. the objective of this study was to analyze the outdoor particle penetration and transport in a multi-zone and multi-story building, and their impact on indoor air. to this end, a contamw simulation was conducted. the study demonstrated that the contamw simulation can be used to analyze the impact of outdoor particles on the indoor environment by identifying key particle transport parameters and through a validated airflow simulation. the results of the airflow simulation in a cold winter show that outdoor air infiltrates through the lower part of building and exfiltrates from the upper part. the results of the particle simulation also indicated that the airflow characteristics, combined with the deposition rates, caused the lower floors of the multi-story building to be exposed to higher fine particle concentrations than the upper floors of the building. to confirm this conclusion, a field test that measures particle concentrations at multiple points in a multi-story and multi-zone building will be required. air pollution and the forests of developing and rapidly industrializing regions relative contribution of outdoor and indoor particle sources to indoor concentrations indoor-outdoor relationships for airborne particulate matter of outdoor origin indoor/outdoor relationships for pm2.5 and associated carbonaceous pollutants at residential homes in hong kong-case study 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approach characterization of particulate matter concentrations and bioaerosol on each floor at a building in seoul personal exposure to airborne particles and metals: results from the particle team study in riverside health effects of particulate air pollution: time for reassessment? multizone modeling of size-resolved outdoor ultrafine particle entry into a test house measurements of children's exposures to particles and nitrogen dioxide in predicting sizeresolved particle behavior in multizone buildings building design and operational choices that impact indoor exposures to outdoor particulate matter inside residences deposition, resuspension, and penetration of particles within a residence a methodology to investigate the particulate penetration coefficient through building shell particle concentrations in inner-city homes of children with asthma: the effect of smoking, cooking, and outdoor pollution airnet-a computer program for building airflow network modeling contamw user guide and program documentation hospital indoor pm10/pm2.5 and associated trace elements in guangzhou the research triangle park particulate matter panel study: modeling ambient source contribution to personal and residential pm mass concentrations numerical simulation of inter-floor airflow and impact on pollutant transport in high-rise buildings due to buoyancy-driven natural ventilation this research was supported by the 2014 research fund of the university of seoul (2014-05159037) for dong hwa kang. also, this research was supported by the korea ministry of environment as "the converging technology project" (2013001650002) for byung hee lee, su whan yee and myoung souk yeo. key: cord-102908-sr7j8z9c authors: mersmann, sophia f.; johns, emma; yong, tracer; mcewan, will a.; james, leo c.; cohen, edward a.k.; grove, joe title: learning to count: determining the stoichiometry of bio-molecular complexes using fluorescence microscopy and statistical modelling date: 2020-07-24 journal: biorxiv doi: 10.1101/2020.07.23.217745 sha: doc_id: 102908 cord_uid: sr7j8z9c cellular biology occurs through myriad interactions between diverse molecular components, many of which assemble in to specific complexes. various techniques can provide a qualitative survey of which components are found in a given complex. however, quantitative analysis of the absolute number of molecules within a complex (known as stoichiometry) remains challenging. here we provide a novel method that combines fluorescence microscopy and statistical modelling to derive accurate molecular counts. we have devised a system in which a given biomolecule is differentially labelled with spectrally distinct fluorescent dyes (label a or b), which are then mixed such that b-labelled molecules are vastly outnumbered by those with label a. complexes, containing this component, are then simply scored as either being positive or negative for label b. the frequency of positive complexes is directly related to the stoichiometry of interaction and molecular counts can be inferred by statistical modelling. we demonstrate this method using complexes of adenovirus particles and monoclonal antibodies, achieving counts that are in excellent agreement with previous estimates. beyond virology, this approach is readily transferable to other experimental systems and, therefore, provides a powerful tool for quantitative molecular biology. the statistical models used in our analysis are available here: https://github.com/sophiamersmann/molecular-counting, the raw data used for molecular counting can be found here: 10.5281/zenodo.3955142. all cellular processes are driven by coordinated networks of dynamically interacting molecular partners. to successfully function, these components typically need to be assembled into specific complexes or clusters. for example, receptor signalling generally requires the co-location of various sensory, regulatory and stimulatory partners; the precise makeup of these assemblies can tune the nature of the signal and resultant physiological output. molecular cell biology research has, thus far, largely focused on determining the identity of the components found in a given complex. however, it is becoming clear that the quantity of any given component is also vitally important. quantifying the number of molecules, or stoichiometry, within an assembly can be used to understand its ultrastructure and, ultimately, to create complete molecular models of entire cellular structures, as has been demonstrated for hiv capsids and the neurological synapse (briggs et al., 2004; wilhelm et al., 2014) . there are various approaches to investigate the number of molecules within a given complex; for example calibrated biochemical analysis or cryo-electron microscopy (cryo-em). however, such methods pose practical and/or technological barriers and, by their very nature, obscure heterogeneity within the sample. single molecule localisation microscopy (smlm) modalities, such as storm and palm, are potentially powerful techniques for counting (lee et al., 2012; coltharp et al., 2012; fricke et al., 2015; veatch et al., 2012; stein et al., 2019) . nonetheless, these approaches typically require detailed a priori knowledge of the experimental system (for instance, a thorough evaluation of the 'blinking' characteristics of the fluorophores (patel et al., 2019) ) and/or a well understood reference sample with which to calibrate the measurement (thevathasan et al., 2019) . these steps need to be performed independently for each different experimental model and microscope set up; this creates a high barrier to implementation and can make these methods vulnerable to experimental variation and artefacts. here we outline an alternative, and potentially complementary, approach that combines (non-smlm) fluorescent microscopy and statistical modelling to extract estimates of molecular numbers within a complex. the method requires differential binary labelling of a constituent (i.e. a protein of interest is labelled with fluorescent dye a or b); by appropriate mixing of the labelled components, any individual molecular complex can be simply scored as being positive or negative for a given label. the frequency of positive complexes is then analysed by statistical modelling to extract estimates of stoichiometry. this approach is simple and requires minimal calibration or a priori understanding of the experimental system. we demonstrate the feasibility and accuracy of this approach by studying the stoichiometry of virus-antibody complexes. adenovirus (adv) is a non-enveloped dna virus; its genome is enclosed within a proteinous shell, called a capsid (nemerow et al., 2012) . the major adv capsid protein is hexon; this assembles into trimeric subunits, that are hexagonal in shape, which in turn arrange to form an icosahedron with 20 triangular faces (a molecular cartoon of the adv particle is provided in figure 3a ). the adv particle has 12 vertices, each of which are occupied by a pentameric subunit (formed of the penton base protein) and a receptor binding 'spike' (formed of the fibre protein). antibodies (ab) that bind sites such as the spike can directly neutralise adv by blocking receptor interactions, therefore preventing the virus particle from entering the cell. however, antibodies targeting the hexon protein (which makes up the majority of the capsid) do not necessarily interfere with the mechanics of virus entry (fender et al., 1995; wohlfart et al., 1985) . nonetheless, anti-hexon antibodies can prevent virus infection by recruiting the intracellular antibody-sensor trim21, which targets the virus for degradation and activates cell-intrinsic immune responses (keeble et al., 2008; mallery et al., 2010) . anti-hexon monoclonal antibody 9c12 inhibits adv infection via this mechanism and is used as a prototypical system to investigate trim21. the stoichiometries of antibody and trim21 recruitment to incoming virus particles remain unclear and are likely to be a determinant of the nature of the resulting cellular response. previous studies, using alternative techniques, have provided estimates of the stoichiometry of adv-9c12 complexes. each adv particle possesses 720 identical hexon proteins, each of these represents a potential binding site for 9c12. however, the hexon subunits are assembled as trimers, and are arranged in a specific geometry (as described above). moreover, antibodie are bivalent, therefore each 9c12 molecule has two hexon binding pockets. consequently, it is highly unlikely that each hexon molecule will be occupied by a single 9c12 molecule (i.e. 720 antibodies per virus particle). analysis by cryo-em, immuno-gold em staining and calibrated fluorescence measurements suggest a true maximum stoichiometry of 100-200 antibodies per particle (mcewan et al., 2012; varghese et al., 2004) ; this maximum is likely determined by the limits to antibody binding and packing enforced by the geometry of the particle. we have applied our counting method to the adv-9c12 complex and generated estimates that are in good agreement with these previous studies, therefore validating our approach. strategy. we used differential binary labelling and statistical modelling to extract estimates of stoichiometry, our strategy is outlined in figure 1 ; note that this approach can be generalised to apply to many other multi-component systems (i.e. how many protein x are found in assembly y?). the hidden truth is the number of antibodies bound to a virus particle; the ab:virus stoichiometry is expected to increase with antibody concentration until it reaches a saturation point where a. the ground truth: the number of antibody molecules (k) per virus particle increases with antibody concentration up to a saturation point (k = nsat). b. extracting truth from data: adv particles (labelled with a green fluorescent dye) are incubated with a defined mixture of two batches of antibody; one batch has received fluorescent label a (magenta), whilst the second has received label b (blue). when viewed by microscopy, every virus particle has received at least one molecule of ab a , whereas only a minority have received any ab b and can be scored in a binary fashion. note, antibody molecules are not drawn to scale the maximum number of abs are bound ( figure 1a .). in our method ( figure 1b ), both components (virus and antibody) are fluorescently labelled, however, two separate batches of, the otherwise identical, antibody are given spectrally distinct dyes (resulting in ab a or ab b ). mixing of the differentially labelled antibody batches at appropriate proportions results in only a minority of virus particles receiving a particular fluorescent dye (b in the example cartoon). therefore, when imaged, we detect three distinct fluorescent signals: each virus particle can be identified by its reference dye (green in the cartoon example), every virus particle has also received antibodies with dye a (magenta), however, very few particles are positive for dye b (blue) and can be scored as positive or negative in a binary fashion. the frequency of virus particles that are positive for ab b is a function of the a:b mixing proportion and the stoichiometry of ab:virus interaction; this relationship between the data and the hidden truth can be modelled. consider a single virus to be capable of binding n sat antibodies at saturation. under the assumption that antibodies bind to the same virus independently from each other, k, the total number of (labelled and unlabelled) antibodies bound to a virus, can be modelled as a binomial random variable where p is the probability of an antibody binding to a particular binding site of the virus. if n sat and p are known, then the expected number of antibodies bound to a single virus is simply e[k] = n sat · p. however, in most cases n sat is not known and p cannot be expressed easily since it depends not only on the antibody concentration used but also the composition of the virus particle and the geometry of interaction, which can be difficult to obtain. to extrapolate an antibody count it is, therefore, necessary to estimate both, n sat and p. as described above, our experimental design utilizes antibody labelled with spectrally distinct dyes allowing binary scoring of individual virus particles as positive if they interact with at least one ab b molecule ( figure 1 ). here, we describe this state as being a bernoulli random variable s that takes the value 1 if the virus is in the positive state, and 0 if it is in the negative state, i.e. where q is the probability a virus interacts with at least one labelled antibody. since q = p (s = 1) = 1 − p (s = 0), we can derive a closed form for q by finding an expression for the probability of a virus not being complexed with any labelled antibody p (s = 0). to this end, we simply sum over all possible virusantibody configurations under the constraint of all antibodies being unlabelled, i.e. a virus could bind zero, one, two, ... up to n sat unlabelled antibodies. the marginal probability of a virus being in a negative state is thus given by where p (s = 0|k = k) is the probability that given the virus binds to k antibodies, exactly zero of them are labelled. the conditional distribution of s given k = k is itself binomial, namely where f l is the proportion of antibodies that are fluorescently labelled. therefore and combining with the probability mass function of k, namely gives a closed form for q directly follows as note that here p (s = 0) is expressed in terms of p and n sat , and will hereafter be referred to as p (s = 0; θ), where θ = (p, n sat ). consider a single experiment (performed at a specific antibody concentration) to describe v viruses with states s = s 1 , ..., s v where v + of these states are positive, i.e. v + viruses have been observed to interact with at least one labelled antibody. assuming independence among viruses, the likelihood of θ is then where q is as given in (1). we are then interested in the posterior distribution of θ given by the central result of bayesian statistics, where π(θ) is a suitable prior for the unknown parameters θ. in most applications, more than one experiment is performed; consider m experiments to be conducted at varying antibody concentrations c 1 , ..., c m , producing m sets of virus state observations s = {s 1 , ..., s m }. parameter inference using the described single-experiment model would entail building m independent models, each estimating p j and n sat,j for experiment j. while estimating concentration-specific antibody binding probabilities is desired, inferring multiple n sat values is unintuitive since n sat is fixed for a particular virus-antibody pair, i.e. n sat should be common to all experiments regardless of the antibody concentration used. it is, therefore, preferable to develop a general model accounting for multiple experiments that estimates all p 1 , ..., p m simultaneously while yielding only a single n sat estimate. such a general model contains a single likelihood l(θ; s), where θ = (p 1 , ..., p m , n sat ). this can be expressed as where l j (θ j ; s j ) is the likelihood function for θ j = (p j , n sat ) as defined in (2). hence, m + 1 unknown parameters are estimated; a probability p j specific to each concentration c j for j = 1, ..., m, and crucially a single n sat shared over all experiments. to sample from the posterior distributions markov chain monte carlo (mcmc) is used, specifically the metropolis-hasting algorithm using pymc (patil et al., 2010) . no prior knowledge is incorporated by imposing a beta distribution beta(1,1) on all p j , j = 1, ..., m, and a uniform distribution with a sufficiently large upper bound, uniform(0,1000), on n sat . the convergence of mcmc is checked by visual inspection of trace and autocorrelation plots for each parameter. model verification via simulations. the proposed method makes use of experimental parameters including the proportion of antibodies labelled (f l ) and the number of viruses sampled (v ). formally, these are not required to comply with specific bounds. however, certain configurations of an experimental set-up are not expected to yield data that lead to a sensible model. labelling almost all antibodies (i.e. f l close to 1), for example, would result in a data set with low information content. to explore how different experimental design choices impact the model's ability to reliably estimate parameters, we analysed simulated data that assumed a range of possible experimental settings. we simulated a single experiment at a time and assumed the number of antibodies bound at saturation to be known. for this we used an upper limit estimate of adv-9c12 stoichiometry that we previously derived from an alternative method, n sat = 205 (mcewan et al., 2012) . in each simulation, the binding probability p of an antibody is thus the only parameter estimated. we considered a range of possible experimental settings by varying the total number of viruses sampled in an experiment (from 100 to 4000) and the proportion of antibodies labelled (from 0.01 to 0.9). for an assumed true antibody binding probability p ∈ [0.1, 0.99], data is simulated by drawing v virus states from s ∼ ber(q) with q as described in (1). the antibody binding probability was then blindly estimated using mcmc and convergence checked using the gelman-rubin statistic (gelman and rubin, 1992) . a total of 1331 simulations were carried out that assess the model's ability to reliably estimate p in various experimental settings. as expected, high proportions of labelled antibodies produced data of low information content, reflected in the model's inability to accurately estimate p, even if the number of viruses used in an experiment was high ( fig. 2a) . by contrast, if f l is less than or equal to 0.1, p was estimated with low bias and low variance ( fig. 2a) . simulations also suggested that for low f l , as a rule of thumb, at least 500 viruses per experiment should be sampled (fig. 2b-c) . however, if the proportion of antibodies labelled is 0.1 (or higher), then the proposed model failed to produce a reasonable estimate of p for most underlying true values; higher number of viruses seemed to compensate this effect to some extent (fig. 2d ). in summary, these simulations put empirical bounds on experimental parameters and show that, if compliant, the method yields sensible estimates. experimental setup. successful implementation of our strategy requires sensitive and unambiguous measurements of individual virus-antibody complexes. we achieved this by immobilising adv particles onto coverslips for analysis by total internal reflection fluorescence microscopy (tirf-m; a detailed description of experimental methodology is provided in the supplementary information). prior to immobilisation, purified adv particles were directly labelled with alexa fluor 488; when observed by tirf-m (figure 3bi ) they appear as monodisperse diffraction limited spots (the particle being ∼180nm in diameter; figure 3a ). to further verify that each object is a single virus particle we used srrf analysis (super-resolution by radial fluctuations (gustafsson et al., 2016) ); for each object we resolved a single maxima of fluorescence that was ∼200nm in diameter (figure 3bii and iii), consistent with the ultrastructure of adv particles ( figure 3a) . immobilised adv particles were incubated with monoclonal antibody 9c12 conjugated to alexa fluor 647 dye, and imaged by tirf-m. each adv particle was positive for antibody, indicating the assembly of virus-antibody complexes ( figure 3c ). moreover, individual adv-9c12 647 complexes could be analysed in a quantitative manner over a >100 fold range in antibody concentration ( figure 3d ). we automated this process to allow measurements of whole populations of virus particles at varying concentrations of antibody ( figure 3e & f). 9c12 647 signal intensity was proportional to antibody concentration and reached a plateau at high values; this indicates increasing virus-antibody stoichiometries up to a saturation point at which maximum antibody binding is achieved (as outlined in figure 1a) . moreover, the populations of virus particles were quite homogenous in 9c12 signal; this suggests a relative uniformity of assembly. in conclusion, we were able to quantitatively analyse the assembly of individual virus-antibody complexes. to achieve differential labelling a second batch of 9c12 was directly conjugated with biotin; this served as the 'b' batch of antibody to be mixed with the 9c12 647 'a' batch ( figure 1) . it may be possible that conjugation with either biotin or alexa fluor 647 has unexpected detrimental effects on antibody binding; therefore, to have confidence in our binary labelling system we needed to demonstrate fair competition between our differentially labelled antibody batches. to test this we incubated immobilised adv particles with a high concentration of antibody (20µg/ml) composed of different proportions of 9c12 647 or 9c12 biotin (e.g. 0.75:0.25, 0.5:0.5). we then monitored the 9c12 647 fluorescence signal under each condition. as the proportion of 9c12 647 dropped we measured a stepwise reduction in fluorescence signal (figure 4a ). if both batches of antibody possess equivalent binding to adv we would expect a linear relationship between the proportion 9c12 647 and fluorescent signal. indeed, when normalised for units, we observed a near perfect linear relationship ( figure 4b , slope = 0.99, r 2 =0.97); indicating a fair competition in binding between our a and b batches of antibody. as depicted in figure 1b , our approach requires detection of single antibody molecules (of b batch) within individual virus-antibody complexes. to explore this we incubated immobilised adv particles with 5µg/ml 9c12 647 spiked with 1% 9c12 biotin (i.e. f l 0.99:0.01). molecules of 9c12 biotin were detected using streptavidin (an ultra-high affinity biotin binding protein) conjugated to a quantum dot (qdot 655 ). the photostability of quantum dots permits signal accumulation over prolonged exposure times (resch-genger et al., 2008; algar et al., 2011) , therefore increasing the sensitivity of detection. analysis by tirf-m revealed that whilst every particle was positive for 9c12 647 only a subset possessed 9c12 biotin -qdot 655 signal ( figure 4c ); this suggests a population of adv particles receiving one, or very few, 9c12 biotin antibody molecules (as outline in figure 1b ). automated analysis revealed well-separated populations of adv particles that were positive or negative for 9c12 biotin ( figure 4d ). note that all particles were positive for 9c12 647 . these data are consistent with the assembly of virus-antibody complexes in which the vast majority of antibody molecules are from batch a (9c12 647 ), but a subset of complexes contain ∼1 molecules of batch b antibody (9c12 biotin ); the proportion of 9c12 biotin positive particles will serve as the output data for statistical modelling. we have demonstrated quantitative analysis of 9c12 interaction with individual adv particles ( figure 3) ; we have confirmed that differential labelling of antibody does not bias binding ( figure 4a & b) ; and that we could detect single molecules of 9c12 biotin allowing discrimination of positive and negative adv-9c12 complexes ( figure 4c & d). therefore, we have satisfied the necessary technical requirements to implement the strategy outlined in figure 1 . we proceeded with a series of experiments to generate data for statistical modelling and stoichiometric estimates. to achieve this we performed four independent overlapping titrations over a >100 fold range in antibody concentration (0.15-20µg/ml). guided by the simulations in figure 2 , we explored a range of 9c12 biotin proportions, from 0.007-0.07 (0.7-7% ), and, where possible, collected >500 particles per sample (the average number of particles collected was >1500). the proportion of positive particles was assessed for each sample, details of data analysis are provided in the supplementary information. supplementary figure 1 provides representative data: scatter plots display adv 488 , 9c12 647 and 9c12 biotin intensities for control samples (treated with unmixed 9c12 biotin or 9c12 647 ) and four representative test samples incubated with a range of antibody concentration, containing 0.007 9c12 biotin . bar charts provide summary statistics for each channel: the particles have uniform adv 488 reference signal, whereas the 9c12 647 fluorescence decreases with antibody concentration; likewise, the proportion of 9c12 biotin positive particles decreases with concentration. this data are consistent with the expected concentration-dependent stepwise reduction in virus-antibody stoichiometry. the four experiments generated 24 sets of binary scores (supplementary figure 1 and table 1 ) which were integrated in to our statistical model, as outlined in the methods. this generated posterior distributions of p (probability of an antibody binding site being occupied) for each sample (supplementary figure 2a ) and an estimated n sat (the maximum number of antibodies that can bind per particle) of 133 (maximum a posteriori (map) estimate; ci (credible interval) [119, 162]; supplementary figure 2b ). absolute antibody numbers for each sample can be derived by multiplying the map estimate of each posterior distribution (for p) by n sat (supplementary figure 2c) . figure 5a displays the mean number of bound antibody at increasing 9c12 concentrations; adv-9c12 stoichiometries range from 29 to 115 across the titration of antibody. for any given sample, alongside the proportion of particles that are positive for 9c12 biotin , the experimental setup provides fluorescent intensity values of 9c12 647 in each adv-ab complex (supplementary figure 1) ; this provides an internal reference for adv-9c12 interaction, therefore, the stoichiometric estimates should correlate with their experimentally-matched fluorescent intensity values. figure 5b demonstrates a near perfect linear correlation between stoichiometry and fluorescence intensities for an example experiment; this would suggest that our statistical modelling faithfully reports the relationship between antibody concentration and adv binding occupancy. the stoichiometric estimates generated by our method derive from an ensemble measurment and, therefore, represent the antibody interactions of the average virus particle; this obscures heterogeneity within the population. however, given the excellent agreement between the estimated antibody counts and 9c12 647 intensity values ( figure 5b ) we used the stoichiometric estimates to calibrate the 9c12 647 signals, therefore allowing us to infer population heterogeneity. to achieve this, for any given sample we matched the median 9c12 647 fluorescence intensity to its associated stoichiometic estimate (generated by the model); extrapolating from this we then converted the 9c12 647 fluorescence values to inferred antibody counts for individual virus particles. figure 5c provides histograms and frequency distributions of inferred antibody counts for a range of concentrations. being slightly skewed to the right the frequency data was best fitted using a log-normal distribution, this is particularly apparent at higher antibody concentrations. this would suggest that a significant proportion of virus particles are binding more antibodies than the average particle. however, no particles bound greater than 230 antibody molecules. we provide an interpretation of these observations in the discussion. in summary we have used statistical modelling to derive stoichiometric estimates of adv-9c12 complexes. this suggests that the most probable antibody binding maximum is 133 molecules (95% ci 119-162). however, using stoichiometric estimates to calibrate fluorescent data revealed population heterogeneity with a small proportion of virus particles binding ∼200 antibody molecules. notably, these values are in excellent agreement with previous estimates that we, and others, have derived using alternative methods (mcewan et al., 2012; varghese et al., 2004 ). various methods permit the investigation of molecular stoichiometries within biological assemblies but technical limitations often make it difficult to obtain reliable estimates. for example smlm, by its very nature, identifies single molecules and, if properly calibrated, can deliver accurate stoichiometries; however, successful counting by smlm requires a very detailed understanding of the photochemical behaviour of the chosen fluorescent dyes. here we outline a robust, and relatively facile, experimental framework for extracting accurate molecular counts using (non-smlm) fluorescent microscopy and statistical modelling labelling of a component of interest with spectrally distinct fluorescent dyes (label a or b), and mixing them at defined ratios such that b-labelled molecules are in the minority, allowed assembled complexes to be simply identified as being either positive or negative. the frequency of positive complexes was then related to the underlying stoichiometry of interaction through statistical modelling. by creating a scheme in which complexes need only be qualitatively scored for a particular label, our method negates the necessity for carefully calibrated measurements and an a priori understanding of the system. the only requirement is that the chosen label is clearly discerned from background; this is easily achievable with bright/stable fluorescent dyes and relatively inexpensive cameras; in this case we used quantum dots for sensitive detection, but many standard fluorescent dyes should also suffice. an obvious limitation is that our method relies on an ensemble measurement and obscures heterogeneity within the population of complexes. however, this information remains accessible via the a-label fluorescent signals measured from each complex. consequently, the ensemble-based stoichiometric estimates of the average complex can be used to calibrate these fluorescent signals and infer approximate molecular counts for individual complexes, therefore, restoring heterogeneity. we were able to make robust measurements of adv-9c12 interactions by fluorescence microscopy and successfully implemented the differential labelling strategy. we performed multiple independent measurements at various antibody concentrations to derive molecular counts for adv-9c12 complexes. our analysis estimated that the average adv particle interacts with a maximum of 133 9c12 antibody molecules. moreover, through examination of population heterogeneity we revealed that a small proportion of adv particles may bind up to 230 antibody molecules. these data are can be reconciled with a molecular model of adv-9c12 complexes. adv particles possess 720 identical hexon subunits, each providing a potential binding site for 9c12, however, previous estimates indicate an absolute maximum of 240 antibody molecules per virion. this would suggest that particle geometry places packing constraints on the arrangement of antibody molecules. cryo-em analyses indicates a complex and heterogeneous interaction network in which particle geometry creates potential antibody clashes and, therefore, prevents binding to every site simultaneously (varghese et al., 2004) . whilst there are consistently five 9c12 molecules at each of the twelve vertices of the adv particle, additional antibody interactions occur through heterogeneous packing across the surface; the pattern of which is likely dictated by the random order in which binding sites become occupied on any given virus particle. as a consequence with optimal antibody packing there is likely to be an absolute maximum binding occupancy of ∼240 molecules. our data indicate that the average particle binds fewer molecules (133) than the threshold dictated by purely geometric limitations. this would suggest that the majority of particles do not achieve optimal antibody packing and saturate at lower occupancies. this model also offers explanation to our observation that no particles bind greater than ∼230 molecules ( figure 5c ). this interpretation of our data exposes another potential flaw in our approach. our modelling strategy assumes that components bind independently, but in this test case the geometry of adv particles create clashes between adjacent 9c12 molecules such that complete saturation is not possible. consequently, antibody binding events can be influenced by prior antibody interactions and, therefore, are not independent. although this may have introduced inaccuracies in our estimates, the molecular counts derived from our approach mean stoichiometric estimates at increasing concentrations of 9c12, error bars indicate standard error of the mean; data fitted with a binding curve, r 2 = 0.69 (graphpad, prism). b. stoichiometric estimates were compared to 9c12 647 fluorescent values from an individual experiment; data fitted with a linear regression, r 2 = 0.98 (graphpad, prism). c. stoichiometric estimates were used to calibrate fluorescent intensity values allowing inference of heterogeneity. histograms display the frequency of inferred antibody counts as a proportion of total particles. the frequency data were fitted with a log-normal distribution, r 2 values were all >0.99 (graphpad, prism). are in good agreement with previous values. moreover, we maintain that the assumption of independent binding is appropriate for a generalisable method that can be applied to other biological assemblies. for example, within virology, we intend on extending this method to investigate the molecular composition and antibody-mediated neutralisation of enveloped viruses such as human immunodeficiency virus, hepatitis c virus and sars-coronavirus-2. beyond the confines of virology, our method could be applied to a variety of other biological assemblies of various scales, for example: bacteria; purified cellular organelles; cellular vesicles, such as exosomes; and supramolecular complexes such as ribosomes or inflammasomes. in conclusion, we have developed a novel and robust method for counting components within biomolecular complexes. this approach has provided accurate counts for a previously characterised system and could be applied in a variety of other contexts. moreover, we expect this system could be integrated with other complementary methods to enhance quantitative analysis; for example differential labelling and statistical modelling may provide a means of internally calibrating smlm-based counting schemes. semiconductor quantum dots in bioanalysis: crossing the valley of death the stoichiometry of gag protein in hiv-1 accurate construction of photoactivated localization microscopy (palm) images for quantitative measurements one, two or three? probing the stoichiometry of membrane proteins by single-molecule localization microscopy fast live-cell conventional fluorophore nanoscopy with imagej through super-resolution radial fluctuations counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (palm) antibodies mediate intracellular immunity through tripartite motif-containing 21 (trim21) regulation of virus neutralization and the persistent fraction by trim21 structure of human adenovirus a hidden markov model approach to characterizing the photoswitching behavior of fluorophores pymc: bayesian stochastic modelling in python quantum dots versus organic dyes as fluorescent labels toward absolute molecular numbers in dna-paint nuclear pores as versatile reference standards for quantitative superresolution microscopy postentry neutralization of adenovirus type 5 by an antihexon antibody correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting interaction between hela cells and adenovirus type 2 virions neutralized by different antisera we would like to thank ricardo henriques for initiating some important conversations and providing technical assistance during image analysis. we would like to thanks niall adams for his help in supervising sfm during the development of the statistical methodology. molecular cartoon images were taken from the wellcome key: cord-009769-cql7go58 authors: chhabra, sudhaker; prasad, ajay k title: flow and particle dispersion in lung acini: effect of geometric and dynamic parameters during synchronous ventilation date: 2011-07-01 journal: j fluids eng doi: 10.1115/1.4004362 sha: doc_id: 9769 cord_uid: cql7go58 the human lung comprises about 300 million alveoli which are located on bronchioles between the 17th to 24th generations of the acinar tree, with a progressively higher population density in the deeper branches (lower acini). the alveolar size and aspect ratio change with generation number. due to successive bifurcation, the flow velocity magnitude also decreases as the bronchiole diameter decreases from the upper to lower acini. as a result, fluid dynamic parameters such as reynolds (re) and womersley (α) numbers progressively decrease with increasing generation number. in order to characterize alveolar flow patterns and inhaled particle transport during synchronous ventilation, we have conducted measurements for a range of dimensionless parameters physiologically relevant to the upper acini. acinar airflow patterns were measured using a simplified in vitro alveolar model consisting of a single transparent elastic truncated sphere (representing the alveolus) mounted over a circular hole on the side of a rigid circular tube (representing the bronchiole). the model alveolus was capable of expanding and contracting in-phase with the oscillatory flow through the bronchiole thereby simulating synchronous ventilation. realistic breathing conditions were achieved by exercising the model over a range of progressively varying geometric and dynamic parameters to simulate the environment within several generations of the acinar tree. particle image velocimetry was used to measure the resulting flow patterns. next, we used the measured flow fields to calculate particle trajectories to obtain particle transport and deposition statistics for massless and finite-size particles under the influence of flow advection and gravity. our study shows that the geometric parameters (β and δv/v) primarily affect the velocity magnitudes, whereas the dynamic parameters (re and α) distort the flow symmetry while also altering the velocity magnitudes. consequently, the dynamic parameters have a greater influence on the particle trajectories and deposition statistics compared to the geometric parameters. the results from this study can benefit pulmonary research into the risk assessment of toxicological inhaled aerosols, and the pharmaceutical industry by providing better insight into the flow patterns and particle transport of inhalable therapeutics in the acini. the acini or parenchyma within the human lungs facilitate gas exchange [1] between the lung and the blood by the action of about 300 million alveoli which together provide approximately 70-80 m 2 of surface area. a complete understanding of pulmonary fluid mechanics and particle transport at the alveolar level is necessary to improve the assessment of the toxicological impact of harmful inhaled particulate matter such as smoke. such information can also benefit the pharmaceutical industry by providing key insights for the design and delivery methods of inhaled therapeutics. macroscopically, the human lung which comprises 24 generations of bifurcations, can be divided into the upper or conducting airways, and the acini or respiratory region. the acini begin at the 17th generation and differ from the conducting airways in that the acinar airways are alveolated [2] . the acinar airways can be clas-sified into three categories [2] : (i) respiratory bronchioles located in the upper acini extending from the 17th to the 20th generation with one or a few alveoli attached to each bronchiole, (ii) alveolar ducts extending from the 21st to the 23rd generation with a higher density of alveoli attached to each bronchiole, and (iii) alveolar sacs which are clusters of alveoli located at the terminus of the acinar tree. although the alveolus can be approximated as a 5/6th spherical cap, the alveolar size and shape vary from upper to lower acini [2] . also, the alveolar size changes with age and height [3, 4] , especially for infants whose airway structure and alveoli size/ shape change considerably during the first 18 months [3, 5] . due to successive bifurcation, the flow velocity magnitude also decreases as the bronchiole diameter decreases from the upper to lower acini. as a result, the associated geometric, and dynamic parameters such as reynolds and womersley numbers, also vary along the acinar tree [2] . the varying parameters affect the local fluid mechanics which in turn cause variations in particle transport and deposition from one generation to the next. the current work focuses on the effect of geometric and dynamic parameters on fluid flow and particle transport in the upper acini (respiratory bronchioles). in the past two decades, numerous studies focused on two-dimensional [6] [7] [8] [9] [10] [11] [12] and three-dimensional [8, [13] [14] [15] [16] [17] [18] [19] [20] acinar models to understand alveolar fluid mechanics and particle transport using numerical [6] [7] [8] [13] [14] [15] [16] [17] [18] [19] and experimental [6, 12] approaches. broadly, two types of acinar models were employed by these researchers: rigid-walled, and flexible-walled/oscillating. within each type, various approximations to the acinar geometry were used: axisymmetric annular or toroidal alveoli with a central duct [6, 13] , a single alveolus attached to a semi-infinite plane [21] , and honeycomb alveoli and duct [17] . we have summarized the key findings of these studies in our previous manuscripts [22, 23] . in this paper, we only discuss the literature pertaining to the effect of geometric or dynamic parameters on acinar fluid mechanics and particle transport. karl et al. [13] employed an axisymmetric, rigid-walled, annular model to study the effect of alveoli aspect ratio (ar) and alveoli-to-bronchiole diameter ratio (d a /d b ) on the resulting flow patterns. their experimental and numerical results showed a single recirculating region occupying the complete alveolar volume for smaller ar, and one large and two small recirculating regions in each alveolus for larger ars. furthermore, they observed a streamline spanning the alveolar opening which separates alveolar and ductal fluids for all aspect ratios. a more sophisticated geometry was used by federspiel and fredberg [6] wherein they numerically modeled a rigid-walled toroidal geometry and varied d a /d b and the alveolar-opening-to-cell-length ratios (al) to study their effect on the resulting flow patterns. they observed greater gas mixing for larger d a /d b and smaller al. tsuda et al. [11] imparted wall motion to their previously used rigid-walled toroidal approximation [9] to study the effect of multiple geometric/dynamic parameters on alveolar flow patterns. they observed that ar and alveolar-to-ductal flow ratios (q a /q d ) had the most noticeable impact on the resulting flow patterns. in contrast to the simpler axisymmetric two-dimensional model, sznitman et al. [16] , and kumar et al. [17] employed a more complex, three-dimensional, rhythmically expanding/contracting, multigeneration numerical model to study the variation of flow patterns along the entire acinar tree. they observed a large recirculation and a saddle point in each alveolus corresponding to the upper acini. the recirculations diminish to yield radially inward/ outward flow in the terminal alveoli. tsuda et al. [7] . numerically modeled multiple generations of the acinar tree to observe alveolar flow patterns resulting from variations in the geometric parameters. furthermore, tsuda et al. [11] extended their numerical work by considering a three-dimensional model to compare the effect of ar and d a /d b between healthy human lungs and immature, developing lungs in infants [24] . since the alveolar geometry, reynolds and womersley numbers, and the resulting flow patterns vary along the acinar tree, fluid mixing and dispersion, and particle transport may exhibit differences across acinar generations. at the same time, the orientation of a given alveolus with respect to gravity may also affect particle transport and deposition. the current work focuses on such geometric, dynamic, and gravity orientation variations in the upper acini. we consider only in-phase flow which mimics synchronous ventilation in healthy human lungs. our experimental model consisted of a single alveolus constructed from highly elastic latex film (elongation limit $800%) mounted over a 16.5 mm hole drilled into the on the side of a rigid plexiglas tube representing the bronchiole (34.8 mm internal diameter, with 1.6 mm wall thickness) as shown in fig. 1 . the alveolus and bronchiole were housed in a sealed, fluid-filled, rectangular chamber which provided the required, refractive index-matched, optical access. the oscillation of the alveolus and the oscillatory flow through the bronchiole were synchronously driven by syringe pumps connected to a stepper motor stage that was computer controlled to provide sinusoidal motion. the working fluid was an 80:20 mixture of glycerol and de-ionized water with a kinematic viscosity of 55 â 10 à6 m 2 /s and the time period of oscillation was on the order of several minutes. to ensure realistic breathing conditions in our in vitro model, three dimensionless geometric parameters were matched to the actual acinar geometry [1] as described in table 1 . in addition, dynamic similarity was achieved by matching model reynolds (re) and womersley ða ¼ d ffiffiffiffiffiffiffiffi x=v p þ numbers to human alveoli. re was calculated using the mean velocity in the bronchiole during one-half of the sinusoidal cycle, and the bronchiole diameter (d) was used as the length scale for determining both re and a. here, x is the breathing frequency, and v is the kinematic viscosity of the working fluid. the physiologically relevant values for these two parameters are compared with our experimental values in table 1 . our experimental model closely replicates the upper acini wherein the model alveolus expands/contracts in-sync with the oscillatory flow through rigid bronchiole. additional details about our model alveolus, experimental conditions, and experimental setup are described in our previous study [22, 23] . the velocity maps for case e in chhabra and prasad [22] are presented as dataset 1 in this paper. processing. in the current work, we acquired piv data to create a total of eight datasets as listed in table 2 . the datasets were designed to isolate the effect of each dimensionless parameter while keeping all other parameters fixed, and to cover a wide range of physiologically relevant geometric and dynamic parameters. however, we were limited to re ! 0.1 and a ! 0.15 due to the extremely small velocities and long experimental durations for smaller re and a values. for each data set, a total of 400 equispaced piv double frames were extracted and interrogated from a complete breathing cycle. the time separation (dt) between piv double frames was 1 s for all datasets except 5 and 6, for which the time separation was 4 s, and 0.5 s, respectively. for all datasets, image acquisition commenced after one complete breathing cycle to ensure fully-developed flow conditions. the particle images were interrogated using 64 â 64 pixel interrogation spots with 50% overlap. the resulting velocity maps were saved for each cycle. the velocity maps from 10 successive cycles were averaged to reduce noise. were computed by solving the equations of motion under the combined influence of flow-induced advection and gravity. the complete particle tracking algorithm is explained in our previous study [22, 23] . a brief description of the particle-tracking algorithm is presented here. the governing equation for particle motion can be written as: as suggested by haber et al. [25] , the governing equation can be rewritten in dimensionless form as follows: whereg is the unit vector in the direction of gravity, g is the acceleration due to gravity, d p is the particle diameter, q p is its material density ($ 1000 kg/m 3 ),ũ p is the particle velocity,ũ f is the local fluid velocity obtained by piv measurements [22] , q f is the air density, l f is the air viscosity, and c c is the cunningham correction factor [26] , x is the breathing frequency ð¼ 2p=tþ, t is the period of the breathing cycle, d is a characteristic length scale ( ¼ alveolus diameter), st is the stokes number, and h is the gravity number. st and h are the only two dimensionless numbers relevant to this problem. if these two numbers are matched between the in vitro model and the actual in vivo alveolus, then all of the particle dynamics will be accurately reproduced. for all d p < 5 lm particles st is small enough that we can ignore inertial impaction (see table 1 ). the particle trajectory can be obtained by integrating eq. (3); a second order runge-kutta method was used to calculate particle trajectories. the particle location at the (t þ 1) time step can be written as: here dt is the time step, u t p is particle velocity, and x t p is particle location at time ¼ t. the particle tracking algorithm described here assumes that the particle loading is small enough that the particles do not influence the fluid velocities (one-way coupling). table 1 physiological and experimental values of geometric and dynamic parameters. the experimental values of the particle transport parameters correspond to the values used in the particle tracking simulations for dp 5 1.5 lm particles in the current work, we calculated trajectories for massless (d p ¼ 0) and one finite-size particle (d p ¼ 1.5 lm) for three gravity orientations for each data set. the massless particle transport isolates the effect of flow advection for a chosen geometric/ dynamic parameter combination, whereas the finite-size particle transport reflects the combined influence of flow advection and gravity. in this section, we will present velocity maps, massless particle maps, and finite-size particle maps for each dataset. next, we will discuss the effect of each parameter on the velocity fields and particle maps. useful information can be obtained by examining the velocity maps corresponding to four time instants within the breathing cycle: (1) peak inhalation (fig. 2) , (2) beginning of transition from inhalation to exhalation (fig. 3) , (3) peak exhalation (fig. 4) , and (4) beginning of transition from exhalation to inhalation (fig. 5 ). as described in chhabra and prasad [22] , during each breathing cycle the bronchiole and alveolar fluid undergo mixing as explained below: the bronchiole fluid is advected into the alveolus during inhalation, after which it mixes with the alveolar fluid during transition from inhalation to exhalation, the mixed fluid is ejected from the alveolus during exhalation, and the fluid retained within the alveolus under-goes another period of mixing during transition from exhalation to inhalation. in the following four subsections we will study the effect of each dimensionless parameter on the velocity maps and fluid mixing. for comparison, our baseline velocity map (dataset 1) corresponds to case e of our previous manuscript [22] . furthermore, we will compare particle transport for massless and d p ¼ 1.5 lm particles in three gravity orientations with those for case e in chhabra and prasad [22] (dataset 1). the particle trajectories after five and 10 cycles are shown in figs. 6 and 7, respectively. [1] which is the focus of our study. a direct comparison between submaps (a) and (b) reveals that the flow topology remains largely unaffected by b: vortex rings are present during transition periods, the flow is slightly asymmetric during transition from inhalation to exhalation, and almost symmetric during transition from exhalation to inhalation. although the fractional volumetric change in the alveolus during expansion/contraction was kept constant at the baseline value of 50%, the absolute volumetric change is smaller for b ¼ 0.5. therefore, the velocities during peak inhalation/exhalation are smaller as well (figs. 2(a), 2(b), 4(a), and 4(b) ). likewise, the velocities associated with the primary vortex rings also decrease with b due to the smaller amplitude of the alveolar wall motion (figs. 3(a) 5(b) ). the differences in velocity maps affect particle transport for both massless and finite particles as explained below. at the end of 5 and 10 cycles, the deposition of massless and finite-size particles for all three gravity orientations increases as b decreases from 0.65 to 0.5 as shown in fig. 6 (a1)-6(d1) and figs. 7(a2)-7(d2). furthermore, the number of suspended particles for b ¼ 0.5 is greater than that for b ¼ 0.65 after five cycle (figs. 6(a1)-6(d1) and 6(a2)-6(d2)). however, the opposite trend is observed after 10 cycles (figs. 7(a1)-7(d1) and 7(a2)-7(d2)) due to additional particle deposition (or rejection for h ¼ p only) for b ¼ 0.5 between 5 and 10 cycles. moreover, the particle deposition is nonuniform but well spreaded on the alveolar wall for b ¼ 0.5 (figs. 6(a2)-6(d2), 7(a2)-7(d2)) while it is limited to a few sites on the alveolar wall for b ¼ 0.65 (figs. 6(a1)-6(d1), 7(a1)-7(d1)). the larger particle deposition observed for b ¼ 0.5 can be attributed to the absence of the secondary vortex ring during transition periods: although the secondary vortex ring enhances fluid mixing, it also keeps the particles in suspension for longer durations. in the absence of the secondary vortex ring for b 5 0.5, the primary vortex ring has a stronger influence near the deep alveolar wall due to which particle deposition is higher. furthermore, although the velocities inside the alveolus increase with b, the proportionality constant is not strictly linear (figs. 2(a) and 2(b) through 5(a)-5(b)). the smaller alveolar size implies that particles need to travel a shorter distance in order to deposit, especially when the particle settling velocity due to gravity is superposed. as a result, the overall particle deposition time decreases and deposition increases for smaller b. transport. the subfigures (a), (g), and (h) in figs. 2-5 display velocity maps for dv/v ¼ 0.5, 0.25, and 0.65, respectively. as before, these values of fractional volumetric change in the alveolus (dv/v) are in good agreement with physiological values in the upper acini. note that a change in dv/v may change b by a few percent which is small compared to the variation in b (23%) considered in the previous section. the flow topology during the complete breathing cycle remains similar for different dv/v similar to the cases for varying b (see sec. 3.1). furthermore, a pair of vortex rings was observed for all dv/v values during both transition periods. also, the flow is symmetric during peak inhalation/exhalation and transition from exhalation to inhalation, and slightly asymmetric during transition from inhalation to exhalation for all dv/v values (parts (a), (g), (h) in figs. [2] [3] [4] [5] . despite an overall similarity between the flow patterns, the alveolar fluid exhibits smaller velocities for smaller dv/v at each time step during a complete breathing cycle: the inward/outward velocities during peak inhalation/exhalation are smaller and show that particle deposition is highest for dv/v 5 0.5 after 5 and 10 cycles. in fact, most of the massless particles are ejected out of the alveolus for the other two dv/v values (figs. 6(a7), 6(a8) and 7(a7), 7(a8)). the transport of d p ¼ 1.5 lm particles reveals that particle deposition is highest for dv/v ¼ 0.5 in each gravity orientation. in contrast, the fraction of suspended particles is highest for dv/v ¼ 0.65 for h ¼ 0 orientation (compare parts (b8) with (b1) and (b7) in figs. 6 and 7) , and for dv/v ¼ 0.5 for the other two gravity orientations (compare parts (c1), (d1), with (c7), (d7), and (c8), (d8) in figs. 6 and 7) . the greater particle deposition for dv/v ¼ 0.5 can be attributed to the moderate expansion/contraction which encourages more particles to enter into the alveolus during the first cycle. a small fraction of these particles leaves the alveolus in at most four cycles while others either remain suspended or deposit on the alveolar wall. in contrast, the small velocities associated with dv/v ¼ 0.25 are unable to advect a reasonable portion of particles during the first cycle. consequently, the fraction of suspended particles is smaller and deposition is low. by this argument, the deposition is expected to be higher for dv/v ¼ 0.65 but in fact, the deposition is small again which can be explained as follows: during transition periods the vortex rings enhance particle transport and deposition into the alveolus. for dv/v ¼ 0.65, the recirculation region is small because the alveolus is small during transition from exhalation to inhalation (fig. 5(h) ). therefore, the particle transport is smaller compared to the baseline case (dv/v ¼ 0.5) and lowers particle deposition. the higher particle suspension for dv/v ¼ 0.65 for h ¼ p orientation can be explained as follows: the gravity reinforces large inward velocities during peak inhalation while counteracting the large outward velocities during peak exhalation. hence, during a complete breathing cycle, a net inward displacement develops but its magnitude is small. therefore, more particles remain suspended for a longer duration. furthermore, the penetration of particles is slow due to the small net inward displacement. the formation of an asymmetric vortex ring during transition from inhalation to exhalation (fig. 3(d) ) is expected due to history effects from the peak exhalation period, as explained in our previous work [22] . during peak inhalation the shear layer formation is promoted by the alveolar expansion. consequently, the rapid bronchiole flow (dataset 3, re ¼ 0.8) causes a tilt in the peak velocities towards the bronchiole flow direction (right side, fig. 2(d) ). in contrast, the influence of the bronchiole fluid motion is suppressed by large outward velocities during peak exhalation. 6 particle maps for d p 5 1.5 lm particles after five cycles for all data sets. each subfigure corresponds to the gravity orientation (indicated at the left of each row) and the specific data set in table 1 (indicated at the bottom of each column). the blue, red, and green boundaries in each subfigure correspond to the alveolar wall location in figs. 3 and 4, and 5, respectively. fig. 7 particle maps for d p 5 1.5 lm particles after ten cycles for all data sets. each subfigure corresponds to the gravity orientation (indicated at the left of each row) and the specific data set in table 1 (indicated at the bottom of each column). the blue, red, and green boundaries in each subfigure correspond to the alveolar wall location in figs. 3 and 4, and 5 , respectively. therefore, the bronchiole fluid is unable to alter the flow inside the alveolus. on the other hand, the flow asymmetry developed during transition from inhalation to exhalation continues to survive until peak exhalation due to a lack of adequate damping ( fig. 5(d) ). furthermore, since the peak exhalation is unable to damp out the asymmetry developed during transition from inhalation to exhalation, this asymmetry continues through transition from exhalation to inhalation as observed in fig. 5(d) . particle maps for re ¼ 0.1, 0.8, and 0.2 are shown in parts (a2)-(d2), (a3)-(d3), and (a4)-(d4), respectively, in figs. 6 and 7. a direct comparison between (a2)-(d2) through (a4)-(d4) in figs. 6 and 7 reveals that particle deposition is higher for re ¼ 0.2 for massless as well as d p ¼ 1.5 lm particles in all gravity orientations. in contrast, re ¼ 0.1 has a greater number of suspended particles after 5 and 10 cycles ((a2)-(d2) in figs. 6 and 7) . particle deposition decreases dramatically as re is increased to 0.8 for massless as well as d p ¼ 1.5 lm particles and does not show much gravity influence until five cycles (fig. 6 (a3)-6(d3)). however, the particle deposition for re ¼ 0.8 increases slightly in 10 cycles for h ¼ p gravity orientation but remains smaller compared to the other two re values (compare (b3) with (b2) and (b4) in fig. 7) . furthermore, the h ¼ p gravity orientation shows a larger fraction of suspended particles compared to the other two gravity orientations after 5 and 10 cycles (in figs. 6(b3) and 7(b3)). as previously noticed, the velocity maps for re ¼ 0.2 exhibit slightly stronger recirculation during both transition periods, compared to re ¼ 0.1 (compare parts (b) with (d) in figs. 3 and 5). the differential between the two recirculations accumulates over multiple cycles and causes a higher particle deposition for figs. 6 and 7) . in contrast, more particles remain suspended for re ¼ 0.1 due to a lower recirculation in this case ((a2)-(d2) in figs. 6 and 7) . as re is increased to 0.8, the velocity field becomes significantly asymmetric during both transition periods (figs. 3(c) and 5(c)). however, the shear layer remains separated from the vortex rings. consequently, the shear layer carries away all the particles introduced near the alveolar opening. hence, particle deposition is lowest for re ¼ 0.8 for both massless and d p ¼ 1.5 lm particles in all three gravity orientations ((a3)-(d3) in figs. 6 and 7) . however, gravity causes streamline crossing for some particles for the h ¼ p orientation due to which some particles enter the recirculation region ((b3) in figs. 6 and 7). consequently, they remain suspended after 5 and 10 cycles and eventually begin to deposit on alveolar wall after 10 cycles (fig. 7(b3) ). similar to re, the effect of a is clearly noticeable at each time step in the breathing cycle. as a is decreased from 0.42 to 0.15, the vortex rings during transition periods become asymmetric which can be observed by comparing (e) with (d) and (f) in figs. 3 and 5. furthermore, the asymmetry is significantly stronger during transition from exhalation to inhalation compared to the other transition period for a ¼ 0.15 (compare figs. 3(f) and 5(f)). in fact, during transition from exhalation to inhalation, the left circulation suppresses the right circulation, merges with the shear layer, and occupies almost the entire alveolar volume. during peak inhalation/exhalation, a small a causes a shift in the largest inward/outward towards the bronchiole flow direction. a similar effect was also observed for large re ( ¼ 0.8) in the previous subsection. the asymmetry in vortex rings during transition periods can be explained as follows: although the fractional volumetric expansion/contraction during inhalation/exhalation is the same for all a values, the rate of alveolar expansion/contraction is significantly different. in fact, a ¼ 0.15 represents 4 and 8 times slower expansion/contraction compared to a 5 0.3 and 0.42, respectively. consequently, the velocities induced by the alveolar wall motion are significantly smaller for the smaller a value. in contrast, the bronchiole flow for all a remains unchanged (re ¼ 0.2). as a result, the bronchiole flow-induced velocities overwhelm velocities caused by the alveolar wall motion and the vortex rings become highly asymmetric. a similar effect causes the largest velocities to shift in the bronchiole flow direction during peak inhalation/ exhalation. parts (a4)-(d4), (a5)-(d5), and (a6)-(d6) in figs. 6 and 7 demonstrate that particle deposition and the fraction of suspended particles decrease with a for massless and finite particles in all gravity orientations. on the other hand, the deposition sites remain unaffected as a is decreased from 0.42 to 0.3 but become fewer for an even smaller a (¼0.15). as previously noticed, the velocity maps and the particle deposition sites are quite similar for a ¼ 0.42 and 0.3 ((a4)-(d4), (a5)-(d5) in figs. 6 and 7) . however, higher inhalation velocities in combination with higher recirculation velocities for a ¼ 0.42 (see (d) and (e) in figs. 2-5) push more particles into the alveolus resulting in greater particle deposition and a higher fraction of suspended particles ((a4)-(d4), (a5)-(d5) in figs. 6 and 7) . when a is reduced to 0.15, the overall flow topology changes (compare figs. 3(f) and 5(f) with 3(d) and 3(e) and 5(d) and 5(e)), i.e., the secondary vortex ring and the shear layer disappear and the primary vortex ring becomes highly asymmetric. consequently, during transition from exhalation to inhalation almost all the introduced particles are swept away by the merged shear layer-asymmetric vortex ring structure (fig. 5(f) ). in summary, our results show that the overall impact on velocity maps of dynamic parameters is stronger than geometric parameters. consequently, the dynamic parameters alter particle trajectories more significantly than the geometric parameters. our velocity maps demonstrate larger velocity magnitudes, greater fluid mixing, and smaller particle deposition for higher values of the geometric parameters parameters employed in this study. we also observed an asymmetric velocity field with greater mixing and larger particle deposition for smaller a. these observations are useful in predicting the variation of flow and particle transport as we travel deeper into the lung acinus which is characterized by increasing values of geometric parameters (b and dv/v), and decreasing values of dynamic parameters (re and a) [27] . therefore, our parametric study indicates that velocity maps would show symmetric flow patterns in the upper acini, and largely asymmetric flow in the lower acini. nonetheless, the upper acini exhibit greater flow mixing yet a smaller particle suspension due to topological differences arising from the associated geometric variations. moreover, our particle maps show that the particle deposition time will be smaller and greater deposition will occur in the upper acinar generations compared to the lower acinar generations. this paper presents alveolar flow patterns and particle transport results for a range of geometric and dynamic parameters within the acinar region of the human lung. specifically, we considered two geometric (b and dv/v), and two dynamic parameters (reynolds and womersley numbers) to study their effect on alveolar flow patterns and particle deposition. we considered massless and d p ¼ 1.5 lm particles for the latter study. the results for b show that the velocities increase with b at each time step during a breathing cycle. the effect of b is more prominent during transition periods due to smaller velocities during these periods. in addition, an alveolus with a smaller b does not show the secondary vortex ring during transition due to its smaller size. although the velocity maps do not exhibit a significant influence of b, the particle maps indicate a significantly higher particle deposition for smaller b. our study also demonstrates that the velocities are strongly influenced by dv/v. furthermore, the fraction of suspended particles and particle deposition is strongly affected by dv/v. the basline value of dv/v ¼ 0.5 enhances particle penetration and deposition while smaller and larger values can reduce particle suspension and deposition. although these results vary slightly with gravity orientation, they are generally true for all particles smaller than 2.5 lm (small and midsize particles). the results for reynolds number show that a higher re causes asymmetric vortex rings during transition periods and shifts the maximum inhalation/exhalation velocities towards the bronchiole flow direction. furthermore, as re is increased, the deposition initially increases before decreasing for all gravity orientations for massless particles. the results for a display the opposite behavior: the velocity maps become highly asymmetric at each time step during the breathing cycle for smaller a. the particle maps show that deposition increases as a is decreased from 0.42 to 0.15. overall, our velocity maps show that the geometric parameters primarily affect the velocity magnitudes but the dynamic parameters distort the flow symmetry while also altering the velocity magnitudes. also, the particle maps display a greater influence of dynamic parameters compared to geometric parameters. our results indicate greater flow mixing and particle deposition but a lower particle suspension for upper acini compared to lower acini. morphometry of the human lung morphometry of the human pulmonary acinus the postnatal development and growth of the human lung. i. morphometry number of alveoli in the human lung structural aspects of postnatal lung development-alveolar formation and growth axial dispersion in respiratory bronchioles and alveolar ducts gas and aerosol mixing in the acinus two-and three-dimensional simulations of aerosol transport and deposition in alveolar zone of human lung effects of alveloated duct structure on aerosol kinetics i. diffusional deposition in the absence of gravity effects of alveloated duct structure on aerosol kinetics ii. gravitational sedmimentation and inertial impaction chaotic mixing of alveolated duct flow in rhythmically expanding pulmonary acinus recirculating flow in an expanding alveolar model: experimental evidence of flow-induced mixing of eerosols in the pulmonary acinus low reynolds number viscous flow in an alveolated duct validation of cfd predictions of flow in a 3d alveolated bend with experimental data three-dimensional convective alveolar flow induced by rhythmic breathing motion of the pulmonary acinus respiratory flow phenomena and gravitational deposition in a three-dimensional space-filling model of the pulmonary acinar tree the effects of geometry on airflow in the acinar region of the human lung particle deposition in the human tracheobronchial airways due to transient inspiratory flow patterns simulation of airflow fields and microparticle deposition in realistic human lung airway models. part i: airflow patterns hamiltonian chaos in a model alveolus shear flow over a self-similar expanding pulmonary alveolus during rhythmical breathing flow and particle dispersion in a pulmonary alveolus-part i: velocity measurements and convective particle transport flow and particle dispersion in a pulmonary alveolus-part ii: effect of gravity on particle transport the finite element 3d reconstruction of the pulmonary acinus imaged by synchrotron x-ray tomography gravitational deposition in a rhythmically expanding and contracting alveolus on the velocity of steady fall of spherical particles through fluid medium pulmonary fluid dynamics this research was supported by philip morris usa, inc., and philip morris international. key: cord-300435-vs0ntcsb authors: katz, al; peña, stephanie; alimova, alexandra; gottlieb, paul; xu, min; block, karin a. title: heteroaggregation of an enveloped bacteriophage with colloidal sediments and effect on virus viability date: 2018-10-01 journal: sci total environ doi: 10.1016/j.scitotenv.2018.04.425 sha: doc_id: 300435 cord_uid: vs0ntcsb four sediments in the colloidal size range: goethite, montmorillonite, illite, and kaolinite, were suspended with the bacteriophage φ6, a model enveloped virus, to determine relative rates of heteroaggregation and the effect of aggregation on virus viability. turbidity was measured on combinations of virus and each sediment type at low concentration to determine aggregation rates. aggregation of sediment with virus occurred regardless of mineral type, and larger fraction of virus is expected to aggregate with increasing sediment concentration leading to higher deposition rates. the negatively charged sediments, aggregated with φ6 (also negatively charged at neutral ph) at a faster rate than the positively charged sediments, yielding turbidity slopes of 4.94 × 10(−3) s(−1) and 7.50 × 10(−4) s(−1) for φ6-montmorillonite and φ6-illite aggregates, respectively, and 2.98 × 10(−5) s(−1) and 2.84 × 10(−5) s(−1), for φ6-goethite and φ6-kaolinite, respectively. this indicates that the interaction between sediments and virus is hydrophobic, rather than electrostatic. large numbers of virions remained viable post-aggregation, despite the fragility of the viral envelope, indicating that small-sized aggregates, which may travel more readily through porous media, may pose an infection risk. the fraction of φ6 that remained viable varied with sediment type, with montmorillonite-φ6 aggregates experiencing the greatest reduction in infectivity at 35%. tem analyses reveal that in all sediment-φ6 combinations, infectivity loss was likely due to disassembly of the viral envelope as a result of aggregation. • sediments interact with enveloped viruses but effects on viability are unknown. • light scattering measured aggregation rates of virus with four mineral types. • heteroaggregation was hydrophobic and faster with negatively-charged sediments. • the greatest reduction in infectivity occurred in virus-montmorillonite aggregates. • aggregation with sediment caused virus disassembly and infectivity loss. a b s t r a c t a r t i c l e i n f o the mechanisms that influence the transport of viruses are important for management of natural systems and freshwater resources. these may include human pathogens such as those introduced by fecal matter, but, more importantly, bacteriophage which serve to regulate bacterial populations (o'brien et al., 2017) . bacteriophage alter biogeochemical cycles through lysis of bacterial cells (díaz-muñoz and koskella and brockhurst, 2014) and impact eukaryotes that depend on bacterial populations, thus having a significant impact on the greater ecosystem. the distribution and residence time of viruses is affected by the chemistry of the water, e.g., dissolved organic matter, metals, and contaminants, but also the suspended materials, such as particulate organic matter (pom) and suspended sediment, primarily clays, which serve as substrate and habitat for biofilm-producing bacteria (alimova et al., 2006; alimova et al., 2009) . in fact, sediment-bearing biofilms have been shown to sequester virus particles in wetlands where they are subsequently concentrated and subject to re-release (flood and ashbolt, 2000) . furthermore, bacteriophages are utilized as subsurface tracers and indicators for a variety of environmental applications (ghanem et al., 2018; keswick et al., 1982; redman et al., 1997) and, therefore, their interaction with sediments may affect their quantification in hydrological experiments. an estimated 10 31 virus particles world-wide are prevalent in both soils and aquatic systems (breitbart and rohwer, 2005; weinbauer and rassoulzadegan, 2004) . in aquatic systems, the length of time in which a virus can interact with potential hosts is largely controlled by sinking rates (fuhrman, 1999) . these rates are strongly influenced by aggregation with suspended particles. sediments are known to influence virus transport and survival in porous media by controlling the potential for viruses to contaminate groundwater (jin and flury, 2002; chu et al., 2000) and to promote lysis of bacteria in soils (kimura et al., 2008) . the mineralogy and size of suspended sediments affects the extent to which viruses will aggregate and sorb to sediments (chu et al., 2000; jin and flury, 2002; mcgechan and lewis, 2002; syngouna and chrysikopoulos, 2015) . with a few recent exceptions, enveloped viruses (block et al., 2016; block et al., 2014) , have been largely omitted from studies of virus-sediment interaction, aggregation, and viability. enveloped viruses possess different structural properties from nonenveloped viruses and include important plant and animal pathogens such as herpesvirus, coronavirus and influenza a and b, all of which are found in natural and wastewaters (batik et al., 1980; gundy et al., 2008; rosenberg et al., 1980; sharp et al., 1975; stallknecht et al., 1990a; stallknecht et al., 1990b) . virus particles typically range from 20 nm to 500 nm in diameter, and are therefore considered colloidal particles. for non-enveloped viruses, the surface charge is determined by the protonation/deprotonation of amino acids in the capsid (elimelech et al., 1995) . however, for enveloped virions, surface charge is determined by envelope proteins and lipids. the colloidal interaction between virus particles and the small fraction of suspended sediments can be described by dlvo theory (derjaguin and landau, 1941; verwey and overbeek, 1948) , in which the potential between colloidal particles is the sum of the attractive van der waals potential and the coulomb potential (repulsive for like-charged particles and attractive for oppositely-charged particles). dlvo theory has been extended to include hydrophobic forces (van oss, 1993; van oss et al., 1990) . previous research has shown that for non-enveloped viruses, sorption is largely influenced by hydrophobicity. puls (1999), (2000) studied the attachment of three, non-enveloped, bacteriophages, t2, ms2 and φx174 to several clay fractions and calculated the electrostatic and hydrophobic contributions to the free energy. their calculations showed that surface hydrophobicity dictates sorption of viruses to clays. chrysikopoulos and syngouna (2012) looked at attachment of the bacteriophages ms2 and φx174 to kaolinite or montmorillonite, and using extended dlvo energy calculations, concluded that the virus-clay attachment was primarily through hydrophobic interaction. a study of heteroaggregation of the non-enveloped cowpea mosaic virus with colloidal hematite revealed that at ph 6, at which hematite carries a positive surface charge and the virus a negative charge, the aggregates accumulated four times as many viruses as hematite particles vilker et al. (1994) . however, at ph 3, in which both particle types are positively charged, the aggregates contained three times as many hematite particles as virus. from these results, they concluded that attraction between the virus and hematite is mostly governed by electrostatic interactions. the above studies all investigated non-enveloped viruses which are structurally different to viruses with lipid envelopes (e.g. influenza, paramyxovirus). in this work, we employ turbidity measurements to investigate the heteroaggregation of a model envelope virus, the bacteriophage φ6, with colloidal goethite and three clay minerals: illite, kaolinite and montmorillonite. φ6 is a member of the cystoviridae family of bacteriophage. the cystoviridae are the only phage family with an outer bi-lipid envelope. φ6 is a dsrna virus whose host cell is pseudomonas phaseolicola, a common plant pathogen. φ6 consists of an icosahedral nucleocapsid surrounded by a bi-lipid envelope and is therefore employed as a model for virus emergence in evolutionary studies and for human pathogens such as coronavirus (dennehy, 2017; mindich, 2004) . the diameter of φ6 is~80 nm. the φ6 virus carries negative charge at neutral ph (block et al., 2014) . montmorillonite and illite platelets both have positive charged edges and negative charged faces with an overall negative charge at neutral ph (gonzález sánchez et al., 2008; van olphen, 1962) . kaolinite platelets have negatively charged faces, and at neutral ph, negatively charged edges (gupta et al., 2011; schroth and sposito, 1997) . goethite is positively charged at neutral ph (gaboriaud and ehrhardt, 2003; zeltner and anderson, 1988) . the effect of the interaction of colloidal sediments and enveloped viruses on virus viability, the nature of that interaction is unknown. we will present aggregation rates as determined by turbidity experiments, the effect of the interaction between φ6 and sediments on φ6 infectivity, and discuss the environmental implications of our findings. in the early stage of colloidal aggregation, suspensions consist mostly of primary particles and aggregation is a bimolecular process in which the kinetics are dominated by the merging of individual primary particles to form doublets: n + n → n 2 (garcíagarcía et al., 2006; garcía-garcía et al., 2007) . the rate equation for the loss of primary particles into doublets can be written as: where n(t) is the number density of particles and k is the aggregation rate constant for doublet formation. the corresponding rate equation for doublet, n 2 , formation is: integration of eq. (1) gives: where n 0 is the number density of primary particles at t = 0. the average number of primary particles in an aggregate is η = n 0 /n(t) and thus in the early stage of aggregation: from eq. (4) it is seen that in the early growth region, aggregate growth is linear in time (kobayashi and ishibashi, 2011; puertas and nieves, 1997) . the turbidity (τ) for a system of monomeric colloidal aggregates is given by (katz et al., 2013) : where l is the optical path length; k = 2π/λ, is the optical wavenumber; c 0 k 4 a 6 is the scattering cross-section of a primary particle; a is the effective primary particle radius; d f is the fractal dimension of the aggregate; and r is the radius of gyration of the aggregate; ηn is the number density of primary particles and is constant for each individual suspension. in the initial stage of aggregation, the aggregates are small and if the primarily particles are much smaller than the optical wavelength, kr b b1. expanding eq. (5), and keeping only the lowest order terms in kr, it is seen that τ ∝ η, i.e. aggregate size, and at early time, τ ∝ kn 0 t. liu et al. (2007) considered measurements of heteroaggregation rate constants by turbidity for dispersions containing colloids of different sizes. they developed a method based on the t-matrix to evaluate changes in turbidity as two primary particles form a doublet during heteroaggregation. they also considered the effects of multiple scattering on turbidity and determined that multiple scattering effects are not significant when the relative volume of the scatterers is b0.1%. in the case of heteroaggregation of two distinct primary particles, e.g. viruses and sediments, three types of bi-molecular particle mergers are possible: sedimentsediment; virusvirus; and sedimentvirus. eq. (2) is modified to include the three possible modes of doublet formation (yu and borkovec, 2002) : in which n s and n v are the number of primary sediment and virus particles in the suspension, respectively; n ss , n vv and n sv are the number of sediment-sediment, virus-virus and sediment-virus doublets, respectively; and k ss , k vv and k sv are the rate constants for sedimentsediment, virusvirus, and sedimentvirus doublet formation, respectively. the light extinction for a system of heteroaggregation is given by a modification of eq. (5): where a eff is an effective primary particle radius, and the scattering cross-section, c 0 , is an average cross-section weighted by the concentrations of each type of primary particle in the aggregate. as in the case of mono-colloidal systems at early times, i.e. early stage of aggregation, kr b b1 and, keeping only terms in kr, τ is proportional to η. purified cystovirus φ6 was prepared following the procedure described in katz et al. (2012) . after purification, the virus was resuspended in buffer a, modified to have a reduced divalent cation concentration (10 mm kh 2 po 4 and 0.5 mm mgso 4 ). it was confirmed by counting plaque forming units (pfu) of p. phaseolicola infection that φ6 survives in the modified buffer for several days at room temperature. the four mineral types chosen for this study were goethite, kaolinite (city college of new york rock and mineral collection); illite, and montmorillonite (wards science, rochester, ny). composition and purity of mineral specimens was confirmed by x-ray powder diffraction (city college of new york). all sediments were first crushed into powders and each washed with a 5% sodium hypochlorite (bleach) solution overnight to remove any possible organic matter or microbial contaminants. sediments were then washed multiple times in distilled water to remove the sodium hypochlorite residue. the small clay fraction (b0.2 μm) was separated by centrifugation in a sorvall ultracentrifuge (thermofisher scientific, waltham, ma) with an ss34 rotor at 5000 rpm (3000 g) for 1440 s (montmorillonite), 1140 s (illite), 1200 s (kaolinite) or 720 s (goethite). the supernatants containing the small fraction were collected and sterilized by autoclaving at 121°c and 100 kpa above atmospheric pressure for 1800 s to eliminate possible bacterial contamination. turbidity measurements were performed at 20°c and ph = 7 with the aggregates in a 1 cm optical path length quartz cuvette. specimens were illuminated with a broadband halogen lamp (hl2000, ocean optics, inc., dunedin, fl.) coupled to an optical fiber. spectra were collected in the wavelength range of 400 nm to 1000 nm. the transmitted light was collected by a second optical fiber coupled to a spectrophotometer equipped with a ccd (hr2000, ocean optics, inc.) which transferred the data to a personal computer. a 1 mm diameter aperture positioned in front of the collection fiber limited data collection to transmitted light while blocking scattered light. a magnetic stirrer was employed to prevent settling of the suspended colloidal aggregates. the distance between the cuvette and aperture was 5 cm, limiting the collection solid angle to 0.00003 sr and thus restricting the intensity of forward scattered light incident on the collection fiber (katz et al., 2013) . for time-resolved measurements, each spectrum was integrated for 250 ms using a high-speed data acquisition mode which allowed rapid collection and transfer of many spectra (3600 spectra for the typical 900 s experiment) to a pc with no dead-time between spectra acquisitions. for the estimation of φ6 concentration by turbidity, spectra were integrated for 30 s. the φ6 concentration was estimated from turbidity, approximating the φ6 virions as spheres with a refractive index of 1.45 and a diameter of 80 nm. the φ6 turbidity is given by (chýlek and li, 1995; xu, 2003; xu et al., 2003) : where c s is the scattering cross section; n v is the φ6 concentration; l is the optical path length; n w is the refractive index of water; n v is the refractive index of φ6; r is the radius of φ6; and λ is the optical wavelength. φ6 is sufficiently small compared to the wavelength of visible light so that higher order terms can be ignored. based on the measured turbidity, the stock φ6 concentration was determined to be 7.8 × 10 11 cm −3 , prior to the 2× dilution incurred when mixed with an equal volume of suspended sediments. this estimate is slightly larger than the estimate from counting pfus (6.8 × 10 11 cm −3 ) but is likely more accurate as it also includes virions rendered non-infectious due to envelope defects. φ6, due to its lower refractive index and smaller size, scatters less efficiently than the selected sediments. therefore, for the heteroaggregation turbidity experiments, the φ6 concentration was adjusted to have a lower turbidity than the turbidity at the lowest sediment concentration. the φ6 stock suspension was diluted to have a turbidity of 0.128 at 750 nm. heteroaggregation experiments were performed by mixing 1 ml of suspended φ6 with 1 ml of each suspended sediment. this resulted in a further 50% dilution of the cation concentration (5 mm kh 2 po 4 and 0.25 mm mgso 4 ). due to both the larger volume of the sediment primary particles (~0.2 μm) and higher refractive index. refractive indices of the three sediment types are (geometric mean of the three axes): goethite: n = 2.18; illite: n = 1.58; kaolinite: n = 1.56; montmorillonite: n = 1.55 (deer et al., 1992; shannon et al., 2002; weidler and friedrich, 2007) compared to φ6 particles (80 nm), the sediment primary particles have a higher scattering cross-section than the virions. therefore, the heteroaggregation experiments were performed using a higher concentration of viral particles than sediments. the sediment concentrations were adjusted to have a turbidity of~0.6 (after 2× dilution by addition of virus suspension) at 750 nma wavelength in which light extinction losses are primarily due to scattering rather than absorption. the concentrations were chosen so that the total volume of the scatterers was b0.1% of suspension volume, thus staying in the single scattering regime and keeping the incidence of multiple scattering small (katz et al., 2013; wind and szymanski, 2002; xu and sun, 2010) , while still providing a reasonable signal-to-noise level. the sediment and virus concentrations after the 2× dilution are summarized in table 1 . after completion of the turbidity measurements, the suspensions were centrifuged at 13,000 rpm (16,000 g) for 600 s. the centrifugation was sufficient to pellet the smallest heteroaggregates (i.e. doublets) and individual sediment primary particles but not individual virions. the pellets were resuspended in buffer and both the resuspended pellets and supernatants were analyzed for the presence of φ6 proteins by sds-page. the separated heteroaggregate pellets and supernatants were analyzed by sds-page (studier, 1973) . the fraction of φ6 in the pellets was estimated by analyzing the relative densities of the p1, p2 and p3 protein bands of the heteroaggregate pellets and supernatants. it is noted that the sds-page experiment does not resolve p1 and p2 due to the closeness of their molecular weight (85 kda and 75 kda, table 1 concentrations of colloidal particles. primary particles cm −3 goethite 0.096 5.3 × 10 9 illite 1.9 9.6 × 10 10 kaolinite 0.66 3.9 × 10 10 montmorillonite 1.6 9.6 × 10 10 φ6 3.9 × 10 11 respectively). p1 is the largest molecular weight protein in φ6 and has a relatively high copy number (120 per virion). thus, p1 is the highest density sds-page band of all the φ6 proteins. viable virus concentrations after aggregation were calculated using the plaque assay technique. the pelleted aggregates were vortexed prior to plaquing to disaggregate the mixture and separate the viruses from the sediments. the supernatants and final, disaggregated, clay suspensions were spot checked on plates with growing p. phaseolicola to determine number of virions in the system. heteroaggregates from the isolated pellets (section 3.3.3) were prepared for tem using standard techniques. samples were negatively stained with uranyl acetate. a minimal amount of stain was employed to reduce the effect of the stain on abundant viral proteins, which would impede identification of viral particles. micrographs of the heteroaggregates were acquired with a zeiss 902 tem operating at 80 kev with a 2048 × 2048 pixel detector. magnification was 85,000× corresponding to 0.512 nm/pixel. φ6 particles are identifiable in the micrographs from size and morphology. micrograph insets presented in the results section were re-sampled 4:1 using coreldraw (version ×8, corel corp, ottawa, can) to reduce pixelation in the enlarged insets. the contrast of the insets were enhanced using coreldraw's autoadjust function. prior work (block et al., 2014) has demonstrated that φ6 does not aggregate in buffer a at neutral ph, therefore k vv = 0 (eq. (6)). it was next confirmed by turbidity measurements that, individually, the four suspended sediments do not aggregate in the dilute buffer. the time evolution of the sediment turbidities at 750 nm, measured over a 900 s time period, are plotted in figs. 1(a-d) . the turbidities remained constant for these measurements, confirming that the cation concentration in the buffer is sufficiently dilute so as to not cause the sediments to aggregate. thus, k ss in eq. (6) can be taken to be ≈0 and eq. (6) simplifies to: i.e. primarily hetero-doublets are being formed at early time rather than virus-virus or sediment-sediment doublets. in this case, from eq. (7), dτ dt ∝k dnsv dt , i.e. the rate of hetero-doublet formation. the turbidity of the heteroaggregates at 750 nm is plotted in fig. 2 (a-d) . the φ6-goethite and φ6-kaolinite heteroaggregates demonstrated a linearly increasing turbidity over the entire 900 s time frame of the experiments, indicating that aggregation remained in the linear range, i.e. early stage, and consisted primarily of doublet formation. the turbidity slopes for φ6-goethite and φ6-kaolinite were 2.98 × 10 −5 ± 1.2 × 10 −7 s −1 and 2.84 × 10 −5 ± 2.2 × 10 −7 s −1 , respectively. the respective linear fits to the turbidity are shown in fig. 2a and c as dotted lines. the least squares linear fit was performed using originpro software (originlab, northampton ma, usa). in both the φ6-illite and φ6-montmorillonite heteroaggregates the duration of the linearly increasing turbidity was significantly shorter (30 s for φ6-illite and 15 s for φ6-montmorillonite). the early stage turbidity changes for illite and montmorillonite, along with linear fits, are plotted in fig. 3 . the corresponding slopes during the linear region (primarily doublet formation) are 4.94 × 10 −3 ± 1.9 × 10 −4 s −1 for φ6montmorillonite and 7.50 × 10 −4 ± 2.9 × 10 −5 s −1 for the φ6-illite aggregates. exact calculations of the heteroaggregation rates from turbidity require knowledge of the scattering cross-sections of both singlets and the doublet (liu et al., 2007; yu and borkovec, 2002 ). the precise shape of the virus-clay platelet doublet is not well known though there is evidence that virusclay platelet doublets occur as both edge-attached and face-attached (block et al., 2014) . although imprecise knowledge of doublet shape complicates calculations of heteroaggregation rates, analysis of the turbidity slope coupled with particle concentrations allows one to determine relative aggregation rates between φ6 and the four sediment types, elucidating the nature of the interaction. from the aggregation rates, it is observed that doublet formation occurs at a much slower rate for goethite and kaolinite compared to montmorillonite and illite. that the two positively charged sediments (goethite and kaolinite) aggregate more slowly with the negatively charge virions than the two negatively charged sediments (montmorillonite and illite), it is likely that the interaction between φ6 and the sediments is not electrostatic in nature but hydrophobic, similar to that observed for ms2 and φx174 (chrysikopoulos and syngouna, 2012) . the sds-page of the pellets and supernatants for the four heteroaggregates are plotted in fig. 4a along with the non-aggregated φ6 control. comparisons of the density profiles of the p1 and p3 protein bands (fig. 4b) reveal that for all four heteroaggregates, most of the virions aggregated with the sediments (montmorillonite 80%; kaolinite 73%; illite 76%; goethite 68%) and the remaining non-aggregated virions in the supernatant. it is expected that formation of larger aggregates resulting from either higher cation concentrations or greater sediment concentrations would result in a greater fraction of the virions aggregating. it should be noted that the sds-page results do not provide information as to virus viability, since the proteins in partially disassembled virions will likely remain intact and thus appear in their respective sds-page bands. φ6 viability in the supernatants and pellets as determined by plaquing is presented in fig. 5a . the distribution of virions (viable and non-viable) is shown in fig. 5b . the viable virus population in the control was 3.5 × 10 11 , in good agreement with our estimates from turbidity (which includes non-infectious virions). φ6 in the montmorillonite aggregates displayed a significant decrease in infectivity for both virions in the aggregates (14% viable) and planktonic cells (35% viable), which is consistent with results from our previous work (block et al., 2014) . in the φ6-kaolinite and φ6-illite aggregates, the virions in the pellets, i.e. aggregated virions, show an appreciable loss in infectivity (14% and 27% of original virus populations remained viable for kaolinite and illite, respectively), while virions that did not aggregate remained close to 100% viable. for the φ6-goethite heteroaggregates, 49% of the virions in the pellets remained viable, while supernatant virion viability was near 100%. the higher viability values for in the φ6-goethite pellet is likely due to fewer virions aggregating as a result of the lower goethite concentration required to achieve approximately equal turbidity as in the experiments with clay minerals to account for goethite's greater refractive index. analysis of tem micrographs confirmed the presence of φ6 in each of the four heteroaggregate suspensions. the viruses were observed to be attached to both platelet faces and edges of the three clay minerals. distortion of the virus morphology was evident in the micrographs (figs. 6a-d). in the φ6goethite heteroaggregates, the virions appear hexagonal with a 55 nm diameter (fig. 6a ). this morphology is consistent with that in micrographs of the icosahedral nucleocapsid (alimova et al., 2015) , indicating that the viral envelope has been disassembled during aggregation with goethite. analysis of tem micrographs of φ6 heteroaggregated with the three clay minerals (figs. 6b. φ6illite; 6c. φ6kaolinite; 6d. φ6montmorillonite) reveal substantial distortion in the φ6 morphology. the viral particle diameters are similar to the 55 nm diameter of the nucleocapsid rather than the 80 nm diameter of the intact virions and the virions appear to be distinctly non-circular. the morphologic changes indicate that the sediments partially disassembled the viral envelopea process likely responsible for the reduced rate of virus infectivity. turbidity measurements on dilute combinations of bacteriophage φ6 and four sediment types were collected to determine the relative rates of heteroaggregation. negatively charged sediments aggregated with φ6 at a faster rate than positively charged sediments, indicating that the interaction is not electrostatic, but hydrophobic in nature. because k, the rate constant, is independent of concentration, the relative rates for aggregation at low turbidity are a good predictor of rates at higher concentrations. for all experiments, most of the virions aggregated with sediments regardless of mineral type. at higher sediment concentrations, we expect an even larger viral fraction to aggregate, resulting in higher deposition rates. nevertheless, at the low concentrations of our experiments, heteroaggregated viruses experienced a loss of infectivity. despite the fragility of the viral envelope, a large number of virions remained viable, indicating that small-sized heteroaggregates may pose an infection risk. while non-aggregated virions in the φ6goethite, φ6 -kaolinite and φ6illite suspensions remained near 100% viable, in the φ6montmorillonite suspensions, a substantial fraction (35%) of the non-aggregated φ6 also lost viability. tem analysis revealed that the observed reduction in infectivity is likely due to partial disassembly of the viral envelope after aggregation. in conclusion, it is expected that viral disassembly and loss of infectivity is likely to be exacerbated with increased contact between virions and sediments which occurs within larger aggregates. the formation of sediment-virus heteroaggregates and subsequent virus inactivation has potential implications for remediation, spread of disease, and management of wastewater particularly during reintroduction via porous media into groundwater systems. our results suggest that soils containing clay minerals, and in particular, montmorillonite, may potentially serve to inactivate viruses possessing a rigid lipid envelopes during colloidal transport. however, our results also show that even in the case of viruses that are not inactivated by association with a solid phase, colloidal sediments could facilitate the transport of viruses into aquifers as small heteroaggregates. furthermore, because multiple virions aggregate with colloidal sediment even in dilute concentrations, forming heteroaggregates in the colloidal size range, this interaction must be considered in conjunction with specific infectivity (ghanem et al., 2018) when phages are used in tracer experiments. hybrid native phosphorescence and fluorescence spectroscopy for cancer detection bacteria-clay interaction: structural changes in smectite induced during biofilm formation the ϕ6 cystovirus protein p7 becomes accessible to antibodies in the transcribing nucleocapsid: a probe for viral structural elements an epidemiologic study of the relationship between hepatitis a and water supply characteristics and treatment disassembly of the cystovirus ϕ6 envelope by montmorillonite clay montmorillonite-mediated aggregation induces deformation of influenza virus particles here a virus, there a virus, everywhere the same virus? forces dictating colloidal interactions between viruses and soil attachment of bacteriophages ms2 and ϕx174 onto kaolinite and montmorillonite: extended-dlvo interactions virus transport through saturated sand columns as affected by different buffer solutions light scattering by small particles in an intermediate region an introduction to the rrock-forming minerals evolutionary ecology of virus emergence theory of the stability of strongly charged lyophobic sols and of the adhesion of strongly charged particles in solution of electrolytes bacteria-phage interactions in natural environments particle deposition and aggregation: measurement, modelling and simulation virus-sized particles can be entrapped and concentrated one hundred fold within wetland biofilms marine viruses and their biogeochemical and ecological effects effects of different crystal faces on the surface charge of colloidal goethite (α-feooh) particles: an experimental and modeling study 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toward an understanding of the turbidity measurement of heterocoagulation rate constants of dispersions containing particles of different sizes sw-soil and water transport of particulate and colloid-sorbed contaminants through soil, part 1: general principles packaging, replication and recombination of the segmented genome of bacteriophage φ6 and its relatives diversity of dna viruses in effluents of membrane bioreactors in traverse city, mi (usa) and la grande motte (france) a new method for calculating kinetic constants within the rayleigh -gans -debye approximation from turbidity measurements filtration of recombinant norwalk virus particles and bacteriophage ms2 in quartz sand: importance of electrostatic interactions the risk of acquiring hepatitis from sewage-contaminated water surface charge properties of kaolinite refractive index and dispersion of fluorides and oxides nature of the surviving plaque-forming unit of reovirus in water containing bromine effects of ph, temperature, and salinity on persistence of avian influenza viruses in water persistence of avian influenza viruses in water analysis of bacteriophage t7 early rnas and proteins on slab gels experimental investigation of virus and clay particles cotransport in partially saturated columns packed with glass beads unit layer interaction in hydrous montmorillonite systems acid-base interfacial interactions in aqueous media dlvo and non-dlvo interactions in hectorite theory of the stability of lyophobic colloids interaction between a plant virus and colloidal hematite determination of the refractive index of particles in the clay and sub-micrometer size range are viruses driving microbial diversification and diversity? quantification of scattering corrections to the beer-lambert law for transmittance measurements in turbid media light extinction and absorption by arbitrarily oriented finite circular cylinders by use of geometrical path statistics of rays evaluation of the uncertainties caused by the forward scattering in turbidity measurement of the coagulation rate anomalous diffraction of light with geometrical path statistics of rays and a gaussian ray approximation distinguishing heteroaggregation from homoaggregation in mixed binary particle suspensions by multiangle static and dynamic light scattering surface charge development at the goethite/aqueous solution interface: effects of co 2 adsorption partial support for this project was provided by psc-cuny award trada-46-247, jointly funded by the professional staff congress and the city university of new york. key: cord-274305-mnyy41po authors: kumar, purnima s; subramanian, kumar title: demystifying the mist: sources of microbial bioload in dental aerosols date: 2020-07-27 journal: j periodontol doi: 10.1002/jper.20-0395 sha: doc_id: 274305 cord_uid: mnyy41po the risk of transmitting airborne pathogens is an important consideration in dentistry and has acquired special significance in the context of recent respiratory disease epidemics. the purpose of this review, therefore, is to examine (1) what is currently known regarding the physics of aerosol creation, (2) the types of environmental contaminants generated by dental procedures, (3) the nature, quantity, and sources of microbiota in these contaminants and (4) the risk of disease transmission from patients to dental healthcare workers. most dental procedures that use ultrasonics, handpieces, air‐water syringes, and lasers generate sprays, a fraction of which are aerosolized. the vast heterogeneity in the types of airborne samples collected (spatter, settled aerosol, or harvested air), the presence and type of at‐source aerosol reduction methods (high‐volume evacuators, low volume suction, or none), the methods of microbial sampling (petri dishes with solid media, filter paper discs, air harvesters, and liquid transport media) and assessment of microbial bioload (growth conditions, time of growth, specificity of microbial characterization) are barriers to drawing robust conclusions. for example, although several studies have reported the presence of microorganisms in aerosols generated by ultrasonic scalers and high‐speed turbines, the specific types of organisms or their source is not as well studied. this paucity of data does not allow for definitive conclusions to be drawn regarding saliva as a major source of airborne microorganisms during aerosol generating dental procedures. well‐controlled, large‐scale, multi center studies using atraumatic air harvesters, open‐ended methods for microbial characterization and integrated data modeling are urgently needed to characterize the microbial constituents of aerosols created during dental procedures and to estimate time and extent of spread of these infectious agents. bacterial pathogens; tuberculosis, and bacterial pneumonia, to name but a few. the proximity of the nasopharynx and lower respiratory tract to the oral cavity creates an open communication channel for movement of viruses and bacteria from these areas into the mouth. in this scenario, aerosol generating dental procedures on patients with infectious respiratory diseases become sources of contagion. in an immunocompetent individual, the risk of spread of infection by aerosolized particles is largely driven by the kinetics of the aerosol, presence of pathogen in the aerosol source, the type of pathogen, frequency of exposure, and the infectious dose. as dental professionals, it behooves us to protect ourselves, our patients and our staff from occupationally acquired diseases. the purpose of this review, therefore, is to examine what is currently known regarding the physics of aerosol creation, the types of aerosols generated by dental procedures, the nature, quantity, and sources of microbiota in these aerosols and the probability of disease transmission from patients to dental healthcare workers. in an attempt to establish context for reviewing the literature on dental aerosols, we begin this review by examining the reasons why definitions of aerosols vary widely. in general, aerosols refer to particles suspended in gas. although aerosols may be generated from a multitude of events, such as combustion, evaporation, industrial work etc., we will focus on aerosols generated in the healthcare environment. in 1934, wells pioneered the concept that airborne infections can be transmitted either as droplets or as aerosols. 1 according to his work, droplets are defined as those with particle sizes > 5 μm and typically carried on heavy colloids like mucus or saliva. droplets cannot remain suspended in air for long or travel long distances, hence, they are spread by close contact with (typically 1 m ) and in the presence of, the host. however, according to wells, droplets < 100 μm dry out before falling ≈2 m to the ground. when these droplets evaporate, they can be carried on airborne vectors and become aerosols. he estimated the particle size in aerosols to be < 5 μm (sometimes called droplet nuclei) and stated that these particles can stay airborne for long periods of time, carry viable pathogen as payload and settle on surfaces distant from the source (which is then referred to as a fomite). the vectors can be natural, namely, mist, fog, and vapor or anthropogenic, for example, smoke, dust, smog, and of particular importance to us, dental aerosol. however, in certain cases, for example, high ambient temperature or high airflow, large droplets can evaporate and acquire aerosol-like properties. because of their size, they can carry larger payloads than droplet nuclei (see below). aerosols have also been classified based on their deposition patterns. for example, using a semi-empirical model, the international commission on radiological protection (icrp) estimated that particles between 1to 10 μm or < 0.5 μm are most likely to deposit in the tracheobronchial and pulmonary regions of the lungs, whereas particles ≤5 μm have the highest probability of entering the lower airways of the average adult during oral inhalation. 2 because the nose offers a greater filtration efficiency than the mouth, only particles ≤3 μm have a high probability of entering the lower airways during nose breathing. particles with diameters between 1 and 3 μm or <0.5 μm have the greatest probability of entering the lung, thereby the highest potential of initiating an infection at this site. the infectious diseases society of america (idsa) has defined "respirable particles" as having a diameter of ≤10 μm and "inspirable particles" as having a diameter between 10 μm and 100 μm, nearly all of which are deposited in the upper airways. 3 other studies on infectious disease transmission indicate that droplets >5 μm are trapped in the upper respiratory tract whereas droplets ≤5 μm can be inhaled into the lower respiratory tract. 4 in this review, we will use the 10 μm diameter to distinguish between aerosolized and non-aerosol particles, because they have important implications for time of settling, penetration depth into airways and requirements for ppe. another important characteristic of aerosolized particles that impacts their definition is settling time. in still air, it has been estimated that particles 0.5 μm take 41 hours to settle over a distance of 5 feet, and that the time exponentially decreases as the size increases. for example, 1 μm sized particles take 12 hours to settle whereas 10 μm take 8.2 minutes and 100 μm take a mere 5.8 seconds. 5 however, this characteristic is heavily influenced by the direction and velocity of air currents (such as those created by foot traffic, opening of doors, position and setting of room air circulation systems etc.), humidity, the forces of attraction/repulsion between aerosolized particles and the size of the agglomerates/coaggregates (see below). in the presence of turbulence, particles nearer the floor continue to follow the settling times described above, but other factors begin to influence those that are two feet or more above the surface, for example, particle impaction, electrostatic forces etc. when vector particles and aerosol droplets collide with each other, they might coalesce or coaggregate, changing the particle size, in which case, the classifications described above do not apply anymore. in certain situations, these aggregates break down into numerous smaller conglomerates, generating a new generation of payload. together, these collisions randomly create a heterogeneous mixture of large and small particles with highly variable electrical charges, aerodynamic diameter, diffusion dynamics, and terminal velocity. 6 it is therefore unsurprising that, in real life scenarios, each aerosol responds in a highly variable manner to gravitational forces. temperature and humidity of the environment, and the superimposition of new aerosol further impact aerosol dynamics. 6 the characteristics and behavior of aerosolized particles are important determinants of defining an aerosol, and for this reason, definitions have to be contextualized. for example, size and penetrability-based definitions have important implications for selecting appropriate face masks, while settling-characteristicsbased definitions are impactful in deciding nature and time of surface decontamination. hence, studies on aerosol transmission must account for these confounding variables in order to be interpreted in the appropriate clinical context. as we shall see below, most studies on aerosol generating medical/dental procedures (agm/dp) have used simplistic calculations, for example, estimating particle size to compute aerodynamic diameter (this has limited use outside of regular sized particles such as inhalable drugs) and applying stokes' law to calculate terminal velocity of a particle in a fluid (the assumptions of stokes' law fail for particles <1 μm). 6 one of the most important considerations in any study is the investigational methodology. early studies employed impaction on solid and liquidized interfaces to measure aerosol volume and properties. 7, 8 advances in visualization technology have enabled greater temporal and spatial visualization of aerosol generated particles and their trajectories. among the various methodologies used to visualize aerosols, laser capture imaging, particle counters, air samplers, and droplet capture methods are the most popular. 9 similarly, methodologies for microbial characterization have demonstrated tremendous advances from the early days of culturing and microscopy to targeted methods such as polymerase chain reaction (pcr) to quantitative pcr to collectively sequencing entire microbial communities. 10-12 a third component is development of computational models of human behavior and predicting patterns and paths of spread. 13 although these advances in pathogen detection, airflow measurement, and disease modeling have had a major impact on understanding the spread of diseases such as ebola 14 and changed our perception of older diseases such as tuberculosis and measles, 15 several questions still remain to be addressed. for instance, although molecular microbiology has allowed us to identify infectious agents earlier and at much lower concentrations, it is not unclear if these doses are clinically relevant, how the relevance is modified by the type of populations (adult versus children, immunocompetent versus compromised, ambulatory versus hospitalized, and individual versus group living) and most importantly, how many of these organisms are viable. 16 similarly, the very act of air-sampling can generate an aerosol as well as destroying the organisms being captured. 17 importantly, computer machine learning relies on large and granular datasets for accuracy, and when studies from the field are unable to capture all the required components, the model is not reflective of real-life scenarios. thus, any investigation of aerosol characteristics must use well-validated methods of aerosol capture, incorporate appropriate positive and negative controls to allow standardization of microbial payload, and be sufficiently powered to reduce the ''noise'' generated by random behavior of aerosol particles. most importantly, they must be quantitative, because pathogen dose is an important element of infectivity. as we will see in the next few sections, much of what we currently know about dental aerosols falls far short of the most basic principles of scientific rigor and reproducibility. until recently, the viral constituents of the oral microbiome had only been examined in the context of their ability to cause disease and spread contagion. we now know that viruses are normal inhabitants of the healthy oral microbiome, 18, 19 and that a diverse population of both dna and rna viruses is found in saliva and subgingival plaque of healthy individuals. 20 the most common oral viruses are cytomegalovirus, herpesvirus one through nine and papilloma virus. 21 the types of viruses that inhabit an individual are highly subject-specific, much more so than the types of bacteria. 22 the oral virome also demonstrates significant gender-specificity. 19 the type of viral exposure an individual has had, and the nature of the shared living environment are two major determinants of individual viral signatures. 23 it is also established that the majority of viral particles are derived from gram-positive and gram-negative bacteriophages rather than free-living viruses. 18 once acquired, these viruses demonstrate remarkable colonization stability in the absence of extraneous influences such as local or systemic disease. 22 studies exploring the role of saliva as a diagnostic tool for viral diseases such as dengue, west nile, sars, chikungunya, mers-cov, ebola, zika, and yellow fever have further expanded our knowledge of non-oral viruses. 24 most of these investigations have reported that whereas viral rna and viable virus were detected in saliva early in the course of disease, viral shedding did not persist after resolution of symptoms. 25, 26 however, influenza a and b were detected in 20 to 60% of asymptomatic individuals. 27 taken together, these studies suggest that (a) the oral viral community is acquired through a non-random process of microbial assembly that is partly dictated by individual genotype (b) viral communities are temporally stable once acquired and (c) exogenous viruses are present in saliva during acute phase infection, but most do not persist following resolution of disease. like viruses, respiratory bacterial pathogens have been detected in saliva during acute and symptomatic phases of respiratory illnesses, 28, 29 as well as in institutionalized and hospitalized, elderly individuals. 30, 31 however, unlike viruses, certain bacterial respiratory pathogens have been identified in the oral cavities of systemically healthy and asymptomatic individuals, especially smokers. 32, 33 for instance, bacteria such as streptococcus pneumoniae can be isolated more frequently and consistently from saliva than from naso-pharyngeal or oro-pharyngeal swabs. 34 these pathogens are known to reside in the subgingival crevice, the buccal mucosa and saliva. 28, [35] [36] [37] [38] [39] however, exogenous pathogens are not dominant members of the oral microbiome, which is one of the most diverse in the human body with over 20 billion microbial cells. 40 moreover, in states of health, a robust interbacterial interaction limits or reduces colonization with exogenous pathogens. for instance, bacteriocins such as ls1 (produced by the oral commensal lactobacillus salivarius) contribute to controlling the growth of s. aureus and s. pneumoniae, 41, 42 and hydrogen peroxide (which is produced by several commensal species) prevents colonization by serratia marcescens, s. agalactiae, s. pneumoniae, haemophilus influenzae and mrsa. 43, 44 in summary, a large body of evidence supports saliva as a potential source of respiratory pathogens, however, many of these studies lack quantitative data. therefore, there is an urgent need for studies that quantify the salivary bioload of these species in non-infected individuals and for investigations on whether these microbial loads are high enough to create a biologically relevant infectious dose. although agm/dp have been implicated in spread of viral contagion, it must be remembered that aerosols are generated during normal physiological activities such as breathing, talking, coughing, and sneezing. studies on healthy volunteers have demonstrated that mouth breathing produces 1-98 particles per liter, 45 with a median diameter of 0.3 μm; with only about 2% of the particles >1μm and none > 5 μm. 46, 47 during speaking, 1 to 50 particles in the 1 μm range are emitted per second (0.06 to 3 particles per liter) 48 ; with some ''super-seeders'' expelling as many at 200 particles per second while speaking loudly. singing creates six times as many droplet nuclei as talking and is equivalent to coughing. 49 sneezing can expel nearly 40,000 droplets between 0.5 to 12 μm at speeds of almost 100 m/sec, while coughing may generate up to 3000 droplet nuclei. 50, 51 collectively, studies such as these demonstrate that healthy individuals generate particles sized between 0.01 and 500 μm, underlining the fact that dispersal of expelled particles does not occur exclusively by airborne or droplet transmission but by both mechanisms concurrently. although healthy and diseased individuals generate aerosols during normal activities, evidence that these aerosols contain an infectious agent is equivocal. for example, streptococcus pneumoniae, staphylococcus aureus, methicillin-resistant staphylococcus aureus (mrsa), escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter baumannii, stenotrophomonas maltophilia, haemophilus influenzae, legionella pneumophila, mycoplasma pneumonia, chlamydia pneumonia, and mycobacterium tuberculosis can be detected in 36% of patients with symptomatic respiratory diseases. 10 however, although 89% of nasal swabs were positive for live virus in 142 patients diagnosed with influenza a, only 39% of individuals exhaled live viral particles in their breath, 52 and the number of particles shed declined significantly within 3 days of onset of symptoms. 12,52 importantly, these particles failed to land on targets placed at a distance of 0.1 and 0.5 m. 53 furthermore, when a patient wore a surgical mask, it reduced the viral shedding in aerosol by 3.4 fold. 54 on the other hand, p. aeruginosa can travel 4 m and persist in the aerosol for 45 minutes subsequent to a coughing episode. 11 wearing surgical masks for 10 to 40 minutes reduced the levels of respiratory pathogens by more than four-fold. 55 collectively, there is a large body of evidence that patients in the acute phase of respiratory infections are capable of disseminating large numbers of airborne microorganisms during activities such as breathing, talking, singing, coughing, and sneezing. this shedding can be mitigated by the simple act of wearing a mask and is effective against viral as well as bacterial pathogens. the sars-1, 2009 h1n1 mers, ebola and zika outbreaks were instrumental in drawing attention to medical aerosols as sources of infection to health-care personnel. two broad categories of agmp have been documented in the literature: those that induce the patient to express the contents of the lower respiratory tract by stimulating cough reflex (sputum induction), and those that mechanically disrupt the contents of the respiratory tract. the latter procedures typically include intubation/extubation, cardiopulmonary resuscitation, bronchoscopy, noninvasive ventilation, tracheotomy, airway suctioning, manual ventilation, and administering oxygen or nebulized medication. 56 all these procedures are conducted on patients who are typically experiencing active disease, and therefore, the aerosols and droplets generated from sites with active pathogen colonization could potentially contain high numbers of respiratory pathogens. however, even though magp have been the subject of at least 400 different studies, questions still remain regarding the amount of aerosols generated, the size and concentration of medically aerosolized particles, and whether such aerosols could transmit viable pathogens to hcp or to other patients. for instance, the review by davies et al. 56 and by o'neil et al. 57 suggests that although the potential for aerosol production exists with agmp, there is little evidence that these procedures actually do create aerosols. during dental procedures, the "wet environment" created by saliva and water coolant combined with high-speed instrumentation generates a large spray which disperses in many forms as dictated by the physics of aerosol creation (see section on characteristics of aerosols, above). thus, the spray can initially take the form of spatter, droplets, droplet-nuclei, a true aerosol, or some combination thereof; and continue to evolve based on room temperature, humidity, airflow dynamics, electrostatic forces etc. the term "dental aerosol", therefore, is somewhat of a misnomer, because it does not encompass the various airborne particles that can be created during an agdp. to avoid confusion, we will use the word spray unless the study specifically measured aerosols. there are four main sources of dental sprays: air-water syringes, ultrasonic instruments, high-speed turbines, and lasers. there is no literature on sprays from air-water syringes, so we will examine the evidence from the rest of instruments below. the quantity of sprays, spatter, or aerosol generated by ultrasonics, the distance travelled by the aerosolized particles and their composition have been studied using air samplers, 58-60 bacterial growth medium placed at strategic locations, 58-67 filter paper strips (with and without dye) on the patient and operator, 68, 69 and heme-detectors. 70 sprays are generated during all types of procedures using ultrasonic instruments, whether it be supragingival scaling, subgingival scaling of periodontally diseased teeth or endodontic instrumentation. the amount of spatter and aerosol generated by sonic, ultrasonic or piezoelectric devices and distance travelled by airborne particles from these devices is similar or comparable. 58, 64, 71, 72 these sprays expose the inhabitants of the operatory to 1.86 × 10 5 particles per cubic meter of space, and the contaminants settle to a great extent on the dominant arm of the operator, and eyewear and chest of the patient and to a lesser extent on the non-dominant arm and chest of the operator and assistant. 66, 68, 69, 73 they can also be detected as far away as 2 to 11 m from the treatment site. 59, 66 however, in the absence of a coolant, the aerosol is limited to an 18 inch radius. 72 the levels of aerosolized particles return to preoperative levels within 30 minutes 68 to 2 hours. 60 in summary, there is unequivocal evidence that some of the spray from all types of ultrasonic devices is converted to aerosol, and while the spatter settles on the person of the operator, assistant and patient, the aerosolized particles can travel much larger distances and settle up to 2 hours after creation. high speed handpieces can generate spatter containing blood and other components, 59, 61, 63, 74, 75 and the amount of microbial bioload varies with the tooth being treated. 74 as well as the caries level of the patient. 65 it has been reported that microbial fallout from restorative procedures can extend up to 1.5 to 2 m, however, this study did not report the type of evacuators that were used during the procedures. 76 when a laser is used to cauterize blood vessels and incise tissue by vaporization, it generates a gaseous material known as surgical smoke plume, which is composed of 95% water. the remaining 5% has been reported to contain blood, particulate and microbial matter. 77 the particle size generated by lasers ranges from 0.1 to 2 μm. all class iv lasers (surgical lasers) carry the risk of plume hazard. although there is no evidence on lasers used in dental operatories, escherichia coli, staphylococcus aureus, human papillomavirus, human immunodeficiency virus, and hepatitis b virus have been detected in surgical laser plumes used in dermatology and otolaryngology. 78 although every single study to date has demonstrated that all forms of mechanical instrumentation in the oral cavity create aerosols and spatter with a significant bioload, critical gaps in knowledge still exist. the first of these is the source of the aerosolized microbiota. it is easy to point to saliva as a source. if this were indeed true, then one would expect a high degree of variability in the clinical studies because of differences in salivary volume, flow rate and composition between patients. however, all the literature detailed in this review report remarkably homogeneous findings in terms of aerosol volume, quantity of contagion, and distance and time of spread. this is in spite of variability in operators, instruments, procedures, subject characteristics, and data collection methods. moreover, if saliva were the source of microbiota in dental aerosols, one would expect a certain level of microbial heterogeneity between the studies. however, the bacteria most frequently identified in all studies were staphylococcus aureus, beta hemolytic streptococci, escherichia coli, spore-forming bacteria, fungi belonging to the genera cladosporium and penicillium, and micrococccus 66, [79] [80] [81] 82 another study documented the presence of high levels (10 5 cfus) of legionella, pseudomonas and non-tuerculous mycobacteria in water lines. 83 thus, there is plausible evidence to suggest that water might contribute to a large fraction of the microbial payload in dental aerosols. this plausibility is further supported by the fact that ultrasonic devices and high-speed handpieces use water as a coolant with a typical flow rate of 10 to 40 ml per minute, 84 whereas the flow rate of saliva during the same time period is 0.4-0.5 ml. 85 thus, the dilution ratio varies between 1:20 to 1:100. that is not to say that saliva does not contribute to the microbial payload in aerosols. in fact, a strong correlation was observed between the number of decayed teeth in a patient and the levels of beta hemolytic streptococci on the operator's mask, 65 and reductions in aerobic and anaerobic colony forming units (cfus) have been reported following pre-procedural mouth rinsing. 65, [86] [87] [88] however, as described above, most the culturable bacteria identified thus far in dental aerosols are of environmental origin, bacterial profiles in aerosols demonstrate remarkably low ''noise'' between studies and the dilution factor because of water coolants is very high. in the absence of evidence demonstrating a salivary source for these bacteria, the microbial similarities between water lines and aerosols is the only evidence that can be brought to bear upon this argument. although the amount to effort invested in studying dental aerosols is commendable, these studies suffer from critical flaws in design and methodology that preclude robust decision making. 8 for instance, none of the studies used a control group where the aerosol was generated in the absence of a patient. this would provide invaluable information on the source of the microbial payload. there is also incredible diversity in the methodologies used. for instance, several studies originating from the indian subcontinent and south east asia have not used any form of aspiration of oral fluids, whereas most studies from europe and the united states have used high or low volume aspirators. because the amount of aerosol directly correlates with the partial pressure of fluid in the mouth, this important variable does not allow for comparisons to be made between studies. perhaps the most important gap in knowledge stems from the use of rudimentary cultivation-based approaches to characterize microbiota. such approaches have created very simplistic views of the microbial contaminants (e.g. gram positive versus gram negative, gross counts of cfu, catalase activity, and other such basic characterizations), have hampered our ability to pinpoint the source of the aerosol and completely ignored the viral, fungal and other constituents of the microbial payload. hence, these studies have allowed room for liberal interpretation of the data, and in some instances, this has served to create a certain level of misinformation. before we examine the statistics on cross-infection in dental settings, it must be acknowledged that lack of reporting poses a huge barrier to obtaining accurate data. an excellent review by volgenant et al. 89 examines the several potential routes of transmitting infections in the dental office. these include blood-borne, contact, and aerosol transmission. several instances of transmission of blood-borne pathogens to patients and health-care personnel have been documented. these are attributable both to poor infection control practices, 90 as well as to blood exposure accidents. 91 however, the risk appears to be very low, with only five cases reported between 2003 and 2016. 92 aerosol-transmitted diseases have been documented, although dental unit water lines appear to be the microbial source. 93, 94 especially, legionellosis has been connected to dental treatment in two case reports. 95, 96 moreover, dentists in certain areas have been shown to have higher antibody levels to legionella when compared to nondental professionals, 97 adding further credence to dental unit water lines as the source of aerosol microorganisms. a careful and contextualized review of the currently available evidence on dental aerosols reveals the following: 1. viral shedding occurs in saliva during acute phases of all respiratory diseases, and influenza viruses have been reported in post-recovery and asymptomatic patients. 2. respiratory bacterial pathogens are present in saliva of asymptomatic individuals; however, their relative abundances are very low. 3. aerosols are generated by all individuals during all times of the day during all types of activities. 4. the microbial payload in physiological aerosols correlates with disease severity for respiratory diseases. 5. aerosols are created during most dental procedures. the four main aerosol emitting devices are ultrasonics, handpieces, air-water syringes, and lasers. 6. there is little evidence to definitively implicate saliva as the primary source of bacteria in these aerosols. although absence of evidence is not evidence of absence, the available evidence currently points to environmental sources, particularly dental unit water lines, as a major basis of aerosol bacteria in the dental environment. large-scale, multi center studies using atraumatic air harvesters and integrated data modeling that is superimposed on a geographic map of the physical space have enabled the medical community to identify patterns of aerosol spread, model disease transmission, and create human and instrument flow paths to reduce risk of infection. 98 similar studies to determine the creation and spread of aerosols during dental procedures and to estimate time and extent of spread are urgently needed. purnima s kumar and kumar subramanian contributed equally to the literature review, writing and reviewing the manuscript. the authors have no conflicts of interest or financial relationships impacting this manuscript. o r c i d purnima s kumar https://orcid.org/0000-0001-5844-1341 international commission on radiological protection. human respiratory tract model for radiological protection preventing transmission of pandemic influenza and other viral respiratory diseases: personal protective equipment for healthcare personnel: update natural 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an air cleaning system during dental procedures the usefulness of the modified extra-oral vacuum aspirator (eova) from household vacuum cleaner in reducing bacteria in dental aerosols do exposures to aerosols pose a risk to dental professionals? bacterial contamination of dental unit waterlines bacterial contamination of dental unit waterlines: what is your dentist spraying into your mouth thermal imaging of ultrasonic scaler tips during tooth instrumentation saliva between normal and pathological. important factors in determining systemic and oral health efficacy of preprocedural mouth rinsing in reducing aerosol contamination produced by ultrasonic scaler: a pilot study reduction of bacteria-containing spray produced during ultrasonic scaling compare the effcacy of two commercially available mouthrinses in reducing viable bacterial count in dental aerosol produced during ultrasonic scaling when used as a preprocedural rinse cross-transmission in the dental office: does this make you ill. curr oral health rep confirmed transmission of hepatitis c in an oral surgery office the prevalence and underreporting of needlestick injuries among dental healthcare workers in pakistan: a systematic review transmission of blood-borne pathogens in us dental health care settings: 2016 update epidemiology of pseudomonas aeruginosa in cystic fibrosis and the possible role of contamination by dental equipment notes from the field: mycobacterium abscessus infections among patients of a pediatric dentistry practice-georgia legionellosis acquired through a dental unit: a case study pneumonia associated with a dental unit waterline occupational risk for legionella infection among dental healthcare workers: meta-analysis in occupational epidemiology nosocomial transmission of emerging viruses via aerosol-generating medical procedures key: cord-010910-hz2vyw6c authors: huang, xinyue; cavalcante, danielle paixão; townley, helen e title: macrophage-like thp-1 cells show effective uptake of silica nanoparticles carrying inactivated diphtheria toxoid for vaccination date: 2020-01-08 journal: j nanopart res doi: 10.1007/s11051-019-4720-1 sha: doc_id: 10910 cord_uid: hz2vyw6c nanoparticles may be used in vaccinology as an antigen delivery and/or an immunostimulant to enhance immunity. porous silica has been identified as an effective adjuvant for more than a decade, and we have therefore investigated the take up rate by an immortalized macrophage-like cell line of a number of mesoporous silica nanoparticles (msnps) with differing diameter and pore size. the msnps were synthesized using a sol-gel reaction and post-synthesis removal of the template. the msnps showed a clear distribution in take up rate peaking at 217 nm, whereas a comparison with solid spherical nanoparticles showed a similar distribution peaking at 377 nm. the msnps were investigated before and after loading with antigen. diphtheria toxoid was used as a proof-of-concept antigen and showed a peak macrophage internalization of 53.42% for loaded lp3 particles which had a diameter of 217.75 ± 5.44 nm and large 16.5 nm pores. optimal msnp sizes appeared to be in the 200–400 nm range, and larger pores showed better antigen loading. the mesoporous silica particles were shown to be generally biocompatible, and cell viability was not altered by the loading of particles with or without antigen. [figure: see text] nanovaccinology involves the use of nanometre sized particles as a means to deliver antigen, in order to enhance antigen processing. a variety of engineered nanoparticles have been developed for vaccine development and while they differ in size, shape and composition, they are typically 25-200 nm in diameter (zhao et al. 2014) . the large surface area to volume ratio of nanoparticles means that the particles may carry a higher proportion of antigen for immune activation. the size of the nanoparticle systems also increases the likelihood that they will accumulate in lymph nodes. this can both increase their effective concentration and reduce systemic reactivity (nuhn et al. 2016) . our previous work compared the antigen loading and unloading capacity of mesoporous silica nanoparticles (msnps), with a variety of different pore sizes, and external diameters (huang et al. submitted) . herein, we investigate the ability of the different particles to be internalized by antigen-presenting cells. the uptake of nanoparticles, and subsequent immune response, may be due to their intrinsic resemblance to natural viruses (chattopadhyay et al. 2017) . since the immune system is primed to recognize viruses, it is not surprising that nanoparticles which can be similar in terms of size, geometry and antigen display could be effective. soluble antigens are well known to have low efficacy in inducing good protective immunity. this is largely due to the insufficient uptake of the antigen by antigen-presenting cells (apcs). attaching soluble vaccine antigens to larger carriers can facilitate recognition and uptake by the apcs (pobre et al. 2014) . materials used for the production of nanoparticles for antigen delivery include lipids, polymers and inorganic compounds. for our study, we have chosen to investigate the use of silica nanoparticles as a carrier for antigen molecules due to a number of favourable properties: silica nanoparticles can be synthesized in a range of sizes and shapes and can be controlled using sol-gel chemistry and is a particularly useful material to work with since nanoscale pores can be easily integrated to generate msnps which have a very large surface area for carrying antigen. in addition to the size of the particles, charge is also an important parameter. some studies have shown that cationic nanoparticles show higher uptake due to the interaction with anionic cell membranes (foged et al. 2005) . however, cationic particles are more likely to induce haemolysis and platelet aggregation than neutral or anionic particles (mccusker et al. 2004 ). the negatively charged silanol groups found on the surface of silica are also beneficial if further modification with additional functional molecules such as those for cell recognition is needed. the attachment of a vaccine antigen to a nanoparticle may be via chemical conjugation or physical adsorption. in this study, we have used physical adsorption which is simply based on the charge or hydrophobic interaction of the antigen and the nanoparticle material. conjugation could potentially result in modification of the antigen as a result of the binding to the nanoparticle surface, potentially leading to a less immunogenic antigen. in addition, silica has been shown to be biocompatible, and the pore size can be carefully controlled to determine the optimal size to adsorb and deliver antigen. while the silica itself can be used as an adjuvant and is capable of inducing strong humoral and cellular immune responses against the relevant antigens, others can also be added as cargo (mahony et al. 2013) . to date, silica has been investigated in formulations for vaccine applications against e. coli, porcine circovirus and hiv (guo et al. 2012; mercuri et al. 2006; cheng et al. 2012) . furthermore, in contrast to other nanoparticles such as gold, silica is also easily degraded (huang et al. 2014) in the body and can then be excreted in the urine. irrespective of the material chosen, to be effective as a vaccine delivery system, it is essential that the microor nanoparticles are internalized by antigen-presenting cells (alshanqiti et al. 2017) . we have therefore investigated the uptake of our library of silica nanoparticles using the human cell line thp-1 as a model system. thp-1 is an immortalized monocyte-like cell line, derived from the peripheral blood of a childhood case of monocytic leukaemia (m5 subtype) (tsuchiya et al. 1980) . these cells can be differentiated into macrophage-like cells using phorbol-12-myristate-13acetate (pma) which activates protein kinase c (pkc). the resulting cells resemble mature macrophages, with increased adherence and loss of proliferative activity (schwende et al. 1996) . the cells can be confirmed to be macrophage-like by the expression of cd-14, a 53-55 kda glycosylphosphatidylinositol (gpi)-linked membrane glycoprotein. as a proof-of-concept, we have loaded the nanoparticles with the inactivated diphtheria toxoid (dt). dt is secreted as a single polypeptide of 58 kda by pathogenic strains of corynebacterium diphtheria. subsequently, the 535 amino acid polypeptide is proteolyzed to two polypeptide chains linked by a disulphide bond. the dt is well characterized and has been used since the 1920s when the first methods to inactivate the toxoids were introduced (ramon 1924 ; glenny and hopkins 1923) . this study therefore investigates the uptake rates of macrophages of loaded and unloaded msnps, with different external diameters and pore sizes. thp-1 cell culture thp-1 cells were donated by dr. paul klenerman (nuffield department of medicine, university of oxford). cells were maintained in rpmi 1640 media supplemented (life technologies) with 10% fetal calf serum (fcs; sigma-aldrich), 2 mm l-glutamine (sigma-aldrich), 100 u/ml penicillin (sigma-aldrich), 0.1 mg/ ml streptomycin (sigma-aldrich) and 0.05 mm 2mercaptoethanol (sigma-aldrich). cells were incubated at 37°c in a 5% co 2 atmosphere. cells were passaged every 4-5 days and discarded after 30 passages. thp-1 cell differentiation thp-1 cells (from 5th generation to 30th) were resuspended in rpmi media at a concentration of 2 × 10 5 cells/ml and treated with 100 ng/ ml phorbol 12myristate 13-acetate (pma; sigma). cells were then plated in a 24-well plate (1 ml per well) and incubated for 72 h at 37°c, 5% co 2 . the pma-containing media was then removed and replaced with fresh rpmi media for 24 h before further experiment. the cd-14 marker on pma-differentiated and undifferentiated cells were compared by flow cytometry since cd-14 is considered a surface marker of macrophages. thp-1 cells were collected and washed with pbs. the fc receptor was blocked by resuspending cells at 1 × 10 4 cells/ ml in 5 μg/ ml igg (sigma) in pbs at a total volume of 100 μl and incubating on ice for 20 min. subsequently, 5 μl of apc-cd-14 primary antibody (biolegend) was added and incubated on ice for a further 20 min. cells were collected by centrifugation and the supernatant removed. cells were washed in 200 μl pbs and then centrifuged and resuspended in 200 μl pbs. controls were also prepared, including undifferentiated thp-1 cells incubated with only igg and undifferentiated cells incubated with both igg and anti-cd14 antibodies. the differentiated thp-1 cells (2 × 10 5 cell/well) were treated with 10 μg/ml fluorescein isothiocyanate (fitc)-labelled msnps for 2 h at 37°c. after the treatment, the cells were resuspended and fixed in 100 μl cold 2% paraformaldehyde for 10 min on ice. the phagocytosis efficacy was evaluated using facs. the negative control was performed on low temperature (4°c) while the other parameters of msnp treatment remain the same. the net phagocytosis efficacy was evaluated by subtracting the result of the negative control for the experiment. fluorescence activated cell sorting fluorescence activated cell sorting (facs) was run on a facscalibur™. all data was collected using cellquest™ pro (bd biosciences). the data was analysed using kaluza™ 1.2. cells were incubated with nanoparticles for 2 h. the particle suspension was then removed and replaced with 100 μl of 0.5 mg/ml mtt (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; life technologies) solution in culture medium. the plate was wrapped in foil and the cells were incubated for a further 2 h, at 37°c. an aliquot of the media (75 μl) was removed and 150 μl of dimethyl sulfoxide (dmso; sigma) was added to the plate. the absorbance was read at 590 nm after solubilization of the precipitate. synthesis of snp01, 02, 03 absolute ethanol (48 ml; fisher scientific) was mixed with 3 ml deionized water and 3 ml ammonium hydroxide (28-30%; sigma) in a flask and placed on a heated stirrer. the temperature was either set to (i) 75°c for snp01, (ii) 48°c for snp02 or (iii) 52°c for snp03. once the temperature was stable, 3 ml tetraethyl orthosilicate (teos; sigma-aldrich) was added. the mixture was then stirred at the same temperature overnight. the particles were subsequently collected by centrifugation and washed three times in ethanol. the particles were then dried in a desiccator and ground using a mortar and pestle. absolute ethanol (30 ml) was mixed with 3 ml of 0.25 mg/ml potassium chloride solution. ammonium hydroxide (28-30%) was added at either (i) 1.5 ml for snp04 or (ii) 3 ml for snp05, and the mixture placed on a heated stirrer at 35°c. in a separate tube, 3.5 ml teos was mixed with (i) 21 ml ethanol for snp04 or (ii) 10.5 ml ethanol for snp05 and vortexed (mix 2). once the temperature had stabilized, either (i) 21 ml for snp04 or (ii) 12 ml for snp05, of mix 2 was added to the first flask. the final mixture was incubated at 35°c for either 4 h (snp04) or overnight (snp05). the particles were subsequently collected by centrifugation and washed three times in ethanol. the particles were then dried in a desiccator and ground using a mortar and pestle. seven different mesoporous silica nanoparticles (msnps) were synthesized with varying external diameter and porosity. the first four had small pores (sp) and were designated sp1, sp2, sp3 and sp4. the latter three had much larger pores (lp) and were named lp1, lp2 and lp3. santa barbara amorphous particles (sba-15) were synthesized by cristália produtos químicos farmacêuticos ltda, brazil. these were included as a comparison since they have been used previously in adjuvant studies in the literature. particles were made after the method of zukal et al. (2007) . in a typical synthesis process, 300 mg of 1methyl-3-octylimidazolium chloride (omimcl), 100 mg of cetyl trimethylammonium bromide (ctab) and 400 mg of sodium silicate (na 2 sio 3 ) were dissolved in 90 ml of ddh 2 o in a 250 ml round-bottom glass flask at room temperature. after the solution cleared by stirring for at least 1 h, 0.5 ml of ethyl acetate was quickly added whilst stirring. after 1 min, the stirring was stopped, and within 10 min at room temperature, a precipitate began to form. the mixture was allowed to stand at 35°c for a further 5 h. the suspension was then heated to 95°c, and the mixture stirred slowly for 48 h. during the ageing process, organic vapour was allowed to escape. the resulting solid phase was recovered by filtration, washed three times with 30 ml ddh 2 o, and then washed a further three times with 30 ml meoh. the template was removed as below. to remove the surfactant, the particles were resuspended in acidic methanol (40 ml methanol; 2 ml 37% hydrochloric acid). the system was then refluxed at 80°c for 24 h. after reflux, the suspension was allowed to cool down to room temperature, and particles collected and washed as in "thp-1 cell culture", with the exception that ethanol was used for washing in place of methanol. the particles were dried in a desiccator under high vacuum for at least 24 h at room temperature. subsequently, the particles were ground to a fine powder using a mortar and pestle. particles were made after the method described in huang et al. (2014) . in a typical synthesis process, 200 mg (ctab) was dissolved in 96 ml ddh 2 o, and 700 μl of 2 m sodium hydroxide (naoh) solution was added, in a 250-ml round-bottom glass flask. the mixture was then stirred and heated to 80°c. once the temperature was attained, 1 ml tetraethyl orthosilicate (teos) was added, and the reaction kept at 80°c for 2 h. the mixture was then allowed to cool to room temperature and the particles collected by centrifugation for 3 min at 12,000 rpm for 3 min. the particles were then resuspended in 30 ml meoh and centrifuged. this washing step was repeated three times. particles were made after the method of he et al. (2009) . in a typical process, 365 mg ctab was dissolved in 99.5 ml buffer solution (comprising 25 mm kh 2 po 4; 14.5 mm naoh; ph 7) and 0.5 ml glycerol under vigorous stirring at 95°c. when the solution became homogeneous, 1.78 ml teos was added slowly into the system. the reaction was maintained at 95°c for 8 h, washed three times with 30 ml ddh 2 o, and then washed a further three times with 30 ml meoh. the template was removed as described above. particles were made after the method of du and he 2010. in a typical synthesis process, 800 mg ctab was dissolved in a mixture of 100 ml ddh 2 o, 0.8 ml nh 4 oh, 20 ml ether and 20 ml ethanol, with constant stirring in a 250-ml round-bottom glass flask at room temperature. after a clear solution was obtained, 2.5 ml teos was added to the solution. after vigorous stirring for 4 h at room temperature, a white precipitate was obtained. the particles were collected by centrifugation and resuspended in 30 ml methanol and centrifuged again. this washing step was repeated twice more. the template was removed as described above. in a typical synthesis, 1 g ctab was added to 15 ml 2 m urea solution and stirred vigorously at room temperature. to the mixture, cyclohexane (25.6 ml) and butan-1-ol (1.234 ml) were added and the system heated at 40°c for at least 1 h before proceeding. teos (2.68 ml) was then added dropwise and stirring continued for 30 min before increasing the heat to 70°c for another 24 h. the system was then cooled to room temperature, and 100 ml ethanol added. the particles were then collected by centrifugation at 12,000 rpm for 5 min. the particles were then resuspended in 30 ml acetone and centrifuged again. this wash step was repeated once, and then repeated twice using water, and then repeated twice using ethanol to wash. the template was removed as described above. in a typical synthesis, 500 mg cetylpyridinium bromide (cpb) was dissolved in 15 ml urea (20 mg/ml) and stirred vigorously at room temperature. cyclohexane (15 ml) was added while stirring, followed by 0.55 ml butan-1-ol. the solution was stirred for 3 h. subsequently, 1.34 ml of tetraethyl orthosilicate (teos) was added dropwise into the system and stirring continued for another 30 min at room temperature. the reaction mix was then maintained at 70°c for a further 16 h. after removal from the heat, 100 ml ethanol was added. the nanoparticles were then collected by centrifugation at 12,000 rpm for 5 min. the particles were then washed with acetone and re-centrifuged, followed by resuspension in water and centrifugation. the template was removed as described above. the protocol was as for lp2 with the exception that butan-1-ol was replaced with 0.65 ml pentanol. absolute ethanol (48 ml) was added to deionized water (3 ml) and ammonium hydroxide (3 ml; 28% to 30%) and the mixture heated to 55°c. teos (3 ml) was added to the system, and the mixture was stirred at 55°c for 16 h. the particles were collected by centrifugation and washed three times with ethanol. the particles were dried in a desiccator and ground using a mortar and pestle. fitc labelling of particles fitc-aptes was prepared in an anaerobic chamber by the addition of 100 μl of 3-aminopropyl triethoxysilane (aptes; sigma) to 25 mg fluorescein isothiocyanate (fitc; sigma) in 5 ml dry ethanol. the mixture was covered to protect from light and stirred overnight at room temperature. the resulting fitc-aptes was stored at 4°c. for labelling, silica nanoparticles were resuspended at 25 mg/ml in ethanol. the suspension was sonicated using a probe sonicator for 2 min, and then 20 μl of fitc-aptes was added to every millilitre of nanoparticle suspension and stirred overnight at room temperature. the labelled nanoparticles were collected by centrifugation, and then washed three times with ethanol. the particles were dried in a desiccator with care to avoid exposure to light. the surface charge of the particles was measured using a zetasizer nano zs (malvern, uk). to determine the electrokinetic potential, or ζ potential, the particles were suspended in distilled water (ph 7) prior to measurement. the suspension was used to fill a dts1070 disposable capillary cell. after 120 s equilibrium time, 30 runs were read before the calculation of zeta potential. the hydrodynamic particle size distributions were determined using a disc centrifuge (dc24000; cps instrument). prior to measurements, a sucrose gradient was built and pvc particle calibration standards were applied (266 nm; pvc000266, analytic ltd.). the diphtheria toxoid was kindly provided by fundação butantan to cristália produtos químicos farmacêuticos ltda. silica nanoparticles were resuspended in pbs at a concentration of 5 mg/ml. the msnp stock suspension was sonicated using a vibracell vc500 sonicator for 1 min (5 s on/ 5 s off). inactivated diphtheria toxoid (dt) added to a final concentration of 2 mg/ml, and mixed well. loading was allowed to proceed for 24 h at 4°c. all data is presented as the mean ± standard deviation, and where appropriate, the student t test was used to determine statistical significance (*p < 0.05, **p < 0.01, ***p < 0.005). to assess the uptake of the different silica nanoparticles, and to determine whether this was affected by size, porosity or loading, we tested the ability of the particles to be taken up into macrophage-like cells. in all phagocytosis assays performed, thp-1 cells were used and differentiated using 100 ng/ml pma for 72 h. the light scatter plot ( fig. 1; panel (i)) shows the changes in the populations of cells before and after treatment with pma to differentiate the cells. the population of cells in the control and after treatment with pma for 72 h shows homogeneous cell populations, whereas treatment for 48 h is an intermediate state with two populations of cells. to confirm the differentiation of the thp-1 cells, cd-14 expression (a surface marker normally overexpressed in macrophages) was evaluated. an apc-labelled cd-14 antibody shows emission in the 660 nm (red) region and was used to assess the expression of the glycoprotein ( fig. 1; panel (ii) ). the median fluorescence intensity increases from 3.95 (control) to 14.73 and 20.98 after incubation with pma for 48 and 72 h, respectively. therefore, pma can be seen to have successfully increased the expression of cd-14 and by implication resulted in the differentiation of the cells into macrophages. as further confirmation, the cells were examined microscopically ( fig. 1; panel (iii) ). the morphology of the cells can be seen to change over the period of incubation with pma and to give the appearance of macrophages after 72 h. solid, spherical nanoparticles were synthesized in a range from 113.25 ± 7.34 to 847.25 ± 27.54 nm in diameter (table 1 ). the nanoparticles were imaged by sem to confirm morphology and the diameter assessed using cps disc centrifuge. multiple batches were prepared for each to ensure that the methodology was robust and repeatable. the solid spherical particles were then tested for their ability to be internalized by macrophages. however, when using fluorescence as a marker on the particles, it is important to be able to differentiate between cell surface-associated particles and those which have been genuinely internalized. to control for this factor, all experiments were performed at room temperature, and then replicated at low temperature (johnstone et al. 2001; gottstein et al. 2013) . active transport of the nanoparticles into cells ceases under low temperature conditions, and therefore, any fluorescence seen would be due to surface attachment and so was subtracted. it can be seen that uptake increases from snp01 to snp03, i.e. in the order 113.25 nm, 275.78 nm and 377.63 nm (fig. 2a) . subsequently, there is no significant increase in the amount of 588.00 nm internalized into the macrophages. once the size reaches 847.25 nm, there is a significant decrease in the amount internalized compared to the peak uptake for 377.63 nm particles. this implies that there may be an optimal size for macrophage uptake of solid spherical silica nanoparticles peaking in the region of 377 nm. although the individual sba-15 is not as large as snp05, the particles are rod-shaped and often severely aggregated, and therefore may not be taken up as readily. while it is useful to look at the different uptakes of solid particles due to their identical morphology, they are unable to carry cargo. we therefore investigated the uptake of a series of mesoporous silica particles (table 2 ). these particles have been previously characterized in terms of their capacity to carry antigen (huang et al. submitted as part 1 of 2). the size of the pores and the zeta potential is also reported in huang et al. submitted as part 1 of 2. it can clearly be seen that a similar pattern of uptake is seen with the msnps with respect to uptake and their external diameter. there is an increase in uptake from sp2 to lp2, i.e. 138 nm, 202.66 nm and 217.20 nm. there is a significant decrease in uptake between lp2 and lp3, lp1, sp3 and sp1, i.e. 217.20 nm and 217.75 nm, 462.33 nm, 496.96 nm and 1509.73 nm. here, the rod-shaped sba-15 particles were taken up to a similar extent to lp1, i.e. 612.75 nm and 462.33 nm, respectively. sp3 is only slightly larger than lp1, i.e. 462.33 nm compared to 496.93 nm, yet there is a very significant decrease in uptake. it is not clear why sp3 has a far lower uptake than sba-15, although the synthesis method is slightly unusual in that it uses glycerol as the co-surfactant and co-solvent [23] . the zeta potential of the particles is all very similar and so not likely to cause significant differences [3] . sp1 has a very large external diameter (1509.73 nm), and this is most likely the reason why this particle has a very low uptake. the macrophage uptake experiments shown in fig. 2b were for msnps without cargo. we subsequently tested whether the addition of antigen to the msnps would affect the uptake (fig. 3 ). due to their very poor uptake without cargo, sp3 and sp1 were omitted from this test (huang et al. submitted as part 1 of 2). the largest particle in this test, lp1 (462.33 nm) showed the lowest uptake (fig. 3a) , similar to results seen in fig. 2b . the particles sp4 and lp2 showed the greatest changes in uptake after loading with diphtheria toxoid (fig. 3b) . sp4 uptake was significantly reduced (p < 0.005) from 56.8 ± 0.5 to 38.2 ± 0.4%, and lp2 uptake was significantly reduced (p < 0.005) from 64.8 ± 1.9 to 42.9 ± 0.3%. it is not clear why these particles would behave so differently since there is no observable difference in table 1 the diameter of the solid spherical nanoparticles was assessed using cps disc centrifugation. zeta potential was determined using a zetasizer nano zs (malvern uk indicates the expression of cd14 on thp-1 cells. panel (iii) microscope images show the morphology of the cells the particle surface charge, surface area or morphology that would explain why these two particles in particular had reduced uptake after loading. we therefore looked at the hydrodynamic diameter of two of the msnps before and after loading see (huang et al. an assessment of mesoporous silica nanoparticle architectures as antigen carriers). lp2 showed a very significant decrease in uptake into macrophages after loading. the size before loading was determined to be 217.20 ± 22.66 nm (fig. 3a) . after incubation with diphtheria toxoid at a ratio of 1:20 for 24 h at 4°c, the size was 330.91 ± 4.86 nm, an increase of just over 50%. in terms of size the increase in size would place it nearer to lp1 (462.33 ± 25.03 nm). unloaded lp1 showed considerably reduced uptake compared to lp2, and so, the size increase could explain the reduction in uptake (fig. 3b) . in contrast, lp3 which was seen to have a modest increase in macrophage uptake after loading only increased in hydrodynamic diameter from 217.75 ± 5.44 nm before loading to 270.61 ± 1.99 nm after loading. once the ability of the macrophages to take up the various different particles had been determined, those which were taken up most readily were further examined for their effect on cell viability (fig. 4) . both naked nanoparticles and those loaded with diphtheria toxoid were assessed. there was no difference seen between loaded and unloaded particles except for lp1 (p < 0.05). the lp1 particles showed the best biocompatibility as measured by survival of the macrophages 24 h after incubation with the particles, 88.1 ± 3.0% and 87.0 ± 4.6% for unloaded and loaded lp1, respectively. none of the particles, however, caused significant cell death with the lowest, dt-loaded sp4, showing a cell viability of 71.9% of the control. given the nanoparticle uptake by the cells (figs. 2 and 3 ) and the relative viability of the cells (fig. 4) after incubation with the particles, the performance of lp2 could be deemed the best, although with the exception of sp3 and sp1 which were poorly taken up by macrophages, all particles performed well. interestingly, we . sba-15 was used in each data set for comparison. data is presented as mean ± standard deviation of triplicate samples and significance was tested using a one tailed t-test (*p < 0.05; **p < 0.01; ***p < 0.005) have previously shown that sp3 and sp1 also showed the lowest loading efficiency for diphtheria toxoid (huang et al. submitted as part 1 of 2). macrophages are cells which form a part of the innate immune system and as such are able to engulf a wide variety of targets, including dead cells, dust, pollen and invading pathogens. bacterial pathogens can differ greatly in their sizes, shapes and surface features, which can all have an effect on virulence (cabeen and jacobs-wagner 2005). this can give indications to the way in which macrophages would interact with man-made materials such as the msnps designed to stimulate the immune system. in order to perform their role in the immune system, macrophages engulf particles by the process of phagocytosis (burke and lewis 2002). phagocytosis involves both structural rearrangements of the cytoskeleton and membrane, and also a complex network of signalling events. the process of phagocytosis is affected by the physical parameters of the particles being engulfed. these can be split into geometric aspects such as the size (pratten and lloyd 1986; ikada 1988a, 1988b; koval et al. 1998) , shape or aspect ratio of the particles (lengerova et al. 1957; champion and mitragotri 2006; gratton et al. 2008; sharma et al. 2010; lu et al. 2010; champion and mitragotri 2009) , surface properties ikada 1988a, 1988b; faraasen et al. 2003; roser et al. 1998; gilberti et al. 2008; ahsan et al. 2002) and mechanical aspects (beningo and wang 2002) . to study the uptake of the particles by macrophages, we used the immortalized cell line thp-1 and used pma for differentiation of the cells. one problem associated with assessing uptake is whether it can be fig. 3 a antigen loaded fitclabelled msnps were assessed by facs for their ability to be internalized by macrophages. b comparison of loaded (light grey) and unloaded (dark grey) particles. data is presented as mean ± standard deviation of triplicate samples and significance was tested using a one-tailed t test (*p < 0.05, **p < 0.01; ***p < 0.005) determined whether particles are attached to the surface of the cells, or have been truly internalized. since the particles were labelled with fitc, they were to be observed via fluorescence. we therefore originally tried the methods which used the fact that trypan blue can quench green fluorescence from fitc (avelar-freitas et al. 2014 ). however, we found that the fluorescence was quenched from both external and internalized particles alike, so could not be used to differentiate uptake. we therefore explored the use of sodium azide, an inhibitor of phagocytosis, as a way to differentiate between particles which were phagocytosed compared to those which were merely surface bound. however, sodium azide has been shown to take up to 3 h of contact with cell culture before they have an effect on phagocytosis (cifarelli et al. 1979 ) and can also inhibit oxidative phosphorylation. since we were concerned that sodium azide could affect cell viability, this method was also not chosen. consequently, we used low temperature to inhibit phagocytosis and subtracted any fluorescence which was detected after such treatment since this would be a result of surface-bound particles. the highly cited silica particle sba-15 was used as a comparison molecule in all our experiments. this is a rod-shaped particle with an aspect ratio of 3:1. our results showed uptake in the region of 30% (fig. 2a) . the low uptake is not unexpected since aspect ratio is known to affect uptake, with very high aspect ratios completely inhibiting phagocytosis (gratton et al. 2008) . size has often been considered the most important factor for uptake, and the optimal size for macrophage internalization has been cited as between 1 and 3 μm (pratten and lloyd 1986; koval et al. 1998; ikada 1988a, 1988b; champion et al. 2008) . this was thought to correspond to the size of a number of large bacteria (kubitschek 1969) . however, our largest particle (sp1; 1509.73 ± 269.93 nm) showed the lowest uptake, and the optimal uptake was for much smaller particles. of course, as has been the case with regard to aspect ratio, the shape of the particles is also important. in 2006, champion and mitragotri first demonstrated that the local shape at the point of contact with the membrane governs whether phagocytosis will be initiated, whilst the size will determine whether the process is completed. in addition to size and shape, the mesoporosity of silica nanoparticles has also been indicated in their biological interactions (asefa and tao 2012) . compared to solid silica nanoparticles, the mesoporosity of the msnps results in lower rigidity which buffers the interaction with biological entities. this also results in lowered interfacial energy between the nanomaterials and the biological surfaces. in our study, the greatest uptake for solid particles was for the snp03 (377 nm) particles, whereas for the mesoporous particles, lp2 (217 nm) had the greatest uptake. however, the uptake for the mesoporous lp2 was slightly higher than for the solid spherical nanoparticle snp03. the size of the pores on the msnps did not seem to affect the uptake, which was related only to external diameter of the particles (fig. 1) . surface charge has also been shown to be an important parameter in the degree of uptake of particles. thiele et al. (2001) found that positively charged particles showed much greater uptake. for non-metallic nanoparticles, there was also a correlation between positive charge and the ability to elicit an immune response. however, negatively charged non-metallic nanoparticles were associated with antigen-specific tolerance (fromen et al. 2016) . despite these studies, there still needs to be further investigation into whether a generalized statement with regard to the charge of the nanoparticle and its influence on the immune response. silica nanoparticles have an inherent negative charge due to silanol groups on the surface. the solid silica nanoparticles synthesized in this study show increasing negative ζ potential with increasing size (table 1) . it is therefore difficult to separate charge and size in determining which factor contributes to the degree of uptake. the behaviour of the particles with regard to uptake by the macrophages altered after the msnps were loaded with dt. the dt has an isoelectric point of 4.1 (pappenheimer 1937) and therefore under low ph conditions, the dt would be positively charged. the silica is negatively charged and so the dt would be electrostatically attracted. this means that not only would loading with dt potentially change the hydrodynamic size, but also the apparent charge of the loaded particle. in addition to the size, shape and charge of the particles, the material may also influence the interaction with the macrophages. the material from which a nanoparticle is made has a direct influence on the functions of the antigen-presenting cells (marques et al. 2017) . gold nanoparticles are one of the most studied in vaccinology (see marques et al. 2017 for summary), but other metals such as iron oxide and nickel have also been investigated. polymers have also been tested and for example, poly (lactic-co-glycolic acid) (plga) delivery systems have shown adjuvant activity (nicolete et al. 2011) , with both humoral and cellular responses (o'hagan and singh 2003; men et al. 1999; carcaboso et al. 2004) . other materials that have been explored include emulsifying wax (cui and mumper 2002) , lecithin-glyceryl monostearate (sloat et al. 2010) , albumin, gelatin (zwiorek et al. 2008) , collage, chitosan and alginate (sloat et al. 2010) . we have chosen to investigate silica since it has the benefit of being easily manipulated to form mesoporous particles which have a very high surface area. msnps have been shown to have intrinsic adjuvant activity and to potentiate antigen-specific t cell immune responses (kupferschmidt et al. 2014; mahony et al. 2013) . furthermore, silica is widely reported to be biocompatible, and the nanoparticles degrade (coppi et al. 2004 ) to the relatively harmless silicic acid (diaconu et al. 2010) . more effective vaccines could benefit from improved, nanoparticulate delivery systems. silica is a biocompatible material which has the potential to both carry antigen and act as an adjuvant. we investigated a range of sizes of silica nanoparticles to determine the effect on uptake by thp-1 macrophage-like cells. it was seen with both solid and mesoporous silica nanoparticles that smaller particles were less effectively taken up by macrophages. optimal sizes appeared to be in the 200-400 nm range. we have previously demonstrated that antigen is stable on silica particles for at least 3 months (huang et al. submitted as part 1 of 2). msnps therefore present a viable alternative to current vaccine compositions. funding information this work was supported financially by a grant from cristalia produtos quimicos farmaceuticos ltda, brazil. conflict of interest xh and het received a research grant from cristalia produtos quimicos farmaceuticos ltda, brazil. dpc is an employee of cristalia produtos quimicos farmaceuticos ltda, brazil. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. targeting to macrophages: role of physicochemical properties of particulate carriers-liposomes and microspheres-on the phagocytosis by macrophages adjuvants for clostridium tetani and clostridium diphtheriae vaccines updating biocompatibility of mesoporous silica nanoparticles trypan blue 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binding and internalization by macrophages strong antibody responses induced by protein antigens conjugated onto the surface of lecithin-based nanoparticles effect of the size and surface charge of polymer microspheres on their phagocytosis by macrophage macrophage phagocytosis of biodegradable microspheres composed of l-lactic acid/glycolic acid homo-and copolymers evaluation of particle uptake in human blood monocyte-derived cells in vitro. does phagocytosis activity of dendritic cells measure up with macrophages? establishment and characterization of a human acute monocytic leukemia cell line (thp-1) preparation of mcm-41 silica using the cationic surfactant blend delivery by cationic gelatin nanoparticles strongly increases the immunostimulatory effects of cpg oligonucleotides key: cord-005112-9mxqwqty authors: zhang, hongpeng; chon, chan hee; pan, xinxiang; li, dongqing title: methods for counting particles in microfluidic applications date: 2009-08-20 journal: microfluid nanofluidics doi: 10.1007/s10404-009-0493-7 sha: doc_id: 5112 cord_uid: 9mxqwqty microfluidic particle counters are important tools in biomedical diagnostic applications such as flow cytometry analysis. major methods of counting particles in microfluidic devices are reviewed in this paper. the microfluidic resistive pulse sensor advances in sensitivity over the traditional coulter counter by improving signal amplification and noise reduction techniques. nanopore-based methods are used for single dna molecule analysis and the capacitance counter is useful in liquids of low electrical conductivity and in sensing the changes of cell contents. light-scattering and light-blocking counters are better for detecting larger particles or concentrated particles. methods of using fluorescence detection have the capability for differentiating particles of similar sizes but different types that are labeled with different fluorescent dyes. the micro particle image velocimetry method has also been used for detecting and analyzing particles in a flow field. the general limitation of microfluidic particle counters is the low throughput which needs to be improved in the future. the integration of two or more existing microfluidic particle counting techniques is required for many practical on-chip applications. particle counting is important and widely used in various areas from environmental (aalto et al. 2005) to biological applications (smolen et al. 1983; amann et al. 1990; yarnell et al. 1991) , such as the following: counting dust particles is required for clean room facility (wu et al. 1989) ; counting debris particles is needed for studies of lubricating systems (miller and kitaljevich 2000) ; counting contaminant particles is the key factor in water purification system (bundschuh et al. 2001; judd and hillis 2001) ; and counting white blood cells is essential for many biomedical diagnostic purposes such as detecting hiv infection (yarnell et al. 1991; kannel et al. 1992; burnett et al. 1999; vozarova et al. 2002) . the particle counting techniques have been developed further to detect single dna molecule (akeson et al. 1999; kasianowicz et al. 1996) and analyze dna contents (sohn et al. 2000) . however, the conventional particle counting methods rely on costly, bulky, and complex instruments and require a large amount of samples and reagents. these are the barriers to many particle counting applications. microfluidic devices have been studied particularly in the past decade and have showed enormous potential for portable and low-cost applications, especially in medical diagnostics (akeson et al. 1999; burnett et al. 1999; kannel et al. 1992; sohn et al. 2000; vozarova et al. 2002; kasianowicz et al. 1996) . lithographic fabrication technique makes possible building inexpensive and small devices integrated with electrodes and sensors, and microfluidic control technologies such as electrokinetics (li 2004 ) are able to control particles and liquid flow in micro-and nano-channels. in addition, microfluidic devices are particularly useful for applications where a very small quantity of samples is available or desired. microfluidics-based particle counting methods have great advantages over the conventional methods and allow the development of accurate, cheap, and portable particle counting devices. in this paper, we review the major advancement of microfluidic particle counting techniques: microfluidic resistive repulse sensors, nanopore sensors, capacitance counters, light-scattering and light-blocking detectors, fluorescent detectors, and micro particle image velocimetry (piv) counters. for each type of particle counters, we also explore various applications and examine the advantages and disadvantages. coulter counter perhaps is the most popular conventional method of particle counting. we review this method first for two reasons: it gives a general perspective of conventional particle counting methods, and its working principle of measuring electric resistive pulse is used in some microfluidic particle counting methods. coulter counter was first invented by wallace h. coulter during world war ii and patented (coulter 1953) . when coulter worked for us navy, he used this technique to count the number of plankton particles that always caused large echoes on sonar. in a coulter counter, a small aperture on the wall is immersed into a container that has particles suspended in low concentration electrolyte solution. two electrodes are placed: one in front and one behind the aperture, and a current path is provided by the electrolyte when an electric field is applied (fig. 1 ) and the aperture creates a ''sensing zone.'' as a particle passes through the aperture, a volume of electrolyte, equivalent to the immersed volume of the particle is displaced from the sensing zone. this causes a short-term change in the impedance across the aperture. this change can be measured as a voltage pulse or a current pulse. the pulse height is proportional to the volume of the sensed particle. if a constant particle density is assumed, the pulse height is also proportional to the particle mass. this technology thus is also called aperture technology. because the coulter counter is simple, highly sensitive, and reliable, it is widely applied in many areas including medical instruments such as counting and analysis of blood cells (horne et al. 2005) , protein (kulp et al. 2004) , and viruses (wahl-jensen et al. 2007 ) besides detecting fine particles and pollen (deblois and bean 1970) . before the coulter counter was invented, complete blood counts (cbcs) were carried out manually with a telescope and static samples, which is time-consuming and inaccurate. with the coulter counter, cbcs can be done for a large amount of samples in a very short period of time. when coulter first demonstrated the coulter counter, it could count red blood cells at a high count rate of 6,000 particles/ s, which revolutionized the science of hematology. within a decade, literally every hospital laboratory in the us had coulter counters, and today every modern hematology analyzer adopts the coulter principle in some way. for example, coulter counters may measure the change of impedance (carbonaro and sohn 2005; jagtiani et al. 2006a, b; zhe et al. 2007; wu et al. 2008a, b) , conductance (sohn et al. 2000; murali et al. 2009 ), and reflected radio frequency power (wood et al. 2005 ) when a particle passes the aperture. after many years of development, the modern coulter counter is versatile and accurate in particle sizing and counting. multisizer tm 4 coulter counter ò (beckman coulter, fullerton, ca, usa), for example, can provide size distribution in number, volume, and surface area in one measurement of particles ranging from 0.4 to 1,600 lm in diameter. its aperture dynamic range can reach to 1:40 by diameter and reproducibility is about 1%. although the coulter counter detects and analyzes particles accurately and reliably, it has still many drawbacks: bulky size, heavy weight, complexity, high power consumption, high cost, and no-portability. especially during outbreak of public diseases such as severe accurate respiratory syndrome (sars) and influenza, there is urgent need for simple, low power, low cost, and portable particle counters. the microfluidic resistive pulse technique applies the basic working principle of the coulter counter to microchannels for counting micro-and sub-micron particles. the resistive pulse sensor (rps) was applied to detect submicron polystyrene beads of 90 nm inside a 0.4-0.5 mm diametric polycarbonate pore by bean in 1970. deblois and wesley (1977) utilized this technique in biological area counter amplifier + fig. 1 schematic of a coulter counter and succeeded in detecting viruses. after the technical advance of micro-fabrication (rogers and nuzzo 2005) , the rps method has been applied to count particles moving in microchannels. many microfluidic rps applications involve manipulating and transporting of particles by electrokinetic flow in microchannels (jagtiani et al. 2006a, b) while some still use traditional flow control by hydraulic pressure. the key advantages of the microfluidic rps include label-free particle detection and simplicity without other peripheral complex instruments other than a simple electric circuit and a micro-or nano-scale sized channel. therefore, it is mostly applicable for portable lab-on-a-chip (loc) devices to detect biopolymers such as dna, protein, and blood cells. however, the flow rate of the microfluidic rps is small and the sensitivity of the microfluidic rps is limited by its aperture size, resulting in poor throughput and sensitivity. to overcome these shortcomings, recent researches on microfluidic rps focus on two main issues: improving of the sensitivity and enhancing of the throughput. the rps throughput is evaluated by particle flow rate at the aperture or the number of the counted particles at a given time. its sensitivity is determined directly by the volume ratio of the detected particles and the aperture and can be adjusted by controlling the amplification gain of the electronic circuit or instrument and the noise reduction from the fluidic network and the electronic sensing system. deblois and bean (1970) were able to detect 90 nm polystyrene spheres, which is equivalent to minimum volume ratio of 0.06% with submicron pores etched in irradiated plastic sheet. to improve the sensitivity, xu et al. (2007) and sridhar et al. (2008) used a relatively wide polydimethyl siloxane (pdms) channel of 16 lm with metal-oxide-semiconductor-field-effect-transistor (mos-fet). they detected particles by monitoring the mosfet drain current modulation instead of the modulation in the ionic current though the sensing channel and achieved ten times smaller minimum volume ratio of 0.006% than that of deblois and bean. recently, wu et al. (2008a, b) developed a microfluidic rps method utilizing a mirror symmetric channel structure and a two-stage differential amplifier (fig. 2) . they could significantly reduce the noise and achieve a much better signal-to-noise ratio. this sensing scheme detected 520-nm diameter polystyrene particles with a 20-lm sensing gate and improved a minimum volume ratio to as low as 0.0004%, which is about ten times more sensitive than the current commercial coulter counter of 0.0037% (beckman counlter ò multisizer tm 4). the principle of the symmetric dual channel design is to make noise levels for the output signals (v d1 and v d2 as indicated in fig. 2 ) from both gate branches identical and hence the noises can be canceled by a subtraction electronic circuit. however, ideal noise subtraction is not possible due to realistic limitation to fabricate the identical dual channels. furthermore, this dual channel method will not be able to detect particles when two particles pass the two apertures at the same time because the two signals with the similar amplitude will be subtracted by each other and cancelled at the second stage of differential amplifier. to improve this microfluidic rps method, wu et al. (2008a, b) solved the signal cancelation problem as mentioned above by using a single sensing gate and two detecting arm channels next to the sensing gate at both ends ( fig. 3) . they coupled the rps with laser fiber-optic fluorescence technique to demonstrate a flow cytometer loc that is able to detect fluorescent and non-fluorescent particles simultaneously, and the rps signal-to-noise ratio is improved significantly. two-stage differential amplification is also used to further increase the signal-to-noise ratio for fluorescent signals to detect 0.9 lm fluorescent particles. this flow cytometer chip showed comparable sensitivity for detecting fluorescent and non-fluorescent particles to commercial flow cytometers with simple, cheap, and compact system on a micro glass slide. drawbacks of the method are the baseline drifting by multiple-stage differential amplification and the low throughput of using single channel detection. the throughput of a single-channel coulter counter is proportional to the square of the diameter of the detecting aperture. when submicron-or nanometer-size particles are to be counted, the size of the aperture has to be scaled down to submicron or nanometer in order to maintain the sensitivity. otherwise, the signal-to-noise ratio will be very low. while the sensitivity of the single-gate microfluidic rps method is much higher, it requires long detecting time due to low flow rate in a microchannel and using of diluted samples to avoid multiple particles flowing together. to overcome this low throughput issue, multi-counting techniques are developed. carbonaro and sohn (2005) first demonstrated the simultaneous immunoassays of two different human antigens by integrate multiple artificial pores and the rps technique on a single chip. coulter and hogg (1976) patented the particle analyzing apparatus and method with multiple sensing apertures. however, it is difficult to integrate the detection circuit and independent power supply of their systems on one chip. zhe group (jagtiani et al. 2006a, b) proposed a multi-aperture coulter counter, which consists of four peripheral reservoirs and a central reservoir (fig. 4a) . each peripheral reservoir is connected to the central reservoir through a miniature channel. their results showed that the sensor can detect and count particles through its four sensing apertures simultaneously. however, the four apertures are the maximum number of apertures that can be built in a single chip due to the configuration limit. furthermore, zhe group (zhe et al. 2007) proposed different high throughput single chip counter using multiple channels, operating in parallel with single common sample reservoir and a power source (fig. 4b) . this counter was capable of differentiating and counting polymethacrylate particles and juniper pollen about three times faster than single channel counter. this fig. 3 the schematic diagram of single channel and two detecting arm channel microfluidic differential rps (a) and the rps counter and fluorescent signals for 0.9 lm nile blue particles mixed with the 0.99 lm dragon green particles (b). the greater rps peaks are the signals of the 0.99 lm non-fluorescent particles concept could be extended to multi-channel microfluidic chips in the future to improve the counting efficiency. a nanopore here is referred to as a small pore of nanometer size in an electrically insulating membrane and a nanopore resistive sensor (nrs) is a nanometer-scale coulter counter, which uses the nanopore as an aperture to detect single molecule. the nrs, the coulter counter, and the rps share the same principle for particle detection; however, the nrs has attracted many researchers since 1990s because the volumetric ratio of the nrs can be high enough to detect much smaller particles such as single molecule by the nanometer sized pores (bezrukov et al. 1996; kasianowicz et al. 1996; akeson et al. 1999 ). there are two types of nanopores according to the nanopore materials: synthetic and natural materials. a typical natural nanopore is a biological protein channel in a lipid bilayer. bezrukov et al. (1996) demonstrated the counting of polymer molecules passing through a single alamethicin pore of 5 and 2 nm in length and diameter, respectively. kasianowicz et al. (1996) showed that they were able to sense single-stranded rna and dna through a 2.6 nm diameter ion channel in a lipid bilayer membrane. this promising result sparked many studies of dna translocation and dynamics in biological nanopores. akeson et al. (1999) detected single dna and rna molecules in an a-hemolysin channel driven by an applied electric field (fig. 5) . since the small-sized pore can hold only one strand of dna or rna at a time, nucleotides within the polynucleotide must pass through the channel/pore in sequential and single-file order during the translocation. in this process, not only counting but also discriminating between pyrimidine and purine segments along a dna or rna molecule can be accomplished. a very important application of this research is the direct sequencing of individual dna and rna molecules with a nanopore. in addition, nanopore method was used to measure small particles like metal ions, nucleic acids, and other types of polymers in a less than 10 nm channel (biance et al. 2006) . the geometrical and chemical properties of biological nanopore can be reproducibly controlled by genetic engineering. however, the biological nanopores are not very robust and not size tunable. even in laboratory environment it can last only several hours. therefore, efforts have been made to fabricate artificial, solid-state nanopores to overcome this limitation. the nanopores based on synthetic material are generally made in silicon compound membranes, such like silicon nitride. manufacturing technique could be focused ion beam (fib) sculpting or electron beam sculpting. martin group did a series of research on synthetic conical nanopores for biosensing applications (siwy et al. 2005; harrell et al. 2006; wharton et al. 2007; sexton et al. 2007 ). they made a conical pore with a 1.5 mm base diameter and a 40-nm tip diameter and sensed a single-stranded phage dna of 7,250 bp and a doublestranded plasmid dna of 6,600 bp. they also detected protein in the same way. alternatively, ito et al. (2004) utilized a 132 nm multiwall carbon nanotube (mwnt) to detect 28-90 nm nano-particles. synthetic nanopores are chemically and structurally stable; however, it is still hard to control the size of these nanochannels. reproducing synthetic nanopores of the same size is hardly possible. additionally, high manufacturing cost is another obstacle to be overcome for further development and applications of synthetic nanopore particle sensors. the capacitance counter uses a similar principle to the coulter counter: it measures the ac capacitance instead of dc resistance when a micron or sub-micron particle passes a sensing gate (aperture). the capacitance counter is particularly useful for detecting particles in liquids of low electrical conductance because the resistance change due to the passage of a particle is difficult to measure in a poor conducting liquid (murali et al. 2009 ). in the past, the capacitance measurements have generally been used to identify bulk materials and to investigate ensembles of biological cells. however, sohn et al. (2000) employed this technique to detect and quantify the polarization responses of dna in the nucleus of single eukaryotic cells. they built an integrated microfluidic device (fig. 6) , and used a syringe pump to deliver the liquid to the device. a capacitance bridge at a frequency of 1 khz across the device was used to detect the capacitance change to determine the dna content of single eukaryotic cell. additionally, they demonstrated the relationship between the capacitance and the dna content of a cell. recently, murali et al. (2009) adopted the capacitance counter to monitor of the wear debris in lubrication oil to avoid catastrophic system failure of machine in real-time. unlike bulk measurement methods, they could scan each individual particle and determine the size of particles without being affected by the change of oil properties. particles from 10 to 25 lm were successfully detected. similar to a microfluidic rps, the capacitance method has advantages in terms of simple sample preparation, cost, size, and robustness. in addition, the capacitance method is sensitive to probe the polarization response of a wide range of materials both organic and inorganic to an external electric field. furthermore, it can monitor changes in dna contents and cell-cycle kinetics so that it may serve as a medical diagnostic device to identify the presence of malignancy in very small quantities of tissue such as tumor cell and monitor in real-time of the effects of pharmacological agents on cell cycle and cell death. the capacitance method, however, has complications resulting from the charge-screening effects at the electrode-conductive liquid interface in an electronic measurement, which prevents the interpretation of the absolute capacitance value. besides, due to ac voltage and frequency required to detect the capacitance, frequency modulation controller is necessary and external support such as a syringe pump is required to deliver a liquid in a channel. these restrictions in flow control and frequency modulation are some of the obstacles for developing compact microfluidic capacitance particle sensors. 6 light-scattering and light-blocking counters a light-scattering particle counter and a light-blocking particle counter are two similar types of light-based particle counters. they both use laser light sources such as laser diodes to illuminate individual particles that pass through the laser beam. the difference between these two counters lies in how the interaction between the light and the particle is measured for counting particles. when light strikes an object generally it will be divided into three parts. some light will pass through the object, some will be reflected, and the rest will be absorbed by the object. the portion of these three components is determined by optical properties of the material composition of the particle. light-scattering counter uses the reflected light of the particle. as shown in fig. 7a , when the source light hits the particle, some of the light is reflected and the reflected light can be detected by a photo-detector positioned at a spot with a certain angle from the light path. in general, the detector is placed at the angle of 20°-40°from the light path to the particle. the strength of detected light signal corresponds to particle size, and the number of pulses of the detected light signal is proportional to number of particles. on the other hand, the light-blocking counter (fig. 7b ) measures the light absorbed or reflected away from the detector by the particle. in this arrangement, the light is focused directly onto the detector; when the particle passes between them, and the photo-detector senses the sudden change in the light intensity by a blocking particle. it is obvious that the larger the particle, the more light it will block. in principle, the light-scattering counter detects light while the light-blocking counter detects the darkness. the light-scattering method is more sensitive than the light-blocking method. this is the same principle that detecting a light in a darkroom is easier than detecting a dark spot in a bright room due to the diffuse reflectance caused by solid surfaces and particles in space. however, in order to focus and detect the scattered light at a specific angle, the light-scattering counter system normally contains more optical elements and complex electronic circuit, which makes the light-scattering counter more expensive than the light-blocking counter. the light-blocking counter is widely used in detecting particles in water (e.g., water treatment application) while the light-scattering counter is mostly used in detecting atmospheric particles. the key disadvantage of these light-scattering and light-blocking counters is the low sensitivity in comparison to the coulter counter because the sensitivity of these light-based particle counters are determined by the particle's surface area while the sensitivity of the coulter counter is decided by the volume of the particle. pamme et al. (2003) researched the counting and sizing of particles and particle agglomerates of c-reactive protein by laser light scattering method. they detected scattering light at two different angles of 15°and 45°. the experiment was carried out on a polymethyl methacrylate (pmma) microchip which consists of three inlet channels of two buffers and one sample channel and one outlet channel. the fluid was driven by syringe pump generating a negative pressure with a flow rate of 1 ll/s to neglect the hydrostatic pressure in the inlet reservoirs. a beam splitter and an objective lens are applied to confine the he-ne laser inside the channel to avoid scattering on the wall. the scattering light signals are sensed by two optical fibers and amplified by a photomultiplier tube (pmt). by this method, the authors realized the particle detection of 2-9 lm. in addition, size discrimination of particles with a diameter ratio of 1:2 was achieved. on the other hand, the standard deviation from the average scattering light intensity for a given particle population was high, up to 30%, and a wide laser beam led to overall lower scattering light intensity and higher background scattering from the channel walls. these cause problems for measurement of smaller particles due to the lower scattering intensity and the lower signal-to-noise ratio. xiang et al. (2005) utilized the light-blocking technique and developed a multifunctional particle detection system with embedded optical fibers in a pdms chip to detect moving micro particles in a microchannel. they developed a pdms-glass microfluidic chip with two pairs of embedded optical fibers and removed the glass cladding layer of the input and receiving fibers. by filling the gap between the fiber and the fiber channel with pdms, the light leakage from the fiber core and the light scattering from the fiber tips were minimized. by using the two-fiber detection method, particle velocity as well as particle counting and size identification were possible. they achieved counting of 10, 20, and 25 lm particles. to get a better signal, both input and receiving fibers must be perfectly aligned. they used larger size of the receiving fiber to improve this problem. however, in real applications, careful handling of the fiber-embedded chip and highly controlled fiber alignment are required. schafer et al. (2009) created an all silicon and glass microfluidic device using femto-second laser ablation and anodic bonding technique and applied it for cell counting. this microchannel fabrication needs just one-step process and shorter fabrication time by femtosecond pulse laser. the optical fibers for light-scattering signal detection directly contact the liquid, causing light focus on the optical detector by removing the optical free space between the detecting fiber and the channel. the incident light was coupled to the fiber with a 209, 0.4 na objective, and the light was delivered through the fiber at the normal direction and collected through the fiber on the opposite side at 14°f rom the normal and then collimated by an objective and focused onto a photodiode. hela cell was detected by scattered light with the reported lowest power. conclusively, the system achieved similar particle-detecting quality with lower laser power of 2 mw and a cheaper photodiode. however, this device has difficulty of controlling of the even depth and the alignment of the grooves used to guide the optic fibers, and has poorer re-productivity than pdms chips. kummrow et al. (2009) developed a microfluidic flow cytometer combined light-scattering detection and fluorescence detection in a pmma chip with integrated optical fibers, mirrors, and electrodes for flow cytometric analysis of blood cells. they used ultraprecision milling technique to fabricate different flow cells featuring single-stage and two-stage cascaded hydrodynamic focusing of particles in horizontal and vertical directions by a sheath flow. as shown in fig. 8 , the first stage decreased the diameter of the sample flow to about 30 mm, the second stage allowed to reduce the diameter down to 5 mm while maintaining stable operation for sample flow rates of up to 20 ll/min. inserted optic fibers were used to excite fluorescence of stained cells and to detect the axial light loss and the light scatter. integrated mirrors were used to image the sample flow in vertical direction, thus proving the efficiency of hydrodynamic focusing in two dimensions. after passing the optical sensor the particles enter the interaction zone for impedance measurements. three multimode optic fibers with angles of 12°, 42°, and 145°from the incident light were used to detect the light scatter. the multimode optic fiber opposite to the incident light served to measure the axial light loss. an argon ion laser was used for forward light at 488 nm and for exciting the fluorescence of stained cells. t-helper lymphocytes labeled by monoclonal antibodies were identified by measuring side scatter and fluorescence. using this cytometer, mono-disperse polystyrene spheres with diameters ranging from 2 to 22 lm were detected. fluorescence is an optical phenomenon in which the molecular absorption of a photon triggers the emission of a photon with a longer and less energetic wavelength. the energy difference between the absorbed and emitted photons ends up as molecular rotations, vibrations, or heat. this fluorescence characteristic has been applied to count particles. lin and lee (2003) proposed a method of fluorescence detection in a micro flow cytometer without on-chip fibers. in this system a pdms microchip is bonded to a 150-lm-thick glass substrate. laser passes a filter cube and optical fiber to excite particles inside the micro-channel. the excited fluorescence is also detected by the same fiber and translated to electrical signals in the photo detector. the lock-in amplifier amplifies the electrical signal and transmits it to computer. embedding the optic fiber into the chip adds difficulty and cost to manufacturing the chip. in this system, however, the fiber is separated by glass substrate and the microchip is disposable. in the experiments, the authors successfully detected and counted 5-20 lm fluorescent particles, white blood cells, and yeast cells. the major advantage of this method is no embedded optic fiber. the glass substrate works as the interface between the detection fiber and fluorescent particle. however, the detected fluorescent signals are not constant as they depend on the distance between the optic fiber detector and particles (the particles moving closer to the detector produce stronger signals). in addition, the precise alignment of the fiber and the channel is still difficult to realize. chen and wang (2009) reported on-chip fluorescence detection and counting system in a pdms microchip. the fluorescence and size information of particle were characterized by combining forward scattering signal and backward fluorescence signal. in the experiments, microparticles of four different sizes with diameters ranging from 3.2 to 10.2 lm were distinguished and counted. the relative percentage of the fluorescence-labeled particles can be analyzed by the ratio of the events of fluorescence signals to forward fig. 8 schematic diagram of a microfluidic system with two stage cascaded hydrodynamic focusing, integrated mirrors, optical fibers, and fluidic connection. multimode fibers serve to detect the axial light loss or scattered light when particles pass the interaction region. orthogonal light scatter and fluorescence is collected perpendicular to the joining plane by a microscope objective scattered signals. similar to lin and lee, this system also adopted the externally installed fiber system for disposable chips. however, without a bulk lock-in amplifier, they could detect 3.2-10.2 lm particle by using an avalanche photodetector and simple electronic filter circuit. in addition, the chip fabrication process is simpler and cheaper by only pdms soft lithography. the same problems of inconsistent fluorescent signals caused by the distance between the photodetector and particles and the difficulty of fiber alignment still existed. in general, fluorescence is the one of the most sensitive methods for particle detection; however, using this method, particles must have fluorescent characteristics and nonfluorescent particles have to be labeled with proper fluorophores. therefore, this technique is widely used to distinguish specific target particles from other particles, and often used in combination with other particle counting techniques (chen and wang 2009; kummrow et al. 2009; murali et al. 2009 ). particle image velocimetry is an optical method used to obtain instantaneous velocity measurements and related properties in fluids. when the small seeding particles flow inside a flowing liquid, the motion of these particles is traced and computed to show the fluid flow by taking sequential images of the positions of the tracing particles. typical piv apparatus consists of a digital camera, a highpower laser, and an optical arrangement to convert the laser output light to a light sheet. a fiber-optic cable often connects the laser to the cylindrical lens setup. the laser acts as a photographic flash for the digital camera, and the particles in the fluid scatter the light. the scattered light is detected by the camera. in the early twentieth century, german scientist ludwig prandtl first used particles to study fluids in a systematic manner (goldstein 1996) , and then the rapid development of lasers and camera technology enabled flow visualization and later on quantifying the whole flow field measurement. modern piv software continues to improve the performance of the piv systems and their applicability to difficult flow measurements. nowadays the piv has become one of the most popular instruments for flow measurements in numerous applications. hirono group developed the image cytometry centrifugation (icc) to count leukocytes (yabusaki et al. 1999 (yabusaki et al. , 2000 and later extended their research to study theoretically and experimentally a microfluidic image cytometry by using micro-piv flow visualization technique (hirono et al. 2008) . in their research, numbers and sizes of particles flowing through a microchannel were measured simultaneously by image sequence analysis. during the experiments, a dilution series of 2 lm polystyrene particle suspensions were measured and compared with the results obtained by conventional burker-turk hemocytometry for validation of the particle counting. for the particle diameter measurements, the diameters of 2, 5, 10, and 20 lm particles were measured and the results agreed well with the reference values. this method may be used to the quantitative study of platelet aggregation in blood flow and become a powerful diagnostic tool in the future. the image processing procedure demands complicated computational work and microscopic instrument, resulting in high cost, additional post processing, and large system. for counting biological or non-biological particles, a traditional coulter counter is still the most popular device because of its high sensitivity and throughput. however, due to the increasing needs for portability and low cost in wide biological applications such as a handheld flow cytometer and a single dna analyzer, many recent researches have focused on micro-or nano-scale particle counting devices using advanced microfluidic technologies to realize portability, low cost, and simplicity. in this paper, among many particle counting techniques, we reviewed only those applicable in microfluidic chips. microfluidic rps has been studied and demonstrated to have significantly higher sensitivity than the commercial coulter counter with low cost. furthermore, the rps technique extends to nano-size by utilizing natural or synthetic nanopore to detect single dna/rna. for applications in liquids of low electrical conductance, microfluidic capacitance measurement has been used instead of resistance measurement for particle counting. the light-scattering and the light-blocking methods can also be applied in microfluidic chips and have a little lower sensitivity than rps technique. however, they are more applicable in applications involving higher density particles. fluorescent detection has an advantage in identifying specific particles among similar sized particles because the fluorescence method has higher specificity than any other electrical measurement. micro piv technique can perform particle counting and analyzing the flow filed simultaneously. in addition to the disadvantages of the individual detecting techniques described in this paper, the low throughput is a major hindrance for the applications of microfluidic particle counting methods. a possible way to overcome this obstacle is to use multiple parallel channels; however, the number of parallel sensing microchannels can be built in single detecting chip is limited practically. therefore, the enhancement of the counting speed as well as stable and sensitive detection over a longer period of time is among the challenging issues. furthermore, in order to meet the demand of the complex analytical detection in real applications, the integration of multiple particle counting methods in a single microfluidic system should be investigated. aerosol particle number concentration measurements in five european cities using tsi-3022 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anodic bonding resistive-pulse studies of proteins and protein/antibody complexes using a conical nanotube sensor protein biosensors based on biofunctionalized conical gold nanotubes neuron numbers in the superior cervical sympathetic ganglion of the rat: a critical comparison of methods for cell counting capacitance cytometry: measuring biological cells one by one experimental characterization of a metal-oxide-semiconductor field-effect transistor-based coulter counter high alanine aminotransferase is associated with decreased hepatic insulin sensitivity and predicts the development of type 2 diabetes temporal analysis of andes virus and sin nombre virus infections of syrian hamsters a method for reproducibly preparing synthetic nanopores for resistive-pulse biosensors highbandwidth ratio frequency coulter counter deposition of submicron aerosol particles during integrated circuit manufacturing: experiments simultaneous particle counting and detecting on a chip microfluidic differential resistive pulse sensors multi-functional particle detection with embedded optical fibers in a poly(dimethylsiloxane) chip wide-spectrum, ultrasensitive fluidic sensors with amplification from both fluidic circuits and metal oxide semiconductor field effect transistors highsensitive method for counting low concentrations of particles in liquids using centrifugation and image analysis fibrinogen, viscosity, and white blood cell count are major risk factors for ischemic heart disease. the caerphilly and speedwell collaborative heart disease studies a micromachined high throughput coulter counter for bioparticle detection and counting acknowledgments the research grants from the canada research chairs program (li) and the canada foundation for innovation (li), and from china 111 project (zhang & pan) are greatly appreciated. key: cord-280272-mn596x1p authors: akhrymuk, ivan; lin, shih-chao; sun, mei; patnaik, anurag; lehman, caitlin; altamura, louis; minogue, timothy; lepene, ben; van hoek, monique l.; kehn-hall, kylene title: magnetic nanotrap particles preserve the stability of venezuelan equine encephalitis virus in blood for laboratory detection date: 2020-01-28 journal: front vet sci doi: 10.3389/fvets.2019.00509 sha: doc_id: 280272 cord_uid: mn596x1p most of the modern techniques used for identification of viral-induced disease are based on identification of viral antigens and/or nucleic acids in patient's blood. diagnosis in the field or in remote locations can be challenging and alternatively samples are shipped to diagnostic labs for testing. shipments must occur under controlled temperature conditions to prevent loss of sample integrity. we have tested the ability of magnetic nanotrap® (nt) particles to improve stability and detection of venezuelan equine encephalitis virus (veev), viral capsid protein, and viral genomic rna in whole human blood at elevated temperature and prolonged storage conditions. nt particles have previously been shown to capture and enrich multiple pathogens including respiratory syncytial virus, influenza virus, coronavirus, and rift valley fever virus. our study indicates that samples incubated with nt particles had detectable levels of infectious veev in blood equal to or greater than samples without nt treatment across all temperatures. viral rna detection was increased in the presence of nt particles at later time points (72 h) and higher temperature (40°c) conditions. likewise, detection of veev capsid protein was enhanced in the presence of nt particles up to 72 h at 40°c. finally, we intranasally infected c3h mice with tc-83, the live attenuated vaccine strain of veev, and demonstrated that nt particles could substantially increase the detection of veev capsid in infected blood incubated up to 72 h at 40°c. samples without nt particles had undetectable capsid protein levels. taken together, our data demonstrate the ability of nt particles to preserve and enable detection of veev in human and mouse blood samples over time and at elevated temperatures. most of the modern techniques used for identification of viral-induced disease are based on identification of viral antigens and/or nucleic acids in patient's blood. diagnosis in the field or in remote locations can be challenging and alternatively samples are shipped to diagnostic labs for testing. shipments must occur under controlled temperature conditions to prevent loss of sample integrity. we have tested the ability of magnetic nanotrap ® (nt) particles to improve stability and detection of venezuelan equine encephalitis virus (veev), viral capsid protein, and viral genomic rna in whole human blood at elevated temperature and prolonged storage conditions. nt particles have previously been shown to capture and enrich multiple pathogens including respiratory syncytial virus, influenza virus, coronavirus, and rift valley fever virus. our study indicates that samples incubated with nt particles had detectable levels of infectious veev in blood equal to or greater than samples without nt treatment across all temperatures. viral rna detection was increased in the presence of nt particles at later time points (72 h) and higher temperature (40 • c) conditions. likewise, detection of veev capsid protein was enhanced in the presence of nt particles up to 72 h at 40 • c. finally, we intranasally infected c3h mice with tc-83, the live attenuated vaccine strain of veev, and demonstrated that nt particles could substantially increase the detection of veev capsid in infected blood incubated up to 72 h at 40 • c. samples without nt particles had undetectable capsid protein levels. taken together, our data demonstrate the ability of nt particles to preserve and enable detection of veev in human and mouse blood samples over time and at elevated temperatures. venezuelan equine encephalitis virus (veev) is one of the most neglected viruses among biowarfare agents. it is classified as a category b biothreat pathogen by national institute of allergy and infectious diseases (niaid), usa, due to its high dissemination rate, minimal infectious dose to induce disease in human, and the requirement for specific and enhanced diagnostic capacities. veev causes disease with symptoms ranging from influenzalike illness to more severe illnesses including myalgia, arthralgia, and neurological disorders that can lead to lethal encephalitis in susceptible hosts. all these characteristics made veev an attractive candidate for weaponization (1) . veev was first isolated from an infected horse brain during a veev outbreak in venezuela in 1938 followed by major outbreaks in venezuela and columbia in 1960s (2) , infecting thousands of people and animals. despite its high contagiousness, veev has drawn little public attention due to only sporadic outbreaks occurring in central and south america since 1995. other reasons for being neglected may be a low mortality rate in humans (<1%) while as high as 90% in horses, as well as no reports of veev outbreaks in the us since the only epidemic outbreak in texas in 1971 (3) . however, the spillover of veev infection from infected horses to humans during epidemics remains a concern. veev is an arthropod-borne epizootic rna virus, belonging to the togaviridae family, genus alphavirus, and is maintained within lower animals (rodents) and mosquitos (4) . the transmission of veev is primarily mediated by mosquitoes, where it replicates in salivary glands and is passed to hosts, such as human and horses with overt symptoms (5, 6) . moreover, aerosolized veev can be directly disseminated and can infect humans or susceptible experimental animal models, causing encephalitis, and possibly limb paralysis (7) (8) (9) . historically, since the late 1930s the former soviet union regarded veev as an operational biological weapon to incapacitate the rear services and reinforcement behind the front line, leading to the spread of the disease among infected individuals with flulike symptoms that are difficult to distinguish from epidemic influenza outbreaks. weaponized veev was not expected to kill the soldiers, but cause panic and ultimately maim the military targets (10) . notably, there are no effective antiviral agents available and approved by the us food and drug administration (fda) and only supportive treatment is available for humans. as a result, it is important to establish a preventive surveillance system based on prompt diagnosis methods. viral stability is an essential factor for diagnosis of veev to confirm the presence of virions or viral rna in a clinical sample. the current diagnostic approaches to confirm veev infection in humans or horses rely on direct detection of viral nucleic acids in serum or spinal fluid samples during the acute-phase of infection using reverse-transcription pcr (rt-pcr) (11) and elisa for veev-specific igm (12) . however, despite high sensitivity of the above-mentioned methods, false negative results can be obtained if samples have been collected during the initial asymptomatic phase of infection where the viral load is low (13) (14) (15) . moreover, the necessity of extra steps of plasma or serum preparation complicates fast virus identification and diagnostic. therefore, virus stabilization directly in collected sample without extra preparation steps is highly desirable. ideally, supplementation of the blood collection device with some type of stabilization agent that can minimize pathogen loss during sample transportation and storage at ambient temperatures is appealing. in view of these challenges, nanotrap r (nt) particles were evaluated for their ability to stabilize veev. nt particles are hydrogel polymer particles comprised of n-isopropylacrylamide (nipam), allylamine (aa), and crosslinked with n,n ′ -methylenbisacrylamide (bis). these particles are functionalized with various dye affinity baits that facilitate capture and retention of analytes from complex biological matrixes (such as blood, saliva, nasal swabs and urine) and concentrate them into a smaller volume (16) (17) (18) (19) (20) . previous work with nt particles has shown the benefit of their use in the enrichment of infectious virus and viral genomic material of rift valley fever virus (rvfv), coronavirus, influenza virus, and respiratory syncytia virus (19, 21) . in this study, we sought to apply new magnetic nt particles that consist of nipam copolymers functionalized with reactive red 120 to evaluate the efficacy of preservation of infectious veev, viral rna, and veev capsid protein in whole blood samples at ambient and elevated temperature as well as at low and high humidity conditions. our results indicate that: (i) magnetic nt particles enhance preservation of infectious veev in whole human blood at 40 • c; (ii) nt maintain significantly higher levels of veev rna in whole human blood at 40 • c; (iii) nt retain their functional activity at both normal and elevated humidity conditions and significantly preserve veev infectivity in such an environment; and (iv) blood samples from veev tc-83 infected animals are better protected from capsid protein degradation if they are incubated with nt. our results demonstrate for the first time the capability of nt particles to stabilize virus in blood at elevated temperatures, the direct interaction of nt particles with veev (via transmission electron microscopy), and the utility of nt particles with viral clinical samples. veev-tc83 viral stocks were produced from electroporation of in vitro transcribed viral rna generated from the ptc83 plasmid [a kind gift from ilya frolov, the university of texas medical branch at galveston] as described (22) . all experiments were performed under bsl-2 conditions. whole human blood and plasma was purchased from bioivt (www.bioivt.com). all of the nt particles were provided by ceres nanoscience, manassas, va (ceresnano.com). veev tc83 was diluted to 1 × 10 6 pfu/ml in whole human blood followed by incubation with nt particle suspension (0.5 mg of nt/ml of sample or 1.25 mg of nt/ml of sample) or without nt particles, rotating for 30 min at room temperature. viral rna was purified using rneasy kit (qiagen). the amount of the viral rna was determined by rt-qpcr using superscript tm iii platinum tm sybr tm green one-step qpcr kit w/rox (thermo fisher scientific) with the following set of primers (integrated dna technologies): 5 ′ -tctgacaag acgttcccaatca-3 ′ and 5 ′ -gaataacttccctccg accaca-3 ′ . the taq-man probe (5 ′ -6-carboxyfluorescein-tgttggaagggaagataaacggctacgc-6-carboxy-n,n,n ′ ,n-tetramethylrhodamine-3 ′ ) was designed against the rna packaging signal as described previously (23) . rt-qpcr was performed using a stepone plus real time pcr system instrument from abi. fold enrichment was calculated based on the enriched viral genomic copy number divided by those in the "without nt particle" group. veev tc83 was diluted to 1 × 10 6 pfu/ml in whole human blood or plasma followed by incubation with or without nt particles at the indicated humidity and temperature conditions for various time points. viral rna was purified using a combination of a trizol r ls from ambion and rneasy mini kit (qiagen). briefly, whole human blood containing viral virions was mixed in 1:3 ratio with trizol ls. hundred microliter of pbs were added to the sample in order to increase the amount of the aqueous fraction. samples were vortexed and 200 µl of chloroform were added to the blood trizol ls mixture. after intensive vortexing and spinning down the upper aqueous fraction containing nucleic acids was collected and used for rna purification by rneasy kit (qiagen) according to the manufacturer's protocol. purified rna was used for cdna synthesis followed by pcr reaction (30 cycles) using a one step rt-pcr kit (thermofisher scientific). for this purpose, the following set of primers was used: 5 ′ -ctg ctc gcc aat gtg acg ttc-3 ′ ; 5 ′ -agc ctg ctc tgt tga cta tag tgt tat acg-3 ′ . to visualize the quantity of viral cdna 10 µl of the pcr product were loaded on 1% agarose gel supplemented with ethidium bromide, followed by gel electrophoresis. samples were visualized on a chemidoc instrument from bio-rad and densitometrically analyzed using image lab software from bio-rad. plaque assay 1 × 10 6 pfu of the veev tc-83 were spiked into whole human blood supplemented (1.25 mg of nt/ml of sample) or not supplemented with nt particles. samples were incubated at the indicated temperature and humidity conditions for the designated amount of time. at the end of the incubation, nt particles containing samples were placed on a magnet rack to separate nt particles from the blood. particles were resuspended in 100 µl of pbs and used for standard plaque assay as described elsewhere (24) . samples that were incubated without nt particles were processed using the same method. veev tc83 was diluted to 1 × 10 6 pfu/ml in plasma followed by incubation with or without nt particles (1.25 mg of nt/ml of sample) at indicated humidity and temperature conditions for various time points. blue lysis buffer (2× novex tris-glycine sample loading buffer sds, t-per tissue protein extraction reagent, 0.5 m edta, complete protease cocktail tablets, 0.1 m na 3 vo 4 , 0.1 m naf, and 1 m dtt) was added to the samples in 1:1 volume ratio, followed by boiling at 95 • c for 10 min. protein lysates were then electrophoresed in a 4-12% of bis-tris sds-page gel and transferred to a pvdf membrane. a 1:2,000 dilution of primary anti-veev capsid antibody (cat#nr-9403, bei, manassas, va usa) in 5% nonfat milk blocking buffer was applied on the membrane at 4 • c for overnight incubation followed by washing with pbs + 0.05% tween 20 three times. horseradish peroxidase (hrp)conjugated anti-goat igg secondary antibody (thermofisher scientific) at a 1:2,000 dilution was incubated with the membrane for 1 h at room temperature. the membrane was washed an additional three times with pbs + 0.05% tween 20 before adding supersignal tm west femto maximum sensitivity substrate (thermofisher scientific) to develop the membrane and image the chemiluminescent signal. samples were visualized on chemidoc instrument from bio-rad and densitometrically analyzed by the image lab software from bio-rad. a total of 27 c3h/hen mice (6-8-week-old) were randomly divided into three groups (n = 9) and veev-tc83 was diluted to 2 × 10 7 pfu/ml with pbs followed by intranasally administrated to anesthetized mice in the amount of 20 µl/mouse. by 2, 3, and 4 days post-infection, 9 mice were sacrificed at each time point and the blood was collected in edta-treated tubes to prevent coagulation. rt-qpcr was performed to quantify the level of viremia and the 6 samples with the highest viremia were pooled together and then incubated with or without nt particles to compare the capsid stability by western blot analysis. experiments were performed in animal biosafety level 2 (absl-2) laboratories in accordance with the national research council's guide for the care and use of laboratory animals (25) and under george mason university iacuc protocol number 0384. the veev structural protein (e1, e2, e3, and capsid) was retrieved from pdb website (id: 3j0c) and the chemical structure of reactive red 120 was downloaded from chemspider website. the heterodimer of e1 and e2 were extracted from the total protein structure with molsoft icm-browser followed by submitting it to swissdock for prediction of possible docking pockets (26) . the docking models were displayed in ball-and-stick style of reactive red 120 and residue surface style of each amino acid on the heterodimer and represented images were selected based on two of the lowest free energy values ( g) on each subunit. inside the laboratory biosafety cabinet (bsc) the nt particle-veev suspension was mixed well with the same volume of 4% glutaraldehyde to achieve a final concentration of 2% glutaraldehyde. virus samples were inactivated with 2% glutaraldehyde inside a bsc for 24 h according to industry standard practice (27) , prior to removal and transfer to the bsl-2 em facility. a drop (8 µl) of the glutaraldehyde treated sample was placed onto a formvar/carbon coated transmission electron microscopy (tem) grid for 10 min in a moist chamber to reduce evaporation. this step ensures that the grid did not dry. using fine forceps to hold the grid, the liquid was wicked away from the grid surface from the side with filter paper. the grid was then washed three times by touching the grid to the surface of drops of deionized (di) water. remaining water was wicked away by touching filter paper to the side of the grid. a small drop of 1% phosphotungstic acid (pta) was applied to the grid and allowed to remain from 10 s to 1 min depending on the sample. the stain was wicked away by touching the edge of the grid to a piece of filter paper. the grid was air dried at room temperature and stored for subsequent tem imaging. tem grids were evaluated on a jeol 1011 transmission electron microscope at 80 kv and all digital images were acquired using an advanced microscopy techniques (amt) camera system. kruskal-wallis test and unpaired t-test were performed throughout this study to calculate the statistical significance among groups with and without nt particles unless indicated elsewhere. nt particles contain affinity baits that enable interactions with analytes through poorly defined characteristics. for example, triazine dyes such as cibacron blue and reactive red 120 bind to proteins through hydrophobic and electrostatic interactions (28) . therefore, the initial step of our study was to screen the panel of magnetic nt particles shown in table 1 , to evaluate their efficacies in enriching veev-tc83. nt particles were mixed with blood-spiked veev-tc83 (0.5 mg of nt/ml of sample) and incubated for 30 min prior to determination of viral rna levels by rt-qpcr. we screened 9 different types of nt particles and found that all the nt particles were capable of enriching veev, but to different levels (figures 1a,b) . to further confirm and test the limit of capture efficiency of the nt particles, we selected three batches of nt particles shown with the best binding efficiencies and enrichment performance, cn3170, cn3160, and cn3080, and mixed nt particles with the blood-spiked veev-tc83 (1.25 mg of nt/ml of sample). all nt particles were capable of enriching veev when used at this concentration, but cn3080 showed the most significant enrichment (bound/unbound = ∼10, figure 2a) , suggesting that the nt particle conjugated with reactive red 120 chemical bait could be the most suitable for the following experiments. we also compared the ability of cn3080 to a non-magnetic version (cn1030) and found that cn3080 was more effective at capturing veev (supplemental figure 1) . to confirm virion capturing by cn3080, we conducted western blot analysis toward the veev-tc83 capsid protein. the capsid protein is only detected in samples that have been incubated with cn3080, but not in samples without nt particles regardless of whether virus was spiked into blood or plasma ( figure 2b) . these data demonstrate the documented ability of nt particles to enrich viral analytes that are below the limit of detection (20) . to characterize the performance of cn3080 we utilized various concentrations of cn3080 ranging from samples without nt particles to a 5 mg of nt/ml of sample to determine the optimal concentration for veev capsid enrichment and detection. to our surprise, the lower concentrations (up to 1.25 mg of nt/ml of sample) seemed to exhibit the most abundant enrichment, but not higher concentrations like 5 and 2.5 mg of nt/ml of sample ( figure 2c ). next, we tested for the optimal incubation time required for nt particle capture of the majority of virions in a sample. thus, we measured the viral capsid concentration at different incubation time-points. as early as after 5 min of incubation, veev capsid protein was detected and by 15-30 min, the capture ability started to reach a saturation level ( figure 2d) . these data show that cn3080 magnetic nt particles provide efficient capture of veev tc-83 virions over short time periods and the binding efficiency of cn3080 to viral samples can be optimized by adjusting both the incubation time and the nt particle concentration. based on these data, we chose an incubation time of 30 min and a nt particle concentration of 1.25 mg/ml of sample for the remainder of our experiments to ensure maximal veev capture. it has been shown in our previous studies that nt particles capture and enrich various viruses, such as influenza virus and rvfv (19, 21, 29) , but it has never been shown if nt particles can directly bind to viruses. using transmission electronic microscopy (tem) we visualized the direct interaction between nt particles and virions. the photo in figure 3 reveals that a single nt particle is capable of capturing multiple virions. our tem data clearly showed that nt particles directly interact with viral particles. therefore, we sought to elucidate the sites of the interaction between the nt particles and veev-tc83. we applied docking simulation via the swissdock website and determined the free energy of binding ( g) toward the affinity bait, reactive red 120, and viral envelope proteins, e1 and e2 (pdb id: 3j0c) (30) . as shown in figure 4 , the docking predictions against the external domains of e1-e2 heterodimer protein reveal that the affinity bait overall interacted with e1 and e2 subunits where the strongest binding was predicted in the groove between e1 and e2 (panel 2a). the free energies of nt binding to each e1 and e2 subunits is summarized in supplemental table 1 . in general, the prediction results suggest that there may be strong interactions between nt particles and veev envelope proteins which may potentially contribute to the viral enrichment exerted by cn3080. previous studies have shown that nt particles are efficient in the preservation of rvfv and nucleoproteins in biologically relevant matrices (19, 29) . however, the effect of nt in the stabilization of virions in a complex matrix such as whole human blood has never been tested before. thus, after determination of the best nt composition that is most efficient in capturing veev tc-83, our next step was to test stability of the virus in whole blood at ambient and elevated temperatures for extended time periods. for this purpose, we spiked 10 6 pfu of veev-tc83 in 0.5 ml of human whole blood and incubated samples at temperatures of 22, 37, 40, and 54 • c for 0, 24, 48, and 72 h followed by measurement of viral infectivity by plaque assays. we observed a gradual drop of virus titers at 37 and 40 • c, whereas viral infectivity remained relatively consistent at 22 • c ( figure 5a) . however, our several attempts to incubate virus at 54 • c for 24 h repeatedly resulted in complete virus inactivation. it should be noted that at 54 • c after 24 h of incubation the blood samples became solidified which made it impossible to completely resuspend samples in buffer for quantitation via plaque assay ( figure 5a) . therefore, we cannot exclude the possibility that some viral particles were captured by coagulated blood and still remained in the sample. as a result of these finding, for most of the following experiments we focus primarily on 37 and 40 • c temperature conditions. we next investigated whether cn3080 can prolong the stability of veev-tc83 in human whole blood. we found that the stability of veev at 37 • c over the time points we tested was not substantially sustained in the presence or absence of cn3080 (figure 5b ) whereas the infectivity of veev tc-83 after 72 h of incubation at 40 • c appeared to be better preserved despite the lack of statistical significance ( figure 5c ). however, it should be noted that only once in four repeats did we observed two plaques developed by virus after 72 h of incubation without nt. in contrast, in all four repeats we were able to detect virus-produced plaques (ranging from 2 to 6 plaques per well) if samples were incubated in presence of cn3080. this indicates that the addition of cn3080 in samples may stabilize the veev virions and preserve infectivity for diagnostic purposes. the primary approach for confirming veev infection in suspected clinical cases is based on detection of the virus specific igm and neutralizing antibodies. reactions based on amplification of the nucleic acid is often used in fatal cases to confirm pathogen presence (5, 19, 31) . rt-qpcr is a very reliable method and highly sensitive for the detection of minimal amounts of viral nucleic acids in the patient samples (11) . however, this method is based on the detection of relatively short fragments of the viral genome and analysis of a longer stretch of viral rna (vrna) would provide a more stringent measure of rna stability. thus, we investigated the stability of both long viral rna (1.5 kb product) by rt-pcr and short vrna (75 bp) by rt-qpcr. veev was spiked in blood and incubated in the presence or absence of cn3080 for the indicated amount of time, followed by rna purification, cdna synthesis and pcr or qpcr reactions. minimal differences in long vrna levels were observed in samples incubated at 37 • c ( figure 6a ). cn3080 overall stabilized long vrna and increased its detection by rt-pcr especially at higher temperature (40 • c) conditions compared to samples that were incubated without nt particles (figure 6b ). densitometric analysis of the pcr products indicated preservation of long vrna in the presence of cn3080 after 72 h of incubation at 40 • c. likewise, rt-qpcr results indicated preservation of short vrna after 72 h at 40 • c (figure 6c) . for comparison, we also monitored the degree of veev capsid protein decay in the presence or absence of cn3080. given the difficulty in analysis of blood samples via western blot, these experiments were performed in blood diluted 1:20 with pbs. as a starting point we tested the ability of cn3080 to prevent capsid protein degradation at standard blood storage conditions. for this purpose, we incubated virus with and without cn3080 at 4 • c and assessed by western-blot analysis the amount of capsid protein in the incubated samples. even though veev capsid was overall more stable at 4 • c, we observed a decrease in capsid protein detection by 72 h, which was rescued by incubation with cn3080 ( figure 7a, bottom panel) . western blot analysis of the samples incubated at 37 and 40 • c also demonstrated that only when incubating with cn3080, capsid can be preserved and more available for detection (figures 7a,b) . thus, nt particles not figure 4 | docking simulation of reactive red 120 and heterodimer of veev e1 and e2 glycoproteins. the heterodimer of e1 (sky blue) and e2 (lemon chiffon) was depicted with molbrowser v3.8 and subdomains of e1 or e2 were labeled as i, ii, iii (e1) and a, b, c (e2). the e1 and e2 subunits were alternatively presented in protein contact surface style in two distinct colors and the reactive red 120 in red was presented in ball-and-stick style. the star and triangle symbols indicated the locations of stem loop and e3 subunit, respectively, and the representative enlarged images were selected based on the lowest delta g free energy provided by swissdock. tm, transmembrane domain. inserted panel d displays one of the strong docking predictions which is located on the rear side of the e1 glycoprotein. only profoundly enriched capsid protein in all samples at all temperature conditions but in their presence the capsid protein degradation was significantly reduced at high temperature. results from the above rt-pcr and protein analyses suggest that cn3080 could efficiently enrich and preserve veev rna and capsid protein against the harsh temperature storage conditions. some studies have shown that relative humidity can affect the stability of airborne viruses including aerosolized veev (32) (33) (34) . therefore, we investigated whether cn3080 could contribute to the blood-spiked veev stability across environmental humidity ranges. we placed the viral samples with or without cn3080 in two incubators with relative humidity at 16 or 98% at 37 • c and incubated them for up to 96 h followed by determination of viral titers. our plaque assay results indicated that viral titers of the blood-spiked veev-tc83 were preserved at both 16% and 98% humidity when incubating with cn3080 for 72 and 96 h post-incubation (figure 8 ). samples incubated in higher humidity conditions were generally more stable, but the addition of cn3080 further enhanced the stability regardless of the level of humidity. our data support the conclusion that nt particles efficiently stabilize infectious virus at elevated and dry humidity conditions for an extended amount of time. to better illustrate the application of cn3080 in clinical samples, we infected c3h/hen mice via intranasal infection of veev-tc83 and measured the viral load in mouse blood at 2, 3, and 4 days post-infection (d.p.i.) followed by supplying cn3080 to demonstrate the preservation capability of nt particles ex vivo. since the mouse blood samples from day 2 showed the highest viral rna concentration among all tested time points (figure 9a) , we used blood harvested from mice at 2 d.p.i., added cn3080, and compared the capsid protein stability with or without nt particles at 40 • c for the indicated amount of time. this experiment was performed with whole blood. in the absence of nt particles, veev capsid was undetectable at all time points (figure 9b) . incubation of mouse blood with cn3080 enable capsid detection at each time point, highlighting the potential use of nt particles in clinical samples. nt particles are made of a hydrogel matrix and can be customized with various affinity baits such as acrylic acid, cibacron blue dye, and reactive red dye by polymerization (35) . as such, they can bind a range of biomolecules. in our previous studies, we have found that nt particles containing a reactive figure 8 | cn3080 preserves infectious veev in humid and dry environments. 2.25 × 10 8 pfu/ml of veev tc-83 was added to blood diluted 1:20 in pbs followed by incubating with (1.25 mg of nt/ml of sample) or without cn3080 at 37 • c in 16 or 98% humidity conditions and preservation was measured by plaque assays with cn3080 or without cn3080. values below the detection threshold were set to 1. data are plotted as the geometric mean ± sem from results of two independent experiments (n = 2). red 120 affinity bait allows nt particles to capture a wide range of rna viruses (19, 29) . using the newly developed magnetic nt particles could simplify workflows allowing the isolation of nt particles in 3-5 s instead of long centrifugation steps. this processing allows the virions present in clinical samples and bound to nt particles to be easily and quickly separated from body fluids using either manual or automated methods. however, one of the obstacles in pathogen identification in whole blood is the natural abundance of host proteins and red blood cells that prevent direct identification of the pathogen from blood and often require additional preparation steps. another inconvenience related to working with whole blood or other body fluid sample is a necessity to keep it at low temperatures during transportation. such obstacles make blood a very difficult matrix for sample preparation and diagnostic analysis. however, despite the existence of the above-mentioned obstacles, we observed efficient separation of veev viral particles from blood by nt particles at various temperature and humidity conditions. moreover, nt particles efficiently preserved infectious veev tc-83 at conditions where it was otherwise undetectable. as much as our nt particle work has shown a significant benefit in preservation and enhancing detection of veev virions confirmed by stabilization of viral capsid protein, viral rna, as well as infectious virions, there are limitations in the use of nt particles. when the environmental conditions are too harsh, the nt particles are only capable of partially alleviating the negative effects. despite the trend showing more preservative via nt particles, not all viral titers at the later time points exhibited statistical differences, suggesting the preservation of nt particles may not be sufficient to prevent the ultimate decay of virions under the most extreme environmental conditions. another limitation for the use of nt particles is the difficulty in separating virions from nt particles. we have previously shown very strong binding of rvfv virions to nt particles (19) . working with rvfv we were able to elute only 5.5% from the total virions bound to nt. for this purpose we used sodium chloride in the highest concentration that does not induce virus degradation (2 m), but even this concentration was not enough to completely elute virions from nt. although strong lysis buffers, such as trizol reagent and ripa buffer, can be utilized for precipitating rna or extracting viral proteins for diagnosis purposes, we have not found a method to elute the virions while retaining the viral infectivity. as shown in our electronic micrograph (figure 3) , a nt particle is potentially bound with dozens of virions, indicating the efficient capturing ability of nt particles. however, the principle of plaque assay is to dilute viral samples until a single virion can form a plaque, so called plaque-forming unit, visualized, and countable by investigators. overwhelming capture affinity of nt particles to virions is an obstacle for the accuracy of the plaque assay method, which might lead to an underestimation of initial viral titers ( figure 5) . therefore, a better way to elute virions needs to be developed to enable more accurate viral titer determination. one possibility for future testing is to competitively elute veev via addition of increasing concentration of the reactive red affinity bait. despite the limitations discussed above, our data indicate that nt particles are a useful innovation to researchers. the nt particles can be customized with various affinity baits to bind different microorganisms or biomolecules. in addition to the viruses, nt particles have been shown to be capable of binding the outer surface protein of borrelia, the bacteria that causes lyme disease, and increasing the specificity of diagnosis for early stage of lyme disease (18) . also, biomarkers in clinical fluid samples or secreted extracellular organelles like exosomes can be captured and the sensitivity of their detection enhanced via nt particles (36) (37) (38) . the ability of the nt particles to preserve veev and its components including capsid protein and genomic rna in such complex matrix such as whole blood highlight an importance to further study the capabilities of the nt particles. overall, the magnetic nt particle presented in this study demonstrated efficient binding affinity to capture one of the biological warfare agents, veev, allowing laboratory technicians to detect this particular pathogen from clinical samples of patients or soldiers in the front line. moreover, the high efficiency in preservation of live pathogen and its proteins and nucleic acids make nt particles an attractive candidate to be used as a stabilization agent in a new blood collection medical device that are currently undergoing development and testing. hence, utilization of the nt particles can be an alternative method to enhance the surveillance system to monitor and prevent the outbreaks. our study provides mechanistic insights for broad use of nt particles. all datasets generated for this study are included in the article/supplementary material. the animal study was reviewed and approved by george mason university institutional animal care and use 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detection of respiratory pathogens with nanotrap particles selective inhibitor of nuclear export (sine) compounds alter new world alphavirus capsid localization and reduce viral replication in mammalian cells conservation of a packaging signal and the viral genome rna packaging mechanism in alphavirus evolution viral concentration determination through plaque assays: using traditional and novel overlay systems guide for the care and use of laboratory animals swissdock, a protein-small molecule docking web service based on eadock dss evaluation of virus inactivation by formaldehyde to enhance biosafety of diagnostic electron microscopy chromatography of plasma proteins on immobilized cibacron blue f3-ga the use of nanotrap particles in the enhanced detection of rift valley fever virus nucleoprotein 4.4 a cryo-em structure of an enveloped alphavirus venezuelan equine encephalitis virus the use of nanotrap particles technology in capturing hiv-1 virions and viral proteins from infected cells effect of relative humidity and temperature on airborne venezuelan equine encephalitis virus effects of temperature, relative humidity, absolute humidity, and evaporation potential on survival of airborne gumboro vaccine virus low ambient humidity impairs barrier function and innate resistance against influenza infection synthesis and characterization of hydrogel particles containing cibacron blue f3g-a core-shell hydrogel particles harvest, concentrate and preserve labile low abundance biomarkers a novel biomarker harvesting nanotechnology identifies bak as a candidate melanoma biomarker in serum exosomes from uninfected cells activate transcription of latent hiv-1 the authors thank kathleen kuehl and rahul raychaudhuri, united states army medical research institute for infectious diseases, for assistance with the tem sample preparation and imaging, respectively. the authors thank erwin berthier and emily welch, tasso inc, for helpful discussions. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets. 2019.00509/full#supplementary-material conflict of interest: ap and bl are employed by the company ceres nanosciences, inc. kk-h is a member of the scientific advisory board at ceres nanosciences, inc. bl is a shareholder at ceres nanosciences, inc. the nanoparticles used in this study were research grade and provided by ceres nanosciences, inc. which are not commercially available products. the authors declare that tasso inc had no role in the design of the study and collection, analysis, interpretation of the data or writing of the manuscript.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 akhrymuk, lin, sun, patnaik, lehman, altamura, minogue, lepene, van hoek and kehn-hall. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-265642-7mu530yp authors: syomin, b. v.; ilyin, y. v. title: virus-like particles as an instrument of vaccine production date: 2019-06-17 journal: mol biol doi: 10.1134/s0026893319030154 sha: doc_id: 265642 cord_uid: 7mu530yp the paper discusses the techniques which are currently implemented for vaccine production based on virus-like particles (vlps). the factors which determine the characteristics of vlp monomers assembly are provided in detail. analysis of the literature demonstrates that the development of the techniques of vlp production and immobilization of target antigens on their surface have led to the development of universal platforms which make it possible for virtually any known antigen to be exposed on the particle surface in a highly concentrated form. as a result, the focus of attention has shifted from the approaches to vlp production to the development of a precise interface between the organism’s immune system and the peptides inducing a strong immune response to pathogens or the organism’s own pathological cells. immunome-specified methods for vaccine design and the prospects of immunoprophylaxis are discussed. certain examples of vaccines against viral diseases and cancers are considered. immunoprophylaxis has long been used to control infectious diseases exerting an enormous impact not only on the global health but also on the safety of numerous populations of agricultural animals. in the past decades, the focus on vaccine production technologies has changed from handling intact pathogens to producing recombinant subunit vaccines based on isolated target antigens [1] . intensive development of recombinant vaccines began in the 1980s when it became possible to clone the desired dna sequences into expression plasmids and produce the target proteins. the construction of a recombinant vaccine takes advantage of the knowledge of the nucleotide sequences of genes encoded by a pathogen, and involves identification of the antigenic determinant, synthesis of the nucleotide sequence encoding the antigen, its cloning into an expression vector, and production of the target peptide in a certain expression system. the expression systems for individual heterologous proteins has been developed based on the prokaryotic and eukaryotic cells, which allow us to produce target proteins both on laboratory and industrial scales [2] . the interest in recombinant vaccines is a consequence of the emergence of new infectious diseases, most often zoonotic ones. among them are the outbreaks of human diseases caused by the ebola virus, zika virus, marburg virus, the middle east respiratory syndrome, and severe acute respiratory syndrome coronaviruses [3] . oncology also places great hopes in recombinant vaccines, which may help to overcome immune tolerance in the case of cancer treatment [4] . moreover, there exists a constant threat of the emergence of new highly virulent strains of well-known viruses due to unceasing mutational processes in virus genomes [5] . against this background, a new scientific concept, vaccinomics [6] , which consists of identifying the minimum subset of antigens, which are able to induce a competent immune response to a pathogen or tumor by their specific interaction with the b and t immune cells, is being actively developed. using this approach it appears possible to design vaccines based on the minimum subset of antigens which most specifically characterize the pathogen in its interaction with the immunome (the set of all immune receptor sequences, which are present in the individual organism) [7] . target detection of epitopes, the regions located on the surface of the protein, is possible with the aid of x-ray analysis, which is rather labor-consuming since it requires obtaining protein crystals. presently, the 3-d protein structure modeling has found broad use. this approach is based on identification of the physicochemical and electrostatic characteristics of the polypeptide chain regions and correlation of these characteristics with antigenic properties. two strategies are currently utilized, namely, modeling by homology and abbreviations: vlp, virus-like particles. udc 636.2+611:619: 616-006.446 de novo modeling. in the first case, the protein data bank (http://www.ncbi.nlm.nih.qov/genbank/) database of 3-d protein structures is used to model the protein. the commonly used modeller [8] , as well as other software such as i-tasser [9] , swiss-model [10] , esypred3d [11] , 3d-jigsaw [12] , phyre [13] , and cphmodels [14] , can be used as homology modeling tools. the limitation of this approach lies in the requirement that the structures of homologous proteins should demonstrate more than 30% identicality [15] . the de novo modeling strategy is based on the computer simulation of the protein folding process (rosetta [16] and tasser [17] software). in this case, all possible variants of polypeptide chain folding are considered, and the most energetically favorable conformation, i.e., the one with the lowest potential energy, is chosen. predicted antigenic determinants are synthesized using genetic engineering vector techniques and heterologous protein expression systems, and then are experimentally tested using, for example, the antibody neutralization assay. using protein expression systems it is possible to produce virus-like particles (vlps), which are made up of monomers, which are able to multimerize into vlps, and display the antigenic determinants of target pathogens on their surface. this point will be addressed further in the review, we will just observe here that even complex epitopes represented by trimers may be obtained using heterologous expression systems. this has been demonstrated, for instance, for the trimeres of the human immunodeficiency virus (hiv-1) envelope glycoprotein [18, 19] and influenza virus haemagglutinin [20] . in order to choose the most specific antigens of the pathological agent of a new infectious disease, it appears inevitable to work with the infected material if only to isolate and sequence the nucleic acids containing the genetic information of the pathogen. however, in order to produce a vaccine based on epitopes characteristic for the target pathogen, there is no need to work with the pathogen itself. therefore, apart from all other advantages of the vaccines of this kind, they appear to be improved in terms of biological safety. vaccines based on the presentation of a subset of antigenic determinants of the infectious agent are characterized by a high level of reproducibility when commercially manufactured and are highly effective [21] , since in this case, the immune response is directed exclusively against the most significant antigenic elements of the pathogenic microorganism or tumor. a number of vaccines specifically interacting with certain immune system receptors have been tested to date. for example, a vaccine containing only a single epstein-barr virus epitope specifically recognized by cd8 + t-cells is proposed for the prophylaxis of mononucleosis in human [22] . this vaccine prevents the development of the disease, although it does not protect the organism from the entry of the virus. another example is classical swine fever virus (csfv). it has been demonstrated that the e2 viral protein produced in the baculovirus expression system induces the synthesis of csfv-neutralizing antibodies in pigs [23] . vaccines are designed to produce immunity to a disease. although a single epitope is able to induce a strong immune response, it appears usually insufficient to induce protective immunity. the in vitro synthesized peptide properly representing the main antigenic determinant of the pathogen is highly likely not to be itself a strong immunogene primarily because of its small size (<10 nm) and high risk of proteolytic degradation. this problem may be overcome by the use of nanoparticles. it has been shown in a considerable number of works that in order to induce a strong immune response, antigenic determinants should be exposed on the surface of nanoparticles whose shape and size (20-150 nm) mimic those of the virus (fig. 1) . in this respect also, vlps, the nanoparticles composed by viral proteins capable of self-assembly (multimerization) into structures morphologically resembling viruses, are the choice selection for the role of a strong immunogene. antigenic determinants multiply repeatedly on the vlp surface and promote complement fixation and b-lymphocyte receptor clusterization leading to the activation of the immune response. additionally, the same antigen multiply displayed on the surface of the particle promotes the multiplication of the pool of autoreactive b-cells, which is the primary objective when designing vaccines against autoimmune diseases and tumors [29] . natural sources for vlp bioengineering and the specific features of their assembly structural proteins of various viruses are capable of autonomous self-assembly into vlps. they interact with the formation of globular, icosahedral, or rodlike structures. so far, the structural proteins of several dozen viruses have been obtained in heterologous expression systems, and almost all of them proved able to form vlps. the vlp size varies from 20 to 200 nm and is similar to the size of the corresponding viruses [30] . being similar to viruses allows vlps to penetrate into the lymph and to be efficiently entrapped by antigenpresenting cells [31] . currently, several heterologous expression systems and the corresponding expression vectors which can be used with them are available. these systems are based on both the bacterial cells (the pet system based on escherichia coli cells is often exploited) and different eukaryotic cells. in the case of eukaryotic cells, the most commonly used systems are yeast expression systems and the ones which rely on drosophila [32] and spodoptera frugiperda insect cell lines [33] . mammalian cells are also utilized, the most pop-ular among them being cho and hek293 [34] . the cost of heterologous protein production in bacterial systems is lower than in eukaryotic cells. however, when studying the functional activity of the protein or vlp associated, for example, with the potential glycosylation sites or interaction with ubiquitin, or other ubiquitin-like peptides [35] , no alternative to eukaryotic expression systems is available. it should be noted that the choice of the expression system is up to the researcher and is driven by the research task. for example, in different laboratories different eukaryotic systems for viral protein expression, including plant cells, are used to produce vlps which are used for vaccination against the hepatitis c virus (hcv) [36] . in most cases, vlps assembled from a virus protein are considered as a candidate vaccine against this very virus, since protein monomers in multimeric configuration (vlp) induce a more potent immune response than protein monomers [29, 30] . the strong immunogenic properties of vlps are determined by several factors. first of all, the dominant epitope of the structural protein is displayed as a part of the particle and is present in a multimeric form as is the case with a native virion [21] . second, vlps stimulate b-lymphocytes and induce t-lymphocytes in the same manner as does the virus infecting the host organism [30] . for example, vlps formed by the main capsid protein l1 of the human papilloma virus of several serotypes are successfully used as vaccines against cervical cancer [37] . a vaccine against hepatitis b virus has been produced which contains vlps in the lipid membrane envelope [38] . the vaccine against coxsackievirus a6 contains vlps assembled from the virus capsid proteins produced in the baculovirus expression system [39] . scientists from china [40] have demonstrated that the capsid protein (vp60) of the rabbit hemorrhagic disease virus (rhdv) efficiently multimerize into vlps, and a single intramuscular injection of the obtained vlp preparation completely protects the immunized rabbits against rhdv infection for at least 180 days. the porcine circovirus capsid protein is also able to efficiently assemble into vlps when synthesized either in the human embryonic kidney cell culture (hek293) [41] , or yeast [42] and baculovirus [43] expression systems, as well as in bacteria [44] . footand-mouth disease vlps are assembled from three structural polypeptides vp0, vp1, and vp3 (naturally produced as a result of processing the p1-2a precursor polypeptide), simultaneously produced in e. coli [45] . it has been recently demonstrated that the polyprotein 2) liposome-based particles [24] , (3) nondegradable spherical nanoparticles (for example, metal nanoparticles) [25] , (4) polymer nanoparticles [26] , (5) graphene nanosheets [27] and nanotubes [28] . antigen of the duck hepatitis a virus produced in the baculovirus expression system assembles into vlps immediately in the cultured spodoptera frugiperda (sf9) cells, while immunization of ducklings with the obtained vlps induces a high level humoral immune response and protects them from developing the disease [46] . the abilities of vlps resulting from multimerization of the cloned virus protein to play the role of an immunogen are not limited just to the presentation of their proper epitopes but may also be taken advantage of to display heterologous proteins. multimerization of capsid proteins into a particle requires the presence of nucleic acid, while for in vitro assembly, a short oligonucleotide (7-10 nt) is sufficient [47] . therefore, vpls formed by capsid proteins are free from the infectious virus rna or dna. moreover, the above-mentioned property, which triggers protein monomer multimerization by nucleic acid, may be taken advantage of to package rna or dna into a particle with two different objectives. hence, antisense rna can be used to suppress virus expression. for example, in the case of the vaccine against foot-and-mouth disease, researchers from china [48] not only displayed the vp1 epitope of the foot-and-mouth disease virus on the surface of vlps but also packaged the antisense rna complementary to the fragment of the viral genomic rna into a particle. another goal of nucleic acid packaging into a particle lies in the presentation of viral nucleic acids leading to the activation of specific immune receptors which induce the synthesis of type i interferons (inf) and other cytokines triggering the antivirus response [49] . it has been demonstrated that long dna and rna molecules may be incorporated into vlps. for example, mrna for the reporter protein, red fluorescent protein, was packaged into particles representing the hepatitis e capsids [50] . the plasmid containing the green fluorescent protein (gfp) gene was encapsidated into the vlps assembled in vitro from the main capsid protein of the hamster polyoma virus [51] . a strategy for the packaging of up to 17 kbp doublestranded circular dna into the particles was developed using the structural protein of the sv40 simian retrovirus expressed in baculovirus system [52] . there exist two fundamentally different approaches for nucleic acid incorporation into vlps: (1) in vitro vlp assembly from protein monomers in the presence of rna or dna, which is to be encapsidated into the vlp; (2) exposure of the assembled vlps to osmotic shock in the presence of nucleic acids. osmotic shock is produced by using a solution with a low ionic strength; nucleic acid enters into a vlp as a result of the shift in the surface structures [53] . however, the incubation of vlps in the presence of nucleic acids without exposure to osmotic stress results in a certain part of the nucleic acids becoming associated with the particles [54] . a far more predictable approach is the in vitro assembly of vlps from the mixture of protein monomers and nucleic acids which should be encapsidated in them. in this case, after synthesis in a heterologous system, the obtained protein monomers are purified to completely remove the contaminating nucleic acids from the protein preparation; then the proteins are denatured in 7-8 m urea, nucleic acids which should be packaged are added, and the proteins are assembled into the particles by eliminating urea from the solution. the use of this strategy was demonstrated, for instance, in the works [47, 55, 56] . protein association into a particle is a reversible process. vlp dissociation can be easily achieved by the addition of a denaturing agent. further, it may be removed and vlps may be reassembled in vitro [47] with the encapsidation of the target rna or dna into the particle. this approach was implemented for the p21 protein of the hepatitis b virus. virus proteins produced in the heterologous expression system were denatured in 7 m urea solution, and their assembly into vlps in the presence of the nucleic acid was further induced [57] . protective immunity is controlled by specific humoral and cellular mechanisms activated by the antigen. posttranslational protein modifications and covalent bonds between the modifying molecules and the functional groups in the polypeptide chains are of great importance for immune homeostasis during the antiviral response. the balance between phosphorylation, ubiquitination, methylation, acetylation, sumoylation, adp-ribosylation, and glutamilation of a certain antigen performs the fine adjustment of the host's antiviral response [49, 58] . the structural proteins for vlp production when synthesized in the eukaryotic expression systems undergo a number of posttranslational modifications. in particular, the gag gypsy monomer proved to be a natural substrate for type 2 casein kinase [59] . moreover, while produced in the eukaryotic cell, gag gypsy monomers become associated with ubiquitin and sumo, the cellular protein partners of the viral structural protein [33] . generally, ubiquitin and sumo can bind with any lysine residue within the protein molecule, although there are preferable binding sites; it should also be noted that phosphorylation determines which of these two partners binds with the protein [60, 61] . additionally, binding with a limited number of the above-mentioned signal peptides, such as monoubiquitination [62] , usually plays a regulatory role and controls the transport of the protein itself, or the particle formed by it. in the case of polyubiquitination, the protein is destined for proteasome degradation [60, 63] . by constructing recombinant polypeptides based on the viral capsid proteins it is possible to obtain vlps bearing several antigens. for example, dna encoding the vp2 capsid protein of the porcine parvovirus was fused with the dna fragment encoding 35 amino acid residues of the main antigen of porcine circovirus (pcv2) nucleoprotein. the resulting hybrid dna was used to produce protein in heterologous cells which further formed vlps. these vlps induced a significantly stronger immune response against pcv2 than the recombinant adenovirus encoding the open reading frame 2 (orf2) of pcv2 [41] . another interesting example is represented by the vlps formed by the influenza virus matrix protein and displaying a toxoplasma gondii antigen on their surface [64] . there exists another approach to designing therapeutic vaccines based on vlps. in this case, the vlp surface displays the variable fragment of an antibody specific to the antigen of the target virus [65] . moreover, knowing which receptor is bound by the virus proteins renders it possible to produce particles possessing tropism to the cells of certain tissues. for example, the pre-sprotein of the hepatitis b virus exposed as a ligand on the vlp surface provided for their specific binding with hepatocytes [66] . vlps assembled from the structural proteins of bacteriophages are also considered as carriers for human and animal vaccine production. for example, the dna fragment encoding foreign protein was inserted into the dna region encoding the n-terminal β-hairpin of the coat protein of the e. coli ms2 phage. it has been demonstrated that the expression of the obtained construct in bacterial cells resulted in the production of the recombinant protein in which a foreign peptide was present in the central part of the hairpin. the obtained chimeric protein monomers were able to self-assemble into particles morphologically similar to the phage capsid both in vivo and in vitro [67] . using the mentioned property of the ms2 coat protein, vlps displaying the ep141-160 epitope of the foot-and-mouth disease vp1 structural protein on their surface were obtained. these chimeric vlps induced string immune response in the animals, which allowed regarding them as a promising base for the development of a prophylactic vaccine [68] . it is worth noting that the exposure of ep141-160 on the vlp surface resolved a long-standing problem of how to make use of the beneficial properties of this peptide. researchers from several laboratories have demonstrated that this antigenic determinant of the footand-mouth disease not only induces the production of neutralizing antibodies but also stimulates t-lymphocytes [69] . however, when isolated, ep141-160 is not able to induce the immune response protecting animals against the foot-and-mouth virus infection [70] . many attempts were made to solve this problem, for example, by combining the antigenic determinant with large molecules, such as for instance, t cell-specific molecules [71] . however, only integration of ep141-160 into vlps resulted in the possibility of using this antigenic determinant as a strong immunogen for vaccine production [68] . practically all structural proteins of phages infecting various species of bacteria are able to autonomously form vlps. for example, salmonella typhimurium phages are able to autonomously multimerize into vlps on whose surface the epitopes of eukaryotic viruses, including the epitopes of the human influenza virus may be exposed [72] . at the same time, however, bacteriophages are apparently incapable of presenting large epitopes, whose length exceeds 24 amino acid residues [67] . structural proteins of retroviruses and retrotransposons have a higher capacity compared to bacteriophage proteins. this means that protein monomers forming the particle may be fused with a longer peptide and still retain the ability to multimerize. in particular, it has been shown for the gag gypsy capsid protein [73] , that at least 26% of its amino acid sequence (more than 100 amino acid residues) is of little importance for multimerization into vlps [55] . therefore, the truncated form of this protein may be fused with a heterologous peptide with the length similar to the length of the deleted fragments and a recombinant protein may be obtained which will retain its ability to self-assemble into vlps. in such a way, the truncated gag gypsy fused with the heterologous peptide formed particles when it was synthesized in bacterial cells. we were also able to assemble particles from the purified protein monomers in vitro [47, 55, 74] . the obtained particles resembled the native gypsy virus by their morphology [75] . the substitution of the deleted region in the dna encoding gag gypsy by the nucleotide sequence encoding the main antigenic determinant of the foot-and-mouth virus vp1 protein (ep141-160) and his 6 -tag resulted in the formation of particles which displayed ep141-160 as the target antigen. the advantage of the technique used is that vlps formed by the protein containing his 6 -tag can be readily isolated and purified by affinity chromatography [76] . chimeric vlps may be obtained through the construction of recombinant dna molecules encoding both the corresponding virus protein and a foreign peptide or protein. another way is vlp pseudotyping. this approach was elaborated using the hamster polyomavirus. the v1 capsid protein of this virus first forms pentamers which further assemble into vlps comprised of 72 pentamers. when v1 is expressed together with the minor capsid protein v2, the latter binds with the central part of each v1 pentamer. it has been demonstrated that the n-terminal part of v2 is not involved in this interaction and therefore may be removed and substituted by the target epitope [77] . another approach is also known. antigen is immobilized on the vlp via covalent binding between the reactive groups of the amino acid residues of the antigen and vlp. this approach proved to be ineffective due to the disruption of the native conformation of the antigen attached to the particle surface [77, 78] . this problem was countered in the following way. glygly-lysglygly sequence was inserted into the monomer subunits for vlp assembly. in this environment, the reactive ε-amino group of lys residue is exposed and ready to interact with the cys-group of any protein in the presence of the cross-linking agent, such as m-maleimidobenzoyl-n-hydroxysulfosuccinimide ester (sulfo-mbs) [79] , or succinimidyl-6-[(β-maleimidopropionamido)hexanoate] (smph) [80] . although the authors claimed that the described system of molecular assembly may be used to induce strong blymphocyte response against most antigens and prospective vaccine prototypes were suggested, this platform became the prototype only for a single prospective preparation based on vlps, the medication for lowering blood pressure in patients with hypertension [80] , due to the development of the new improved techniques for antigen immobilization of the vlp surface, which will be discussed below. one of them takes advantage of the ability of his 6tag to bind with multivalent tris-nitrilotriacetic acid (trisnta) which in turn binds with a broad range of molecules, in particular, with biotin. the possibilities of this elegant technique for the functionalization of noninfectious viral nanoparticles were demonstrated in the case of vlps formed by the norovirus (nov) structural proteins. using the baculovirus expression system, the authors obtained nov vlps, displaying his 6 -tag on their surface, which was first used to purify vlps, and further to bind trisnta molecules conjugated with a fluorescent dye, or biotin which in turn successfully bound streptavidin [81] . notwithstanding some authors suggesting the described technique to be promising for vlp vaccine production, this approach continues to be only a prototype and has not advanced further than model experiments. the search for techniques providing efficient and stable immobilization of the antigen on the vlp surface led to the development of a versatile platform which allows us to covalently attach large antigens to the vlp surface [82] . this molecular assembly system uses the tag-catcher conjugation system which was derived from the cnab2 domain of fibronectin-binding protein fbab from streptococcus pyogenes. as a result a highly reactive spytag peptide (13 amino acid residues) was obtained which efficiently interacted with the spycatcher protein with the formation of isopeptide bonds in a broad range of buffer solutions [83] . the spytag peptide was inserted into the vlp-forming polypeptide of the acinetobacter-infecting phage (these particles were called ap205), so that each monomer forming ap205 displayed two spytag peptides. a recombinant antigen consisting of the target protein and the spycatcher peptide was produced using bacterial or baculovirus expression systems [84] . the obtained antigen efficiently bound with the vlp surface and induced a very strong immune response. almost the same effect was observed in the case of the reverse combination of the conjugating peptides, that is when spycatcher was incorporated into vlps, and spytag was fused to the antigen. this system is already utilized to design different types of vaccines, including the vaccines against tuberculosis and malaria [84, 85] . a recent report has demonstrated that it was successfully used to develop a vaccine against breast cancer [86] . this work is remarkable due to the fact that the high density of the her2 antigen (human epidermal factor receptor 2) synthesized in the cultured drosophila melanogaster cells could be obtained on the ap204 vlp surface [32] . the obtained particles induced a strong immune response to the antigen in the patients, which rendered it possible to overcome the immune tolerance to her2 known for patients with the her2-dependent breast cancer [85, 86] . in summary, the analysis of the discussed published data allows us to conclude that the nature of the vlp-forming protein is not as important for vlp functionalization as the technique which makes it possible to obtain high antigen density on the surface of vlps (table 1) . the product range a number of vlp vaccines are available on pharmaceutical markets in many countries. the most wellknown are the cervarix® and gardasil® vaccines against cervical cancer, which have been successfully used for prophylaxis of this disease in girls for more than ten years. both vaccines are based on vlps formed by the main capsid protein l1 of the human papilloma virus belonging to several serotypes [37] . engerix and recombivax hb vaccines against the hepatitis b virus representing vlps enveloped in a lipid membrane [38] , as well as hecolin and xiamen innovax vaccines against hepatitis e [88] , were developed and commercialized. the anti-malaria vaccine malaria rts has been certified, which represents the c-terminal part of the cs protein of plasmodium falciparum attached to the surface antigen of the hepatitis b virus (hbsag), which is also used in the certified vaccines against hepatitis b. to stabilize recombinant vlps, the fused protein is expressed together with the hbsag protein in saccharomyces cerevisease [89] . a vaccine against the porcine circovirus representing vlps assembled from the vp2pcv2 capsid protein synthesized in a heterologous system has also been commercialized [41] . an increasing number of works have appeared that report on the development of vaccines based on nanoparticles, including vlps and/or replicationinactive viruses. among the apparent advantages of vaccines of this kind are their high specificity, efficiency, and good pharmacokinetic characteristics. it has been demonstrated that in an organism vlps reach lymphatic nodes in less than 10 min, while the particle mixture bearing different antigens may be pro-cessed by the same antigen-presenting cells simultaneously [90] . it should be noted that nanoprticle-based vaccines offer new possibilities in the development of immunoprophylaxis strategies, especially, the development of injection-free formulations, in particular, intranasal vaccines and inhalers. the administration of these vaccines may not involve humans, which is especially important in the case of industrial animal breeding in large agricultural enterprises characteristic of presentday russia [19] , since it proves to be extremely difficult to administer identical injections to a large number of animals at the same time. this problem may be resolved by nebulizing aerosol vaccines in the area where animals are kept. the potential of this approach was demonstrated for the first time in 1947 with mice [92] ; then, in the 1950s-1970s, attempts were made to introduce this approach into animal and poultry farms in many countries. starting from the 1960s, attempts were also made in the soviet union [93] . however, at that time, the method of inhaled administration of vaccines was not introduced in practice since protective immunity could only be obtained when the tolerable dose was exceeded many times. for example, the inhaled administration of the vaccine against the newcastle disease led to 10-20% lethality in birds [94] . however, due to the introduction of vaccines based on the exposure of the epitopes of a pathogen on the surface of nanoparticles, including recombinant vlps, together with the development of innovative approaches to aerosol production, inhaled vaccines again became a topical issue. for example, biodegradable polymer nanoparticles formed by poly(glyceroladipate-co-ω-pentadecalactone), pga-co-pdl, proved themselves as an effective antigen carrier for inhaled vaccination [95] , while the vaccine based on the adenovirus with the particles ranging from 4 to 10 µm induced stable protective immunity when administered via inhalation to monkeys [96] . lastly, a method for vlp-vaccination via inhalation was proposed as protection against the human papilloma virus [97] . one of the most important properties of vlps is mimicking virus particles and the consequent ability to induce a strong immune response to the antigen which they demonstrate irrespective of the source of the monomers which multimerize into vlps, these being either insect viruses, in particular the gypsy virus [56, 61] , or plant viruses [98] . vlps based on the structural proteins of plant viruses produced in plants [99] make it possible to obtain vaccines with another nonconventional way of administration, edible vaccines [100] . vaccines of this type may be synthesized directly in plant forage, with the oral vaccination of this kind inducing an immune response. expression vectors for foreign protein production in plants have been developed based on plant viruses, which allows obtaining plant-producing recombinant viruses or vlps displaying the target antigen on their surface [101, 102] . vlps assembled from the structural proteins of plant viruses are also used to design functionalization platforms for antigen binding. these platforms are based on the strategies described above. in particular, they take advantage of the reactivity of the lys ε-group to immobilize the antigen on the surface of the plant's vlps [102] [103] [104] [105] . using the tobacco mosaic virus [106] and potato x virus [104] , it has been shown that a considerably large antigen can be immobilized on the surface of the corresponding vlps via the formation of the streptavidin-biotin complex. finally, based on the structural protein of the plant virus, the platform for antigen immobilization using the tag-catcher conjugation system is being developed [107] . it should be noted that among the drawbacks of vlp vaccines manufactured using proteins which are not specific for mammals, for example, the plant virus proteins, include the development of the immune response to the structural component of the particles, which is the plant virus protein. undoubtedly, there is no therapeutical benefit in developing such immunity; however, whether there are adverse effects will be demonstrated by further studies. model molecules including biotin, alexa 488 (fluorescent dye), and gfp protein conjugated with trisnta [81, 87] isopeptide bond formation between spytag (integrated into vlp monomers) and spy-catcher (fused with antigen) peptides vaccine against breast cancer (her2 antigen presentation). it is also implemented to develop vaccines against malaria and tuberculosis [82] [83] [84] [85] [86] the use of vlps for vaccination in medicine is becoming increasingly widespread. this point is reinforced by the list of clinical trials of vlp vaccines prepared by the united states national institute of health. currently, more than hundred vlp vaccines, which are directed against human and avian influenza viruses, the norwalk virus, norovirus, hiv-1, and the chikungunya virus, the foot-and-mouth disease virus, as well as against melanoma, adenocarcinoma, papillomatoses, and cervical cancer, are undergoing clinical trials (http://clinicaltrials.gov/ct2/search/index). it may be expected that the variety of nanoparticle vaccines against different cancers and viral infections will grow steadily, and vlps which can be used for immunization against microorganisms belonging to different taxons as well helminthes will also be developed. we thank the designers oleg vasil'ev and galina podzolkova (institute for statistical studies and economics of knowledge (issek), national research university higher school of economics) for their assistance in preparing the illustrations. the article was prepared within the framework of the basic research program at the national research university higher school of economics (hse) and supported within the framework of a subsidy by the russian academic excellence project 5-100. the authors declare that they have no conflict of interest. this article does not contain any studies involving animals or human participants performed by any of the authors. 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lettuce-based edible vaccine expressing the cysteine proteinase of fasciola hepatica for fasciolosis control in livestock a launch vector for the production of vaccine antigens in plants rotavirus vp6 expressed by pvx vectors in nicotiana benthamiana coats pvx rods and also assembles into virus-like particles chemical conjugate tmv-peptide bivalent fusion vaccines improve cellular immunity and tumor protection potato virus x as a novel platform for potential biomedical applications incorporation of tetanus-epitope into virus-like particles achieves vaccine responses even in older recipients in models of psoriasis, alzheimer's and cat allergy. npj vaccines. 2, 30 modified tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications use of plant viruses and virus-like particles for the creation of novel vaccines key: cord-032161-sbtu48c2 authors: xu, zhonglin title: particle and size distribution date: 2013-08-07 journal: fundamentals of air cleaning technology and its application in cleanrooms doi: 10.1007/978-3-642-39374-7_1 sha: doc_id: 32161 cord_uid: sbtu48c2 the purpose of air cleaning technology is to get rid of airborne particles as much as possible. gaseous medium containing dispersed airborne particles is one kind of dispersed systems, which is termed as aerosol. 1. visible particle. visible with eyes and the corresponding particle diameter is larger than 10 μm. 2. microscopic particle. visible with the microscope and the corresponding particle diameter is 0.25-10 μm. 3. supermicroscopic particle. visible with supermicroscope or electronic microscope and the corresponding particle diameter is smaller than 0.25 μm. it should be noted that particles with diameter 0.1-5 μm are classified as fine particles; those <0.1 μm are superfine particles, and those >5 μm are big particles. in the technical field of aerosol, terms as "dust," "smoke," and "mist" are frequently used. some definition and concept in air cleaning technology are also usually referred to these terms (such as "dust" concentration in the air and oil "mist" meter). these are the common classifications for particles: 1. dust. it includes all the solid dispersed particles. the movement of these particles in the air is influenced by the combined effect of gravity and diffusion. it is almost ubiquitous in air cleaning technology. 2. smoke. it includes solid coagulated particles, particles formed by coagulation effect from liquid and solid particles, and particles from the transition from liquid particle to crystallized particle. according to iso definition, smoke is clearly defined as "aerosol formed by solid particles during the metallurgy process. it is the gaseous condensate from the vapor generated after the evaporation of melted material. during the formation process, chemical reaction such as oxidation usually occurs." in the usual circumstance, particles from smoke with diameters much less than 0.5 μm are mainly controlled by brownian motion effect in the air, which makes the particles strong disperse capacity, and they are difficult to settle down in still air. smoke stream generated by smoke generator is usually used to detect the leakage on air filters in air cleaning technology field and to perform the experiment for the flow visualization. 3. fog and haze. fog includes all the dispersed liquid particles and coagulated liquid particles. according to iso definition, mist means "the general term for liquid airborne system in air. in meteorology field it means the airborne system containing droplets which causes the visibility length less than 1 km." particle size differs with formation state, and it is between 0.1 and 10 μm. stokes law mainly dominates their movement. for example, sulfate mist generated from so 2 gas will be transformed into oil mist by the combined effect of heat and compressed air, and oil mist could be the standard aerosol for testing air filter. the combination of fog and fine solid particles is termed as haze. coagulated particles. the size ranges from several tenth micrometers to dozens micrometers, such as the combined system formed by coal dust, so 2 , co, and water vapor, which exist in the air of industrial zone (the typical case is the london mist which is the mixture of smoke and mist, and the feo smog generated in iron plant). however, according to iso definition, smog usually represents "visible aerosol generated during combustion process" and "no water vapor included," which means there is a little difference between smog and mist. particle size is usually used to represent how big the particle is. however, not all the particles, especially dust particles, have regular geometric shape such as spherical or cubic. therefore the term "particle size" used frequently does not mean the real diameter of sphere. in the field of aerosol and air cleaning technology, the meaning of "particle size" is some length dimension inside particles, without indication of the meaning of regular geometric shape. when particle scale is analyzed, "particle size" does have this indication. exactly speaking, particle size could be divided into two categories: the first one was measured and defined according to particle's geometric characteristic, such as microscopic method used to determine the particle size. for example, when particles were observed with optical microscopic after the dust sampling, the dust sample was moved towards one direction and through micrometer, where particle was projected onto this scale meter, and the length between two cutting edges by the meter represents the particle size. particles were measured in a sequential and random way, so the long length was determined when the longer side of particle approached, and the short length was measured when the shorter side approached (shown in fig. 1.2) . the long and short sides were called directional tangential cutting edge or random diameter. when the number of measured particles was large enough, results could reflect the average cross section of particle sample properly. in this way, it is 2 one method to determine particle size convenient to take measurements. however, in some cases only the maximum projected distance was adopted as particle diameter, such as several standards about cleanroom in the usa. obviously micrometer must be rotated during measurement, and the location for maximum distance could not be fixed precisely. so the japanese standard "measurement of airborne particles in cleanroom" (japanese industrial standards, jis) suggests no need to rotate micrometer, and instead only the projected maximum length is estimated, which was thought to cause small error. for cubic particles, the diagonal is also used to represent particle size, so it equals with the multiplication of the side length and ffiffi ffi 3 p . for particles whose projected area is rectangular, the average of long and short side lengths is used, and also the diagonal length could be used based on the short side length. in the following paragraph, nacl particles will be introduced, and particle size determined by diagonal method is used since the projected area of their crystal is usually quadrate. the other group was based on the indirect measurement and definition of some physical property of particles. precipitation method and optic-electrical method are used to determine the particle size. the value obtained is actually an equivalent diameter. in the former federal german standard vdi-2083, particle size was defined as the equivalent diameter according to the measuring method, which means some physical property and parameter of referenced particles are equivalent with the counterparts of these particle cloud based on this diameter value. for example, when light scattering air particle counters are used, "particle size" represents the comprehensive effect, i.e., the geometric size range, by comparing the equivalent light scattering intensity between the tested particle and standard particle (such as psl particles). setting velocity of particles could be measured, and when the settling velocity in still air calculated by stokes law in chap. 6 is equal to that of particles with same density, the spherical diameter of latter is called settling diameter, also stokes diameter, which is labeled as d st , and it is smaller than other diameter value. assume the density is 1, the diameter of spherical particles whose settling velocity is the same will also be called aerodynamic diameter, which is widely used in environmental science and labeled as d a . it is known from eq. (6.7) that d 2 st á ρ p ¼ d 2 a á 1. so the following correlation can be obtained: where ρ p represents the particle density. american federal standards 209c and 209e allowed to use either of (1) maximum visible linear length of particle or of (2) equivalent diameter measured by automatic meter. therefore, one method to reflect some characteristic by the average particle size from all the particles must be found, and the average value is called "average particle size." this special method is used to represent a hypothesized particle diameter according to some certain characteristic. assume the actual particle sizes are d 1 , d 2 , . . ., d n , respectively, which is shown in fig.1. 3. these values of particle size were determined by the before-mentioned method. some certain characteristic (such as light scattering property) could be represented by f(d 1 ), f(d 2 ),. . ., f(d n ), so the property of the particle group f(d ) has the following relationship with that of individual particle: suppose another particle group contains particles with the same particle size d and its property (such as light scattering property) is the same as that of the actual particle group, so we could get where the particle size d means the average particle size of this particle group corresponding to some certain property. d could be defined as the side length of the hexahedron or the diameter of sphere. the latter is usually used, which means the particle group could be treated as a group of spheres with the same diameter. the simplest case is to assume that the group consists of particles with the same diameter d 1 , so the total length of all diameters is the same as that of actual particle group. according to eq. (1.3), we could obtain d 1 d n actual particle group conceived particle group fig. 1.3 conceived particle group and average particle size this is the arithmetic average diameter. in this equation, d i is the particle size by any measuring method; m i is the number of particles with diameter d i ; n is the total particle number. if the summation of assumed particle areas (projected area or surface area) is the same as that of the actual particle group, we could get the following equation based on eq. (1.3): this diameter is termed as average surface diameter. if the specific length area (i.e., the cross section area per unit length) of the assumed particle group is the same as that of the actual particle group, we could get the following derivation: this diameter is termed as specific length diameter. the same method could be utilized to obtain other average diameters. the cross section could be circular (sphere) or rectangular. moreover, average diameter could be determined according to the particle size frequency distribution. by comparison of these diameters, we could get the following sequence: it should be noted for the average particle size names listed in the table. in literature, terms with converse sequence could be encountered. for example, some use "average area diameter," while others use "area average diameter." therefore, the meaning could be clearly understood only when the expression is known. but if we start from the definition, it's easier to understand the meaning. for example, in table 1 .1, "average area" obviously means all the areas averaged by some quantity the diameter corresponding with the maximum proportion in the samples. the minimum diameter calculated with the frequency distribution obtained from the summit on the distribution curve of particle frequency medium diameter the number of particles with diameter lager than this value is the same as that with diameter less, and this is called the number medium diameter. the mass of particles with diameter lager than this value is the same as that with diameter less, and this is called the mass medium diameter. all the diameters related to the number are smaller than that related to mass obtained at 50 % of the cumulative distribution curve of particle number (or mass) it is a kind of arithmetic average values and is the habitually most popular diameter. but small particles occupy the majority in the particle group. so even though the mass may be small, the calculated average diameter will also be greatly reduced. so there is great limit between the real size of particle group and the physical property of this particle group where n i is the particle number at each size and σn i is the total particle number it is the division between the projected particle area and the summation of diameters, which is the average diameter per unit length it is the division of the total volume of particles to the summation of cross-sectional area, which is the average diameter per unit area it is obtained with the surface area per unit mass, namely, the average diameter of unit mass (or volume). it is larger than any other diameter (such as particle number), so the area is in the numerator. "area weighted" ("specific area") obviously means per unit area, so the area is in the denominator. as long as we keep in mind of this standard, we will not be confused. as for which average diameter is reasonable, it depends on the purpose of the work. if the gravimetric method is used to measure the particle concentration, diameter d v which is related to mass should obviously adopted; when the property of light scattering is under investigation, it's better to use average area diameter d s or average volume diameter d v , because for different particle size range, the quantity it is the area weighted diameter according to the particle number it is the volume (or mass) weighted diameter according to the particle number it is the average of the logarithmic diameters • expressed with natural logarithm or it is the nth root of the multiplication of several values or it is the diameter with the maximum frequency from the lognormal distribution, and it is equal with the medium diameter of the particle number. it is always equal to or less than the arithmetic average diameter • for unclassified data • for classified data • the summit point can be obtained from the lognormal distribution curve of light scattering probably depends on the particle area or volume; when the problem is related to the light refraction property, arithmetic average diameter d 1 should be used, because this property depends on the dimension of particle length. now let's calculate the average diameter of particles. during the measurement of particle concentration with the sodium flame method, electronic microscopic could be used to measure the short side length of nacl particles from 823 samples obtained from the supply air, and we assume the amplification ratio is 30,000 (i.e., include the amplification ratio of electric glass and amplification ratio of reader microscopic for reading values on sem figures); the lower and upper limits for the group of short side could be calculated by where d p is the upper/lower limit of particle size range a is the measured value of upper/lower limit of short side results for a are as follows: <0.87 0.87-<1.7 1.7-<3.5 3.5-<6.9 6.9-<10. 4 according to the data from table 1 .2, we could obtain the arithmetic average diameter: when geometric mean diameter is calculated, we know hence we get reading on the photoelectric flame photometer used in the sodium flame method is proportional with the mass concentration of sodium chloride. therefore, it is better to use mass ratio diameter d 4 to represent average diameter of sodium chloride particles. in the above example, d 4 ¼ 0.513 μm, while the designated value in foreign standard is 0.6 μm. a lot of data on particle size will be frequently obtained in the field of air cleaning technology. after sampled on chemical porous membrane, particles with various shapes could be visible with microscope. different samples will be obtained even though they are sampled simultaneously. although particles are distributed randomly on the membrane surface, useful "information" is embedded behind those disorderly data. if arrangement and analysis are performed on these data, useful "information" could be extracted fully and correctly, which could be used as the basis for technical measurement adopted in dust measures, dust proof, and dust control. the purpose of data arrangement and analysis is to find the distribution law of particle number with particle size or density. a functional relationship could be used to describe this kind of law. however, there is no special theoretical basis that which kind of law is suitable for particle distribution. it is mainly dependent on experience. it is not enough to represent particle characteristic only with calculated average diameter. particle characteristic depends to a great extent on the law of particle size distribution. distribution of particle size also means the dispersity of particles, which represents the number or mass percentage of particles at different size within the group of particle clusters. if particle percentage in the size distribution is introduced with particle number, it is called number frequency distribution (or simplified as frequency distribution). particle number is usually a big number, so frequency distribution is preferred instead of number distribution in the study of particle size distribution. if particle mass is used for size distribution, it is called mass distribution. if particle surface area is used, it is called surface area distribution. dispersity of a particle group will be higher for the case of particle cluster with higher percentage of smaller particles and vice versa. dispersity represents the extent of fragmentation of dispersed material. when dispersity of a particle group is needed, particle size distribution should be obtained. in the figure of particle size distribution curve, the number of particles within certain range of diameter values, or the ratio (or frequency) of particles in various diameter range segments, will be presented. the curve is obtained by smoothing the frequency distribution in the histogram. δd (%) it is described with the percentage ratio of certain physical parameter (e.g., number δn ) of particles with diameters between d p and d p + δd p to the same parameter (e.g., total number δn ) corresponding to the particle group: at first particle diameter is divided into several groups as needed or according to measurement method. it is better to set the diameter distance equally. the upper limit diameter of each group coincides with the lower limit diameter of the neighboring group with lager diameter. if one diameter value equals with the threshold value, it belongs to the larger diameter group. for example, when the diameter group of 0.2-0.4 and 0.4-0.6 are considered, particles with diameter 0.4 μm enters into the second group. secondly, particle numbers in each diameter group are countered, and frequency numbers are obtained. when frequency number is plotted as ordinate and diameter as abscissa, histogram could be acquired after rectangles with the height of frequency number are draw. if the ordinate is replaced with frequency (particle number within certain diameter range divided by the whole particle number), the histogram of frequency distribution is acquired. figure 1 .4 is the nacl aerosol particles used in the sodium flame method. figure 1 .5 illustrates the situation after its sedimentation and coagulation. figure 1 .6 presents the shape after deformation when wetted [2] . table 1 .3 shows the number of another group of nacl aerosol particles with sem figures, from which we could get the number averaged diameter d ¼ 0:192 μm. in table 1 .3, the cumulative mass frequency is obtained from the mass frequency distribution which could be calculated by the product of cubic of mean diameter in the diameter range and particle number. relative frequency distribution histogram shown in fig. 1 .7 could be depicted by this table, where the dashed line means the fitted particle distribution curve on the histogram. figure 1 .8 shows the stereo of nacl particles [3] . particle size distribution curves for most particle group are asymmetrical, and they are inclined towards larger diameter. this is almost an intrinsic feature for dust particles since particles with small diameter occupy most portions of the whole dust particles. this kind of distribution is called "right-handed inclination" distribution. furthermore, there are also "left-handed inclination" distribution and "symmetrical distribution," which are both presented in fig. 1.9 . ϕðdþ (% · μm à1 ) it is described by the frequency distribution corresponding to the unit particle size range (e.g., 1 μm), i.e., when the particle size ranges, especially those in the middle of data, are not equal, the fitted curve from histogram of frequency distribution will not be smooth, and it deviates greatly from the reality. take a look at table 1 .4 [4] ; we could see the great discrepancy exists between particle size ranges. if histogram is plotted with the frequency data, we could get fig. 1.10 . when the frequency density is used, which is the proportionality of it is expressed as the percentage of certain parameter of all particles with diameter larger than d p when compared with the same parameter of whole particle group, i.e., rðdþ ¼ it is expressed as the percentage of certain parameter of all particles with diameter smaller than d p when compared with the same parameter of whole particle group, i.e., for some aerosol particles, there will be two or more than two peak values in the frequency distribution curves. the distribution function is comparatively complex, so it will not be introduced here. some examples are given for reference. 1. oil mist aerosol. oil mist is produced by the processes of oil heating, nozzle spraying, and condensation after evaporation. according to the research report [5] , this kind of oil mist aerosol has the feature of bimodal distribution, where the main summit located at the diameter 0.133 μm and the second summit at 0.024 μm. the curve shape is independent of the amount of big particles separated. figure 1 .14 shows the curve shape. the reason to form this kind of bimodal distribution is that there are many components and a few nonvolatile materials in the test oil (turbine oil) and these components have respective distribution summit values. 2. cold dop aerosol (which can be seen in sect. 17.2). it is also called pressurized dop aerosol, which is the liquid mist sprayed from the laskin nozzle after compressed air goes through the dop liquid. according to some research report [6] , the generated particles have the characteristic of bimodal distribution, no matter how much the compressed air pressure is. the particle size at valley between two summits is equivalent to the median diameter 0.275 μm. figure 1 . 15 shows the frequency distribution measured by the channels of laser optical particle counter (every channel corresponds to a certain particle size range, and in the figure the data of particle size were calculated with the channel data from las-x particle counter in the test). it is shown that one channel exists between the second peak and the valley and the second peak value is corresponding to 0.3 μm (median diameter). it should be noted that the above experiment was carried out with the laser particle counter which was able to detect the particles with diameter smaller than 0.1 μm (0.09 μm). if the particle counter with the incandescent light was used and the lower detect limit is 0.3 μm, there would be no bimodal distribution [7] , and the resultant number median diameter would obviously be smaller than 0.3 μm, which is different from the above experimental result. therefore the appearance of bimodal distribution is related to the test method, which needs further investigation. normal distribution curve is symmetrical in the middle which is shown in fig.1.9 . there is a highest point in the curve. the curve decreases monotonically towards two sides when the abscissa value of this point is considered as the center. the concept of normal distribution is one of the most important distribution concepts in statistics. in natural and engineering applications, it is the most widely used distribution for continuous data. now the key points useful for study of particle size distribution will be introduced. it is known from figs. 1.9 and 1.10 that a comparatively smooth curve could be obtained from the histogram. besides, the smooth curve could also be obtained when the sample number increases and the distance of subrange decreases, which is called probability density curve and is usually expressed as y ¼ f ðxþ where y means the probability density, which is equal to the frequency value per unit abscissa value, and x means the abscissa value, which is the obtained data. normal probability density function could be used to represent the normal distribution curve: where d t is the particle size; d is the average size, and it is usually the arithmetic mean diameter, so in mathematics it is the average value or expectation; for the case of normal distribution, d ¼ d m ¼ d mod ; σ is the standard deviation of a certain particle group. since particle number of the particle group sample is very large, it could be expressed as it is known with calculation that after many times of measurement, the probability for particles with measured diameter in the range of d ae σ is 68.3 %, and for particles with d ae 2σ, it is 95.4 %, while for particles with d ae 3σ, it is 99.7 %. at the same time, the value of σ could be used to represent the extent of "slim" for the curve. the curve is "fat" with large value of σ, which means data are scattering and vice versa. figure 1.16 gives the schematic diagram of both situations. for the case of normal distribution when the abscissa of the highest point on the curve equals with 0 and the standard deviation is 1, it is termed as the standard normal distribution and labeled as n(0, 1). standard normal distribution curve could be obtained when any nonstandard normal distribution curve is translated, as shown in fig. 1.17 . cumulative distribution function f(x) is obtained by integrating probability density function, and it is as follows: (1.14) the relationship between ϕðxþ and f(x) is that of derivative and integration. this is the same for other statistical distribution, and it will be used in the process of sampling and measurement. one special coordinate paper is called normal probability paper shown in fig. 1.19 , where its abscissa uses normal scale while the ordinate is drawn according to the normal distribution feature. if particle diameters follow normal distribution, a straight line will be obtained when the abscissa represents the particle size and the ordinate means the cumulative distribution frequency. this is one simple method to check whether particle size distribution is normal distribution. normal distribution could be used to depict specially generated monodisperse aerosols and pollen particles with single diameter, while most particle diameters do not follow normal distribution but close to it. for the case shown in fig. 1.11 , the cumulative distribution curve on the normal probability paper is not straight as noted in fig. 1.19 . the reasons are: 1. in normal distribution, the variable is continuous data (such as length measurement), while only discrete data are available in the particle sizing process. as detection means develops, the grouping range will be closer and the measured data will be more continuous, so it will approach the normal distribution. fig. 1.19 normal probability paper 2. the normal distribution curve is symmetrical. so it is possible that part of the curve goes through the abscissa zero point and part of particle diameter values is required to be negative, which is absolutely impossible. 3. due to the right inclination distribution characteristics of the aforementioned particle swarm. if the abscissa of fig. 1.11 is converted into logarithmic coordinate and the channel frequency distribution method is used, table 1 .5 could be obtained from the data in table 1 .4. in such a way, a curve more symmetrical and closer to the normal distribution will be obtained. this kind of curve is illustrated in fig. 1 .20. this distribution is termed as lognormal distribution. when the particle size distribution of the before-mentioned oil mist aerosol with bimodal distribution characteristic is plotted on logarithmic graph paper, the main peak is also close to the lognormal distribution as shown in fig. 1.21 . it should be noted that in the process of drawing frequency distribution curve, the extreme left on the logarithmic scale of the abscissa is not starting at 0 but at the smallest unit of this channel. for example, if the first channel is <0.2 μm, the left side could start at 0.1 μm, which means the value of δlgd p is lg0.2 à lg0.1 ¼ 0.301, not lg0.2 à lg0. in some cases other suitable starting point could be considered according to the situation of the instrument. for another example, when the 3030type electrostatic aerosol analyzer is used to measure the particle size, the particle size channel represents the nominal diameter, so for the first channel 0.0237 μm which includes the range between 0.0178 and 0.0316 μm, the value of δlgd p is lg 0:0316 0:0178 . when the points from the curve, which is basically in line with the lognormal distribution, are plotted on lognormal probability graph paper (i.e., normal probability graph paper with the logarithmic scale for the abscissa), a straight line could be obtained although no straight line is got at normal probability graph paper as shown in fig. 1.19 . the straight line (3) in fig. 1.22 is made from fig. 1.11 (or table 1 .4). this is a simple method to check whether the particle size distribution fits lognormal distribution. line (2) in fig. 1 .22 is plotted with the data in table 1 .2. although the data set distances are unequal, the characteristic of lognormal distribution could also be displayed on lognormal probability graph paper. fig. 1.19 . although they deviate too much from the straight line, it is still reasonable to consider that the data basically fits lognormal distribution. because both ends of the scale on lognormal probability graph paper are actually amplified, the error range of cumulative frequency at both 5 and 95 % is four times that of 50 %. in addition, grouping and measurement of both ends are generally coarse, so requirement for points with cumulative frequency smaller than 5 % or larger than 95 % is not so stringent, while it is enough to pay attention to the linear feature between 20 and 80 %. experiments have shown that particle size distributions of some dispersed aerosol and aggregated aerosol follow lognormal distribution. from the assumption of the characteristic during solid particle rupture process, particle size distribution was found to approach towards lognormal distribution. although the reason is (1) (1) heretofore not clear, lognormal distribution has more theoretical meaning than other distribution [8] . if particle size distribution is quite different from normal distribution, such as an extreme case shown in fig. 1 .23 which represents the sampled particle size distribution curve in the cleanroom air, large deviation could be found when the lognormal probability graph paper is plotted. line 1 on fig. 1.22 is the resultant line. experiment has shown that some particle size distribution curves on log-log graph paper are close to be straight and they follow the rule of negative exponent, so they are also termed as negative exponential distribution. for the special case of finer particles, the linear dependence is well presented. attenuation distribution gives the information about the change of particle number with the change per unit particle size, which is equivalent with the previous frequency distribution when the ordinate means the particle number per unit particle size spectra instead of the frequency per unit particle size spectra. this is often used in the study of atmospheric aerosol, which will be introduced in chap. 2 in detail. this means the change characteristic of particle number for the particle size equals with or larger than a certain value. for airborne particles with various diameters in the air of clean environment, their distribution curves of particle size on log-log graph paper are almost parallel straight lines with quite the same slope (especially those particles with diameter larger than 0.1 μm). figure 1 .24 is an example of a cleanroom which is the same cleanroom shown in fig. 1 .23. the feature of the line in fig. 1 .24 is more obvious than that of fig. 1 .23, so it is more useful for practice use. this is used in the classification of cleanness class, which will be illustrated later. atmospheric particles also have this feature, which is usually used in the air cleaning technology. for detailed information, please refer to chap. 2 about atmospheric particles. particles are not uniformly distributed in terms of density (particle number per unit space or area). for example, when 1 l air is sampled from the cleanroom, the particle number measured by particle counter is 10#. however, particle number is actually not always 10# when 1 l air is sampled. for all that, there is a law existed in the sampled particle number per unit volume of air when particle counter is used. in general case, the density distribution obeys the law of poisson distribution. density data could only be positive integers such as 1, 2, 3, 4, . . ., instead of continuous one, so density does not follow normal distribution. since the most important law for discrete data (counting value) is poisson distribution, particle density distribution should also accord with poisson distribution. in addition, four conditions are satisfied for the general cleanroom space, which are also required by poisson distribution: 1. the measured space is much larger than the sample volume, even 10,000 times (the minimum is 100 times) 2. the probability of single particle appearing in the volume of every sample is very small such as several ten thousandths (the minimum is several hundredths) 3. it is incompatible that particle will fall into the sample volume and not. 4. since particle concentration in the space is quite low, particle number during each sampling process is a small finite value (e.g., less than 10). the probability of different particle number with poisson distribution could be expressed as where p(ξ ¼ k ) means the probability of k particles where k must be positive integer including 0 and λ means the average time of the appearance of incidence, and it also represents the average concentration -average particle number included in the sampling volume. figure 1 .25 shows the change of poisson distribution curve when λ increases. the distribution curve for discrete data could only be polygonal line. when λ > 5-10, the curve is more symmetric and close to the normal distribution. according to the probability theory, the probability for the appearance of more than k particles could be obtained from eq. (1.15): the probability for the appearance of less than k particles is people may be afraid that the unidirectional flow in the cleanroom has almost no dilution effect (please refer to the cleanroom principle in chap. 8), which will change the distribution law of particle size, so the poisson distribution may not be applicable. but the poisson distribution does not require uniform distribution of the particles. for example, during the investigation of flaw spots on the cloth and rejecting rate of piles of mechanical parts, they are both not uniformly distributed. but when enough random sampling time is performed, the conclusion of the law will be roughly in line with the actual situation. using the measured data from a cleanroom with unidirectional flow, the above conclusion could be validated. table 1 .6 shows 440 measured data in a cleanroom with unidirectional flow where the original concentration is comparatively high and the efficiency of air filter is low. the sample volume of each test is 0.1 l. the test shows λ ¼ 1.69. table 1 .6 also lists the data by theoretical distribution with the value of λ. for example, the probability for the appearance of no particle can be calculated so the times of appearance are 0.1845 â 449 ¼ 81.2. results show that except for the influence by external interference (data labeled with *), agreement exists between calculation and test. comparison between them could be clearly seen in fig. 1.26 . in the following part, several test examples were performed in cleanrooms with unidirectional flow where the initial cleanness is high. tests were performed at fixed position. table 1 .7 presents cases in china. ¼ 0.3048 m) . the resultant conclusions are consistent. in the measurement examples both in and outside china, except for the calculated value of nonzero time in the third example of table 1 .7, which is quite different from the measured value, others agree well with measurements. this further proves that poisson distribution is suitable to describe the particle distribution in terms of particle number density. poisson distribution is also valid for the problem of studying the particle deposition number on a surface in cleanroom, which also meets the above four conditions. we have applied this conclusion in the study of the relationship between the yield rate and particle number in air, and agreement was found between calculation and experiment [13] . this conclusion will also be useful for the sample of microorganism particles, and detailed information will be introduced later. statistical analysis of the measurement data of colony in biological cleanroom also support the above conclusion [14] , which is shown in fig. 1 .27. in a cluster of particles, it will be called monodisperse when particle sizes approach a single value. its concentration degree is the maximum. on the contrary, it will be called polydisperse when particle sizes are scattering. its concentration degree is the minimum. however, particle sizes in practice are not likely to be single value, but may be in the narrow range around the two sides of average diameter. therefore, concentration degree of particles is defined as the percentage between particle numbers corresponding to particles with a certain diameter and the whole particle number. it is obvious that concentration degree of particle sizes is not an antonym of scattering degree of particle sizes introduced in sect. 1.3. if small particles with one or two kinds of diameters occupy quite a large number of a particle group, e.g., 90 % of the whole particle number, the scattering degree of this particle group is high, while it does not mean the concentration degree is low. on the contrary, since 90 % of particles have diameter concentrated in a small range, the concentration degree is very high. it is shown in fig. 1.16 that the value of standard deviation σ represents the degrees of concentration and scattering of particle sizes. so σ could also be used to stand for the concentration degree or monodispersity of particles. we have such experiences that the absolute error for measuring comparatively large things is normally big, and it is small when tiny thing is measured. σ is only an indication of absolute fluctuation of particle size data. evaluation of the concentration degree and monodispersity of standard latex particles, the standard particles used for calibrating royco particle counters, made by american dow chemical company is a good example, which is illustrated in table 1 .9. error is related to the absolute value of particle size. in order to avoid the influence, it is more reasonable to use the relative fluctuation value to represent the scattering degree of data. relative standard deviation, also called change coefficient, is usually used to represent the concentration degree of particle sizes. however, since particle size distribution is usually close to lognormal distribution, it is more reasonable to use lgσ g instead of σ. σ g is called geometrical standard deviation. when the scattering degree of particle sizes is small and suppose they are near from one certain particle size d 1 , we could get the following expression from when d is replaced by d g and the logarithmic coordinate is used, we could get (1.20) we know ln 1 þ x ð þ%x when à 1 < x 1, so we can obtain this is why the following expression is obtained [3] : fuchs (н.а.фукс) defined monodisperse particle as those particle group with α 0.2 (i.e., lgσ g 0.087, or σ g 1.22) [15] . this means the proportion of particles within the diameter range d 1 ae 0.2 d 1 will be 68.3 % where d 1 is the arithmetic diameter of a particle group and α ¼ 0.2. for example, tsi co. ltd from the usa adopts σ g as the concentration degree of standard particles which were still manufactured by dow company. they consider standard particles with σ g < 1.06 as monodisperse particles. in practice, simplified method could also be used, which uses the proportion of particle numbers within certain diameter range to the whole particle number to represent the concentration degree. for example, if particle number of those with diameter 0.3 μm occupies 70 % of whole number, 70 % represents the concentration degree of these particles. it is obviously shown from the above discussion that although the method to determine the concentration degree is simple, it varies with different size range, so it is preferable to judge based on the value of α. for modern optical particle counter which will be introduced in chap. 17, the minimum channel is 0.1 μm, and the largest variation of average diameter in this channel is < 0:2d , which means the ratio between this variation and average diameter is <0.2. the ratio for particles within larger channels will be smaller. therefore, if we describe the concentration degree with the percentage of particle number in each channel, the ratio will be smaller than 68.3 % mentioned before, and usually it is 60 %. the concentration degree will be higher when it reaches 70-80 %. if particle size distribution follows the lognormal distribution characteristic, logarithmic probability graph could be used to determine the concentration degree of particle sizes and various kinds of average diameter. if particle size distribution curve on the logarithmic probability graph is a straight line, particle size distribution will follow the lognormal distribution, which is shown in fig. 1 table 1 .1 it is known that lg d i ¼ lg d g where d g is the geometric average diameter. for the case of normal distribution, d ¼ d 50 , so d g ¼ d 50 for lognormal distribution, and the above equation could be written as this function represents the relative number of particles with diameter d t . if the total number is 100 %, the percentage of particle number of those with diameter smaller than d t is we know that eq. (1.26) could be written as [16] y this is the normal distribution n (0,1) and the standard deviation is 1. from eq. (1.27) we know it is obvious that it is easy to solve lgσ g when t is supposed to be 1 and σ g ¼ d i d 50 . refer to the normal distribution table and fig. 1 .28, the percentage of particle number with diameter d t when t ¼ 1 is y 84:13 % or that of diameter >d t is y ! 15:87 %. therefore in a similar way we could get the following expression for t ¼ à1: the value of calculated lgσ g could be used to determine the concentration degree of particle group and thus to judge if it is monodisperse. suppose the actual particle size distribution measured by electronic microscope on monodisperse psl spherical particles with claimed diameter 0.8 μm is shown in table 1 .10. therefore it is reasonable to consider this particle group as monodisperse, and 68.3 % of particle diameter will be in the range of 0.784 ae 0.108 â 0.784 ¼ 0.784 ae 0.085 μm. as mentioned before, it is quite difficult to calculate the average diameter. but for the case of lognormal distribution, data could be plotted on the lognormal distribution probability graph, and then it is easy to calculate the median diameter d 50 , geometrical standard deviation σ g , and various average diameters. here only the results of various average diameters calculated are shown [16] . process of data plotting could be referred to fig. 1.29 . the above results are close to the calculated results from the data of table 1 .2. the more the linear proportionality on the lognormal probability graph is, the less difference between them it becomes. the abovementioned particle size distribution represents the particle number distribution corresponding to the particle size. for particle mass distribution on particle size, it represents the percentage of particle mass corresponding to certain particle size in the whole mass. for particle area distribution on particle size, it represents the percentage of particle area corresponding to certain particle size in the whole area. they can be derived from the particle size distribution, and measurement on mass or area is not needed. since each weighted distribution of lognormal distribution is also lognormal, they are parallel straight lines with the same σ g . their relationships are lg d v 50 ¼ lg d 50 þ 6:908lg 2 σ g (1.39) lg d s 50 ¼ lg d 50 þ 2:303lg 2 σ g (1.40) where d v 50 is the particle size at 50 % in the mass distribution curve, i.e., the mass median diameter; when lognormal distribution is valid, it is called the mass geometric average diameter; d s 50 is the particle size at 50 % in the area distribution curve, i.e., the area median diameter; when lognormal distribution is valid, it is called the area geometric average diameter. from the example of fig. 1 .29, we could knowd v 50 ¼ 0:458 μmandd s 50 ¼ 0:24 μm from calculation, and straight lines which represent area distribution and mass distribution and which are parallel to the particle number distribution could be plotted. it should be noted that in order to fit the scale on probability graph paper well, only data group of those "<0.6" is needed at one edge, while those between 0.6 and 1.0 could be considered as data of "!0.6," which means the proportion of data <0.6 is 98.4 % while that of !0.6 is 1.6 %. if more precise data are needed, subdivision of two edges could be made. as for the case of surface contamination, area distribution is preferred; while for the hygiene problem, mass distribution is used, where mass of particles have direct implications for the problem. when we are dealing with the problem in the cleanroom, particle number distribution with particle size is used. statistic calculation of particle size is needed for the abovementioned particle distributions. it is a basic statistic problem that how many particles should be sampled in the particle group of each sample so that the error of the measured particle size is minimized. from the principle of statistics, standard deviation of average value equals with the ratio of standard deviation σ to the root of sample number, which is shown in eq. (1.41): where n is the particle number. the real size of particle under the 95 % confidence level is is the relative error of particle size. when it is set to be δc, the following requirement should be met: the above expression was used in ref. [17] to obtain the necessary particle number for counting of monodisperse standard particles. according to the definition of monodisperse aerosol, we know σ d 0:2 for calibration of particle counter, the requirement is much stricter, so it could be assumed 3 %. with the above data, eq. (1.43) becomes n ! 2 â 0:2 0:03 2 ¼ 178 during the counting process, the abovementioned problem of relative error exists. when particle number is too less, man-made error will exist because of inadequate randomness. when it is too much, the workload of counting process is heavy. when particle counter is used, to count 178 particles is not difficult, so 300-500 standard psl particles are needed for calibrating particle counter in the national standard "calibration method of the performance of particle counter," which will result in error smaller than 3 %. for polydisperse aerosol, σ is unknown, so is σ d . but for common aerosol particles, the value of σ could be assumed. now we assume σ in table 1 for the usual test, the relative error of 5 % is acceptable. therefore the particle counting in table 1 now we know from the table that p n i ¼ 823, which meets the requirement. moreover, ref. [18] presents the guideline to measure the particle number needed. since it is generally introduced, we will not introduce in the book. for those readers who need the information, please find the original paper. air pollution control engineering sampling technique and measurement of dispersity of nacl aerosol guideline for the generation method of aerosol for pollution control aerosol technology (trans: sun yufeng institute of hvac at china academy of building research, people's liberation army no. 57605 of the people's republic of china characteristic of aerosol generator with cold dop aerosol technical report for the development of jl leakage detection apparatus. institute of havc at china academy of building research, liaoning hongbo radio factory the mechanics of aerosols (trans: gu zhenchao) calculation of the minimum sampling volume for particle counter in environments with different air cleanliness levels discussion of the deviation phenomena of statistical average value of particle number during measurement performance of ulpa filter in ultra cleanroom acceptance inspection of cleanroom with class 1 (trans: du chunlin, proofread by chen changyong). compiler group for code for construction and acceptance of cleanroom relationship between the yield and the air cleanliness level in the clean environment discussion of the bacterial deposition method to measure the air cleanliness level in cleanroom generation of smoke introduction to aerosol technology methods for testing the performance of airborne particle counter aerosol handbook (trans: zhou jinqin) key: cord-332255-7sxioviw authors: narita, m.; fukusho, a.; shimizu, y. title: electron microscopy of the intestine of gnotobiotic piglets infected with porcine rotavirus date: 1982-10-31 journal: journal of comparative pathology doi: 10.1016/0021-9975(82)90011-1 sha: doc_id: 332255 cord_uid: 7sxioviw abstract five gnotobiotic piglets inoculated orally with porcine rotavirus developed an enteric lesion. electron microscopy of the mucosal epithelium 12 h after inoculation showed that the virus penetrates into the absorptive cells between microvilli, possibly by a pinocytic mechanism. afterwards, virus particles were most often seen within dilated cisternae of the rough endoplasmic reticulum (rer). these infected cells showed a range of changes, such as disruption of the microvilli, loss of cytoplasmic density and deposition of lipid droplets. subsequently, most of the epithelial cells were desquamated from the villi. the interaction of virus and intestinal cells thus indicates that rotavirus is pathogenic for the epithelial cells. porcine rotavirus is an enteropathogen for neonatal piglets (fukusho, shimizu and ito, 1981; lecce and king, 1978; mcnulty, pearson, mcferran, collins and allan, 1976) . mortality is high in pigs infected during the first 3 days after birth, but it is reduced as the pig increases in age (pearson and mcnulty, 1977) . the pathogenesis of porcine rotavirus in gnotobiotic piglets was studied by light microscopy (lm) , fl uorescent antibody technique (fa), scanning electron microscopy (sem) and transmission electron microscopy (tem), and it was demonstrated that clinical signs were correlated with viral replication and with induced virus lesions in the small intestine (mcadaragh, bergeland, meyer, johnshoy, stotz, benfield and hammer, 1980; narita, fukusho, konno and shimizu, 1982; pearson and mcnulty, 1979; theil, bohl, cross, kohler and agnes, 1978; torres-medina and underdahl, 1980) . however, there are as yet no reports about the penetration of rotaviruses into the epithelial cells. the present paper describes the morphological changes in infected intestinal cells of 2-day-old piglets inoculated orally with porcine rotavirus and discusses the penetration of virus into the absorptive cells. this was prepared as previously described (narita et al., 1982) . briefly, a faecal suspension was prepared as a bacteria-free 20 per cent intestinal contents filtrate after one gnotobiotic piglet passage. no virus other than rotavirus was detected in the suspension by electron microscopic examination. piglets were inoculated orally with 2 ml of the faecal filtrate. .4nimals five p-day-old gnotobiotic piglets were used. they were kept at 26 "c in individual metal cages with positive pressure ventilation. each was given 2 ml of the rotavirus suspension and they were killed at 6, 12, 18, 24 and 48 h after inoculation. samples of the small intestine were removed under general anaesthesia from 5 sites adjacent to those from which samples had been taken for histopathology. samples were fixed in 2.5 per cent glutaraldehyde in 0.1 m phosphate buffer, ph 7.2, for 2 h at 4 "c. they were post-fixed in 1 per cent osmium tetroxide in phosphate buffer for 1 h at 4°c. the tissues were dehydrated in a graded series of ethanol and embedded in epon 812. sections were cut with glass knives, stained with uranyl acetate and lead citrate, and examined with a nihon denshi tem-ioocx electron microscope. jejunal and seal epithelial cells from piglets 6 and 12 h after inoculation showed no morphological alteration detectable by ultrastructural examination; there was no sign of detachment or desquamation of the villi. microvilli were intact and contained a prominent nucleus, abundant mitochondria, and rough endoplasmic reticulum (rer). however, virus particles ranging from 65 to 75 nm in diameter were detected in a few absorptive cells of a sample ofjejunum collected at 12 h. particles were observed on the microvillar border or between the microviili where they were often located in rows ( fig. 1) . individual or small numbers of particles were found inside vesicles located in the terminal web area or in the upper region of the apical cytoplasm ( fig. 2) . eighteen hours after infection most of the absorptive epithelial cells in the jejunum and ileum were slightly swollen. most of the microvilli of the brush border were normal in length, but some were short, thick and irregular. these infected cells had lost most of their staining density and contained many virus particles within the distended rer. in some sections, effete cells containing viral particles were found in the process of being destroyed (figs 3 and 4) . iz few goblet cells also contained virus particles (fig. 5) . at 24 h after infection, some of the absorptive epithelial cells were becoming degenerate. in the jejunum, the degenerating cells were rounded, swollen, and had lost some of their staining intensity. they had large lipid droplets and an rer distended by vacuoles. these degenerated cells contained few viral particles (fig. 6) . in some sections, the infected cells appeared to be detaching from adjacent epithelial cells and being shed into the lumen (fig. 7) . man) viral particles were seen within the distended rer. they were classified as 3 types; enveloped, non-enveloped particles and nucleoids. enveloped particles were 70 to 80 nm in size and some appeared to have acquired an outer shell by, budding through the endoplasmic reticulum. non-enveloped particles were 50 to 65 nm in size. both types of viral particles had an electron-dense core measuring 20 to 30 nm. in the cytoplasm of the infected cells, non-membrane bound, granular matrices containing an electron-dense virus core were present (fig. 8) . in the ileum, most of the epithelial cells were desquamated from the villi and a small number of neutrophils was observed in the submucosa. at 48 h after infection, disruption and desquamation of the cells were still present in a few areas of villi. at this time the short villi were covered with a type of cell which was ultrastructurally different from any of those seen on tht villi in the earlier stages. they were flat to cuboida1, contained oval nuclei lying parallel to the axis of the villi and were closely applied to the basement membrane. their :te. (fig. 9 ). viral particles 'were not detected'&%& apparently newly produced and undifferentiated cells. the present em study has established that the porcine rotavirus becomes localized within the epithelial and goblet cells of the small intestine of piglets. the viral particles, in the form of double-shelled or enveloped particles (70 to 85 nm), single-shelled particles (60 to 70 nm), and small cores or nucleoids (25 to 30 nm), were readily seen in the epithelium of the piglets killed 18 h after infection. thereafter, infected cells were only rarely observed. these observations agreed with previous reports by adams and kraft (1976)) chasey i 1977)) esparza, gorziglia, gil and romer ( 1980) , pearson and mcnulty (1979) and saif, theil and bohl (1978) ) but detection of virus within goblet cells is a new finding. 4t low magnification the infected cells were visible, with many viral particles present in the rer and the apical cytoplasm. the least severe and probably the earliest changes consisted of shortening and loss of the microvilli. at a more advanced stage the infected cells were rounded, had large lipid droplets and an rer distended with vacuoles. subsequently, most of the absorptive cells appeared to be lost due to either cell desquamation or disruption. therefore, the interaction of virus and intestinal cells, and the resulting absorptive cell degeneration, indicates that rotavirus is pathogenic for the epithelial cells of the small intestine. these tem changes correlated well with those previously found by lm, sem and fa examination by lecce, king and mock (1976), mcadaragh et al. (1980) , mcnulty (1977), theil et al. (1978) and torres-medina and underdahl (1980) . concerning the entry of the virus into cells, pensaert, haelterman and hinsman (1970) and wagner, beamer and ristic (1973) reported that transmissible gastroenteritis (tge) virions are taken up by pinocytosis and then transported into the epithelial cytoplasm. electron micrographs of cells taken 12 h after inoculation with rotavirus revealed viral particles between microvilli and in the apical tips, and vesicles in the terminal web areas of some otherwise apparently normal epithelial cells. these findings resemble those with tge virus infection and therefore they strongly suggest that porrinc rotavirus is also taken up by pinocytosis. the present em observations have provided additional information on the mode of virus release from host cells into the lumen. there seem to be 2 processes involved; one is the desquamation of the infected cells of the villous . . cplthehal lining, and the other is the destruction of the luminal plasma membrane, with consequent discharge of cytoplasmic contents. these processes are commonly seen in small intestine infected with adenovirus (takeuchi and hashimoto with other enteric viruses the same release mechanisms from the epithelial cells into the lumen. five gnotobiotic piglets inoculated orally with porcine rotavirus developed an enteric lesion. electron microscopy of the mucosal epithelium 12 h after inoculation showed that the virus penetrates into the absorptive cells between microvilli, possibly by a pinocytic mechanism. afterwards, virus particles were most often seen within dilated cisternae of the rough endoplasmic reticulum (rer). these infected cells showed a range of changes, such as disruption of the microvilli, loss of cytoplasmic density and deposition of lipid droplets. subsequently, most of the epithelial cells were desquamated from the villi. the interaction of virus and intestinal cells thus indicates that rotavirus is pathogenic for the epithelial cells. intestinal changes in gnotobiotic piglets experimentally inoculated with porcine rotavirus pathological changes in the small intestine of neonatal pigs infected with a pig reovirus-like agent (rotavirusj transmissible gastroenteritis of swine: virus-intestinal cell interactions we wish to thank dr s. konno for suggestions, and mrs m. osada and y. andow for assistance with electron microscopy. journal of general virology, 37, 443-451. esparza, j., gorziglia, m., gil, f., and ramer, h. (1980) . multiplication of human rotavirus in culture cells: an electron microscopic study. journal of general virology, 47, 46 l-472. fukusho, a., shimizu, y., and ito, y. (1981) key: cord-032165-nyj72o21 authors: xu, zhonglin title: movement of indoor fine particle date: 2013-08-08 journal: fundamentals of air cleaning technology and its application in cleanrooms doi: 10.1007/978-3-642-39374-7_6 sha: doc_id: 32165 cord_uid: nyj72o21 only when indoor airborne particles move towards the nearby of the precision product and then deposit onto the sensitive area, damage may be caused for the product. so it is important to understand the mechanism of particle movement and deposition for the control of environment. cleanroom comparatively. as for the movement of particles in the electric field, it has been introduced in the chapter about air filter, so both the electrostatic force and other kinds of forces with little significance are not discussed in this chapter. for the airborne particle shown in fig. 6 .1, it will be influenced by the gravity f 1 , the buoyancy f 2 , and the resistance f 3 by the medium. for spherical particles, the gravity is: the buoyancy is equal to the weight of the medium with the same volume, i.e., 2) the resistance equals to the multiplication of the relative velocity between the particle and the airflow, the projected area of particles, and the drag coefficient, i.e., where m p and m a are mass of particle and air, respectively (kg); ρ p and ρ a are density of particle and air, respectively (kg/m 3 ); v is the relative velocity of particle (m/s); d p is the particle diameter (m); ψ is the drag coefficient. the unit of various kinds of force is n. when forces are acting on the particle, it deposits simultaneously. the settling velocity increases during the deposition process. when the resistance, the buoyancy, and the gravity are balanced, i.e., f 1 à f 2 ¼ f 3 , it becomes the uniform settlement. then, the speed is v ¼ v s , which is called the settling velocity or stokes velocity. it can be calculated with the following expression: the drag coefficient ψ depends on the flow state where particle is suspended, i.e., the laminar flow or the turbulent flow. it is also dependent on the shape of particle. the flow state is decided by the reynolds number re of the particle with the relative movement. where μ is the gas viscous coefficient, pa · s. it is also called dynamic viscous coefficient, which is different from the kinematic viscous coefficient υ, υ ¼ μ ρ a . with the legal unit, for air with temperature 20 c, μ ¼ 1.83 â 10 à5 pa · s and ρ a ¼ 1.2 kg/m 3 . for the movement of particle, re is usually less than 1. when re is smaller than 1 especially smaller than 0.5, the resistance for spherical particle can be calculated with the following formula: this is the famous stokes formula, where the resistance has the opposite direction to the movement. 1/3 of this resistance is the component of shape resistance for the particle, and 2/3 is the component of the frictional resistance for the particle. with eq. (6.3), the drag coefficient can be derived: therefore, the resistance is strictly related with the particle velocity. for the flow with larger re, the value of ψ is shown in table 6 .1 [1, 2] . for nonspherical particle, the drag coefficient should be multiplied by a correction coefficient β. this means the drag coefficient for nonspherical particle is ψ 0 ¼ βψ . value of β is shown in table 6 .2. when eq. (6.6) is substituted into eq. (6.4), and let ρ p -ρ a % ρ p , then the settling velocity is: when square is performed on both sides, it becomes: two points should be paid attention to during the application of this equation: 1. in the field of aerosol technology, particle density is generally assumed ρ p ¼ 1,000 kg/m 3 , while for atmospheric dust, particle density is generally assumed ρ p ¼ 2,000 kg/m 3 . 2. the unit of μ is very confusing. in some literature, when the wrong unit is used, the calculated result can differ by ten times, where complete opposite conclusion can be obtained (for detailed information, please refer to the later chapter about isokinematic sampling). in this book, the legal system with international unit is adopted. the difference between this kind of unit and the engineering unit in the past is shown as follows: when the found value of μ is based on cgs unit, the corresponding value with legal unit is the found value divided by 10. when the found value is based on the engineering unit, the corresponding value with legal unit is the found value multiplied by 9.81. when the found value of μ is based on kms unit, the corresponding value with legal unit is the found value divided by 3,600. so, for atmospheric dust, the relationship between the settling velocity (m/s) and particle diameter with normal temperature 20 can be obtained with eq. (6.7): v s % 0:54 2; 000 â d p â 10 à6 à á 2 1:83 â 10 à5 m s = ð þ ¼ 590 â 10 à7 â 10 2 d 2 p cm s = ð þ or v s % 0:6 â 10 à2 d 2 p cm=s ð þ (6.8) the calculation result is also shown in fig. 6 .2. it is shown that for particles with diameter 1 μm, v s is only 0.006 cm/s. that means it only takes 4 h for the deposition of particles from the working area (0.8 m above the floor) to the floor. but for particles with diameter less than 0.5 μm, the diffusional distance is even larger than the settling distance, and this is why it is not easily deposited. it should be noted that slip correction should be considered for particles with diameter less than 1 μm. according to the aerosol mechanics, stokes formula is derived with the continuous flow condition, which assumes that there is no velocity jump on particle surface. that is to say, there is no relative velocity between the flow particles and the infinite thin medium layer attached to the surface, or there is a boundary layer on particle surface without relative velocity. for small particles, when the radius is close to the mean free path of gas molecule or the gas pressure is comparatively small, the movement of particle has the characteristic of molecular. particles will be so small that they will "slide" between gas molecules, namely, the relationship between the settling velocity and the particle diameter existence and movement of particle will neither influence the gas velocity distribution nor cause any airflow in gas medium. so the abovementioned boundary layer with zero velocity does not exist. conversely, there is a speed jump layer on the particle surface, namely, air slippage phenomena occur on the surface of the moving particle. obviously, the resistance caused by medium should be reduced, which is favorable for the stokes settling velocity. this is the reason to consider the slip correction for small particles. the decisive parameter for the flow in the slip flow range or others is called knudsen number: the specific division of flow range is shown in fig. 6 .3 (table 6. 3). let c the slip correction coefficient, which is also called cunningham correction coefficient, the settling velocity after correction becomes: table 6 .4 shows the value of c with normal temperature and normal pressure by eq. (6.11). it is found that for particles with diameter 1 μm, the corrected settling velocity will be faster by that without correction by 16 %. the movement of particles under the action of inertia force means particle moves with inertia with initial velocity after the external force disappears. for example, particles on human body and equipment are affected by the mechanical force because of the activity of people and the movement of equipment. suppose initial horizontal velocity is obtained after particles leave the human body or equipment, the applied force disappear (now the airflow force and gravity are neglected), then particles decelerate with the inertia. according to the newton's law, the movement of particles with initial horizontal velocity v 0 can be described with the following equation: where f is the external force. when only inertia force exists, here the slip correction should be considered. the expression of f 3 with stokes equation becomes then, eq. (6.12) becomes: after integration, we get: 14) the distance of movement in time t is: when t tends to infinity, the movement of particles becomes stable. the distance of inertia movement, expressed as s r , can be calculated with the following expression: inserting the expression of τ into eq. (6.16), the calculation results are listed in table 6 .5. it is shown that τ has the dimension of time, and it is called the "relaxation time" in aerosol dynamics. it is an important parameter to describe the movement of particles, and it is also termed as the characteristic time. it is the time needed for the transition from initial stable state to another stable state. for example, it is known from eq. (6.16) that after the force is removed, the velocity of particle with diameter 1 μm will reach 1/3 of the initial velocity within the time t ¼ τ. so when t > τ, the movement status of particles will change a little. from table 6 .5, because of the rapid decline of velocity, the horizontal movement distance of a particle with initial velocity 1,000 cm/s is very short. it is impossible to be suspended with this mechanical force. due to the impact of air molecules with brownian motion, significant uneven displacement of airborne particles will occur, and the disorderly movement will be shown, which is shown in fig. 6.3 . after the collision, the movement direction and speed of molecules change suddenly. the trajectory is composed of many segments of straight lines. because the mass of particle is much larger than that of the air molecule, after the impact with air molecules, the velocity of particle can be reduced to be small enough to ignore. only after several times of impact, the direction and velocity of particle will change significantly. so the trajectory of particle is almost smooth curve. this kind of phenomena about disorderly movement for particle is called diffusional movement. although the displacement of particle in all direction with diffusional movement is random, pure linear displacement will occur when t >> τ and t ¼ 1 s is enough. so the absolute value of average displacement during 1 s in the given direction can be obtained with the following expression [3] : where t is time (s); d is the diffusional coefficient of particle (cm 2 /s). figure 6 .4 gives the relations between d and particle size. table 6 .6 gives the diffusional movement distance with different sizes. from the table, the movement distance of particle with diffusion is insignificant. usually it is thought that only diffusional deposition exists. this kind of diffusional deposition includes molecular diffusion and convection diffusion. for the room without air supply, indoor air will not keep stagnant because of the existence of convection. with the effect of convective diffusion, particles approach to the surface gradually at first and then deposit onto the surface with the molecular diffusion within a very thin layer near the surface, which is shown in fig. 6 .5. for the particle with a certain particle size, the molecular diffusion coefficient d is known, while the convective diffusion coefficient is unknown. fuchs solved this problem with a simplified method [4] . in any deposition mechanism, the concentration variation of particles because of deposition is linearly proportional to the particle concentration n, i.e., the negative sign represents the decrease of concentration. after integration, the above expression becomes where n 0 is the original concentration (pc/cm 3 ); n is the concentration at the moment t after deposition (pc/cm 3 ). then, the problem is attributed to find out the value of β related to the diffusional deposition mechanism. there is convection in the room without air supply. it means outside of the molecular diffusion layer, concentration becomes uniform with the effect of convection. and the concentration decreases continuously with time (with air supply, n is a constant). therefore, the particle number deposited onto the vertical surface with unit area within unit time is where v d is the velocity of diffusion deposition (m/s); n is the concentration which decreases continuously; i is the deposition rate [pc/(cm 2 ·s)]; δ is the thickness of molecular diffusion layer. although it is difficult to specify the value, it is about 20 μm on the order of magnitude according to the experimental results [1] . so the variation of particle number in the space because of diffusion deposition within time dt is where v is the space volume (cm 3 ); s is the area of vertical surface (cm 2 ). inserting eq. (6.21) into eq. (6.18), we obtain: when it is inserted into eq. (6.19) and simplification is made, the number of particles deposited on vertical surface with unit area by diffusional deposition is: in order to have an idea about the value of n, an example is made as follows: assume s v ¼ 1 4;000 cm à1 , t ¼ 3.6 â 10 3 s, d ¼ 6.2 â 10 à7 cm 2 /s (for particle with diameter 0.5 μm), and the number of these particles is n 0 ¼ 1 pc/cm 2 , so: n g ¼ 4; 000 â 1 à e à0:00028 à á â 1 ¼ 4; 000 â 0:00028 â 1 ¼ 1:12 pc cm 2 à á deposition of particles on undersurface includes the settlement and the diffusion. and diffusion also includes the molecular diffusion and the convective diffusion. in fuchs' opinion, the convective diffusion coefficient is close to 0, because the convective velocity approaches zero when it is near the undersurface. it is only the molecular diffusion that plays a role in the very small distance from the undersurface, which will influence the particle concentration distribution near the bottom, but will not influence the total deposition rate. therefore, the number of particles settled down onto the bottom with area 1 cm 2 within time t is: as mentioned before, although concentration keeps uniform in the space of the room without air supply, it varies with time t. the reduction of particle number in the air column with height h is consistent with the number of deposited particles, which can be express as: when it is inserted into eq. (6.24), we know: with the same data of the example in previous section (for particles with diameter 0.5 μm, v s ¼ 0.0015 cm/s, and assume h ¼ 200 cm), fumiko and susumi proposed the expression for the deposition of particles on the interior surface in the room with air supply [5, 6] : where f is the sedimentary area; t is the settling time; h s is the room height; h is the distance between the settling plane and the ceiling; n is the air change rate. since nh s v s is far greater than 1 for 0.5 min à1 , the above expression can be simplified as it should be noted that eq. (6.27) is valid for the extreme case with room height h s ! 1 for the room without air supply. only when the height is infinite ideally, the indoor particle concentration can be considered without variation because of deposition and considered as a constant. but there is no practical significance for the condition h s ! 1. in other words, this equation is not valid for the room without air supply. although eq. (6.26) is derived for the room with air supply, author thought it is not comprehensive to consider the particle deposition only, especially for the local plane. in the room with air supply, there are several ways for the deposition of particles onto plane, so the method to estimate the deposition efficiency for these ways is given. inertial deposition of particles on the plane is shown in fig. 6 .6. the inertial deposition efficiency is: figure 6 .7 shows the relationship between η st and st [8] . st is the inertial parameter, which has been introduced in chap. 3. table 6 .7 presents the exact value of η st . in the table, u is the air velocity and d p is the particle diameter. interception deposition of particles on the plane is shown in fig. 6 .8. for the condition with large reynolds number (equivalent with the situation of cleanroom) with unknown re value, only the upper limit of interception deposition efficiency can be calculated, namely, table 6 .8 gives the value of η r . r is the interception parameter, which has been introduced in chap. 3. sedimentation deposition of particles on the plane is shown in fig. 6 .9. the sedimentation deposition efficiency is where v s is the sedimentation speed; u is the air velocity. table 6 .9 gives the value of η g for the general circumstance. for horizontal plane, there is a very thin layer close to it, where the height and the temperature gradient are extreme small. its vertical component of convective velocity is much less than the vertical component to the plane. it tends to zero, which means the convective flux is approaching zero. so the corresponding molecular diffusion flux towards the plane is greatly reduced. when the dispersity of particles is larger, the number of particles deposited on the vertical plane with fig. 6 .9 inertial deposition of particles on the plane table 6 . diffusion deposition is larger, while that on the horizontal plane is less. this means the diffusion deposition efficiency on horizontal plane is less than that on vertical plane, or the maximum number of deposited particles can be considered with vertical plane. when n ¼ 1 pc/cm 3 and the diffusional coefficient for particles with diameter 1 μm is d ¼ 3 â 10 à7 cm 2 /s, we can obtain the value of i is 0.54 pc/cm 2 within 1 h according to eq. (6.20) . so the diffusion deposition efficiency for the velocity 0.3 m/s is [5] for particles with diameter 5 μm, of course, the value of η d for horizontal plane should be smaller than the above calculation result, or the maximum value can be determined with vertical plane. usually it could be ignored. when the circular monocrystalline silicon wafers with diameter 3 and 30 cm which is used for the production of integrated circuit are made as an example, the above deposition efficiency can be listed as follows: for particles with diameter 1 μm on the plane with diameter 3 cm on the plane with diameter 30 cm on the plane with diameter 3 cm on the plane with diameter 30 cm from the above sequence, the probability of sedimentation deposition is the largest in the room with air supply. others should also be considered appropriately. but the larger the plane area is (such as the example with diameter 30 cm), the lower the efficiency of both the interception deposition and the inertial deposition is (for the plane with diameter 30 cm, η r % 0 for particles with diameter 1 μm, and η r < 3 â 10 à5 for particles with diameter 5 μm). therefore, in order to make the estimation simple, the sedimentation deposition can only be considered with a deposition correction factor α. from the above sequence of efficiencies, it is known that for the circular silicon wafer with diameter 3 cm, α can be 1.3 for particle with diameter 1 μm, and it can be 1.1 for particle with diameter 5 μm, while it can be about 1 for particle with diameter 7.5-10 μm. for the circular silicon wafer with diameter 30 cm, α can be 1 for particle with diameter larger than 1 μm. with the above method, the value of α for particles with diameter 1 μm when the plane diameter !30 cm is shown in table 6 .10. for particles with the same diameter, values of α are different if the air velocity is different. the above data correspond to the air velocity 0.3 m/s. if the air velocity becomes 0.6 and 0.15 m/s, values of α are 1.6 and 1.15, respectively, for particles with diameter 1 μm on the plane with diameter 3 cm. that means when the benchmark is based on the air velocity 0.3 m/s, the deposition rate will increase by 1.23 times in the room with the same particle concentration, if the air velocity increases to two times. when the air velocity decreases by half, the deposition rate can also be reduced to 88 % of the original value. so we have the following correction formula: except for α, the influence factors of settlement amount include air velocity, particle settling resistance, particle density, and equivalent diameter. further correction can be made for the above express so that it can reflect the real situation well [9] . the formula can be rewritten as: where α is the deposition correction coefficient; ω is the air velocity correction coefficient, which considers the correction of α by air velocity. (the previous calculation is based on the air velocity 0.3 m/s in cleanroom. when the air velocity differs, the value of α will change. so ω ¼ 1 for the air velocity 0.3 m/s. when the air velocity increases to two times, the value of α will increase by 1.23 times, so ω ¼ 1.23. in the room without air distribution, there is still flow movement, so the corresponding air velocity can be 0.15 m/s. meanwhile, the value of α will decrease to 88 % of the original value, so þ is the settlement resistance correction coefficient, which considers the influence of particle shape on the settling velocity during the natural sedimentation process. it is obtained by eq. 6.4. (generally v s is calculated with spherical particle. but in reality dust particles are not completely spherical, and their surfaces are irregular. so the correction coefficient β considering the particle shape should be used. v s is linear proportional to the root of correction coefficient. for irregular particles, β is 2.75-3.35, and generally it is 2.75. correction is not needed for the unnatural sedimentation process.) ρ 0 p =ρ ð þ is the density correction coefficient. it can be obtained with eq. (6.7). (as mentioned before, the density for atmospheric dust is usually ρ p ¼ 2. but in the place where people density, activity, and dust are a lot, the particle density ρ 0 p may be 2-2.5. in some experiment such as the liquid droplet, ρ 0 p ¼ 1. so this kind of correction is not needed for the general situation.) it is easy to determine the value of v s in eq. (6.32) when particles are monodisperse. when airborne particles are polydisperse, it should be calculated with some kind of average diameter. the amount of deposited particles is dependent on the frontal resistance, which is related to the cross-sectional area. so the area weighted diameter should be adopted to describe the average diameter of whole particles, which is used to estimate the deposition amount. when the particle concentration of cleanroom is n ¼ 1,000 pc/l ¼ 1 pc/cm 3 , the area weighted diameter for airborne particles with diameter larger than 0.5 μm is d s ¼ 0.98 μm [9] . that means the deposition amount for particles with diameter ! 0.5 μm can be considered as the deposition amount with particle diameter 1 μm. so the total deposition amount of particles on the surface with area 1 cm 2 per hour can be obtained in the cleanroom, where the airborne particle concentration is 1,000 pc/l and the air velocity is 0.3 m/s. it is also called the unit deposition rate, which is the deposition concentration mentioned in chap. 2. as mentioned before, the calculated result only represents the possibility of the deposition amount of particles. it may be possible for the resuspension after deposition or no deposition because of the disturbance on the ground. the above data can only show the maximum probability of deposition, which is related to the surface exposure time and the actual dispersity of specific particles. table 6 .11 shows the comparison of measured data and calculated data about the deposition amount of particles on the surface with unit area in the room with air supply. the experimental data are from the report of tan dade from institute of havc at chinese academy of building science. it was obtained with the microscope, when the total number of particle with diameter !5 μm is counted on the steel disk. in the table, the calculated deposition rate was obtained with the standard particle size distribution for particle diameter 5-10 μm. the particle size distribution is as follows: other rooms are omitted. this result is much closer to reality compared with the calculation result with the average diameter as the equivalent diameter performed by author [7] . with the calculation results in previous sections, the ratio of particle deposition amount on the vertical surface to that on the undersurface is very small. so it is unnecessary to use the material such as the advanced stainless steel for the wall in the cleanroom, and the requirement for the hygienic cleaning of the vertical wall is lower than that of the floor. from the discussions in previous sections, with the effect of gravity, inertia force (mechanical force), and diffusional force, the velocity and the distance are very small. for particles with diameter 1 μm, the movement distances are 0.006, 0.006, and 0.0004 cm, respectively. the indoor air velocity (including the air velocity with heat convection) is usually more than 0.1 m/s. in the flowing air, small particles will follow the movement of airflow with the same velocity [10] . semiempirical equation can be used to calculate the flow in circular pipe, and the calculation result is well agreed with the experimental data. for the single spherical particle with diameter d p and density ρ p in the flowing air with density ρ p , when it follows the airflow completely, the force acting on the particle equals with that on the fluid whose volume is occupied by the particle, i.e., d dt where u a is the velocity of the fluid in the space which is occupied by the particle; t is the time. in fact, particles will not completely follow the airflow. the force exerted on the particle equals with the force component f r by the relative movement between particles and the fluid subtracted from the above force. so the governing equation for the movement of the particle is d dt where v p is the particle velocity; f r is equivalent to the resistance on the particle with velocity v pu a in the still viscous flow. the solution process of this equation is so complex that it will not be cited here. the study result is given: ω is the fluctuation frequency of turbulent flow (khz). except the semiempirical equation for the flow in circular pipe where both the calculation and the experimental results agree well with each other, so far there is no good result in this aspect for the cleanroom. but compared with the calculation result in circular pipe, the order of ω is only thousands hz in the cleanroom. so particles with diameter d p ¼ 5 μm and this means the relative difference between the velocity of particle following the airflow when the diameter d p ¼ 1 μm and the air velocity is less than 10 à3 . this can be further verified by the research about the particle trajectory and the streamline trajectory at the control site (such as the bench) which was performed by shuji et al. [11] . the obstacle at the control site will cause influence on the particle and generate the energy by the turbulent flow. this influence is larger than that at other places. these influences include the inertial force, the diffusional force, the buoyant force, and the electrostatic force. only when the gravity was considered, the x-and y-components of the velocity were calculated with the movement equation of the particle by fujii [11] . the change rate was obtained with the integration in the small time δt (1 â 10 à5 s), and the movement coordinates of the particle is then calculated. figure 6 .10 shows the calculation result about the particle trajectory. the initial condition of calculation is that the particle velocity is the same as the air velocity. the calculation terminates at the height with ordinate à0.05 above the control site. the particle sizes used were 0.621 and 1.004 μm. it is shown from the figure that δm x and δm y are the difference between the particle trajectory and the streamline trajectory in x-and y-directions. the values are shown in table 6 .12. it is shown in the table that for particle with diameter 1.004 μm, when u a is less than 0.3 m/s, the relative deviation between the particle trajectory and the streamline trajectory is not possible to be larger than 10 à3 , which agrees with the above velocity. so the conclusion of this research was that the particle trajectory at the control site is approximately the streamline trajectory. so in other places of the cleanroom, it is reasonable to consider that particle moves with the airflow together. from the above result, it can be thought that even for the case v p u a < 0:9, the air velocity is much larger than that caused by settlement, diffusion, and inertia of particles. so the following velocity of particle is still the main factor that influences the distribution of particles, but there is a lag of time for the airflow. it will not cause any problem for the investigation, and it should be considered only for the study of laser doppler velocimetry. therefore, it is the air distribution that mainly influences the airborne particle distribution. indoor particles will be affected by the airflow from air supply (including the primary air and the secondary air), the flow caused by occupant's walk, and the flow by heat convection. except for the primary air from the supplied air, the influence of other kinds of flow will be introduced in this section. it is a concern for the people that under what kind of situation, resuspension of particles by the airflow will occur, after they deposit onto the surface. because resuspended particles may be taken away by vortex and then cause damage. this is the migration effect of airflow. when particles are assumed spherical, the force to suspend particles with the effect of horizontal airflow is a function of the weather area of the particle, i.e., where u c is the surface velocity, namely, the air velocity flowing along the surface of the particle (m/s); φ is the suspension coefficient. when the suspension force is larger than the particle weight, it can be expressed as: the suspension coefficient is an experimental value, which is difficult to determine. but for spherical particles, the suspension coefficient is approximately same as the resistance coefficient. so when re < 1, φ ¼ 24 re can be inserted into eq. (6.36). when ρ a is ignored, we can obtain there is a boundary layer when air flows along the surface. the air velocity above the boundary layer is much larger than that within the boundary layer, and usually it reaches more than three times. so the air velocity to suspend particles should be when the air velocity reaches u, the migration of particles is formed in the following way. as shown in fig. 6.11 , with the effect of gravity, particles in the airflow will deposit on the bottom gradually and rotate forward and slide with the frontal airflow. when air passes through the rotating particles, vortex will be formed at the bottom and the side of the particle, which increases the pressure relatively. the pressure at the top of the particle reduces by the airflow. with the difference of the pressure between the top and the bottom, particles are suspended. when particles suspend to the height where the air velocity at the top is the same as that at the bottom, particles begin to deposit again with the gravity. particles near the surface undergone the migration process of deposition-rotationsuspension with the enough intensity of the horizontal airflow, and it repeats continuously. the migration velocity calculated with eq. (6.36) is very small. but experiment has shown that it is not easy for the very small particle on the plate to be blown away by the airflow, and the reason is that the molecular force of the interaction between the particle and the wall surface, as well as between particles, is not considered. it is shown in eq. (6.36) that the velocity u c needed for the suspension of the particle will be large when this kind of molecular force is added. but detailed experimental data in this aspect are very rare, and it is very difficult to determine the magnitude of this molecular force. however, the influence of the molecular force can be estimated from the experimental curve in fig. 6 .12 [12] . in the figure, the abscissa represents the particle radius. the curve shows that the air velocity needed to suspend particles with radius less than 50 μm will increase. the solid line in the figure is the experimental data, and the dashed line for extrapolation is added by author. after suspended by the airflow, particles with diameter larger than 10 μm will deposit soon. while for particles with diameter less than 10 μm, the air velocity needed to suspend them is bigger, namely, these particles are difficult to be suspended. therefore, when air velocity should be controlled that particles will not be suspended, the control diameter can be 10 μm. in the experiment, the grit was used. for the grit with diameter 10 μm, u is about 32 cm/s. if particles with the density which is 2/3 of the grit, u can be 20 cm/s. this means the air velocity along the surface (mainly the floor) in the cleanroom should not be larger than 20 cm/s (it is not limited for the horizontally unidirectional flow air cleanroom). for example, the backflow velocity for the side air supply mode should be determined with this consideration. from the analysis above about the particle deposition and suspension, for the deposited particles on the horizontal plane such as the floor in the cleanroom, big particles are more likely to suspend and migrate, which generate the secondary float. this is opposite with the common opinion that the smaller particles are likely to be blown away from the floor. for cleanroom, one large particle is more hazardous than one small particle. on the other hand, large particles are likely to deposit, and the number of deposited particles on the floor is large. so the cleaning work on the horizontal surface in the cleanroom cannot be neglected. surface cleaning treatment must be performed for the object entering the room, since the air velocity along the surface is much larger and particles on the surface are more likely to suspend than that on the floor. since particles follow the airflow almost with the same velocity of the air, except for the supply air, other local airflows will also have influence on the movement and distribution of particles. heat convection airflow is one kind of important factors. for example, the buoyant flow near the shadowless lamp can be 0.6 m/s [13] , but its influence on the particle distribution in the cleanroom has not been noticed and investigated. there are three kinds of situations for the buoyant flow generated by heat convection airflow. the following part will introduce the method to determine the velocity for this kind of the airflow. the surface temperature of the heat wall is higher than that of the ambient air. convection will be generated near the wall surface because of the temperature difference. after the rise of the air, it will stretch out, which will promote the dispersion of particle pollution [14] . author has ever observed the fluorescent lamp with 40 w vertically installed on the wall (the bottom of the lamp was 0.62 m above the floor) as shown in fig. 6 .13 [15] ; the buoyant flow in a certain range of two ends was measured with thermometer velocimetry when no air supply was provided. at the top, the air velocity at the place 2 cm from the lamp surface reached 0.22 m/s, and the thickness of the layer with obvious air velocity reached 20 cm. when smoke was released to observe along the whole length of the lamp surface with 15 cm away, the buoyant flow appeared. outside the layer of this buoyant flow and until about 1 m away, backflow appeared with both the method of the silk thread and the smoke release. so it is advisable to consider the airflow within 15 cm away from the surface turbulent, and that in the range between 15 cm and 1 m is eddy with uncertain direction. in ref. [16] , it is also pointed out that there is usually three layers of airflow in the direction perpendicular to the vertical heat wall. except for the above two layers, the most inner layer is laminar flow with very thin thickness, which is difficult to distinguish. the combined thickness of laminar and turbulent flow is much thinner than the third layer. figure 6 .14 shows the situation of parallel flow with air supply velocity 0.25 m/s. the two flow patterns observed in the above experiment near the fluorescent lamp the above observed phenomena show that: 1. the buoyant flow near the vertical heat wall such as vertically installed fluorescent lamp is very strong, and the influence range is very large. 2. under the situation of air supply with a certain velocity, the influence will be limited. when the particle source is 0.5 m outside of the lamp surface, the particle distribution will not be affected by this buoyant flow. experiment with the particle source located in the room center where is 1.5 m away from the lamp surface shows that even though the air supply velocity is as small as 0.105 m/s, particles released from the source will appear in the range 1 m below the lamp. ref. [10] also pointed out that if the pollution source is in the stagnant region of the lee side of the object, its influence is much larger than that in the backflow region. if there is also no pollution source in the backflow region, no influence will be generated. this is consistent with the above observed case when pollution source is far from the backflow region. since it is difficult to install the lamp at the ceiling of the unidirectional flow cleanroom and it also occupies the air supply area, someone has ever proposed to install the lamp on the vertical wall. from the above analysis, when there is air supply with a certain speed in the room where the indoor area is very small, the buoyant flow generated will cause very large influence, when the fluorescent lamp was installed at a relative high position of the vertical wall (such as above 1.5 m). fuchs gave the equation to calculate the buoyant flow velocity along the heat wall [17] , (table 6.13): where l is the height from the bottom of the heat wall (m); t s and t a are the surface temperature of the wall and air temperature, respectively (k); β is the air expansion coefficient, which equals with 1/t a ; g is the gravitational acceleration (m/s 2 ). but according to the experimental data provided by батурин [18] , eq. (6.40) should be modified to: in the environment with normal temperature 23 c, the surface temperature of the fluorescent lamp with 40w was measured to be about 40 c, namely, the temperature difference is 17 c. table 6 .14 shows the calculated velocity of the buoyant flow with eq. (6.39) and the measured velocity. they are close to each other. so it is appropriate to use eq. (6.41) to calculate the buoyant flow velocity near the heat wall. the velocity of the buoyant flow on the heat surface reaches the maximum at 1-2 cm above the surface [16] . in author's opinion, human body can be treated as the vertical heat wall approximately [15] . the relationship between surface temperature of the human body and the room temperature is shown in fig. 6 .15 [19] . when the temperature difference is 5 and 7 c, the calculated value of u with eq. (6.40) is 0.18 and 0.22 m/s, respectively. according to the measurement abroad [13] , the velocity of the buoyant flow near the surface of human body reaches 0.2 m/s, which is consistent with the calculated result. it is clearly shown in fig. 6 .16 that even with the down supply of table 6 .13 velocity of the buoyant flow along the vertical heat wall (m/s) (wall height 2.74 m, human body located at two both sides with the white color. the white part in the middle represents the airflow. the white part below means the operating table parallel flow, backflow will appear near the surface of human body [20] (of course the surface of human body is uneven). this explains the influence of the buoyant flow on the air distribution. as for the buoyant flow along the wall in the room with air-conditioning system in summer, the temperature difference between room and inner wall is about 2 c, so the buoyant flow along the wall is weaker than that along the human body. лыков gave the equation to calculate the velocity of the buoyant flow along the heat object with a certain volume [14] : where l is the characteristic length, i.e., the length of the airflow around a body (m). for example, l ¼ l 0 (l 0 is the thickness of the plate) for the plate, l ¼ ðπd=2þ for sphere, and l ¼ h for the cube where its height is h. it is obvious that compared with the pure vertical heat wall, the velocity of the buoyant flow along the heat object with the same height is much larger, which can be explained by the fact that the coefficient in eq. (6.24) is much larger than that in eq. (6.41). figure 6 .17 shows the electric furnace with two pipes. the velocities of the buoyant flow at the intervals of 20 cm above the shell were measured, and the measuring positions are shown in the figure. the velocity of the buoyant flow increases and reaches the maximum (0.75 m/s) at about 1.2 m above the shell and then reduces gradually. it is not easy to choose the value of the parameter in eq. (6.42) when it is used for calculation. since the value of l is only several centimeters (when the porcelain tube is concerned) and the temperature difference is several hundreds, the order of magnitude for the calculated result is equivalent with that of measurement. it is obvious that eq. (6.42) is not suitable to describe this problem. эльтерман has performed experimental study [18] , and куница derived the theoretical expression which agrees well with experiment [21] : where z is the vertical height from the heat source surface (m); u z is the air velocity at z (m/s); δt is the temperature difference between surface and ambient medium ( c); r y is the equivalent radius of planar heat source (m). for rectangular heat source where a is the length of long side on the rectangle (m); b is the length of short side on the rectangle (m). for circular heat source r y ¼ rðmþ when z % 1.8r y , the above equation can be simplified as the above equation shows that the velocity of the buoyant flow above the heat source increases from zero to the maximum which is at about z % 0.43 m and then reduces gradually when it is further above the heat source. figure 6 .18 shows the curve by experiment abroad [22] . it represents the boundary of the pollution (namely, the boundary of the particle distribution). the larger the air supply velocity is, the lower the height of the pollution boundary is. data on the right are the data added by author with the above equation. when the temperature of hot surface reaches 200 c in the room without air supply, the maximum velocity of the buoyant flow is 0.47 m/s, which appears at z ¼ 0.43 m. at the height of 1.2 m, the measured velocity is 0.375 m/s, while the calculated value is 0.4 m/s. so the calculated result is close to the measure value. in the common references, people in cleanroom should not walk too fast. usually it is about 3.6 km/h (1 m/s), because the secondary flow caused by people's walk will drive particles to move together. but there is no study about how much is the fig. 6 .19, and the following conclusions were obtained: 1. the maximum velocity of secondary flow. this value was not obtained with the velocity sampling at some place when people pass by (the intersection place "o" in the figure) but was the measured velocity at the place where people stop (a certain distance from the sampling place). meanwhile, the closer the distance from the human body is, the larger the velocity of secondary flow is. 2. the relationship between the maximum velocity of secondary flow and the speed of people's walk. figure 6 .20 shows the measured velocities along x-and ydirections. although the relationship between the maximum velocity v max of secondary flow and the speed v of people's walk is not apparent, the boundary for the maximum value can be estimated with the following equation: the above measurement and analysis of the secondary flow caused by people's walk can be the basis to determine the lower limit of air velocity in horizontally unidirectional flow cleanroom. this will be discussed in chap. 8. airborne particles will become big particle because of the mutual collision and adhesion during the relative movement process (that caused by brownian motion, gravity, or aerodynamic force). this phenomenon is called coagulation of particles. with particle coagulation, particle size will become large, which is good for observation, measurement, and removal. the coagulation process can be classified as thermal coagulation and movement coagulation (the movement resulted from airflow movement and acoustic vibration). next the phenomenon of thermal coagulation will be briefly introduced. according to fuchs' derivation [23] , the average time interval between two collisions is 1 4πdd p n . the relative dispersion coefficient of two contacting particles equals with the summation of dispersion coefficients of two particles. when two particles have the same size, it becomes 2d. so the number of particles which contact with one particle per unit volume in the time period t ¼ 1 s is because there are n particles in total per unit volume, the time of collisions will be 1 2 n8πdd p n . the coefficient 1/2 was introduced because two particles will coagulate and become one particle when they collide with each other. the collision rate ðdn=dtþ will be negative with the following expression: since d p d can be approximately constant, the change rate of particle concentration will become where k 0 is called coagulation coefficient. it can be calculated with the following expression: when integration is performed on eq. (6.47), we obtain: so we get: where n 0 is the initial concentration; n t is the concentration at the moment t, which can be obtained with the following expression: table 6 .15 shows the value of k 0 under standard condition. table 6 .16 shows the variation of concentration during the thermal coagulation process for monodisperse particles when k 0 ¼ 5 â 10 à10 cm 3 /s [24] . from the above data, when initial concentration is less than 10 6 pc/cm 3 , the influence of coagulation can be neglected during measurement with time period 10 min. if the measurement time is 2 days, the concentration should be less than 10 3 pc/cm 3 so that the coagulation effect can be ignored. this aspect should be noticed during the experimental study, leakage detection of air filter, and selfpurification time with smoke release. the above sections have discussed various movement situations of single particle and the influence generated by local airflow. now the further study is presented about the particle distribution with particles released by the source in the flow field. the simplest case is the particle distribution with the point source in the parallel flow field. the specific aim is to determine the boundary of particle distribution, namely, the range of pollution. here it is called the enclosure line of pollution. in practice, there is the particle source which can be treated as point source, such as the spray of the leakage hole in one direction or several directions or the release of the leakage from the unsealed equipment. these particle sources exist in clean environment, and their size is very small; otherwise, they are not allowed to be inside the room. the meaning to study this problem is to find the basis for the air velocity which can be used to control pollution from various directions. this will also be discussed in chap. 8 about the lower limit of air velocity in unidirectional flow cleanroom. figure 6 .21 shows the case with point pollution source in parallel supply air. the flow field in the room is the combination of the parallel supply air and the flow field by point pollution source. now the spherical coordinate is adopted. r is the radial distance from point pollution source. θ is the intersection angle between r and z axis, and the anticlockwise direction is positive. the stream function of parallel flow field is: the stream function of point source flow field is: the stream function of the stack flow field is: where v 1 is the air velocity of parallel supply air (m/s); q is the source strength, which is equal to the flow rate of polluted flow (m 3 /h). when the air velocity equals with the velocity of polluted air, the polluted air along z axis direction is restrained at the position a (shown in fig. 6.22 ), which can be called the stagnation point. polluted air in other directions will be restrained at positions b and c. when the supplied air arrives at position a, it begins to turn round and moves forward when passing positions b and c (it is the same for other directions). this means when the polluted air flows through the lines a, b, and c, the component of velocity along z direction disappears. when the influence of molecular dispersion is not considered and that of flow fluctuation is very small, the polluted air cannot pass through this line and is strained under this line. therefore, this line can be called the enclosure line of pollution [15] . so the enclosure line of pollution is the line passing through points a, b, and c. for position a, θ ¼ 180 , so the stream function is: when the above two equations are combined, we obtain: cos θ (6.55) in fact, pollution source is not a geometrical point; instead, it should have a certain volume. suppose the radius is r and the velocity on the spherical surface with radius r is v (q ¼ 4πr 2 v), the above expression can be simplified as this is the trajectory equation for the enclosure line of pollution which passes through points a, b, and c. now take the actual distribution of particles released by point pollution source simulated in parallel flow as an example, the test rig is shown in fig. 6 .23 [15] . the pollution source was the table tennis with radius r ¼ 2 cm and many perforation on it. compressed air was used to spray the smoke from five cigarettes. the average pollution concentration supplied into the room is shown in table 6 .17. particle counter was used to measure the cross-sectional concentration in the center of the room at the working area height 0.8 m above the floor, when different conditions the data in the bracket is the flow rate (l/min) 6.8 enclosure line of point pollution in parallel flow with parallel air velocity and particle generation rate were provided. figures 6.24, 6.25, 6.26, 6.27, and 6.28 are several cases. in the figure, the abscissa represents the sampling position. the measured concentrations by particle counter were labeled. the sampling position without data means the measured value is zero. this means the average concentration in these positions is extremely low, so these places are not polluted. it is observed in the experiment that for pollution flow with velocity v ¼ 1.27 m/s from the pollution source, it disappears once leaving the hole. from the figure of concentration field, pollution can barely be measured at the same height of the pollution source. for pollution source with velocity 2.5 m/s, the elevated height of the polluted buoyant flow was observed only several centimeters. from the figure of concentration field, pollution can only be detected at the height of the pollution source. for pollution source with velocity 2.7 m/s, the increase of pollution concentration becomes larger. the elevated height of the buoyant flow was observed 10 cm. it is shown from the figure of concentration field that pollution was detected at the height 10 cm above the pollution source. in short, when the ratio of the polluted air velocity to the supply air velocity is less than 20, the elevated height of the polluted flow is within 20 cm (in the figure of concentration field, there is not sampling point at 10 cm below). experiment has also shown the situation of the buoyant thermal flow from the fluorescent lamp on the wall. the left of the concentration field is the situation when the lamp is turned on. since the downwards supply air velocity above the lamp is between 0.16 and 0.3 m/s and the dust source is 1.5 m away, polluted particles are not detected near the region above the lamp. the right of the figure shows that the downwards supply air velocity above the lamp is less than 0.1 m/s. three rows of measured data at 0.5 m away from the lamp show the obvious increase of pollution when the lamp is turned on. the pollution height reaches 1.5 m (shown in fig. 6 .25). this is consistent with the previous case about the buoyant flow near the wall. difference is large between the calculated enclosure line with eq. (6.54) and the boundary of particle distribution by experiment. the range of the former case is relative narrow, while that of the latter case is much spacious. it can be found with the analysis of experimental concentration field that: 1. although the decrease rate of pollution flow is very fast, it is still slower than the calculated result with spherical coordinate. 2. the crosswise stretching range of polluted air is much larger than the range in the enclosure line. as for the first conclusion, the main reason is that the actual pollution source is not a uniform spherical dispersion source, and it has the characteristic of jet flow from small pore. the decline of velocity is very slow. after a certain distance, it declines like the spherical surface. as for this problem, there is no special investigation, and the data of perforated spherical air supplier performed by baturin can be referred [17] , which is shown in fig. 6 .29 and table 6.18. as for the second conclusion, it has also been shown in the experiment performed by baturin, which was specially pointed out in fig. 6 .29. this kind of velocity field in different directions with θ is not uniform, and the crosswise stretch is much faster. the feature of the velocity field with the perforated spherical air supplier should be investigated further. a correction coefficient related to θ should be added for eq. (6.56). according to the in-site tested concentration field, the value of this correction coefficient can be ð1 à cos θþ à1:5 . so eq. (6.56) becomes the following semiempirical equation: r ¼ 5:7 1 à cos θ ð þ à1:5 r ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi v v 1 1 à cos θ ð þ r (6.57) where r is called pollution radius. curves in figs. 6.24, 6.25, 6.26, 6.27, and 6.28 are the enclosure line calculated with this equation. as mentioned before, with the influence of thermal convection caused by the fluorescent lamp and the influence of the eddy caused by the frame between air filter and side wall (refer to chap. 8 about the unidirectional flow cleanroom), the buoyant flow along the wall will drive the polluted flow towards two sides, so the two end of the pollution region from experiment is wider than the width of the calculated envelope line. it is also the case under the height of the return air grille. but compared with the calculated envelope line, it is still consistent for the pollution region above the return air grille. particles released from the pollution source are within the range of the envelope line. only when v 1 v is larger than 20, some particles will go outside of the envelop line. it can be estimated that the theoretical result will match well with practice for the standard parallel line by full return air on the floor. the further discussion about eq. (6.57) will be performed in chap. 8 about the lower limit of air velocity. the mechanics of aerosols (trans: gu zhenchao) dust removal in the crush and sieve workshop the mechanics of aerosols (trans: gu zhenchao) the mechanics of aerosols (trans: gu zhenchao) abstracts of the academic lectures by japan building institute (kanto) measurement of sterile environment relationship between the yield and the air cleanliness level of the environment impaction of dust and small particles on surface and body collectors exploration of the relationship between the deposition bacterial method and the airborne bacterial method following characteristic of discrete particles in turbulent flow investigation on the design of laminar flow cleanroom. abstracts of the academic lectures by the mechanics of aerosols (trans: gu zhenchao) influence of obstacle and thermal plume in laminar flow room theoretical basis of building thermal physics (trans: ren xingji, zhang zhiqing) lower limit of air velocity in parallel flow cleanroom indoor air dynamics (trans: zhou moren) the mechanics of aerosols (trans: gu zhenchao) industrial ventilation principle (trans: liu yongnian) вопросы лучистого отопления fundamental and clinical study of vertical flow device in cleanroom конвективные струи над нагретыми поверхностями environmental control in electronic manufacturing the mechanics of aerosols (trans: gu zhenchao) aerosol technology (trans: sun yufeng key: cord-286878-5nv3ec5l authors: simpson, j.p.; wong, d.n.; verco, l.; carter, r.; dzidowski, m.; chan, p.y. title: measurement of airborne particle exposure during simulated tracheal intubation using various proposed aerosol containment devices during the covid‐19 pandemic date: 2020-06-19 journal: anaesthesia doi: 10.1111/anae.15188 sha: doc_id: 286878 cord_uid: 5nv3ec5l the covid‐19 pandemic has led to the production of novel devices intended to protect airway managers during the aerosol‐generating procedure of tracheal intubation. using an in‐situ simulation model, we evaluated laryngoscopist exposure of airborne particles sized 0.3 ‐ 5.0 microns using five aerosol containment devices (aerosol box; sealed box with and without suction; vertical drape; and horizontal drape) compared with no aerosol containment device. nebulised saline was used as the aerosol‐generating model for 300 seconds, at which point, the devices were removed to assess particle spread. primary outcome was the quantity and size of airborne particles measured at the level of the laryngoscopist’s head at 30, 60, 120, and 300 seconds, as well as 360 seconds (60 seconds after device removal). airborne particles sizes of 0.3, 0.5, 1.0, 2.5 and 5.0 microns were quantified using an electronic airborne particle counter. compared with no device use, the sealed intubation box with suction resulted in a decrease in 0.3, 0.5, 1.0 and 2.5 micron, but not 5.0 micron, particle exposure over all time‐periods (p = 0.003 for all time periods). compared with no device use, the aerosol box showed an increase in 1.0, 2.5 and 5.0 micron airborne particle exposure at 300 seconds (p = 0.002, 0.008, 0.002, respectively). compared with no device use, neither horizontal nor vertical drapes showed any difference in any particle size exposure at any time. finally, when the patient coughed, use of the aerosol box resulted in a marked increase in airborne particle exposure compared with other devices or no device use. in conclusion, novel devices intended to protect the laryngoscopist require objective testing to ensure they are fit for purpose and do not result in increased airborne particle exposure. the coronavirus 2019 (covid19) pandemic has highlighted the urgent widespread necessity for adequate personal protective equipment (ppe) for healthcare providers. coronavirus 2 (sars-cov-2), the virus that causes covid-19, is found in highest concentration in sputum and upper airway secretions. although the sars-cov-2 coronavirus itself ranges in size from 0.075-0.160 microns, it requires a water and mucous envelope to spread [1] . respiratory secretions, consisting of mucus and water, provide these envelopes. the size, accumulation and volume of virus-containing envelopes determine the size of respiratory droplets. droplets can be categorised as either large (> 60 microns in diameter) or small (10-60 microns in diameter). large droplets tend to fall on surfaces closer to the patient (< 2 m) and small droplets, with thinner mucus and water envelopes, tend to fall on surfaces further away [2] . envelopes < 5-10 microns in diameter are not called droplets, but airborne particles or infectious droplet nuclei. airborne particles will remain suspended in the environment for a period of time depending on a number of factors including air circulation, humidity and atmospheric pressure [2] . aerosolisation, such as that produced during aerosol-generating procedures produce airborne particles as well as both small and large droplets [2, 3] . aerosol-generating procedures are thought to increase the risk of infection to healthcare providers [4] . tracheal intubation is one of the highest risk aerosolgenerating procedures performed due to direct exposure to the airway and potential patient coughing during induction [4, 5] and is of particular concern to frontline anaesthetic, emergency, and intensive care teams. this has created a race to manufacture aerosol containment devices including improvised protection strategies for use during tracheal intubation. there has been significant promotion of these devices on social media, resulting in rapid proliferation and discussion globally [5] [6] [7] [8] [9] [10] . whilst these innovations aim to reduce aerosol dispersal, many have not been tested and are presented as viable options with only short reports and correspondence being cited in peer review literature. currently, the use of aerosol containment devices is not recommended by any international ppe guideline [11] [12] [13] [14] [15] . the consequences of promotion of such untested devices include either a false sense of security using these devices, or paradoxical increase in healthcare workers exposure [16, 17] . some devices claim to protect against both large and small droplets as well as airborne particles in small simulation experiments. using a simulated cough producing fluorescent droplets, canelli et al. [16] compared the contamination of a laryngoscopist with and without an aerosol box. they concluded that the device reduced the macroscopic contamination of both the laryngoscopist and their immediate surroundings. however, their simulation did not test for air turbulence and flow direction, nor were they able to visualise small droplets accepted article or airborne particles contained in aerosols. however, this did not demonstrate any quantifiable reduction in either particle dispersal or subsequent risk of infection. begley et al. [17] performed a simulated crossover study assessing the effect of two aerosol boxes on tracheal intubation performance and found the safety of the laryngoscopist and patient could be compromised by an increased time to intubation and potential damage to ppe. to guide our institutional protocols for the airway management of patients with suspected or confirmed covid-19, we sought to test whether different aerosol containment devices confer any protective advantage to the laryngoscopist specifically with respect to airborne particle dispersal. we also aimed to examine the pattern of airborne particle dispersal in the room upon removal of these devices. our primary research question was how aerosol containment devices (aerosol boxes and plastic drapes) placed over a patient during tracheal intubation compare to no intervention with respect to exposure of the laryngoscopist to airborne particles? our secondary research question was to measure the size and distribution of the particles for each device and how effective they are, or not, at reducing airborne particle dispersion over a 5-min time period, and at 60 s post-removal of the aerosol containment device. this was a single centre, prospective non-blinded in-situ simulation study performed at a major metropolitan teaching hospital in melbourne, australia. consent was obtained from participants for both research and publication of images. simulation was performed in a self-contained, intensive care unit (icu) room measuring 5.2 x 4.2 x 2.7 m. the room was pressure-equilibrated with the rest of the icu, with an estimated negative pressure of -10pa and 18 room air changes per hour of (300 l.s -1 ). seven healthy adult (> 18 y) volunteers, four male and three female, participated in this study, each volunteer acting as both patient and laryngoscopist in random order. each patient lay supine on a pillow (10° inclination) on a standard icu bed. another volunteer laryngoscopist stood at the head. the room doors were closed. to conserve ppe, the laryngoscopist wore only a surgical facemask and gloves. a lighthouse 3016iaq airborne particle counter (fremont, ca) was positioned on an intravenous pole preset at head height (75 cm above the laryngoscopist's head) immediately in front of the laryngoscopist. this height of the airborne particle counter on the intravenous pole was maintained throughout the duration of the experiment. the airborne particle counter, typically used for indoor air quality testing in semiconductor cleanrooms, research laboratories, and operating theatres, utilises a laser diode and photo detector to count particles by collecting scattered light from particles as they pass through a sample inlet ( fig. 1) . the device counts airborne particles within of 0.3, 0.5, 1.0, 2.5 and 5.0 microns. airborne particle counter flow rate was set to 2.83 l.min -1 , with detection of the ambient air occurring in 1 s sweeps. the full specifications for the airborne particle counter used are included in online appendix s1. four aerosol containment devices and no aerosol containment device were trialled once by each of seven laryngoscopists in a random order. the four aerosol containment devices tested were: a locally manufactured aerosol box with similar specifications to those reported in the literature attached to wall suction (fig. 2a) ; a clear plastic drape suspended vertically above the patients' head as a barrier between the patient and the laryngoscopist, with the laryngoscopist's hands placed under the drape (fig. 2b) ; a clear plastic drape forming a horizontal tent above the patients' upper torso, with the laryngoscopist's hands placed under the drape (fig. 2c) ; and a sealed aerosol containment box with openings through a neoprene sheet and rubber gloves, tested using two configurations, with and without wall suction, each connected to a heat/moisture exchange viral filter (fig. 2d) . aerosol containment devices were tested against no device use (fig. 2e ). in total, six configurations were tested with seven different laryngoscopists totalling 42 trials performed. specifications regarding aerosol containment devices can be found in online figures s1 and s2). to simulate aerosolisation, 5 ml of saline was nebulised at 6 l.min -1 , using a standard hudson rci "micro mist" small volume nebuliser (teleflex, wayne, pn, usa) without the facemask, and held beneath the patient's mouth. the patient coughed every 30 s for the duration of the 5-min trial. the aerosol containment device was then removed at the 300 s time-point and the airborne particle counts were also measured for 1 min after device removal. following this time, the room doors were opened, and particle counts in the room were allowed to passively return to under 500 particles of 0.3 micron size between trials. particle counts were zeroed to the baseline environmental reading at the start time of each trial. this article is protected by copyright. all rights reserved the sample size calculation for this experiment was based on an initial pilot experiment showing a two-to three-fold increase in particle counts at 0.3 micron at 30 s. to detect a mean difference of 12,000 particles (sd 8000) from a baseline level of 8000 particles with a two-sided significance level of 1% and power of 80%, an estimated seven participants were needed per device tested. all values were normalised against the background particle count present in the intensive care room at the start of each individual trial. time series graphs reflect median values of 5 s rolling averages of particle counts for each trial. differences between groups that showed heterogeneity of variance were calculated using a kruskal-wallis 1-way analysis of variance (anova) at 30 s, and 1-min intervals. a two-sided p value of < 0.05 was used for statistical significance of the anova. post-hoc analysis between these groups was performed using a mann-whitney u test and as multiple testing was conducted, a bonferroni correction was used with a pvalue of 0.01 considered to be significant. all statistical analysis was performed using ibm spss v23 (ibm). the median 5 s rolling average of the total particle counts of all five particle sizes over the 5-min trial period for each device can be seen in figure 3 . there was no significant difference with the horizontal or vertical sheet compared to no device use. only the sealed aerosol containment box on continuous suction demonstrated an airborne particle count similar to baseline. a kruskal-wallis anova testing the hypothesis that there was a difference in median airborne particle counts between the six interventions over the 5-min trial period at each time-point and at each particle size showed significance (one-way anova p < 0.001). median and iqr for each intervention at each particle size as well as a total count across all sizes at 0, 30, 60, 120, 180, 240, 300 and 360 s can be found in the online supporting information (table s1) . post-hoc comparisons were then performed to compare these median airborne particle counts for each device at each time-point to no device use (table 1) . compared with no device use, the sealed box with suction resulted in a decrease in 0.3, 0.5, 1.0 and 2.5 micron, but not 5.0 micron, particle exposure over all time periods beyond 30 s (p = 0.003 for all time periods) ( table 1 ). the sealed box without suction showed a decrease in 2.5 micron particles compared to no device use, but only at 300 and 360 s (p = 0.004, p = this article is protected by copyright. all rights reserved 0.007) ( table 1) . compared with no device use, the aerosol box showed an increase in median particle count at 300 s for 1.0, 2.5 and 5.0 micron particles (p = 0.002, p = 0.008, p = 0.002) (fig 4a-e, table 1 ). both horizontal and vertical drapes showed no difference in airborne particle size exposure of the laryngoscopist compared with no device use. apart from no intervention compared to the sealed box, the difference in particle spread of 0.3 micron particles with a device when compared to no intervention was not significant (table 1, figure 4a ). during testing, it was also noted by investigators that clouds of aerosolised particles escaped from the arm openings of the aerosol box towards the laryngoscopist with each patient cough. for every particle size, the number of particles measured was consistently higher with the aerosol box compared to all other devices and with that of no device use (fig. 3 ). there were visible spikes in particle counts when the volunteers coughed. the aerosol box was also the only intervention to record 5.0 micron particle counts above environmental baseline (p = 0.001). removal of the devices did not result in a statistically significant increase in airborne particles during this study. the race to generate sustainable equipment to protect healthcare workers during tracheal intubation procedures in patients with suspected or proven covid-19, particularly in settings where ppe supply is limited, has flooded the scientific community and social media with a variety of novel devices meant to contain potentially infectious aerosols produced by patients. evidence for the safety and efficacy of these devices is lacking. many international organisations have released consistent recommendations in the appropriate use of ppe based on the modes of viral transmission. none of these recommendations include these novel devices [11] [12] [13] [14] [15] . the dispersal of droplets and airborne particles from the patient depends on the aerosol (size, flow rates, turbulence, physiochemical properties) and patient (position, lung function) characteristics [18] [19] [20] . it is not clear in the manufacturing specifications of these novel devices that variables such as these have been fully considered in addition to the variability of employing these devices. the use of such devices adds to the complexity of an already complex procedure (tracheal intubation following local covid-19 protocols including airborne ppe use) with the potential to compromise the safety of both the laryngoscopist and the patient [21] . multiple methods of producing in situ aerosols in order to assess potential healthcare provider exposure are described in the literature. techniques include the use of tracer particles of nebulised liquid droplets in a cloud or solid particles in smoke [1, [22] [23] [24] . these droplets are detected using optical particle accepted article detection techniques, such as particle counters and electron microscopy [22] . we selected an established and reproducible aerosol dispersion method that would maximise aerosol generation across multiple particle sizes. the number of airborne particles produced via nebulisation of saline may far outnumber that produced during coughing and following paralysis of the patient for tracheal intubation. we selected nebulisation to generate large amounts of airborne particles to better discriminate the protective benefit, if any, of various aerosol containment devices compared to one another and to that of no device use. despite small numbers of volunteers, our study demonstrates a statistically significant difference in the quantity of airborne particles between the various devices described, with only a fully sealed box on suction demonstrating airborne particle quantity similar to the icu room at baseline. we were surprised to find airborne particle contamination of the laryngoscopist increased substantially using the aerosol box compared with all other devices and to no device use. spikes of airborne particles were clearly seen on the time series graph, coinciding with patient coughing. we hypothesise that these represent particles escaping from the arm access holes in the aerosol box as a result of the bernoulli principle. this was demonstrated in a recent simulation study by dalli et al. [24] where photographic images showed that substantial amounts of air moved out of aerosol boxes into the operating theatre during coughing. these data may be extrapolated to assess the utility of aerosol containment devices for tracheal extubation, where coughing is far more likely. however, this was not the objective of this study and further trials would need to be conducted. equally concerning is the increased exposure of the laryngoscopist at 300 s to 1.0, 2.5, and 5.0 micron particles when using the aerosol box, compared to not using any aerosol containment device (table 1) . when compared with all other proposed aerosol containment devices and to no device use, exposure of the laryngoscopist to 5.0 micron particles was significantly greater with the aerosol box (fig. 4e) . the sealed box with suction appears to maintain airborne particle count at baseline but only with the use of ongoing suction. we hypothesise that the efficacy of suction depends on the negative pressure generated, relative to the volume of the sealed box, the potential leak of the sealed box, and the length of time it takes for the air within the sealed box to become saturated with aerosol. while the sealed box was effective at maintaining environmental airborne particle counts, its design renders tracheal intubation mechanically impossible. it does, however, demonstrate the degree of enclosure necessary to eliminate particle contamination. this article is protected by copyright. all rights reserved wall suction has been proposed in many experimental setups [6] [7] [8] over high volume extraction as it is present in most intubating areas and is readily available. it should be noted that the flow of most wall suction is intentionally limited to reduce the risk of trauma to patients [25, 26] , is not connected to any particle filter, and was never intended to be used for the purposes of removal of airborne particles. we acknowledge limitations with this small prospective in-situ simulation study. there was significant variability in the airborne particle in the aerosol box, no intervention group, and vertical sheet setups compared to the other methods. this suggests that the level of laryngoscopist exposure is unpredictable and likely influenced by the aerosol containment device set-up and the volume, negative pressure generated from suction and frequency of patient coughing. to better assess variability, an increased number of subjects would be required, yet such variability suggests an inability to fully predict airborne particle contamination based on device alone. this experiment also did not assess the particle count for individuals at the side of the bed or elsewhere in the room. given airborne particles are known to spread over distances greater than 1.5 m [1, 2, 23] , this is especially pertinent for the laryngoscopist's assistant, who is often standing very close to the patient's head during tracheal intubation and extubation. in conclusion, this study demonstrates that devices such as the aerosol box confer minimal to no benefit in containing aerosols during an aerosol-generating procedure and may increase rather than decrease airborne particle exposure. a sealed box with suction appears to decrease airborne particle exposure of the laryngoscopist, although whether it hinders assistance or execution of tracheal intubation remains a point of study. further large-scale studies are needed to examine aerosol containment devices in this context, as well as others, such as tracheal extubation. the use of any aerosol containment device has been eliminated from our intubation protocols until their safety can be properly established. this article is protected by copyright. all rights reserved appendix s1 lighthouse 3016-1aq handheld particle counter the role of particle size in aerosolised pathogen transmission: a review personal protective equipment (ppe) for both anesthesiologists and other airway managers: principles and practice during the covid-19 pandemic detection of sars-cov-2 in different types of clinical specimens aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review safety testing improvised covid -19 personal protective equipment based on a modified full-face snorkel mask taiwanese doctor invents device to protect us doctors against coronavirus keusch glass inc. creates intubation boxes for medical personnel australian doctors design and make life-saving equipment needed for coronavirus pandemic covid19: minimising risk to healthcare workers during aerosol producing respiratory therapy using an innovative constant flow canopy reducing droplet spread during airway manipulation: lessons from the covid-19 pandemic in singapore consensus guidelines for managing the airway in patients with covid-19: guidelines from the difficult airway society, the association of anaesthetists the intensive care society, the faculty of intensive care medicine and the royal college of anaesthetist consensus statement: safe airway society principles of airway management and tracheal intubation specific to the covid-19 adult patient group recommendations for endotracheal intubation of covid-19 patients rational use of personal protective equipment for coronavirus disease 2019 (covid-19) expert recommendations for tracheal intubation in critically ill patients with novel coronavirus disease 2019 barrier enclosure during endotracheal intubation the aerosol box for intubation in covid-19 patients: an insitu simulation crossover study personal protective equipment during the coronavirus disease (covid) 2019 pandemica narrative review generation of aerosols: barc nebulizer and others. international nuclear information system droplet fate in indoor environments, or can we prevent the spread of infection? should we use an aerosol box for intubation? airflow and droplet spreading around oxygen masks: a simulation model for infection control research turbulent gas clouds and respiratory pathogen emissions evaluating intubation boxes for airway management suction devices the principles of vacuum and clinical application in the hospital environment the study was approved by the hospital research and ethics committee (hrec lrr20/040). no external funding or competing interests declared. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord-282858-zikoui4h authors: graudenz, gustavo silveira; degobbi, cristiane; saldiva, paulo hilario title: sars-cov-2. long distance airborne transmission and its public health implications date: 2020-11-02 journal: clinics (sao paulo) doi: 10.6061/clinics/2020/e2343 sha: doc_id: 282858 cord_uid: zikoui4h nan by the end of july 2020, more than 15 million confirmed cases of and more than half a million deaths from coronavirus disease , caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), had been recorded. of these, more than 2.5 million cases and 85,000 deaths were recorded in brazil. as it is difficult to sustain measures of social isolation, more focused and effective preventive measures against viral transmission are urgently needed to prevent the spread of the disease. until recently, the world health organization (who) endorsed only two modes of viral transmission. the first mode is via inhalation of large respiratory droplets generated from coughing, sneezing, or breathing by an infected individual, wherein the infective potential is short-ranged. the second mode is via direct contact with contaminated surfaces (fomites), wherein the virus is carried by the hands to the nose, mouth, or eyes. considering this, frequent hand washing, maintaining a social distance of 1 m, good respiratory hygiene, and avoiding crowds (no specifications) are considered main preventive measures (1) . until now, covid-19 has not been considered an airborne disease. the only diseases recognized as belonging to this category are tuberculosis (mycobacterium tuberculosis), chicken pox (varicella-zoster virus), measles (morbillivirus), and invasive aspergillosis (aspergillus sp.) (2) . the category is restricted to diseases transmitted by small viable and infectious particles that are able to travel long distances, stay for long periods in suspension, are strongly influenced by air currents, and are able to penetrate deep into the lungs of individuals who have had no previous direct contact with or have not been in close proximity to an infected individual. despite some controversies regarding the penetration of particles into the respiratory system, it is widely accepted that small particles with an aerodynamic diameter of less than 10 mm are able to pass the glottic barrier and penetrate up to the thoracic level. particles ranging from 5 to 10 mm have the potential to transmit disease over both long and short distances, and particles less than 5 mm in diameter can penetrate up to the alveolar level (3). in contrast, particles larger than 20 mm in diameter have a more ballistic behavior, meaning they are more under the influence of gravity than wind currents. the particles generated through breathing, coughing, and sneezing are between 4 and 8 mm in diameter (4). more recent and precise studies that take into account the age-dependent decay process of the particles report that most exhaled particles measure less than 1 mm and are generated during speaking and not only during coughing or sneezing (5) . in addition, exhaled particles are not necessarily isolated in muco-salivary droplets. during the exhalation process, multiphase turbulent gas clouds are created. these clouds retain their temperature and humidity and allow the contained droplets to evade evaporation for much longer than isolated droplets can. under these conditions, the lifetime of a droplet could be considerably extended, by a factor of up to 1000, from a fraction of a second to minutes. as a result, these clouds can carry particles 7 or 8 m away from the infected individual, after which the particles undergo dehydration and aging, resulting in smaller particles that are able to travel even longer distances (6) . according to roy and milton (7), airborne infections can be largely underestimated because of methodological limitations. they suggest the following classification for airborne diseases: 1. mandatory: when the pathogen can only be transmitted through the airborne route (e.g., tuberculosis). 2. preferential: when the pathogen can be transmitted by other routes but the airborne route is the main route (e.g., measles, smallpox). 3. opportunistic: when the pathogen is mainly transmitted by other routes but can be transmitted by the airborne route under certain circumstances. currently, there is no clear evidence of airborne transmission of sars-cov-2. this is a matter of intense debate. its predecessor, sars-covid-1, the agent that caused severe acute respiratory syndrome (sars) in hong kong in 2003, showed strong evidence of opportunistic airborne transmission in different environments, such as collective housing environments (8) , indoor environments such as airplanes (9), and health service institutions (10) . similarly, some researchers considered airborne transmission of another coronavirus, middle east respiratory doi: 10.6061/clinics/2020/e2343 copyright & 2020 clinics -this is an open access article distributed under the terms of the creative commons license (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited. no potential conflict of interest was reported. syndrome coronavirus (mers-cov), which has been described as unable to infect the upper airways but as having a preferential affinity toward the lower airway, with infective particles less than 10 mm in diameter (11) . the consideration of airborne transmission of sars-cov-2 by the (who) was based on evidence from a study by ong et al. (12) that suggested transmission of sars-cov-2 through infected surfaces and contaminated individual protection equipment as well as long distance environment contamination. the negative results from the air samples were interpreted as effective room disinfection and air renovation measures. despite the air samples testing sars-cov-2-negative, samples from the recirculation system tested positive, revealing that small particles were carried over longer distances. considering this, new studies have been conducted, and evidence of airborne transmission has started increasing. in a hospital designated to treat patients with covid-19, airborne viruses were detected in sub-micrometric particles and were more concentrated in used protective apparel of health professionals (13) . in another study in a hospital treating covid-19 patients in nebraska, usa, the largest viral concentrations were detected in the ventilation grills, indirectly indicating long-distance infective particle transportation into the building ventilation system (14) . in an experimental study conducted in an aerosol chamber, potentially infective and stable particles lasting for up to 3 h were identified in a controlled contamination experiment using particles with less than 5 microns in diameter (15) . considering the reported evidence and the many similarities among these coronaviruses (sars-cov-1, mers-cov), it is likely that sars-cov-2 is transmitted via the air. however, some questions remain unanswered, for instance, the origin and area as well as the infectious dose and infection rates after exposure to particles smaller than 10 mm (16). despite the controversies, experts recommend precaution in all indoor environment-related situations. measures such as increasing air exchange rates, avoiding air recirculation, downwind positioning relative to other individuals, and reducing the number of people sharing the same indoor environment should be considered, among the general measures aimed at reducing the infection risk (17) . in health care settings, the center for disease control's recommendations for prevention of airborne transmission include maintaining a negative pressure environment, fine filtering of exhaust air from infected patients' rooms, maintaining high air exchange rates (12 air exchanges per hour), shutting recirculation ducts, and establishing pressure cascades (2) in these settings until further evidence of long distance transmission is obtained unfortunately, these precautionary measures have not yet been applied in most health care facilities in brazil. even when it is not feasible to establish negative pressure or pressure cascades using ventilation systems, a simple alternative could be to use natural ventilation to increase air exchange rates and thus ensure pathogen dilution. it is important to avoid the use of a ventilation system without air exchange such as split systems in these settings. in our scenario, scientific data reinforces that indoor air quality standards for health care in brazil, the nbr 7256 (18) , must be updated to protect health personnel dealing with diseases with a high potential for being contagious, such as covid-19. recently, a call for action supported by 239 scientists from different parts of the world was published that indicated that information on warnings and precautionary measures for the control of potential aerial transmission of sars-cov-2 should be conveyed by health authorities (19) . considering the risk of airborne transmission of covid-19 and taking its preventive measures is particularly important at this moment when several countries, including brazil, are progressively increasing their economic and social activities and gathering people is inevitable. so, it is important to stress the necessity of maximizing air renewing and mandatory use of face masks, not only in health care settings but also in commonly shared indoor environments, like workplaces, schools and public transportation. modes of transmission of virus causing covid-19: implications for ipc precaution recommendations. scientific brief guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings preventing transmission of pandemic influenza and other viral respiratory diseases: personal protective equipment for healthcare personnel: update the numbers and the sites of origin of the droplets expelled during expiratory activities size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities turbulent gas clouds and respiratory pathogen emissions: potential implications for reducing transmission of airborne transmission of communicable infection--the elusive pathway evidence of airborne transmission of the severe acute respiratory syndrome virus transmission of the severe acute respiratory syndrome on aircraft a major outbreak of severe acute respiratory syndrome in hong kong recognition of aerosol transmission of infectious agents: a commentary surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient aerodynamic characteristics and rna concentration of sars-cov-2 aerosol in wuhan hospitals during covid-19 outbreak transmission potential of sars-cov-2 in viral shedding observed at the university of nebraska medical center aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 is the coronavirus airborne? experts can't agree ventilation control for airborne transmission of human exhaled bio-aerosols in buildings tratamento de ar em estabelecimentos assistenciais de saúde (eas) -requisitos para projeto e execuc¸ão das instalac¸ões. abnt it is time to address airborne transmission of covid-19 key: cord-297377-yy1dq81q authors: poostchi, ali; kuet, mong-loon; pegg, kate; wilde, craig; richardson, patrick s.; patel, moneesh k. title: efficacy of slit lamp breath shields date: 2020-05-12 journal: eye (lond) doi: 10.1038/s41433-020-0940-y sha: doc_id: 297377 cord_uid: yy1dq81q nan the use of enlarged breath shields has been suggested as part of a wide range of infection control measures implemented during the covid-19 pandemic. breath shields have long been a standard feature of slit lamps and act as a physical barrier between the examiner and subject but there is an absence of evidence on their effectiveness in reducing droplet transmission and respiratory infections. sars-cov-2 shares many of the features of other respiratory viruses including sars-cov-1 and is thought to be commonly spread though respiratory droplets (>5 μm) and fomites [1] . fomites are formed either from droplets settling on surfaces or through direct contamination from touching mucosal surfaces. smaller aerosolised droplet nuclei (≤5 μm) can travel further and remain in air longer. they have been shown to carry viable virus particles in experimental conditions [2] but are not thought to be a common mode of transmission of covid-19 [1] . the risk of transmission from tears is also thought to be low [3] . we sought to examine the efficacy of facemasks and standard and augmented slit lamp breath shields using a breathing simulator. these have been described previously and generally comprise of a particle source, commonly a nebuliser attached to a bellows or air tank and a particle detector which can consist of a laser particle counter [4] or an impinger from which viral particles can be sampled from air, cultured in cells and detected as plaques [5] . direct visual inspection of sprayed dye droplets has also been described as a way to test eye protection [6, 7] . we experimented using nebulised fluorescein 2% but were unable to capture sufficient dye to determine the patterns of droplet distribution. we used a mouthpiece nebuliser (galemed corp, taiwan) containing 5 ml of 0.9% saline as our particle source and attached it to a 500 ml paediatric bag valve mask that was manually compressed 12 times per minute to simulate normal adult tidal breathing. the device produces a range of particles from 1 to 25 μm with median mass aerodynamic diameter of 3.8 μm. we used a met one a2400 optical particle counter (hach co, loveland, co) operating at a flow rate of one cubic foot per minute to detect particles that reached the eyepiece over a 1-min period. this was initially performed without any shielding, and then repeated with the standard (11 × 11 × 0.2 cm) and augmented (45 × 44 × 0.2 cm) acrylic shields attached to the slit lamp objective lens (fig. 1) . we then tested the effect of placing a fluid resistant surgical facemask (barrier 4313, mölnlycke healthcare, sweden) over the nebuliser mouthpiece alone and in combination with the large shield. the slit lamp arm was offset to 60°throughout and each barrier was tested five times. linear regression was used to determine the effect of shield type and particle size on particle count. all analyses were performed using stata v14. with no shield in place, the mean log particle count was 3.59 (95% ci: 3.48-3.70). there was a significant reduction to 3.01 (95% ci: 2.90-3.13, p < 0.01) with the standard fig. 1 photograph of the experimental setup. the nebuliser particle source and optical particle detector can be seen either side of the slit lamp. shield, 2.63 (95% ci: 2.52-2.75, p < 0.01) with the augmented shield and 2.62 (95% ci: 2.50-2.73, p < 0.01) with the facemask secured in front of the nebuliser. with the mask and large shield both in place, the mean log count dropped to 2.47 (95% ci: 2.36-2.59, p < 0.01). we found that the barriers we tested were all effective at reducing transmission of particles > 3 μm. the surgical facemask and large shield offered the best protection, while the small standard shield seemed to be effective for larger particles (fig. 2) . the benefits of larger shields should be weighed against the increased risk of fomite transmission, especially where they cause the user to touch the sides in order to perform tonometry or manipulate the slit lamp arm. barrier protection, while helpful, should be seen as only one part of a combined approach with hand hygiene and effective cleaning essential to limit viral spread. there is ongoing debate over the use of masks. evidence from a large-clusterbased randomised control trial showed no benefit from using n95 masks compared to surgical facemasks to reduce influenza and other respiratory viral illnesses [8] . subsequent meta-analyses have shown similar results with the benefits of filtering facepiece (ffp) masks thought to be offset by discomfort with their prolonged use leading to more frequent manipulation and/or reduced compliance [9] . there may be a similar tradeoff between different sizes of shield with an increased risk of contamination through contact with larger shields. the protocol we describe has helped us to reduce some of the uncertainty that surrounds our use of protective equipment and can be adapted to different scenarios. most of the components we used were readily available and the particle counter was not difficult to source. particle counters are routinely used in our trust to monitor sterility of theatres and pharmacy clean rooms and more recently to test the fit of ffp masks. a limitation of our approach is that we did not distinguish between infectious and noninfectious particles or their source. we performed our study in a room used for intravitreal injections to limit the number of background particles but this remains a potential confounder. we found it reassuring that the standard measures in place were effective and that the larger shield and facemask appeared to offer some additional protection. despite the close proximity of slit lamp examinations, the effectiveness of shielding together with the short contact time and lack of aerosol generation, leads us to expect the risk of transmission from this route to be low. viruses spread fear and uncertainty along with disease. through research, consultation and pragmatic use of evidence, we hope to help build a shield to this as well. report of the who-china joint mission on coronavirus disease 2019 (covid-19). geneva: world health organization aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 assessing viral shedding and infectivity of tears in coronavirus disease dispersion and exposure to a cough-generated aerosol in a simulated medical examination room effectiveness of surgical masks against influenza bioaerosols eye protection in orthopaedic surgery. an in vitro study of various forms of eye protection and their effectiveness barrier enclosure during endotracheal intubation n95 respirators vs medical masks for preventing influenza among health care personnel: a randomized clinical trial effectiveness of n95 respirators versus surgical masks against influenza: a systematic review and meta-analysis acknowledgements we are grateful to stuart lawrie for the construction and installation of the augmented acrylic breath shields in our department. conflict of interest the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-274780-fmnro0kw authors: hoshino, y.; zimmer, j. f.; moise, n. s.; scott, f. w. title: detection of astroviruses in feces of a cat with diarrhea date: 1981 journal: arch virol doi: 10.1007/bf01320252 sha: doc_id: 274780 cord_uid: fmnro0kw astroviruses were detected by electron microscopy in the feces from a 4 month old kitten with diarrhea. the mean diameter of the viral particles was 28.7 nm, and they showed characteristic fiveor six-pointed star-shaped surface configurations. the clinical disease manifested by the cat and the observed morphology of the viral particles are consistent with previous reports on astroviruses of other species. astroviruses were detected by electron microscopy in the feces from a 4 month old kitten with diarrhea. the mean diameter of the viral particles was 28.7 nm, and they showed characteristic five-or six-pointed star-shaped surface configurations. the clinical disease manifested by the cat and the observed morphology of the viral particles are consistent with previous reports on astroviruses of other species. . viral gastroenteritis is one of the leading causes of morbidity and mortality in neonatal and young animals, birds and man throughout the world (2, 3, 5, 17, 19) . the development of methods for identification of viruses in fecal samples by direct electron microscopy (em) has led to the discovery of a number of previously unrecognized enteric viruses, including astroviruses. astroviruses are isometric, approximately 28 nm in diameter viral particles which, when negatively stained, give the impression of a white, five-or six-pointed star superimposed on the particle. astroviruses have been detected in the feces of humans (1, 8, 9, 12, t3, 15, 18) , calves (23) , lambs (20) and turkeys (16) with diarrhea. since attempts to propagate the mammalian and avian astroviruses in cell cultures have been unsuccessful, their size and morphology as determined by em are the primary criteria by which they are identified. viral antigen was detected by indirect immunofluorescent staining within the cytoplasm of cultured cells infected with fecal material of human (10, 11) and bovine (23) astroviruses. however, these infections were abortive in y. hos~l-¢o, j. f. zi~iet~, n. s. molse, and f. w. scott: that no specific fluorescence was seen in passaged celt cultures, t~ecently, t-ierrii~g et al. (4) reported that ovine astrovirus has a single-stranded rna genome which resembles that of the picornaviruses and ealiciviruses but the polypeptide composition is unlike that of either of these groups. we report here the detection by direct. em of astroviruses in feces from a 4 month old kitten with diarrhea. an intact female 4-month-old kitten was presented to the small animal hospital at the new york state college of veterinary medicine, cornell university, on october 4, 1979. the chief complaint was that the kitten had had diarrhea, characterized as loose and watery, for approximately one week. a tentative diagnosis of parasite infestation and a diet-induced diarrhea was made, and the cat was vaccinated for feline panleukopenia, rhinotracheitis, and eaticivirus. the cat was returned five days later (october 9) with the report that the diarrhea had continued and that the cat had been anorectie for three days. the diarrhea was reportedly more severe than on the initial visit. physical examination revealed slight dehydration, poor body condition, gas-distended loops of small intestine, and a green, watery diarrhea. a fecal sample was submitted for em examination for viral particles. two days later (october 11), the kitten was hospitalized because of more severe dehydration and diarrhea. the results of a hemogram were normal, but the kitten was mildly acidotic and hypokalemic. during the subsequent nine days of hospitalization, the kitten's feces improved in consistency. the cat was discharged on october 20. re-examinations one week and 3 months after discharge indicated that the kitten improved steadily and made an uneventful recovery. the feces had become normal, and the appetite was good. fecal sample was examined by em for ~5ral particles using the procedures reported previously (6) . a 20 percent (w/v) fecal suspension was prepared in eagle's minimum essential medium (mem) with antibiotics and without serum. this suspension was centrifuged at 1,200 × g for 10 minutes (crude fecal suspension), and the supernatant was then ultra.centrifuged at 10,000 × g for 10 minutes (clarified fecal suspension). one to 4 drops of crude fecal suspension were mixed with 20 drops of distilled water, one drop of 0.1 percent bovine serum albumin and three to four drops of 4 percent phosphotungstie acid adjusted to ph 7.0. this mixture was applied to a carbon-parlodion-coated copper grid with an all-glass nebulizer and examined in a philips 201 electron microscope at 80 kv. direct em examination demonstrated the presence of astrovirus-like particles, mostly in aggregates, in the fecal suspension (fig. 1) . these viral particles were roughly spherical in outline, ranged in size from 27.5 nm to 29.4 nm in diameter and h ad a mean diameter of 28.7 nm. the surface structure had the appearance of characteristic five-or six-pointed stars (7fig. 1, arrows). this star-shaped configuration was not always apparent on all particles. bridging structures between virus particles were frequently seen (fig. t, arrowheads) . these structures have been observed on human (14) and ovine (20) astroviruses. it is speculated that these bridging structures may be surface fibers similar to those on adenoviruses. attempts at viral isolation with cell cultures were carried out as previously reported (7) . confluent monolayer cultures (16 × 125 mm culture tubes) of first transfer cells of feline kidney and an established fetal rhesus monkey kidney celt line (ma 104) were inoculated with 0.1 ml of clarified fecal suspension. after one hour adsorption at 37 ° c, cell cultures were washed once with phosphate buffered saline, p h 7.2, fed with 1.0 ml of maintenance medium (eagle's mem supplemented with 0.01 percent laetalburnin hydrolysate, 0.075 percent sodium! bicarbonate, antibiotics and no serum), and incubated at 35 ° c. three blind passages were made at one week intervals. attempts to propagate this feline astrovirus in cell cultures have been unsuccessful. no calicivirus or other eytopathogenic virus was isolated in cell cultures. experimental transmission experiments with mammalian astroviruses indicate that. they induce mild, constitutional symptoms (10, 20, 21, 23) . studies on the pathogenesis of astrovirus infection in lambs have shown the site of virus multiplication to be the mature villous epithelial cells of the small intestine (21, 22) . there is a clinical impression that astrovirus-associated gastroenteritis in man and animals is generally milder than that associated with rotaviruses. asymptomatie excretion of astrovirus particles has been reported in man (1, 12), calves (23) and lambs (20) . studies on the pathogenicity of feline astrovirns have been hampered by a shortage of material. as this is the first report of the identification of a feline astrovirus and since no serological test. is available, it is unknown how widespread and how significant this virus is. since eats frequently have close contact with humans, it will be important to study the relationship between feline and human astrovirnses. astrovirus-associated gastroenteritis in children the role of viruses in acute diarrhoeal disease viral intestinal infections of animals and man. comp. i m m u n . microbiol purification and characterization of ovine astrovirus viral gastroenteritis coronavirus-tike particles in the feces of normm cats isolation and characterization of feline rotavirus small spherical viruses in faeces from gastroenteritis patients astrovirus-associated gastroenteritis in a children's ward astrovirus infection in volunteers astroviruses detected by immunofluorescence (letter) 1 ). : viruses in infantile gastroenteritis (letter) 28-n~ particles in feces in infantile gastroenteritis (letter) comparison of the features of astroviruses and caliciviruses seen in samples of feces by electron microscopy viruses in the stools detection of astroviruses in turkey faeces by direct electron microscopy the tecumseh study. x i . occurrence of acute enteric illness in the community coronavirus et "astrovirus" observ@s d ans ies selles d'enfants atteints de gastroentdrites recent advances in viral gastroenteritis detection and transmission of 30-nm virus particles (astroviruses) in faeces of lambs with diarrhoea pathogenesis of diarrhoea caused b y astrovirus infections ultrastructure of the small intestine in astrovirus isolation of small viruses resembling astroviruses and ealiciviruses from acute enteritis of calves key: cord-031037-up0z1yaf authors: edwards, david; hickey, anthony; batycky, richard; griel, lester; lipp, michael; dehaan, wes; clarke, robert; hava, david; perry, jason; laurenzi, brendan; curran, aidan k.; beddingfield, brandon j.; roy, chad j.; devlin, tom; langer, robert title: a new natural defense against airborne pathogens date: 2020-07-07 journal: nan doi: 10.1017/qrd.2020.9 sha: doc_id: 31037 cord_uid: up0z1yaf we propose the nasal administration of calcium-enriched physiological salts as a new hygienic intervention with possible therapeutic application as a response to the rapid and tenacious spread of covid-19. we test the effectiveness of these salts against viral and bacterial pathogens in animals and humans. we find that aerosol administration of these salts to the airways diminishes the exhalation of the small particles that face masks fail to filter and, in the case of an influenza swine model, completely block airborne transmission of disease. in a study of 10 human volunteers (5 less than 65 years and 5 older than 65 years), we show that delivery of a nasal saline comprising calcium and sodium salts quickly (within 15 min) and durably (up to at least 6 h) diminishes exhaled particles from the human airways. being predominantly smaller than 1 μm, these particles are below the size effectively filtered by conventional masks. the suppression of exhaled droplets by the nasal delivery of calcium-rich saline with aerosol droplet size of around 10 μm suggests the upper airways as a primary source of bioaerosol generation. the suppression effect is especially pronounced (99%) among those who exhale large numbers of particles. in our study, we found this high-particle exhalation group to correlate with advanced age. we argue for a new hygienic practice of nasal cleansing by a calcium-rich saline aerosol, to complement the washing of hands with ordinary soap, use of a face mask, and social distancing. airborne transmission of infectious disease by the very small droplets we emit from our airways on natural breathing, and that accumulate in poorly ventilated indoor environments, has been observed for a range of respiratory diseases, including tuberculosis, measles, chicken pox, influenza and severe acute respiratory syndrome (sars) (li et al., 2007) . recent observations (zhang et al., 2020) place the novel coronavirus sars-cov-2-carried by the small airborne droplets exhaled by covid-19 infected individuals (liu et al., 2020) and reported stable in aerosol form (van doremalen et al., 2020) for beyond 3 h-in the family of airborne transmitted infectious diseases as well. the assessment of sars-cov-2 as an airborne pathogen clarifies the nature of the fight against the covid-19 pandemic (fauci et al., 2020) . while major international scientific efforts advance toward the development of drugs and vaccines in response to the covid-19 pandemic, less attention has been given to new ways to prophylactically combat airborne viral and bacterial threats. social distancing, washing of hands, and wearing of face masks, while each effective and necessary, do not prevent the transmission of pathogens that travel through the air within droplets smaller than 1 μm in diameter. such small particles are not only too small to be filtered effectively by conventional masks (bunyan et al., 2013; leung et al., 2020; zhang et al., 2020) , but also too small to settle by gravity within the 2-m threshold of social distancing. submicron droplets happen to be the majority of particles we emit from our mouths and noses when we naturally breathe. they emerge from our respiratory systems either by the necking of airway lining fluid (alf) that occurs with the expansion and contraction of the lungs (scheuch, 2020) , or by the rapid movement of air through upper airways, as occurs during natural breathing, coughing, sneezing, and speaking (watanabe et al., 2007) . whether high or low in the airways, shedding of small droplets from infected lungs can carry viral and bacterial pathogens into the environment, promoting disease spread (edwards et al., 2004; leung et al., 2020) . when this shedding occurs in the upper airways, it can promote movement of pathogen deeper into the lungs, and self-infection. the delivery of saline to the respiratory system has been observed to diminish the exhalation of these very small particles (edwards et al., 2004) . this diminution is due to electrostatic interactions between salt cations and mucin and mucin-like proteins on the surface of alf. these increase the viscoelasticity of the surface of alfs (watanabe et al., 2007) , reducing the formation of airborne droplets (watanabe et al., 2007) . calcium chloride (cacl 2 ), far more than sodium chloride (nacl), has been found to increase the surface elasticity of alf, potentially promoting even more substantial suppression of airborne particle generation in the airways, while enhanced surface plasticization also further resists pathogen penetration through the mucus layer, strengthening its biophysical barrier to infection (watanabe et al., 2007) . calcium and sodium salts appear to have antimicrobial properties as well. high concentrations of extracellular calcium (krisanaprakornkit et al., 2003) , as can be achieved by the aerosol delivery of calcium salts, promote the secretion of β-defensin 2 from nasal epithelial cells (alp et al., 2005) . human β-defensin 2 is an endogenous antimicrobial peptide that conjugates with receptorbinding domains of many viruses, including coronaviruses, to promote expression of antiviral and immune-inducing molecules as well as chemokine recruiters of leukocytes (kim et al., 2018) . human β-defensin 2 has been shown to be effective as an antiviral adjuvant due to its binding to the spike protein of middle east respiratory syndrome coronavirus (mers-cov) (zhao et al., 2016) , and mouse beta defensin 4 derived peptide has shown activity against sars-cov-1 (kim et al., 2018) . chloride salts have been shown to diminish viral replication as far back as the 1960s (speir, 1961) . chloride ions promote antiviral activity by the induction within cells of hypochlorous acid, the active constituent of bleach (ramalingam et al., 2018) . chloride salts induce innate immune response of epithelial cells in the presence of nacl (ramalingam et al., 2018) . sodium hypochlorite, the sodium salt of hypochlorous acid, has particularly demonstrated effectiveness as a disinfectant against coronavirus (dellanno et al., 2009; geller et al., 2012) . high concentrations of chloride, delivered via hypertonic saline to nasal epithelial tissues, have been found to diminish viral infections associated with the common cold (adam et al., 1998; šlapak et al., 2008; ramalingam et al., 2019) . in an open-labeled randomized controlled human study of 68 subjects with common cold infections including rhinoviruses and coronaviruses, as well as enterovirus and influenza a virus, nasal delivery of 2-3% hypertonic saline 2-8 times a day (median thricea-day) significantly lowered duration of illness, as well as use of over-the-counter medications, household transmissions, and viral shedding (ramalingam et al., 2019) . nasal administration of 3.5% hypertonic seawater has similarly shown indications of efficacy against common cold symptoms in other human clinical trials (adam et al., 1998; šlapak et al., 2008) . to address the need for a broad prophylactic and anti-contagion defense against respiratory viral and bacterial infections, we designed salt compositions for hygienic and therapeutic applications by incorporating three ions that are abundant in human tissues: calcium, sodium, and chloride. we hypothesized that an aerosol combining calcium and sodium salts would improve the barrier function of the mucus lining to protect against infection and promote natural innate immune disinfectant properties suited to a range of bacterial and viral infections, while diminishing bioaerosol formation in the lungs and nasal passages. given the hygienic practice of nasal salines, we decided to evaluate the nasal delivery of our physiological saline with a specially designed nasal mist, as a practical, efficacious, and safe personal hygiene intervention, complementary to masks. we assumed that this might prove of particular utility to the immediate fight against the current covid-19 pandemic. compositions and cell models: influenza, rhinovirus, and pneumonia we sought to establish optimal compositions and mechanism of action for physiological salts suited to antimicrobial activity against viral and bacterial pathogens with in vitro systems. formulations were prepared with compositions across a range of cacl 2 and nacl ratios. these formulations were aerosolized by nebulizer and delivered onto air-liquid interface (ali) cultures formed by calu3 in order to identify compositions of optimal efficacy and clarify mechanism of action, we evaluated the ability of these formulations to lower viral titers collected (in the case of the calu3 ali model) in an apical wash 24 h after administration and (in the case of the alf mimetic model) to lower rate of progression across alf. we found that exposing the calu3 cells via nebulization to solutions of 1.29% (0.115 m) cacl 2 or 12.9% (1.15 m) cacl 2 in saline (0.9% nacl) reduced viral titers in a cacl 2 dependent manner (fig. 1a ). this study was repeated with magnesium chloride (mgcl 2 ) to determine whether increased tonicity or specific ionic composition determined efficacy; the mgcl 2 was dissolved in saline at identical molar concentrations as the cacl 2 . as shown in fig. 1b , viral titers did not significantly change with mgcl 2 exposure, suggesting calcium, more than chloride or sodium, was most responsible for the reduction in viral titer. we further examined relative contributions across a range of tonicities. the results, shown in fig. 1c , show that ion type and not tonicity itself is critical to effectiveness, and that calcium, with a strong synergistic effect of nacl, was the primary active ingredient. cacl 2 alone was also less effective, suggesting perhaps a secondary chloride role, with chloride concentration titrated independently of calcium by sodium. we sought to determine optimal ratios of cacl 2 and nacl based on antiviral efficacy through a series of experiments with the calu3 alf influenza model. this led to the identification of three optimal compositional zones as shown in fig. 1d . we chose in our subsequent studies to explore three unique (fast emergency nasal defense, fend) composition ranges of cacl 2 and nacl, all with hypertonicity ranges well tolerated in the pulmonary treatment of cystic fibrosis. the constituents (cacl 2 and nacl) and tonicity ranges of these formulations are particularly common to currently marketed nasal salt products: • fend1: a 2âisotonic composition [0.12 m cacl 2 , 0.15 m nacl] (1.29% cacl2, 0.9% nacl); fig. 1 . the in vitro antiviral efficacy of calcium and sodium salt formulations. (a) calcium chloride (cacl 2 ) reduced influenza infectivity in a dose-dependent manner. calu3 cells exposed to no formulation were used as a control and compared to calu3 cells exposed to formulations of different concentrations of isotonic saline or cacl 2 dissolved in isotonic saline. the of virus released by cells exposed to each aerosol formulation was quantified by 50% tissue culture infectious dose (tcid 50 ) assay. bars represent the mean and standard deviation of triplicate wells for each condition. (b) calu3 cells exposed to no formulation were used as a control and compared to calu3 cells exposed to formulations of magnesium matched to that of calcium on a molar basis. the concentration of virus released by cells exposed to each aerosol formulation was quantified by tcid 50 assay. bars represents the qrb discovery 3 the first is a cacl 2 augmentation of saline, the second near the salinity of sea water, and the third a composition with a salinity in the upper range of what has been observed as effective in previously published studies of antiviral nasal saline (adam et al., 1998; dellanno et al., 2009; ramalingam et al., 2019) . these formulations were tested in calu3 cells against a variety of influenza a and influenza b strains and shown to be effective in all cases with increased cacl 2 concentration (fig. 1e) . we sought to evaluate the calcium-mediated mechanism of action initially with normal human bronchial epithelial cells (nhbecs). we evaluated the effect of treatment on viral titer using the same ali approach as described above for calu3 cells. these data indicate that treatment of human bronchial epithelial cells with fend formulations reduces viral titer (fig. 1f ). in addition, we evaluated mrna expression of 26 genes following application of cacl 2 formulations compositions (supplementary material). we found that 7 of the 26 genes consistently expressed three-fold or greater expression after treatment. these included: β-defensin 2 (bd2), interleukin 8 (il-8), interleukin 6 (il-6), 10-kda interferon-γ-induced protein 10 (ip10), mucin 5ac, mucin 5b, and regulated on action, normal t expressed and secreted protein (rantes). these results pointed to the ability of the calcium and sodium salt compositions to enhance innate immunity and notably expression of bd2 a powerful antimicrobial potent against viral pathogens including coronaviruses (fig. 1g) . we performed a second set of experiments to assess calciuminduced retardation of microbial movement across simulated alf mimetic systems as may relate to diminished infection rates in time-constrained viral infection systems such as the nose or the ali systems in our studies. we first evaluated the speed of bacterial pathogen progress across the alf mimetic. we topically applied by nebulizer aerosol saline or fend1, then added the gram-positive and gram-negative pneumonia bacteria. the results are summarized in fig. 2a -e. in all cases calcium interactions with the alf mimetic increase the viscoelasticity of the alf-as has been observed and explained elsewhere (edwards et al., 2004) -and lower rate of passage of the bacterial pathogens across alf. this same effect was observed with influenza and rhinovirus as shown in figs 2f,g, notably at concentrations of calcium in the range of the fend2 and fend3 compositions. optimal combinations of cacl 2 and nacl as typified by fend1, fend2, and fend3 all show potential for antimicrobial activity by a combination of stimulating secretion of antimicrobial peptides and by slowing pathogen movement across alf. we carried these compositions forward in animal and human studies. to evaluate the therapeutic (antimicrobial) and hygienic (airway particle cleansing) properties of fend in animals we examined mice and porcine models. we first explored fend antimicrobial efficacy in a mouse pneumonia model. mice nasal and pulmonary exposure of an estimated 6.4 mg/kg was achieved by nebulizer either 2 h after (treatment) or 2 h before (prophylaxis) infection with streptococcus pneumoniae (serotype 3; atcc 6303) with fend1 or a saline control. as shown in fig. 3a both in the case of treatment and prophylaxis bacterial burden is significantly lowered by the delivery of the calcium and sodium salts relative to the control. to verify that the efficacy of the fend composition was driven by calcium per se and not by sodium, chloride, or the di-valency of calcium, we repeated the experiments with magnesium replacing cacl 2 and with nacl alone. fig. 3b shows that no significant decline in bacterial burden follows nasal and pulmonary delivery of mgcl 2 relative to the untreated control, while aerosolization of cacl 2 alone produces a significant decline in bacterial burden relative to nacl alone. we next studied the hygienic properties of fend in a large animal (swine) model of influenza. piglets were randomly assigned to infected or naive groups (n = 8 total; 4 per group) in each study and each group was housed in a separate clean room. following infection of untreated animals, naïve animals were exposed to the exhaled breath of the infected animals. exposures were performed such that the infected animals were housed in the bottom of the exposure chamber and the naïve animals in the upper portion of the chamber to avoid contact transmission and allow only transmission via exhaled breath. infected and naïve animals were each fitted with anesthesia masks and connected by >3 00 of tubing. once connected, naïve animals were exposed to the exhaled breath of the infected animals for 1 h/day at 48-and 72-h post infection. in a separate group of piglets (n = 8 total; 4 per group), anesthetized animals were administered fend1 aerosols at 48-and 72-h post infection to reduce airborne particle emission. in the first experiment, treatment largely eliminated infection as measured in terms of lung consolidation (fig. 4a ). in the second experiment, 10 min following treatment, naïve animals were exposed to the exhaled breath of the infected animals in the same manner as described above. exhaled breath from untreated infected animals caused infection in naïve animals with a 75% transmission rate (fig. 4b) , suggesting transmission by bioaerosol. treatment by fend1 aerosol (48 and 72 h post infection) with an estimated 0.5 mg/kg deposited dose reduced the infection rate in the exposed animals. fend1 treatment of infected animals completely blocked transmission (0% transmission) to noninfected animals ( fig. 4b) . ▲ mean and standard deviation of triplicate wells for each condition. (c) the effect of multiple formulations of cacl 2 , magnesium chloride and nacl on viral titers in influenza infected calu3 cells, as quantified by tcid 50 assay. bars represent standard deviation of triplicate wells for each condition. data were analyzed statistically by one-way anova and turkey's multiple comparison post-test and indicated that cacl 2 in a nacl solution was the optimal combination of salts. (d) data from multiple independent studies were pooled and the change in rate constant for each is plotted. the greatest reduction in rate constant was observed for calcium concentrations between 0.575 and 1.14 m and sodium concentration between 0.075 and 0.3 m. three areas with darker the shades of blue are shown by the batched red boxes and indicate the greatest reduction in viral replication rate constant, optimizing the ratio of ca + and na + in the fast emergency nasal defense (fend) formulations at an 8:1 ratio. (e) anti-viral efficacy of the fend formulation was evaluated across a range of influenza virus a and b strains. moi 0.1 to 0.01. (f ) normal human bronchial epithelial cells (nhbecs) exposed to no formulation were used as a control and compared to cells exposed to fend1 and fend3. the concentration of virus (influenza a/panama/2007/99) released by cells exposed to each aerosol formulation was quantified by tcid 50 assay. bars represent standard deviation of triplicate wells for each condition. data were analyzed statistically by one-way anova and turkey's multiple comparison post-test. ( g) nhbe donor cells were exposed to low and high cacl 2 fend formulations, then harvested 8, 24, and 30 h post treatment. bd-2 mrna expression relative to the air (untreated) control was elevated at all time points after exposure to the high cacl 2 formulation. in addition, there was a time-dependent increase in bd-2 protein in the apical surface wash between 4 and 30 hours after exposure in cells exposed to the high cacl 2 fend formulation relative to the air control. given these results, we decided to pursue preclinical safety studies of both fend1 and fend2. we performed 14-day studies of fend1 and 28-day studies of fend2 via daily nose only inhalation exposure in rats and daily nasal inhalation exposure in dogs. all aerosols had mass median aerodynamic diameters (mmad) of <5 μm, to facilitate aerosol exposure to the entire respiratory tract, including nose and lungs. in the 14-day repeat dose nonclinical safety studies, we exposed groups of animals to vehicle (saline) control and three doses of fend1. for fend1, no observed adverse effect level (noael), expressed in terms of cacl 2 dose, for pulmonary exposure in sprague dawley rats and in beagle dogs, respectively were determined to be 13.5 and 8.77 mg/kg/day. while no adverse events were basolateral samples were serially diluted in saline and plated on blood or chocolate agar plates at each time point to quantify the number of bacteria. the percentage of the total number of bacteria passing through the mimetic in 4 h following saline exposure (maximum colony forming units [cfu] recovered from each assay) was calculated for each time point and the area under curve (auc) of each curve was determined. in each case, the exposure of mimetic significantly reduced the number of bacteria crossing the mimetic compared to the control condition (p < 0.05 for t test of auc between saline and fend1 treatment group for each pathogen). (f-g). sodium alginate mucus mimetic was treated with the indicated formulations and the movement of influenza a/wsn/33/1 or rhinovirus (rv16) was assayed. influenza was assayed from the basolateral buffer 4 h after treatment by quantitative polymerase chain reaction (qpcr) and rhinovirus was assayed over time by 50% tissue culture infectious dose (tcid 50 ) assay. (f-g). sodium alginate mucus mimetic was treated with the indicated formulations and the movement of influenza a/wsn/33/1 or rhinovirus (rv16) was assayed. influenza was assayed from the basolateral buffer 4 h after treatment by qpcr and rhinovirus was assayed over time by tcid 50 assay. (f ) data depict the mean ae standard deviation of replicate runs of the qpcr reaction and are representative of three independent experiments. data was analyzed by one-way anova and tukey's multiple comparison test (*p < 0.05 and **p < 0.001 compared to the saline control). (g) data are representative of two independent experiments. the limit of detection of the tcid 50 assay was 1.2 log 10 tcid 50 /ml. observed associated with nasal exposure, noaels for nasal exposure of fend1 based on the pulmonary adverse events in sprague dawley rats and in beagle dogs, respectively were determined to be 56.38 and 8.77 mg/kg/day. in 28-day inhalation toxicology studies for fend2, rats and dogs were administered vehicle (saline) control and three doses of fend2. again, no adverse events were observed for nasal exposure. noaels, expressed in terms of cacl 2 dose of fend2 and based on pulmonary adverse events, for both pulmonary and nasal exposure in sprague dawley rats and in beagle dogs, respectively were determined to be approximately 10.2 and 18 mg/kg/day. the results of our studies are summarized in table 1 (see also supplemental materials). given these results, we decided to pursue human studies to evaluate human safety, and the potential of fend to suppress exhaled bioaerosol when delivered to the entire respiratory system versus delivery preferentially to the nose. a total of eight (n = 8) human subjects were administered ascending pulmonary doses of fend1 (12.9, 38.7, and 77.4 mg cacl 2 ), in the target efficacy range of 0.25-1.0 mg/cacl 2 /kg. doses were delivered using a pari lc hand-held nebulizer with mass median aerodynamic particle size of 3.1 μm suited to deposition through the respiratory tract and the deep lungs. all eight subjects received each dose and one dose of placebo (0.9% nacl) on separate dosing days throughout the course of the trial. safety assessments included adverse event (ae) monitoring and concomitant medication as well as clinical laboratory evaluations, vital sign measurements, electrocardiogram (ecg), oxygen saturation, and pulmonary function test. all 8 subjects completed all 4 treatment days (1 placebo day and 3 days with escalating doses of the study drug). there were no serious aes (saes) reported in this study and no subjects were discontinued by the investigator due to an ae. a total of 17 treatment-emergent aes (teaes) were reported by 7 (88%) of the 8 subjects dosed in this study, as summarized in the supplemental material. of the aes observed in pulmonary treated subjects, all were self-limited and resolved spontaneously without specific treatment. we measured bioaerosol expiration (total particle counts per liter, and separately particles below and above 1 μm in size) in all of the subjects before and after treatment and compared with a placebo saline control. total exhaled particle counts per liter varied significantly between the individuals (table 2 ). some subjects exhaled many more than other subjects, consistent with previous observations of "super producer" subjects (edwards et al., 2004) . prior to placebo or treatment, five of the eight subjects exhaled on average 151 ae 87 particles per liter, while three of the individuals (subjects 2, 6, and 7) exhaled on average 1,201 ae 1,715 particles per liter. suppression of bioaerosol by delivery of the fend aerosol relative to placebo control occurred particularly at the high dose (see supplemental material for lower doses), as shown in fig. 5a , with six of the eight subjects breathing out over the duration of 12 h post treatment, a significantly lower mean particle count per liter relative to placebo (p < 0.05). this included two of the three high-producer subjects pneumoniae and pretreated with nacl aerosol for 15 min 2 h before infection have a higher bacterial burden than animals pretreated with cacl 2 aerosol. pooled data from multiple experiments are shown. each data point represents the data obtained from a single animal. the bar for each group represents the geometric mean of the group. the data were statistically analyzed using a mann-whitney u test (ns = not significant). david edwards et al. (6 and 7), who alone accounted for over 80% of the total exhaled bioaerosol particles of the group. duration of effect was particularly strong between 2 and 6 h post treatment. as further shown in fig. 5b , suppression of bioaerosol expiration at the high dose of fend1 is notably significant for bioaerosols in the 300-1,000 nm range. these small particles represent the majority of bioaerosol particles (77%) measured in our study. such submicron particles are not successfully captured by surgical masks (leung et al., 2020) , and are most prone to travel great distances, being too large to be particularly diffusive in the air and too small to settle by gravity. the nose being a major compartment for viral infection, and with the high nasal prevalence of the human angiotensinconverting enzyme (ace2) by which sars-cov-2 entry is mediated (hou et al., 2020) , we chose to develop a nasal delivery system to pursue a human nasal hygiene study and compare our pulmonary administration results with nasal delivery. we designed a hand-held nebulizer (nimbus) for fend capable of delivering nasal doses of around 1-2 mg calcl 2 (see supplementary material). we integrated vibrating meshes (see supplementary material) with a 6 μm pore size to produce, on tipping of the device, an aerosol cloud with a particle size distribution optimal for delivery within the nose through natural nasal inspiration. the particle size distribution of the aerosol cloud reveals a median volume particle diameter of 9-10 μm (see fig. 6a ), an optimal size for nasal and upper airway deposition of aerosol following a natural tidal inspiration through the nose and with relatively uniform distribution of deposition from the anterior to the posterior of the nose (calmet et al., 2019) . the nimbus particle size distribution is significantly smaller than that produced from a standard nasal pump spray (fig. 6b) . on tipping (fig. 6c) , nimbus produces 57 ae 2 mg within a 10 s actuation, after which power ceases until tipped back upright and again overturned. we designed the device to deliver a controlled dose of approximately 33 mg (i.e., 1.56 mg calcl 2 for fend2 or 0.43 mg calcl 2 for fend1) by filling an empty 6-oz glass with the cloud for the internally programmed 10-s actuation of the device and then inspiring the cloud directly from the glass into the nose (fig. 6d) . uncontrolled dosing can also be achieved by creating the cloud before the nose and direct natural deep nasal inspiration (fig. 6d) . we decided to pursue a human volunteer study with nimbus to evaluate the effectiveness of fend for suppressing exhaled aerosol particles following nasal administration in comparison to our observation of the effectiveness of fend on pulmonary delivery. (b) each bar represents the percent lung consolidation from the most affected lobe from a single animal. naïve animals (n = 4 for each replicate) were secondarily exposed to the exhaled breath of primary infected untreated animals (black bars, n = 4) or primary infected treated animals (red bars, n = 4). exhaled breath from the four infected swine was combined and delivered to individual naïve swine using a custom-designed exposure system designed to prevent direct contact transmission. naïve swine were exposed to exhaled breath from infected animals for 1 h/day on day 2 and 3 post infection. influenza transmission was observed between primary untreated animals and secondary animals (left black to gray bars). primary infected treated animals (red bars) showed evidence of reduced infection rates and evidence of transmission was not observed (right red to light red bars). ten healthy volunteers were recruited in st augustine, florida and boston, ma. each signed informed consent to participate in the several-hour nasal saline hygiene study. five of these subjects were older than 65 years (70, 75, 82, 83, and 88) and five younger than 65 years (30, 40, 59, 60, and 63) . subjects with severe respiratory illnesses were excluded from the study, while two of the subjects (ages 30 and 63) were cigarette smokers. all subjects began the study by breathing into an apparatus that measured expired aerosols. following a baseline assessment of exhaled aerosol particle count subjects drew two deep nasal inspirations via nimbus of fend2. subsequent to fend administration, subjects breathed into the airborne particle detector at intervals for up to 6 h post dosing. subjects also self administered a commercial (isotonic nacl) simple saline cleansing spray (cvs nasal saline). subsequent to nasal administration of the placebo control, subjects breathed into the airborne particle detector at intervals for up to 2 h. baseline exhaled particles per liter per subject age are shown in fig. 7 . two of the ten subjects in the older age (>65) group exhaled very high numbers of particles per liter of air (24,088 ae 9,413 and 7,180 ae 1,250) (fig. 7a) while the other eight (fig. 7b ) exhaled between approximately 10 and 1,200 particles per liter. in the latter group, two individuals (ages 30 and 63), were smokers. there is a strong correlation between high numbers of exhaled particles and age with the group older than 65 exhaling on average 6,641 particles per liter while the group younger than 65, on average, exhaling 440 particles per liter. in all subjects over 95% of the baseline exhaled particles were less than 1 μm in size with most smaller than 500 nm (fig. 8a,b) . following fend administration, exhaled particle numbers diminished for up to several hours as shown in fig. 8 , presented on a log scale. this diminution relative to baseline is statistically significant (p < 0.05) for all 10 subjects. duration of effect continued up to the last data point several (2-6) hours after administration for all of the subjects other than subjects b and e, each of whom were very small producers of particles. administration of the simple saline control has a minor suppressive effect on exhaled particles for two of the subjects in the first hour following administration while in the other subjects, we observed no suppressive effect (fig. 9) . using the lowest exhaled particle number following fend administration as a measure of suppression effect, diminution of bioaerosol ranged from a low of 45% (subject age 75) to a high of 99% (subject ages 83 and 70), with overall suppression of aerosol for the group (99%) predominantly related to the dramatic effect of fend on suppression among super producing individuals (subject ages 83 and 70). hypertonic cacl 2 and nacl solution delivered to the respiratory system appears to have potential as both hygienic and therapeutic biodefense against airborne pathogens. hygienically, these physiological salts coat the surfaces of alf to diminish breakup and clear away the submicron bioaerosol droplets (fig. 5c ) that are not effectively captured by masks (fig. 4b) . by potentially boosting natural immunological defenses-strengthening the barrier function of the alf (fig. 2) and promoting the secretion of β-defensin 2 from nasal and bronchial epithelial tissues (fig. 1g )-these salts may also act therapeutically for antimicrobial prophylaxis or treatment. while our results suggest that cacl 2 and nacl salt combinations may be therapeutically useful against bacterial and viral infections including influenza, rhinovirus, and pneumonia, our results point to an immediate hygienic value in the fight against any airborne infectious disease, including covid-19, by cleaning the airways of the small airborne droplets that carry airborne infection. our finding that nasal inspiration of fend in a group of 10 healthy human subjects reduces exhaled particles between 45 and 99% by way of an aerosol too large to penetrate the lower airways (figs 8 and 9) , suggests that the upper airways are a primary source of expired bioaerosol. the high velocity airstreams created during natural breathing (often reaching turbulent air flow conditions) in the trachea and main bronchi disturb the surfaces of alf in the way of wind passing over the sea to generate sea mist (wattanabe et al., 2007) . such phenomena are highly sensitive to compositional variations in the underlying fluid, making exhaled bioaerosol a sensitive measure of alf and introducing variability within and between subjects. in our study, fend substantially cleared away exhaled particles, most being less than 1 μm in size. that particles in the range of 300-500 nm were the most predominant observed in the exhaled breath of subjects can be explained by the fact that such particles are both too small to deposit in the lungs by gravity or inertia, once generated, and too large to be deposit by diffusion. these are the submicron particles most likely to remain suspended in the atmosphere essentially indefinitely. possibly, most important in terms of their ability to transmit infection-and deposit on surfaces including airways of the infected or naive individuals-are those particles in the 500-1,000 nm range, a significant fraction of the exhaled particles of the super spreader individuals. these particles are also substantially eliminated by fend treatment. in our study, most of the airborne particles were exhaled from 2 of 10 "super producing" individuals. this super production of bioaerosol promotes the phenomenon of super spreaders (stein, 2015) . super spreading events for covid-19 have been reported in china (so-called patient 31), india (the punjab outbreak), south korea, and many other regions, and are suspected to be a primary mode of transmission of the disease (kupferschmidt, 2020). among key correlates of super spreading are suppressed immunity and infected lungs (stein, 2015) -two particular vulnerabilities of the exhaled bioparticles per liter of the 8 human subjects prior to fend or placebo treatment were counted by light scattering in four size bins, or categories (0.3-0.5, 0.5-1.0, 1.0-5.0, and >5.0 μm). subjects were required to wear a nose clip and breathe into a mouthpiece for three deep breaths followed by approximately 3 min of targeted tidal breathing at each time point. prior to each subject's measurement at each time point, the detection system was calibrated, providing background noise estimation for the filtration of the particles in the room environment. abbreviation: fend, fast emergency nasal defense. most aged. indeed, older subjects in our study exhaled many more particles than younger subjects-suggesting the possibility that seniors, while among the most vulnerable to covid-19 infection, may also be those most likely to spread the disease, and underscoring the extreme risk seniors face today in nursing homes. as nasal hygiene, fend is easy to administer (fig. 6 ), rapid (one or two deep nasal inspirations) and lasts long (at least 6 h in those expiring the largest numbers of particles). it might be easily administered to individuals on entering environments, where they are likely to encounter others, including hospitals, nursing homes, prisons, fig. 5 . suppression of expired bioaerosol from human lungs following treatment by fast emergency nasal defense (fend)1 at the high clinical trial dose. (a) exhaled bioparticles per liter versus hours post dosing (relative to baseline) of the eight human subjects following fend1 treatment or following saline placebo control. bioparticle sampling from exhaled air was performed at the following time points: prior to dosing and at 0.5, 1, 2, 4, 6, 8, 10, and 12 h post dose for each period, and prior to release from the clinic on period 4, day 2. each subject was treated with a placebo (saline) control and with each of the three doses of fend1 at 24 h intervals. data from each subject's high dose recording are compared with each subject's placebo control. (b) expired bioparticles per liter (relative to baseline) in the size range of 300-500 nm from each of the human subjects are shown relative to placebo versus hour relative to dosing. each subject was treated with a placebo (saline) control and with each of the three doses of fend1 at 24-h intervals. triplicate measurements were performed twice on pure water yielding (9.5 μm average diameter) and on fend2 composition yielding a mass mean aerodynamic diameter of 9.23 μm ae 0.60 and 8.84 μm ae 0.45. (b) triplicate measurements were performed twice on a nasal pump spray triplicate measurements with fend2 composition yielding a mass mean aerodynamic diameter of 107 μm. (c) tipping of nimbus actuated mesh vibration and generates an aerosol cloud for dosing. (d) fend can be administered by nimbus with a deep nasal inspiration either in an uncontrolled fashion before the nose or in a controlled fashion by containing the cloud in a glass. fig. 7 . exhaled particles from the 10 human volunteers prior to fast emergency nasal defense (fend) dosing. exhaled particles per liter of air are shown within three size distributions -between 300 and 500 nm, 500 and 1,000 nm, and 1,000 and 5,000 nm. (a) two of the human subjects (ages 63 and 70) exhaled greater than 25,000 and 7,000 particle per liter, respectively, the majority of these particles between 300 and 500 nm, and a large minority of the particles between 500 and 1,000 nm. (b) the other eight individuals breathed out on average several hundred particles per liter. schools, offices, factories, stadia, restaurants, and museums. the use of fend as an "invisible mask" supplement to traditional masks administered prior to close encounters with others in public and private spaces in order to clean the air of the small particles that masks do not block appears a prudent addition to current hygienic practices in the face of the covid-19 pandemic. more research is meanwhile needed to assess the consequences of calcium-enriched physiological salt nasal hygiene on airborne infection and transmission rates within environments at high-risk of covid-19 and other airborne infection diseases. the therapeutic potential of these nasally and pulmonary delivery salts should also be explored beyond the scope of the in vitro and animal studies reported here. the goal of our studies was to explore the potential of a purely saltbased natural aerosol to promote innate immunity and dampen bioaerosol shedding with application to bacterial and viral airborne pathogens. calu3 cells were cultured on permeable membranes (12 mm transwells; 0.4 μm pore size, corning lowell, ma) until confluent. ali fig. 8 . exhaled particles from the 10 human volunteers following fast emergency nasal defense (fend) dosing. the exhaled particles per liter per human subject are shown on a log scale prior to dosing (t = 0) and at various times post dosing up to 6 h post dosing. in all cases statistically significant suppression of exhaled aerosol is observed while the effect is dramatically significant for the largest "super producing" subjects (ages 63 and 70), whose overall exhaled particle counts diminish more than 99% for 6 h following fend nasal inspiration. cultures were established by removing the apical media and culturing at 37°c/5% co 2 . cells were cultured for >2 weeks at ali before each experiment. prior to each experiment the apical surface of each transwell was washed 3â with 500 μl/well phosphate buffered saline (pbs) and the basolateral media (media on the bottom side of the transwell) was replaced with 1.5 ml/well fresh media after aerosol exposure. nhbe cells were obtained from multiple donors from the university of north carolina. cells were cultured as describedin the supplemental materials. to establish ali cultures, cells were cultured on permeable membranes similar to calu3 cells. nhbe cells were cultured on transwell permeable supports under ali conditions for a minimum of 4 weeks before experiments. cells were exposed to nebulized aerosols using a customized sedimentation chamber that exposed triplicate wells of cells to the same aerosol. aerosols were generated using series 8900 nebulizers (slater labs). following the delivery of formulations to cells, a second plate was exposed to the same formulations to quantify the delivery of total salt or calcium to cells. following nebulization, each well of the second plate was washed with dh 2 o and salt concentration was assayed by osmometry. following treatment, cells were infected apically with 10 μl/well of influenza a/wsn/33/1 at a multiplicity of infection of 0.1-0.01. four hours after infection, the apical surfaces were washed with 500 μl/well pbs to remove excess formulation and unattached virus and cells were cultured for an additional 20 h at 37°c/5% co 2 . twenty-four hours after infection, virus released onto the apical surface of infected cells was collected in 250 μl/well in plaque assay media (ham's f12 plus supplements) and subsequently further diluted in 300 μl of plaque assay media before tittering. assays with other influenza strains were conducted in the same manner. influenza titers in recovered washes were determined by 50% tissue culture infectious dose (tcid 50 ) assay. madin-darby canine kidney cells (mdck) cells were plated in 96-well flat bottom tissue culture plates (costar) at 10,000 cells/well 24 h prior to use. samples were serially diluted on mdck cells in media containing tpcktreated trypsin and cultured for 48-72 h at 37°c/5% co 2 . infected wells were quantified by hemagglutinin assay using 0.5% chicken erythrocytes. plates were incubated for 2 h at 4°c, positive wells were visually scored and tcid 50 was calculated according to the spearman-karber method. a rate constant was used as a means of normalizing data across studies such that the difference between placebo and active treated samples in a given study over the actual duration of bacterial of viral growth is calculated for each formulation in order to normalize across different studies. the apical surface of airway epithelium is lined by alf that contains a mucus layer and a pericilliary layer. the alf is rheologically complex and functions as a barrier for passage of pathogens and environmental particles protecting the epithelium. we developed a model system to mimic the mechanical (rheological) barrier of mucus following techniques previously described by wattanabe et al. (2007) . our mucus mimetic was comprised of 4% alginate solution prepared from 2.0 ae 0.0005 g sodium alginate and 50 ml milli-q water (di h2o). the alginate samples were dissolved by adding a small amount to an aliquot of the water in a centrifuge tube, vortexing, and increasing step-wise the amounts of water and alginate. they were left to mix on the rotating wheel and stored at 4°c until homogenous. the alginate sample was used only when there was no visible bacterial growth. since bacterial growth is known to lower the alginate's viscosity, a strain sweep was also run at the beginning of each test. in all samples, 2.5 ≤ tan (δ) ≤ 3.0 at 3 rad/s. the tan(δ) variable is the ratio of the viscous to elastic modulus g 00 /g 0 , such that a decrease in tan(δ) indicates a relative increase in elastic (vs. viscous) response. the increase in the viscous versus elastic response can be interpreted as an increase in the material's overall "stiffness" in oscillatory conditions. the tan(δ) is measured using a rheometer with du noüy ring to measure the tension between it and the surface of a sample using rotational motions with variable rate of oscillations and strain applied to a meniscus. the rate of oscillation is measured as rad/s over a range of 0.6283-15 rad/s and the strain is assessed over a range of 0.02-40%. the sodium alginate sample is poured into the sample dish and any large air bubbles drawn out by pipette. the du noüy ring is lowered into the sample's bulk and raised again to a level slightly above the surface, so that a small, durable meniscus was visible. the strain sweep is run with a known oscillation rate to ensure that the viscosity is within the range of the normal mucus layer before treatment with the formulations and reassessment. see supplemental materials. for gene expression studies, total rna was isolated from hbecs exposed to aerosolized formulations using the qiagen rneasy plus mini kit according to the manufacturer's animal cell protocol. expression levels of selected genes were quantified by quantitative polymerase chain reaction using the δδct method and gapdh as an internal reference for each sample (see supplemental materials). secreted bd-2 protein levels were determined in apical washes of nhbe cells by elisa (peprotech inc). specific pathogen-free female c57/bl6 mice (6-7 weeks, 16-22 g) were obtained from charles river laboratories and housed in the animal facility at harvard school of public health according to their guidelines. mice were given access to food and water ad libitum. for infections, s. pneumoniae (serotype 3; atcc 6303) were streaked onto blood agar plates and grown at 37°c plus 5% co 2 overnight. prior to infection, animals were anesthetized by intraperitoneal injection of a mixture of 50 mg/kg ketamine and 5 mg/kg xylazine. single colonies of s. pneumoniae were resuspended in sterile saline to od 600 = 0.3 and subsequently diluted 1:4 in saline. colloidal carbon was added to 1% and 50 μl of the resulting solution (~1 â 10 6 colony forming units [cfu] ) was instilled into the left lobe of anesthetized mice. following infection, the bacterial titer of the inoculum was determined by serial dilution and plating on blood agar plates. after 24 h, mice were euthanized by exposure to isoflurane and the bacterial burden in lungs of infected animals was determined by plating serially diluted lung homogenate on blood agar plates. a whole-body exposure system using a custom-designed high output nebulizer was utilized to deliver salt aerosols to a piechamber exposure system. each pie chamber exposure chamber was modified such that a single tube delivered aerosol to a central manifold and ultimately to one of 11 mouse holding chambers via 4 inlet ports in each chamber. the total flow through the system was 11.7 l/min and animals were exposed to cationic aerosols for 15 min. mice were randomly assigned to different study groups on the day of the infections. different aerosol exposure times relative to the time of infection were utilized to test the effect of aerosols in both prophylaxis and treatment regimens. for each exposure, mice were loaded into a customized wholebody pie chamber system in which aerosols were delivered to a central manifold and subsequently to each individual animal. aerosol exposures consisted of 15 min exposures, which delivered an estimated dose of 6.4 mg/kg/day of cacl 2 . after 24 h of infection, animals were euthanized by isoflurane inhalation and the lungs were surgically removed and placed in sterile water. lungs were homogenized using a glass mortar and pestle until no large tissue fragments were visible. cfu were enumerated by serially diluting lung homogenates in sterile water and plating on blood agar plates. plates were incubated overnight at 37°c plus 5% co 2 and cfu counted the following day. normal, 4-week-old piglets were obtained from the penn state swine herd and allowed to acclimate for 1-2 weeks prior to study. nasal swabs from piglets were screened for naturally occurring infections including prrs (porcine reproductive respiratory syndrome), influenza, mycoplasma and salmonella and the piglets underwent a 1-week course of antibiotic therapy prior to influenza infection to limit confounding co-infection. preinfection serology was performed to identify previous influenza exposure and only piglets with a hemagglutinin titer of <1:10 were used in studies. for influenza infections and aerosol treatments, animals were anesthetized with a mixture of tiletamine (telazol, 2 mg/kg), zoleazepam (2 mg/kg), and xylazine (4 mg/kg). animals were inoculated intranasally with swine influenza a (h1n1) virus grown for 48 h in 11-day-old embryonated eggs. the inoculum was prepared to deliver 10 7 eid50 in 0.5 ml pbs administered intranasally. animals were monitored twice daily for feed intake, rectal temperature, attitude, appetite, breathing rate, and evidence of dyspnea. anesthetized piglets were exposed to aerosols via a snout-only system via anesthesia masks in a custom-built holding chamber. aerosol was generated from a custom-built high output ultrasonic nebulizer and delivered into a central manifold to which each individual animal was connected. individual animals fitted with canine anesthesia masks were exposed to aerosol from the central manifold via one-way valves; animals similarly exhaled via one-way valves to maintain a dynamic flow. animals were treated with fend1 for 22 min on days 2 and 3 after infection. body temperatures and clinical signs were recorded throughout the postinfection period. at day 9 post infection, animals were euthanized, lungs were removed and the percentage of the lung that showed consolidation as quantified. for transmission studies, piglets were randomly assigned to infected or naive groups (n = 8 total; 4 per group) and each group was housed in a separate clean room. following infection, anesthetized animals were treated with fend1 on days 2 and 3 after infection as in treatment studies. ten minutes following treatment, naïve animals were exposed to the exhaled breath of the infected animals. infected and naïve animals were each fitted with anesthesia masks and connected by >3 00 of tubing. once connected, naïve animals were exposed to the exhaled breath of the infected animals for 1 h/day on days 2 and 3 after primary infection. the study protocols were reviewed and assessed by the animal care committee (acc) of itr, montreal, qc, canada. animals were cared for in accordance with the principles outlined in the current "guide to the care and use of experimental animals" as published by the canadian council on animal care and the "guide for the care and use of laboratory animals", an nih publication. studies were conducted in compliance with good laboratory practice (glp). sprague dawley rats (n = 10/sex/group) were exposed to fend1 or fend2 by nose-only inhalation once daily for up to 120 min per exposure. doses were achieved by exposing animals to different durations of each formulation. control animals received isotonic saline for 120 min. aerosol was produced using two sidestream nebulizers in tandem. the airflow rate through the exposure system was monitored and recorded manually during the aerosol generation and was controlled using variable area flow meters. determinations of aerosol concentration, particle size distribution, oxygen concentration, relative humidity, and temperature were performed on test and control atmosphere samples collected from a representative port of the exposure chamber. in the fend1 study, rats were treated for 14 days at doses of 4.79, 13.5, and 56.4 cacl 2 /kg and in the fend2 study, rats were treated for 28 days at doses of 3.05, 10.3, and 32.7 cacl 2 /kg. mmad in the fend1 study ranged from 1.3 to 2.0 μm with geometric standard deviations (gsd) of 1.63-2.03. in the fend2 study, mmads were 1.5-1.9 μm with gsd of 2.20-2.37. in-life observations were made starting 1 week prior to dosing and continued through the dosing period. these included clinical observations, body weight, food consumption, ophthalmoscopy, clinical pathology, hematology, clinical chemistry, and urinalysis. at necropsy, gross pathology was conducted on internal exam and tissues were preserved in neutral buffered formalin for histopathological analysis and microscopic analysis of hematoxylin and eosin stained slides. beagle dogs (n = 3/sex/group) were exposed to fend1 or fend2 by orthonasal inhalation once daily for up to 120 min per exposure using a facemask exposure system. aerosol generation and monitoring was conducted similar to the rat study, with adjustments made for the dosing system. in the fend1 study, dogs were treated for 14 days at doses of 1.15, 3.24, and 8.77 mg cacl 2 /kg and in the fend2 study, dogs were treated for 28 days at doses of 1.11, 4.16, and 18.0 cacl 2 /kg. in the fend1 study, mmads were between 0.9 and 1.2 and gsds ranged from 1.67 to 1.97 across groups. in the fend2 study, mmads were 1.2-1.3 μm with gsd of 1.90-2.05. in-life observations with similar to the rat studies, with the additional inclusion of ecg on the first and last day of dosing. emitted size distributions from the fend nasal nebulizer and fend nasal spray were determined via laser diffraction using the spraytec spray analysis system (malvern panlytical ltd, uk) in an open-bench configuration. the emitted size distributions of purified water and the fend2 formulation were assessed from each delivery system. the delivery devices were affixed approximately 2 00 from the measurement beam. data collection occurred at a 1 khz acquisition rate over the duration of the spray event, with reported results representing the time-averaged size distributions. all experimental conditions were assessed in triplicate. a phase l, double-blind, placebo-controlled, randomized study was conducted to evaluate the safety and tolerability of single ascending doses of fend1 and to assess the pattern of exhaled bioparticles following fend1 inhalation. the trial enrolled eight healthy subjects who consented to receive a single dose of fend1 or placebo (isotonic saline) once daily in four periods with four dosing sequence groups. doses of 12.9, 38.7, or 77.4 mg cacl 2 were administered in ascending order with two subjects receiving placebo on each day of dosing. dose escalation to the next dose level was only permitted if, in the opinion of the principal investigator, adequate safety and tolerability were demonstrated at the previous lower doses. subjects were confined to the clinic from the evening prior to the first dose until 24 h after the final dose in period 4. subjects returned to the clinic approximately 7 days following the final dose, for follow-up safety assessments. primary endpoints were clinical signs and symptoms from physical examination, aes, laboratory safety (hematology, serum chemistry, and urinalysis), vital signs (blood pressure, heart rate, temperature, and respiratory rate), ecgs, oxygen saturation, and pulmonary function test. the secondary endpoint was exhaled bioparticle measurements. exhaled bioparticles were counted and measured for size distribution. exhaled bioparticle samples were analyzed for number and size distribution using a proprietary bioaerosol expiration particle counter. particles were counted and sized by light scattering into 4 size bins, or categories (0.3-0.5, 0.5-1.0, 1.0-5.0, and >5.0 μm). exhaled breath profiles were collected and passed through a laser particle counter and a flow meter measured the full inhalation and exhalation airflow rate. software combined these two measurements and calculated all relevant parameters. primary measurements were total particle counts/liter, and the particle counts/liter that fell into the four size bins (bin cut points in μm): 0.3-0.5, 0.5-1.0, 1.0-5.0, and >5.0 counts/liter. exhaled bioparticles were counted and measured for size distribution. exhaled bioparticle samples were analyzed for number and size distribution using a proprietary bioaerosol expiration particle counter. particles were counted and sized by light scattering into four size bins, or categories (0.3-0.5, 0.5-1.0, 1.0-5.0, and > 5.0 μm). exhaled breath profiles were collected and passed through a laser particle counter and a flow meter measured the full inhalation and exhalation airflow rate. software combined these two measurements and calculated all relevant parameters. primary measurements were total particle counts/liter, and the particle counts/liter that fell into the four size bins (bin cut points in μm): 0.3-0.5, 0.5-1.0, 1.0-5.0, and >5.0 counts/liter. to establish the dose for our nasal administration human volunteer study, we determined a fend efficacious dose level for bioaerosol mitigation per surface area of airway tissue of 0.00015 mg/cm 2 , based on a human lung surface area of 50 m 2 based on the finding from our human pulmonary-administration study that inhalation of fend1 produces significant suppression of expired bioaerosol at the highest total lung dose only. for nasal administration, this translates into a dose range from 25 μg calcl 2 to 2 mg calcl 2, with the lower end of the range based on our observed exhaled aerosol suppression and a standard nasal surface area of 181 cm 2 and the upper end of the range based on our observed antimicrobial activity as observed in mice and pigs. in our nasal administration human volunteer study exhaled particles were measured by a particle detector (climet 450-t) designed to count airborne particles in the size range of 0.3-5 μm. the particle detector was connected to standard nebulizer tubing and mouthpiece that filters incoming air through a hepa filter. each standard nebulizer tubing and mouthpiece was removed from its sealed packaging before each subject prior to the subject's first exhaled particle detection. on subsequent counting maneuvers, the same mouthpiece and tubing was replaced into the particle counter system to insure oral hygiene. subjects breathed at normal tidal breathing through a mouthpiece, while plugging their noses over at least 2 min-beginning with one or two deep breaths. over this time frame, particle counts per liter diminished from the ambient particle count to a lower number representing the particles emitted solely from the subject's airways. once the lower plateau of particle counts was reached subjects continued to breathe normally. three to six particle counts (average values assessed over 5 s) were then averaged to determine the mean exhaled particle count and standard error. fend was administered to each subject by tipping into an empty 6-oz glass. after 10 s actuation, nimbus delivers approximately 33 mg (i.e., 1.56 mg calcl 2 ) from the glass into the nose and upper airways. dosing is accomplished by subjects immediately inspiring through the nose the cloud directly from the glass. subsequent to fend administration subjects breathed into the airborne particle detector at intervals from 10 min post dosing to 6 h post dosing. a clinical trial of hypertonic saline nasal spray in subjects with the common cold or rhinosinusitis expression of beta-defensin 1 and 2 in nasal epithelial cells and alveolar macrophages from hiv-infected patients presumed asymptomatic carrier transmission of covid-19 nasal irrigation: from empiricism to evidence-based medicine. a review respiratory and facial protection: a critical review of recent literature nasal sprayed particle deposition in a human nasal cavity under different inhalation conditions remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov-2 t replication in vitro the antiviral action of common household disinfectants and antiseptics against murine hepatitis virus, a potential surrogate for sars coronavirus inhaling to mitigate exhaled bioaerosols covid-19-navigating the uncharted well-differentiated human airway epithelial cell cultures human coronaviruses: insights into environmental resistance and its influence on the development of new antiseptic strategies saline irrigation for allergic rhinitis sars-cov-2 reverse genetics reveals a variable infection gradient in the respiratory tract human β-defensin 2 plays a regulatory role in innate antiviral immunity and is capable of potentiating the induction of antigen-specific immunity intracellular calcium in signaling human beta-defensin-2 expression in oral epithelial cells (2020) clinical and virological data of the first cases of covid-19 in europe: a case series. the lancet infectious diseases respiratory virus shedding in exhaled breath and efficacy of face masks role of ventilation in airborne transmission of infectious agents in the built environment -a multidisciplinary systematic review aerodynamic analysis of sars-cov-2 in two wuhan hospitals pilaipan puthavathana (2010) influenza a viral loads in respiratory samples collected from patients infected with pandemic h1n1, seasonal h1n1 and h3n2 viruses antiviral innate immune response in non-myeloid cells is augmented by chloride ions via an increase in intracellular hypochlorous acid levels lynn morrice & aziz sheikh l (2019) a pilot, open labelled, randomised controlled trial of hypertonic saline nasal irrigation and gargling for the common cold spread of influenza virus and sars-cov-2 by breathing only analyzing real-time pcr data by the comparative c(t) method efficacy of isotonic nasal wash (seawater) in the treatment and prevention of rhinitis in children effect of several inorganic salts on infectivity of mengo virus super spreaders in infectious diseases pathogenicity of influenza virus aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 covid-19 may transmit through aerosol why inhaling salt water changes what we exhale guidelines on hand hygiene in health care: first global patient safety challenge clean care is safer care zheng, b (2016) a novel peptide with potent and broad-spectrum antiviral activities against multiple respiratory viruses j (2020) sars-cov-2 viral load in upper respiratory specimens of infected patients acknowledgments. our research was performed over a period of several years from our first experiments at harvard, mit and penn state university-at pulmatrix, and in a group of collaborating labs in the united states, canada, england, and northern ireland. many pulmatrix scientists and technicians participated in the execution of these studies. jérôme edwards performed statistical analysis on the human bioaerosol data. funding for all of the in vitro and in vivo pulmonary studies was provided by pulmatrix, funding for all of the in vitro and in vivo nasal studies was provided by sensory cloud, and all fend compositions have been licensed from pulmatrix to sensory cloud. key: cord-329137-5pw07qje authors: dryden, kelly a.; tihova, mariana; nowotny, norbert; matsui, suzanne m.; mendez, ernesto; yeager, mark title: immature and mature human astrovirus: structure, conformational changes, and similarities to hepatitis e virus date: 2012-10-05 journal: journal of molecular biology doi: 10.1016/j.jmb.2012.06.029 sha: doc_id: 329137 cord_uid: 5pw07qje abstract human astroviruses (hastvs) are a major cause of gastroenteritis. hastv assembles from the structural protein vp90 and undergoes a cascade of proteolytic cleavages. cleavage to vp70 is required for release of immature particles from cells, and subsequent cleavage by trypsin confers infectivity. we used electron cryomicroscopy and icosahedral image analysis to determine the first experimentally derived, three-dimensional structures of an immature vp70 virion and a fully proteolyzed, infectious virion. both particles display t =3 icosahedral symmetry and nearly identical solid capsid shells with diameters of ~350å. globular spikes emanate from the capsid surface, yielding an overall diameter of ~440å. while the immature particles display 90 dimeric spikes, the mature capsid only displays 30 spikes, located on the icosahedral 2-fold axes. loss of the 60 peripentonal spikes likely plays an important role in viral infectivity. in addition, immature hastv bears a striking resemblance to the structure of hepatitis e virus (hev)-like particles, as previously predicted from structural similarity of the crystal structure of the astrovirus spike domain with the hev p-domain [dong, j., dong, l., méndez, e. & tao, y. (2011). crystal structure of the human astrovirus capsid spike. proc. natl. acad. sci. usa 108, 12681–12686]. similarities between their capsid shells and dimeric spikes and between the sequences of their capsid proteins suggest that these viral families are phylogenetically related and may share common assembly and activation mechanisms. human astroviruses (hastvs) are an important cause of gastroenteritis in children, the elderly, and immunocompromised adults. the virus was first identified in 1975 in infants who developed diarrhea in hospital nurseries in the united kingdom. 1, 2 hastv is a small, single-stranded, positive-sense rna virus with an~6.8-kb genome. the 5′-terminal two-thirds of the genome [open reading frame (orf)-1a and orf-1b] encodes two major nonstructural proteins, a serine protease whose structure has been solved by x-ray crystallography 3 and an rnadependent rna polymerase. the 3′-terminal onethird of the genome (orf-2) encodes the structural protein vp90, which is translated as an 87-to 90-kda capsid precursor protein. vp90 has a highly conserved n-terminal domain and a variable c-terminal domain among astrovirus serotypes. to date, eight serotypes have been identified of which type 1 is the most prevalent, and type 8 has been studied extensively. while their genomes are fairly well conserved, 4-6 the mature virions of different serotypes vary somewhat in the exact size and number of structural proteins that form the capsid (reviewed by krishna 7 ) . the current consensus is that morphogenesis of infectious particles relies on a series of proteolytic cleavages of the capsid precursor protein (fig. 1 ). vp90 assembles in infected cells and undergoes c-terminal cleavages by caspases to generate vp70 (a 70-to 79-kda protein) for release from the cells as immature virus particles. 8 generation of infectious particles is dependent upon further trypsin cleavages usually resulting in three structural proteins, vp34, vp27, and vp25, each of which range in actual size and name depending on the serotype. 9-11 vp34 contains the conserved n-terminal domain and is ascribed to the capsid, while the overlapping vp27 and vp25 subunits contain the variable c-domain and are ascribed to the viral spikes. 7,12 vp25 is generated by additional trimming at the n-terminus of vp27. 11 astroviruses were initially named for the distinctive star-like appearance of their viral surface observed in~10% of the fecally shed viral particles evaluated by negative-stain electron microscopy (em). em ultrastructural analysis of infectious hastv generated in cell culture showed spherical particles with a surface that was studded with spikes and an external diameter of~410 å. 13 a recent milestone was determination of a highresolution x-ray structure of the p-domain of the astrovirus spike vp25 (ordered residues 430-645). 12 similarities in sequence and domain organization of hepatitis e virus (hev) and hastv enabled the building of a homology model of hastv. in this study, we sought to gain direct insight into the structure and assembly of astrovirus by electron cryomicroscopy (cryoem) of immature and proteolytically activated particles. we examined (1) immature hastv-8 particles composed of the 70-kda intermediate protein, which has undergone caspase but not trypsin cleavage and is therefore not infectious, and (2) fully cleaved, mature infectious particles of hastv-1. the sequences of the two strains are extremely similar, with 83% identity in the conserved domain and 60% identity in the variable domain. therefore, we expect that our three-dimensional (3d) reconstructions are representative of the astroviridae family. although the capsid shells of both immature and mature hastvs are nearly identical, there is a striking difference in the apparent stoichiometry of the surface spikes. immature, uncleaved particles, which are strikingly similar in appearance to hev-like particles (hev-lp), 14 trypsin cleavage of the coat protein between the conserved (white boxes) and variable (shaded boxes) domains is required for viral maturation. the specific pattern of peptide products varies between serotypes. in these experiments, we examined uncleaved particles of hastv-8 formed by vp70 and fully cleaved particles of hastv-1. have 90 spikes, whereas mature, cleaved particles only display 30 spikes, all located at the icosahedral 2-fold axes. loss of the 60 peripentonal spikes likely plays an important role in viral infectivity. astrovirus particles have t = 3 icosahedral symmetry purification of hastv has been very challenging, and sufficient material was only available to perform cryoem once for immature hastv-8 and mature hastv-1. icosahedral image analysis of electron cryomicrographs (fig. 2 ) yielded 3d maps at~25 å resolution ( fig. 3a and b ). both particles display two concentric layers of density that conform to t = 3 icosahedral lattice symmetry. a nearly identical inner layer forms a solid capsid shell 45 å thick with an outer diameter of~350 å. this capsid shell has a chiseled appearance assembled from 180 protein subunits in which trimeric facets form a plateau ( fig. 3a and b, bottom) . the trimeric facets are in close contact across the icosahedral 2-fold (2f) symmetry axes, with depressions at the 5-fold (5f) and 3-fold (3f) symmetry axes and a groove across the local 2f axes. distal from the solid capsid shell is an outer layer of globular spike-like densities. the spikes are~40 å in diameter, with a somewhat elliptical shape, yielding an outer particle diameter of~440 å. the stoichiometry suggests that the spikes are dimers with contributions from adjacent trimer facets (fig. 3a , color code and labels). thin linker densities connect the spikes to the capsid shells and are visible in 3d reconstructions of negatively-stained particles ( fig. s1 ) or when the isosurface is lowered significantly in the cryoem maps. the lack of well-defined linker density may be the result of the narrowness of the linker, minor heterogeneity in maturation cleavage, or wobbling of the spike head, which would obscure the connections during icosahedral symmetry averaging. although the capsid shells of both immature and mature hastvs are nearly identical, there is a striking difference in the apparent stoichiometry of the surface spikes. immature, uncleaved particles have 90 spikes (fig. 3a, top) . thirty are located at the icosahedral 2f symmetry axes, and sixty surround the 5f vertices, centered at the local 2f axes. mature, cleaved particles only display 30 spikes, all located at the icosahedral 2f axes (fig. 3b , top). the packing of the viral proteins displays two arrangements for the dimer interaction of the spikes-one that places the dimer densities in close proximity (cc dimers) and one that spans nearly twice the distance between the contributing subunits (ab dimers) (fig. 3a , bottom labels). this difference in separation may expose different cleavage sites during maturation. proteolytic cleavage is a common mechanism for activation of both enveloped and nonenveloped viruses, including influenza virus, 15, 16 coronavirus, 17 rotavirus, 18 reovirus, 19 and alphaviruses. 20 a recurring theme is that cleavage triggers a conformational change in a surface spike or other cell-attachment domain. for astrovirus, the uncleaved, immature t = 3 particle contains 180 copies of vp70, in which the viral capsid is attributed to the n-terminal domains and the 90 spikes are attributed to dimers of the variable c-terminal domain. similarly, trypsin cleavage of the immature particles produces three products, of which we correspondingly attribute vp34 to the capsid shell and the two different c-terminal products, vp27 and vp25, to the spikes. sds-page suggests that trypsin cleaves virtually all of the vp70 molecules (fig. s2 ). in addition, the 3d reconstruction in fig. 3b suggests that trypsin cleavage results in release of two-thirds of the spikes in the mature particles. however, we note that the bands of vp27 and vp25 on sds-page gels display intensities similar to that of vp34, 10 suggesting that trypsin cleavage might lead rather to conformational flexibility and that the products of proteolysis, or a significant percentage of them, could remain noncovalently attached to the capsid shell. limitations in material have precluded careful quantitative analysis to resolve this question. however, we note that the trypsintreated particles in fig. 2b have noticably less surface density than the particles in fig. 2a . if the cleaved proteins remain noncovalently associated with the capsid shell, then one would still expect to see surface density in the images of individual particles, even if the polypeptides do not conform to icosahedral symmetry. consequently, our working hypothesis is that trypsin cleavage leads to loss of 60 peripentonal spikes, which confers infectivity, possibly by exposing additional receptor binding sites on the capsid surface. the structures of immature hastv-8 and hev-lp are remarkably similar the size, shape, and architecture of immature hastv-8 are very similar to those reported for a t = 3 hev-lp 14 (fig. 3c) . in the case of hev-lp, a t = 1 x-ray crystal structure allowed detailed modeling of the protein subunits, which led to the proposal that the capsid is assembled from dimers around the 5f axes and across the icosahedral 2f axes. the hev capsid protein is divided into three domains, defined as the shell (s-domains), intermediate (m-domains), and protruding densities (p-domains). dong et al. identified the hev p-domain as structurally similar to their crystal structure of the astrovirus dimer (construct p2 412-646 ), which represents most of vp25 by dali alignment, and from which they proposed a phylogenetic relationship. 12 they also used the hev t = 1 x-ray structure to create a homology model of the astrovirus core sequences (residues 82-406) and docked their crystal structure of residues 430-645 in place of the hev p-domain. our experimental results support the proposed relationship between hastv and hev. both in silico 12 was docked into the 3d cryoem density map of hastv-1 (b). the close-up view at the right shows a 20-å-thick central slab of the density maps. with no scaling or translation, the maps align very well, except that the hastv spikes extend to a higher radius (arrow) than those of hev. density maps are in gray scale, and for clarity in the enlarged views, the interior density was set to zero at a radius of 120 å. the backbone chains are color coded by domain as defined in (a) for hev; s-domain (green), m-domain (blue), and p-domain (red). atomic models match exceedingly well with the hastv density maps and dock without any scaling: the hev-lp model into our vp70 immature structure (figs. 3c and 4a ) and the hastv model into our mature structure (figs. 3d and 4b) . the similarity reinforces the supposition that vp34 forms the capsid shell and that vp27/vp25 complexes comprise the spikes. the one noticeable difference between the structures is that the astrovirus spikes extend to a radius greater than that of hev-lp (fig. 4a, arrow) . in comparison, structures from the caliciviridae family, many of which are also t = 3 viruses that assemble as dimers, do not dock nearly as well, with differences in capsid features and orientation of their p-domains (data not shown). furthermore, although the conservation is low, the top score for viral proteins from a naïve national center for biotechnology information blast (basic local alignment search tool) search of the protein database for the hastv capsid protein identified similarity with the hev capsid protein (e = 2e − 3 ) (fig. 5) . the sequence in the hastv conserved domain, amino acids 72-256, has 21% identity with the capsid s-domain of hev. aaa45736) . a pair of proline residues in hastv were aligned with the hev hinge (red ellipse), and a long spacer inserted for hastv to account for the greater distance from the capsid observed for its spike. yellow highlights identical residues, and orange highlights similar residues. regions of the hastv vp90 sequence can also be aligned to the hev m-and p-domains with moderate identity (~13%) (fig. 5b) . when the hev capsid protein is used as the reference sequence, the search identifies the conserved domain from multiple astrovirus species, with the strongest scores for bat and bovine astroviruses (e b 1e − 8 ). it is also interesting to note that, while most astroviruses cause gastroenteritis in their host, duck astrovirus causes fatal hepatitis. 21 possible similarities in assembly and activation of hastv and hev although the astrovirus polyprotein is larger bỹ 120 residues, there may also be biological similarities between hastv and hev. for instance, in vitro assembly of expressed hev capsid protein requires deletion of the 52 c-terminal residues, which may be analogous to caspase cleavage of vp90 and removal of~125 amino acids to generate vp70 (fig. 1) . as there is no cell culture system currently capable of propagating hev, little is known about its infectivity. nevertheless, it is reasonable to speculate that a cleavage cascade similar to hastv may be required for both assembly and activation. furthermore, by analogy with the hev-lp model, we may be able to infer that the differences between the ab and cc dimers of immature astrovirus is relevant for assembly, in addition to activation. in the case of hev-lp, there is a measureable difference in the hinge angles between the ab and cc dimers, which may explain the similar differences observed in hastv. it was proposed for hev that the flatter angle of the cc dimers is required to make t = 3 rather than t = 1 particles and only happens in the presence of rna. 14, 22 the need for rna as a structural component may account for the inability, to date, to assemble stable astrovirus-like particles from expressed proteins. both systems may provide insights for the other. production and purification of hastv-8 the astrovirus strain yuc8 was isolated from a natural infection and adapted to grow in caco-2 cells. 4 to propagate the virus, we washed the cells twice with minimal essential medium without fetal bovine serum and inoculated them with yuc8, previously treated with 200 μg/ml of trypsin for 1 h, followed by a 1-h treatment with soybean trypsin inhibitor at the same concentration. infected cells were incubated at 37°c and harvested 1 day post-infection. the cells were lysed by three freeze-thaw cycles and extracted once with an equal volume of genetron 113 (trichlorotrifluoroethane). from this point, every step was carried out at 4°c, and solutions were maintained in an ice-water bath. the aqueous phase obtained after centrifugation at 5000g for 30 min was pelleted via ultracentrifugation for 4 h at 85,000g (beckman sw28 rotor). the pellet was resuspended in tne buffer [50 mm tris-hcl (ph 7.4), 0.1 m nacl, and 10 mm ethylenediaminetetraacetic acid] and layered onto a cesium chloride solution (1.36 g/ml). a density gradient was formed during ultracentrifugation for 16-18 h at 110,000g (beckman sw50.1 rotor), and the opalescent band corresponding to viral particles was collected and diluted in tne buffer. the final pellet was isolated by ultracentrifugation of this suspension for 2.5 h at 150,000g (beckman sw50.1 rotor), which was resuspended in tne buffer and stored at 4°c. infectivity assays and protein analysis of trypsin-treated virus were performed to ensure the identity of the purified particles. 11 production and purification of hastv-1 monolayers of a continuous line of colon carcinoma cells (caco-2) were infected with hastv serotype 1 (oxford strain) that was initially adapted to grow in a continuous line of monkey kidney epithelial cells (llcmk2). virus propagation required the addition of trypsin (10 μg/ml, type ix; sigma) to the culture medium (rpmi 1640). at 48 h post-infection, cells were lysed by three freeze-thaw cycles, and the lysates were partially purified by fluorocarbon extraction and differential centrifugation. the viral pellets were solubilized in tnmc buffer [50 mm tris-hcl (ph 8), 100 mm nacl, 10 mm mgcl 2 , and 10 mm cacl 2 ]. the suspensions were then layered on a cesium chloride density gradient (1.37 g/ml). following isopycnic centrifugation, the fraction containing peak enzyme immunoassay activity corresponded to a density of 1.37 g/ml. this fraction was diluted 10-fold with tnmc buffer, and viral particles were concentrated by ultracentrifugation for 2.5 h at 4°c (150,000g). samples were prepared for cryoem by standard methods. in brief, 3-μl aliquots from each viral preparation were applied to freshly glow-discharged, holey carbon grids, blotted to near dryness, and flash frozen in a slurry of liquid ethane. grids were maintained at liquid nitrogen temperatures in a gatan 626 cryo-holder and examined using an fei cm120 electron microscope (eindhoven) under low electron dose conditions as previously described. 23 images were recorded on kodak so-163 low-density film at nominal magnifications of 35,000 or 45,000×. micrographs with minimal astigmatism and drift were digitized on a zeiss microdensitometer (z/i imaging) at a 7-μm sampling interval corresponding to 2.0 or 1.56 å resolution at the specimen. particles were digitally extracted from the micrographs using the program x3d. 24 a linear background gradient was subtracted from the masked particle images, and the contrast was normalized. underfocus values were calculated using robem ‡ and ranged from − 1.45 to −1.85 μm. correlation methods (ppft) were used to derive particle orientations, and the 3d reconstructions were generated by fourier-bessel inversion. 25 initial models were determined from 10 cycles of the random model calculation. 26 additional rounds of image processing restricted the data search to the outer radii (r =150-230 å), but definition of the surface spikes did not improve. the data sets were randomly divided into two groups of particle images, and the two resulting maps were compared by fourier shell correlation analysis, from which the resolution for both hastv-1 and hastv-8 maps was estimated to bẽ 25 å using a correlation coefficient cutoff value of 0.5. the final 3d hastv-8 and hastv-1 maps were calculated from 332 (of 451) and 397 (of 583) particles, respectively, with selection based on their correlation coefficients. the handedness of both maps was set by matching the capsid features to those of the hev structure. the amino acid sequences of hastv-1 (genbank id: caa80132), hastv-8 (genbank id: caa91443), and human hev (genbank id: aaa45736) were analyzed using the programs blast § and clustalw2∥. blast searches were run using the default selections and with multiple database options [nonredundant protein sequences, protein data bank (pdb) sequences, and swiss protein sequences]. density maps calculated from pdb models pdb files containing full t = 3 icosahedrally related copies of the hev (pdb id: 3iyo 14 ) or hastv (kindly provided by dr. yizhi tao 12 ) asymmetric unit (or spiketruncated sequences) were generated using the viperdb utility generateoligomers. 27 density maps of the models were then calculated from the pdb files with the eman program 28 pdb2mrc using a step-size equivalent to the cryoem maps and truncated at 25 å resolution. the hev cryoem map (emd id: 5173) was low-pass filtered with the eman program proc3d. contour levels were set to encompass the expected volume of 180 copies of vp70 (hastv-8), or 180 copies of vp34 plus 60 copies of vp27, based on a protein density of 1.35 g/cm 3 . figures of the maps and docking of pdb coordinates were performed using chimera. 29 letter: viruses and gastroenteritis in infants letter: 28 nm particles in faeces in infantile gastroenteritis structural and biochemical analysis of human pathogenic astrovirus serine protease at 2.0 å resolution molecular analysis of a serotype 8 human astrovirus genome subgenomic rna sequence of human astrovirus supports classification of astroviridae as a new family of rna viruses the complete sequence of a human astrovirus identification of structural domains involved in astrovirus capsid biology caspases mediate processing of the capsid precursor and cell release of human astroviruses characterization of a human astrovirus serotype 2 structural protein (vp26) that contains an epitope involved in virus neutralization proteolytic processing of the astrovirus capsid proteolytic processing of a serotype 8 human astrovirus orf2 polyprotein crystal structure of the human astrovirus capsid spike ultrastructure of human astrovirus serotype 2 structure of hepatitis e virion-sized particle reveals an rnadependent viral assembly pathway activation of influenza a viruses by trypsin treatment enhancement of the infectivity of influenza a and b viruses by proteolytic cleavage of the hemagglutinin polypeptide cleavage of spike protein of sars coronavirus by protease factor xa is associated with viral infectivity proteolytic enhancement of rotavirus infectivity: molecular mechanisms proteolytic digestion of reovirus in the intestinal lumens of neonatal mice membrane fusion process of semliki forest virus ii: cleavage-dependent reorganization of the spike protein complex controls virus entry astrovirus-like particles associated with hepatitis in ducklings structure of the hepatitis e virus-like particle suggests mechanisms for virus assembly and receptor binding three-dimensional structure of the rotavirus haemagglutinin vp4 by cryo-electron microscopy and difference map analysis methods for reconstructing density maps of "single" particles from cryoelectron micrographs to subnanometer resolution a model-based approach for determining orientations of biological macromolecules imaged by cryoelectron microscopy ab initio random model method facilitates 3d reconstruction of icosahedral particles viperdb2: an enhanced and web api enabled relational database for structural virology eman: semiautomated software for high-resolution singleparticle reconstructions ucsf chimera-a visualization system for exploratory research and analysis we gratefully acknowledge dr. yizhi tao for providing her model of astrovirus vp25. we also thank dr. barbie ganser-pornillos for helpful discussions and yunuen acevedo for technical support. molecular graphics images were produced using the ucsf chimera package from the resource for biocomputing, visualization, and informatics at the university of california, san francisco [supported by national institutes of health (nih) p41 rr001081]. this work was supported by funding from the austrian science fund (j1348) to n.n., dgapa-unam (219910) and conacyt (79574) to e.m., nih r21 ai43513 to s.m.m., and nih r01 gm066087 to m.y. supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jmb.2012.06.029 key: cord-279716-kxfc4npg authors: blachere, francoise m.; lindsley, william g.; slaven, james e.; green, brett j.; anderson, stacey e.; chen, bean t.; beezhold, don h. title: bioaerosol sampling for the detection of aerosolized influenza virus date: 2007-10-22 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2007.00020.x sha: doc_id: 279716 cord_uid: kxfc4npg background influenza virus was used to characterize the efficacy of a cyclone‐based, two‐stage personal bioaerosol sampler for the collection and size fractionation of aerosolized viral particles. methods a collison single‐jet nebulizer was used to aerosolize the attenuated flumist® vaccine into a calm‐air settling chamber. viral particles were captured with bioaerosol samplers that utilize 2 microcentrifuge tubes to collect airborne particulates. the first tube (t1) collects particles greater than 1.8 μm in diameter, while the second tube (t2) collects particles between 1.0 and 1.8 μm, and the back‐up filter (f) collects submicron particles. following aerosolization, quantitative pcr was used to detect and quantify h1n1 and h3n2 influenza strains. results based on qpcr results, we demonstrate that aerosolized viral particles were efficiently collected and separated according to aerodynamic size using the two‐stage bioaerosol sampler. most viral particles were collected in t2 (1‐1.8 μm) and on the back‐up filter (< 1 μm) of the bioaerosol sampler. furthermore, we found that the detection of viral particles with the two‐stage sampler was directly proportional to the collection time. consequently, viral particle counts were significantly greater at 40 minutes in comparison to 5, 10 and 20 minute aerosol collection points. conclusions due to a lack of empirical data, aerosol transmission of influenza is often questioned. using flumist®, we demonstrated that a newly developed bioaerosol sampler is able to recover and size fractionate aerosolized viral particles. this sampler should be an important tool for studying viral transmission in clinical settings and may significantly contribute towards understanding the modes of influenza virus transmission. influenza infections are a public health concern accounting for more than 30 000 deaths and 100 000 hospitalizations annually. 1 the primary populations at risk for infection include young children, elderly adults, and immunocompromised subjects. during influenza outbreaks or pandemics, healthcare workers are at an elevated risk for acquiring influenza infection due to the prolonged periods of exposure to influenza viruses within healthcare settings. the mechanisms of influenza virus dissemination and transmission are poorly understood. experimental studies have shown that influenza viruses can be transmitted via contact with respiratory secretions, large droplets, and aerosolized small particles ⁄ droplet nuclei. 2 mucous membrane contact of large droplets expelled from the respiratory tract are thought to be the predominant mode by which influenza infection is transmitted, but small particle aerosols and droplet nuclei are also of concern due to the potential for prolonged airborne suspension. 3, 4 given the epidemic potential and public health concern of newly emerging diseases such as avian influenza (h5n1) and severe acute respiratory syndrome (sars), 5, 6 it is important to develop methods to study aerosolized viral particles. such measures would not only enable improved monitoring and detection of viruses, but more importantly, would help prevent widespread transmission. to date, several bioaerosol samplers are available that use impaction or impinger sampling methodologies for viral aerosol detection. 7, 8 however, limitations exist within each of these methods such as poor collection efficiency, limited sampling time, and inability to separate particles based on size. further complicating the study of viral aerosols is that in many environments where it would be desirable to sample for viral aerosols (e.g., hospitals, airports), the level of other biologically relevant particles such as bacteria, fungi, and pollens can also be very high. based on a one-stage, cyclone-based bioaerosol sampler developed at the national institute for occupational safety and health (niosh), 9 a two-stage, cyclone-based bioaerosol sampler was recently fabricated. 10 the two-stage bioaerosol sampler is a lightweight device that can be used either as an area sampler (e.g., in a hospital room) or as a personal breathing zone air-sampling device worn on the lapel of the subject (e.g., a healthcare worker). this bioaerosol sampler contains two microcentrifuge tubes and a back-up filter, which separate airborne particulates based on their aerodynamic diameter. 10 the first tube (t1) of the sampler collects particles that are greater than approximately 1.8 lm in diameter, while the second tube (t2) collects particles from 1.0 to 1.8 lm in diameter and the back-up filter (f) collects submicron particulates. because the sample is deposited directly in microcentrifuge tubes, the sampler facilitates the direct processing of samples for downstream diagnostic applications such as quantitative polymerase chain reaction (qpcr) and enzyme-linked immunosorbent assay. the bioaerosol sampler potentially eliminates several limitations associated with other air sampling devices such as sample loss, contamination, and degradation. in this proof of concept study, we characterized the utility of the two-stage bioaerosol sampler for fractionating viral particles and separating them from other particulates. the influenza virus vaccine flumist ò 2005-2006 formula was purchased from medimmune vaccine, inc. (gaithersburg, md, usa). flumist ò is a live, trivalent vaccine, composed of the a ⁄ new caledonia ⁄ 20 ⁄ 99 (h1n1), a ⁄ california ⁄ 7 ⁄ 2004 (h3n2), and b ⁄ jiangsu ⁄ 10 ⁄ 2003 (b ⁄ shanghai ⁄ 361 ⁄ 2002-like) strains. these strains are genetically altered to attenuated, cold-adapted, and temperature-sensitive phenotypes, which limits viral replication to the nasal pharynx. each 0.5 ml dose has been formulated to contain approximately 10 7 tcid 50 (10 6.5 -10 7.5 median tissue culture infectious dose) per viral strain. viral aerosols (flumist ò ) were collected using 4 two-stage bioaerosol samplers and two vertical reference samplers placed at the bottom of a calm air aerosol settling chamber, as described previously. 11 the bioaerosol samplers were connected to personal air sampling pumps (model 224-pcxr4; skc, eighty four, pa, for aerosolization experiments, one dose (0.5 ml) of flu-mist ò was diluted with 49.5 ml of saline (0.9% nacl). one milliliter of this solution was initially drawn for assay, and the remainder placed in a collison single-jet nebulizer (bgi incorporated, waltham, ma, usa). the solution was aerosolized at 138 kpa (20 lbs ⁄ in 2 ) air pressure, passed through a diffusion drier (model 3062; tsi incorporated, shoreview, mn, usa), and mixed with 26 l ⁄ minute dry filtered air. the dry aerosol then flowed into the top of the calm air chamber. to avoid possible degradation of the virus by ozone, an unavoidable by-product, the bipolar ionizer employed in earlier studies 10 was not used. instead, all conducting lines and the settling chamber itself were metal and grounded. previous tests with the calm air chamber indicated that these precautions were sufficient to avoid electrostatic aerosol deposition. 11 the concentration and size distribution of the diluted aerosol was recorded using an aerodynamic particle sizer (aps model 3321; tsi), which drew air from a vertical probe placed at the same height as the bioaerosol sampler inlets. at the start of each experiment, the nebulizer was operated for 10 minutes to allow the aerosol concentration to reach equilibrium. after 10 minutes, the sampling pumps and vacuum source were activated simultaneously for all samplers. for aerosol collection studies, sampler pumps were switched off after 5, 10, 20, and 40 minutes, while reference samplers were switched off after 20 and 40 minutes. after aerosol collection, the exterior of each sampler was disinfected (conflikt; decon labs, king of prussia, pa, usa) and the collection tubes and filters removed for analysis. a 1 ml aliquot of the solution remaining in the nebulizer was also removed for analysis. the particle size-segregation characteristics of the twostage sampler were reported previously for a range of particle sizes. 10 using this information, the particle size and number data from the aps, an estimate was made of the relative amounts of viral material that should have collected in the first and second tubes and on the back-up filter. this estimate was compared to the actual proportions of viral material found in each stage of the sampler as measured by qpcr (below) to indicate if the two-stage sampler was separating the aerosol particles based on size as expected. viral rna was extracted using the magmax tm viral rna isolation kit (ambion, austin, tx, usa). briefly, the supplied lysis ⁄ binding solution supplemented with carrier rna was used in the extraction of viral rna from either the neat flumist ò (qpcr standards) or experimental samples (t1 and t2). the back-up filters (f) from the bioaerosol samplers were aseptically removed, immersed in 1 ml of the supplemented lysis ⁄ binding solution and vortexed at moderate speed for 2 minutes. viral lysis, rna binding, magnetic capture, washing, and drying of the rna-bound beads were all carried out according to the manufacturer's instructions. cdna was generated by reverse transcription of the isolated viral rna using taqman tm reverse transcription reagents (applied biosystems, foster city, ca, usa). in brief, 40 ll of viral rna was added to the rt-pcr mixture containing 10 ll of 10x rt buffer, 22 ll of 25 mm mgcl 2 , 20 ll of 10 mm dntp mix, and 5 ll of 50 mm random hexamers. rnase inhibitor (2.0 ll, 20 u ⁄ ml) and 2.5 ll of multiscribe reverse transcriptase (50 u ⁄ ml) were added finally, for a final volume of 100 ll. samples were briefly centrifuged, placed in a thermo hybaid thermocycler (ashford, uk) and run under the following conditions: 25°c for 10 minutes, 48°c for 30 minutes, and 95°c for 5 minutes. a control without template was run for each experiment. as a pilot study to simulate the more biologically complex aerosols that one might encounter in field situations, flu-mist ò was mixed with fungal spores and then aerosolized and collected. aspergillus versicolor (atcc 44408, american type culture collection) was grown on malt extract agar for 16 days at 24.9°c and the spores isolated as previously described. 10 spore concentrations were determined by hemacytometer count. for co-aerosolization, one dose of flumist ò was diluted with 49.5 ml of 0.9% nacl containing 10 7 a. versicolor spores and 2 ml of rnasecure reagent (ambion) to inhibit viral rna degradation. the aerosol was generated using a collison single-jet nebulizer, passed through a diffusion drier and mixed with dry filtered air using the same experimental procedure as described above. the aerosol was collected with 4 two-stage samplers and two reference samplers. two of the two-stage samplers and one reference sampler collected the aerosol for 10 minutes, while the remaining samplers collected the aerosol for 40 minutes. genomic dna was extracted from a. versicolor-containing samples as described by griffin et al. 12 briefly, 400 ll of ap1 buffer supplemented with 4 ll rnase a (dneasy plant mini kit; qiagen, valencia, ca, usa) was added to 10 7 spores (qpcr standard) or experimental samples. filters were eluted in 1 ml of the supplemented ap1 buffer, vortexed for 2 minutes, and transferred to a microcentrifuge tube. approximately 250 mg of 0.1 mm zirconia ⁄ silica beads (biospec products, inc., bartlesville, ok, usa) was added to each sample. samples were bead-beaten at maximum speed in a mini-beadbeater-8 (biospec products, inc.) for 1 minute and chilled on ice for 2 minutes. the bead-beating ⁄ cooling steps were repeated twice. samples were centrifuged for 10 minutes at 11000 g and protein precipitation, washing, and drying of fungal dna was carried out according to the manufacturer's instructions. primer ⁄ probe design and optimization 13 real-time pcr primers and probes were specifically targeted against influenza surface glycoproteins and designed using primer express software (applied biosystems). sequence information for a. versicolor was obtained from the u.s. environmental protection agency (http://www.epa.gov/microbes/moldtech.htm). synthesis of all primers and probes was performed by applied biosystems. table 1 lists the sequence and dye labels for primers and probes used in qpcr detection. to determine the optimal primer concentrations for qpcr analysis, an optimization qpcr matrix was performed for each primer ⁄ probe combination (singleplex). optimal primer and probe concentrations used in qpcr detection ranged from 250 to 900 nm. for detection of influenza or a. versicolor in aerosol samples, 10 ll of the generated viral cdna or fungal genomic dna, respectively, was added to 25 ll applied biosystem's taqman tm universal pcr master mix. the appropriate concentration of primers and tamra-labeled probes was added, and brought to the final reaction volume of 50 ll with pcr-grade water. all reactions were run using the applied biosystem's 7500 real-time pcr system at the following conditions: 50°c for 2 minutes, 95°c for 10 minutes, 40 cycles at 95°c for 15 seconds, and 60°c for 1 minute. in order to quantify the relative amount of viral particles or fungal spores collected at each stage of the bioaerosol sampler, qpcr was performed in parallel using either serial 10-fold dilutions of cdna generated from a single dose of non-aerosolized flumist ò containing approximately 10 7 tcid 50 per influenza strain or genomic dna isolated from 10 7 spores. a negative control without template was included in all qpcr reactions. statistical analysis was conducted on the experimental setup as repeated-measures anova, with main effects of stage and time. stage is a three-level categorical variable with factors tube 1, tube 2, and back-up filter. to determine the effect of time, measurements were taken at 5, 10, 20, and 40 minutes. proc mixed in sas v9.1 (sas institute, cary, nc, usa) was used to analyze the data for significance and to calculate regression parameters. proc mixed was also used to insure similarity of experiments by testing variability between replicates. results were considered significant if p < 0.05. standard curves were generated for influenza strains h1n1 and h3n2 using serial 10-fold dilutions of cdna isolated from a single dose of flumist ò (10 7 tcid 50 ⁄ viral strain). for qpcr detection of a. versicolor, a standard curve was generated using genomic dna isolated from 10 7 spores and serially diluted 10-fold. the standard curves for h1n1, h3n2, and a. versicolor were linear over a 4-log range of 1.0 · 10 3 -1.0 · 10 6 particles. we were unable to generate a reproducible signal for the b strain virus using the specific primers that were designed. standard curves were used to extrapolate relative viral or spore numbers in unknown samples using the 10 7 titer reported by the manufacturer or hemacytometer counts, respectively. when flumist ò was aerosolized the virus-laden aerosol in the calm air chamber typically contained about 17 000 particles ⁄ cm 3 with a median aerodynamic diameter of 0.8 lm and a geometric standard deviation of 1.33. as part of methods development, the optimal back-up filter composition for virus collection was determined. four bioaerosol samplers were fitted with different types of filters -gelatin, glass fiber, ptfe, and polyvinyl chloride. following a 40 minute collection, several gelatin filters were found to be fractured. during rna extraction, the glass fiber filters released a high number of particulates that presumably interfered with qpcr detection. based on an initial experimental run, the ptfe filter was found to be optimal for the recovery of viral particles ( figure 1 ). all subsequent aerosol experiments were performed with the ptfe filters. to characterize the collection efficiency of the bioaerosol sampler, three identical experiments were performed. figure 2(a, b) illustrates the collective results for viral aerosols collected at 5, 10, 20, and 40 minutes. for both h1n1 and h3n2, collection of viral particles with the two-stage sampler was linear up to 40 minutes. viral particle counts were significantly greater at 40 minutes in comparison with the 5, 10, and 20 minute collection times (p < 0.05). furthermore, as predicted by the aerodynamic particle size data, the greatest proportions of the viral particles were detected in t2 and the back-up filter f, regardless of the collection time. for strain h1n1 at 40 minutes, 75% of the viral particles were detected in stage t2 and f, while only 25% of particles were detected in t1. likewise, with strain h3n2, 77% of the particles were detected in stages t2 and f with 23% of the particles detected in stage t1. these results were consistent throughout all experiments and demonstrate the ability of the bioaerosol sampler to separate particles based on aerodynamic size. while a similar distribution of h3n2 particles was recovered, for unknown reasons the absolute particle numbers were significantly lower. to examine the effectiveness of the bioaerosol sampler to separate a mix of aerosolized particles, we co-aerosolized flumist ò with a. versicolor spores (aerodynamic diameter 2.3 lm). following 40 minutes of aerosolization (figure 3 ), the bioaerosol sampler was able to separate these particles of differing size. both qpcr and spore counts confirmed that stage t1 retained 96% of the a. versicolor spores while stage t2, with 2% of the spores, was found to contain the greatest amount of viral particles (48%). an overall shift in the deposition of viral particles toward t1 was observed when co-aerosolized with a. versicolor spores (24%-42%). significantly fewer viral particles were detected on the back-up filter f (10%) in comparison with the back-up filter f using flumist ò alone (38%), suggesting some interaction or aggregation of the particles during aerosolization. the need for rapid and accurate methods for detecting airborne viruses has increased in recent years, particularly following the reported outbreaks of avian influenza (h5n1) and sars. various aerobiological monitoring studies have shown a high degree of variability with capturing, recovering, and detecting aerosolized viral particles in environmental and clinical samples. 7,14-16 using the niosh bioaerosol sampler, we were able to overcome some common viral particle sampling limitations and fractionate aerosolized influenza particles from an artificially generated aerosol. bioaerosols vary in size, concentration, composition, and settling times. 3,17 an aerodynamic particle sizer was used to monitor the concentration and size distribution of the flumist ò aerosol within the settling chamber. using the bioaerosol sampler, it could be demonstrated that capture, recovery, and subsequent detection for each sampler stage (t1, t2, f) were consistent with the expected values based on particle size. as anticipated, qpcr results confirmed that a significant number of the aerosolized viral particles were localized within stages t2 and f. these results are in agreement with the sampler cutoff size of 1.0 and <1 lm for stages t2 and f, respectively. the small number of aerosolized particles detected in stage t1 can be attributed to the collection efficiency of the bioaerosol sampler. the engineering design of the bioaerosol sampler is critical in the size-fractionation of bioaerosols, but sampling time must also be taken into consideration. qpcr results of aerosolized viral samples collected at 5, 10, 20, and 40 minutes confirms that a sampling time of 40 minutes yields the highest quantity of viral particles; however, this does not necessarily represent an ideal environmental sampling time as the quantities of aerosolized influenza virus expelled from patients have not yet been evaluated. in aerobiological studies using different viral strains, 18, 19 it was found that prolonged sampling periods resulted in decreased viral recovery. likewise, because influenza viruses are stress sensitive, the possibility exists that lengthier sampling times may result in the desiccation of viral-laden aerosol and consequently compromise stability. future studies will aim at addressing the effects of prolonged sampling time on viral recovery and stability. using the bioaerosol sampler, the collection of aerosols within disposable microcentrifuge tubes limits sample loss and contamination. moreover, samples are directly processed within the respective tubes and further analyzed by different diagnostic methods including immunoassays or molecular detection techniques such as qpcr. currently qpcr is the preferred methodology for the rapid and sensitive detection of viruses; 20 however, several limitations still exist. the sensitivity and specificity of qpcr detection is limited to the targeted template. in this study, primers and probes were designed to selectively amplify the ha and na genes from strains h1n1 and h3n2, respectively. interestingly, the results showed a considerable difference in the number of viral particles that were detected for strains h1n1 and h3n2. the flumist ò vaccine is formulated so as to contain an equivalent concentration of each viral strain using the median culture infectious dose (tcid 50 ) assay 21 but does not account for non-viable viral particles. qpcr can detect viral cdna from both viable and non-viable viral particles, and perhaps explain this discrepancy. as for the qpcr detection of strain b, erratic results were obtained. as qpcr detection was successful for both strains h1n1 and h3n2, this may not be due to the presence of inhibitors in the reaction but instead is more likely due to poor primer or probe design or the presence of secondary (10 7 ) and flumist ò were co-aerosolized together into the calm air chamber. samples were collected at 40 minutes and analyzed for influenza viruses using qpcr and a. versicolor using qpcr and hemacytometer counts. data for each stage is presented as the percentage of total number of particles collected in the three stages. spore counts were the average of eight replicate hemacytometer counts. values for the flumist ò with no fungal spores were taken from the previous experiment presented in table 2 and represent the combined average values for h1n1 and h3n2 strains. structures in the influenza b rna, resulting in poor reverse transcription and insufficient target template. environmental bioaerosols vary considerably and may consist of a number of different microorganisms including viruses, bacteria, and fungi. recent findings by lindsley et al 10 demonstrated that the two-stage bioaerosol sampler was effective at collecting and separating aerosolized fungal spores and fragments. results of the current study provide supporting evidence that the bioaerosol sampler is able to successfully recover and size-fractionate viral-laden aerosols. the culmination of these results suggests that the bioaerosol sampler would be an ideal air-sampling device for the aerobiological monitoring of various microorganisms within occupational environments. however, preliminary findings from a co-aerosolization study suggest fractionation of environmental samples may be more complex. namely, an overall shift in the viral particle deposition in the presence of a. versicolor spores was observed. the shift in viral particle deposition may be attributed to the attachment of viral particles to a. versicolor spores. aps data (not shown) further suggest that particles adhere to one another. given that the viral and spore-laden solution is pre-mixed prior to aerosolizing, it remains unclear as to whether the binding may occur before, during or after aerosolization within the calm-air settling chamber. future studies will further elaborate on the use of the bioaerosol sampler to capture and effectively size-fractionate co-aerosolized particles of different species. currently, there are conflicting views as to whether influenza viruses are spread by direct contact with secretions, large droplet transmission, or aerosol transmission. due to the lack of virtually any environmental data, aerosol transmission of influenza viruses is often overlooked as a possible mode of transmission. in this study, by aerosolizing flumist ò , we demonstrate the recovery of aerosolized viral particles using the bioaerosol sampler and detection of influenza by qpcr. whether aerosolized influenza particles are a significant contributor to influenza transmission in work environments and the community remains to be determined. future experiments will focus on studying the dissemination of viral-laden aerosols utilizing an artificial cough generator to simulate cough dispersal of influenza viruses within a room-sized aerosol chamber. these findings would significantly contribute toward understanding the transmission of aerosolized influenza viral particles. furthermore, we will test the bioaerosol sampler in a healthcare setting to monitor the prevalence of airborne influenza viral particles and to study the transmission of influenza via the inhalation of aerosolized viral particles. such studies will help to further elucidate the routes of transmission of influenza and would ultimately contribute to better patient management as well as improve infection control guidelines and decrease worker health risk. avian flu to human influenza transmission of influenza: implications for control in health care settings toward understanding the risk of secondary airborne infection: emission of respirable pathogens review of aerosol transmission of influenza a virus the origins of pandemic influenza-lessons from the 1918 virus the severe acute respiratory syndrome sampling methodologies and dosage assessment techniques for submicronmetre and ultrafine virus aerosol particles new personal sampler for viable airborne viruses: feasibility study development of a personal sampler for collecting fungal spores a two-stage cyclone using microcentrifuge tubes for personal aerosol sampling design and use of a settling chamber for sampler evaluation under calm-air conditions a rapid and efficient assay for extracting dna from fungi the value of a database in surveillance and vaccine selection long-term sampling of viable airborne viruses collection efficiencies of aerosol samplers for viruscontaining aerosols optimization of a sampling system for recovery and detection of airborne porcine reproductive and respiratory syndrome virus and swine influenza virus on-line monitoring and identification of bioaersosols air sampling for the detection of exotic newcastle disease virus in poultry houses spincon based air sampler foot and mouth disease virus test report. cambridge: lares corporation molecular diagnosis of influenza a simple method of estimating fifty percent end points this work was supported by internal funds from the health effects laboratory division. the findings and conclusions in this report have not been formally disseminated by the niosh and should not be construed to represent any agency determination or policy. key: cord-009825-6cargkwy authors: nazaroff, william w title: indoor bioaerosol dynamics date: 2014-12-27 journal: indoor air doi: 10.1111/ina.12174 sha: doc_id: 9825 cord_uid: 6cargkwy inhaling indoor air is the primary means by which humans are exposed to bioaerosols. considering bacteria, fungi, and viruses, this study reviews the dynamic processes that govern indoor concentrations and fates of biological particulate material. bioaerosol behavior is strongly coupled to particle size; this study emphasizes the range 0.1–10 μm in aerodynamic diameter. the principle of material balance allows concentrations to be determined from knowledge of important source and removal processes. sources reviewed here include outdoor air introduced by air exchange plus indoor emission from occupants, occupant activities, and moldy materials. important mechanisms that remove bioaerosols from indoor air include air exchange, deposition onto indoor surfaces, and active filtration. the review summarizes knowledge about size‐dependent particle deposition in different regions of the respiratory tract, techniques for measuring indoor bioaerosols, and evidence for diseases caused by airborne exposure to bioaerosols. future research challenges and opportunities are highlighted. in our daily lives, we humans move through a sea of microbial life that is seldom perceived except in the context of potential disease and decay (feazel et al., 2009) . it is by now well established that most humans spend most of their time indoors. furthermore, it is also well known that the indoor environments occupied by humans contain abundant material of microbial origin. however, we have gained until now only partial understanding of the complexity and richness of the indoor microbiome and its significance for human well-being. while acknowledging the limitations, in this article, i seek to summarize what is known or what can reasonably be inferred about an important subtopic of the microbiology of the built environment. the title 'indoor bioaerosol dynamics' is meant to imply that we seek a mechanistic, quantitative description of the processes and outcomes regarding the microbiology of the built environment, centering on what is, was, or will be airborne. there are many reasons to be interested in the microbiology of the built environment. the context for this study derives from three primary concerns. first, exposure to bioaerosol material can cause or can contribute to many important diseases. second, adverse respiratory symptoms correlate well with indicators of indoor dampness (fisk et al., 2007) . it is a reasonable hypothesis, although not yet well established, that the underlying cause of these associations is microbial. third, the microbiology of spaces we occupy may influence the human microbiome in ways that could confer health benefits. evidence for this last point is admittedly thin. there is growing evidence that people influence the microbiology of the spaces they inhabit (e.g., lax et al., 2014) . emerging evidence also indicates that some aspects of the human microbiome are important for health (grice and segre, 2012) . we further know that aspects of our personal microbiome can be influenced by individual factors such as diet and illness. so, it is not a large stretch to imagine that elements of our personal microbiome that matter for health might in fact be influenced by attributes of the spaces we inhabit, including their microbiology generally and their bioaerosol aspects specifically. hanski et al. (2012) reported evidence supporting an analogous suggestion: 'rapidly declining biodiversity may be a contributing factor to . . . the rapidly increasing prevalence of allergies and other chronic inflammatory diseases among urban populations. . .. ' following the introduction, the study is organized into two main sections. in 'framing the issues,' i define a framework for considering indoor bioaerosol dynamics. that section also defines the scope and limitations of this study. it presents some empirical evidence about indoor bioaerosols to help establish a context for studying dynamics. the section outlines the use of a material balance as a first-principles method for linking process to outcome for indoor bioaerosols. the size of particles is described as a primary variable of concern. regional deposition of bioaerosol particles in the respiratory tract is described as a process linking concentrations and exposure to dose. opportunities and limitations for progress in understanding indoor bioaerosol dynamics are strongly influenced by measurement technology, so some of the key measurement methods are summarized. evidence regarding infectious disease transmission by airborne routes is also assembled. the second primary section ('dynamic processes') summarizes available information on many important processes that affect the concentrations and fates of indoor bioaerosol particles. this section begins by reviewing the state of knowledge regarding building air exchange, an important removal mechanism for indoor bioaerosols and also a means by which outdoor bioaerosol particles are brought indoors. then, the article proceeds to discuss several additional processes that can affect indoor bioaerosol levels: deposition onto room surfaces, bioaerosol intrusion from outdoor air, indoor emission sources, and other factors, including bioaerosol control, airborne growth and decay, and indoor transport and mixing. in the conclusion, important challenges facing further studies are described along with several opportunities for near-term progress advancing knowledge about indoor bioaerosol dynamics. such progress is fundamental to efforts to better understand how the microbiome of indoor environments interacts with human health and well-being. framing the issues scope, limitations, and approach bioaerosols are usually defined as aerosols or particulate matter of microbial, plant or animal origin. . .. bioaerosols . . . may consist of pathogenic or nonpathogenic live or dead bacteria and fungi, viruses, high molecular weight . . . allergens, bacterial endotoxins, mycotoxins, peptidoglycans, b(1?3)-glucans, pollen, plant fibers, etc.douwes et al. (2003) 'bioaerosol' is a contraction of 'biological aerosol', and 'aerosol' refers to a suspension of particles in a gas. the exact boundary for what should be included or excluded in the term 'bioaerosol' is challenging to define. for the purposes of this study, the central focus will be defined more narrowly. all microbes are included: viruses, bacteria, and fungi. also included are microbe-associated chemicals such as endotoxins and mycotoxins. however, although they are part of bioaerosols, this article shall not explicitly address either pollen or pet-or pest-associated allergens such as cat dander or fecal pellets of dust-mites. this review also emphasizes the human-bioaerosol nexus in indoor environments. the indoor environments to be considered are those ordinarily and commonly occupied by humans: residences, offices, schools, and other settings that are occupied a high proportion of the time, or in which occupant density is high. not included are industrial environments that have high associated occupational exposure potential, such as those associated with food systems. much of the literature concerning microbiology in indoor environments focuses on dust as the sampled matrix (rintala et al., 2012) . favoring this approach is that dust reflects a longer term average condition in the environment, whereas bioaerosols are more dynamically variable and therefore more challenging to sample in a manner that is representative of time-averaged conditions. the primary counterargument is that the relationship between dust and human exposure is much less clear than is the relationship between bioaerosol particles and inhalation exposure. notwithstanding its prominence in the literature, the microbiological composition of indoor dust is not a primary focus of this study. the overall approach employed in this review is to adapt and apply concepts from indoor aerosol science (e.g., as described in nazaroff, 2004) . in particular, material balance is utilized as a core principle. particle size is a primary determinant of indoor bioaerosol behavior. we seek a mechanistic understanding because it provides a powerful basis for extrapolating from limited empirical evidence. we also seek to develop quantitative insight, because knowledge of the scale of processes and outcomes is essential as a basis for separating the important from the trivial. some prior scholarly reviews cover similar topics (burge, 1990; douwes et al., 2003; gregory, 1971; spendlove and fannin, 1983) . however, none of these earlier reviews is as strongly grounded in indoor aerosol science as the present article. to provide context for the process-oriented discussion to follow, it is instructive to consider some of the important empirical evidence concerning indoor bioaerosols. in the summaries to follow, i highlight several field-sampling studies whose results provide important clues about bioaerosol concentrations, associated particle-size distributions, and potential influencing factors. the largest published survey of indoor bioaerosol levels involved more than 12 000 fungi samples measured indoors and outdoors in~1700 buildings in the united states (shelton et al., 2002) . the analysis was culture based, so the results reflect number concentrations of viable airborne spores. little information was reported on the sampling protocol; however, it seems likely that the measurements were based on short-term sample collection. also, little information was reported about the buildings in which these measurements were taken. the cumulative probability distribution functions ( figure 1) show that the data are well fit by lognormal distributions. the geometric mean outdoor concentration (493 cfu/m 3 ) is about 6 9 higher than the geometric mean indoor concentration (79 cfu/ m 3 ). the indoor levels show considerably broader range of concentration than do the outdoor levels (gsd of 5.5 compared to 3.3). these data support an important general finding that 'in the majority of cases, indoor fungal aerosols are controlled by outdoor concentrations' (burge, 2002) . note that the ratio of geometric mean concentrations, 0.16, lies well within the range 0.04-0.46 predicted by riley et al. (2002) to apply as the fraction of coarse-mode (2.5-10 lm) particles that would be found in indoor air when outdoor air was the sole source. culture-based analyses measure only a small portion of bioaerosols. figure 2 presents data from a personal monitoring study of elementary school teachers in finland (toivola et al., 2004) . for each of the 81 subjects, air was sampled through filters throughout two 24-h periods using personal sampling pumps. particles were extracted from the filters and analyzed for both fungi and bacteria, using culture-based methods and also using microscope-assisted visual counting. a key point is that the total number concentrations of fungal spores and bacterial cells determined microscopically were a few orders of magnitude higher than the corresponding number concentrations of colony forming units, as determined by culture-based assessment. if (shelton et al., 2002) . in all, 9619 indoor and 2407 outdoor samples were collected during indoor air quality investigations although it is common for fungi concentrations to be higher in outdoor air than indoors, in occupied spaces the reverse is commonly true for bacteria (bartlett et al., 2004; fox et al., 2005; hospodsky et al., 2015) . figure 3 (chen and hildemann, 2009a) illustrates this point. the plotted results are geometric mean concentrations based on filter samples of 9-12 h duration collected inside and outside of ten occupied residences in california. summing across all particle sizes, the gm level of endotoxin (associated with gramnegative bacteria) was about 50% higher indoors than outside, whereas the gm level of (1-3)-b-d-glucans (a marker of fungi) in outdoor air was almost twice the corresponding indoor level. an important point is that human occupancy and activities are major factors influencing indoor microbiology. humans are important primary sources of certain bacteria and viruses. even for fungi, for which humans do not appear to be a major primary source, human activities play an important role, for example, in shedding particulate matter from our clothing or in suspending settled dust that can contain materials of fungal origin. figure 4 illustrates this point using samples collected in the same ten houses as discussed in connection with figure 3 . in this case, only indoor air samples are considered. the ten houses are divided into two equal-sized groups, sorted according to degree of occupancy (chen and hildemann, 2009b) . occupancy is used here as a surrogate measure of the intensity of human activity. across all measures displayed in the figure-that is, airborne concentrations of particulate matter, protein, endotoxin, and glucans -the geometric mean level in the high activity homes was considerably higher than in the low activity homes. measurement of airborne viruses in indoor environments has lagged behind measurement of bacteria and fungi. the recent development of quantitative polymerase chain reaction (qpcr) and other dnabased measurement technologies has facilitated studies that measure pathogenic viruses in indoor air. data from one study targeting the influenza a virus are presented in figure 5 . during the flu season, sizeresolved particle samples were collected on filters in three different environment types: a day care center, a health center, and (three) airplanes. in all, 16 samples were collected by means of sampling at a rate of 9 l/min for periods of 6-8 h. eight of these samples (50%) contained influenza a virus, with concentrations ranging from 5800 to 37 000 genomes per m 3 , and a substantial proportion of the detected virus was associated with fine particles (< 2.5 lm) that can remain airborne for extended periods and that can also penetrate and deposit deeply in the respiratory tract when inhaled. a fundamental principle that is usefully applied in quantitative, mechanistic studies of indoor environmental quality is material balance: stuff is conserved. on a time-averaged basis, the sum of the rates of supply of a bioaerosol component to the indoor air must balance the sum of the rates of removal. this quantitative balance provides a basis for connecting rates of processes to concentrations of bioaerosol components. figure 6 presents schematic representations of indoor environments that can be used to formulate material balance equations. consider a residential space ( figure 6a ). in this representation, three processes can add bioaerosol material to the indoor air: (i) natural ventilation through designed openings (at rate q n 9 c o ); (ii) infiltration through leaks in the building envelope (p 9 q l 9 c o ); and (iii) direct indoor emissions (e). (to the extent that tracked-in microbes contribute to the indoor bioaerosol load, this process would be included in the direct indoor emission term, e.) three processes can remove bioaerosol material: (i) ventilation airflow out of the building ([q n + q l ] 9 c); (ii) filtration in the recirculating airflow (g r 9 q r 9 c); and (iii) deposition onto room surfaces (bvc). some important observations should be made about equation (1). first, the symbol '~' is used instead of an equal sign because the expression is only approximately true. among the considerations that limit its strict applicability are that some of the parameters on the right-hand side are time dependent. if applied over short time intervals, one must also be concerned that accumulation, that is, the increase or decrease in the amount of bioaerosol in the indoor space, is not incorporated in equation (1). also, on a time-averaged basis, the equation is only approximately correct because it does not account for the possibility that time-varying parameters may correlate in such a way that the average of the product is not the same as the product of the averages. a second key point is that some of the processes exhibit strong dependence on particle size. this aspect is addressed in detail in subsequent sections of the study. a third feature of this equation is that it implicitly assumes that the indoor space can be represented as well mixed. that is not always the case. finally, the equation is specific to the particular schematic representation of the indoor environment depicted in figure 6a . this configuration accommodates some common conditions in residences, such as air exchange by a combination of natural ventilation schematic representation of indoor environments for quantitatively relating influencing processes to resulting concentrations of bioaerosol parameters: (a) residence; (b) commercial building. symbols (associated units) are as follows: c o -outdoor concentration of bioaerosol parameter (quantity/m 3 ); cindoor concentration of bioaerosol parameter (quantity/m 3 ); v -interior volume (m 3 ); q n -natural ventilation rate (m 3 /h); q l -ventilation rate associated with infiltration (m 3 /h); q r -recirculation airflow rate (m 3 /h); q m -mechanical ventilation supply flow rate (m 3 /h); p-penetration efficiency of bioaerosol parameter associated with infiltration (-); g r -single-pass filtration plus deposition efficiency in recirculating airflow (-); g m -single-pass filtration plus deposition efficiency in mechanical ventilation supply flow (-); b-rate coefficient for deposition on indoor surfaces (h à1 ); and e-emission rate from indoor sources (quantity/h) (yang et al., 2011) . in all, eight of sixteen samples collected (3 of 9 in a health center, 3 of 4 in a day care center, and 2 of 3 in an airplane) tested positive for influenza a. abbreviation: gcn = genome copy number (q n ) and infiltration (q l ), and the potential presence of a central air distribution system (with flow rate q r ) for heating and cooling. on the other hand, the schematic and the resulting equation would need to be modified to accommodate mechanical ventilation. for a commercial building space, the schematic representation in figure 6b is more common, with mechanical ventilation (q m ) and no natural ventilation. the appropriate material balance in this case is presented in equation (2). similar caveats as in equation (1) apply. particle size most indoor airborne microbial material is found on particles in the diameter range 0.1-10 lm. this range of diameters corresponds to six orders of magnitude in particle mass. in part because of the large range, size is a major determinant of indoor airborne particle behavior (nazaroff, 2004) . the behavior of larger particles is strongly influenced by their mass. gravitational settling and inertial impaction are important deposition mechanisms. the smaller particles in this size range follow airstreams more closely. transport mechanisms leading to the departure of smaller particles from air streamlines include brownian motion. generally, the efficiency of filtration, the likelihood of deposition somewhere in the respiratory tract, and the rate of deposition onto indoor surfaces are all considerably smaller for particles with diameters in the range 0.1-1 lm as compared with particles with diameters in the range 1-10 lm. whole microbial agents vary widely in size and mainly follow this pattern: viruses are much smaller than bacterial cells or endospores, which are smaller than fungal spores. for example, the influenza a virion is~0.1 lm in size (noda, 2012) . the cells of staphylococcus aureus, a common opportunistic pathogenic species, have diameters of~1 lm (http://textbookofbacteriology.net/staph.html). common indoor fungal spores have been measured to have aerodynamic diameters of~1.8 lm (cladosporium cladosporioides), 2.2 lm (aspergillus fumigatus), and~2.7 lm (penicillium melinii) (reponen et al., 1996) . spores of other species such as alternaria alternata and epicoccum nigrum are considerably larger (mcginnis, 2007) . bioaerosol particles can be larger or smaller than the size of whole microbial agents. for example, bacteria have been observed to occur in clusters or attached to other material such as fragments of human skin (davies and noble, 1962) . fungal fragments have also been measured in indoor air (g orny et al., 2002) . based on samples of outdoor air, yamamoto et al. (2014) found that the geometric mean size of fungal dna associated with particular taxa was considerably larger than individual cultured spores. theoretical and experimental evidence support an expectation that microbial airborne particles behave similarly to abiotic particles of the same aerodynamic size. consequently, powerful tools and theories from aerosol mechanics can be applied to study indoor bioaerosols. for bioaerosol particles, arguably the most important exposure pathway is inhalation followed by deposition in the respiratory tract. the probability of deposition varies with particle size, with lung morphology, and with breathing characteristics. figure 7 , which is based on semi-empirical modeling originally developed for radiological protection (yeh et al., 1996) , illustrates some of these features. in these model calculations, the respiratory tract is divided into three zones: the head region (nopl), the tracheobronchial or conducting airways (tb), and the pulmonary or gas-exchange region (p). the information presented in this figure reflects two dominant characteristics of the system. first, the three regions of the respiratory tract are exposed to bioaerosol particles sequentially. for the largest particles considered, the high deposition effifractional particle deposition in different regions of the respiratory tract in relation to particle size. the results are from the ncrp/itri semi-empirical model (yeh et al., 1996) . particle density is assumed to be 1 g/cm 3 . the results assume nose breathing, tidal volume of 0.77 l, breathing frequency of 13/ min, and a functional residual capacity of 3 l. in each frame, the deposition fraction is referenced to particle concentrations in the inhaled air. total deposition fraction in the respiratory tract for a given particle size would be obtained by summing results for the three regions ciency in the head protects the distal airways from exposure. second, two different mechanism classes control particle deposition. the behavior of the larger particles is dominated by their inertia. larger particles have a higher mass-to-drag ratio, and so the larger the particle, the more efficient the deposition. however, for submicron particles, inertial processes are relatively unimportant. for the smallest particles in this figure, brownian diffusion is the dominant transport mechanism. this is a slow process, important only in the smallest airways: deposition efficiency of 0.1-1 lm particles is small in the head region, yet substantial in the pulmonary region. similarly, the deposition efficiency increases with decreasing particle size when brownian diffusion dominates. worth noting is that the combination of these effects leads to two important modes of particle deposition in the pulmonary region. not only are the smallest particles deposited with reasonable efficiency, but there is also an important mode that peaks in efficiency at about 3 lm in diameter, a size that is important for bacterial and fungal bioaerosol particles. measure what is measurable, and make measurable what is not so.-galileo galilei many important aspects of indoor bioaerosols must be determined by experiment rather than from theory. experimental capabilities are intrinsically linked to technologies available for measurement. while many methods have been developed for mea-suring bioaerosol attributes, the availability of suitable methods remains an important constraint on research progress. table 1 provides a summary of many methods that have been used for bioaerosol sampling and analysis. (see also the reviews by henningson and ahlberg, 1994 and verreault et al., 2008) . the most widely applied methods have been culture based. these are subject to the limitations noted in connection with figure 2. culture-based methods offer the virtues of being relatively inexpensive, well developed, quantitative, and taxon specific. disadvantages include that only viable organisms are measured and only a subset of airborne organisms is culturable. a commonly employed method of direct impaction onto an agar substrate utilizes a short sampling period, which provides only a snapshot of the viable organism concentration at the time of sampling. relating these results to longer term exposure conditions is challenging because of the high degree of temporal variability, for example, associated with occupancy and occupant activities. effective bioaerosol sampling and analysis for studies of indoor environmental conditions must address two key challenges: specificity and temporality. no method is well suited to address both of these challenges well. the nature and significance of these issues varies according to the specific concern. in studies of airborne infection, specificity is essential. pathogenic strains may be closely related to nonpathogenic organisms of the same species. some of the chemical analytes that can be used in bioaerosol studies are of interest because of their direct potential for adverse health consequences. examples include endotoxin and (1?3)-b-d-glucans. other analytes, such as ergosterol or muramic acid, are not of direct health concern, but rather can be valuable as quantitative indicators of broad bioaerosol categories. dynamic processes can influence indoor bioaerosol concentrations by an order of magnitude or more over time scales that are as short as minutes. consequently, it is important to have measurement tools that permit sampling and analysis with high time resolution. there are no methods for bioaerosol sampling and analysis that are suitable for routine research application, that are highly specific, and that exhibit good time resolution. because of these limitations, process-oriented studies that are discussed in the next section have largely used abiotic particles as surrogates. the recent advent of real-time fluorescence-based instruments enhances capabilities for studying dynamic behavior of bioaerosols. these instruments offer excellent time and size resolution, but typically provide no biological specificity, even at the level of differentiating between bacteria and fungi. diseases associated with bioaerosol exposure . . .there is a growing body of data in support of the conclusion that air transmission within enclosed spaces plays an important role in the communication of many bacterial and viral diseases, especially those of the respiratory tract.robertson (1943) there are two noteworthy points to make about the quote from robertson: (i) it was published more than 70 years ago and (ii) the role of airborne routes in the transmission of disease remains controversial today for many infectious agents. table 2 provides a list of many diseases for which there is published evidence indicating that airborne exposure indoors makes a meaningful contribution to the occurrence or spread of disease. this section summarizes evidence concerning the dynamic processes that influence indoor bioaerosol levels. the emphasis is on those processes depicted schematically in figure 6 . building air exchange is the replacement of indoor air with outdoor air. air exchange is needed to limit the accumulation of carbon dioxide and other bioeffluents from human occupants. it is also used to limit the concentrations of pollutants emitted from inanimate indoor sources. when outdoor air is uncontaminated, then increasing the air exchange rate consistently improves indoor air quality. however, when climate conditions are not comfortable, then the air exchange rate is limited to avoid excessive energy use. in many circumstances, outdoor air is polluted to levels that exceed health-based standards. in these cases, by introducing pollutants from outdoor air, air exchange can be an important contributor to indoor pollution. building air exchange may be divided into three modes: infiltration refers to the uncontrolled leakage of air through the building envelope; natural ventilation occurs through windows and other designed openings; and fans induce mechanical ventilation. buildings generally leak, so infiltration is regularly a contributor to air exchange. although practices vary and are changing with time, it is common in the united states for air exchange in single-family dwellings to occur by a combination of natural ventilation plus infiltration. mechanical ventilation plus infiltration is common in commercial buildings. the air exchange rate (aer) is an important metric for characterizing air quality aspects of buildings. this measure is the volume-normalized flow rate of air from the building to outdoors. as depicted in figure 6 , the aer would be (q n +q l )/v for the residential schematic (i) and (q m +q l )/v for the commercial schematic (ii). figure 8 presents a summary of aer data from two large studies conducted in the united states. the measurements in residences ( figure 8a) show a median in the approximate range of 0.5-1 per hour. considering individual households, most of the data lie within a range that spans about an order of magnitude, from 0.2 to 3 per hour. the base study of~100 commercial buildings (figure 8b) shows a similar central tendency and a somewhat larger range, especially at the high end of the distribution. each of these data sets conforms reasonably well to a lognormal distribution. the aer sets a time scale for one of the main removal processes for impurities in indoor air. an aer of 0.5-1 per hour means that any airborne species removed primarily by air exchange will have a characteristic residence time of one to two hours in the building air. across the aerodynamic diameter range of 0.1-10 lm, particle deposition onto room surfaces is an important fate. in equations (1) and (2), the deposition loss rate is parameterized by a size-dependent rate constant, b. the importance of deposition as a removal mechanism for airborne bioaerosol particles can be explored by comparing b to the air exchange rate. figure 9 presents some data on size-dependent particle loss rates by means of deposition to room surfaces. these data show that, for particles in the size range 0.5-1 lm, the deposition loss rate coefficient is~0.2-0.3 per hour, a value that is comparable to the lower end of range of air exchange rates discussed in the previous subsection. under low air exchange conditions, deposition of these submicron particles is competitive with air exchange as a removal process, but in indoor for each house, one or two measurements of air exchange rate were taken over a few-day period using perfluorocarbon tracers. analysis is based on treating each house as a single unit; in cases where two aer measurements were taken, the results were averaged. yamamoto et al. (2010) have also published an analysis of these data. fig. 9 particle deposition loss rate coefficient (b) measured in a 14-m 3 room as a function of particle diameter. three of four experiments were conducted with four small fans operating to induce different degrees of air motion. as noted in the key, the mean measured speed in the room with the fans on ranged between 5.4 and 19.1 cm/s. the room was furnished with carpeting, chairs, table, bookcase, and curtains. source: thatcher et al. (2002) spaces with high air exchange rates, deposition of these smaller particles is less important than air exchange. for larger particles, in the range 3-10 lm in diameter, the deposition loss rate coefficient is much higher, in the range 2-10 per hour. for these larger particles, deposition is an important mechanism influencing the fate of bioaerosols even for buildings with relatively high air exchange rates. the data in figure 9 also illustrate that enhanced air movement increases the rate of particle deposition, at least over the range of conditions studied (up to air speeds of about 20 cm/s). the effect of such air movement is to more rapidly deliver particles to surfaces to which they deposit. these air speeds are too low to cause particles to be resuspended from surfaces onto which they had previously deposited. most of the particle loss reflected in figure 9 is attributable to gravitational settling onto upward facing surfaces. however, some deposition also occurs to vertical and even downward facing surfaces. figure 10 shows results from a study that measured particle deposition to chamber walls under stirred conditions. the deposition velocity, plotted on the vertical axis of the figure, multiplied by the airborne concentration yields the deposition flux. so, for example, if we consider as a typical indoor air value for viable fungal spores of 80 cfu/m 3 (figure 1 ) and assume they are associated with 6-lm particles for which the deposition velocity is 30 mm/h (= 0.03 m/h), then the resulting deposition flux to vertical walls would be 80 9 0.03 = 2.4 cfu/m 2 /h. this process would represent a small contribution to the total loss rate of fungi from indoor air, but could be an important step in the process of fungal colonization of walls. such colonization would also require sufficient quality and quantity of nutrients, appropriate surface ph, and sufficient water activity in the substrate (pasanen et al., 1992) . bioaerosol sources: outdoor air . . .the atmosphere is thronged with travellers: microbes using the wind, speaking teleologically, as a convenient transport from one place to another. travellers mostly performing quite short journeys.gregory (1971) it is worthwhile to differentiate among the sources that contribute to indoor bioaerosols. such differentiation can improve the basis for understanding concentrations, exposures, and influencing factors. it also serves as a basis for engineering interventions to alter exposures. among the major categories that can contribute to indoor bioaerosols is air exchange-induced supply from outdoor air. from equations (1) and (2), the rate of supply of bioaerosol material from outdoors is represented either by the term (q n + pq l )c o (for the residential schematic, figure 6a ) or by the term [(1àg m ) q m + pq l ]c o (for the commercial building schematic, figure 6b ). we have discussed the various air exchange flow rates and-to an extent-the outdoor bioaerosol concentration, c o . the new terms to address here are the fractional penetration of bioaerosols along with infiltration flow, p, and the efficiency of bioaerosol particle removal in the mechanical ventilation supply flow, g m . an important point is that both of these efficiency terms are expected to exhibit significant particle-size dependence. consequently, the relationship between outdoor bioaerosol concentrations and the source of indoor bioaerosols can vary with particle size. the main principles that govern p and g m are well understood. however, evaluation of proper values of these parameters for any particular building remains a challenge because uncontrolled and unknown details of construction and operation can influence the outcomes. consider particle penetration through leakage paths. as air flows into a building through a leak in the envelope, particles suspended in that airstream may contact a surface bounding the leak, adhere, and be lost from the airstream. the penetration factor, p, represents that portion of particles in the outside air that make it through the leaks to enter the indoor environment. large particles may deposit because of gravitational settling or inertial impaction. small particles may deposit because of brownian motion. figure 11 presents the results of model calculations showing how the fig. 10 deposition velocity (v d ) measured to the vertical wall of a 2-m 3 chamber as a function of particle diameter (d p ) (lai and nazaroff, 2005) . the deposition velocity is linked to the loss rate coefficient as follows: b w = v d s w /v, where b w is the contribution to the total loss rate coefficient attributable to deposition on the walls (h à1 ), s w is the wall area, and v is the room volume. the model equations are linear regressions to the log-transformed data, utilizing three measured points (v d1 ) and six measured points (v d2 ), respectively penetration factor varies with particle size for idealized crack geometry. different parameter values are assumed for the indoor-outdoor pressure drop (4 or 10 pa) and the height of the crack (0.1, 0.4, and 1.0 mm). an important message from this figure is that cracks must be quite fine for any meaningful attenuation of the airborne particles during infiltration. specifically, penetration is essentially complete across the full diameter range 0.1-10 lm for any crack whose minimum dimension exceeds~1 mm (given a 4 pa or higher pressure drop and assuming that the flow channel through the crack is no longer than 3 cm). the distribution of leak dimensions in any real building is not known. however, it seems likely that a normal case would feature most of the infiltrating air flowing through cracks larger than 1 mm in minimum dimension. hence, there is an expectation that p~1 for bioaerosol particles. in the case of a mechanically ventilated building, a second important parameter is the efficiency of a particle filter in the flow path connecting outdoor air to the air supply registers. figure 12 illustrates two important points about filter efficiency. first, it is highly variable with filter quality, ranging from low for filters with a merv 4 rating to high for merv 13 or merv 16 filters. second, filter efficiency can vary markedly with particle size. across the range that is pertinent for bioaerosols, the filtration efficiency tends to be higher for larger particles than for smaller particles. a mechanically ventilated building with a high mechanical ventilation to infiltration flow rate ratio (q m /q l ≫ 1) and a high-efficiency filter (1àg m ( 1) can provide a high degree of protection of the indoor environment from outdoor bioaerosols. in mechanical ventilation systems, some bioaerosol deposition can occur on surfaces other than the filters, including ducts and heat-exchanger fins. evidence suggests that such deposition is size dependent and much higher for the larger bioaerosol particles than for the smaller ones (sippola and nazaroff, 2003; waring and siegel, 2008) . this deposition process might contribute to meaningful rates of removal from airstreams in some circumstances. however, a more significant concern is the risk of fouling and the degradation of system hygiene. an important and challenging feature of indoor bioaerosol dynamics is characterizing indoor emission sources. from a systematic research perspective, a core advantage of source characterization is that it is likely to provide more generalizable information than would phenomenological studies of concentrations or other outcome variables. the information sought in source characterization would include these factors for any particular bioaerosol analyte: the quantity emitted per time, the size distribution of particles with which the emitted analyte is associated, and the important parameters that influence the emission rate. depending on the particular source, experiments to investigate emissions might suitably be conducted in a bench-scale laboratory apparatus, in a room scale chamber, or through controlled field monitoring. there are many potential indoor sources of bioaerosols. research that characterizes emissions is still in a relatively early stage of development, with limited specified minimum single-pass particle removal efficiency of fibrous filters used in ventilation systems as a function of particle size (three shading styles) and merv rating (horizontal axis). source: azimi and stephens (2013) quantitative information available for most sources. in the following paragraphs, several studies that have investigated indoor bioaerosol sources are highlighted. primary goals include indicating the breadth of source types that have been investigated and providing entry points into the literature for those interested in deeper study. among the merits of quantifying emissions from interior sources are these. first, such quantification allows for the assessment of the relative importance of indoor vs. outdoor sources as contributors to the indoor burden. examine the numerators of equations (1) and (2), and note that in each case, there is a term that is proportional to the outdoor concentration and a term (e) that reflects indoor emissions. if, for a particular bioaerosol component of concern, the indoor emissions term is small compared to the outdoor source, then we can safely focus our attention on the outdoor environment transported via air exchange as the dominant contributor to indoor levels. conversely, if the indoor emission source greatly exceeds the term associated with the outdoor level, then we can focus on indoor emissions and scale down our attention to the outdoor air as a significant contributor. second, if the indoor emissions are well-characterized, then (provided we have adequate additional information about the indoor environment) we can estimate the contribution of the indoor emissions to airborne concentrations. equations (1) and (2) illustrate the relationship, and the additional required information is that needed to quantify the appropriate denominator. human occupants. adult man carries 10 12 microbes associated with his epidermis and 10 14 microbes in his alimentary tract. . . . the 10 13 cells in his body are a distinct numerical minority of the total being that we call man. if we abandon anthropomorphism for the microbic view, we must admire the efficiency of these microbes in using man as a vehicle to further their own cause. -luckey (1972) in the context of better understanding and controlling airborne infection, bioaerosol emissions from humans have been a topic of concern since at least the 1940s. for example, duguid (1946) experimentally assessed the size distribution of particles and droplets emitted by sneezing, coughing, and talking. the likelihood that such particles would contain bacteria was estimated based on their prevalence in respiratory fluids. duguid and wallace (1948) experimentally investigated the 'bacterial contamination of air produced by liberation of dust from the skin and personal clothing during bodily movement'. using culture-based analysis methods, they found that dust particles carrying bacteria were liberated at a rate of about 1000 per minute from a 'person making slight movements' and that marching liberated culturable bacteria about 10 9 more intensely. bernard et al. (1965) observed that the shedding of airborne bacteria from humans was markedly elevated during a period 10-45 min after showering. the application of lanolin or alcohol to the skin reduced the effect, as did wearing a tightly woven fabric. hall et al. (1986) did not find this showering effect to occur. however, they did observe that 'men dispersed many more bacteria than women' and that the emissions rate could be considerably lowered through the application of skin lotion. they also noted that friction between skin and clothing appeared to be an important factor inducing the release. a second mechanism by which human activities may contribute to bioaerosol loading is through resuspension of biological particles that had previously settled on flooring or on other upward facing surfaces . using a mechanical stepping apparatus that mimicked walking combined with artificially seeded flooring materials, tian et al. (2014) found that the fractional resuspension of abiotic particles was 10 à5 and~10 à4 per step for particles with diameters 1-3 lm and 5-10 lm, respectively. goebes et al. (2011) documented that foot traffic contributes to measureable concentrations of airborne aspergillus; resuspension from flooring was demonstrated to be an important mechanism. a few recent studies have aimed to quantify sizeresolved biological particle emission rates associated with human occupants using modern analytical methods. qian et al. (2012) studied a university classroom with bioaerosol sampling using a cascade impactor followed by quantitative pcr applied with universal bacterial and fungal primers. by applying material balance to the indoor and outdoor data under room-occupied and unoccupied conditions, they inferred the per-occupant effective emission rates of bacteria and fungi. for example, the bacterial emission rates were determined to average 37 million genome copy numbers per hour per occupant. particle size conformed reasonably well to a lognormal distribution with a geometric mean aerodynamic diameter of 4.4 lm and a geometric standard deviation of 1.39. bhangar et al. (2014) used realtime particle detection to quantify size-resolved emissions of fluorescent biological aerosol particles also in a university classroom. they concluded that the modal size was in the range 3-4 lm and that the average emission rates were 1.6 million particles per person per hour during the main portion of lecture classes plus 0.8 million particles per person emitted during transitions between classes. advanced analytical methods and ongoing concerns about the spread of infectious respiratory diseases have motivated a renewed effort to study bioaerosol release from the nose and mouth. fennelly et al. (2004) measured the size distribution of coughgenerated particles containing culturable mycobacte-rium tuberculosis emitted from patients with pulmonary tuberculosis. stelzer-braid et al. (2009) collected respiratory emissions from 50 subjects while breathing, talking, and coughing. they detected one or more of nine respiratory viruses in 21 of 33 subjects who had symptoms of upper respiratory tract infections and only in four among 17 asymptomatic subjects. gralton et al. (2013) conducted an analogous study that focused on breathing and coughing, included children among the subjects, and investigated the size distribution of the emitted particles and droplets. they concluded that 'individuals with symptomatic respiratory viral infections produce both large and small particles carrying viral rna on coughing and breathing'. overall, human occupants are important contributors to the bioaerosol burden of indoor environments. they shed bacteria along with their skin; they emit viruses from their respiratory tract; and they resuspend particulate material that contains biological agents from floors and other surfaces that they contact. moldy materials. a second potentially important emission source category for indoor bioaerosols is moldy building materials. dampness and mold is common in buildings. for example, spengler et al. (1994) reported that half of surveyed households in 24 us and canadian cities had a dampness-related condition (water damage, water in basement, and/or mold or mildew). several laboratory studies have investigated emissions of bioaerosols from moldy materials. for example, g orny et al. (2001) characterized the release of fungal spores-aspergillus versicolor, cladosporium cladosporioides, and penicillium melinii-from ceiling tiles in relation to the air speed above the surface and the vibration of the contaminated material. seo et al. (2008) investigated the release of (1?3)-b-d-glucan from moldy ceiling tiles and gypsum board. in many buildings, moisture intrusion or condensation occurs in wall cavities or in other hidden spaces that may be coupled by airflow pathways to the occupied building interior. muise et al. (2010) demonstrated experimentally that mold spores could penetrate effectively through wall service outlets. that finding is consistent with expectations (see figure 11 ) as most fungal spores are smaller than 10 lm in diameter and as cracks and gaps between a wall cavity and the indoor space would commonly be larger than 1 mm in minimum dimension. housekeeping and hygiene. a third bioaerosol source category that is potentially important indoors is related to housekeeping and hygiene. several examples are briefly mentioned here. davies and noble (1962) demonstrated that bedmaking increased the airborne concentrations of skin scales and bacteria. wouters et al. (2000) reported increased levels of microbial markers in house dust in homes in which household organic waste was separately stored indoors. floor dust or settled dust can be richly microbial (rintala et al., 2012) , and this can serve as a bioaerosol source through resuspension . in a laboratory study, substantial bacteria and mold emission rates were observed from the operation of household vacuum cleaners (veillette et al., 2013) . this finding is not a surprising: vacuum cleaner use was already demonstrated to be a potent short-term generator of airborne particulate matter, both in the exhaust air (because of incomplete filtration) and through mechanical agitation of the floor (corsi et al., 2008; knibbs et al., 2012) . the net effect of vacuuming on indoor bioaerosol levels is not clear, however, as presumably vacuum cleaner use would also reduce the floor load of microbes available for later resuspension. indoor surfaces that are frequently moist can harbor microbial growth; examples related to household hygiene include shower curtains (kelley et al., 2004) and sink drains . bollin et al. (1985) showed that showerheads and hot-water faucet use could produce aerosols containing legionella pneumophila. thomson et al. (2013) found non-tuberculous mycobacteria (ntm) in shower aerosols in homes of patients with pulmonary disease caused by ntm, a finding broadly consistent with evidence from feazel et al. (2009) of enhanced ntm prevalence in showerhead biofilms. toilet flushing can produce aerosol droplets, and as fecal material is rich in microbes, the toilet is a potentially important source of indoor bioaerosols, especially in connection with diarrheal diseases (johnson et al., 2013) . bioaerosol exposure control. let us acknowledge that the goal should not be to make indoor environments sterile. at the same time, elevated levels of airborne pathogens are to be avoided, as are excessive levels of many bioaerosol attributes. and in certain circumstances, we should be particularly concerned about protecting vulnerable people from even ordinary bioaerosol exposure, such as individuals who are immunocompromised. how can bioaerosol control be achieved? conceptually, in the context of the material balance described earlier, there are two broad options: (i) reduce sources and/or (ii) increase removal rates. equations (1) and (2) provide a basis for quantitatively estimating the benefit of a control measure. among the options for source control are to keep indoor environments dry, to maintain good hygienic conditions in ventilation systems, to apply effective filtration on mechanical supply ventilation, and to use masks in the event of respiratory illness. regarding removal processes, the primary control alternatives are three, which appear in the denominator of equations (1) and (2): (i) increase the outdoor air ventilation rate (typically either q n or q m ); (ii) use recirculating air filtration (i.e., introduce or enhance g r q r , where g r is the filtration efficiency, as illustrated in figure 12 , and q r is the recirculating flow rate through the filter; or (iii) enhance the rate of deposition or degradation of the bioaerosol attribute (thereby increasing b). miller-leiden et al. (1996) explored the effectiveness of in-room recirculating filtration for controlling the transmission of tuberculosis. cheng et al. (1998) have evaluated the use of portable air cleaners for reducing indoor concentrations of fungal spores. ultraviolet germicidal irradiation can be applied to reduce the infectivity of air without actively removing the bioaerosol particles (reed, 2010) . the control measures described in the preceding paragraphs all aim to reduce the airborne concentration of the bioaerosol agent, denoted c in equations (1) and (2). a complementary approach is to provide susceptible individuals with personal protective equipment, which-if performed well-can reduce the inhalation intake by an order of magnitude or more for a given airborne concentration. nicas (1995) presents an illustrative example for the case of respiratory protection of healthcare workers against mycobacterium tuberculosis bacilli. airborne growth and decay. the embodiment of the material balance principle in equations (1) and (2) does not account for microbial reproduction or for any other bioaerosol growth process during the period of suspension in the indoor air. there is some evidence supporting the possibility of airborne microbial life in the atmosphere, as reviewed by womack et al. (2010) . however, indoors, where the airborne residence time is limited to about a few hours or less, such processes have not been demonstrated and seem unlikely to be important. infectious agent viability may decay at a significant rate when airborne (weber and stilianakis, 2008) . incorporating the effects of such processes into model equations can be achieved through the decay parameter, b, in equations (1) and (2). transport and mixing. throughout this study, it has been assumed that an indoor environment can be represented as a well-mixed space. at the level of an individual room, the size of a typical bedroom or private office, that description is often but not always reasonable. an entire residence, or a large building, might be appropriately represented as a network of well-mixed rooms or zones, interconnected by airflow paths (feustel, 1999) . for some situations, the well-mixed conceptualization is inappropriate. for example, while much of mechanical ventilation practice uses air diffusers designed to promote rapid mixing, other concepts aim to deliberately exploit incomplete mixing as a basis to improve efficiency. such methods include displacement ventilation (novoselac and srebric, 2002) and personalized ventilation (melikov, 2004) . understanding how sources relate to concentrations in the breathing zone of occupants in cases like these cannot be accurately accomplished using a well-mixed analysis framework; instead, more sophisticated methods are required, such as approaches based on computational fluid dynamics (chen, 2009 ). experimentally, investigations in such conditions require methods that can accommodate spatially varying contaminant concentrations (bjørn and nielsen, 2002; brohus and nielsen, 1996) . although study of the normal human lung microbiome is still in its early stages, the bulk of published evidence demonstrates that phylogenetically diverse microbial communities in the lungs of healthy humans can be detected using high throughput sequencing. -beck et al. (2012) in a recent review, grice and segre (2012) articulate important ways in which microorganisms modulate human health: 'the human microbiome is a source of genetic diversity, a modifier of disease, an essential component of immunity, and a functional entity that influences metabolism and modulates drug interactions'. they then summarize what has recently been learned about the microbiology of the human gastrointestinal tract, the oral cavity, the reproductive tract, and the skin. however, they do not comment on the microbiology of the respiratory tract. beck et al. (2012) , in their review, note that, 'although the lungs were classically believed to be sterile, recently published investigations have identified microbial communities in the lungs of healthy humans'. lax et al. (2014) document that the microbes found in a home are distinctively related to the people who live in that home and that 'after a house move, the microbial community in the new house rapidly converged on the microbial community of the occupants' former house, suggesting rapid colonization by the family's microbiota'. what is not yet clear, but seems plausible, is whether aspects of the human microbiome are strongly influenced by microbiological and other conditions in inhabited indoor spaces. indoor bioaerosol behavior might play an important role in these stories. almost certainly, the most important exposures of the human lung to environmental microorganisms occur via inhalation of bioaerosols. furthermore, most of the air that is inhaled by humans is indoor air. bioaerosols also are important vectors transporting microorganisms from outdoors to indoors and from one indoor surface to another. it is feasible that airborne transport to and deposition on the human envelope influences skin microbiota . relative to the complexity and importance of the subject of indoor bioaerosol dynamics, our understanding is not yet mature. one might anticipate fundamental paradigm shifts to occur as our knowledge grows. our ability to ask and answer incisive questions should improve. although the gap between what we know and what we would like to know is quite large, our current knowledge is substantial. for example, in considering the dynamic behavior of airborne biological particles in buildings, indoor aerosol science provides a good starting point. mechanistically, bioaerosol particles behave like their abiotic counterparts. particle size is a primordial determinant of behavior, and bioaerosol particles are mainly found in the aerodynamic diameter range 0.1-10 lm. aerosol science has developed powerful tools and theories regarding emission processes, airborne behavior, and fate. much of the understanding developed from aerosol science can be applied to indoor bioaerosol dynamics. as we proceed in studying the microbiology of the indoor environment, we should maintain a central focus on people. human occupants are a major source of indoor bacteria. our activities influence the emissions and fate of other bioaerosols as well. the outcomes of primary concern are centered on human health and welfare. technological advances enable the acquisition and analysis of microbial data of phenomenal richness. as we conduct research with the new tools, it is important that we not lose sight of the knowledge gained by prior generations of scholars. public health engineering studies conducted in the 1940s through 1970s, for example, on the theme of infection control in healthcare settings, contain particularly important insights that remain relevant to our current research agendas. the diversity and complexity of the system will continue to pose great challenges for studies of indoor bioaerosol dynamics, especially in efforts to link microbiological abundance to exposure and to health outcomes. the measurement limitations continue to be daunting. dust is an attractive sampling medium of questionable exposure relevance. culture-based analysis methods have limited scope. microscopic methods, dna-based analysis, and methods using chemical markers are best suited for making time-integrated measurements with sampling periods of hours. these methods are not well suited for studying dynamic processes. the fluorescent particle-sampling and analysis methods have the advantage of offering excellent time and particle-size resolution. however, these methods lack specificity. in recent years, we have seen benefits from efforts to fuse concepts and approaches from the indoor environmental sciences with the rapidly developing techniques and evolving knowledge of microbial ecology. we can anticipate continuing opportunities from cooperation between scholars from these domains. as we gain empirical knowledge, it will be important to seek generalizable understanding from the specifics of particular investigations. we will never measure everything! research that focuses on processes and that is framed in the context of well-established mechanistic knowledge can be a valuable way to proceed. in this review, the principle of material balance has served to structure a relationship between bioaerosol processes and indoor concentrations, an important intermediate outcome. further studies can be fruitfully pursued to better understand each of the input parameters that appear in the material balance equations. research could also be undertaken to test the accuracy of and to refine as necessary the model equations themselves. benefits would especially be anticipated from studies to better characterize and quantify indoor bioaerosol emission sources and the influencing factors. this particular process has especially large influence on outcomes, it is difficult to characterize without direct experimental measurement, and it is subject to enormous variability. the ultimate goal for improving knowledge about indoor bioaerosol dynamics is to contribute to a stronger knowledge base for the design, construction, operation, and maintenance of healthful buildings. this review has focused on the natural science and engineering aspects of the theme. this review has not addressed the complementary and comparably complex theme of how human health is influenced by bioaerosol exposure. because we are at an early stage of understanding both themes and because of their complementarity, progress toward the ultimate goal would benefit from the development of synergistic interactions between the respective research communities. that is a challenging proposition for several reasons, including largely independent educational tracks and funding agencies that-at least in the united states -do not embrace the nexus between the built environment and health. the first steps to overcoming any challenge are recognizing its importance and making a commitment to the attempt. perhaps the recent efforts to improve knowledge about the indoor microbiome and about the relationship between the human microbiome and health are establishing fertile ground for future synergistic efforts to better understand the relationship between the indoor microbiome and health. singapore-berkeley building efficiency and sustainability in the tropics (sinberbest) program. bears has been established by the university of california, berkeley, as a center for intellectual excellence in research and education in singapore. thanks to naomichi yamamoto, jordan peccia, and two anonymous reviewers for constructive comments and suggestions that contributed to improvements in the final article. the diversity and distribution of fungi on residential surfaces hvac filtration for controlling infectious airborne disease transmission in indoor environments: predicting risk reductions and operational costs evaluation and determinants of airborne bacterial 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metagenomic framework for the study of airborne microbial communities evidence of airborne transmission of the severe acute respiratory syndrome virus partial support for this work was provided by a grant from the alfred p. sloan foundation in support of the berkeley indoor microbial ecology research consortium (bimerc). additional support was provided by the republic of singapore's national research foundation through a grant to the berkeley education alliance for research in singapore (bears) for the key: cord-285077-okwck5sv authors: sayahi, tofigh; nielson, christopher; yu, yuan; neuberger, kaden; seipp, michael; firpo, matthew a.; kelly, kerry; park, albert h. title: airborne aerosolized mouse cytomegalovirus from common otolaryngology procedures: implications for covid-19 infection date: 2020-09-15 journal: otolaryngol head neck surg doi: 10.1177/0194599820957966 sha: doc_id: 285077 cord_uid: okwck5sv objectives: to determine whether common otolaryngology procedures generate viable aerosolized virus through a murine cytomegalovirus (mcmv) model for infection. study design: mcmv model of infection. setting: university of utah laboratory. methods: three-day-old balb/c mice were inoculated with mcmv or saline. five days later, each mouse underwent drilling, microdebrider, coblation, and electrocautery procedures. particle size distribution and pm(2.5) (particulate matter <2.5 µm) concentration were determined with a scanning mobility particle sizer and an aerosol particle sizer in the range of 15 nm to 32 µm. aerosolized samples from these procedures were collected with an aerosol devices biospot sampler for viral titer based on polymerase chain reaction and for viable virus through viral culture. results: as compared with the background aerosol concentrations, coblation and electrocautery showed statistically significant increases in airborne aerosols (tukey-adjusted p value <.040), while microdebrider and drilling at 30,000 rpm did not (.870 < tukey-adjusted p value < .930). we identified viral dna in samples from coblation and drilling procedures, although we did not identify viable viruses in aerosol samples from any of the 4 procedures. conclusion: coblation and electrocautery procedures generate >100-fold increases in aerosol concentrations over background; only coblation and drilling produce aerosolized viral dna. the high concentration of aerosols from coblation and electrocautery suggests the need for appropriate safeguards against particle exposure to health care workers. the presence of viral dna from drilling and coblation procedures warrants the need for appropriate protection against droplet and aerosol exposure. results. as compared with the background aerosol concentrations, coblation and electrocautery showed statistically significant increases in airborne aerosols (tukey-adjusted p value \.040), while microdebrider and drilling at 30,000 rpm did not (.870 \ tukey-adjusted p value \ .930). we identified viral dna in samples from coblation and drilling procedures, although we did not identify viable viruses in aerosol samples from any of the 4 procedures. conclusion. coblation and electrocautery procedures generate .100-fold increases in aerosol concentrations over background; only coblation and drilling produce aerosolized viral dna. the high concentration of aerosols from coblation and electrocautery suggests the need for appropriate safeguards against particle exposure to health care workers. the presence of viral dna from drilling and coblation procedures warrants the need for appropriate protection against droplet and aerosol exposure. 1 this infection has significantly affected society through quarantines, stay-at-home orders, 2 business closures, and travel prohibitions. in april 2020, the us unemployment rate jumped to 14.7%, the highest level since the great depression. 3 a key priority during this crisis has been the need to minimize disease transmission to health care personnel. as millions of people have been staying at home to minimize viral transmission, health care workers have been doing the opposite in going to hospitals and clinics. several reports indicate that the sars-cov-2 virus particles reside with extremely high concentrations in the oral cavity and nasopharynx and can be a significant source of transmission. [4] [5] [6] this characteristic property of the virus places health care professionals who examine and work in these areas at particular risk. otolaryngologists and ancillary staff are especially vulnerable to viral transmission directly through mucus, blood, and aerosolized particles when examining or operating in these areas. there have been anecdotal reports from china, italy, and iran that otolaryngologists are among the highest-risk group contracting the virus while performing upper airway procedures and examinations if not using appropriate personal protective equipment. [7] [8] [9] this dilemma puts otolaryngologists in a difficult situation when presented with patients with time-sensitive and emergent problems that require surgery. two studies evaluated several endonasal and otologic procedures. using fluorescein solution and digital image processing in a cadaveric head model and clinical setting, the authors noted that only high-speed drilling produced significant aerosol contamination. 10, 11 we proposed to build on these studies by measuring particle size and concentration and by trying to detect aerosolized viral dna and viable virus during common otolaryngology procedures, using a murine model for cytomegalovirus (cmv) infection. recombinant murine cmv (mcmv; strain k181 mc.55 [ie2-gfp1]) expressing green fluorescent protein (gfp) was used. virus purification was carried out by virapur as previously described. 12 animals inbred balb/c mice were used for these experiments. mice were housed and bred under specific pathogen-free conditions under controlled temperature and humidity at the central animal facility at the university of utah. the university of utah institutional animal care and use committee approved all procedures in accordance with the standards established by the us animal welfare act. mice were injected via an intracerebral route with recombinant mcmv (strain k181 mc.55 [ie2-gfp1]) expressing gfp at postnatal day 3 as previously described. 12 control animals (uninfected) received the same volume of normal saline. the injections were completed with a 5-ml hamilton syringe with a 30g needle. mice underwent the following procedures in order: drilling (at 30,000 rpm), microdebrider, coblation (setting at 7 for coblate), and electrocautery (with and without suction) at 8 days of age. animals were anesthetized with xylazine/ketamine. the hair over the cranium was shaved. a 1-cm incision was made to expose the skull, which was removed with an otologic drill and saline irrigant (acumed). the brain was then removed with a 3.4-mm tricut blade microdebrider (medtronics). the remaining soft tissue in the head and neck, chest, and abdomen was removed with a coblator (smith & nephew) and then with a monopolar bovie set (ethicon megadyne) at 15 w. each procedure was performed for 6 minutes 45 seconds. an additional 4 minutes 30 seconds elapsed before the next procedure to ensure that aerosolization dropped to background levels. this step was verified by real-time measurements of aerosol concentration. particle size distribution and concentration were measured with a scanning mobility particle sizer and an aerosol particle sizer (tsi smps 3081-tsi aps 3022; smps-aps) in the range of 15 nm to 20 mm. each smps-aps scan required 2 minutes 15 seconds, and 3 scans were performed for each procedure (total, 6 minutes 45 seconds). in addition, a grimm 1.109 aerosol spectrometer provided pm 2.5 (particulate matter \2.5 mm) mass concentration and size distribution in the range of 0.225 to 34 mm in 31 class sizes in 6 seconds. a tsi dusttrak ii 8530 provided additional pm 2.5 mass concentration measurements in 6 seconds. the measurements were collected in the breathing zone within 25 cm of the procedure. background measurements were collected 15 minutes prior to each procedure, during each procedure (6 minutes 45 seconds), and 15 minutes postprocedure, which allowed us to determine the aerosol decay time. we collected 1 liquid sample for each surgical procedure and each mouse for a total of 17 samples for each procedure with a liquid spot sampler (sll 110a; aerosol devices). the liquid contained 2% bovine serum albumin in 1% sterile phosphatebuffered saline (thermo fisher), and these samples were analyzed for mcmv. the spot employs a condensation growth tube and allows for .90% virus collection efficiency and improved virus viability as compared with traditional samplers, which have virus collection efficiencies of \10%. [13] [14] [15] detection of mcmv genome by qpcr the presence of viral dna was assessed in aerosol condensates and tissue culture by quantitative polymerase chain reaction (qpcr) with the cmv immediate-early response gene 1 (ie1) as the amplification target, as previously described. 16 dna was purified from samples with the quick-dna miniprep kit (zymo research). each sample was assayed in duplicate with the taqman gene expression master mix (life technologies), ie1 primers and ie1 prime time qpcr probe (integrated dna technologies), and the applied biosystems quantstudio 12k flex real time pcr system (life technologies). quantification of ie1 was achieved by comparing the crossing threshold of the unknown samples to that of an ie1 standard curve. the ie1 standard curve was created by extracting dna from a solution of stock mcmv in 2% bovine serum albumin/phosphate-buffered saline and quantified with qubit fluorometric quantification (thermo fisher). a copy number log dilution series standard curve was generated to calculate amplification efficiency relative to the theoretical maximum. the observed efficiency was applied to the lowest reproducible dilution to assign the limit of detection. to assess the presence of infectious cmv viral particles, cultured cells were inoculated with aerosol condensates and monitored for gfp expression by fluorescent microscopy. nih-3t3 cells (atcc) were seeded onto 96-well plates and grown overnight to 70% confluence. ten microliters of each condensate sample and positive control standards were applied in duplicate and incubated for 2 hours at 37°c and 5% co 2 . the standard consisted of 4 log dilutions per well (63,000, 6300, 630, and 63 pfu). after the 2-hour incubation, 70 ml of fresh media was added, and the plates were incubated for 24 hours before microscopic examination. imaging was performed on a nikon a1 confocal microscope with a gfp laser and brightfield imaging (cell imaging core, university of utah). the presence of infectious cmv viral particles was also monitored by qpcr. twenty-four-well plates were seeded with 5 3 10 5 nih-3t3 cells and grown overnight to achieve 70% confluence. all but 100 ml of media was removed from the 24-well plates, and 10 ml of sample was applied in duplicate wells. after 2-hour incubation at 37°c and 5% co 2 , 900 ml of fresh media was added, and the plates were then incubated for an additional 72 hours. prior to dna extraction, the covered plates were examined with an evos microscope and gfp laser for fluorescence (cell imaging core, university of utah). in 1 experiment, culture supernatant was removed and viral dna quantified in the adherent cells by qpcr. in a separate experiment, viral dna was assessed in cells and supernatant to capture signal from any shed virus. in both cases, adherent cells were freed from the wells with 0.25% trypsin (gibco). dna was extracted as described earlier. the analyses were performed in python 3. the measurements from each instrument were averaged over the period for each mouse and each procedure (6 minutes 45 seconds). student's t test was used to determine the statistical differences between the uninfected and infected tests. tukey's test was performed to evaluate the statistical differences between the blank test and each procedure. 17 the aerosol sampling instrumentation measures particle size and concentration with accuracies of 5% and 2%, respectively. 18, 19 the spot sampler has a standard deviation of 593 pfu/l of viable ms2 virus. 14 we anticipated that the spot would have similar standard deviation for cmv. we examined the difference between aerosol generation in infected and uninfected mice. figure 1 and table 1 show that, in general, the total count and pm 2.5 concentration differences between the uninfected and infected mice were not statistically significant (.067 \ p values \ .970, except for microdebrider grimm total counts and drilling smps-aps total counts, which were close to the background counts). therefore, we analyzed the aerosol measurements from all 17 mice for each procedure, regardless of infection status. two procedures, coblation and electrocautery, generated the highest concentrations of aerosols ( table 2 ; supplemental figures s1 and s2, available online). for example, the smps-aps showed 364-and 1202-fold increases in total particle counts for coblation and electrocautery, respectively, as compared with background. during electrocautery, suction reduced the total count by 26.8%; however, this reduction was not statistically significant (p = .082). the results also indicated that drilling and microdebrider did not cause statistically significant increases in aerosol concentrations and total counts when compared with background (.870 \ tukey-adjusted p value \ .930; figures 2 and 3 , table 3 ). figures 4 and 5 show the particle size distribution of the aerosols generated from each test. the results demonstrate that when compared with background, with a mean 6 sd diameter of 208 6 40 nm (table 2) , the heat-generated particles from coblation and electrocautery procedures yielded smaller particle sizes, with mean diameters of 120 6 14.1 nm and 103 6 10.5 nm (tukey-adjusted p values \.023), respectively. drilling and microdebrider, with mean diameters of 227 6 50.8 nm and 149 6 42.3 nm, did not show statistically significant differences as compared with background (tukey-adjusted p values ..432 ). since surgical masks are manufactured to filter particles .5 mm, we then calculated the relative percentage of particles \5 mm by figure 1 . comparison of smps-aps total particle counts between 3 uninfected and 14 infected samples. the bar denotes the mean total count. smps-aps, scanning mobility particle sizer. abbreviations: pm 2.5 , particulate matter \2.5 mm; smps-aps, scanning mobility particle sizer and an aerosol particle sizer. procedure. based on the smps-aps, .99.99% particles were \5 mm for all procedures. we also calculated the relative percentage of particles .7 mm, which is the limit of protection for n95 masks. more than 99.99% particles were also .7 mm for all procedures. abbreviations: pm 2.5 , particulate matter \2.5 mm; smps-aps, scanning mobility particle sizer and an aerosol particle sizer. figure 2 . smps-aps total particle counts for each procedure. the bar denotes the mean total count. smps-aps, scanning mobility particle sizer and an aerosol particle sizer. figure 6 shows the time required after each procedure to reach background aerosol concentrations (decay time). the electrocautery procedure required the longest time, approximately 3 minutes, to reach background aerosol concentrations. viral dna was detected in 3 of the 12 condensates collected during coblation and 3 of the 16 condensates during drilling of tissue from cmv-infected mice (supplementary table s1 ). viral dna was not detected in condensate samples collected from noninfected mouse tissue for either technique. no viral dna was detected in condensates collected during microdebrider or electrocautery use. the fact that viral dna was not detected for microdebrider samples that had been injected with 6.3 3 10 4 pfu of cmv immediately prior to the procedure suggests that the technique itself might be responsible for degrading the viral dna. infectious particles were not detected by gfp expression in tissue culture (figure 7 ) or by gfp expression or cmv qpcr of culture cells after 3 days following any procedure. tissue culture experiments resulted in confluent cell growth for all condensate-treated wells ( figure 7e and 7f) , whereas positive control wells treated with cmv-gfp virus showed lower cell density ( figure 7a and 7b) , indicating cell loss due to cmv infection. similarly, positive control wells treated with cmv-gfp resulted in amplification of viral dna by qpcr at levels well above input levels. these data indicate that infectious particles would have been detected in condensate-treated wells, if present at levels similar to or above the lowest levels of cmv included in positive controls. the risk for patient transmission during an otolaryngology procedure will depend on the probability that sufficient quantities of viable virus will infect the health care worker. the results from our study demonstrate that a number of these procedures can generate relatively large concentrations of aerosolized particles and that a significant percentage are small enough to pass unimpeded through conventional surgical and even n95 masks. the highest concentration of aerosolized particles was found from electrocautery and coblation. the 1202-fold increase in aerosolized concentrations from electrocautery over background is consistent with the carr et al study that measured the particle number concentrations of children who underwent electrocautery tonsillectomy. 20 in an unpublished communication, an edge electrosurgical button switch pencil (medtronics) and ent coblation wand (smith & nephew) were tested on fresh bovine thymus and myocardial tissue to determine quantity of aerosolized particles. 21 it reported a 240-fold reduction in particle concentration when the coblation wand was compared with the electrocautery device. we noted a much more modest reduction, which may be due to the lower-wattage setting that we used for our electrocautery experiments and the wider range of particle sizes measured with our instrumentation. the report cited a p-trak ultrafine particle counter with a range of 20 nm to 1 mm. our smps-aps measures a wider distribution of aerosols, between 15 nm and 20 mm. elmashae et al reported that electrocautery exhibited a peak in particle size between 60 and 150 nm, which agrees with our study's peak for this procedure at 89.9 nm. 2 we also found a modest reduction in aerosol concentrations when suction was applied in conjunction with electrocautery. a surgical mask requires food and drug administration clearance and protects the wearer from larger particles, .5 mm. 22 a n95 mask is cleared by the national institute for occupational safety and health and food and drug administration and will prevent aerosolized particles .0.7 mm. 23 our results indicate that 99.9% of particle counts for all the procedures are .7 mm. these results are consistent with others who reported that these particles can range from 10 nm to 1 mm. 2, 24 mowbray et al searched the cochrane database, medline, pubmed, embase classic, embase, and metaregister of controlled trials and found 5 of 20 studies showing that diathermy or laser can produce ultrafine particles that are respirable in size. 25 a powered airpurifying respirator (papr) may be a preferred option when performing some of these procedures in patients who are covid-19 positive. a papr filters out contaminants in the air and uses a battery-operated blower to provide the user clean air through a tight-fitting respirator, hood, or helmet. 26 it has a higher assigned protection factor than n95 or other air-purifying respirators, by a factor 25. an assigned protection factor is the ratio of pollutants outside the device (environment) to those inside the device (inhaled component). 26 this approach would be difficult to use for otologic surgery, and there may be challenges with verbal communication. bischoff et al reported that despite passing fit-testing, 10% of n95 respirator users encountered breakthrough with exposure to influenza virus as compared with full protection provided by a papr. 27 however, there have been no controlled clinical trials comparing the efficacy of pars with other modalities for sars-cov-2 or even for earlier pandemics (eg, sars-cov-1, ebola, or mers). a systematic review of sars-cov-2 observational and simulation studies failed to demonstrate differences in health care worker infection in cohorts using paprs versus other appropriate respiratory protection. 26 a clinical trial to address this question is urgently needed. in contrast to electrocautery and coblation, we did not detect significantly elevated concentrations of aerosols from microdebrider procedures. these results are consistent with workman and colleagues' assessment of microdebrider or cold instrumentation. 10 they performed these procedures on 2 fresh-frozen heads in a dedicated surgical laboratory. our results with drilling did not show a significant difference as compared with background levels. workman et al also considered only particles between 1 and 10 mm. perhaps their background aerosol concentrations were lower than those in our experiments. in addition, a human cadaveric sphenoid rostrum would contain much more bone stock than what could be drilled from the skull of an 8-day-old mouse, and this may have resulted in more mechanically generated particles in the 1-to 10-mm range. an important component in viral transmissibility from these procedures is its viability as an aerosol. we were able to detect viral dna in 3 of the 15 condensates for coblation. one might have expected to detect viral dna from the electrocautery procedures given our reported high concentrations. unlike coblation, which produces a plasma field at temperatures between 60°c and 70°c, electrocautery generates temperatures as high as 400°c to 600°c. 28 this much higher temperature can denature any viral dna. johnson and robinson measured aerosols from electrocautery and cooler drilling administered to known hiv-1-inoculated blood. 29 infectious hiv-1 was detected in the aerosolized collected viral culture media from drilling but not from electrocautery. sawchuk et al reported aerosolized dna following removal of plantar warts with electrocautery. 30 these samples were obtained at a relatively low 6 w and with a sampling only 2 cm from the surgical site. despite the reassuring low evidence of viral transmission from electrocautery, the elevated concentrations and composition of the surgical plumes are concerning. these plumes may contain as much as 3 to 51 ppm of hydrogen cyanide, a known cardiotoxic compound, and 0.15 to 0.69 ppm of 1,3-butadiene, a known carcinogen. 31 this surgical smoke can be toxic to patients as well as health care workers. dobrogowski et al measured benzene and toluene in the urine of patients who underwent laparoscopic cholecystectomy and detected significantly higher concentrations after surgery than before, presumably from the absorption of surgical smoke. 32 in the context of cigarette smoking, tomita et al noted that electrocautery removal of 1 g of tissue has the mutagenic potential of smoking 3 to 6 cigarettes. 33 thus, surgeons, operating room staff, and patients should be aware of the harmful effects from surgical plume. lower wattage and smoke evacuation systems should be used whenever possible. 20 somewhat surprising was our detection of viral dna during the drilling procedures. this finding suggests the importance of an aerosol temperature that is not too high to denature the virus. several studies have reported measurable aerosols following drilling procedures. 34, 35 our results are particularly relevant given a recent publication confirming the presence of sars-cov-2 rna detected in the middle ear and mastoid in 2 of 3 patients with covid-19. 36 our findings would support the recommendation to consider covid-19 preoperative screening and the use of appropriate precautions for potential aerosol and droplet generation for any middle ear or mastoid procedure. 36 other options to consider include modified drapes to reduce aerosol exposure and betadine irrigation during drilling to inactivate virus. chari et al reported that a second suction and a barrier drape reduced the average measured aerosol to baseline levels. 37 on the basis of an in vitro study demonstrating inactivation of sars-cov-2 and a small case series showing a significant reduction in covid-19 viral loads in 2 of 4 patients using a povidone-iodine mouthwash, we have started to use 0.5% povidone-iodine irrigation during our mastoidectomy procedures. 38, 39 samplers that are commonly used for bioaerosol sampling are not designed to collect nanosized viral aerosols. 14 hogan et al collected bacteriophages with 3 bioaerosol samplersthe agi-30, the skc biosampler, and a frit bubbler-and noted that the collection efficiency for each system was \10% for particles in the range of 20 to 100 nm. 40 we used a laminar-flow, water-based condensational particle growth tube collector. this device condenses water vapor onto a viral particle, creating droplets 2 to 5 mm in diameter. using this same method, pan et al demonstrated much greater collection of virus particles and efficiency of collecting viable virus-specifically, 10 to 100 times better than standard biosamplers. 14 despite the greater sensitivity with this growth tube collector, we were not able to detect any viable virus. we believe that this lack of viable virus reflects the very small quantities of aerosolized virus generated from these procedures. this observation is confirmed by the relatively low copy numbers in the detectable viral dna and may mean that these procedures are low risk for transmitting infection. a limitation of our study, however, is that the collection time for each procedure was only 6 minutes 45 seconds. this duration was based on the limited tissue available for surgery from an 8-day-old mouse. a longer procedure may have enabled detection of more viral dna and viable virus. we used a neonatal mouse infected with cmv because of our familiarity with this model and a mcmv virus, since it is not pathogenic to humans (biosafety 1 designation). 12, 16, 41 the mcmv strain expresses a gfp during transcription that is readily noticeable during active infection. tom and mina suggested that patients with covid-19 whose symptoms have resolved and who have a crossing threshold .34 are likely not to have meaningful or transmissible disease. 42 our studies indicate that these higher crossing thresholds were found in all our procedures. future studies-perhaps with a more clinically relevant model, as in a hamster or mouse infected with sars-cov-2-may provide insight into the transmissible potential from aerosolizing procedures to health care workers. coblation and electrocautery procedures generate .100-fold increases in aerosol concentrations over background; yet, only coblation and drilling produce aerosolized dna samples. the absence of any viable infectious particles from all procedures is reassuring and may indicate a low potential for viral transmission. the high concentration of aerosols from coblation and electrocautery suggests the need for appropriate safeguards against particle exposure to health care workers. the presence of viral dna from drilling and coblation procedures warrants the need for appropriate protection against droplet and aerosol exposure. additional studies with this model in a sars-cov-2 animal model may provide insight into the relative risk from these procedures in patients infected with covid-19. tofigh sayahi, performed the experiments, organized and designed the studies and contributed to data analysis, drafted the first version of the manuscript and contributed to editing and approval of the final manuscript; christopher nielson, performed the experiments, maintained the breeding colonies, organized and designed the studies, contributed to the data analysis and contributed to editing and approval of the final manuscript; yuan yu, performed the experiments, maintained the breeding colonies, contributed to editing and approval of the final manuscript; kaden neuberger, performed the experiments, contributed to editing and approval of the final manuscript; michael seipp, performed the experiments, organized and designed the studies, contributed to the data analysis, to editing and approval of the final manuscript; matthew a. firpo, organized, designed the studies, contributed to the data analysis, to editing and approval of the final manuscript; kerry kelly, performed the experiments, organized and designed the studies, contributed to data analysis, editing and approval of the final manuscript; albert h. park, performed the experiments, organized and designed the studies, contributed to the data analysis, drafted the first version of the manuscript and contributed to editing and approval of the final manuscript. surgical smoke simulation study: physical characterization and respiratory protection us unemployment rate soars to 14.7 percent, the worst since the depression era high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa covid-19 indirect contact transmission through the oral mucosa must not be ignored laboratory diagnosis of emerging human coronavirus infections-the state of the art covid-19 and the otolaryngologist: preliminary evidencebased review covid-19 in otolaryngologist practice: a review of current knowledge guidance for otolaryngology health care workers performing aerosol generating medical procedures 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immunodeficiency virus-1 (hiv-1) in the vapors of surgical power instruments infectious papillomavirus in the vapor of warts treated with carbon dioxide laser or electrocoagulation: detection and protection composition of volatile organic compounds in diathermy plume as detected by selected ion flow tube mass spectrometry chemical composition of surgical smoke formed in the abdominal cavity during laparoscopic cholecystectomyassessment of the risk to the patient mutagenicity of smoke condensates induced by co2-laser irradiation and electrocauterization bloodcontaining aerosols generated by surgical techniques: a possible infectious hazard mastoidectomy and trans-corneal viral transmission sars-cov-2 virus isolated from the mastoid and middle ear: implications for covid-19 precautions during ear surgery aerosol dispersion during mastoidectomy and custom mitigation strategies for otologic surgery in the covid-19 era. otolaryngol head neck surg comparison of in vitro inactivation of sars cov-2 with hydrogen peroxide and povidone-iodine oral antiseptic rinses is povidone iodine mouthwash effective against sars-cov-2? first in vivo tests sampling methodologies and dosage assessment techniques for submicrometre and ultrafine virus aerosol particles cytomegalovirus (cmv) infection causes degeneration of cochlear vasculature and hearing loss in a mouse model to interpret the sars-cov-2 test, consider the cycle threshold value competing interests: none. funding source: support for dr kelly's participation in this research was provided by a grant from the national institute of environmental health sciences, national institutes of health (5k25es027504-02). additional supporting information is available in the online version of the article. key: cord-261908-olcuq6tm authors: lai, ka-man; bottomley, christian; mcnerney, ruth title: propagation of respiratory aerosols by the vuvuzela date: 2011-05-23 journal: plos one doi: 10.1371/journal.pone.0020086 sha: doc_id: 261908 cord_uid: olcuq6tm vuvuzelas, the plastic blowing horns used by sports fans, recently achieved international recognition during the fifa world cup soccer tournament in south africa. we hypothesised that vuvuzelas might facilitate the generation and dissemination of respiratory aerosols. to investigate the quantity and size of aerosols emitted when the instrument is played, eight healthy volunteers were asked to blow a vuvuzela. for each individual the concentration of particles in expelled air was measured using a six channel laser particle counter and the duration of blowing and velocity of air leaving the vuvuzela were recorded. to allow comparison with other activities undertaken at sports events each individual was also asked to shout and the measurements were repeated while using a paper cone to confine the exhaled air. triplicate measurements were taken for each individual. the mean peak particle counts were 658×10(3) per litre for the vuvuzela and 3.7×10(3) per litre for shouting, representing a mean log(10) difference of 2.20 (95% ci: 2.03,2.36; p<0.001). the majority (>97%) of particles captured from either the vuvuzela or shouting were between 0.5 and 5 microns in diameter. mean peak airflows recorded for the vuvuzela and shouting were 6.1 and 1.8 litres per second respectively. we conclude that plastic blowing horns (vuvuzelas) have the capacity to propel extremely large numbers of aerosols into the atmosphere of a size able to penetrate the lower lung. some respiratory pathogens are spread via contaminated aerosols emitted by infected persons. further investigation is required to assess the potential of the vuvuzela to contribute to the transmission of aerosol borne diseases. we recommend, as a precautionary measure, that people with respiratory infections should be advised not to blow their vuvuzela in enclosed spaces and where there is a risk of infecting others. aerosols play an important role in the spread of communicable diseases [1, 2] . aerosol transmission can be airborne, where contaminated droplet nuclei exhaled by an infected individual are inhaled by a susceptible individual. a second route of infection is when deposited droplets are carried to the mouth or nose through physical contact, often by hand [3] . airborne aerosol transmission is believed to make a major contribution to the spread of diseases such as tuberculosis and measles [4, 5] . aerosols have also been implicated in the transmission of diseases such as the common cold, chickenpox, rubella, influenza, pneumococcal disease and severe acute respiratory syndrome (sars) [3, 6, 7, 8, 9, 10] , although their contribution is less clear cut as non aerosolized respiratory secretions also contribute to the spread of these diseases. some airborne pathogens are extremely contagious; in the usa an outbreak of measles was traced to a sporting event where transmission was found to have occurred between an athlete in the arena and spectators in the stadium, with no evidence of close contact [11] . spread of respiratory disease is of particular concern in large crowds and at international gatherings [12, 13] . this includes the annual muslim pilgrimage to mecca and associated sites, during which respiratory infections are the most common cause of hospitalization [14] . the reported infections include tuberculosis and influenza and the saudi ministry of health recommends wearing of protective face masks by those attending the hajj [13, 15] . the emergence of epidemic strains of flu have also caused concern; in 2009 fears around the spread of influenza h1n1 resulted in a temporary ban on public events in some countries [16] . aerosols are created and expelled into the atmosphere during coughing, sneezing, singing or talking. if the person has a respiratory infection a proportion of the aerosols may carry pathogenic particles [17, 18, 19] . the size of a contaminated aerosol droplet is crucial in determining its ability to transmit disease. whereas large drops (.100 microns diameter) will rapidly fall to the ground smaller droplets may remain suspended in the air where evaporation can occur resulting in the formation of tiny 'droplet nuclei' that can stay airborne for hours or days [20, 21] . these particles can be breathed in by susceptible individuals who may then become infected. the fate of the droplet nuclei on inhalation also depends on their size; particles greater than five microns are likely to remain in the upper airways but smaller particles are more likely to deposit in the alveoli and so may transmit infections of the lower respiratory tract such as tuberculosis [22, 23] . the vuvuzela is a plastic blowing horn that has been adopted by sports fans to provide audible support for their team. it is used in several countries in asia and africa and is particularly popular in south africa where it figured prominently during the recent fédération internationale de football association (fifa) world cup. the instrument is typically 60 cm in length, tapering from a bell end 11.5 cm in diameter to a mouthpiece of 2.5 cm. vuvuzela playing requires forceful and sustained blowing. air from the lungs is expelled from the mouth through vibrating lips held against the plastic mouthpiece and out through the instrument. we speculated that the mode of action may facilitate the propagation and dissemination of aerosols from the respiratory tract of the person blowing the instrument. the instrument is frequently used in crowded situations and it was therefore important to determine the extent of aerosol production to assess whether blowing the vuvuzela might assist in the spread of aerosol borne diseases. to assess the number of aerosols propagated during blowing the vuvuzela eight volunteers (4 male and 4 female) were each given an instrument and asked to blow enthusiastically, as if they were attending a football match. to enable comparison with other activities undertaken at sporting events each volunteer also shouted into a paper cone constructed to have the same 115 mm diameter exhale opening as the vuvuzela ( figure 1 ). particles exiting the vuvuzela or shouting cone were assessed using a laser particle counter and enumerated in six categories according to their diameter. the velocity of air as it exited the devices was measured with a hot-wire anemometer and peak airflows were recorded. the duration of playing was recorded with a stopwatch. triplicate experiments were undertaken for each individual tested. airborne particles exiting the instrument were measured every second throughout the experiment and reported as particles per litre. the mean concentration of particles recorded from playing the vuvuzela and shouting were 658610 3 and 3.7610 3 per litre respectively. to compare the number of particles emitted by an individual when shouting to the number emitted when playing the vuvuzela, the data were log 10 transformed and the difference was calculated for each individual. the mean log 10 difference was 2.20 (95% ci: 2.03,2.36; p,0.001). men expelled particles at a higher mean concentration than the women when playing the vuvuzela (741610 3 vs 575610 3 per litre) although this was not statistically significant (p = 0.69).when shouting there was no difference in the numbers of particles captured (male:female; 3.4610 3 vs 3.9610 3 per litre, p = 0.89). aerosols were enumerated in six size categories according to the diameter of the particle: 0.5-0.7 mm; 0.7-1.0 mm; 1.0-3.0 mm; 3.0-5.0 mm; 5.0-10.0 mm and .10.0 mm. the distribution of particles by size category is presented in figure 2 . the great majority (97%) of particles captured from both the vuvuzela and the shouting cone were between 0.5 and 5 microns in diameter and small enough to enter the lower respiratory tract. the geometric mean (gm) particle diameter was calculated for each experiment and is presented in table 1 . slightly larger particles were emitted when playing the vuvuzela compared to shouting the mean duration for vuvuzela playing events was 2.1 sec (range: 1.25-3.90 sec) and the shouting lasted for an average of 2.2 sec (range: 0.96-4.72 sec). the peak velocity of air exiting the vuvuzelas was higher than from the shouting cone with a mean of 0.59 ms 21 (range:0.12-1.80 ms 21 ;) compared to a shouting mean of 0.18 ms 21 (range: 0.07-0.32 ms 21 ) and this difference was statistically significant (p = 0.03). this was equivalent to airflow of 6.1 and 1.8 ls 21 respectively, for the vuvuzela and shouting. although the duration in playing vuvuzelas between females and males were similar (2.1 sec), the mean peak airflow was nearly double in males compared to females, 7.9 compared to 4.3 ls 21 (this difference was not statistically significant p = 0.19). the difference between females and males in shouting was not as apparent, although males also had a higher peak airflow compared to females, 2.1 compared to 1.6 ls 21 (p = 0.37). we have estimated the numbers of aerosols exiting the vuvuzela when blown by male and female adults. in triplicate experiments from eight individuals the mean concentration of particles exiting the vuvuzela was 658,000 per litre. the mean peak volume of air exiting the instrument was 6.1 litres per second. thus we estimate that approximately 4 million particles per second were being disseminated from the vuvuzela at peak blowing times. for shouting we estimated a peak aerosol concentration of 3,700 per litre or 7,000 particles per second (assuming peak flow volume of 1.8 ls 21 ). the data we obtained for shouting is in broad agreement with a recent study of particles exhaled by healthy adults during normal to deep breathing (tidal volume range: 20-80%) where between 5 and 5,000 droplets per litre were recorded [24] . the differences we observed between male and female volunteers might be explained by differences in their lung capacities, however this was not measured [24] . our results suggest that the vuvuzela is an efficient means of propagating large numbers of aerosols. the great majority of particles measured were of a size that could remain suspended in the air as droplet nuclei and would be capable of entering the alveolar airspaces of the lung. during normal (resting) breathing an adult inhales approximately 7 litres of air each minute, of which 5 litres reaches the respiratory bronchioles [25] . when attending a sporting event and surrounded by vuvuzela players a spectator could expect to inhale large numbers of respiratory aerosols over the course of the event. actual exposure would be affected by the proximity of the vuvuzelas and ambient ventilation which would serve to dilute the stream of particles. the large number of aerosols emitted by the vuvuzela raises the possibility that, if used by persons with an infection of the respiratory tract, they could act a conduit for the spread of infectious particles. for ethical and safety reasons we only examined healthy volunteers during this study; assessment of pathogenicity of aerosols disseminated by the vuvuzela will require further study using patients with known respiratory infections. aerosols can be created at various locations within the respiratory tract [26] and carriage of pathogens by exhaled aerosols depends on the site of infection and the quantity pathogenic particles in the airways [18] . we speculate that aerosols propagated while blowing the vuvuzela may originate in either the lower or upper respiratory tract, or the mouth. to obtain the desired trumpet sound when blowing the vuvuzela air is forced through the lips into the opening of the instrument which may serve to create further aerosols, or alter the size of droplets produced elsewhere in the respiratory tract. in addition to the manner in which the instrument was blown the number of contaminated particles expelled will vary according to the pathogen, the site of infection and the extent of disease. some infections may result in inflammation and physiological changes within the respiratory tract that would affect the person's capacity to blow the vuvuzela [27] . in addition, some conditions are associated with changes in the rheology of respiratory secretions that might affect aerosol formation [28, 29] . studies of cough aerosols from pulmonary tuberculosis patients and cystic fibrosis patients with bacterial infections found that the concentration of infectious particles varied widely between patients [30, 31] . to attain an accurate assessment of the vuvuzela's potential to disseminate infected aerosols, sample sizes will need to be increased to include individuals having a range of upper and lower respiratory tract infections. symptomatic and non symptomatic carriers should be assessed. in addition to counting the number and size of particles, the viability of infectious particles should also be assessed. for bacterial infections this might be achieved by modification of a cough aerosol sampling system previously used to assess tuberculosis patients [30] . coughing, sneezing, singing and talking can all produce aerosols capable of transmitting airborne respiratory diseases [17, 18, 30, 32] . reports from earlier investigators suggest that coughs may produce up to 5,000 droplet nuclei and a sneeze may generate as many as 900,000 particles [21, 33] . the data we present suggests that blowing the vuvuzela for even a short time period has the potential to create more droplet particles than either coughing or sneezing. there were some limitations to this study that may have had an impact on the results. the particle counter used to assess the concentration of particles recorded measurements at one second intervals and it is possible that the peak values recorded were not the maximum level of particle produced. as it was not possible to assess variation in flow rates over the blowing period the total number of particles expelled during a blowing or shouting event could not be estimated. the performance of individuals and production of aerosols may have been influenced by their respective lung capacities [24] , this factor was not assessed in the experiment. the use of a paper cone to assess the droplets from shouting was not ideal as the surface areas and shape of the paper cone may increase the chance that particles attach to the surface rather than remain in the airstream, affecting the number and size of particles reaching the counter. as exhaled air cools and mixes with ambient air condensation droplets may form. although ambient air temperature and humidity remained similar in all experiments, the difference in shape between the cone and the vuvuzela may have affected the mixing and rate of formation of table 1 . exhale duration, peak air velocity, particle concentration and mean particle diameter recorded during playing the vuvuzela and shouting by four male and four female volunteers. these transient droplets. a further consideration is that only healthy individuals were recruited for this study, and as described above, it is possible that people with respiratory illness with impaired lung function would perform differently when blowing the vuvuzela. nonetheless we have demonstrated that these plastic trumpets provide an excellent means of propagating respiratory aerosols, exceeding both sneezing and coughing as a means of disseminating droplet nuclei and we conclude that their potential to spread respiratory diseases requires further investigation. the frequency, duration, and vigor of vuvuzela playing will vary considerably from person to person, depending on the occasion and their expertise at blowing and we are unable to comment on the number of aerosols produced during an entire sporting event. a further factor is the environment in which they are used; open situations with a strong draft or breeze will serve to rapidly dilute the aerosols produced but transmission risks may be considerably higher in enclosed arenas. a further risk factor for disease transmission will be the density of vuvuzela players and the prevalence of respiratory infections in the population. as far as we are aware this is the first report in the scientific press regarding the issue of aerosol dissemination by the vuvuzela and no epidemiological data regarding impact of the instrument on disease transmission have been reported. similarly there have been no reports of disease transmission from sharing vuvuzelas, or from transfer of non aerosolized respiratory secretions that collect inside the instruments. the vuvuzela has become popular in south africa, a country with the highest urban prevalence of tuberculosis in the world and that recently experienced a measles epidemic [34] . it has been used at domestic soccer games for the past decade and was adopted by many visiting fans during the 2010 fifa world cup competition. the tournament was held during late june and early july and coincided with the annual flu season. surveillance reports show an increase in the proportion of influenza b compared to previous years, but evidence to link this to the presence of visiting spectators is not presented [35] . similarly a number of measles cases were confirmed amongst visitors from other countries but evidence as to the source of their infections is not available [36] . the plastic vuvuzela is believed to have emerged as a child's toy, before being adopted by sports fans in africa and parts of asia, where it is a multi-million dollar industry. in africa it has become a symbol of the soccer industry but vuvuzelas are also blown by fans of cricket and rugby football. they have been banned from a number of sports grounds due to the volume of noise emitted and safety concerns arising from their ability to nullify public address systems. studies have previously suggested that vuvuzela playing poses a risk of noise induced hearing loss [37, 38] . we recommend that consideration is taken of their propensity to disseminate respiratory aerosols and that persons with respiratory infections be advised not to blow their vuvuzela in places where they risk infecting others. this should include enclosed spaces and crowded venues such as large sporting events. we also recommend that research be commissioned to determine the risks to public health posed by the vuvuzela. this study was undertaken at the healthy infrastructure research centre at university college london. ethical approval was obtained from university college london research ethics committee and informed consent was obtained in writing from all participants. eight healthy volunteers, 4 males and 4 females working in the research centre participated. the experiments were conducted in a closed room free from drafts. the study subjects were in the age range 20 to 45 years and all self reported as being free from illness. to avoid cross contamination a new vuvuzela instrument was provided for each participant (boogie blast co, johannesburg, sa). the velocity of air leaving the instrument was measured using a hot-wire anemometer (testo, ukflow) and duration of playing recorded with a stopwatch. our initial measurements showed that the average time of playing the vuvuzela was about 2 sec. to enable comparison with other activities undertaken at sporting events each individual tested was requested to also shout into a paper cone constructed to have the same diameter exhale opening as the vuvuzela (figure 1 ). subjects were requested to hold their shout for about 2 sec (not compulsory) and to shout the word ''go''. particles exiting the vuvuzela or the shouting cone were measured using a six channel laser particle counter (lighthouse 5016, uk). particles were enumerated in six categories according to their diameter: 0.5-0.7 mm; 0.7-1.0 mm; 1.0-3.0 mm; 3.0-5.0 mm; 5.0-10.0 mm and .10.0 mm. a 0.047 litre sample of air was tested every second and the number of particles recorded. analyses are based on either the average or peak concentration observed during the vuvuzela or shouting event. triplicate experiments were undertaken for each individual tested. volume of the airflow was estimated by multiplying the peak air velocity recorded in the anemometer with the duration of playing and shouting and the surface area of the exhale opening of the vuvuzela and paper cone. in each experiment (in which an individual either shouted or blew on a vuvuzela) data were collected on particle concentration every second. these data were summarized in one of two ways: i) as the concentration observed in the 2 nd second after the start of the experiment, which usually corresponded to the peak concentration or ii) as the average concentration over the length of the shout or vuvuzela blow. all analyses were carried out using both peak and average concentrations. however, since both yielded similar results, we restrict our presentation to the analysis of peak concentrations. the geometric mean size (gm) of particles was calculated for each experiment by fitting a log normal distribution to the particle size data at peak concentration. estimates were obtained by maximum likelihood, allowing for interval-censoring (particle sizes were recorded using the categories 0.5-0.7 mm; 0.7-1.0 mm; 1.0-3.0 mm; 3.0-5.0 mm; 5.0 -10.0 mm and .10.0 mm). each individual shouted and blew the vuvuzela three times. statistical analysis of particle concentrations and gm particle size were based on the means of these triplicate measurements; this was done to eliminate dependence in the data arising from repeat measurements on the same individual. to compare the number of particles emitted when shouting to the number emitted when playing the vuvuzela, we logtransformed each individual's average concentration (averaged over the three measurements) when shouting and when playing the vuvuzela and calculated the difference between these logtransformed values. the confidence interval for the mean difference and p-value were based on a paired (one-sample) t-test. differences in the average (gm) particle size between vuvuzela and shouting were similarly assessed using a paired t-test (although the data were not log-transformed in this instance). comparisons between men and women of particle concentration and airflow were made using permutation tests (based on the wicoxon rank sum statistic) rather than t-tests owing to the small numbers (4 men and 4 women). the mechanism of transmission of pathogenic organisms affecting the respiratory tract factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises mechanisms of transmission of rhinovirus infections strategies for minimizing nosocomial measles transmission aerial dissemination of pulmonary tuberculosis. a two-year study of contagion in a tuberculosis ward viruses as agents of airborne contagion airborne transmission of communicable infectionthe elusive pathway review of aerosol transmission of influenza a virus an epidemic of pneumococcal disease in an overcrowded, inadequately ventilated jail airborne transmission of chickenpox in a hospital an outbreak of measles at an international sporting event with airborne transmission in a domed stadium pandemic influenza: mass gatherings and mass infection health risks at the hajj pattern of admission to hospitals during muslim pilgrimage (hajj) bacteria and viruses that cause respiratory tract infections during the pilgrimage (haj) season in makkah, saudi arabia questions raised over response to influenza a outbreak singing and the dissemination of tuberculosis expulsion of pathogenic organisms from respiratory tract airborne infection on air-borne infection study ii. droplets and droplet nuclei the size and the duration of air-carriage of respiratory droplets and droplet-nuclei distribution and deposition of inhaled particles in respiratory tract on the mechanics of droplet nuclei infection. ii quantitative experimental air-borne tuberculosis in rabbits characterization of exhaled particles from the healthy human lung-a systematic analysis in relation to pulmonary function variables respiratory physiology upate in anaethesia: world federation of societies of anaesthesiologists size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities pulmonary function during and after common respiratory infections macrorheology of cystic fibrosis, chronic obstructive pulmonary disease & normal sputum airborne infectious disease and the suppression of pulmonary bioaerosols cough-generated aerosols of mycobacterium tuberculosis: a new method to study infectiousness cough-generated aerosols of pseudomonas aeruginosa and other gram-negative bacteria from patients with cystic fibrosis epidemiology of primary tuberculosis in an industrial school characterization of expiration air jets and droplet size distributions immediately at the mouth opening global tuberculosis control:who severe acute respiratory illness (sari) surveillance: influenza report fifa soccer would cup, south africa: communicable disease risks and surveillance. pretoria: national institute for communicable diseases vuvuzela sound measurements vuvuzela -good for your team, bad for your ears we would like to thank hector altamirano-medina for providing technical support. we are grateful to the volunteers who gave their breath in this study. key: cord-319221-x834qscq authors: abuhegazy, mohamed; talaat, khaled; anderoglu, osman; poroseva, svetlana v. title: numerical investigation of aerosol transport in a classroom with relevance to covid-19 date: 2020-10-01 journal: phys fluids (1994) doi: 10.1063/5.0029118 sha: doc_id: 319221 cord_uid: x834qscq the present study investigates aerosol transport and surface deposition in a realistic classroom environment using computational fluid-particle dynamics simulations. effects of particle size, aerosol source location, glass barriers, and windows are explored. while aerosol transport in air exhibits some stochasticity, it is found that a significant fraction (24%–50%) of particles smaller than 15 µm exit the system within 15 min through the air conditioning system. particles larger than 20 µm almost entirely deposit on the ground, desks, and nearby surfaces in the room. source location strongly influences the trajectory and deposition distribution of the exhaled aerosol particles and affects the effectiveness of mitigation measures such as glass barriers. glass barriers are found to reduce the aerosol transmission of 1 µm particles from the source individual to others separated by at least 2.4 m by ∼92%. by opening windows, the particle exit fraction can be increased by ∼38% compared to the case with closed windows and reduces aerosol deposition on people in the room. on average, ∼69% of 1 µm particles exit the system when the windows are open. transmission of covid-19 occurs primarily through sars-cov2-laden droplets and aerosol particles inhaled directly or transmitted from contaminated surfaces. 1 effective mitigation measures necessitate clear understanding of droplet and aerosol transport, surface retention, and evaporation kinetics in different environments and conditions. 2 aerosols are generated during exhalation, talking, coughing, sneezing, and other activities. 3, 4 in indoor environments, some of the generated particles exit the system through ventilation, some deposit on surfaces in the room and may settle or re-enter the air, and others may be directly inhaled. of primary interest to mitigation measures is maximizing the fraction of particles that exit the system and minimizing aerosol deposition on people to reduce disease transmission. 5, 6 aerosol transport within a control volume is primarily affected by inertial forces due to airflow and drag on the particle, and gravitational sedimentation. 7 the forces acting on a particle primarily depend on particle size and its position in the flow field. for smaller particles (<0.5 μm), brownian force can play a significant role in aerosol transport but becomes less important with increased particle size. 7, 8 the velocity field of the fluid (air) under known boundary conditions can in principle be estimated by numerically solving navier-stokes equations through direct numerical simulations (dns), or more practically by numerically solving reynolds-averaged navier-stokes (rans) equations with approximate turbulence closures such as k-ϵ and k-ω closures. 9, 10 as particle properties significantly affect aerosol and droplet transport within a system, it is necessary to consider accurate particle shape, size, and evaporation kinetics. the distinction between aerosols and droplets is rather arbitrary with no general agreement on a particle size threshold or suspension time threshold. 3 however, droplets are typically considered to be larger particles where evaporation kinetics is rapid leading to the production of smaller aerosols with slow evaporation kinetics. 3 aerosol particles and droplets released from activities such as exhalation, talking, or coughing are polydisperse in nature. exhalation and talking release particles mostly <1 μm, 11 and coughing releases larger particles typically <10 μm, 12 while sneezing was found in one study to release particles characterized by a bimodal size distribution with peaks edge, no studies have investigated aerosol transport in a classroom environment although classroom sizes, the air conditioning layout, and aerosol source distribution are characteristically different than hospital care units and other indoor spaces discussed in the literature. while a typical 900 sq. ft classroom can fit 18 students and an instructor, guidelines for re-opening schools have restricted the number of students to less than 10 students with 6 ft minimum spacing between the students. the effectiveness of these measures is dependent in part on aerosol transport within the classroom's air conditioned environment, which remains under-characterized. other strategies for covid-19 mitigation may include the use of glass screens as barriers to reduce aerosol transport between people in the room, opening windows, and redistributing students in classrooms, but the ability of these measures to reduce aerosol transmission from one person to another needs to be carefully evaluated. the objective of the present work is to investigate aerosol transport and surface deposition in a model classroom environment using computational fluid-particle dynamics (cfpd) simulations. particularly, it is of interest to estimate the fraction of particles that exit the system, deposit on students, and deposit on surfaces such as desks, ground, walls, and ceiling. the effects of particle size, aerosol source location, glass barriers, and windows are investigated. aerosol deposition on different students from different sources is compared to qualitatively explore the risk posed to individuals in the room due to their position with respect to an infected student. a three-dimensional model of a classroom consisting of nine students and an instructor was developed. the model uses realistic classroom dimensions and air conditioning. the classroom shown in fig. 1 is 9 × 9 m 2 in area and 3 m in height. the distance between each student is 2.4 m (7 ′ 10 ′′ ), which is greater than the recommended 6 ft separation distance for covid-19 mitigation. the model includes desks (with glass screens and without them) and windows. all students are represented similarly and have the same dimensions. each student consists of a cuboid body (0.5 × 0.25 × 1 m 3 ) and a cuboid head (0.16 × 0.15 × 0.2 m 3 ) with a rectangular mouth surface (0.06 × 0.03 m 2 ) through which particles and air are injected into the system. the simplified human model is inspired by models used in a numerical investigation of cross-transmission in hospitals. 6 no chairs are considered in the model due to the extensive variability in chair sizes and shapes. students are assumed to be exposed to aerosols in order not to underestimate deposition on students. an instructor is defined in the front, as shown in fig. 1(a) , and is assumed to be 1.7 m in height. independent surfaces are defined in the model for each object for tracking the aerosol deposition on objects and students, respectively. air conditioning of the classroom follows ashrae 62.1 ventilation standards for acceptable indoor air quality. 26 the air conditioning system consists of five supply diffusers and four return air diffusers distributed as shown in fig. 1(a) . the cubic feet per minute (cfm) required for adequate ventilation was found to be ∼1230 cfm. the supply diffusers (1, 3, 5, 7, and 9 ) supply air at a 37 ○ angle from the horizontal surface with an inlet flow area of 0.294 m 2 and a diffuser inlet vertical air velocity of 0.395 m/s based on ashrae recommendations. 27 in the present work, the effect of opening windows while the air conditioning system is running on particle removal is explored. for this purpose, the model includes 3 windows (2.2 × 1.3 m 2 ) that can be opened up to 50% in 10% increments. an unstructured, tetrahedral mesh is used, as shown in fig. 1 . the mesh was generated using ansys icem 19.1. the mesh consists of 3.3 × 10 6 mesh cells with a minimum cell size of 0.5 cm and maximum cell size of 10 cm with gradual transition, maximum skewness of 0.823 (a mean value of 0.593), and maximum aspect ratio of 3.21 (a mean value of 1.43). the grid is refined near surfaces to maintain a wall y + <10 during the simulations. each case of the 20 cases simulated in this work consumed ∼9 h running on four computer cores. the present study uses the commercial cfd code, ansys fluent 19.2, to simulate the airflow and particle transport. the continuity and momentum equations of the continuum phase (air) are solved in the beginning independent of the discrete phase using the steady state reynolds averaged navier-stokes (rans) incompressible solver. the present simulations use the re-normalization group (rng) k-ε model. 28 the choice of the rng k-ε model is motivated by the work of ramponi and blocken who investigated the influence of turbulence models on cross-ventilation for a generic isolated building, and it was found that the rng k-ε model was suitable for their application and operation conditions, which, in part, resemble the current application. 29 the simple algorithm implemented in ansys fluent is applied for pressure velocity coupling with pressure interpolation of first order. the convection and viscous terms of the governing equations were discretized utilizing the second-order discretization scheme. the solution is assumed to be converged when all the scaled residuals stabilize and approach a minimum of 10 −5 for k, ε, x, y, and z momentum equations as well as 10 −4 for the continuity equation. once the continuum phase solution converges, the flow field is then frozen and is used to transport the discrete phase (aerosol particles). the effect of the particles on the flow of air is negligible. one-way coupling between the continuum phase and the discrete phase is used given the low concentration of the aerosol particles in air. the particle trajectory is determined by solving the equation of motion for the particle in a lagrangian framework. the where v i is the velocity of the particle, m is the mass of the particle, → f drag is the drag force between the air and the particle, → fg is the gravity force, and → fa represents the other additional forces including the pressure force, virtual mass force, basset force, brownian force, and saffman's lift force. the particles used in the present work are sufficiently small to neglect pressure and virtual mass forces and sufficiently large to neglect brownian force. 7, 8, 30 as the particles are much smaller than the mesh elements, it is necessary to use drag models. the present work uses the stokes-cunningham drag model. therefore, the equation of motion of the particles could be written more explicitly as follows [eq. (2)]: where ui is the velocity of the flow, f is the drag factor, 31 τp is the particle reaction time, and cc is the cunningham correction factor. 32 the present simulations use 96 000 particles, which is a reasonable number of particles for sound statistics and is greater than those used in another study of aerosol removal in hospital care units. 15 the turbulent dispersion of particles and the random effects of turbulence on particle dispersion were taken into account using the discrete random walk method implemented in ansys fluent. since the particles are small enough to stick to surfaces, the trap boundary condition is used for the particles over all solid surfaces. in reality, some of the particles will be reflected and others may re-enter the air after deposition. however, re-entry and reflection are difficult to account for as they are affected by particle properties, surface properties, and flow conditions. 33 an escape boundary condition is employed for the diffusers and mouths. air flow from mouths is assumed to be exhaled at 20 l/min specified as a velocity inlet boundary condition (0.185 m/s) for a mouth inlet area of 0.0018 m 2 . the particles are released with the same velocity normal to the mouth surface. the base case uses 1 μm particles, student 5 as the source, no glass barriers, and windows closed. the choice of 1 μm particles is in the range of the particle size of aerosol particles released in exhalation and talking. 11 student 5 is used as the source for the base case due to their location far away from vortices at the edges of the room. the present study investigates the effects of particle size, source position, glass barriers, and windows on the fate of the exhaled aerosol particles. parameters of the base case are varied to investigate these effects. particle sizes studied are 1 μm, 4 μm, 10 μm, 15 μm, 20 μm, and 50 μm. aerosol sources considered are students 1, 2, 5, 8, and 9 and are studied with and without 70 cm high glass barriers/screens placed on top of the desks. the effect of windows is explored by comparing aerosol deposition and transfer in the classroom with 0%, 10%, 20%, 30%, 40%, and 50% open windows. as three windows are available in the room, 10% open windows implies that 10% of each of the three windows is open. the effect of windows is explored while the air conditioning system is running. while this can increase the cooling/heating load and decrease the energy efficiency of the air conditioning system, the present work is concerned with the effect on particle removal. table i summarizes the parameter combinations investigated in the present work. the velocity field of the continuum phase and the distribution of turbulent kinetic energy and vorticity are of fundamental importance to aerosol transport. figure 2 shows the turbulent kinetic energy distribution, velocity magnitude distribution, and velocity vectors of air across a two-dimensional slice going through students 2, 5, and 8. in this slice, air is injected into the system through the supply diffuser in the middle (inlet 5) at a 37 ○ angle with the ceiling. return diffusers 2 and 8 are shown at the sides. the turbulent kinetic energy is more significant at the edges of the room (especially at the outlets) and close to student 8 by the virtue of their location with respect to air conditioning [ fig. 2(a) ]. the velocity magnitude is strongest at the inlets and outlets, but the air is not stagnant in the rest of the room due to air conditioning [ fig. 2(b) ]. the velocity vectors [ fig. 2(c) ] demonstrate the recirculation near the edges of the room and near student 8's head. vortices can partially trap aerosol particles that are transported to those regions and increase deposition on neighboring surfaces. particle transport in the classroom environment due to an impulse aerosol source is a transient process. for the purposes of characterizing the dynamics and the fate of exhaled aerosol particles, a single-release impulse source is used. figure 3 shows the distribution of 1 μm aerosol particles in the classroom at different points in time since particle release. figure 3 (a) illustrates the transport of particles released from student 5. after 1 s of release, the aerosol particles exhibit a parabolic distribution at the front of the particle swarm. the particles slowly disperse and rise up during the first 50 s. once the particles reach the downstream of the air conditioner, the particles are rapidly transported to different parts of the room. as air flows from the supply diffusers to the return diffusers, the particles that reach the downstream of the air tend to follow the flow and exit the system. overall, there are significantly more 1 μm particles in the upper half of the room than the bottom half due to the flow of air to the return diffusers that are located in the ceiling in the present model. figure 3 (a) highlights the significance of the flow velocity distribution on aerosol transport in the room. therefore, the results of the present work are applicable to classrooms with comparable air conditioning. figure 3 (b) illustrates the transport of 1 μm particles released from student 8. aerosols released from student 8 [ fig. 3(b) ] exhibit a substantially different distribution than aerosols released from student 5 [ fig. 3(a) ]. at 1 s, the particle swarm curves downward and much of the particles deposit on the source student (student 8). this is a result of the position of student 8 with respect to the air conditioning system. as shown in fig. 2 , the velocity magnitude near student 8 due to air conditioning is strong compared to that near student 5. student 8 is also present near a region with recirculation and strong vortices compared to the rest of the room. the particles disperse slowly, and even after 5 min, most of the particles are present in the back half of the room. particle size is of fundamental importance to aerosol transport. 7 the present work considers spherical aerosol particles in the 1 μm-50 μm size range. figure 4 shows the effect of particle size on the fraction of aerosol particles released from student 5's mouth that deposit on different surfaces in the room, such as ground, ceiling and walls, desks, and students, or escape from the outlet of the air conditioning system. no significant difference is observed between 1 μm and 4 μm particles [ figs. 4(a) and 4(b) ]. nearly 50% of 1 μm and 4 μm aerosol particles exit the room through the air conditioning system after 15 min. roughly 15% of the particles deposit on the ceiling, and ∼10% deposit on the walls of the classroom, which is comparable to 14%-15% deposition on the ground [figs. 4(a) and 4(b) ]. this suggests that gravity does not play a significant role in the transport of 1 μm and 4 μm particles in the timescale of 15 min. in the case of 10 μm particles [ fig. 4(c) ], deposition on the ground increases to 27% compared to 14%-15% in 1 μm and 4 μm desks, and students. the rest of the particles deposit on the ceiling and walls, exit the room through the air conditioning system, or remain in the air for longer than 15 min. figure 4 also shows that ∼15 min is adequate for 1 μm-50 μm particles to have at least one interaction with a surface or exit the room. in the case of 50 μm particles, the particles deposit rapidly in less than a minute and mostly on the source student. the extensive deposition of 50 μm particles on the source student is due to gravitational settling and the simplified, rectangular geometry of the student modeled (figs. 1 and 2) . much of these 50 μm particles would deposit on the ground if not for the simplified student geometry. the position of the initial aerosol source in the fluid flow field affects the trajectory of the released particles [eqs. (1) and (2)]. the location of the student with respect to air conditioning influences the local flow field and particle dynamics (figs. 2 and 3) . it is, therefore, of interest to understand the extent of the effect of source location on the fate of the exhaled particles. figure 5 compares the aerosol deposition on various surfaces originating from different sources (students 1, 2, 8, and 9) using 1 μm particles. the aerosol deposition for the student 5 source was shown earlier in fig. 4(a) . the results in fig. 5 show that the effect of source location on aerosol transport can be substantial as in the case for student 8. the deposition distribution in the case of student 1 [ fig. 5(a) ] is similar to that of student 5 [ fig. 4(a) ] except for very low aerosol deposition on the ground compared to student 5 (3.9% vs 13.7%) and the increased aerosol deposition on the walls and ceiling (∼38% vs 26%). the deposition results for student 9, who is positioned in the back corner, also suggest increased deposition on the wall and ceiling to ∼44% of exhaled aerosol particles. in the case of student 1 and student 9, the increased deposition on walls and ceiling can be explained in part by proximity to walls and in part due to the vortex structures present near the edges of the room (fig. 2) . student 2 who is positioned in the front-middle, far from walls, experiences increased deposition on the walls and ceiling compared to student 5 [ figs. 5(b) and 4(a) ]. this increase in deposition on the wall may be explained by the vortices present in the flow in front of student 2 (fig. 2) . the deposition on the ground appears somewhat stochastic due to the vortices, but in general, it is <20% for 1 μm particles. the fraction of particles that exit through the air conditioning system is consistently >30% except for student 8 [ fig. 5(c) ]. the case of student 8 is special due to their unique position with respect to the air conditioning system (fig. 2) , which directs the particles downward and onto themselves (fig. 3) . less than 10% of the particles exhaled by student 8 exit the room through the air conditioning system. one of the commonly used measures to reduce covid-19 transmission is the use of sneeze guards in the form of glass or plastic barriers. the efficiency of barriers is not independent of the flow field where they are employed, which depends on air conditioning and the geometry of the surroundings. therefore, it is necessary to evaluate its effectiveness in the classroom environment especially for small particles such as 1 μm particles which can diffuse for long distances in the room. figure 6 shows the deposition distribution of 1 μm particles released from different student sources in the presence of 70 cm tall glass barriers on top of the desks [ fig. 6(f) ]. the deposition of the particles on the screens varies significantly from one source to another. the fraction of 1 μm particles deposited on the screens is very small (<1.5%) for students 5, 8, and 9 [figs. 6(c)-6(e)]. more significant deposition on the screens is observed in the case of student 1 (9%) and student 2 (∼51%), as shown in figs. 6(a) and 6(b). differences in the aerosol deposition compared to the case with no barriers (fig. 5) are also observed. the differences can be attributed to the modulation of the local flow field as a result of the barriers, which further depends on the position of the barrier in the flow field. notably, the inclusion of barriers decreases the total fraction of particles deposited on the students by ∼63% on average compared to the case with no barriers. however, barriers appear to slow down aerosol removal and deposition. for instance, ∼20% of the particles remain in the air after 15 min in the case of student 9 when barriers are used, while only ∼3% of particles remain in the case with no barriers. it is difficult to assess the effectiveness of glass barriers in reducing aerosol transmission based on figs. 5 and 6, which do not discriminate between the source student and receivers. for a clearer comparison, fig. 7 shows source-receiver maps for 1 μm particles in the absence and presence of screens. the sources considered are student 1, student 2, student 5, student 8, and student 9. self-deposition is indicated in a box next to each student, and the fraction of aerosol deposited on other students is marked by arrows from the source to the receiver. a threshold of 0.01% (∼10 particles) is applied to the maps. the use of a threshold is to ensure that only statistically meaningful numbers are reported. on average, the total fraction of aerosols transmitted from a source student to others in the classroom decreases by ∼92% in the case with screens. in the presence of screens, very few aerosol particles (<0.01%) are transmitted from student 1 to the others in the room, and self-deposition is significantly reduced from 1.5% to 0.3%. in the cases of student 2 and student 5, aerosol transmission to others and self is consistently reduced with the exception of increased transmission from student 5 to student 9 (from 0.04% to 0.08%). in the case of student 8, self-deposition increases from ∼47% to ∼60% and deposition on students 6 and 9 increases, but deposition on others decreases significantly. in the case of student 9, total aerosol transmitted to others is reduced by ∼74%. however, transmission from student 9 to student 5 increases from 0.02% to 0.3% and that from student 9 to student 7 increases from 0.05% to 0.18%. overall, the addition of screens substantially reduces aerosol transmission from one student to another, but it does not eliminate particle transmission between students. the effect of opening windows while the air conditioning system is running is investigated in order to understand its impact on particle removal compared to the case with windows closed. a typical sliding window can be opened up to 50% of its total width. the present work considered cases with 0%, 10%, 20%, 30%, 40%, and 50% open windows using 1 μm particles. the source student is assumed to be student 5. figure 8 shows the effect of opening windows on aerosol deposition and removal. the total fraction of particles that exit the system through the windows and air conditioning outlet is increased on average by ∼38% [ figs. 8(a)-8(f) ]. the fraction of particles that exit the system through air conditioning is reduced by ∼60%. this is advantageous as fewer particles may be able to transfer to other rooms bypassing the air conditioning filters. the fraction of particles that exit through the windows appears to be affected by the extent to which the windows are open. the results shown in figs. 8(a)-8(f) suggest that there may be an optimal configuration such that the fraction of particles that exit the system is maximized although no systematic trend is observed. the fraction of particles that exit the system for 0%, 10%, 20%, 30%, 40%, and 50% open windows is 50%, 87%, 51%, 92%, 36%, and 78%, respectively. on average, ∼69% of particles exit the system when windows are open at all, compared to 50% with windows closed. with the exception of 40% open windows, opening windows increases the fraction of particles that exit the system. the results demonstrate that a large fraction (24%-50%) of smaller particles (<15 μm) exit the room without interacting with any surfaces in the room. this finding highlights the need for efficient filtering in the air conditioning systems. the aerosol released from students disperses in the room, and its concentration decreases. the concentration of the aerosol particles increases again as they enter the air conditioning system. the transfer of a larger fraction of exhaled particles to the air conditioning return diffuser, although beneficial to individuals in the room, may pose greater risk to individuals in other rooms as air conditioning systems often use recycled air. it is also found that a 2.4 m separation distance between students is inadequate to eliminate particle transmission between students with the exception of 50 μm particles. the fraction of particles that exit the system without interacting with any surfaces depends on the source location. interestingly, students closer to the supply diffusers such as student 1, student 5, and student 9 are associated with greater particle exit fractions than students closer to outlets such as student 2 and student 8. the position of the student in the flow field significantly affects particle transport. significant aerosol deposition (∼47%) on student 8 is observed due to the aerosol they released. this is due to their unique position in the flow field near a vortex region close to the edge of the room and close to an outlet. an important implication of this increased aerosol deposition on student 8 is that it suggests the presence of mixing hotspots in the room where aerosol deposition can increase by as much as tenfold. in such a hotspot, if two students are present, the chances of aerosol transmission between the two will be significantly higher than elsewhere in the room. this highlights the need for thorough characterization of aerosol transport in different environments to identify and avoid hotspot areas. sneeze guards/glass barriers were found to effectively reduce the transmission of 1 μm aerosol between students by ∼92% on average. while the fraction of particles deposited on the screens directly is small in most cases studied, the screens appear to modulate the local flow field resulting in less aerosol transmission between students. screens, however, do not completely eliminate transmission of 1 μm particles between students and their effectiveness depends on source location within the classroom with respect to the air conditioning system. nevertheless, the 92% reduction in aerosol transmission is highly beneficial. opening windows was found to increase the fraction of particles that exit the system by ∼38% compared to the case with closed windows. the fraction of aerosol particles that deposit on students (including the source) decreased from 2.3% to an average of 0.45% when windows are open at all suggesting that opening windows reduces aerosol deposition on students by ∼80%. the present study only investigated one source (student 5) for cases with open windows. however, the results suggest that opening windows while the air conditioning system is running reduces aerosol transmission between students and increases the fraction of particles that exit the system. the present work is subject to many limitations. first, deposition of aerosol particles on contact with solid surfaces is assumed. reflection and re-entry are not considered. this is, however, justified as most of the simulations conducted in this study are of 1 μm particles. particles <50 μm in diameter can stick to surfaces through van der waals forces. 34 adhesion forces acting on 1 μm particles can exceed gravitational force acting on the particle by factors greater than 1 × 10 6 . 34 adhesion forces, however, depend on particle properties, surface properties, and environmental factors. 33 second, the present work does not investigate the synergy between the different factors considered. for instance, the effect of opening windows on aerosol removal and deposition is not necessarily independent of particle size. nevertheless, investigating the synergy between the different variables would necessitate extensive computational resources not available to the current project. the current study is rather focused on identifying what factors are important for aerosol transport in a classroom in order to inform other studies that may further investigate the interactions between the different factors. third, the deposition fraction is assumed to be a single deterministic value. statistical characterization of the deposition fraction would be of interest especially because of the existence of recirculation and vortices near the edges of the classroom. fourth, classrooms are subject to extensive variability in sizes, air conditioning, student distribution, and student age/, which would affect aerosol deposition and removal. effective mitigation strategies should consider multi-layer approaches including using masks, redistributing students, using glass barriers, opening windows, optimizing the air conditioning system for maximum particle removal, and improving air conditioning filters. understanding aerosol transport in different environments is of critical importance to covid-19 mitigation measures. the present study investigated aerosol removal and surface deposition in a realistic classroom environment using computational fluidparticle dynamics (cfpd) simulations. a model classroom that included nine students and a teacher was constructed. air conditioning of the classroom followed ashrae 62.1 ventilation standards for acceptable indoor air quality. four different factors were considered: particle size (1 μm-50 μm), source location (students 1, 2, 5, 8, and 9), presence of barriers/sneeze guards, and opening windows (10%-50% of window width). the following points highlight the main findings of this work and the implications of these findings: (a) aerosol distribution in the room is not uniform and is strongly influenced by the air conditioning layout. (b) even with only 9 students in the room and 2.4 m distance between students, the aerosol (1 μm-20 μm) is transmitted in significant quantities between students and from one student to other students' desks with aerosol transmission between two neighboring students reaching 0.9% of exhaled particles in some 1 μm particle cases. studies have estimated that ∼20 000 particles in the 0.8 μm-5.5 μm range are released and that over 100 000 virions are emitted per minute of physics of fluids article scitation.org/journal/phf speaking. 35, 36 therefore, particles transmitted between neighboring students separated by a 2.4 m distance in a classroom may exceed 180 particles per minute. the transmission of particles from one student to other students' desks highlights the need for hand sanitization even without contact with other students' belongings. (c) the effect of source location on aerosol transport is significant. student 1 in the front corner transmitted ∼0.55% of exhaled 1 μm aerosol particles to other students, while student 5 in the middle transmitted ∼2.1% of exhaled particles to others. removing the middle student seat (student 5) may help reduce the risk of infection to others. furthermore, student position appears to affect the likelihood of receiving aerosol particles from others. students 7 and 9 in the back corners received 2 to 3 times less particles on average than most other students in the room. therefore, students at risk of covid-19 complications may be placed in positions with a lower chance of receiving particles. (d) opening windows while the air conditioning system is running, while not recommended from an hvac point of view, significantly increases particle exit fraction by ∼38% and reduces transmission between students by ∼80%. (e) glass screens reduce aerosol transmission from one student to another and should be used. the extent of their effectiveness depends on the source location with respect to the air conditioning system. (f) particles disperse in the room and re-concentrate at the return ducts of the air conditioning system. a large fraction of exhaled particles end up in the air conditioning system, which highlights the need for effective filtration and sterilization systems within air conditioners. finally, the results of this work should be interpreted under the context of the air conditioning layout and student distribution used. other classrooms may employ different air conditioning standards and might necessitate aerosol transport investigations tailored to the specific classroom. each case of the 20 cases simulated in this work consumed ∼9 h running on four computer cores. notably, this runtime was enabled by freezing the continuum solver upon convergence of the residuals. only the discrete phase transport is solved for a simulation time of 15 min. mr. ibrahim el-hagali at pennsylvania state university is gratefully acknowledged for providing computational resources used in this work. the data that support the findings of this study are available from the corresponding author upon reasonable request. transmission of covid-19 virus by droplets and aerosols: a critical review on the unresolved dichotomy the flow physics of covid-19 review of indoor aerosol generation, transport, and control in the context of covid-19 visualizing speech-generated oral fluid droplets with laser light scattering how can airborne transmission of covid-19 indoors be minimised? an air distribution optimization of hospital wards for minimizing cross-infection effectiveness of direct lagrangian tracking models for simulating nanoparticle deposition in the upper airways the numerical computation of turbulent flows two-equation eddy-viscosity turbulence models for engineering applications characteristics of exhaled particle production in healthy volunteers: possible implications for infectious disease transmission quantity and size distribution of cough-generated aerosol particles produced by influenza patients during and after illness characterizations of particle size distribution of the droplets exhaled by sneeze a cfd modeling study in an urban street canyon for ultrafine particles and population exposure: the intake fraction approach removal of exhaled particles by ventilation and deposition in a multibed airborne infection isolation room radiation dosimetry of inhaled radioactive aerosols: cfpd and mcnp transport simulations of radionuclides in the lung visualization of local deposition of nebulized aerosols in a human upper respiratory tract model sneezing and asymptomatic virus transmission on respiratory droplets and face masks virus transmission from urinals can a toilet promote virus transmission? from a fluid dynamics perspective modelling aerosol transport and virus exposure with numerical simulations in relation to sars-cov-2 transmission by inhalation indoors transport and trajectory of cough-induced bimodal aerosol in an air-conditioned space american society of heating, refrigerating, and air-conditioning engineers american society of heating, refrigerating, and air-conditioning engineers development of turbulence models for shear flows by a double expansion technique cfd simulation of cross-ventilation for a generic isolated building: impact of computational parameters comparison of indoor aerosol particle concentration and deposition in different ventilated rooms by numerical method an investigation of particle trajectories in twophase flow systems slip correction measurements of spherical solid aerosol particles in an improved millikan apparatus deposition of micron-sized particles on flat surfaces: effects of hydrodynamic and physicochemical conditions on particle attachment efficiency the airborne lifetime of small speech droplets and their potential importance in sars-cov-2 transmission size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities key: cord-304048-wncau6zm authors: sun, ke; lu, lin; jiang, hai title: a numerical study of bend-induced particle deposition in and behind duct bends date: 2011-12-23 journal: build environ doi: 10.1016/j.buildenv.2011.12.009 sha: doc_id: 304048 cord_uid: wncau6zm this paper investigated the microparticle deposition and distribution due to the presence of duct bends by employing the eulerian approach with reynolds stress turbulent model and a lagrangian trajectory method. the air velocity, particle velocity and particle deposition velocity were validated with available experimental data. several particle deposition ratios were proposed to describe the particle accumulation due to bends. particle deposition velocities in and behind bends were analyzed numerically. it is found that bend walls with surfaces of higher capture velocity tend to accumulate more contaminant particles as seen with an increased factor of 1.2 times on particle deposition velocity. particle deposition reaches a maximum value near bend outlet, e.g. 15.2 times deposition ratio for particles of d(p) = 23 μm, and decay exponentially to a status of fully developed deposition in approximately 10d length. compared to traditional consideration of sole deposition in bends, a new general concept of total deposition including that in bends and behind bends is proposed to better describe the particle deposition induced by bends since the enhancement deposition ratios behind bends compose 42–99% in the total ratios for particles of d(p) = 3–23 μm. furthermore, models of fast power and exponential decay trend are demonstrated to uncover the relationship among enhancement factor of deposition velocity behind bend, dimensionless distance behind bends and particle stokes number. the present study can contribute to the understanding and controlling of contaminant aerosol flow behavior in ducts, e.g. particle sampling, removal and associated epidemiologic study between particle and human health. contaminant particles suspending in a room may cause health problems to the occupants [1e5], for example, robust associations between ambient pm concentrations and human morbidity and mortality [6] and the transmission of severe acute respiratory syndrome (sars) [7] . nowadays, to provide a comfortable built environment, central ventilation systems are commonly installed in modern commercial, institutional and public buildings, and therefore the ventilation ducts become one main way to transport particle-laden airflow [8] . in real ventilation ducts, one of the main parts is the bend for flow direction adjustment and pollutant aerosol deposition [9] . additionally, there are other practical applications of particle flowing through bends, including the removal of particulates from industrial duct bends [10] and extractive sampling of pollutants in gas flow [11, 12] . to construct the ventilation ducts, many kinds of materials can be adopted [13, 14] . several kinds of steel and aluminum with different coatings are used widely, and materials with special usage such as plastic, glass and masonry could also be found. construction materials of ducts play significant roles in ventilation systems [13, 15] . in previous research, however, only sippola and nazaroff [16] investigated the deposition in 90 ventilation bends related to typical commercial or public mechanical ventilation systems. peters and leith [10] studied industrial pipe bends with grease coating which was assumed to capture all particles approaching it. in the reported studies of mcfarland et al. [11] and pui et al. [12] , the deposition surface materials were not considered. few research was conducted to investigate the particle distribution and deposition in 90 bends regarding different wall materials. in view of the experimental approach, particle distribution and deposition are affected by many factors [17e19], hence the experimental analysis of the particle behavior throughout duct bends becomes quite challenging. the other method, computational fluid dynamics (cfd), has become an efficient and robust tool to investigate the airflow and particle behavior in ventilation ducts according to previous studies [20e23] . however, little research adopted the cfd method including a discrete trajectory approach to study particle deposition and distribution throughout bends. in our previous studies [24, 25] , the cfd approach together with an improved particleewall impact model was successfully applied to predict the particle distribution and deposition in 90 bends. furthermore, experimental studies of the enhanced particle deposition behind bends have been reported recently [16, 26] . miguel et al. [26] conducted the study of aerosol deposition and distribution in bifurcating duct system which corresponded to human respiratory system. it was found that particles accumulated mainly around the bend segment. sippola and nazaroff [16] investigated the mechanical ventilation system with bends and reported sharp increase of deposition velocity behind a bend about one to two order of magnitude. however, related research about the sole bend effect on the succeeding long straight duct was seldom found. therefore, this work aims to further study the particle deposition and distribution caused by bends by initiating the concept of deposition ratio and enhancement factor. the airflow phase and particle phase are solved by the eulerian method with reynolds stress turbulent model and a lagrangian discrete tracking approach with a particleewall collision model, respectively. the improved cfd approach is then used to analyze the particle deposition ratio, deposition velocity and associated distributions inside and behind bends. in these perspectives, four demonstrating decaying equations are proposed to describe the enhanced deposition behind bends. the air phase which was governed by eulerian conservation equations was predicted by employing the generic cfd approach, fluent [27] . the reynolds stress model (rsm) was adopted to solve the airflow turbulence along with two-layer enhanced-wall model. this is attributed to the reason that this rsm model performs reasonably well for two-dimensional flows [28] because it accounts for the effects of curvature, swirl and rotation and can resolve the near-wall flow features. the ability of this model together with two-layer model has been verified clearly to prediction particle deposition in straight ducts accurately [29] . the flow boundary conditions were given as follows. at the flow outlet, neumann boundary condition was adopted, i.e. mass conservation conditions for velocities and zero gradient boundary conditions for other variables. the duct walls were assumed to be smooth. in order to impose fully developed turbulence profile, the velocity profiles at flow inlet of y direction were defined according to the following 1/7th power law equations, where u ave is the averaged velocity in the straight duct before bend, d is the cross dimension accordingly and x d is nearest distance from one of the two inlet duct walls. the turbulent kinetic energy k was given as, where r a is the air density, the constant c 0 equals 0.09 and f is the fanning friction factor. this inlet condition was integrated into the cfd code by programming a user-defined function (udf). with regard to numerical scheme, the finite volume method (fvm) together with simple algorithm [30] was adopted within the cfd code. the rsm model employed the second-order upwind scheme for all the variables except pressure. the discretization of pressure was based on a staggered presto! scheme [27] . scaled convergent residuals were less than 10 à5 for velocity components, turbulent quantities and reynolds stress variables. the solid and spherical particle flow was assumed to be diluted enough to ignore the interaction of particle to particle and particle to air. therefore, all the computational results were obtained by utilizing one-way coupling method. depending on these assumptions [9] , the lagrangian formulations of particle motion were formed as follows, where u pi stands for particle velocity in the ith direction (m/s) and u i is the air velocity. the first term on the right is the drag forces exerted on the particle (per unit particle mass) in the ith direction (m/s 2 ), and the second one is the gravitational force per unit particle mass. the third term means other potential forces. these forces were studied by zhao et al. [22] , and saffman force was suggested to be important for large particles. therefore, only this lift force was added into the third term of eq. (5). since the particle motion trajectory can be influenced by the instantaneous fluctuating airflow velocity, 'eddy lifetime' model was utilized [31] . this particleewall impact model initiated by brach and dunn [15] accounts for the particleewall interaction including material dissipation, friction and adhesion in a convenient algebraic formula. the coefficient of restitution, e, can be written as follows, where, v p 1;n and v p 2;n are particle normal incident and reflected velocities, respectively, r pw for the coefficient of restitution without adhesion, r pw as the coefficient of adhesion. if this coefficient satisfies the relation r pw ¼ 1, particles will be all trapped on a surface; if r pw equals 0, it means there is no adhesion effect. these two coefficients can be expressed as, where, the parameters k, l, a, b, and v c (capture/critical velocity) are determined by experimental method [15] . due to the limited numbers of experimental surfaces, only three types of experimental surfaces were selected to analyze the material effect on particle deposition and distribution near the wall surfaces. these surface parameters are listed in table 1 . the capture velocities for the three pairs of copper, mica and molybdenum surfaces are 0.29, 1.10 and 1.47 m/s respectively. it means that the particle motion near copper surface possesses the lowest capture velocity and that near molybdenum hold the highest. the particles are assumed to be captured on a surface if the incident normal velocity falls below the capture velocity. to incorporate the particleewall impact model into the cfd code, a udf program in bends was developed. three particle size groups (3, 5 and 7 mm) were studied with three different surface materials to conduct material comparisons considering the available narrow range (3e7 mm) of particle diameter provided by existing experimental data [15] . for copper surface, another four particle sizes (10, 16, 20 and 23 mm) were also investigated to estimate the particle deposition velocity, and to compare the prediction result with available experimental data. this study presented the following two methods for the investigation of the particle deposition results in bends: 1) by demonstrating the particle deposition amount at different reflection angle from q ¼ 0 to 90 . the deposited particle number n dq from the bend entrance to a specific angle q was expressed as, where n 0 and n q are the particle number traveling through bend entrance and reflection angle q, respectively. in order to interpret the deposition amount increase caused by bend, a concept of "bend-induced deposition ratio throughout a bend" (r db ) was proposed as follows, where n d and n f are the particle number deposited in a bend and those in the straight duct of fully developed flow section with a length d, respectively. in this article, n f is selected as the average deposited particles between the reference sections of 3de2d and 2de1d before bend inlet. r b and d are bend radius and the cross dimension of the straight duct before bend, respectively. the symbol a stands for the total reflection angle of the bend, and in this paper it is p/2. in this formula, the deposition in the bend is treated as "equivalent length of straight duct" with fully developed flow. this deposition ratio can show the average deposition amount enhancement due to bend-induce flow turbulence and direction change. 1) by employing the correlation between dimensionless particle deposition velocity v þ d and particle relaxation time s þ p , which are determined by the following equations, where, v d means the deposition velocity, u w is the friction velocity, c c is the cunningham slip correction factor, r p stands for the particle density, d p is the particle diameter, m and n are dynamic and kinematic viscosity respectively of air. in this proposed lagrangian tracking approach, the particle deposition velocity onto a bend surface can be generally estimated by calculating the number of deposited trajectories in a time period as below, where, t b is the flying time scale for particles through a bend; n 0 and n d are the numbers respectively of the particles that enter the entrance of the bend and those deposited within the bend; and, a and v are the area and volume of deposition bend respectively. similar method to eq. (13) has been successfully employed before by previous researchers [18, 32] to predict particle deposition in straight duct flow. to investigate bend-induced particle deposition behind bends, this paper gives two methods as follows: 1) by adopting the relationship between dimensionless particle deposition velocity v þ d and dimensionless particle relaxation time s þ p , which has been expressed in eqs. (11) and (12) . the particle deposition velocity v da behind bends was estimated by the following equation, where u b stands for the bulk flow velocity at the domain inlet, n in is the particle number entered into a specific straight duct section, n out is the particle number escaped from the straight duct section, d is the duct cross dimension, l is the length of the straight duct. this approach has been proved before in sole straight duct particle deposition [9] , and it is applied here to study the deposition behind bends with developing turbulence [16] . the enhancement factor of deposition velocity behind bends (e da ) can be calculated by, where v df is the deposition velocity in straight duct between the reference sections of 3de2d and 2de1d before bend inlet, where the flow is assumed to be fully developed. this deposition velocity is also estimated by eq. (14) . 2) by introducing a concept of "bend-induced deposition ratio behind a bend" (r da ) as follows, where n da is the deposited particle number behind a bend at a specific straight duct section. following this expression, the increased degree and trend of particle deposition can be clearly seen due to the bend-induced turbulence behind the bend. table 1 condition and information for example particle impacts [15] . combining eq. (10) and eq. (16), "total bend-induced deposition ratio per cross dimension d" (r dt ) can be expressed as, where s is the distance away from bend outlet. this equation reflects the total particle deposition amount increase due to bend. furthermore, relatively to the deposition in fully developed flow section, "total bend-induced enhancement deposition ratio per cross dimension d" (r dte ) can be determined by subtracting the deposition of fully developed section in the equation above, this definition shows the total ratio of the additional deposition increase attributed to the existence of bend. the geometry and mesh of a two-dimensional 90 bend are shown in fig. 1 . with regard to this bend, the width d, radius r b and curvature ratio r o are 0.1 m, 0.17 m and 3.4, respectively. four grid systems were tested to check the mesh independence as shown in table 2 . all the grids were denser near the wall areas to solve boundary flows with the first near-wall grid from 0.0044 to 4.2 for the four grid systems. the mesh numbers in bends were respectively 42 000, 15 400, 9744 and 5850 from grid system 1 to 4 as stated in table 2 . the results in fig. 2 show similar results are obtained from the different grid systems with 97% agreement. therefore, a relatively coarser grid system, i.e. system 3, was chosen to benefit the computational efficiency. sixty thousand monodisperse spherical particles uniformly distributed at the entrance of the computational domain were released as lagrangian discrete tracking approach was employed to solve the particle motion equation. the influence of this particle number on particle motion was checked with two hundred thousand particles, and subtle difference was found. in addition, the densities of the particles and the air are 2990 kg/m 3 and 1.225 kg/ m 3 , respectively. the air viscosity is 1.7894 â 10 à5 kg/m/s. present simulation results are shown together with previous results of laser doppler velocimetry (ldv) experiment [33] in figs. 3 and 4. the air and particle velocities were normalized by the bulk air velocity u b , and these dimensionless velocities were plotted against dimensionless wall distance r* for comparison as shown in fig.1 . the comparisons indicate that the present model achieve more than 95% consistency with experimental data. this result shows that twodimensional flow is accurate enough and relatively efficient. since current numerical study focuses on introducing and studying the concept of bend-induced deposition in and behind bends, therefore, the simplified two-dimensional bend is utilized in this article. fig. 5 demonstrates the detailed particle numbers deposited from bend inlet to locations at the four deflection section q ¼ 0 e90 with three surfaces for d p ¼ 3e7 mm particles. the deposited particles are calculated by eq. (9). the deposition particle number increases slowly from q ¼ 0 to 60 and obviously from q ¼ 60 e90 . when particle diameter d p increase from 3 mm to 7 mm, the particle number at the bend entrance decreases considerably, especially for molybdenum surfaces. with much larger particles in the experimental parameters, this decreased degree would be much higher, especially for large capture velocity surfaces. take d p ¼ 7 mm for example, the deposited particles in bends are 1.7 times on molybdenum surfaces to those on copper surfaces. fig. 6 summarizes previous experimental data and predicted particle deposition velocities against particle relaxation time in 90 [11, 12, 16] in 90 bends were collected in the figure. the experimental data from liu & agarwal [34] was also presented to compare the deposition velocities in bends with those in straight ducts. it can be seen that predicted deposition velocities in bends are much larger than those in straight ducts by one or two orders of magnitude. sippola and nazaroff [16] also drew a similar conclusion when comparing their experimental data in bends with those in their straight ducts. among the three experiments in bends, result data also change by about one order of magnitude due to different experimental conditions. simulated particle deposition velocities from s þ p ¼ 1.2 to 32.9 fall within the experimental data [11, 12, 16] . the surface effect on particle deposition velocity can be clearly seen from fig. 7 . the deposition velocity with copper surface is the lowest. this observation can also be seen in fig. 5 where deposited particle numbers with different surfaces are presented. the reason of a low deposition velocity with copper surface could be due to the low capture velocity near such surface. between particle relaxation time s þ p ¼ 1:2 and 6:4, the particle deposition velocity with molybdenum surface is 0.7e1.2 times larger than that of a copper surface. given more experimental data about particle collision behavior onto different surfaces with a wide particle diameter range, more deposition velocities could be predicted following the proposed method in this work. q ¼ 0 e90 with three surfaces: (a) d p ¼ 3 mm; (b) d p ¼ 5 mm; (c) d p ¼ 7 mm. since a bend in the flow stream can cause the change of flow pattern and lead to flow turbulence behind bends, the particle deposition behind bends would be enhanced due to the bend. to describe the deposition change along the straight duct behind a bend, dimensionless distance (s*) is expressed as s* ¼ (la à x)/d, which is shown in fig. 8 . in the present work, the dimensionless distance is an integer number. in the equation, la is the total straight duct length behind bends, which equals 1.2 m in this paper, and x is the horizontal coordinate. in addition, the predicted results in the following discussion were simulated based on the wall surfaces of the lowest capture velocity. fig. 9 shows the comparison of average particle deposition velocity behind 90 bends for d p ¼ 3e23 mm particles from s* ¼ 1 to 12. correspondingly, the stokes numbers of these particles are from 0.016 to 0.89. each particle deposition velocity at an s* value is the average deposition velocity based on the deposition particle numbers from s* à 1 to s*. the calculation method can be found in eq. (14) . as shown in the figure, the magnitude of the dimensionless particle deposition velocity changes from 0.0317 to 1.332, which is much higher than those illustrated in figs. 6 and 7 . this behavior provides quantitative evidence through simulation that the deposition behind the bend is enhanced due to bend-induced turbulence. along with the increasing of dimensionless particle relaxation time from s þ p ¼ 1:21 to 67:8, the deposition velocity increases sharply by 4.8e10 times. current results of the enhanced particle deposition behind bends and the changing trend could explain and support previous experimental visualized observations and reports behind bends qualitatively [16, 26] . however, when the distance increases away from the bend outlet, i.e. from s* ¼ 1 to 12, the particle deposition velocity decrease. the maximum deposition velocity is located at s* ¼ 1, which means the areas between bend outlet and one cross dimension distance for particles of specific stokes number. from s* ¼ 1 to 2, the particle deposition velocity reduces tremendously, which reflects the rapidly decreasing influence of bend-induced vortices. the reduction degree from s* ¼ 2 to 5 is moderate and from s* ¼ 6 to 12 is minor. the decreasing deposition could be probably attributed to the turbulent level reduction when the distance increase behind bends as shown in fig. 10 . from s* ¼ 1 to 5, it can be seen in the figure that large vortices are produced behind the bend, while from s* ¼ 6 to 12, turbulent levels are rather low. to describe the quantity of particle deposition along the straight duct behind bends, the bend-induced deposition parameter r da is demonstrated in fig. 11 . in the presentations of the deposition ratio and the following enhancement factor, eqs. (1e4) were adopted at the domain inlet to guarantee the airflow was fully developed in straight duct ahead of bends. the results range from r da ¼ 0.91 to 15.2, which indicate the quantitative increase of particle deposition caused by bend. particle accumulation is very high near the bend outlet. deposition ratios increase from 4.61 to 15.2 times for particle diameters increasing from d p ¼ 3e23 mm. the deposition is reduced sharply from areas near the bend outlet to those further away from it. from s* ¼ 10 to 12, the deposition ratios are close to unity 1. this phenomena means that the flow disturbance due to bend is weakened to a marginal level at these areas, and the length of increased deposition due to the presence of the bend is fig. 11 . bend-induced deposition ratio distribution along the distance behind bend for different particle diameter. 10 times as long as the cross dimension (d) of straight duct. this interpretation is partially supported by the experimental measurements of particle deposition in developing turbulent flow in the studies of sippola and nazaroff [16] . fig. 12 illustrates the bend-induced enhancement deposition ratio in and behind bends for present simulation domain. the enhancement deposition ratios in bends and behind bends correspond to the first and second term in eq. (18), respectively. these ratios are calculated based on eqs. (16)e (18) . they can be also called as additional deposition caused by bend. for small particles, for example, d p ¼ 3 mm, the enhancement deposition ratio is rather low because the ratio considers the bend as equivalent straight duct and the stokes number indicates that small particles are easy to follow fluid flow. in other words, smaller particles are hardly to impact onto bend walls. however, the flow pattern change by bends also leads to deposition enhancement behind bends, and the total ratio is mainly composed of the enhancement deposition behind bends, approximately 99% for particles of d p ¼ 3 mm. this behavior is probably due to that eddies behind bends are adaptable to catch smaller particles onto wall surfaces as demonstrated in fig. 12 . for larger particles, the major composition of the total ratio is reversed, i.e. in bends. however, the deposition behind bends are still of a great portion, approximately 42% for particles of d p ¼ 23 mm. therefore, when describing the particle deposition induced by bends, the general concept of total deposition including that in bends and behind bends is much better than sole particle accumulation in bends. it is interesting to find that the decay trend of particle deposition ratio behind bend in fig. 11 . this decay trend satisfies two conditions: (1) it peaks at s* ¼ 1; (2) when s* ! n, the deposition ratio asymptotically approaches unity 1. the second condition means the particle deposition is in a fully developed flow. therefore, subtracting the deposition in a fully developed flow, the "enhancement deposition ratio behind a bend" (r dae ) would be infinitely small at s* ! n. take the particles of d p ¼ 20 mm for example, as shown in fig. 13 , the relationship between the enhancement deposition ratio (r dae ) and dimensionless distance (s*) can be built as follows, r dae ¼ 11:5ðs*þ à1:67 ; at st ¼ 0:674 (19) the r 2 value is as high as 0.982, which shows a well-fitting between the simulation data and the proposed equation. in addition, this fitting can also transformed as exponential correlation between the two variables. for other particle diameters, similar fittings can be obtained through this method. enhancement factor of deposition velocity behind a bend (e da ) varies with dimensionless distance (s*) and particle diameter or stokes number (st), as shown in fig. 11 . a three-dimensional surface fitting code (tablecurve 3d, jandel scientific, san rafael, ca) was adopted to develop least square fits of the current simulation data [11] . therefore, a non-linear fitting of r 2 ¼ 0.994 could be expressed as follows, decreasing relationship only with dimensionless distance and particle stokes number. this relationship is valid for s* ¼ 1e12 and st ¼ 0.016e0.89. combining eq. (15) and eq. (20) , the dimensionless deposition velocity behind bend ðv þ da þ is deducted as, which means the dimensionless deposition velocity in the developing turbulent flow behind bend can be associated with the reference dimensionless deposition velocity v þ df by the enhancement factor. substituting the dimensionless parameter st with s þ p , the function y st becomes, this equation reveals that the dimensionless deposition velocity behind bend (v þ da ) is related to dimensionless particle relaxation time as shown in fig. 9 . furthermore, the curves of s* ¼ 6e12 shown in fig. 9 exhibit similarity to develop a more universal relationship than that in eq. (21) . by averaging seven dimensionless deposition velocities at each dimensionless relaxation time, fig. 15 is obtained together with the standard deviation of the averaged values. using a polynomial fitting of r 2 ¼ 0.999, the relationship is expressed as, under current simulation conditions, this relationship reveals that dimensionless deposition velocity depends only on dimensionless relaxation time for s* ¼ 6e12 as demonstrated in fig. 15 . therefore, eq. (23) is easy to use in the applications. this paper investigated the microparticle deposition in and behind 90 bends. particle deposition distribution and deposition velocity in 90 ventilation bends with different surface materials were analyzed numerically, and the simulation results were also validated with experimental data. bend-induced deposition ratio and deposition velocity were also discussed and fitted quantitatively. to summarize, the following conclusions can be drawn: a) predicted particle deposition velocities in bends are much higher than those in straight ducts by one to two orders of magnitude. between the predicted particle relaxation time 1.2 and 6.4, the particle deposition velocities with surfaces of highest capture velocity can be 1.2 times larger than those of lowest capture velocity. this behavior indicates that the duct bend wall with surfaces of high capture velocity is easy to accumulate contaminant particles. b) current simulation provided quantitative evidence that the deposition behind a bend is sharply enhanced due to bendinduced turbulence. particle deposition had a peak value near bend outlet. along the straight duct behind bends, the depositions decreased exponentially toward a deposition pattern of fully developed turbulent flow since the bend-induced eddies are weakened. c) it is much better to adopt the general concept of total deposition including that in bends and behind bends to describe the particle deposition induced by bends. deposition behind bends forms a large portion of the total bend-induced deposition, especially for smaller particles. d) power decay trend was obtained between the enhancement deposition ratio (r dae ) and dimensionless distance (s*). nonlinear exponential decay trend was also given among enhancement factor of deposition velocity behind a bend (e da ), dimensionless distance (s*) and particle stokes number (st). these fitting equations are valid according to the current simulation condition for the ranges of s* ¼ 1e12 and st ¼ 0.016e0.89. finally, two fitting equations of dimensionless deposition velocity behind bend were developed to model the deposition trend easily and universally. this work proposed new concepts of bend-induced deposition which includes the deposition both in bends and behind bends. quantitative discussion was given to evaluate the two location depositions. the decaying correlations were proposed based on current results. this paper contributes to the understanding of pollutant particle deposition and distribution in and behind ventilation duct bends, which can be applied on contaminant particle sampling, removal and associated epidemiologic study between particle and human health through ventilation system. this work was financially supported by the hong kong polytechnic university through research grants a-pj61 and a-pj12. length of the straight duct behind a bend, m la total length of the straight duct behind a bend, m k turbulent kinetic energy, kg ms à1 n 0 number of particles flew across bend entrance n d number of particles deposited on bend wall surface n da number of particles deposited behind a bend at a specific straight duct section n f number of particles deposited on straight duct of fully developed flow section ahead of a bend n in number of particles entered into a specific straight duct section behind a bend n out number of particles escaped from a specific straight duct section behind a bend n q number of particles flew across bend reflection angle q r 1 , r 2 radius of the inner and outer wall of the bend, respectively, m r* dimensionless wall distance r o bend curvature ratio r b bend radius, m r da bend-induced deposition ratio behind a bend r dae bend-induced enhancement deposition ratio behind a bend r db bend-induced deposition ratio throughout a bend r dt total bend-induced deposition ratio per cross dimension d r dte total bend-induced enhancement deposition ratio per cross dimension d r pw coefficient of restitution in the absence of adhesion t time, s s distance behind bend outlet, m s* dimensionless distance behind bend outlet, integer in this paper st stokes number t b flying time scale for particles through a bend, s u b air bulk velocity, ms à1 u a, u air velocity, ms à1 u p , u pi particle velocity, ms à1 u w friction velocity, ms à1 v c capture velocity, ms à1 v p 1;n , v p 2;n particle normal incident and reflected velocity, respectively, ms à1 v volume of the domain, m 3 v d deposition velocity, ms à1 v da deposition velocity in straight duct behind a bend, ms à1 v df deposition velocity in straight duct of fully developed flow section ahead of a bend, ms à1 v þ air quality guidelines for particulate matter, ozone, nitrogen, dioxide and sulfur dioxide large eddy simulation of inhaled particle deposition within the human upper respiratory tract modeling particle dispersion and deposition in indoor environments characterization of expiration air jets and droplet size distributions immediately at the mouth opening nanoparticle-laden flows via moment method: a review epidemiology of particle effects researchers get to grips with cause of pneumonia epidemic role of ventilation in airborne transmission of infectious agents in the built environment e a multidisciplinary systematic review modeling deposition of particles in typical horizontal ventilation duct flows particle deposition in industrial duct bends aerosol deposition in bends with turbulent flow experimental study of particle deposition in bends of circular cross-section ashrae handbook e heating, ventilating, and air-conditioning systems and equipment (i-p edition) particle deposition in ventilation ducts models of rebound and capture for oblique microparticle impacts particle deposition in ventilation ducts: connectors, bends and developing turbulent flow research on the transport and deposition of nanoparticles in a rotating curved pipe prediction of particle deposition onto indoor surfaces by cfd with a modified lagrangian method particle deposition in indoor environments: analysis of influencing factors comparison of different k-epsilon models for indoor air-flow computations modeling particle deposition and distribution in a chamber with a two-equation reynolds-averaged navierestokes model comparison of indoor aerosol particle concentration and deposition in different ventilated rooms by numerical method a study of particle rebounding characteristics of a gas-particle flow over a curved wall surface a computational investigation of particle distribution and deposition in a 90 bend incorporating a particleewall model experimental study and numerical investigation of particle penetration and deposition in 90 degrees bent ventilation ducts aerosol particle deposition and distribution in bifurcating ventilation ducts ansys fluent 12.0 users guide evaluation of various turbulence models in predicting airflow and turbulence in enclosed environments by cfd: part 2-comparison with experimental data from literature particle deposition in turbulent duct flows e comparisons of different model predictions numerical heat transfer and fluid flow turbulent dispersion of particles using eddy interaction models aerosol particle transport and deposition in vertical and horizontal turbulent duct flows ldv measurements of a turbulent air-solid 2-phase flow in a 90-degree bend experimental observation of aerosol deposition in turbulent flow key: cord-356116-c40e3zp7 authors: savory, eric; lin, william e; blackman, karin; roberto, matthew c; cuthbertson, lauren r; scott, james a; mubareka, samira title: western cold and flu (wecof) aerosol study – preliminary results date: 2014-08-23 journal: bmc res notes doi: 10.1186/1756-0500-7-563 sha: doc_id: 356116 cord_uid: c40e3zp7 background: influenza virus is responsible for annual deaths due to seasonal epidemics and is the cause of major pandemics which have claimed millions of human lives over the last century. knowledge about respiratory virus transmission is advancing. spread is likely through the air, but much work remains to be done to characterize the aerosols produced by infected individuals, including viral particle survival and infectivity. although coughs have been characterized, little work has been done to examine coughs from infected individuals. the wecof project aims at providing evidence to support prevention measures to mitigate person-to-person influenza transmission in critical locations, such as hospitals, and during pandemics. findings: a novel experimental cough chamber facility – the flugie – has been developed to study the far-field aerodynamics and aerosol transport of droplets produced by the coughs from humans naturally-infected with influenza. the flow field of each cough is measured using particle image velocimetry (piv). a preliminary study involving 12 healthy individuals has been carried out in order to quantify the strengths of their coughs at a distance of 1 m from the mouth. the spatially averaged maximum velocity was determined and the average value was 0.41 m/s across 27 coughs of good data quality. the peak value of velocity was also extracted and compared with the average velocity. conclusions: preliminary results show that there is significant air motion associated with a cough (on the order of 0.5 m/s) as far away as 1 m from the mouth of the healthy person who coughs. the results from this pilot study provide the framework for a more extensive participant recruitment campaign that will encompass a statistically-significant cohort. we have gained extraordinarily detailed knowledge in the past decade about the molecular nature of influenza virus and other respiratory viruses yet surprisingly little is known about how respiratory viruses are transmitted from person to person. mathematical modelling of households, containing infected individuals, showed aerosol transmission to be more significant than contact transmission for influenza virus and that airborne transmission may be a significant contributor [1, 2] . recent reviews of the literature support this important possibility [3, 4] . aerosols consist of particles in a range of sizes. traditionally droplets of >5 μm diameter have been implicated in short range (<1 m) spread, and droplet nuclei of <5 μm are believed to be responsible for longer range or airborne transmission (>1 m) [5] . however, larger-sized particles may be responsible for wider pathogen spread depending on other factors. for example, particles of the same size may travel different distances, depending on the velocity of the jet propelling them [6] . these particle dynamics remain undefined in the clinical setting, and the implications are significant from an infection prevention and control perspective. in addition to its effect on dispersion, particle size has implications for the inhaling host. larger particles (>10 μm) will be deposited by impaction in the upper respiratory tract and smaller particles (0.003-5 μm) may penetrate the tracheobronchial and alveolar regions, principally through sedimentation and diffusion [7] . by studying the aerosols produced by infected individuals, we hope to precisely characterize how long virus infectivity persists in suspended aerosol droplets of various sizes. recent work has begun to address this question for bacterial transmission by patients with cystic fibrosis [8, 9] . the question of droplet survival duration pertains to droplet size distribution and, on this front, there are discrepancies across the literature which are likely due to the varying measurement methodologies and techniques. solid impaction with micrometry, a method that is insensitive to smaller droplets, revealed particle sizes from 1 to 2,000 μm [10] . size distribution analysis using an optical droplet counter [11] [12] [13] is reportedly less accurate for particles >2 μm [14] but is useful in the submicron size range. other previous techniques to measure size distribution include aerodynamic droplet sizer [15, 16] , scanning mobility particle spectrometer [15] , andersen six-stage cascade impactor [17] , electrical low pressure impactor [18] , interferometric mie imaging [19] and laser diffraction [13, 20] . aside from the dependency on initial size, the change in droplet size due to evaporation and condensation is also directly related to their chemical composition [21] . recent findings suggest a tri-modal size distribution during speech or voluntary coughing, with bronchiolar fluid film burst and laryngeal modes both contributing to a distribution peak centred near 1 μm size and with an oral mode yielding a distribution peak centred near 100 μm size due to droplets produced between the epiglottis and the lips [22] . measurements near the mouth (0.17 m distance) of healthy non-smokers, giving best-effort voluntary coughs, indicate that 99% of expelled droplets are inhalable (<10 μm) [20] . for infected individuals, significant influenza rna also appears to be contained in droplets with diameters in the respirable size range -35% of influenza rna was found in droplets of greater than 4 μm diameter, 23% in droplets of 1 to 4 μm diameter and 42% in droplets of less than 1 μm diameter [23] -and symptomatic subjects appear to emit more particles [18] . however, few results are available for the relationship between droplet size and infectivity for influenza virus. the viability of airborne pathogens also depends on other factors, including environmental humidity, temperature and the presence of ultraviolet light. the relationship between these factors and infectivity is poorly understood. a recent study using a breathing mannequin and bioaerosol samplers indicated that high relative humidity (rh) was associated with reduced infectivity of influenza virus [24] . earlier studies using small settling chambers, influenza bioaerosols and guinea pigs found inactivation at high rh [25] [26] [27] [28] . evaporation and particle shrinkage are expected since particles typically enter an environment at lower rh than the respiratory tract [14] . the evaporation process is fast; particles of 10 mm diameter or less typically evaporate in less than 0.5 seconds [21] . at low rh, droplets evaporate more quickly and remain suspended longer compared to droplets generated under conditions of higher rh, thereby increasing the probability of ensuing inhalation [6, 21, 29] . temperature has also been shown to enhance or interrupt transmission at low (5°c) or high (30°c) temperature, respectively, for all values of rh [28] . assessment of data in the literature suggested a relationship between absolute humidity (ah) and influenza survival. when extended to a human population level study, negative local daily deviation of ah from its 31 year mean was found to be associated with the start of influenza outbreaks during the winter [30, 31] . studies on the effect of ultraviolet light irradiation showed avian influenza a (h7n9) virus was inactivated after at least thirty minutes of exposure [32] , while adenoviruses appeared to be uv-resistant [33, 34] . the vaccinia virus was found to be less susceptible to ultraviolet radiation at high rh than at low rh [35] . the precise mechanisms through which rh, ah, temperature and uv light exposure affect virus transmission and survival require clarification. since the severe acute respiratory syndrome coronavirus (sars cov) outbreak in 2003 [36] , the scientific and medical communities, as well as the general public, have gained an appreciation of the public health importance of understanding respiratory virus transmission. although cough droplet sizes have been characterized, more research is needed to examine cough flows from infected individuals. much is known about airflow rates during coughing [37] [38] [39] , including parameters such as cough peak flow rate (cpfr), peak velocity time (pvt) and cough expired volume (cev), that is the area under the flow rate versus time curve. a study of 12 female and 13 male subjects showed that the non-dimensional airflow rate (flow rate/cpfr) versus non-dimensional time (time/pvt) curve could be defined by two gamma-probability functions based on the medical parameters of cpfr, pvt and cev that were themselves related to height, weight and gender [40] . also, a sequential cough was found to be a combination of two single coughs, with the first being approximately the same as a single cough, whilst the second was a scaled down version of the first. visualizations [40] showed that the airflow direction did not vary greatly amongst the subjects and, although mouth opening remained constant during a cough, there was a considerable variation of area across the subjects with no correlation to other parameters such as height. their research examined the bulk parameter of flow rate, whilst earlier studies examined the flow field qualitatively using strobe photography [41] or thermal plume imaging by schlieren [42] . more recently, quantitative analysis of shadowgraph images indicated that this technique requires a significant temperature difference (10°c) between exhaled air and room temperature and that coughs were detectable out to a maximum distance of 0.6 m from the source [43] . quantitative analysis of highspeed video images of a cough were limited to a similar range whilst also requiring the cougher to expel cigarette smoke as a tracer substance [44] , which is problematic to apply in the study of humans with respiratory illness due to the unacceptable possibility of causing harm to participants. accurate velocity measurements at greater distances in the far-field of a cough require a different measurement technique and approach. particle image velocimetry (piv) velocity measurements have been undertaken using an artificial cough flow simulator [45, 46] , a thermal mannequin with simulated breathing [47, 48] and healthy human subjects [19, [49] [50] [51] . such measurements have revealed a peak cough velocity of 6 to 22 m/s, with an average of 11.2 m/s [51] , but usually mouth area is merely assumed and not measured [50] [51] [52] . kwon et al. [53] reported average initial cough velocities of 15.2 m/s (males) and 10.6 m/s (females), with the angle of exhaled air being 38°(males) and 32°(females), although it is doubtful whether this difference in angle is statistically significant. singh et al. [39] found the peak flow rate produced by women to be 60% that of men and chao et al. [19] reported the maximum cough velocity of women to be approximately 77% that of men. on the other hand, vansciver et al. [49] found no significant difference in maximum cough velocity related to sex and weight of the cougher. furthermore, they noted that, from the fluid dynamic point of view, a cough may be considered as a short-duration transient jet, being notably unlike a very-short-duration jet in which much of the cough would be entrained in a single vortex ring. their piv data showed a wide range of maximum cough velocities (1.5 to 28.8 m/s) and that the self-similarity of flow profiles associated with a transient jet was not applicable to coughs, such that it is necessary to develop an envelope of cough profiles rather than attempting to define a "typical" cough. the measurements by zhu et al. [51] showed that some saliva droplets produced during a cough can travel further than 2 m and (using the lagrangian equation) that the transport characteristics of expelled saliva droplets change with size. furthermore, a recent study of patients with cystic fibrosis emitting cough aerosols, which were collected with an anderson impactor in a wind tunnel of modest cross-sectional area, reported that viable bacteria can travel 4 m from the patient or remain aloft for up to 45 minutes [9] . these findings call into question the "3 feet/1 metre rule" or "6 feet/2 metre rule", which have been considered to be safe separation distances for preventing droplet transmission [54] , and motivate further study of virus-laden bioaerosols and the velocity field at extended distances from the source. indeed, all previous piv flow measurements have been taken near the mouth, where velocities are highest, rather than far downstream near the limits of possible person-toperson transmission. the novelty of the current collaborative research projectthe western cold and flu (wecof) aerosol studylies in the fact that the fluid dynamics of the jet aerosols produced by a minimally-confined cough is being examined concurrently with the biological processes associated with virus droplet formation and transmission, using human subjects when they are naturally infected by influenza virus and, again, when they return to health. this is in contrast to previous fluid dynamic studies that have measured the velocity field using artificial aerosol sources or only healthy subjects. ultimately, the research aims of the present wecof project are; 1. to understand the penetration of viral droplets into the ambient environment, 2. to rigorously test the "3 feet/1 metre" and "6 feet/2 metre" rules and 3. to identify host determinants of individuals who emit higher quantities of virus which disperse further, all of which are important for implementation of future transmission prevention measures. the measured data will also be of use to other researchers who are attempting to develop realistic theoretical [55] or computational fluid dynamics (cfd) [50, 56] models for cough jets/plumes and virus transmission. such models require reliable modeling of the transport of aerosol droplets and virus particles. in addition, data from human subjects may be used to test simpler models that use the spatial distribution of expiratory aerosols and the viability functions of airborne viruses to estimate exposures to airborne viruses in the indoor environment, where previously such models were based on artificial puff sources, e.g. [57] . this introductory review has covered issues such as cough droplet sizes and the influence of environmental parameters, notably temperature and relative humidity, which will be studied as part of the wecof project. however, the present paper focuses on the experimental facility and methods and presents the results from the initial experiments using healthy human volunteer subjects. a novel experimental facility (the flugie cough chamber) has been constructed at western and protocols developed for its use. biosafety and research ethics approvals have been obtained for studies involving human participants who are naturally-infected with pathogens such as influenza virus. figure 1 shows a diagrammatic layout of the facility for piv measurements, with the subject seated outside the chamber. since the entrainment of ambient air into the cough jet is important to the development of that jet, a solid barrier with only a small opening for the mouth (such as the 5 × 3 cm hole used by [51] ) is inappropriate. in the flugie, the opening is pear-shaped such that the participant's nose and mouth area are unobstructed whilst a cough is directed into the enclosed test chamber. the major vertical axis of the pear-shaped opening is 15 cm high and the base of the opening, where the participant's chin rests, is 67 cm above the chamber floor. the minor horizontal axis of the pear-shaped opening is 10.5 cm wide. this chamber inlet has a cover, which is only opened when a cough is introduced into the chamber. the position of the participant's head is fixed by a chin rest and a forehead rest, such that the angle of the cough is horizontal and consistent over multiple trials. although it may be argued that a more natural cough would be observed by permitting unrestricted head motion, it is likely that such freedom would permit a significant cough velocity component due to forward translation of the participant's upper body during the expulsive phase, as well as introduce a greater unpredictability to the cough flow trajectory, which would be problematic for any experimental technique with a limited measurement site or window. from the perspective of achieving the present research aims in a controlled laboratory experiment, it is acceptable to examine the cough velocity produced by pulmonary effort alone. an open bench set-up [20] may be distinguished from a study of a confined cough but, in essence, all coughs in an indoor setting are the latter type. the salient point is to allow sufficient separation between the cough and all solid boundaries, such that the flow does not exhibit any significant deviation from a cough in fully open surroundings. even though a participant may be coughing at an open bench towards an open fume hood, the distance from the mouth to the fume hood is of interest, as well as whether the fume hood fan is operating and possibly affecting the cough flow. hence, a chamber of ample size is preferable as a quiescent environment in which a cough flow may be studied without flow disturbances from uncontrolled surroundings. the internal dimensions (1.81 m length, 1.78 m width and 1.81 m height) for flugie were sized for separation distances to be greater than participant mouth diameter by more than an order of magnitude. at the measurement window, the separations decrease to less than an order of magnitude due to the lateral spreading of the cough flow with distance from the mouth, yet remain several times the lateral extent of the cough. the test chamber is raised by 0.25 m above the laboratory floor and mounted upon casters to allow measurement at various streamwise positions. in order to quantify the viral content of the aerosols produced during coughs, particles are sampled by collection onto polytetrafluoroethylene (ptfe) membrane filters of 1.0 μm pore size and 37 mm diameter. as shown by figure 2 the identity of the pathogen acquired by each study participant is confirmed by asking for a self-collected midturbinate swab (mts) and these specimens are interrogated by multiplex polymerase chain reaction (multiplex-pcr) assay for a panel of respiratory viruses (rvp fast, luminex). the viral content from the membranes is quantified using a virus-specific monoplex quantitative real-time pcr assay and calculated using quantitative curves and number of litres of air sampled. separate measurements are performed to quantify the cough flow field. optical access areas into the test chamber are outlined in light blue in figure 1 . a beam of green light (532 nm wavelength) horizontally emitted from the laser head (120 mj, nd:yag crystal) is redirected to vertical by a 45°-angled mirror and expanded into a narrow light sheet (~1 mm thickness) with cylindrical and spherical lenses. the light sheet enters the test chamber through a glass window in its floor, and illuminates a centreline plane from the test chamber floor to the test chamber roof. the vieworks va-4 m32 camera is a charge-coupled device (ccd) with a resolution of 10.0 pixels per mm and a sensor array of 1,752 pixels by 2,336 pixels, where the longer side is oriented vertically for this experiment. the camera is focused upon the light sheet at the chamber centreline and optical access is through a glass window on a chamber wall. the test chamber is seeded with titanium dioxide particles (rutile mineral form). the product specifications indicate a particle size distribution ranging from 0.15 to 0.47 μm, where 69% of the particles are in the 0.34 to 0.43 μm size bin and 29% of the particles are in the 0.27 to 0.34 μm size bin. the titanium dioxide (tio 2 ) powder is dried in a vacuum-oven, stored in a vacuum container to minimize clumping and aerosolized using a customcrafted version of the pitt 3 aerosol generator [58] . this device consists of a cylindrical drum with small inlet and outlet ports near its bottom and top ends, respectively. the drum is filled with tio 2 powder, which is carried up and out of the drum by the flow driven by a 30 kpa air line attached to the inlet port. the drum is placed on top of a loudspeaker, which generates sound waves to vibrate and break up the powder. from the outlet port of the aerosol generator, the aerosolized particles enter a settling chamber mounted on top of the test chamber through a tube with perforations. the flugie settling and test chambers are separated by a fine mesh, which permits tio 2 particles, under the action of gravity and local airflow, to gently enter the test chamber along its centreline. the cough jet generated by the participant disturbs the tio 2 particles which are imaged to obtain quantitative information of the flow field. thus, this setup achieves an intersection of the tracer particles, the light sheet illuminating the tracer particles, the focused field-of-view of the camera recording the illuminated tracer particles and the cough flow, over a sizable region of space and time (400 cm 2 and 5 s, respectively). a pulse generator (berkeley nucleonics corporation, model 555-4c) is used to control the timing and synchronizing of the laser and camera. image pairs are captured at a rate of 16.7 hz, from which instantaneous velocity fields are calculated using commercial software (tsi incorporated, insight3g) that cross-correlates the image pairs. in the tests reported here the field-of-view of the camera is centred at 1.00 m from the cough inlet. for each study participant, following the aforementioned cough droplet sampling, piv is performed for another three independent single coughs with a settling time of 30 s between each cough. a preliminary study involving healthy individuals has been carried out in order to assess the performance of piv for measuring the far-field region of this transient and turbulent air flow. in addition, this work has provided the framework for a more extensive campaign that will encompass a statistically significant cohort. the velocity fields associated with the coughs from 12 healthy young adults were quantified at a distance of 1 m away from the mouth. this limited study with healthy volunteers leads into the recruitment and study of individuals who are naturally-infected with influenza virus. written in an ongoing pilot study at a university student health clinic (student health services at western university), a small cohort of undergraduate students, who were naturally-infected with influenza, are being referred by clinicians to the wecof aerosol study. written informed consent is being obtained from all participants in this ongoing study. the hsreb reviewed and approved this study. a germicidal lamp, which produces continuous light in the ultra-violet b range, is used to disinfect the test chamber between study participants. in addition, an outlet has been retrofitted with a hepa filter through which chamber air can be withdrawn to further reduce the risk of viral contamination between subjects. the experiments are repeated several weeks later, with the same participants, after recovery from the respiratory illness to permit an assessment of the differences in the coughs between an infected and a healthy person. preliminary experiments have been carried out involving 12 healthy volunteer participants (9 male, 3 female, ages 20 to 32). since they were healthy, cough airflow measurements using piv were conducted without viral aerosol sampling. each participant produced 3 coughs with the piv system recording image pairs prior to, during and after the cough. these were then processed to yield instantaneous velocity vector arrays within the field of view. figure 3 (left) shows that the field of view was located 1.00 m downstream of the entry to the flugie cough chamber and encompassed a region of 174.8 mm in the streamwise direction, centred at the 1.00 m location. the field of view extended over 233.1 mm vertically and was below the level of the cough to take into account the fact that, even if the cough was initially directed horizontally forward by the study participant, most of the coughs had drifted downwards at the 1 m location, contradicting the importance of buoyancy in a proposed model based on visualizations out to 0.70 m from a participant in an open laboratory setting [55] . an example of a processed vector field is shown in figure 3 (right), where the green arrows are vectors that have passed validation by the insight3g processing software. red vectors are spurious values, which were typically attributable to the reduced light sheet intensity at the edges of the field-of-view, whose size approached the upper limits of this piv equipment. based on the selected interrogation window size (i.e. the area from which a vector was calculated), the maximum possible number of vectors in each processed image was 3,816. it should be noted that the 12 participants had their coughs recorded on different days. for 3 participants, the results were poor due to low particle seeding levels and low numbers of validated velocity vectors (only about 0.8% of the total number of vectors were considered to be accurate, representing approximately 30 vectors per image). three other participants were recorded in another session and showed the best results in terms of percentage of validated vectors, with values around 90% (their data are shown in figure 4 ). the other 6 subjects were recorded on a separate day and showed acceptable validation levels in the 70 to 80% range. the findings for each of the three coughs by each of the twelve participants are given in table 1 , where the first character in the participant identifier (id) indicates participant gender (f for female and m for male) and the second character is an integer index for each individual of that gender. the magnitude of each vector in each image pair was extracted with this quantity averaged (i.e. spatially) over the entire number of such validated vectors to produce a value representing the average air velocity within the field of view and the maximum mean value, occurring in each individual cough event, is shown in table 1 . a representative measure of processed data quality is included, together with observations. furthermore, the peak value of velocity from each image pair was extracted and plotted against time for that cough. the time-histories of spatially-averaged and peak velocity values for three subjects are shown in figure 4 . it may be seen that, in all cases, the motion of the cough through the field of view, located a metre away from the cougher, is clearly defined with an initial rapid increase of velocity followed by a slower decay. it is also evident that there is no single characteristic shape for a cough velocity profile and, thus, it is necessary to define an envelope of cough profiles based on the measurement and analysis of a larger number of coughs than those examined in these initial trials. as would be expected from the limitation on the piv window size and the variable physical traits of study participants, it was found that there was considerable variation in location and strength of each cough, with some coughs missing most of the imaged field of view entirely. the distribution of values of the spatially-averaged maximum velocity magnitude is illustrated in figure 5 . the average value across all 36 coughs is 0.52 m/s, but with the data from the three poor quality experiments removed, the average across the remaining 27 tests is 0.41 m/s. these values indicate that there is significant air motion during a cough, of the order of 0.5 m/s, at a location as far away as 1 m from the person who is coughing. during the set-up phase for this preliminary study, a single volunteer produced 5 coughs, with a 180-mm-wide piv field being located at different distances from the mouth for each cough. the participant attempted to produce a series of coughs each of the same strength. although the quality of the resulting vector field was not sufficient to provide quantitative data, it was possible to identify the arrival of the cough front at each location and, thereby, estimate the velocity of the cough front at the centre of each field of view. the results are shown in figure 6 , illustrating the rapid decrease in velocity in the near-field of the mouth, as would be expected. at 1 m from the mouth the cough front velocity has a magnitude in agreement with the average of the spatially-averaged maximum velocity magnitude from our preliminary study with 12 healthy participants. note that the fitted curve is based on an approximation of a linear growth of the cough jet diameter with distance from the mouth. a novel experimental facilitythe flugiehas been designed to study the far-field aerodynamics of human coughs produced by subjects naturally-infected with respiratory viruses, together with measurement of the viral content of the droplets produced by those coughs, in order to quantify the factors relating to person-toperson airborne transmission of virus. a preliminary study involving 12 healthy individuals has been carried out in order to quantify the strengths of their coughs at a distance of 1 m away from the mouth. the velocity fields were measured using particle image velocimetry and the results indicate an important finding, namely that there is significant air motion during a cough, of the order of 0.5 m/s, even at a location 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normal human cough dispersion on particle image velocimetry (piv) measurements in the breathing zone of a thermal breathing manikin anisotropy in the breathing zone of a thermal manikin particle image velocimetry of human cough investigation into airborne transport characteristics of air-flow due to coughing in a stagnant room environment study on transport characteristics of saliva droplets produced by coughing in a calm indoor environment numerical study of transport of droplets or particles generated by respiratory system indoors study on the initial velocity distribution of exhaled air from coughing and speaking basic infection control for health care providers violent expiratory events: on coughing and sneezing a review of dispersion modelling and its application to the dispersion of particles: an overview of different dispersion models available a methodology for estimating airborne virus exposures in indoor environments using the spatial distribution of expiratory aerosols and virus viability characteristics an aerosol generator for the resuspension of cotton dust western cold and flu (wecof) aerosol study -preliminary results the facility development and preliminary study were funded by the ontario thoracic society and the workplace safety and insurance board. thanks are due to university machine services at western for the construction of the flugie facility and to the 12 anonymous experimental participants. dr. sydney siu provided key assistance with the medical research ethics and safety approvals. the piv system was kindly made available by dr. kamran siddiqui. dr. qiuquan (charles) guo provided physical assistance in the laboratory and autoclave training. authors' contributions es, sm and js conceived of the wecof study and overall methodology and arranged the funding and required ethics and safety approvals. wel designed the facility with technical advice from js and es. data were acquired by wel, kb and mcr. es guided the data analysis by wel and mcr. es interpreted the results and drafted the technical note with assistance from sm and lrc for the literature review and wel for technical descriptions, figures and tables. sm and wel critically revised the draft. all authors read and approved the published version and agreed to be accountable for the accuracy and integrity of the entire work. key: cord-253922-qjsd3whg authors: hamilton, r. c.; drane, d. p.; smith, h. v. title: shedding of “virus-like” particles in canine faeces date: 1995-09-30 journal: veterinary microbiology doi: 10.1016/0378-1135(95)00095-r sha: doc_id: 253922 cord_uid: qjsd3whg abstract diarrhoeic faeces from about 500 dogs were examined by negative stain electron microscopy. as well as parvovirus, and some of the other recognised viral causes of gastroenteritis, unusual “virus-like” particles were observed in about 8% of the samples. the particles were spherical, 100 nm to 300 nm in diameter, and surrounded by a thick wall penetrated by numerous pores. an additional 74 samples of normal faeces yielded no “virus-like” particles. we do not know the nature of these particles. canine parvovirus was identified in 1978. one of the methods used in the diagnosis of canine parvovirus was negative stain electron microscopy of diarrhoeic canine faeces to detect parvovirus particles. using this technique astroviruses, coronaviruses, coronaviruslike particles, papova-like viruses, parvoviruses, picomaviruses, rotaviruses and small round viruses have been detected (williams, 1980; carmichael and binn, 1981; hamilton et al., 1982: hammond and timoney, 1983; marshall et al., 1984) . we have used this technique to investigate parvovirus disease and to evaluate a diagnostic test (drane et al., 1994) . during this work we observed unusual "virus-like" particles similar to those recorded by hamilton et al. ( 1982) . here we report further observations on these particles. about 500 faecal samples or swabs supplied by veterinarians or from our own studies were suspended in either phosphate buffered saline or distilled water and clarified by low particle (arrow ). bar= 100 nm speed centrifugation. a drop of sample was applied to a formvar-coated copper electronmicroscope grid which was then inverted onto 2% ( w/v ) noble agar. after the grid had settled onto the agar. it was picked up then floated on 3% ( w/v) ammonium molybdate for thirty seconds at room temperature. the grid was picked up and excess stain removed with filter paper. three grids were prepared from each sample. the grids were examined with a philips em301 electron microscope, at an instrumental magnification of 50 000 x all virus or "virus-like" particles that were observed were photographed. about 8% of the abnormal faecal samples, and none of the normal faecal samples, contained "virus-like" particles. the "virus-like" particles were usually spherical and had a thick wall which was penetrated by numerous pores (fig. i ) . this wall appears to be rigid as the particles are not readily distorted on the grid. when damaged particles were observed they appeared to be cracked open ( fig. 2) . the particles did not disintegrate into a cluster of subunits as has been observed with a number of other viruses. indented particles were observed (fig. 3 ) often the particles were penetrated by stain but when they were not penetrated the detailed surface texture became apparent (fig. 1) . when the particles were penetrated by stain, no regular internal component was observed, but they did not appear to be completely empty ( fig. 2) . 3 . "virus-like" particles of two different size\. larger particle (thick arrow) smaller particle ( thin arrow) bar equal5 100 "tn. the particles ranged from 100 nm to about 300 nm. usually in any one sample there were particles of one size but occasionally particles of two different diameters were present in one sample ( fig. 4 and fig. 8) . the particles were usually scattered singly or in small groups throughout the sample ( fig. 1) but, at times, larger groups or groups of over a hundred particles were observed bound within membranes (fig. 5. fig. 6 and fig. 8 ). the particles often absorbed to gut bacteria (fig. 7) . parvovirus particles were observed in some samples containing the viruslike particles ( fig. 8) we have observed these "virus-like" particles in diarrhoeic canine faeces from 198 1 to 1994. similar particles have been observed in canine faeces in portugal (alves de matos, 1988, personal communications) which indicates the particles have a wide temporal and geographic distribution. other comprehensive negative-stain electronmicroscopy studies of canine faeces, however, have not reported on these particles (williams, 1980: carmichael and binn, 198 1; hammond and timoney, 1983; marshall et al., 1984) . initially we thought that the particles may be a new family of viruses (hamilton et al.. 1982) . we now think that this is unlikely. although the particles have a regular patterned r. hamilton et al. / veterincq microhiolog~ 46 (1995) 307-313 fip. 7. "virus-like" particles (thick arrows) adsorbed to a bacterium. ( thin arrow ) bar equal\ io0 nm surface they do not appear to be composed of typical viral sub-units nor do they appear to be composed of "spikes" inserted into a membrane. perhaps the particles are botanical or mineral components of dog's food. perhaps the "virus-like" particles are some structures associated with bacteria or parasites in the gut of dogs. the fact that the particles are found enciosed within membranes would suggest this. they may be spore or egg structures that are formed within a membrane, usually released from the membrane within the gut then shed in the faeces. we do not know if these particles are involved in any disease process but they are found in diarrhoeic faeces. however, the faeces often also contained parvovirus, a well known cause of gastroenteritis in dogs. they have also been observed associated with picomaviruses (alves de matos, 1988, personal communication). we do not know the true nature of these "virus-like" particles. new enteric viruses in the dog evaluation of a novel diagnostic test for canine parvovirus viruses in suspected canine parvovirus specimens an electron microscopic study of viruses associated with canine gastroenteritis viruses and virus-like particles in the faeces of dogs with and without diarrhoea astrovirus-like. coronavirus-like and parvovirus-like particles detected'in the diarrhoeal stools of beagle pups we thank m.b. walker for her assistance with the electron microscopy and photography. key: cord-325915-dw989txm authors: wolf, michael w; reichl, udo title: downstream processing of cell culture-derived virus particles date: 2014-01-09 journal: expert rev vaccines doi: 10.1586/erv.11.111 sha: doc_id: 325915 cord_uid: dw989txm manufacturing of cell culture-derived virus particles for vaccination and gene therapy is a rapidly growing field in the biopharmaceutical industry. the process involves a number of complex tasks and unit operations ranging from selection of host cells and virus strains for the cultivation in bioreactors to the purification and formulation of the final product. for the majority of cell culture-derived products, efforts focused on maximization of bioreactor yields, whereas design and optimization of downstream processes were often neglected. owing to this biased focus, downstream procedures today often constitute a bottleneck in various manufacturing processes and account for the majority of the overall production costs. for efficient production methods, particularly in sight of constantly increasing economic pressure within human healthcare systems, highly productive downstream schemes have to be developed. here, we discuss unit operations and downstream trains to purify virus particles for use as vaccines and vectors for gene therapy. production procedures for viral vaccines and gene therapy vectors. one striking example is the development of cell culture-derived influenza vaccines. while conventional production processes rely on egg-based systems, optimized cell culture systems are currently being established to cope with sudden demands for pandemic vaccines and increasing supply of seasonal vaccines. with advances in upstream procedures to increase yields and harvest volumes for influenza vaccines, as well as for other vaccines and viral vectors, downstream processing (dsp) is becoming an important factor in the race for higher overall productivity and decreased cost of goods. the general aim of dsp is the recovery and purification of biological products from process-and product-related impurities. process-related impurities might originate from cell culture reagents and additives (e.g., antibiotics, bovine serum albumin and benzonase ® [merck kgaa, darmstadt, germany]), from the purification process (e.g., extractables and leachables in chromatography), or from the cell substrate (e.g., host cell protein, nucleic acids, proteoglycans and glycosaminoglycans). examples for virus particle-related impurities include free envelope proteins, virus aggregates or empty capsids, and virus particles that contain nucleic acid sequences other than the intended virus particles are currently used for medical [1] [2] [3] [4] [5] , analytical and scientific applications [6] [7] [8] [9] , and as bioinsecticides [10, 11] . recently, medical applications are gaining an increasing interest owing to the growing markets for viral vaccines (table 1 ) and the potential broad usage of viral gene therapy vectors. vaccines, administered to prevent or treat viral diseases, are mainly based on attenuated or killed viruses, membrane fractions derived from purified virus particles, or recombinant viral proteins expressed in various hosts. examples of successful attenuated or killed virus vaccines are influenza, measles, mumps, rubella, rotavirus, yellow fever and varicella [3, 4, 12, 13] . gene therapy involves the transfer of genetic information to cells or tissues of individuals to achieve a therapeutic effect [14] . therefore, required genes are largely delivered by viral vector systems based, for example, on the herpes simplex virus, adenovirus, adeno-associated virus (aav), retrovirus (e.g., lentivirus) and vaccinia virus [1, 15] . currently, numerous clinical gene therapy trials are conducted to investigate the treatment of diseases such as cancer, cystic fibrosis, alzheimer's, parkinson's, hemophilia and hiv/aids [5, 16, 17] . the broad spectrum of these applications and the current expansions of medical markets underline the ongoing efforts to improve review genome [18] [19] [20] . naturally, the requirements on product purity and product safety depend on the particular application. vaccines and viral vectors need to meet the stringent guidelines of regulatory authorities such as the us fda and ema. viruses are complex bioparticles with varying size, shape, composition and surface structure. the virus surface defines their individual physicochemical characteristics including number and distribution of charges, hydrophobic residues and post-translational modifications (i.e., glycans) of surface proteins. purification of virus particles based on these unique characteristics and removal of contaminants according to the regulatory guidelines can only be achieved by a combination of different unit operations. this article provides an overview of individual unit operations currently used for dsp of viral vaccines and gene therapy vectors. this includes methods such as precipitation, flocculation, extraction, centrifugation, microfiltration, ultrafiltration, bead-based and membrane-based chromatography, and the use of monoliths. in addition, it addresses issues concerning the use of continuous chromatography methods, that is, simulated moving bed chromatography, and the utilization of kits for small-and medium-scale purifications and concentrations of virus particles and vectors for gene therapy. finally, examples for complete purification trains are presented for dsp of virus particles for therapeutic applications and vaccine manufacturing. owing to the complexity of large bioparticles and the necessity to maintain the specific immunogenicity and infectivity of virus particles and viral vectors, precipitation and flocculation are rarely used in dsp. even for purification of recombinant proteins, use of these methods is considered problematic owing to potential losses in biological activity. however, the latest advances in upstream processes for recombinant proteins in terms of high cell density cultures and drastically improved product levels triggered the re-evaluation of these methods, particularly for purification of monoclonal antibodies [21, 22] turkey coronavirus was precipitated with ammonium sulfate but with low recoveries compared with ultracentrifugation (60% sucrose cushion) [25] . other virus particles were precipitated with varying combinations of polyethylene glycol (peg) and sodium chloride. in particular, peg precipitation of bovine rotavirus particles resulted in an approximately tenfold better yield than pelleting by high-speed centrifugation based on results determined by the tissue culture infective dose (tcid 50 ) assay [27] . this could be owing to increased damage of virus particles during high-speed centrifugation [27] or an irreversible aggregation of virus particles leading to overall reduced tcid 50 measurements. furthermore, precipitation of aav vectors via peg can lead to an enhanced purity compared with the classically conducted cesium chloride (cscl) gradient ultracentrifugation [19] . an alternative to the precipitation of the target virus is the removal of contaminating host cell dna or protein from crude culture harvests. a recent study focusing on the precipitation of host cell dna from a clarified cultivation broth of influenza virus particles has been conducted by kröber et al. [28] . they investigated cationic reagents with respect to their ability to selectively precipitate host cell dna and observed successful dna reduction applying the cationic polymers protamine sulfate and polyethyleneimine [28] . however, as discussed by the authors, the acidic isoelectric point of influenza virus particles (table 2 ) may have resulted in co-precipitation of virus particles with cationic polymers [28] , and therefore product losses. in the past effective purification of encephalomyocarditis virus from cellular components [29] , and the removal of cellular dna from poliovirus produced in continuous cell lines [30] as well as from preparations of inactivated vaccines against tick-borne encephalitis [31] by protamine sulfate precipitations was demonstrated. considering the continuous efforts to increase harvest volumes and cell concentration in vaccine and gene therapy vector production, precipitation represents an interesting approach for reduction of the high loads of host cell dna and proteins. however, process robustness (i.e., the specificity of the applied precipitants) needs to be improved, and nontoxic compounds for precipitation processes need to be identified for economic applications in pharmaceutical production. [170, 174] hepatitis c virus flaviviridae 30-60 na spherical yes (+)ssrna [175] herpes simplex virus yes dsdna [170, 176] influenza virus orthomyxoviridae 80-120 (h2n2) a2/singapore/57: 5.0 pleomorphic, spherical yes (-)ssrna [177] [178] [179] [180] [181] [182] [183] (h3n1) a/pol/l/71: 6.5-6.8 [170, 188] retroviridae 80-120 na spherical yes (+)ssrna [189] papillomavirus papillomaviridae 52-55 papillomavirus: 5.0 icosahedral no dsdna [170, 190, 191] poliovirus picornaviridae 28-30 pv-1: 7.4 and 4.0 icosahedral no (+)ssrna as for precipitation, the system's complexity, which is mainly dependent on the structural and physicochemical diversity of virus particles, constrains this technique to become widely used. further drawbacks of applying extraction methods for the purification of virus particles are the possible losses in viral infectivity, immunogenicity or transduction efficiency, high levels of co-extracted contaminants and the high cost for recycling of the utilized organic solvents [32, 33] . nevertheless, extractions via aqueous two-phase systems with peg, dextran, salt or polyvenyl alcohol are used for the separation of virus particles from cultivation medium. examples for these applications are the purification or concentration of adenovirus [34] , bovine leukemia virus [35] , feline leukemia virus [36] , and human and simian immunodeficiency virus [37] . the number of publications [24, [38] [39] [40] [41] [42] [43] [44] [45] [46] and patents [301] [302] [303] describing the purification or concentration of virus particles by centrifugation methods demonstrates that these procedures are extensively used at industrial-and small-scale levels for viral vectors and vaccine production processes. for industrial scales only continuous flow centrifugation methods can be considered as they allow handling of larger volumes. however, these methods require high investment costs and suffer in some cases from losses of infectivity [33, 38, 47] , leading to an increased usage of ultrafiltration techniques particularly outside of laboratory scales. a clear advantage of centrifigation methods is their potential to separate some assembled viral vectors from their empty capsids, which is commonly challenging with other separation techniques [38] . another advantage is in the dsp of viruses with frequently changing strains (e.g., influenza) as these centrifugation methods are robust in relation to viral strain differences, which resulted in a rediscovery of this technology for a number of processes. the most common centrifugation method for the purification of macromolecules and virus particles is density gradient centrifugation using cscl, iodixanol, or sucrose gradients. these can be classified into two categories: rate-zonal separation and isopycnic separation. the latter is based on the buoyant density of the particle and the rate zonal separation on the particle size and mass. examples of virus particles that were purified by gradient centrifugation techniques are feline leukemia virus [48] , gibbon ape lymphoma virus [49] , hepatitis b virus [50] , influenza virus [51] , japanese encephalitis virus [52] , mammalian type c virus [53] , mouse mammary tumor virus [54] , mouse oncorna virus [55] , mumps virus [56] , murine leukemia virus [57] , tick-borne encephalitis virus [58] , turkey coronavirus [25], rabies virus [59] and vaccinia virus [60] . an alternative centrifugation method is the differential centrifugation where pelleting (epstein-barr virus [61] and hepatitis a virus [62] ) or simple sedimentation onto a sucrose cushion (moloney murine sarcoma virus [63] ) is done once or repeatedly at different centrifugation speeds. current examples for the concentration or purification of virus particles via centrifugation are: adenovirus and aav [38, 39] , bromovirus [24], hepatitis c virus [40] , herpes-like virus [41] , retrovirus [42] [43] [44] , rotavirus [45] and vaccinia virus [46] . microfiltration is commonly used for clarification in biotechnological production processes as an alternative to centrifugation to remove microcarriers, producer cells, cell debris and organelles. no dsrna [170, 195] rubella virus togaviridae (rubivirus) 50-75 na quasispherical yes (+)ssrna [198, 199] semliki forest virus [46, [202] [203] [204] [205] [206] [207] connaught: 4.9 lister: 5.1 lister: 3.9 lister (egg): 3.7 lister (rabbit): 3.0 wr: 4.8 yes dsdna [170] † data on isoelectric points of different viruses taken from michen and graule [208] . ‡ this is a brick-shaped viral particle therefore the dimension are shown in terms of axis x-y-z. d: diameter; l: length; na: not available. [147] 500 kda 71 300 kda 96 tangential flow filtration (millipore; pellicon ii filter module) polyethersulfone; flat sheet 500 kda not provided [209] tangential flow filtration (millipore; ultra-15 centrifugal filter unit) regenerated cellulose; flat sheet 100 kda 57 [210] tangential flow filtration (millipore; pellicon ii filter module) polyethersulfone; flat sheet 1000 kda not provided [82] 300 kda not provided tangential flow filtration (individual setup) polysulfone (ge-healthcare) 300 kda 83 [211] polysulfone frequently, membranes with pore sizes in the range of 0.45-1 µm are applied, although this technique involves the risk of viral losses [44, 64, 65] . recoveries depend on the initial membrane pore size, type of membrane (material), quality of the crude stock, virus (i.e., size), virus subtype and its aggregation behavior, applied buffer conditions, filtration rate, nature and level of protein concentration in the filtrate, and extent of pore obstruction as demonstrated for retroviral vectors [65] . virus losses are mainly attributed to mechanical disruption, particularly for active virus particles (i.e., gene therapy vectors), exclusion of virus particles [64] and membrane entrapment as well as unspecific virus adsorption. losses due to pore obstruction can be reduced by applying membrane cascades with decreasing pore sizes or filter capsules with dual membranes to minimize membrane fouling [50, 66, 67] and applying larger membrane areas (i.e., frequent changes of the filtration units or usage of larger units) to limit the volume of supernatant per membrane surface. ultrafiltration is one of the preferred methods for virus removal in biological production methods [68] [69] [70] and for virus concentration and buffer exchanges in vaccine and viral gene transfer vector manufacturing processes (table 3 ). here, virus particles are usually enriched or maintained in the retentate while water and small-molecular-weight molecules are removed with the permeate. although ultrafiltration has a low resolution, the size difference of virus particles (table 2 ) compared with soluble biopolymers (i.e., nucleic acids and proteins) allows a relatively efficient and economic separation of the virus. the key parameters for an ultrafiltration process are the transmembrane pressure (tmp), feed (retentate) and permeate flux as well as the nominal molecular weight limit, also sometimes referred to as molecular weight cutoff (mwco). pore sizes defining the mwco are normally distributed about the mean pore size, which varies depending on the production method for the membrane, and therefore also between the manufacturers. in order to minimize product losses via the permeate, the mwco has to be significantly smaller than the virus particle. however, if the mwco selected is too small, permeate fluxes are reduced,, which leads to longer filtration times, increased tmp and decreases the possibility of reducing the amount of contaminating proteins and nucleic acids. furthermore, membrane fouling, which decreases the permeate flux, can often be greater for larger pore size membranes than for smaller ones. here, small virus particles but also cell and virus review debris are able to enter the pores where they are eventually trapped in constricted channels. asymmetric ultrafiltration membranes with the finer filtering surface facing the feed suspension should be applied where possible to reduce internal fouling. important for concentration of active virus particles is a gentle processing for which tmp and retentate flux have to be optimized for low wall shear rates during filtration. the filtration performance and in particular the permeate flux is heavily affected by the type or components of the cultivation medium, thereby underlining the importance of a tight interaction between the up-and down-stream development. nowadays, serum containing growth media are rarely used for production processes. serum-free media often contain defined proteins such as bovine albumin and insulin, but also protein hydrolysates. protein-free media only include defined polypeptides and amino acids. the higher the concentration of proteins, target virus, and nucleic acids and the larger the size of contaminating dna fragments in the retentate, the higher the sample viscosity, leading to a continuously decreasing permeate flux at constant tmp. additionally, high retentate protein concentrations result in an increased degree of concentration polarization, which also reduces the permeate flux [71] . finally, ionic strength, type of ions and the ph in the suspending buffer influence the sieving coefficient of proteins [72] [73] [74] [75] and probably also macromolecular biomolecules and virus particles. ultrafiltration can be carried out by a wide variety of filtration devices (table 3 ). for small scales (up to 10 ml) centrifugal filtration devices are well suited, and for small to medium volumes (10-300 ml) stirred cell tanks are ideal. larger volumes are usually concentrated by tangential flow filtration. there are three types of membranes available: tubular membranes, flat sheets (cassettes) and hollow-fiber membranes from which the latter two are commonly used in the biopharmaceutical industry. generally, flat-sheet and tubular tangential-flow membrane modules have higher mass transfer coefficients at low cross-flow rates compared with hollowfiber modules. the latter have wide feed flow paths, providing laminar flow with low shear rates [76] , which are crucial processing life vaccines and gene therapy vectors. high shear rates can significantly reduce their efficacy and overall yield. a general advantage of hollow-fiber membrane modules compared with flat sheets is the high membrane surface-to-module volume ratio. however, individual modules of flat-sheet tangential-flow membrane modules are easier to clean and replacement of defective membrane elements is uncomplicated. overall, the type of membranes eventually used for a specific process depends largely on the viscosity, solid content and volume of the feed as well as the product stability (shear-sensitivity). thus, for the concentration or diafiltration of active virus particles, hollow-fiber membranes are superior to flat-sheet membranes. a potential improvement of the classical tangential-flow filtration is the high-performance tangential-flow filtration, which achieves a superior separation factor [71, 77] . in high-performance tangential-flow filtration, part of the permeate is circulated cocurrent to the feed on the permeate side of the module, resulting in a more constant tmp throughout the module due to an axial pressure drop along the permeate flow channel [78] . this allows, by careful optimization of the operating conditions, a more stable transmembrane flux over time and in some cases leads to an improved selectivity for the purification of virus particles (table 3 ). in summary, the main advantages of ultrafiltration compared with other methods are their high-throughput and (for the concentration of active virus particles) the gentle processing at optimal operating conditions [43, 47] that results in improved efficacies for purification of viral vectors for gene therapy. another advantage of utilizing size differences of virus particles to other soluble components in the culture medium for their purification and concentration is its broad applicability among different types of viruses, virus subtypes or different recombinant forms of a virus. this opens the path for the establishment of generic platform technologies. considering a complete purification train for the production of vaccines or gene therapy vectors (figure 1) , current improvements of the dynamic binding capacities in chromatography media might facilitate the removal of the initial concentration step within the downstream process. however, later concentration steps and buffer exchanges for final formulations will continue to be done via ultrafiltration. chromatography is the most popular methodology for largescale purification of vaccines and viral gene therapy vectors. chromatographic separation is based on differences in the interaction of target virus and other components to the applied stationary phase. the specific characteristics of the virus particles exploited are described by the physicochemical properties of the outer particle surface, electrostatic properties of the whole particle, or their large hydrodynamic volume compared with other soluble process components. there are four main points of chromatography that have to be considered for a successful purification [79] : • physical structure of the stationary phase including the morphology (i.e., beads, membranes and monoliths), pore size, porosity, mechanical and chemical properties (e.g., resistance to pressure and surface charge of the mobile phase), and specificity for resin particle size and particle distribution; • surface chemistry of the stationary phase and the composition of the mobile phase; • mode of operation such as adsorption or partitioning, type of elution; • column/housing and system hardware. for each application a balance between these individual aspects must be found to obtain an efficient chromatographic procedure. in the following sections, however, we will focus on the first two points. chromatographic purification of virus particles is mainly achieved by use of three different types of stationary phases: packed beds, membrane adsorbers and monoliths. in the case of packed beds the matrix consists of resins (beads), which are filled into a column. for bioseparations polymer and inorganic resin materials are commonly used. polymer-based beads either consist of natural polymers (agarose, agarose-dextran composites, cellulose and dextran) or synthetic polymers (polyacrylamide, polystyrene divinylbenzene, derivatives of polyacrylamide derivatives and polymethacrylate) [80] . examples for inorganic matrices used for the purification of virus particles are hydroxyapatite [81] [82] [83] , silica [84] and controlled-pore glass [85, 86] . beads can be spherical and nonspherical shaped and are available as porous, nonporous and solidcore resins. pore sizes of conventional porous beads range from 10 to 100 nm [79] , although specific particles with pore diameters of up to 400 nm are available [33] . virus particles vary generally from 30 nm to greater than 300 nm (table 2) . hence, the material transport of virus particles is limited by pore-diffusion or pore-exclusion effects. even small virus particles, which are able to enter large pores, diffuse through the intra-particular channels two-to 100-fold slower than proteins [87] . in fact, the majority of chromatography resins currently available are optimized for purification of proteins rather than virus particles, which primarily adsorb to the outer bead surface [88] . furthermore, the inter-particle porosity for packed beds can reach values of maximally 40%, whereas for stationary phases such as monoliths this value can be up to 90% or more [89] . monoliths are continuous stationary phases of homologous columns, where interconnecting channels allow for convective flow of the mobile phase through the entire matrix [90, 91] . commercially available monoliths are made of polymethacrylate copolymers (bia separations, ljubljana, slovenia; dionex sunnyvale, ca, usa), polyacrylamide (bio-rad, hercules, ca, usa; protista biotechnology ab, lund, sweden), polystyrene (dionex, sunnyvale, ca, usa), polystyrene-divinylbenzene (dionex, sunnyvale, ca, usa), modified cellulose (sepragen, hayward, ca, usa) and silica (merck, darmstadt, germany; phenomenex, torrence, ca, usa) [92, 93] . depending on the manufacturer they are sold as disks, tubes and rods with pore sizes ranging from 13 nm (silica, merck) [94] to 6 µm (polymethacrylate, bia separations). membrane materials for chromatographic bioseparations include cellulose, polyamine, polysulfone, hydrazide and composite membranes such as polyethylene oxide and polyethersulfone coated with hydroxyethyl-cellulose [95, 96] . there are three types of chromatographic membrane adsorbers used for the purification of bioproducts: flat sheets (e.g., pall, dreieich, germany; sartorius, göttingen, germany), hollow fibers (e.g., kinetic systems inc., boston, ma, usa) and radial-flow devices (e.g., 3m, neuss, germany) [96, 97] . the advantages of hollow-fiber membrane adsorbers are the high surface area and the reduction in particle accumulation near the pore entrance owing to the cross-flow principle used during separation. however, the breakthrough of target compounds is generally broadened, leading to poor adsorber utilization compared with flat sheets [97] . furthermore, eluted products are heavily diluted and internal mixing within the fiber housing results in low resolutions, and the application of gradient elution is very limited. radial-flow adsorbers have flat-sheet membranes spirally wound over a cylindrical core [97] whereby the available surface area can be significantly increased. thus, the volume-to-surface area for spiral-wound membrane adsorbers is better than for the majority of hollow-fiber adsorbers. still, products are diluted and gradient elutions are accordingly challenging. flat sheets are mainly used as stacks of numerous individual sheets providing a larger adsorbent volume. in flat-sheet membrane adsorbers the liquid is commonly introduced vertical to the membrane adsorber surface, allowing a minimum dead volume in an optimally designed membrane module. thus, these membrane adsorbers do not suffer from diluted product fractions and can be used for gradient elution procedures. the main advantages of membrane adsorbers and monoliths for the separation of virus particles are the predominance of convective material transport with nearly no diffusion limitations, [79, 90, [96] [97] [98] [99] owing to large pores, and well-interconnected channels with small constrictions and high dynamic binding capacities [79, 99, 100] , whereby the latter is sometimes lower for membrane adsorbers than for comparable monoliths [101] . the pore structure of membrane adsorbers and monoliths facilitates the accessibility of large virus particles to bound ligands in contrast to typical resins where they are excluded from the internal bead pores [98] . except for monoliths, eddies are formed in the majority of chromato graphic matrices including membrane adsorbers. as a result, labile virus particles can be deteriorated and separation impaired by peak broadening. stacks of membrane adsorbers can ac (sulfated cellulose) sulfated cellulose ma 73-94 [105] ac sartobind zn-ida ma 75 [104] ac different ligands immobilized to monoliths ¶ not provided [156] parvoviridae (aedes aegypti densonucleosis virus) sartobind q, d sartobind s, c not provided [230, 231] parvoviridae (adeno-associated virus, aav1-gfp) [100] retroviridae (vector; momlv-derived vsv-g-pseudotyped retroviral vector) ac fractogel emd heparin (s) 61 [66] filtration, uf, df, ac, sec fractogel emd heparin (s) sepharose cl-4b retroviridae (vector; momlv-derived vsv-g-pseudotyped retroviral vector) ac fractogel emd heparin (s) 61 [106] retroviridae (vector; momlv-derived rd114-pseudotyped retroviral vector) ac fractogel emd heparin (s) 43 [106] retroviridae (vector; vrx496 a vsv-g pseudotyped human immunodeficiency virus type 1-derived vector) aec mustang q 65 [235] sec sephacryl s500 70-80 retroviridae (vector; vsv-g-pseudotyped retroviral vector) sec sepharose cl-4b 70 [236] uf, sec, uf/df sepharose cl-4b 19 retroviridae (vector; his 6 -tagged retroviral vector) ni-nta agarose 56 [114] retroviridae (vector; biotin-tagged retroviral vector) ac fractogel emd streptavidin ‡ ‡ 17 [237] steptavidin-monolith § § 8 [109] retroviridae ( review be considered as very thin slices of monoliths. pores between membrane layers are connected by a void space between individual membrane slices, leading potentially to eddy formations. flow distributions in hollow-fiber and radial-flow membrane adsorbers are less well controlled compared with either monoliths or packedbed columns. in addition to eddy formations this could result in reduced dynamic binding capacities and dispersions. the latter does not only affect negative-mode adsorption chromatography, but also reduce the resolution of positive-mode applications. the majority of membrane adsorbers are designed for singleuse applications, supporting current efforts in the pharmaceutical industry to reduce costs for validation, cleaning and sanitization. monoliths are currently following this trend. for beads, however, it remains to be seen if disposable approaches can be realized economically. nevertheless, a wider diversity of adsorptive surface chemistries is currently available for resins than for convective alternatives, but the product range for membrane-based separations constantly expands. currently, five different modes are used for the chromatographic separation of virus particles: size-exclusion chromatography (sec), ion exchange chromatography (iec), affinity chromatography (ac), hydrophobic-interaction chromatography (hic) and mixed-mode chromatography (table 4 ). the action principles and possibilities for process optimization are limited by the stability of the individual viruses. extreme running conditions in terms of ph, osmolarity, ion compositions or certain organic solvents might influence the efficacy of vaccines or gene therapy vectors. hence, normal or reversed-phase chromatography requiring organic solvents is usually not applied for whole-virus separations. however, for split vaccines or subunit vaccines, where virus membrane integrity does not necessarily have to be conserved, organic solvents and thus reversed-or normal-phase chromatography might be used depending on the solvent effects on the target proteins. partition chromatography such as sec can only be achieved by porous particles but not by membrane adsorbers or monoliths. for sec, virus particles are generally recovered in the column void volume allowing the separation of bulk proteins and small molecular contaminants without buffer changes under gentle operating conditions, thereby maintaining virus infectivity and immunogenicity. owing to the large difference in hydrodynamic volume of intact virus particles and soluble contaminants, the sec exclusion limit can easily be optimized not to distinguish between morphologically similar viruses, allowing robust process conditions and broad applicability where different strains are routinely required (e.g., for influenza vaccines). the main disadvantages of sec are the low capacity, product dilution and the poor pressure resistance of the matrix, requiring low flow rates. despite these disadvantages, sec represents a valuable method in addition to adsorptive chromatography, but high-throughput options for ultrafiltration processes will challenge the application of this unit operation for the separation of virus particles in the future. industrial chromatography processes are usually optimized for high dynamic binding capacities, particularly for the primary capturing step, and the elimination of residual contaminants during the polishing step (figure 1 ). high capacities are attained by large accessible surface areas, optimized ligand densities and high selectivity. the latter is important if highly contaminated loads (i.e., feedstocks) are applied. in addition to the dynamic binding capacity, the ligand density of adsorption matrices such as iec, ac, and hic also contributes to the overall adsorption strength. this is mainly due to the multivalent interactions of virus particles with the matrix ligands. thus, ligand density needs to be carefully selected taking into account both of these aspects. furthermore, the desorption conditions need to be considered, selecting optimal ligands and ligand densities, and allowing elution conditions that do not affect the virus immunogenicity or transfection efficiency of vectors. strong adsorptions, requiring harsh desorption conditions for the target virus particles, are often observed for iec or hic [102] . adsorption matrices are commonly used both in positive mode (i.e., virus particles adsorbing to the chromatography medium) and negative mode (i.e., contaminants adsorbing to the matrix and virus particles eluting). positive-mode applications are usually used for capturing or intermediate purification and negative modes for the final polishing. ion exchange chromatography exploits the charge-to-charge interaction between the virus surface and immobilized ion exchange groups on the matrix. depending on the overall isoelectric point (table 2 ) of the individual virus particles, cation-or anion exchange chromatography (aec) matrixes are utilized. different variations of iec have been applied for the purification of a variety of viruses (table 4) . adsorbed virus particles are in general displaced by an increasing ionic strength. alternatively, they are desorbed by a change in buffer ph, leading to an unfavorable overall charge of the virus surface or the matrix, at which the virus particles do not bind anymore. iec has a relatively low specificity resulting in a reduced dynamic binding capacity for the target virus and often results in co-elutions of contaminants (e.g., host cell dna for aec). differential elution between the target virus and contaminants can sometimes be achieved by linear-or stepgradient elutions. if high salt concentrations or nonphysiological ph conditions are required for purification, it is crucial to monitor immunogenicity or transfection efficiency of virus particles [47, 304] . hence, iec matrices have to be carefully selected in terms of the type of charged groups, ligand density, and applied buffer conditions with respect to the virus type, product application and utilized assays for process characterization. affinity chromatography of virus particles relies on a specific and reversible adsorption and subsequent recovery of the active target virus from a ligand immobilized onto the chromatography matrix. the specific interactions between ligand and virus are based on individual structural properties, and the virus particles are commonly eluted by an altered conformation via a changed buffer ph or ionic strength or competitive displacement. however, the latter is generally not economic for large-scale production processes, but is frequently applied at smaller scales. applied ligands or matrices for the purification of virus particles are immobilized metal ion affinity [103, 104] , sulfated carbohydrates (e.g., heparin) [100, [105] [106] [107] , specific antibodies [108] or antibody fragments, biotin streptavidine system for recombinant viral vectors [109] , lectins [110, 111] , dna aptamers [112] and peptides (table 4 ). for the majority of viruses, no specific ligands are known, resulting in the application of lessspecific ligands such as lectins or sulfated carbohydrates (i.e., heparin). lectins interact with accessible carbohydrate moieties of the surface glycoproteins according to their specificity. heparin is primarily a weak and strong cation exchanger containing sulfo and carboxyl groups as well as numerous hydroxyl groups [113] . desorption of the virus particles from heparin matrices is done by increasing the ionic strength. however, it often requires a higher ionic strength than for classical cation exchangers owing to the multivalent interactions of the branched linear structure of heparin and the formation of hydrogen bonding between the target virus and heparin [113] . an alternative to laborious ligand screens or the application of less specific pseudo-affinity ligands are engineered viral vectors containing affinity tags on the virus surface [109, 114] . crucial for specific affinity ligands is the consideration of their dissociation constants. for recombinant proteins, dissociation constants of 10 -4 -10 -10 m are usually considered a good working range [115] . optimal affinity ligands allow fast binding kinetics for high flow rates but mild elution conditions to avoid reduction in immunogenicity or activity. therefore, it is essential to consider the overall binding strength of large virus particles, which is due to the already discussed multivalent interactions. if proteins or larger biomolecules are used as ligands, their potential toxicity or immunogenicity has to be considered and, if applied, assays have to be developed for their detection and quantification. furthermore, for many proteins, high costs are associated and the low stability of these ligands towards sanitizing agents in some cases prohibits their application for large-scale processes [116] . in some cases, additional drawbacks of specific ligands are their restricted application as platform technology and limited process robustness for vaccines with frequently varying subtypes (e.g., influenza vaccines). furthermore, for viruses with varying progenies (e.g., vaccinia virus) [117] , some virus particles might be excluded, thereby affecting the overall yield. on the other hand, the applied specificity allows the separation of specific progenies. ligands targeting post-translational modifications on the virus surface are heavily susceptible to the cultivation conditions and host cells [118] . hence, target epitopes for ac matrices on the virus surface have to be carefully selected, considering their stability during the entire process. nevertheless, matrices with highly specific affinity ligands and high dynamic binding capacities are very well suited to capture virus particles after clarification from low concentrated cultivation broths. hydrophobic-interaction chromatography separates biomolecules dependent on the differential interaction of these compounds with hydrophobic ligands on the surface of the stationary phase. it is routinely used for bioseparations of proteins [119] [120] [121] [122] , and has also been applied for virus purification [100, 113] since it is an orthogonal separation technique to purification methods based on ionic interactions. however, many factors such as type of salt and ionic strength of buffer, ph, temperature, ligand and ligand density influence the performance of hic, making its development and optimization a difficult and time-consuming endeavor. the two most commonly used hic ligands are butyl and phenyl [113] . interactions with these ligands are relatively strong and have been frequently cited to denature labile proteins, potentially reducing their efficacy. however, often proteins subjected to these methods revert upon elution to their native structure [113] . even so, if used for purification of gene transfer vectors or vaccines, their transfection efficiency or immunogenicity has to be closely monitored, just as for the high salt applications for the iec methods. stronger hic ligands such as hexyl and octyl are commonly destructive to proteins, and most likely also to virus particles or gene therapy vectors if applied in positive mode. furthermore, hic requires the application of high concentrations of kosmotropic salts, which potentially also reduces virus efficacies. also, the application of high salt concentrations influences virus aggregation in the majority of cases, affecting further downstream processes and often process analytics. in addition, disaggregation of the formed aggregates usually impacts virus transfection efficiency and immunogenicity. nevertheless, it was recently shown that hic can be successfully applied to remove residual dna after ac for purification of active vaccinia virus particles (mva-bn ® ; bavarian nordic a/s, kvistgaard, denmark) (table 4 ) [100, 305] . mixed-mode chromatography exploits a multimodal functionality that allows virus particles or contaminants to adsorb to the stationary phase by a combination of ionic interactions, hydrogen bonds and/or hydrophobic interactions. a typical example for a mixed-mode chromatography media is hydroxyapatite, which supports metal affinity interactions through its hydroxyapatite calcium groups, and cation exchange interactions through its hydroxyapatite phosphate groups [81] . successful applications of mixed-mode matrices for the purification of gene therapy vectors have been described for adenoviruses [82] , aav [123] , moloney murine leukemia viruses [124] and retroviruses (table 4 ) [83] . continuous chromatographic processes, such as the simulated moving bed (smb) technology is today well established for the separation of binary mixtures of petrochemicals, sugars and small molecule pharmaceuticals [125, 126] . smb significantly improves the volumetric throughput as well as the purity and concentration factor relative to batch chromatography. however, purification of complex biological mixtures is still challenging because most systems are operated as a binary fractionator, providing only two exit streams [127] . however, new operation modes such as the threefraction smb [128, 129] , solvent gradient smb, and cleaning in-place smb are important steps leading the way for its successful application in bioseparations. finally, the complexity of the equipment and the experimental setup -that is, the large number of valves required -poses a serious challenge for process validation. the design of chromatographic purification processes is challenging owing to the multitude of unit operations and process variables, that is, a constantly growing number of available matrices, different chromatography modes and diverse operating conditions. as a result, an extremely large amount of design freedom exists. so far, the selection of unit operation and the choice of process conditions relies mainly on practical experiences or on trial and error approaches. in order to save valuable time and resources, miniaturized parallel screening tools have been developed [130] , that can be combined with additional kinetic studies (e.g., surface plasmon resonance [spr] spectroscopy) [98, 131] , thermodynamic measurements and dynamic light-scattering ana lysis [132] to guide the early stage process development and optimization. for the selection of the most appropriate experiments and parameters, the screens are commonly planned via experimental design software tools (e.g., design-expert, modde, statistica), implementing the concepts of 'quality by design'. furthermore, software tools for computer-aided process design, production scheduling and process debottlenecking is often used [133, 134] . to date, high-throughput process developments have been mainly applied to production processes for recombinant proteins. however, these methods can also be applied for the dsp of viral vaccines and gene therapy vectors. here, progress will largely depend on the establishment of reliable and robust analytical assays, which can cope with the high number of samples. a particular problem is the determination of product titers, which requires provision of suitable standards, biological and technical replicates, and assay validation on a logarithmic range. the latter typically results in comparatively large errors. typically, standard deviation of methods for virus titer assays in the range of 0.3 to 0.5 log 10 have to be accepted. screening and ana lysis of well-proven operating conditions are one point, to further facilitate efficient downstream development, good modeling tools have to be established and applied. for instance, vicente et al. combined the steric mass action model of ion exchange [135] with a standard chromatographic column model to simulate and predict adsorption and elution conditions for virus-like particles on anion-exchange (aex) membrane adsorbers [136] . the same research group recently described the effect of ligand density on aex membrane adsorbers for the purification of recombinant baculoviruses by comparing model strategies based on spr spectroscopy with diethylaminoethyl (deae) ligand densities of membrane adsorbers. both methods indicated that a lower ligand density increased the overall yields by over 20% [98] , demonstrating that spr technology can be successfully used to model membrane adsorption processes. their studies were further aided by a theoretical model to predict process conditions for dsp improvement [98] . overall these studies demonstrate how chromatographic models combined with parallel screening techniques and rationally designed experiments can streamline the design and optimization of viral dsp in the future. single-use components for individual unit operations in dsp have been used for years in the pharmaceutical industry, mainly for filtration and buffer/media storage. over the last decade, the relevance of disposable concepts has been extended to other unit operations including chromatographic applications for which matrices such as membrane adsorbers, monoliths and more recently resins are used. the driving force for the increasing interest in single-use concepts for the production of viral vaccines is the fast and highly flexible set up of production processes, which is expected to improve the response time for increasing vaccine demands such as for pandemic preparations. in addition, disposable concepts for limited production campaigns are economically attractive considering that under current good manufacturing practice (cgmp) standards, raw material and equipment in direct contact with the product have to be dedicated to the particular production process [137] . furthermore, single-use concepts commonly reduce or eliminate time-consuming and cost-intensive process steps such as cleaning and sanitization and their validation. however, the economic application depends heavily on the scale and requirements of the individual product, in addition to the available production facilities. hence, the use of disposable concepts always has to be considered on a by-case basis, taking into account investment costs, the entire variable (running) costs of the particular manufacturing facility and individual processes, and the product requirements [137] . during recent years, kits for small-and medium-scale purifications and concentrations were introduced for adenoviruses (adenoviridae), aav (parvoviridae) and lentiviruses (retroviridae). in the ease of operation they are comparable to plasmid purification kits and require approximately 2 h for the entire process. the majority of manufacturers rely on adsorption membrane technology based on centrifugal devices (small scale), syringe capsules (small scale) or larger capsules for fast protein liquid chromatography systems. purification of lentivirus and aav particles is mainly achieved by the use of aex and cation-exchange (cex) membrane adsorbers. according to the manufacturer's handbooks, the maximum capacities of the largest units currently available are approximately 1 × 10 12 and 3 × 10 13 total virus particles for adenovirus and aav, respectively, and 2 × 10 8 infectious particles for lentivirus. the required culture volumes range from 20 to 500 ml, allowing recoveries of approximately 60-80% of the total particles depending on the culture conditions and virus. for the best results, it is important to consider the virus type for which the respective kit has been optimized. however, the kits can be used for other virus species after adapting the operating conditions. importantly, nucleic acids (i.e., host cell dna and proteoglycans) will also bind to aex membrane adsorbers and potentially co-elute. hence, kit procedures often include nuclease treatment of the harvest to reduce nucleic acid contaminations and to improve the flow rates downstream processing of cell culture-derived virus particles and thus process time (i.e., kits for adenovirus purification). in addition, a digestion step using chondroitinase abc, an enzyme that degrades glycosaminoglycan side chains of chondroitin sulfate proteoglycans [138] , is sometimes included to remove proteoglycan contaminations. certainly, the purity of the resulting virus fractions heavily depends on culture conditions, virus species, type and strain, as well as the applied kit. therefore, no detailed information on virus purity can be provided by the manufacturers. it should also be pointed out, however, that viruses purified by this method can only be used for research purposes and are not intended for clinical studies or medical applications. current downstream processes aim to combine individual unit operations to an overall purification train taking into account the virus type (morphology, specific surface characteristics), the final product type (e.g., subunits vaccines, split vaccines, inactivated or active virus particles), application requirements (contamination levels), product yields and size of the production batch. for the development of new purification schemes it will be equally important to develop and optimize new technologies, as to design efficient dsp trains based on optimal combinations of these new technologies with currently used methods. unfortunately, very few complete purification schemes, from culture supernatant to clinical grade virus product, have been described in detail with overall recoveries and degree of contaminations in the literature. hence, a precise evaluation of the respective methods is nearly impossible. in general, however, overall viral recoveries of greater than 30% are currently considered acceptable for human pharmaceutical products. a particularly challenging issue for the dsp of human vaccines and the purification of gene therapy vectors is the removal of host cell dna. current guidelines for newly licensed human inactive vaccine products from continuous cell lines stipulate that residual dna levels exceeding 10 ng per dose are not acceptable [139, 140] . levels of contaminating host cell dna for gene therapy applications are even lower. here, levels of 10-100 pg of residual host cell dna per parenterally administered dose are considered acceptable by most medical agencies [141] . for many applications these levels can only be achieved via nuclease treatments such as benzonase. the clear advantage of such a treatment is the elimination of potential oncogenes or other functional dna sequences [142] even in very low amounts of host cell dna and the cleavage of nucleic acids adsorbed to the target virus. in addition, nuclease and condroitinase treatment frequently results in improved efficacies for gene therapy vectors. for the purification of gene therapy vectors, the removal of empty capsids or virus particles containing nucleic acid sequences other than the intended genome is crucial for their efficacy. however, owing to the small differences in specific surface characteristics or in virus morphology this is fairly difficult. successful separations were described via density centrifugation (adenovirus) [38] , iec (aav) [20, 143] , and partial depletions by hic and immobilized zinc-chelating interaction chromatography (adenovirus) [144] . however, the possible application of the methods and their optimization has to be considered on a by-case basis. a further substantial challenge for dsp of viral vaccines, gene therapy vectors and other biotechnological products is batch-tobatch variations from the upstream processes. cell culture harvests can vary considerably not only in product concentration and quality but also in the type and amount of contaminations. for efficient and successful process development, all the aforementioned issues have to be addressed within a tightly coupled up-and down-stream process. therefore, critical parameters of the cultivation process effecting the dsp should be closely monitored and the subsequent purification methods adapted accordingly [145] . in the following paragraphs, we summarize unit operations and purification trains for different virus types that have been described in the last 5 years (table 4 ). a thorough review has been published by lusky et al. on the gmp production of adenoviral vectors for clinical trials [146] . another overview by burova and loffe deals with the chromatographic purification of recombinant adenoviral and aav vectors [38] . in general, aec is the driving force for the described processes for adenoviruses [147, 148] . eglon described a purification via aec in combination with sec from clarified and benzonase-treated cell lysate, which yielded total virus recoveries of 36% compared with 27.5% via the classical cscl-purification method [149] . duffy et al. compared a novel membrane-based technology for the purification of adenovirus and aav to conventional techniques [150] and peixoto et al. described a downstream process for adenoviral vectors based on membrane technology [147] . this purification train comprising clarification, ultrafiltration, aex membrane adsorber, ultrafiltration and sterile filtration resulted in a recovery of 52% of the infectious particles [147] . another very important aspect, namely the virus aggregation and association of dna to virus particles, was addressed by konz et al. [82] . wright reviewed, among other issues, vector quality characteristics such as aav-related impurities (e.g., aav-encapsidated dna impurities) and their pharmacological impact as well as gmp considerations for clinical aav vector production methods [18] . a chromatographic purification train for aav using a combination of hydroxyapatite, deae-sepharose and cellufine ® (chisso america incorporated, rye, ny, usa) sulfate with an overall yield of 30% has been described by o'riordan et al. [123] . more recently okada et al. described the use of a combination of cex and aex membrane adsorbers for the purification of aav serotype 1 and 8 with average recoveries of 17.4 and 49%, respectively [151] . downstream processing strategies for purification of retroviral vectors are summarized in at least two excellent reviews from segura et al. [47] and andreadis et al. [116] , the latter being published in 1999, ahead of the former. rodrigues et al. described the purification of murine leukemia virus-derived gene therapy vectors by means of membrane separation and aec with overall review recoveries of 26% and a purity of greater than 99% relative to the protein concentration [152] . moreover, lesch et al. illustrated a scalable capture step for lentiviral vectors generated in 293t cells with baculoviral vectors [153] . capturing based on deae monolithic columns reached a 65% recovery. ausubel et al. described the cgmp large-scale production of an oncolytic recombinant vesicular stomatitis virus based on a downstream process composed of a primary clarification step (filtration), benzonase treatment, aec, dia-/ultra-filtration and sterile filtration. the obtained yields of individual unit operations for infectious virus particles ranged from 59% to approximately 100% after sterile filtration [5] . the fact that full recovery was obtained after the complete purification of some batches suggests that some of the titers, which were estimated via plaque forming assays, might be overestimated. however, the overall process is described comprehensively. working et al. discussed critical points affecting the development and production of clinical grade oncolytic adenoviruses, describing the up-and downstream process in detail [154] . they illustrated a downstream process composed of cell lysis (triton), clarification, benzonase treatment, aec capture, ultra-/dia-filtration, sec, ultra-/dia-filtration (final formulation), followed by a final sterile filtration. however, the achievable purities and yields were not discussed in detail. downstream processes for purification of influenza virus particles were recently reviewed, summarizing applied purification trains for egg-and cell culture-derived influenza virus particles by wolff and reichl [155] . motivated by the recent pandemic threats of influenza, numerous research laboratories worked on the optimization of influenza vaccine production processes. for the downstream side the center of attention was on specific chromatography media to substitute the frequently applied cellufine sulfate for the capturing of influenza virus particles [105, 156] and on the use of chromatographic matrices with improved volumetric throughputs (i.e., monoliths [156, 306] ) and membrane adsorbers [102, 104] . wolff et al. described a downstream process for vaccinia virus based on a combination of pseudo-affinity and ion-exchange membrane adsorbers as well as pseudo-affinity and hic matrices [100, 117] . the most promising downstream train resulted in an overall yield of 34% (infectious virus particles). furthermore, depletion of total dna to 0.01% of the starting material and a total protein amount of less than 25 µg per dose was achieved [100] . however, batch variations of the starting material resulted in significant variations in the product yield and purity, and need to be further addressed. the success story of vaccination against major infectious diseases rests on a 200-year-old history [157] . gene therapy in contrast is a relatively new but promising technology with the revolution of molecular genetics in the 1970s paving the way into successful medical applications [158] . vaccines currently comprise a rapidly growing market within the biopharmaceutical industry and gene therapy vectors have the potential to follow this line. nevertheless, for some vaccines, manufacturing capacities are still limited (i.e., for pandemic influenza vaccines). in general these problems are related to both, upsteam processing and dsp, and can only be efficiently solved if both are considered together. in the last 10 years, upstream production processes of some vaccines moved away from their respective classical systems to the usage of diploid and continuous cell lines. however, downstream purification trains were in many cases only adapted to the new requirements but still lack further optimization. main improvements for vaccine purifications have to be governed by enhanced throughputs, capacities and potentially specificities of individual unit operations, as well as an enhanced linking of applied unit operations and the reduction of the number of purification steps. process development for gene therapy vectors could significantly benefit from the experience accumulated in vaccine production, especially with vectors derived from vaccines. nevertheless, there are still several technical problems to be solved for both product classes such as the benzonase-free removal of host cell nucleic acids. specific difficulties for the purification of gene therapy vectors are: • high final vector titers; • removal of transduction inhibitors from producer cells (proteoglycans, glycosaminoglycans [159] [160] [161] ); • elimination of free envelope proteins, defective-virus particles and empty capsids. removal of host cell proteins, medium proteins and peptides is usually less problematic but equally important owing to potential allergic reactions. of note, the reduced starting levels of process-related protein contaminations are mainly due to increased usage of serum-free, protein-free or even fully defined media for upstream cultivations -an excellent example for improved overall process economics via a tighter coupling of up-and down-stream methods. in summary, for the downstream processes described in table 4 , overall yields of virus particles of greater than 30% for production of human vaccines or gene therapy applications should be considered satisfactory. for other purposes, higher yields can be achieved depending on the requirements on product purity, the type and the amount of contaminants in the starting material, and the exertion. constant pressure towards improved product quality and safety as well as tighter timelines for the development of bio pharmaceuticals will certainly push for more efficient process development. current advances in the field indicate that increased application of highthroughput automated scaled-down models in combination with good mathematical models for experimental design and process development will support these efforts. certainly, these advances will also require the establishment of high-throughput assay • the application of parallel screening techniques, rationally designed experiments and the development of chromatographic models needs to be implemented to support downstream processing (dsp) development. • development and application of dsp platform technologies to improve the flexibility and response time of production processes is also required. • overall process economics need to be enhanced via a tighter coupling of upstream and downstream methods. • specific capturing of virus particles from bioreactor harvests via affinity chromatography can optimize the overall dsp by combining the primary concentration and purification step. • utilization of modern chromatography matrices such as membrane adsorbers and monoliths in place of classical resins enables an improved productivity in numerous dsp for viral particles. platforms for precise and reliable process characterization. as a result, unit operations should be available that have not only higher specificity and increased volumetric throughput but also enable design of purification trains with a reduced number of steps. advances in dsp will be most likely involve a broader use of filtration techniques (e.g., tangential flow filtration and diafiltration), the availability of new chromatography matrices such as membrane adsorbers, monoliths or bifunctional beads, and the identification of new, specific ligands. furthermore, single use concepts and the introduction of continuous dsp methods will play an important role as well as tighter process integration in terms of the up-and downstream process. furthermore, the establishment of platform technologies will have high priority whereas a large step towards platform technologies has already been accomplished by the development of downstream purification kits for gene therapy vectors. for additional readings, pedro et al. recently published a detailed summary dealing with the purification of bionanoparticles [162] , gagnon published a comprehensive description on the chromatographic purification of virus particles [113] and segura et al. published an overview of current scalable methods for purification of viral vectors [43] . 32 hammar l, gilljam g. extraction of hiv-1 in aqueous 2-phase systems to obtain a high-yield of gp120 aids res. hum. retroviruses 6(12), 1379-1388 (1990). 33 morenweiser r. downstream processing of viral vectors and vaccines. gene ther. 12, s103-s110 (2005). been highlighted as: • of interest •• of considerable interest the use of aqueous 2-phase systems to concentrate and purify bovine leukemia-virus outer envelope protein gp51 concentration and purification of feline leukemia-virus (felv) and its outer envelope protein gp70 by aqueous 2 phase systems purification of simian immunodeficiency virus, sivmac251, and of its external envelope glycoprotein, gp148 chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications extensive overview on methods and implications on the chromatographic purification of adenoviral and adeno-associated viral vectors adenovirus vector production using low-multiplicity infection of 293 cells construction of the vero cell culture system that can produce infectious hcv particles purification of a herpes-like virus from abalone (haliotis spp.) with ganglioneuritis and detection by transmission electron microscopy production and concentration of pseudotyped hiv-1-based gene transfer vectors overview of current scalable methods for purification of viral vectors purification and characterization of retrovirus vector particles by rate zonal ultracentrifugation a rapid method to produce high yields of purified 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transmitted as germinal provirus in the c3h and riii mouse strains physical and chemical properties of an oncornavirus associated with a murine adrenal carcinoma cell line large-scale purification of animal viruses in the rk-model zonal ultracentrifuge: iii. semliki forest virus and vaccinia virus biochemical characterization of the amphotropic group of murine leukemia viruses preparation of a highly purified vaccine against tick-borne encephalitis by continuous flow zonal ultracentrifugation purification, potency and immunogenicity analysis of vero cell culture-derived rabies vaccine: a comparative study of single-step column chromatography and zonal centrifuge purification infectious poxvirus vectors have capacity for at least 25000 base pairs of foreign dna new method for large-scale growth; and concentration of the epstein-barr viruses structure of the hepatitis a virion: peptide mapping of the capsid region antigenic differentiation of m-msv(o) from mouse, hamster, and cat c-type viruses purification of retroviral vectors for clinical application: biological implications and technological challenges •• comprehensive review on the purification of retroviral vectors clinical retroviral vector production: step filtration using clinically approved filters improves titers a novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography harvesting and concentration of human influenza a virus produced in serum-free mammalian cell culture for the production of vaccines antibody production virus removal by filtration: points to consider virus removal from factor ix by filtration: validation of the integrity test and effect of manufacturing process conditions high-performance tangential flow filtration using charged membranes tangential flow filtration for virus purification electrostatic and electrokinetic interactions during protein-transport through narrow pore membranes effect of solution ph and ionic-strength on the separation of albumin from immunoglobulins (igg) by selective filtration protein fractionation using electrostatic interactions in membrane filtration bioprocess membrane technology high performance tangential flow filtration ultrafiltration of parvovirus polymethacrylate monoliths for preparative and industrial separation of biomolecular assemblies •• compares conventional chromatography media and monoliths for industrial bioseparations chromatographic media for bioseparation •• detailed overview on chromatographic media used for bioseparations hydroxyapatite for biomolecule purification development of a purification process for adenovirus: controlling virus aggregation to improve the clearance of host cell dna describes the development of a purification process for adenoviruses (ad5) screening anion-exchange chromatographic matrices for isolation of onco-retroviral vectors capture and release of viruses using amino-functionalized silica particles the rapid concentration and purification of influenza virus from allantoic fluid controlled-pore glass as a stationary phase in chromatography direct visualisation of plasmid dna in individual chromatography adsorbent particles by confocal scanning laser microscopy chromatographic approaches in the purification of plasmid dna for therapy and vaccination noninvasive methods for characterization of largevolume monolithic chromatographic columns dispersion effects in preparative polymethacrylate monoliths operated in radial-flow columns large volume monolithic stationary phases: preparation, properties, and applications macroporous elastic polyacrylamide monolith columns: processing under compression and scale-up flow-dependent entrapment of large bioparticles in porous process media monoliths for fast bioseparation and bioconversion and their applications in biotechnology review: purification of proteins by membrane chromatography membrane processes in biotechnology: an overview • comprehensive review on membrane processes in biotechnology protein separation using membrane chromatography: opportunities and challenges • detailed overview on protein separations via membrane adsorbers impact of ligand density on the optimization of ion-exchange membrane chromatography for viral vector purification the emerging generation of chromatography tools for virus purification outlines the advantages of monoliths as chromatography tools for virus purification purification of cell culturederived modified vaccinia ankara virus by pseudo-affinity membrane adsorbers and hydrophobic interaction chromatography monoliths emerge as key purification methodology direct capture of influenza a virus from cell culture supernatant with sartobind anion-exchange membrane adsorbers engineering adeno-associated virus for one-step purification via immobilized metal affinity chromatography purification of cell culture-derived influenza virus a/puerto rico/8/34 by membrane-based immobilized metal affinity chromatography sulfated membrane adsorbers for economic pseudo-affinity capture of influenza virus particles exploiting heparin-binding properties of momlv-based retroviral vectors for affinity chromatography purification of porcine reproductive and respiratory syndrome virus from cell culture using ultrafiltration and heparin affinity chromatography removal of poliovirus type 1 from a protein mixture using an immunoaffinity chromatography column affinity capture of a biotinylated retrovirus on macroporous monolithic adsorbents: towards a rapid single-step purification process concanavalin a affinity chromatography for efficient baculovirus purification lectin-affinity chromatography for downstream processing of mdck cell culture derived human influenza a viruses one-step selection of vaccinia virusbinding dna aptamers by monolex chromatographic purification of virus particles tagging retrovirus vectors with a metal binding peptide and one-step purification by immobilized metal affinity chromatography protein adsorption dissociation constants in various types of biochromatography large-scale processing of recombinant retroviruses for gene therapy capture of cell culture-derived influenza virus by lectins: strain independent, but host cell dependent development and scale up of preparative hic for the purification of a recombinant therapeutic protein high-throughput screening of chromatographic separations: ii. hydrophobic interaction recent advancement in application of hydrophobic interaction chromatography for aggregate removal in industrial purification process new method for preparation of hydrophobic interaction chromatographic stationary phases based on polymer-grafted silica and their chromatographic properties scaleable chromatographic purification process for recombinant adeno-associated virus (raav) purification of a functional gene therapy vector derived from moloney murine leukaemia virus using membrane filtration and ceramic hydroxyapatite chromatography step gradients in 3-zone simulated moving bed chromatography: application to the purification of antibodies and bone morphogenetic protein-2 simulated moving-bed chromatography and its application to chirotechnology theoretical study of multicomponent continuous countercurrent chromatography based on connected 4-zone units two-fraction and threefraction continuous simulated moving bed separation of nucleosides analysis of a simulated moving bed operation for three-fraction separations (3f-smb) high-throughput screening of chromatographic separations: i. method development and column modeling one article of a series (four publications) covering in detail high-throughput screening of chromatographic separations modeling protein binding and elution over a chromatographic surface probed by surface plasmon resonance modeling electrostatic interactions of baculovirus vectors for ion-exchange process development the role of process simulation in pharmaceutical process development and product commercialization • describes the application of batch process simulators to expedite the development of pharmaceutical products advanced production scheduling for batch plants in process industries steric mass-action ion exchange: displacement profiles and induced salt gradients anionexchange membrane chromatography for purification of rotavirus-like particles disposables in downstream processing chondroitinase abc promotes corticospinal axon growth in organotypic cocultures issues associated with residual cellsubstrate dna in viral vaccines dna removal from a purification process of recombinant hepatitis b surface antigen who study group on cell substrates for production of biologicals removal of empty capsids from type 1 adeno-associated virus vector stocks by anion-exchange chromatography potentiates transgene expression empty capsids in column-purified recombinant adenovirus preparations. hum rapid highperformance liquid chromatographic analysis of adenovirus type 5 particles with a prototype anion-exchange analytical monolith column good manufacturing practice production of adenoviral vectors for clinical trials • overview on gmp production processes for adenoviral vectors including a section focusing on gmp regulations in the usa and europe towards purification of adenoviral vectors based on membrane technology • describes the purification of adenoviral vectors based on a combination of filtration techniques and membrane adsorbers production and formulation of adenovirus vectors purification of adenoviral vectors by combined anion exchange and gel filtration chromatography purification of adenovirus and adeno-associated virus: comparison of novel membrane-based technology to conventional techniques scalable purification of adenoassociated virus serotype 1 (aav1) and aav8 vectors, using dual ion-exchange adsorptive membranes. hum scateabte purification process for gene therapy retroviral vectors production and purification of lentiviral vectors generated in 293t suspension cells with baculoviral vectors meeting product development challenges in manufacturing clinical grade oncolytic adenoviruses • describes the up-and down-stream process in detail of clinical grade oncolytic adenoviruses downstream processing: from egg to cell culture-derived influenza virus particles summary on downstream processing methods and purification trains for egg-derived and cell culture-derived influenza viruses development of a strategy of influenza virus separation based on pseudoaffinity chromatography on short monolithic columns comptes rendus de l academie des sciences serie iii-sciences de la vie from the molecular genetics revolution to gene therapy: translating basic research into medicine removal of proteoglycans increases efficiency of retroviral gene transfer proteoglycans secreted by packaging cell lines inhibit retrovirus infection analysis of 4070a envelope levels in retroviral preparations and effect on target cell transduction efficiency purification of bionanoparticles purification of densonucleosis virus by tangential flow ultrafiltration gene therapy: promises and problems isoelectric-focusing of parvoviruses the structure of nuclear polyhedrosis viruses: ii. the virus particle the complete dna-sequence of autographa-californica nuclear polyhedrosis-virus polyethylenimine coating to produce serum-resistant baculoviral vectors for in vivo gene delivery bromovirus isolation and rna extraction in: methods in molecular biology: plant virology protocols: from virus isolation to transgenic resistance fields virology properties and classification of hepatitis-a virus vaccine 10 isoelectric focusing of hepatitis a virus in sucrose gradients hepatitis b: the virus and disease the hepatitis c virus: overview electron cryotomography reveals the portal in the herpesvirus capsid the genome sequence of an h11n2 avian influenza virus from a thick-billed murre (uria lomvia) shows marine-specific and regional patterns of relationships to other viruses les virus influenza chez l'homme et les animaux the rab11 pathway is required for influenza a virus budding and filament formation isolation of a2-singapore-57 influenza virus v and s antigens by isoelectric focusing studies on the adaptation of influenza-virus replication at low temperature. 5. isoelectric-focusing studies study of the electrophoretic mobility of a1 (leningrad) and a3 (leningrad) influenza-viruses electrophoretic studies on pr8 influenza virus overview: japanese encephalitis morphological, chemical, and biological characterization of japanese encephalitis virus virion and its hemagglutinin gene delivery by lentivirus vectors an overview the interferon-inducible rna helicase, mda-5, is involved in measles virusinduced expression of antiviral cytokines high-throughput analysis of mumps virus and the virus-specific monoclonal antibody on the arrays of a cationic polyelectrolyte with a spectral spr biosensor functions of the 5´ leader of murine leukemia virus genomic rna in virion structure, viral replication and pathogenesis, and mlv-derived vectors human papillomavirus (hpv) type distribution in females with abnormal cervical cytology. a correlation with histological study the ph stability of the papilloma virus protein the removal of phages t1 and pp7, and poliovirus from fluids with hollow-fiber ultrafilters with molecular-weight cutoffs of 50000, 13000, and 6000 behaviour of a guanidinedependent strain of poliovirus 1 in sucrose density and ph gradients characterization of type 1 poliovirus by electrophoretic analysis electrofocusing of viruses and sensitivity to desinfection viral aggregationeffects of salts on aggregation of poliovirus and reovirus at low-ph epidemiological survay of different rabies virus strains in iran morphological characteristics of rubella virus mapping the rubella virus subgenomic promoter biology and application of alphaviruses in gene therapy alphaviruses: semliki forest virus and sindbis virus vectors for gene transfer into neurons the complete genomic sequence of the modified vaccinia ankara strain: comparison with other orthopoxviruses cryo-x-ray tomography of vaccinia virus membranes and inner compartments identification of orthopox virus by isoelectrofocusing in a granulated gel micro-electrophoresis of pox viruses in molar sucrose micro-electrophoresis of cowpox and vaccinia viruses in molar sucrose measurement of the electrokinetic properties of vaccinia and reovirus by laser-illuminated whole-particle micro-electrophoresis isoelectric points of viruses • detailed collection on isoelectric points of viruses development of a novel adenovirus purification process utilizing selective precipitation of cellular dna purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration pilot-scale adenovirus seed production through concurrent virus release and concentration by hollow fiber filtration efficient purification of bromoviruses by ultrafiltration purification of anatid herpesvirus 1 particles by tangential-flow ultrafiltration and sucrose gradient ultracentrifugation a simple method for purifying the white spot syndrome virus using ultrafiltration downstream processing of mdck cell-derived equine influenza virus tangential flow microfiltration and ultrafiltration for human influenza a virus concentration and purification purification of the densonucleosis virus by tangential flow ultrafiltration and by ion exchange membranes purification of minute virus of mice using high performance tangential flow filtration increased viral titer through concentration of viral harvests from retroviral packaging lines improved methods of retrovrral vector transduction and production for genetherapy concentration of live retrovirus with a regenerated cellulose hollow-fiber optimisation of a multipartite human immunodeficiency virus based vector system; control of virus infectivity and large-scale production development and optimization of an adenovirus production process purification of adenoviral vectors using expanded bed chromatography high yield purification of functional baculovirus vectors by size exclusion chromatography ion-exchange membrane chromatography method for rapid and efficient purification of recombinant baculovirus and baculovirus gp64 protein hepatitis c virus and hepatitis b virus bind to heparin: purification of largely igg-free virions from infected plasma by heparin chromatography purification of cell culturederived human influenza a virus by size-exclusion and anion-exchange chromatography impact of adsorbents selection on capture efficiency of cell culture derived human influenza viruses binding aedes aegypti densonucleosis virus to ion exchange membranes densonucleosis virus purification by ion exchange membranes a simplified baculovirus-aav expression vector system coupled with one-step affinity purification yields high-titer raav stocks from insect cells serum-free production and column purification of adeno-associated virus type 5 purification of recombinant adenoassociated virus type 8 vectors by ion exchange chromatography generates clinical grade vector stock large-scale purification of a lentiviral vector by size exclusion chromatography or mustang q ion exchange capsule size-exclusion chromatography purification of high-titer vesicular stomatitis virus g glycoproteinpseudotyped retrovectors for cell and gene therapy applications. hum affinity recovery of moloney murine leukaemia virus purification of rabies virus produced on vero cells using chromatography techniques the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.no writing assistance was utilized in the production of this manuscript. key: cord-311486-auuyjct6 authors: cook, t. m. title: personal protective equipment during the covid‐19 pandemic: a reply date: 2020-06-04 journal: anaesthesia doi: 10.1111/anae.15158 sha: doc_id: 311486 cord_uid: auuyjct6 nan i thank professor murphy for his interest [1] in my article [2] . there is a great danger that an anaesthetist steps well 'outside their lane' when discussing respiratory particle physics and fluid dynamics. these are complex sciences in their own right, in which i have no training. that said, i think we largely agree that respiratory secretions vary in size over several magnitudes and this will affect their behaviour when expelled from the respiratory tract. for clarity, i neither stated that particles above 5 µm were large nor referred at all to whether these are visible or not and i am unclear why these are referred to in professor murphy's letter. particles of around 5 µm diameter are especially important because this is the (perhaps historical) cut-off used by most sources for defining behaviour as a droplet (>5 µm) or an aerosol (<5 µm) and because particles of these size are of the appropriate size to reach the alveoli rather than depositing higher up in the respiratory tract [2] . however, the behaviour of particles is highly complex, not dependent only on size and much debated: as i stated in my article "the dichotomy into> 5 and < 5 µm particles leading to droplet or airborne spread, respectively, is likely to be simplistic, with aerosols being maintained over a wider range of particle size". i infer that professor murphy's concerns are that larger particles than described contribute to aerosols and therefore that airborne transmission is a significant risk, and that the 2 m 'droplet zone' is insufficient. royal united hospitals bath nhs foundation trust, bath, uk email: timcook007@gmail.com no external funding or competing interests declared. personal protective equipment during the coronavirus disease (covid) 2019 pandemic -a narrative review personal protective equipment during the coronavirus disease (covid) 2019 pandemic -a narrative review the role of particle size in aerosolised pathogen transmission: a review modes of transmission of virus causing covid-19: implications for ipc precaution recommendations centers for disease control and prevention. interim infection prevention and control recommendations for patients with suspected or confirmed coronavirus disease corona virus-infection-prevention-and-control/transmission-characte ristics-and-principles-of-infection-prevention-and-control key: cord-326409-m3rgspxc authors: lai, alvin c.k.; chen, f.z. title: comparison of a new eulerian model with a modified lagrangian approach for particle distribution and deposition indoors date: 2007-03-24 journal: atmos environ (1994) doi: 10.1016/j.atmosenv.2006.05.088 sha: doc_id: 326409 cord_uid: m3rgspxc understanding of aerosol dispersion characteristics has many scientific and engineering applications. it is recognized that eulerian or lagrangian approach has its own merits and limitations. a new eulerian model has been developed and it adopts a simplified drift–flux methodology in which external forces can be incorporated straightforwardly. a new near-wall treatment is applied to take into account the anisotropic turbulence for the modified lagrangian model. in the present work, we present and compare both eulerian and lagrangian models to simulate particle dispersion in a small chamber. results reveal that the standard k–ε lagrangian model over-predicts particle deposition compared to the present turbulence-corrected lagrangian approach. prediction by the eulerian model agrees well with the modified lagrangian model. gaining understanding of particle transport and deposition indoors has numerous engineering applications. there has been considerable interest over the past decade in exposure assessment of fine particles inhalation and its influence on public health. the anthrax mailing accidents following the terrorist attacks of 11 september 2001 have generated enormous concern in design and application of ventilation strategy for protecting indoor environment against the intentional release of biological agents. phrases like aerobiological engineering or immune-building technology are coined recently to reflex the importance of studying aerosol behaviors indoors. better understandings of the aerosol dynamics in terms of mixing time and dispersion rate are vital to decide the positioning of air toxic sensors which have became an important element for monitoring buildings (gadgil et al., 2003) . improper placement of the sensors can impair the ability of the sensors to provide the first response decision. in addition, understanding aerosol dispersion and transport is very essential in the prevention of nosocomial transmission of airborne pathogens (cole and cook, 1998; li et al., 2005) . www.elsevier.com/locate/atmosenv 1352-2310/$ -see front matter r 2007 published by elsevier ltd. doi:10.1016 ltd. doi:10. /j.atmosenv.2006 after the outbreak of severe acute respiratory syndrome (sars) in south east asia 2003, there is increasing research interest in studying transport and control of airborne bacteria or viruses in indoor environments (beggs et al., 2005; nicas et al., 2005) and in confined environment like flight cabinet (mangili and gendreau, 2005) . while conducting in situ experiment can provide detailed information on microorganisms transport and survival in enclosed environments, potential danger and cost involved should be carefully considered. computational fluid dynamics (cfd) provides a very cost-effective way to perform parametric studies to investigate the microorganism dynamic behavior prior to full-scale experiments. there are two modeling approaches for twophase flow problems; namely the eulerian-eulerian model (hereafter refers to eulerian model) and the eulerian-lagrangian model (hereafter refers to lagrangian model). the eulerian method considers the particle phase as another continuum. governing equations derived from the mass (species) conservation condition are solved to give details of the particle concentration field. however, some studies treated the particle phase as scalar species (no inertia) and the results should be inferred cautiously (lu et al., 2005; noakes et al., 2004) . gravitational settling was considered in some eulerian cfd models (murakami et al., 1992; holmberg and li, 1998; zhao et al., 2004) , nevertheless, the deposition rate in those studies was only estimated empirically or simply ignored. it has been shown that, in certain circumstances, ignoring the deposition flux may result in numerical instability problems. theoretical model needs to be developed to evaluate deposition rate according to local turbulent flow condition. the second approach is the lagrangian method, which treats the dynamics of a single particle by the trajectory method. under this framework, generally speaking, the flow field is obtained by applying reynolds averaged navier-stokes (rans) turbulent models. the flow and other quantities obtained are ensemble-averaged components. near-wall fluctuating velocities are highly anisotropic with the component normal to the wall substantially smaller than those in the other two directions. proper treatment for the fluctuation components is critical to particle deposition modeling. however, many previous rans approaches for indoor environments ignored this effect and lead to incorrect deposition rates. equation of motion resulting from various forces exerting on an individual particle is solved to acquire the single-particle trajectory. a large number of sample particles should be analyzed before statistical conclusions can be drawn. a number of lagrangian simulations have been carried out for particle transport and deposition in ventilated single-zone room (zhao et al., 2004) , two-zone chamber (lu et al., 1996) and multi-zone chamber (chung, 1999) . unfortunately, the effect of turbulence on particle phase was overlooked in most of those models, even though the airflow fields were simulated with turbulence models. recently, large eddy simulation (les) has applied to simulate particle transport and deposition for simple singlezone geometries bouilly et al., 2005) . recently, the present authors have proposed a new eulerian model which takes external drift forces into account (chen et al., 2006) . gravitational settling has been incorporated as an external drift velocity and the results agree well with the experimental measurements. lately, a new lagrangian model has been developed to improve turbulent intensity in the vicinity of the wall (lai and chen, 2007) . in the present work, we compared particle distribution and deposition rates for a small model chamber by the two approaches. the geometry of the single-zone model room is shown in fig. 1 . the room dimension are length (x) â width (y) â height (z) ¼ 0.8 m â 0.4 m â 0.4 m. its inlet and outlet are of the same size, 0.04 m â 0.04 m. their centers are located at x ¼ 0, y ¼ 0.2 m, z ¼ 0.36 m and x ¼ 0.8 m, y ¼ 0.2 m, z ¼ 0.04 m, respectively. the symmetrical plane at y ¼ 0.2 m is referred as the center plane in the following discussion. two inlet velocities, 0.225 and 0.45 m s à1 (corresponding to air exchange rates of 10 and 20 h à1 , respectively), are tested. the room air temperature is set as 27 1c. due to the high computational cost for continuously particle tracking for lagrangian models, particles were injected only once instead of continuous injection adopted in the eulerian approach. this is a very common practice for almost all lagrangian simulations; nevertheless, due to the different nature of particle injection, it imposes a key constraint comparing to the eulerian models. the main objective of the present work is to highlight and compare the two approaches on the prediction of particle phase dispersion in a chamber, thus the air phase model is not mentioned here and the approach can be found elsewhere (chen et al., 2006; lai and chen, 2007) . in brief, the airflow field was resolved by a rng k-e turbulence model for both eulerian or lagrangian approaches. the simulations were performed with the aid of the commercial cfd code fluent (2005). a simplified eulerian drift-flux model has been developed to take full advantage of the extremely low volume fraction of indoor particles. the term ''drift-flux'' (or drift velocity) stands for particle flux (or velocity) caused by effects other than convection, i.e. gravitational settling and diffusion for the current work. as the convective velocity of the particle phase is the same as the air phase, the complexity of the two-phase flow system is greatly reduced. the governing equation for particle transport in turbulent flow field is given as where u is the air phase velocity vector, c i is the particle mass concentration, kg m à3 (or number concentration, m à3 ) of particle size group i (hereafter in this paper, the subscript i denotes particle size group), v s;i is the particle settling velocity, e p is the particle eddy diffusivity, and d i is the brownian diffusion coefficient and s c i is the mass concentration source term. for coarse particles, the loss by deposition (i.e. sedimentation) must be properly treated. in the current approach, the concentration field is divided into two regions: the core region and the concentration boundary layer. the methodology adopted here is to get the distribution of particles in the core region with the three-dimensional mass conservation eq. (1), while within the concentration boundary layer, the particle wall flux is determined with a one-dimensional semi-empirical particle deposition model (lai and nazaroff, 2000) and the results are substituted into eq. (1) as the boundary condition. the equation of motion of a small aerosol particle can be written as where u p;i is the velocity of the particle, t is the particle relaxation time, n i (t) is the brownian force per unit mass, r p and r are the particle and air density, respectively, and g i is the gravitational acceleration. in a stochastically modeled turbulent flow, the instantaneous fluid velocity can be expressed as which is the sum of the mean velocity component, u i , from the rng k-e model and the fluctuating velocity component, u 0 i , which will be described in fig. 1 . schematics of the model room. section 2.3. the brownian force per unit mass is important for submicron particles. the brownian force is modeled as a gaussian white noise random process as described by li and ahmadi (1992) . the procedure for simulating the brownian force is to generate a white noise process with the noise intensity, and the spectral intensity s 0 is given by where g i is a zero-mean, unit variance-independent gaussian random number and k b ¼ 1.38 â 10 à23 j k à1 is the boltzmann constant. the third terms on the rhs of eq. (2) represents gravity force exerting on the particle. it has been reported by the present authors that the requirement for a careful grid independence test is more stringent for a lagrangian simulation (lai and chen, 2007) . to model particle deposition accurately, the grid should be able to resolve not only the turbulence field, but also the flow field properties along particle paths. if the near-wall boundary layer is not properly resolved, the predicted deposition rate may depart from the actual solution severely. in the present model, the near-wall turbulence is deliberately damped. if the near-wall cell center is out of the viscous sublayer, the normal velocity at the point may not be negligible and consequently the interpolated normal velocity at the particle position may be sufficiently large to drive the particle to impact onto the wall directly. hence, the near-wall grid should be fine enough to resolve the important deposition boundary layer. three different grid systems are tested and details of them are listed in table 1 . taken into account of both computational resources requirement and result accuracy, grid system 2 with 181,976 hexahedral cells is chosen for the present simulation (lai and chen, 2007) . to take into account of anisotropic turbulence, a new correction scheme, which is essentially a hybrid combination of the methods of he and ahmadi (1998) and matida et al. (2004) , is proposed. the quadric relation used by he and ahmadi is adopted, written as ffiffiffiffiffi ffi where a ¼ 0.008 (bernard and wallace, 2002) is used by fitting the dns results of kim et al. (1987) . in order to implement this correction to the isotropic k-e turbulence models, the method of matida et al. is used to simplify the system by forcing the streamwise and spanwise normal reynolds stress components equal to the normal component, i.e. ffiffiffiffiffiffi a new turbulent kinetic energy for particle tracking calculations can then be defined as in this method, only the turbulent kinetic energy in the near-wall area needs to be corrected, and the resultant turbulent time scale will be updated accordingly. it should be emphasized that the optimized turbulent kinetic energy in eq. (8) still remains isotropic. there are several salient features of this hybrid scheme. the method can be conveniently applied to various conditions, as the modification to the model is minimal. the prediction of the normal reynolds stress component is remarkably improved, thus it can result in a more reasonable particle deposition rate. on the other hand, the turbulent intensities in the rest two directions are underestimated undesirably. this under-estimation is unlikely to incur significant error when the particle deposition rate is the parameter of concern, as particle deposition is not directly affected by the rest two components and, the mean flow velocities in these two directions overwhelms the fluctuation counterparts. (all particle sizes are referred to aerodynamic particle diameters). a noticeable observation is that the submicron particle concentration can achieve fairly uniform in just 300 s, and in 30 min the particle concentration is virtually homogeneous for the entire zone. the ultrafine particles exhibit similar dispersion characteristic to that observed for 0.3 mm particles (results not shown). the dispersion rate decreases with the particle size as concentration gradient is distinctively observed for coarse particles. for 7 mm particles concentration homogeneousness cannot be achieved (even beyond 1800 s). to represent the non-uniformity of a concentration field, the coefficient of variation, cv i ðtþ, of concentration is defined as (mage and ott, 1996) where c i ðtþ is the volume averaged concentration, c i (t) is the concentration at elapsed time t at each sample point and n is the number of sample points. in case of non-uniform mixing, a useful parameter, the mixing time, is used to quantify the time needed to reach a well-mixed state. it is defined as the time at which the coefficient of variation becomes o10% permanently since the release of pollutant. fig. 3 presents the coefficients of variation of the three particle sizes. inferring from the results, it can be shown that cv strongly depends on the particle size and airflow; small size and high airflow favor mixing. one thing should be noted is that the mixing characteristics of 0.3 and 1 mm are very close whereas very distinct difference can be observed for 1 and 7 mm particles. as the gravitational settling magnitude is a function of particle size with an exponent of 2, sedimentation influences the mixing and dispersion rates (fig. 2) in a non-linear way; in the results presented, the distinct behavior of 7 mm particles is observed. for lagrangian model, the results presented are very different. since particles are treated as a discrete and not a continuum phase, concentration contour does not exist. another factor further complicates the issue is the computer resources required for lagrangian model. due to the current limitation of computational resources, almost all lagrangian models inject particles momentarily and track the individual particle based on the forces exerted. because of the transient in nature and the validity of the well mixed (discussed above), these pose limitation for presenting the results obtained from lagrangian approach. in the literature, the parameter decay rate loss coefficient is often used to characterize the particle removal rate in an enclosure. the decay rate coefficient is derived from the mass balance principle where well-mixed condition is assumed. this has a significant implication on the application of particle decay rate coefficient for quantifying the fate of particles in a non-well-mixed enclosures and it must be used cautiously (bouilly et al., 2005) . hence for lagrangian framework, another common approach, which counts the number of particles remaining in the domain versus time, is adapted . the particle deposition fraction, z, is defined as where n in and n out are the numbers of particles released at the inlet and the number of particles exited from the outlet, respectively. as observed in fig. 4 , the original eim without correction overpredicts particle deposition rate noticeably, particularly for 0.3 and 1 mm particles at the high ventilation rate. the turbulent intensity is stronger for the high-velocity case. if the correction scheme is not applied, the original eim tends to give more severe over-prediction for the high-velocity case. generally, the deposition fractions predicted with the correction are closer to the semi-empirical equation (lai and nazaroff, 2000) . thus far we have highlighted the main features of two models developed recently and results are presented separately. it is very valuable to quantify the simulation results by a same parameter. in velocities are shown. overall speaking, the results modeled by the two approaches agree well with each other; as the particle size increases, the deposition fraction increases. for submicron particles, the deposition fractions predicted by lagrangian (without near-wall turbulent correction) is higher than those predicted with correction and eulerian drift flux prediction follows. deposition fraction for 0.45 m s à1 is also higher than the case of 0.225 m s à1 . this is because for submicron particles, the deposition is significantly affected by turbulent diffusion and it is stronger for the high-velocity case. for supermicron particles scenario, the discrepancy between eulerian and lagrangian increases with particle size, however, is not significant. one thing should be noted is that as the particle size increases, the influence of the turbulent correction diminishes. these observations can be explained by the dominant fate mechanism for this size range of particles. as particle size increases, deposition by gravitational settling increases significantly and the other effect, i.e. turbulent diffusion, becomes less important. it must be careful when direct comparison is attempted to be made. as discussed above that the injection type for eulerian is continuous while for lagrangian, the injection is momentarily. for steady-state condition such as eulerian model, the parameter deposition velocity can be used to quantify the deposition rate in a more rigorous way, while for lagrangian framework, using deposition velocity as a parameter is not an obvious means. on the other hand, the deposition fraction cannot be used to quantify the deposition onto various orientated surfaces, however, it can be used to characterize both models. the current study focuses on an empty chamber so some comments on practical applications should be addressed. the author has published one article regarding the effects of room furnishing on particle deposition rates indoors (thatcher et al., 2002) . the experimental results showed that increasing the surface area from bare to fully furnished increased the deposition loss rate with the latest increase seen for 0.5 mm particles. it can be attributed to the additional surfaces available for diffusion loss. the current models can be applied to both empty and furnished room with proper boundary conditions. knowledge of particle dispersion and deposition indoors improves human exposure assessment and provides insights for better pollutant control measures. cfd has become a virtual tool for studying particle dynamics. we compared a new drift-flux eulerian and a modified lagrangian models for a single-zone chamber geometry. one key feature of the drift-flux model is to encapsulate external forces into the formulation and present the results in a continuous domain. near-wall anisotropic turbulence correction is properly applied in the lagrangian model. inferring from the results, it is shown that non-corrected turbulent scheme over-predicts particle deposition significantly for submicron particles while supermicron particles are not sensitive to the correction scheme. the paper also highlighted the results obtained by eulerian and lagrangian models. the two models agree very well for submicron particles. as the size increases, the discrepancy increases moderately. it is not straightforward to conclude which model is better than the other as each model has its own fundamental assumptions imposed. the results shown here reveal that the two present models are comparable. turbulent flow: analysis, measurement and prediction methodology for determining the susceptibility of airborne microorganisms to irradiation by an upper-room uvgi system using large eddy simulation to study particle motions in a room effect of ventilation strategies on particle decay rates indoors: an experimental and modelling study modeling particle distribution and deposition in indoor environments with a new drift-flux model three-dimensional analysis of airflow and contaminant particle 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nonuniform mixing and human exposure in indoor environments transmission of infectious diseases during commercial air travel improved numerical simulation of aerosol deposition in an idealized mouth-throat diffusion characteristics of airborne particles with gravitational settling in a convection-dominant indoor flow field toward understanding the risk of secondary airborne infection: emission of respirable pathogens development of a numerical model to simulate the biological inactivation of airborne microorganisms in the presence of ultraviolet light effects of room furnishings and air speed on particle deposition rates indoors comparison of indoor aerosol particle concentration and deposition in different ventilated rooms by numerical method key: cord-306371-jmaqkovs authors: jensen, ditte marie krohn; cun, dongmei; maltesen, morten jonas; frokjaer, sven; nielsen, hanne mørck; foged, camilla title: spray drying of sirna-containing plga nanoparticles intended for inhalation date: 2010-02-25 journal: j control release doi: 10.1016/j.jconrel.2009.10.010 sha: doc_id: 306371 cord_uid: jmaqkovs local delivery of small interfering rna (sirna) to the lungs constitutes a promising new area in drug delivery. the present study evaluated parameters of importance for spray drying of sirna-loaded poly(d,l-lactide-co-glycolide) (plga) nanoparticles (nps) into nanocomposite microparticles intended for inhalation. the spray drying process was optimised using a statistical design of experiment and by evaluating powder characteristics upon systematic variation of the formulation parameters. concentration, carbohydrate excipient (trehalose, lactose and mannitol) and the ratio of np to excipient were varied to monitor the effects on moisture content, particle morphology, particle size and powder yield. the identified optimum conditions were applied for spray drying of sirna-loaded nanocomposite microparticles, resulting in a product with a low water content (0.78% w/w) and an aerodynamic particle diameter considered suitable for inhalation. the use of mannitol in the formulation allowed a significantly lower moisture content than trehalose and lactose. the inclusion of 50% (w/w) or higher amounts of nps resulted in a marked change in the surface morphology of the spray-dried particles. importantly, the integrity and biological activity of the sirna were preserved during the spray drying process. in conclusion, the present results show that spray drying is a suitable technique for producing nanocomposite microparticles comprising sirna-containing plga nps for potential use in inhalation therapy. the concept of rna interference (rnai), mediated by small interfering rna (sirna), provides an opportunity for a whole new class of drugs directed towards disease-associated genes. however, before rnai can be used to its full potential for treatment of a variety of diseases, challenges regarding the delivery of sirna need to be overcome [1] . a promising approach to overcoming delivery challenges related to stability and cellular targeting of sirna is local delivery of sirna directly onto the diseased tissue using a delivery system capable of protecting the sirna from degradation [1, 2] . poly (d,l-lactide-co-glycolide) (plga) nanoparticles (nps) have been proposed for delivering macromolecules, such as sirna, to the cytosol of cells in vitro [3, 4] . the nps have been shown to be taken up by endocytosis and can escape the endosomes to a certain degree before degradation [4] and subsequently release the drug, although the mechanism of endosomal escape is unclear at present. once present in the cytosol, the plga is suggested to be degraded slowly, sustaining the release of encapsulated sirna, a property which might be very useful for therapeutic purposes [3] . in this perspective, the airways are interesting sites for local delivery of sirna. the lungs are exposed to a variety of pollutants on a daily basis and are therefore also susceptible to a wide range of diseases. examples of airway-located diseases, which would be treatable with sirna, are cancer and infections like severe acute respiratory syndrome (sars) and influenza [2, 5] . diseases may affect different parts of the airways, which branch out approximately 17 times before ending up in the alveoli [6] . however, many infections are localized in the lower airways [7, 8] , so efficient delivery systems must be developed to treat airway diseases. even though the total alveoli surface area of 100-150 m 2 in adults provides a large surface for drug deposition [9] , the branching, mucociliary clearance and breathing are mechanisms that prevent particles from entering the lower airways. to overcome these barriers and achieve particle deposition in the lower airways, the size distribution and aerodynamic behaviour of the particles intended for inhalation are of great importance. the aerodynamic diameter can be used to predict the behaviour of a given particle in an airstream and thereby predict the site of impact with airway epithelium. larger particles with an aerodynamic diameter above 5 µm will most likely impact with the tissue of the mouth, throat or upper lung mucosa and be eliminated from there, whereas particles smaller than 1 µm will often suffer from being exhaled and thus not be deposited in the alveoli. when formulating drugs for pulmonary delivery, another issue to be considered is the clearance mechanism for particles deposited in the deeper areas of the lungs. the clearance in the deeper areas of the lungs is mostly provided by pulmonary macrophages, which are mobile phagocytic cells monitoring the inner surface of the alveoli by ingesting and degrading pathogens and particles. even though microsized particles will be processed by the pulmonary macrophages, nps with a diameter smaller than approximately 260 nm might escape macrophage clearance [10, 11] . to overcome the macrophage clearance and still benefit from a particle size suitable for deep lung deposition and sustained release, the present study employs nanocomposite microparticles consisting of co-spray-dried plga nps and a carbohydrate excipient. the methodology of spray drying enables formation of particles of a size suitable for inhalation comprising both nps and an excipient [12] [13] [14] . furthermore, nanocomposite microparticles composed of plga nps and sugars, such as trehalose and lactose, have been shown to decompose in the lung lining fluid, releasing the nps from the formulation [12] [13] [14] . the spray drying process is widely applied for production of inhalable particles, and allows for a large degree of variation of the final product. spray drying has previously been used for a number of applications, including the preparation of dry powder protein particles intended for inhalation [15, 16] , as well as dry powder containing cationic plga nanoparticles, the latter to prevent aggregation and loss of transfection potential when subsequently complexed to pdna [17] . during spray drying, a feed solution, suspension or emulsion of the compound(s) is dispersed into fine droplets, which are then dried by a hot air-stream. several parameters in the drying process itself, as well as the composition of the feed formulation, greatly influence the characteristics of the dry end product [18, 19] . carbohydrate excipients function as bulking agents that enable production of stable particles of a size suitable for inhalation and allow a rapid release of the nps in the lung lining fluid upon inhalation. also, the excipients are added to the formulation to provide some degree of protection of the nps and the encapsulated drug (in this case sirna) during the process of spray drying, in particular against shear forces and increased temperatures. excipients like mannitol, trehalose and lactose are commonly used in spray drying with satisfying end products [13, 20] . these carbohydrates are dissolvable in water [21] and possess good safety profiles and are thus good choices for the production of the nanocomposite microparticles, which should decompose quickly in the lung lining fluid. the objective of the current study was to identify optimal formulation variables and process parameters for spray drying of sirnaloaded plga nps. the success criterion was obtaining a product suitable for inhalation without compromising the integrity and biological activity of the active compound in the production. to the authors' knowledge, this is the first time that spray drying of a delivery vehicle containing sirna intended for pulmonary delivery has been performed. enhanced green fluorescent protein (egfp) and luciferace (fluc) dicer substrate asymmetric duplex sirnas were provided by integrated dna technologies bvba (leuven, belgium) as dried, annealed, purified and desalted duplexes. sequences were as follows: egfp, sense 5′-pacccugaaguucaucugcaccaccg-3′, antisense 5´-cgguggugca-gaugaacuucaggguca-3´, fluc sense 5′-pgguuccuggaacaauug-cuuuuaca-3′, antisense 5´-uguaaaagcaauuguuccaggaaccag-3 [ 22] . lower case letters are 2´-deoxyribonucleotides and underlined capital letters are 2´-o-methylribonucleotides. plga (lactide:glycolid molar ratio of 75:25, m w : 20 kda) was purchased from wako pure chemical industries, ltd (osaka, japan). polyvinylalcohol (pva) 403 with an 80.0% degree of hydrolysis was provided by kuraray (osaka, japan). all other general chemicals were obtained commercially at analytical grade. two different techniques were employed to prepare sirna-loaded and non-loaded plga nps. the use of the two different methods was required since it was not possible, in the small-scale lab available, to produce larger amounts of non-loaded nps required for process optimization by the double emulsion solvent evaporation (dese) method. however, nps produced by the two methods had similar characteristics and are thus considered to be comparable (results not shown). the dese method was used for production of sirna-loaded nps. in brief, 250 µl of an 8 µm sirna solution in te-buffer (10 mm tris-hcl, 1 mm edta, ph 7.5) was added to 500 µl of a 60 mg/ml solution of plga in dichloromethane, and the mixture was sonicated for 90 s to obtain an emulsion. a volume of 2 ml 2% (w/v) pva in diethylpyrocarbonate (depc)-treated water was added to the emulsion, and a second sonication of 1 min was performed, giving a double emulsion. the double emulsion was subsequently diluted with 4 ml of 2% (w/v) pva in depc-treated water and left for 3 h under stirring to evaporate the dichloromethane. for isolation of nps, the dispersion was centrifuged for 12 min at 4°c and 18,000 ×g. the supernatant was discarded, and the np pellet was redispersed in depc-treated water. centrifugation and redispersion of the nps were repeated three times. the "modified spontaneous emulsification solvent diffusion" (modified-sesd) method was used for the production of non-loaded plga nps, employed in the process optimization [23] . a mixture consisting of 7.5 ml acetone and 5 ml methanol was used to dissolve 500 mg of plga. the polymer solution was then added dropwise to 50 ml 4% (w/v) aqueous pva solution at a rate of approximately 2 ml/min. the pva solution was continuously stirred by a eurostar basic propeller mixer (ika labortechnik, germany) at 400 rpm concomitant with addition of the polymer solution. nps were isolated as described above but with centrifugation at 41,000 ×g for 12 min. the nanocomposite microparticles were prepared by spray drying of a plga np suspension in different aqueous carbohydrate solutions using a büchi b-290 spray dryer (büchi labortechnik ag, postfach, switzerland). the spray dryer was a co-current model, equipped with a nozzle atomizer using nitrogen as the atomizing gas. the nozzle orifice diameter was 0.7 mm. dry particles were separated from the airstream by centrifugal forces using a high-performance cyclone (büchi labortechnik ag, postfach, switzerland). the applied process parameters (table 1) were chosen on the basis of pilot experiments providing a dry powder which could be reconstituted into a np suspension without any marked change in the np size distribution (data not shown). the formulation parameters evaluated by design of experiment were i) type of carbohydrate excipient, ii) ratio of nps to excipient and iii) total concentration of carbohydrate excipient and np in the feed solution ( table 2) . a factorial 2 × 2 × 3 experimental design with 6 centre points was set up using the software sas jmp ® (sas institute inc., cary, nc, usa).the complete design is shown in table 3 . analysis of the results was carried out in sas jmp ® using the standard least squares approach. the dry powder yield was determined as the difference in weight of the sample vial before and after product collection. the weight difference was compared to the total dry mass used for the specific sample, and the yield in % was calculated. the moisture content in the dry powder was not considered when calculating the yield. the water content of the powder was examined by thermogravimetric analysis, using a tga 7 (perkin elmer, waltham, massachusetts, usa) with nitrogen purging. samples were heated to 110°c at a constant rate of 10°c/min. the temperature was kept at 110°c until a stable weight was obtained. the weight loss in percent due to water evaporation was subsequently calculated and defined as the moisture content. the surface morphology was examined using a scanning electron microscope (sem) of the type jsm-5200 scanning microscope (jeol ltd., tokyo, japan). a powder sample was sprinkled onto a sem stub covered with double side carbon tape and subsequently sputter coated with gold prior to examination. the aerodynamic particle size was determined using an aerodynamic particle sizer spectrometer 3321 equipped with a small-scale powder disperser (tsi incorporated, shoreview, mn, usa) used to generate the aerosol. the instrument employed a time of flight principle, measuring the velocity of the individual particles where the aerodynamic diameter was defined as the diameter of a spherical particle with the same velocity and behaviour as the analyzed particle. the resulting particle size distribution accounted for the density, was number based and was afterwards converted to a mass based particle size distribution by the software provided with the instrument. a few mg of spray dried powder was placed on the turntable inside the small-scale powder disperser and introduced into the aerodynamic particle sizer by a venturi aspirator. the flow rate and the rotation rate of the small-scale powder disperser were controlled to yield a particle concentration in the range of 0.3-3.0 mg/m 3 and analysis was carried out continuously for 20 s. it was assumed that the shear forces present in the small-scale powder disperser were sufficient to deagglomerate the spray dried particles, which might not be the case for pharmaceutical devices used for inhalation therapy. for powders dispersed with metered dose inhalers the aerodynamic particle size obtained with the aerodynamic particle sizer spectrometer 3321 correlated well with the aerodynamic particle size obtained with cascade impactors [24, 25] . the formulation in the present study was spray dried and the dry powder was assumed to be chemically homogeneous making the chemical analysis of the cascade impactors less important. in the following text, the aerodynamic particle size is given as the mass median aerodynamic diameter (mmad). the content of pva in the nps was examined by uv-spectroscopy, employing the method described by barman et al. [26] . in brief, 5 ml of 1 n naoh was added to a sample of 10 mg nps (10 mg of plga as reference sample) and left while rotating until no solid was visible. 0.9 ml 37% (v/v) hcl was added. to 1 ml of sample solution, 0.9 ml of 3.7% (w/v) boric acid was added to form the borate salt, and subsequently 0.1 ml ki/i 2 stock solution (3.32 g of ki and 2.54 g of i 2 in 200 ml of deionized water) was added. the od 620 nm was measured on a cary ® 100 bio uv-visible spectrophotometer (varian inc., palo alto, ca, usa) and the value for the reference sample was subtracted. the concentration was determined from a standard curve in the linear range of 0.01 mg-1 mg pva. to evaluate the ability of the nanocomposite microparticles to decompose into plga nps, 1 ml of water was added to approximately 1 mg of powder sample. after mixing, the sample was centrifuged for 12 min at 4°c and 18,000 ×g and washed three times with water. the nps were redispersed in water, and the size was determined by dynamic light scattering using the photon correlation spectroscopy (pcs) technique. the measurements were performed on diluted samples (n = 3) at 25°c using a malvern nanozs (malvern instruments, worcestershire, uk) equipped with a 633 nm laser and 173°d etection optics. malvern dts v.5.10 software (malvern instruments, worcestershire, uk) was used for data acquisition and analysis. a polystyrene size standard (220 ± 6 nm, duke scientific corp., duke, nc) was used to verify the performance of the instrument. the integrity of the sirna extracted from the spray-dried nps was evaluated by gel electrophoresis to investigate the effects of the spray drying process on sirna loaded into nps. the biological activity was determined in transfection assays using the human epithelial nonsmall cell lung cancer cell line h1299 stably transfected with egfp (a gift from prof. jørgen kjems, university of aarhus, denmark). sirna was extracted from the nps before and after spray drying by dissolving the plga matrix in chloroform. briefly, an amount of powder corresponding to approximately 1 mg of np was weighed. samples consisting of nanocomposite microparticles were dissolved in 1 ml of water, the nps were separated by centrifugation for 12 min at 4°c and 18,000 ×g and the supernatant discarded. a volume of 200 µl chcl 3 and 500 µl te-buffer was added, and the samples were rotated for 90 min. the two phases were separated by centrifugation for 20 min at 18,000 ×g and 4°c, and the aqueous phase was collected. the sirna concentration in the extracts was determined using the ribogreen ® rna quantitation reagent (invitrogen a/s, taastrup, denmark). the concentration was determined using a standard curve ranging from 0 to 50 ng/ml, and samples were diluted to obtain values in this range. the fluorescence of the samples was measured using a fluostar optima fluorescence plate reader (bmg labtech, offenburg, germany) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. the integrity of extracted sirna was evaluated on a 4-20% polyacrylamide gel containing tbe buffer (0.089 m tris base, 0.089 m boric acid, and 2 mm sodium edta, ph 8.3). electrophoresis was carried out in tbe buffer at a constant voltage of 100 v for 1 h. sirna bands were visualized using an image station (kodak image station 1000, eastman kodak company, or, us) after staining for 40 min with a 1:10,000 dilution of sybr-green ii rna gel stain (invitrogen a/s, taastrup, denmark) in depc-treated water. 2.6.3. biological activity of spray-dried sirna 2.6.3.1. cell culture. h1299 cells stably expressing egfp were maintained in rpmi 1640 medium (fisher scientific biotech line, slangerup, denmark) supplemented with 100 u/ml penicillin, 100 µg/ ml streptomycin, 2 mm l-glutamine (all from sigma-aldrich, brøndby, denmark), 0.2 mg/ml geneticin (invitrogen a/s, taastrup, denmark) and 10% (v/v) fetal bovine serum (fbs) (paa laboratories gmbh, pasching, austria). cells were grown in an atmosphere of 5% co 2 /95% o 2 at 37°c, growth medium was replaced every 48 h, and the cells were sub-cultured approximately 1:5 twice a week. prior to sirna addition, h1299 cells were detached from culture flasks by incubating the cells for 5 min at 37°c with trypsin/edta solution. the cells were seeded in 24-well tissue culture plates in 0.5 ml growth medium, at a density of 8 × 10 4 cells per well, and kept at normal culturing conditions for 24 h. 2.6.3.2. transfections and flow cytometry. after 24 h (approximately 80% confluency), the cells were incubated with 300 µl of pre-mixed samples in fresh rpmi 1640 + 10% (v/v) fbs for 48 h at 37°c. the samples consisted of either 2 nm sirna extracted from nps or mps at a concentration of 1 mg/ml tested before or after spray drying and with or without the addition of lipofectamine 2000. in the case of the "extracted" control non-loaded samples, sample volumes were identical to sirna-containing samples. lipofectamine 2000 (invitrogen a/s, taastrup, denmark) was added in amounts recommended by the manufacturer. after incubation, the cells were washed with 500 µl pbs, and 100 µl of tryple tm express (invitrogen a/s, taastrup, denmark) was added. after incubation with tryple tm express for 5 min, 1 ml of growth medium was added, the cells were transferred to tubes and centrifuged for 10 min at 1076 ×g and 4°c. the supernatant of each tube was removed, and the cells were redispersed in pbs containing 10% (v/v) fbs followed by a second centrifugation, after which the cells were redispersed in ice-cold pbs containing 10% (v/v) fbs (1 ml) and 1 µg/ml propidium iodide (pi) (invitrogen a/s, taastrup, denmark) for staining of dead cells. the cells were analysed on a facscan flow cytometer (becton dickinson, nj, usa) using the cellquest software (becton dickinson). dead cells were excluded based on the pi-staining. measurements were performed in triplicate, unless otherwise stated. values are given as means ± sd. the statistical significance of the results obtained from cell transfections was determined using one-way analysis of variance (anova) employing a confidence interval of 95%. the present study addresses the influence of different formulation and processing parameters on the characteristics of spray-dried nanocomposite microparticles. the results were evaluated to determine the optimal formulation for spray drying of plga np-based nanocomposite particles. further, sirna-containing plga nps were spray dried using the optimal formulation, and the sirna extracted from the nps was evaluated with respect to content, integrity and biological activity. in table 3 , the design of experiment is listed along with the responses. the samples were spray dried using non-loaded nps for optimisation purposes. the product yield after the spray drying process varied between 19.3 and 56.4%. the moisture content for all samples was relatively low, and the samples containing mannitol displayed considerably lower moisture content than the other samples. the majority of the samples showed a mmad within the desired range of 1-5 µm, and only a few had a diameter slightly above 5 µm. all samples with a np to excipient ratio of 0.2 appeared as particles with a smooth surface morphology, while samples having a ratio of 0.5 or 0.8 displayed corrugated and raisin-like surfaces (fig. 1) . the average diameters of the nps before and after spray drying, as well as the polydispersity indexes, are shown in the supplementary data section. all samples could be redispersed completely into nps with no marked change in the np size, as compared to the size before spray drying. statistical evaluation was performed on data presented in table 3 . the analysed output values were moisture content, yield and mmad. fig. 2 shows the impact of the different formulation parameters on the measured responses. it is apparent from fig. 2 , that the products produced with lactose and trehalose possessed similar characteristics. mannitol changed the effects exerted by the ratio and concentration somewhat as compared to the two other excipients. table 4 shows the statistically significant parameters for each of the three evaluated responses. the scaled estimates provide an overview of the magnitude of the exerted effects found. in brief, the moisture content was affected significantly by the type of excipient, and mannitol provided the lowest water content of the three examined excipients (p < 0.0001). the ratio of nps to excipient also have a significant effect on the water content (p = 0.0001), since a high ratio resulted in a decrease in the water content. factors showing a significant effect on the aerodynamic diameter of the nanocomposite microparticles were ratio (p = 0.0023) and concentration (p = 0.0005) of the sample. high ratios provided particles of slightly smaller mmad, whereas high concentrations resulted in higher mmad. the dry powder yield was significantly affected by the ratio of nps to excipient (p = 0.0063), as increasing the ratio lead to a lower yield. also, the yield was dependent on the concentration of total dry substance in the feed formulation (p = 0.0016), since a higher yield was obtained at increased concentrations. however, the effect of concentration on the yield could also be due to a reduced loss in the collection process, since larger amounts were used with increasing concentrations. to ensure that no bias was imposed on the result by the use of different batches of nps, the effect of the batch of nps on the different outcomes was examined. no significant difference was observed from the variation of the np batches (data not shown). the optimum formulation found to provide a low water content, a sufficient yield and a small particle size was with mannitol as the carbohydrate excipient, a total dry substance concentration of 30 mg/ ml and a ratio of nps to excipient of 0.2. therefore, this formulation was used in the following experiments. nps containing sirna were produced for spray drying into nanocomposite microparticles. the nps had an average diameter of 262 ± 6.5 nm with a polydispersity index of 0.186 ± 0.019, and the encapsulation efficiency of the sirna was 32 ± 5.5% corresponding to 381 ± 66 ng sirna/mg nanoparticles with this particular production method. the resulting powder of nanocomposite microparticles had a mmad of 4.99 ± 0.15 µm and a water content of 0.78 ± 0.067% (w/w, n = 2). the nps were redispersible after spray drying into nanocomposite microparticles upon addition of depc-treated water (data not shown). the content of residual pva in the sirna-containing nps was 6.5 ± 0.5% (w/w, n = 2), corresponding to a level of 1.3% w/w in the nanocomposite microparticles. this suggests that the pva is relatively strongly bound to the nps, which was also suggested in previous reports [23] . the amount of pva present in the nanocomposite microparticles is relatively low, and should thus not present an obstacle for future inhalation therapy. the procedure of production of nps and nanocomposite microparticles has been repeated several times with similar results. the surface morphology of the sirna-containing microparticles was examined by sem. the microparticles displayed a relatively smooth surface morphology (fig. 3) , as expected from the previous investigations, since the employed ratio of nps to carbohydrate was 0.2. the content of sirna in the nanocomposite microparticles was extracted, and the total concentration was determined to be 232 ± 18 ng sirna/mg microparticles, corresponding to 60% of the content in the nps before spray drying. on investigation of the ratio of nps to carbohydrate before and after spray drying, no difference was found, indicating that the loss of carbohydrate and nps in the spray dryer was equal, and the calculations of the concentration of sirna in the nanocomposite microparticles are valid. to ensure that the integrity of the sirna was maintained during the spray drying process, nondenaturing page was performed. the sirna extracted from the nanocomposite microparticles appeared intact and of the same size as before the encapsulation and spray drying (fig. 4) . to prove that the sirna in the nanocomposite microparticles maintained the biological activity, h1299 cells were transfected with sirna extracted from the nanocomposite microparticles (fig. 5a) . the results showed that there was no significant difference between the activity of the sirna extracted from nanocomposite microparticles and the positive control sirna. no significant difference was observed between the blank and the negative control or the non-loaded nanocomposite microparticles (fig. 5a) . nps containing sirna were evaluated for their biological activity in the h1299 cell line. the evaluation of the gene silencing efficiency of the np was done both before and after spray drying as well as with and without the addition of lipofectamine 2000 to the samples. importantly, nps mixed with lipofectamine 2000 provided 68% knockdown of egfp expression in h1299 cells (fig. 5b ). none of the tested samples showed significantly higher cell death than the negative control, as determined from the pi staining (results not shown). the effect of different variables of the spray drying process on the outcome of the nanocomposite microparticles was investigated using non-loaded nps. a statistical design of experiment approach was used in order to extract as much information as possible from the results. the nanocomposite microparticles were all shown to be of acceptable quality, proving that the chosen fixed parameters were suitable. however, nanocomposite microparticles containing mannitol showed a significantly lower water content than the other formulations. the low water content in the powder is highly desirable, since it will decrease interparticulate cohesion and thereby increase the respirability of the powder [15, 27] . a high water content might also have a negative effect on the stability of the product [15] . for these reasons, mannitol was chosen for all succeeding studies. the ratio of nps to excipient was also shown to have an effect on the water content; increasing the ratio provided a lower moisture content. however, this effect was minor compared to the effect of using mannitol and was thus not taken into consideration. since all samples containing mannitol had acceptable water content, no further attempts were made to reduce the water content. the concentration and the ratio of nps to excipient had a significant effect on the particle size. the concentration of total dry substance in the feed formulation had a positive effect on the particle size, as the particle size increased with a higher concentration. this effect has previously been described in literature [16, [28] [29] [30] and is thought to be caused by a larger amount of dry substance in each droplet after atomisation in the spray dryer. the ratio had a small negative effect on the particle size, as the particle size decreased when the amount of nps in the feed formulation was increased. this could be correlated to the observation of corrugated particles appearing when higher amounts of nps were used. the corrugated surface morphology changes the aerodynamic properties of the particles and thus also the mmad. the ratio and concentration in the feed formulation were also shown to have an effect on the yield of dry powder. increased concentrations resulted in a higher yield, whereas a high ratio reduced the yield. an increase in yield at higher concentrations has previously been reported [16, 19] and corresponds well with the results. since the cyclone used for product recovery relies on centrifugal forces for collection of the final product, larger particles will have a higher recovery rate. as it has already been shown that increased concentrations lead to larger mmad, the observation that increasing concentrations increase the yield corresponds well with this. the finding that increasing the ratio provides lower yields also corresponds with this, since the increased ratio has been shown to decrease the mmad. besides providing the optimal formulation, the experimental setup for determination of the optimal factors proved that the fixed spray drying parameters were suitable for the formulation of nanocomposite microparticles with a size range applicable for inhalation, since almost all samples were of the appropriate size and with moderate water content. thus, based on the results of the statistical evaluation a high concentration and a low ratio combined with mannitol as excipient were chosen for further studies. as mentioned above, it has been reported previously that mannitol, when present in larger amounts, can have a negative effect on the respirable fraction of spray-dried particles, probably due to formation of particle aggregates [20] . this effect is not seen from the values measured in this study. particles containing mannitol in this study have very low water content, and this may be the reason for the different results, since the particle aggregation will be significantly reduced. the analysis method in this study differs from the method used for measuring the respirable fraction in the study by bosquillon et al. [20] , where the methods of tap density and electrical zone sensing on spray-dried powders suspended in saline was combined to provide an aerodynamic diameter. the aerodynamic behaviour was investigated using an andersen cascade impactor. the difference in analysis methods could also explain the difference in results, but it is very likely that the low moisture content seen in this study provides a significant reduction in particle aggregation leading to the different conclusions. the redispersibility of non-loaded nps from the spraydried powders were successfully demonstrated for the developed method of spray drying for each of the three different excipients, and it is highly likely that the results for sirna-loaded nps spray dried with mannitol will also apply for nanocomposite particles with the remaining two excipients. the surface morphology of the produced nanocomposite microparticles was also investigated. there was a difference in surface corrugation when the content of nps was varied. some degree of surface corrugation has been shown to be beneficial for particles intended for inhalation, and it is suggested that the improved aerosolization properties of corrugated particles is not only due to the lower aerodynamic particle size but also to a reduction in interparticulate cohesion [31, 32] . surface corrugation has also been reported to reduce the influence of the inhaler device on the aerosol generation [32] . even a small degree of corrugation is sufficient to obtain the described effects [31] . corrugated particles might also be more appropriate for dissolution in the lung fluid due to a larger surface area. this could be an advantage in the case of the nanocomposite microparticles since the rapid release of the nps into the lung fluid is necessary to avoid macrophage clearance. the discovered possibility of modifications of the surface corrugation in the nanocomposite microparticles represents an interesting possibility for further optimisation of the system for delivery by inhalation. in addition, since the sirna-containing nps were produced by a modified version of the double emulsion solvent evaporation method, the size of the nps can easily be modified to provide smaller particles by making small changes to the production method. smaller nps could possibly minimize loss by macrophage clearance of released nps following inhalation [10, 11] . importantly, the data confirms that the sirna is preserved during the spray drying process. the sirna extracted from the microparticles was shown to be of the same size as before spray drying when analysed by page. to ensure that the biological activity of the sirna was preserved, two cell transfection studies were performed. the first study showed that the biological activity of the sirna was retained after spray drying by evaluating the gene silencing effect of sirna extracted from nps combined with the commercial transfection reagent lipofectamine 2000 (fig. 5a) . the second study evaluated the silencing efficiency of the nps before and after spray drying and with or without the addition of lipofectamine 2000 (fig. 5b) . the nps displayed a good gene silencing efficiency in combination with lipofectamine 2000. however, there was no observable effect with the nps without lipofectamine. preliminary results indicate that the lack of silencing activity for the nps alone might be due to a very slow sirna release, which can be accelerated in the presence of lipofectamine 2000, resulting in the release of a dose sufficient for gene silencing within the 48 h of the in vitro experiment. no cellular toxicity was observed upon addition of lipofectamine 2000. in addition, the nps seem to be capable of endosomal escape, which would therefore not be the cause of the lack of silencing in this shortterm cell study. we are currently testing the effect of modified plga matrixes on the release profile and the gene knock-down efficiency of the sirna-loaded nps. such modifications should ideally not compromise the favourable safety profile of the polymer, and still enable the preparation of nanocomposite microparticles with optimal characteristics for inhalation therapy. although there seems to be a small degree of degradation during the spray drying process, the remaining sirna is intact and functional. at least two mechanisms are suggested to be responsible for this protection: 1) the plga matrix protects the sirna during the spray drying process, and 2) the excipients enhance the in-process stability of the sirna. the ratio of nps to excipient was assessed after spray drying in order to eliminate an uneven loss of nps and carbohydrate excipient as a cause of the decrease in sirna content. the ratio was not affected by the spray drying, suggesting that the decrease in sirna content must be due to degradation. a method has been optimised for the spray drying of nanocomposite microparticles consisting of mannitol and plga nps containing sirna. the nps used for spray drying were easily redispersed when the nanocomposite microparticles were dissolved in water. the sirna encapsulated inside the nps could be extracted and successfully used for gene knockdown in a cell transfection study using a commercial transfection agent. also, the nps showed a significant knockdown when mixed with lipofectamine 2000, probably due to a change of the release profile making the silencing measurable in a short term in vitro study. the ratio of nps to excipient was found to have a significant effect on the surface morphology of the spray-dried nanocomposite microparticles. this discovery could provide the basis for further optimisation of the nanocomposite microparticles with respect to aerosolization properties. this study has proven for the first time that it is possible to obtain a spray-dried formulation containing sirna with characteristics known to be optimal for inhalation therapy, without compromising the activity of the sirna. future work will address the preparation and in vivo test of this type of nanocomposite microparticles. harnessing in vivo sirna delivery for drug discovery and therapeutic development inhalable sirna: potential as a therapeutic agent in the lungs sirna drug delivery by biodegradable polymeric nanoparticles rapid endo-lysosomal escape of poly(dl-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery non-viral sirna delivery to the lung mechanisms of macromolecule absorption by the lungs pathology of human influenza revisited time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars inhalation, deposition, and fate of insulin and other therapeutic proteins investigation of the proinflammatory potential of biodegradable nanoparticle drug delivery systems in the lung fundamentals of pulmonary drug delivery preparation and properties of inhalable nanocomposite particles: effects of the size, weight ratio of the primary nanoparticles in nanocomposite particles and temperature at a spray-dryer inlet upon properties of nanocomposite particles preparation and properties of inhalable nanocomposite particles: effects of the temperature at a spray-dryer inlet upon the properties of particles nanocomposite particle physical characteristics and aerosolization performance of insulin dry powders for inhalation prepared by a spray drying method quality by design -spray drying of insulin intended for inhalation spray-drying preparation of microparticles containing cationic plga nanospheres as gene carriers for avoiding aggregation of nanospheres spray drying in practice spray-dried powders for pulmonary drug delivery influence of formulation excipients and physical characteristics of inhalation dry powders on their aerosolization performance merck research laboratories functional polarity is introduced by dicer processing of short substrate rnas preparation of poly(dllactide-co-glycolide) nanoparticles by modified spontaneous emulsification solvent diffusion method method for quantifying the sample collected by an andersen cascade impactor using total organic carbon analysis evaluation of a new aerodynamic particle sizer spectrometer for size distribution measurements of solution metered dose inhalers two methods for quantifying dna extracted from poly(lactide-co-glycolide) microspheres the role of particle properties in pharmaceutical powder inhalation formulations physicochemical and in vitro deposition properties of salbutamol sulphate/ipratropium bromide and salbutamol sulphate/ excipient spray dried mixtures for use in dry powder inhalers droplet and particle size relationship and shell thickness of inhalable lactose particles during spray drying particle formation and capture during spray drying of inhalable particles how much particle surface corrugation is sufficient to improve aerosol performance of powders? use of solid corrugated particles to enhance powder aerosol performance we are grateful to the alfred benzon foundation (d.c.), the danish research council for technology and production sciences [grant no. 26-02-0019 (d.m.k.j.)] and the danish agency for science, technology and innovation for the financial support. we also acknowledge the alfred benzon foundation and drug research academy for cofunding the fluostar optima plate reader. dorthe kyed ørbaek and maria laessøe pedersen have been of great assistance in the practical work with the sem and cell cultivation, respectively. in addition, this paper has been carried out with the financial support from the commission of the european communities, priority 3 "nanotechnologies and nanosciences, knowledge based multifunctional materials, new production processes and devices" of the sixth framework programme for research and technological development (targeted delivery of nanomedicine: nmp4-ct-2006-026668). it does not necessarily reflect its views and in no way anticipates the commission's future policy in this area. key: cord-022504-tk7v4hoj authors: nan title: environmental and safety issues with nanoparticles date: 2012-03-16 journal: nanoparticle technology handbook doi: 10.1016/b978-0-444-56336-1.50007-2 sha: doc_id: 22504 cord_uid: tk7v4hoj nan since nanoparticles have superior surface activity and can be applied to the production of particles with various functions, they are extremely important for the future development of sophisticated material technologies. on the other hand, this superior activity of nanoparticles is a cause of trouble from the perspective of safety, and does not always have a positive influence on the environment. attention must also be paid to impact on health. nevertheless, all technologies have negative aspects, and overcoming these kinds of problems, we will be able to utilize the superior characteristics of nanoparticles for practical purposes. to achieve this goal, it is necessary to fully understand the influence of nanoparticles on the environment and the relevant safety issues. this chapter evaluates the relationship between nanoparticles and the environment, and also describes the trouble caused by nanoparticles as well as the safety issues. the relationship between nanoparticles and the environment will be clarified from the viewpoint of what kind of influence nanoparticles generated either artificially or naturally have on the environment. the influence on the indoor environment, where nanoparticles are produced, will also be clarified. the safety of nanoparticles will be clearly described from the perspective of the trouble caused by the superior surface activity of nanoparticles; the effect of the compositional characteristics of nanoparticles, and also the influence on health. a method for assessing the influence of nanoparticles using quantum dots is also explained. in the final section, methods for removing nanoparticles from gas and liquid are described as technology to control the influence of nanoparticles on the environment. in our atmospheric environment, particles ranging from several nanometers to several tenth micron orders are suspended. they are emitted into the atmosphere at the rate of 2.5 billion tons every year. emission sources are classified as either natural or artificial. natural particles occupy 60% of total particles, consisting mainly of salt particles (ϳ1 billion ton) from the sea and soil particles (ϳ0.5 billion ton) from the land. on the other hand, the latter particles are brought about by human activities. although occupying only 16% of the total emitted particles, their size is mostly of submicron order and because they contain hazardous chemical components such as nitrates, sulfates, hydrocarbons, heavy metals, etc. in high concentration, their effects on the ecosystem are serious. fig. 7 .2.1 shows an overview of the size and concentration ranges of various aerosol particles. as it can be seen, the number concentration of atmospheric aerosol which we inhale every day ranges from several thousand particles per cm 3 in clean area to several hundred thousands in dusty areas, and the size range lies between 10 nm and several tens of micrometer. fig. 7 .2.2 shows mass-based size distribution of atmospheric aerosol particles. since the size distribution in the nanosize range appears only when the sources of particle generation exist, the size distribution is usually bimodal with peaks in the size range of a few to 10 micron and submicron. the former peak consists of naturally generated coarse particles such as soil dust, sea salt spray, and so on. on the contrary, the latter contains plenty of artificially generated particles, some of which grow from molecules (in most cases vapor state) exhausted by human activities through chemical reaction, condensation, and coagulation. particle growth rarely leads to particles larger than 1 m unless high concentration of vapors or particulate matters which cause the above-mentioned growth mechanisms exist in the atmosphere. as it can be seen from the differences in the particle generation process, fine particles generated from molecules or nanoparticles are much more complicated in their chemical component than the coarse particles, and sometimes have serious adverse health effects. such fine particles are called pm2.5, which is defined for particles less than 2.5 m including nanosized particles. recent epidemiologic investigation reports that the concentration of pm2.5 showed a positive correlation to the mortality due to pulmonary diseases [1] . various research techniques are used in order to understand the process of particle growth and to trace back to the source of pollution. an example is shown in fig. 7.2.3 where a characteristic function of sulfur dioxide is shown taking into account all possible factors related to particle growth. where f is the characteristic function that expresses particle size, particle concentration, particle composition, and so on [2, 3] . particulate materials in water are present in the form of colloids. these colloid particles are classified into inorganic colloids. examples of the former are oxides of aluminum, silicon and other substances, and typical examples of the latter are substances such as humic acid and fulvic acid. while the structure and molecular weight of particles vary depending on the area of water, it is known that what are usually present in water are comparatively small colloids (particles smaller than 500 nm). the number concentration of colloid particles in ground water, or a typical water area environment, ranges from 10 11 to 10 20 (number / m 3 ) and varies significantly depending on the geochemical conditions of the aquifer. it is known that in moving water, colloid particles sometimes act as a medium in conjunction with water and in some cases move faster than water. homogeneous porous layer such as a sand layer, and most of the colloid particles are trapped. the mechanism of partical trap in this layer is explained by the sand filtration theory. c and d are a gravel layer and a rock bed, respectively, and both have high water permeability with large gaps and cracks. particles can also pass through easily. safety and movement characteristics of colloid particles have a significant influence on the movement of materials such as ionized molecules in aquatic environments. since fine particles such as nanoparticles in particular are highly stable as colloid particles, it will be very important in the future to understand their influence. at the same time, these characteristics are considered to have a high potential to be developed for further application of nanoparticles. in most cases, nanoparticles in exhaust gases are studied from the viewpoint of the influence of total particulate matters on the environment. the term "nanoparticles" is used only in a few cases, "fine particles" is usually used for investigation. since nanoparticles are part of fine particles, this section will be described from this perspective. major sources of combustion exhaust gases are stationary large-scale combustors and diesel engines for stationary and portable use. for stationary combustors, fuels such as coal, oil, and gas are used. lighter fuels have a lower rate of particulate emission, but have a higher fine particle content including nanoparticles. fig. 7 .2.5 [1] shows the frequency distribution in combustion of coal and heavy oil. fig. 7 .2.5a and b are the distributions on a number and mass basis, respectively. as these figures clearly show, the total weight of particles of a size of 1 m or smaller is extremely low, while their total number is, on the contrary, very large. it is clear that, while the total quantity of particulate material is far larger in coal combustion than in oil combustion, the difference is less when it comes to particles 1 m or less in diameter, including nanoparticles. most of the particles contained in pulverized coal combustion exhaust gases are considered to be formed as particulate materials directly from ash content, which is originally contained in coal and also includes some unburned carbon. particularly, almost all large-size particles are considered to be this type of particle. on the other hand, fine particles include two types. one type is formed in the process by which low boiling point metal contained in coal ash is evaporated and vaporized in a high-temperature combustion field and then becomes particles in the exhaust gas cooling process. the other includes carbon particles formed in the gas phase, or so-called soot, which is generated due to the delay in oxygen supply for combustion of evaporated volatile matter in the initial stage. fig. 7 .2.6 [2] shows the relationship between the trace metal content in coal ash and the particle diameter. aluminum with a high boiling point has a constant concentration regardless of the particle diameter. however it is obvious that in the case of metals with a lower boiling point, the smaller the particle diameter, the larger the content. with regard to particles with sizes 1 m or smaller in the nano domain, it has been clarified that the generated amount is increased rapidly by reducing combustion air supply or by weakening the oxidation atmosphere in the volatile matter combustion area, for example, when air supply from a burner is reduced in twostage combustion. this also demonstrates the significant contribution of carbon particles formed in the gas phase. also in the case of ash from heavy oil combustion, there are large particles of a carbon residue type generated from sprayed liquid particles and particles formed in the gas phase as well. as in the case of coal, trace metal contained in heavy oil with a low boiling point is concentrated into fine particles and discharged. also in the case of the combustion of liquefied natural gas, carbon particles formed in the gas phase are generated, albeit in trace amounts. in contrast, only in the case of diesel engines, fuel is injected into the high-temperature and highpressure atmosphere produced by compressing only air to induce spontaneous ignition, and combustion continues with a heterogeneous mixture of fuel and air in the combustion chamber. therefore, particulate materials mainly consisting of unburnt carbon are generated due to incomplete combustion. fig. 7 .2.7 [3] shows changes in the diameter of particles according to changes in the diesel engine load. it is obvious that the overall concentration of particles increases with the increase in the load rate of the engine. according to observations using sem, fine particles in diesel engine exhaust gases have also been found to comprise fine primary particles of a size several tens of nanometers, and coarse particles with carbon hydride condensed on the surface of secondary aggregates of primary particles. influence of particle diameter on trace element contents. the volume of industrial and domestic wastewater is increasing significantly year by year with the change in the lifestyle based on mass consumption and mass disposal brought about by the dramatic development of the economy and industry. effective advanced wastewater treatment is required because wastewater contains a variety of constituents such as particles, organic materials, and emulsion depending on the resource. inorganic nanoparticles are not generally stabilized in the liquid because they form aggregates of some sort more or less. for wastewater reclamation and reuse, these nanoparticles can be removed from the liquid by the advanced treatment processes such as membrane filtration following biological treatment processes. organic materials such as macromolecules are regarded as soft nanoparticles judging from their sizes, in contrast with hard inorganic particles. chemical mechanical polishing (cmp) is one of the fastest growing processes in semiconductor industry, and it has become an integral part of the state-of-the-art fabrication line for the multilayer wiring board of large-scale integrated circuit (lsi). besides the semiconductor devices, cmp is widely applied to the magnetic head, the magnetic memory, and the imaging devices. the process is primarily used for polishing the device side of a semiconductor wafer through the mechanical downforce of slurry abrasive in combination with chemical oxidation of wafer surface. in general, colloidal silica is used as abrasive slurry to planarize the oxide wafer surface. particles in slurry are highly charged to avoid aggregations between particles or between particles and wafer surfaces. during the process, large volumes of ultrapure water are consumed to clean the surface of the wafer, which generates large quantity of cmp wastewater typically having high solid content resulting from slurry abrasive particles of sio 2 , al 2 o 3 , or ceo 2 , depending on the nature of the cmp application. the quantity of cmp wastewater generated is expected to increase proportionally with the growing needs of the cmp processes. as a result, the treatment and reuse of cmp wastewater has become increasingly necessary. the cmp wastewater has been generally treated with the conventional chemical coagulation-sedimentation process, producing large quantity of sludge. currently, a membrane filtration process coupled with chemical pretreatment is used to separate the nanoscale particles from the cmp wastewater to reclaim the water [1, 2] . wastewater of nanosized metal colloid, which is hard to be removed by coagulation-sedimentation process, is discharged in such diverse fields as metalworking factory, electronic components factory, and pigment-manufacturing factory [3] . it is reported that various trace elements of heavy metal are contained in wastewater discharged from a pulp production plant [4] . a spent emulsion, which contains nanosized copper colloid, is discharged from plants manufacturing copper cables for electrical industry, and the treatment for purification of effluents is examined by the integrated membrane system based on ultrafiltration (uf) and nanofiltration (nf) [5] . a glass company generates the wastewater containing fine clay and glass particles from the grinding process of glass surfaces during production of crt glass used for tvs and monitors. separation of fine clay and glass particles by microfiltration (mf)/uf is examined in order to treat glass industry wastewater for reuse in the manufacturing process [6] . the colored substances are free from regulatory constraint of water quality so far because they are not considered hazardous substances. however, water color is being recently used as a standard for the judgment of the purity in water because the removal of color becomes important for wastewater reclamation and reuse. dye works are scattered across the country as the industry with local tradition. the dyehouse effluent is discharged in large quantity, and it has extremely complex composition because it contains not only dye but also dyeing aid and finishing agent. in general, dye cannot be removed by standard biological treatment because of its low environmental biodegradability. dye wastewater is treated by coagulation-sedimentation and activated sludge processes, and nanoparticles produced in the course of the treatment are released into the environment [7] . the color is often imparted by organic substances, predominantly humic substances. aquatic humic substances including humic and fulvic acids are a term referring to a broad class of naturally occurring mixture of organic compounds, ubiquitous in surface waters, ground waters, and soil pore waters. they are a complex mixture of heterogeneous organic materials in terms of elemental composition, chemical functionality, and molecular size distribution since humic substances can be derived from any organic materials, including plant and animal debris, microfauna, biowaste, pesticide, and others. the molecular weights of humic acids range from several thousands to several tens of thousands daltons, and those of fulvic acids range from several tens of thousands to several hundred thousands daltons. because of this versatility, humic substances are known to significantly affect the behavior of some pollutants in natural environments, such as trace metal speciation and toxicity, solubilization and adsorption of hydrophobic organic compounds, and disinfection by-product formation [8] . melanoidins are natural polymeric compounds of dark brown color, and they are closely related to humic substances. they are produced by a set of consecutive and parallel nonenzymatic reactions taking place between amino compounds and carbohydrates during a maillard reaction [9] . they are contained in the molasses wastewater from alcohol distillery, sugar processing and refinery industry, and glutamate processing industry. such wastewater containing melanoidins has frequently caused a coloration problem of water environment, and thus the suitable decolorization treatment is required in many fermentation and sugar industries using molasses. treatments by flocculation, ozonation, and electrolysis are promising in color removal [10] . food-processing wastewater usually contains a variety of organic materials in varying degree of concentration. in cheese-making in the dairy products industry, only ϳ10% of the initial milk volume becomes product, cheese, and the other 90% becomes by-product, liquid cheese whey. since cheese whey is a protein-and lactose-rich by-product of cheese production, its cost-effective utilization is becoming increasingly important. recent developments in membrane technology have provided exciting new opportunities for large-scale protein and lactose fractionation in whey treatment [11] . in textile industry, typically it takes over 100 l of water to process just 1 kg of textile material. not only the washing water must be treated to recover important by-products such as lanolin, but bleaching and dyeing chemicals must also be removed before discharge back to the rivers [12] . surfactants are a primary constituent of the detergent used in the household routinely, and also they are widely used in industry and agriculture because they have several functions such as washing, emulsification, and dispersion. the surfactants are usually present in the solution in the form of the micelle, and large amounts of surfactant wastewater are discharged in the rivers [13] . pesticides whose molecular weight ranged from 200 to 400 da (ϳ1 nm) have been used in great quantities not only for agricultural use but also in golf links and resort. therefore, the wastewater and effluent treatments have become an important issue, and pesticide separation by nf membranes is found to be very efficient [14] . the potential reclamation of high-quality water produced by the advanced treatment of the secondary effluent of the municipal sewage has come a long way in recent years. the sewage contains various components such as virus [15] , pharmaceutical substances [16] , and endocrine disrupting compounds derived from zoonotic excretory substances [17] . the advanced treatment of such chemical contaminants at low level becomes increasingly important. as mentioned above, the removal of nanoparticles contained in wastewater is stringently required to recycle the reclaimed wastewater in a wide variety of industries such as chemical industry, textile industry, pulp and papermaking industry, food-processing industry, dairy products industry, and pharmaceutical industry. also for domestic wastewater, the reuse of the reclaimed wastewater for nonpotable purposes is becoming more and more important, and this is expected to raise awareness of the behaviors of nanoparticles contained in wastewater in order to upgrade the water treatment processes. in recent urbanized lifestyles people tend to spend more time in enclosed buildings or residences than outdoors. therefore, it is of great importance to characterize indoor particles and correlate between indoor and outdoor ones from the viewpoint of evaluating the influence of indoor air quality (iaq) on human health. as shown in table 7 .2.1, indoor nanoparticles originate from the several sources such as products of chemical reactions, nonvolatile residues (nvrs) of liquid droplets, printers/photocopiers, combustion, bioaerosols, and infiltration of outdoor air. 7.2.5.1 secondary particle formation by gas phase ozonolysis for particle formation resulting from chemical reaction via ozone, the reaction of terpenes is very common in indoor environments as well as atmospheric ones. terpenes are emitted from fragrance-containing vegetable oils such as pine oil and citrus oil, and wooden materials including woody furniture [1] . meanwhile, sources of emission of ozone are air cleaners, air-conditioners, laser printers using corona discharge, and infiltration of outdoor air. terpenes are generic terms of unsaturated organic compounds that are composed of isoprene as unit (e.g. -pinene and limonene). these compounds used for household applications readily react with ozone because they have one or more double bonds. it has been proposed that, as shown in fig. 7 .2.8, the reaction mainly proceeds to form less volatile pinonic acid via pinonaldehyde of intermediate [2] . furthermore, the acid-catalyzed reaction allows the products to convert into higher molecular weight compounds by the polymerization via carbonyl groups in the aldehydes and the aldol condensation [3] . it has been reported that the resultant generated particles have a size distribution with a peak diameter of about 30 nm, and that the products by terpenes ozonolysis irritate human airways [4] . nanoparticles are also generated from air humidifiers or negative air-ion generators in which water is atomized. in general, humidifiers are mainly categorized into vaporization type and atomization type [5] . the former does not entrain impurities in water when it is fed into indoor spaces of interest. meanwhile, the latter has the drawback that nvrs are suspended in spaces to be humidified by feeding water via spraying and sonication. the nvrs in tap water include colloidal particles and soluble fraction such as silicates, sulfates, carbonates, and chlorides. the size of nvr particles, d p _ r can be estimated from the following equation: where dp_ m is the droplet size, c the mass fraction of nvr in the droplets, m the droplet density, and r the nvr particle density, respectively. assuming that 2 m-sized droplets ( m ϭ 1,000 kg/m 3 , r ϭ 2,500 kg/m 3 ) are formed by an ultrasonic nebulizer, and the mass fraction of nvr in city water is 100 ppm (10 ϫ4 ), the nvr particle size, d p_r is estimated to be 68 nm. recently, a wide variety of negative ion generators using the lenard effect, corona discharge, uv/photoelectron emission and electrospray have been commercialized and attention has been focused on features such as air purification and physiological activation [6] . among them there are the ion generators that atomize water based on the lenard effect and electrospray form the nvr particles as by-product in addition to ion products if the supplied water contains nonvolatile impurities. fig. 7 .2.9 shows an example of electrical mobility distribution for ions generated by the electrospray method (positive in this case) [7] . this method atomizes liquid fed to a tip of a capillary electrode to form fine droplets with large amounts of charge by applying high voltage between the tip and the downstream counter electrode. when water in the generated droplets evaporates and their surface charge density attains the charge limit called "rayleigh limit", this phenomenon induces their self-fragmentation followed by the formation of a high concentration of cluster ions. in this figure, the high-mobility peak on the right side corresponds to the cluster ions. these ions are 2 nm in size assuming that they are singly charged. meanwhile, another peak on the figure results from nvr in water and then its height increases with the increase in the fraction of tap water in the fed liquid. the electrical-mobility-equivalent size of nvr particles measured by differential mobility analyzer (dma) -condensation nucleus counter (cnc) method ranges from 10 to 20 nm and their concentration is on the order of 10 4 particles/cm 3 . comparing the forementioned electrical mobility distribution with the particle size one, the nvr nanoparticles are estimated to hold about 100 charges. accompanying the recent proliferation of computers, the use of inkjet printers and electrophotographic machines such as laser printer and photocopier is becoming common in homes as well as offices. it has been reported that these devices emit various sorts of pollutants. the eco-friendlinessoriented standards such as blue angle standard [8] regulate the maximum permissible limits of benzene, styrene, total volatile organic compounds (tvoc), ozone and particles. as the regulation of particles is based on the emitted mass per hour, mainly the relatively coarser particles such as toner and dust adhering to paper have been targeted. however, some reports have revealed that nanoparticles are emitted from inkjet printers or laser printers [9] . fig. 7 .2.10 depicts the size distribution of particles emitted from a laser printer measured by a scanning mobility particle sizer (smps). as seen in the figure, nanoparticles with a peak diameter of around 30 nm are generated in printing mode, whereas the emission in the case of feeding paper without printing is about one third of the normal printing mode. furthermore, these particles were dried by passing them through a diffusion dryer because they are thought to originate from the nucleation of water vapor emitted from papers in the fixation process. as a result, it was found that most particles formed in the paper feed mode evaporated and then vanished, while particles in the printing mode contained nonvolatile components as well as water. from these results it is anticipated that the particles are derived from styrene remaining in the toner even though their composition has still not been identified. meanwhile, it is thought that during ink discharge ink-jet printers emit not only the main ink droplets but also their satellites (about 1 m) to result in nanometer-sized nvrs during printing. one of the most significant source of indoor nanoparticles relevant to combustion is cooking such as frying and sautéing [10] . some reports said that over 90% of the particles by number were in the ultrafine fraction range during cooking with bimodal peaks at 60 and 10 nm, attaining the number concentration on the order of 10 5 particles / m 3 and the emission rate of 10 14 particles / h. owing to lifestyles in asian countries, cigarette smoke, incense, and mosquito coils also contribute to indoor nanoparticle levels [11] . it was reported that especially in the indian subcontinent the combustion of biofuels such as straw and dried cattle manure used for cooking could have a significant impact on climate change in the south asian region [12] . sidestream cigarette smoke also contains nanoparticles, having a concentration distribution with the main peak between 0.1 and 0.2 m [13] . in addition, it was found that nanoparticles a peak size of 30 nm formed by the nucleation of vapor fraction in filtered sidestream smoke immediately after burning when the dilution of smoke by air was insufficient [14] . attention should be paid to air cleaners when a high concentration of cigarette smoke has to be treated by the cleaners using a single unit air filter. airborne virus particles or virions are typically in the 20-400 nm size range, and are a good example of nanoparticle bioaerosols. smaller viruses typically contain one subunit, which consists of an outer protein capsid, internal nucleic acid (e.g. dna and rna), and other internal proteins. corona virus that causes sars and influenza are good examples of them. the viruses most often are transmitted through direct contact with an infected person, such as by shaking hands, hugging or kissing, while sometimes it is spread by nasal droplets. however, it is still unknown how these virus particles behave in the case of airborne infection. recently, the studies that attempt to elucidate the behavior have been progressing [15] . this section describes the sources of nanoparticle generation in industrial processes by categorizing them into specific processes where a cleanroom is used and other general ones. the sources of emission of unwanted nanoparticles in general workplaces are categorized as fumes from hot processes (e.g., smelting, refining, and welding) and from (incomplete) combustion processes. favorable conditions required for the generation of nanoparticles are found in workplaces where there is (1) presence of vaporizable material, (2) sufficiently high temperature to produce enough vapor, followed by condensation to form an independent aerosol, and (3) rapid cooling and a large temperature gradient. there have been so many studies on occupational exposure to fine particles in the field of public health. in general, high spikes of nanoparticle concentration are observed during active operations, followed by a gradual decay after the operation, primarily because of coagulation, evaporation, dilution, and/or deposition. the fraction of the total number of nanoparticles generally decreases, whereas that of the number of submicrometer particles increases with time and distance from the point of emission. in order to accurately estimate exposure, the effects of spatial and temporal changes will need to be evaluated. therefore, it is important to identify the time required for the concentration to decline to the normal or background levels. as an example of reports on grinding processes, fig. 7.2.11 shows the case where a steel substrate was ground upon using a high-speed grinder [1] . from the figure the distribution of concentration of generated particles has a distinct bimodality, one with the finer peak at around 10 nm and the coarser one at around 1 m. the former results from within the grinder motor and the volatilization or combustion of amenable ground substrate and/or grinding materials, the latter from the mechanical abrasion and attrition. however, the resultant total concentration on the order of 10 5 particles/cm 3 is not so high. 7.2.6.2 industrial processes with cleanrooms cleanrooms and associated controlled environments (e.g., in the case of an iso class 3 cleanroom, the maximum permissible airborne particle concentration is less than 10 3 particles/m 3 for particles with the size of 0.1 m or larger, while the airborne particle concentration in ordinary indoor environments is on the order of 10 9 particles/m 3 or higher) are usually adopted to avoid particle contamination in industrial processes where precision products such as engineered nanoparticles, semiconductors, and other electronic or optical devices are fabricated because the deposition of particles onto product surfaces causes their yield reduction and quality deterioration. the emission sources in cleanroom environments are tabulated in table 7 .2.2. since some of the listed emission sources emit trace amounts of nanoparticles, these nanoparticles are not regarded as particulate contaminant but as chemical or molecular one. in this section, these nanometer-sized solid substances formed on solid surfaces by chemical reaction are also included. size distribution of nanoparticles generated when a steel plate was ground with a high-speed grinder. (1) air exhaled by humans emissions from human bodies are a minor contribution in ordinary indoor situations because airborne particle concentration in such places is quite high, whereas the emission cannot be seen as negligible in cleanroom environments. the major human emissions are thought to be atmospheric dust deposited on clothes and skin fragments, and most of these particles are submicrometer in size. meanwhile, particles in exhaled air are composed of fine liquid droplets from spittle (99.5% of water), and then evaporate to form nanoparticles of nvr. in fig. 7 .2.12 an example of size distributions of particles in exhaled air before and after smoking is shown [2] . when measuring particles in exhaled air, the air was introduced into a measuring device after drying them by passing them through a diffusion dryer. the size distribution of particles in exhaled air before smoking (n db ϭ 0) has a bimodality, one with the peak size of 0.2 m, and the other of 20 nm or smaller. the former peak comes from atmospheric aerosols as it decreases with the increase in number of deep breaths in a clean booth (n db ). the latter originates from nvr particles of spittle droplets. incidentally, since smoking induces the rapid increase in number concentration of particles 0.1 m or larger by 10 4 times or more and for nanoparticles by about double, special attention should be paid to the management of personnel's clothes such as face mask when they enter a cleanroom after smoking. (2) emission from ionizers ionizers are commonly used in cleanrooms to eliminate electrostatic charge on substrates for precision electronic devices. the most popular ionizer is a corona-discharge type. corona-discharge type ionizers are categorized into the following three groups; ac, dc and pulsed-dc types. the issues of emission of contaminants such as ozone, no x and particles have been pointed out [3] . these issues are also applicable to air cleaners using a corona discharger. among these problems is that the particle emission has a potential for particle contamination onto product surfaces and eventually decline in product yield. the particle emission, which has been studied since the 1990s, is caused by foreign particle deposition onto electrodes, electrode erosion, and gas-to-particle conversion. the issue of electrode erosion can be solved by the improvement of electrode materials, whereas for the issue of gas-to-particle conversion, the airborne molecular contamination (amc) control to be ionized has to be made. it was reported that fundamentals ch. 7 environmental and safety issues with nanoparticles corona-discharge ionizer (e.g. gas-to-particle conversion of low-molecular-weight cyclosiloxane) boron-containing particles from borosilicate glass fibers of hepa filter haze by chemical reaction on solid surfaces (precipitation of ammonium salt and silica) watermark on wafer surfaces at drying leakage from thin film and nanoparticles processing equipment silicon-containing compounds that precipitated on the electrodes result from the gas-to-particle conversion of low-molecular-weight cyclosiloxane (lmcs) from silicone sealant via corona discharge [4] . (3) boron-containing particles from hepa filter with borosilicate glass fibers the use of hepa and ulpa filters made of borosilicate glass fibers prevails in the most cleanrooms. it has been known that owing to chemical reaction in equation (7.2.2) bf 3 vapor is formed from glass fiber filters by passing hf gas leaking from wet cleaning equipment through the filters. boron, which is a dopant element for semiconductors, has been thought to be a contaminant that might cause failure in semiconductor devices if it comes from the surroundings. in addition, it has been revealed that trace amounts of boron in the form of boric acid (h 3 bo 4 ) are also formed from the fibers via the reaction with moisture in the surrounding air (equation (7.2.3)). fig. 7 .2.13 depicts the change in volatilized boron mass from various filters in terms of airborne boron concentration. especially, at the initial stage just after the initiation of ventilation, the volatilized boron mass increases with increase in relative humidity [5] . boric acid, which is solid at room temperature, is surmised to form in the particulate form. however, its existence was identified only by chemical analysis because it is present only in trace amounts. haze might form on glass surfaces of lenses and mirrors for optical instruments if they are exposed for a long time to cleanroom environments where amcs are not controlled properly. the haze is more likely to bring about the insufficient light delivery onto a surface to be exposed in photolithographic processes. one of the reasons is that ammonium sulfate ((nh 4 ) 2 so 4 ) is formed, which then precipitates on the glass surfaces via chemical reaction of sulfur dioxide (so 2 ) with ammonia or amines. for another reason, hexamethyldisilazane (hmds) used as additive in resist coating or lmcs from silicone sealant is adsorbed, and then decomposed to form silica precipitates on glass surfaces by photochemical reaction during laser irradiation, followed by the unwanted decline in laser penetration [6] . as another example a report said that tiny projections, which are also known as "haze", with a size of 0.2 m or smaller were formed on silicon wafer surfaces owing to the adsorption of organosilicate compounds in thin film formation processes with cvd. it is similarly caused by the precipitation of sio 2 [7] . (5) watermarks on solid surfaces during drying when a silicon wafer surface is cleaned with deionized water and then dried in air, a watermark is formed on it via the mechanisms demonstrated in fig. 7 .2.14. oxygen in air is dissolved and diffused into water droplets or adsorbed water on a wafer surface, followed by the formation of silicate compounds via silanol reaction. the watermark on a wafer surface is detected in the form of nanometer-sized particles by an electron microscope [8] . (6) leakage from nanoparticle production processes in regard to the risks to processing equipments by nanoparticle leakage from production processes, the vdi report in germany [9] has been described in detail. the production of engineered nanoparticles can be generally categorized into two approaches. one is a "top-down" approach that is initiated with a bulk material and then breaks it into finer pieces using some form of energy such as etching, ball milling, sputtering, and laser ablation. the other approach is to synthesize materials from the atomic or molecular level by growth and assembly to form the desired nanoparticles. processes included in this "bottom-up" category are sol-gel, chemical vapor deposition, flame synthesis laser pyrolysis, and so on. most of these processes are performed in a closed reaction chamber installed in a cleanroom or associated controlled environment. human exposure to these engineered particles does not take place during synthesis unless there is an unexpected system failure (e.g. rupture of a seal). human exposure is more likely to occur after the manufacturing when opening the reaction chamber, drying the products, or in the post-process handling of the products. the release of nanoparticles during production chamber cleaning operations is another critical point. cleaning typically involves using water or some solvent. brushes, sponges, or tissues used in the cleaning will carry nanoparticles into the waste stream. disposal of the waste and wastewater may become a source of nanoparticle release into the environment. further, conditioning of nanoparticles such as compression, coating, and composition to form final products may also result in the release to the environment and resultant exposure although very few studies have been carried out on this subject. recent studies [10] to evaluate the aerosol discharge during the handling of carbon nanotubes showed that the generation of nanoparticles occurred under vacuum to remove spilled nanotube materials or vigorous mechanical agitation. however, they reported that the concentrations were very low. in addition, measurements of nanoparticle levels during final packaging of carbon black, which is a typical engineered nanoparticle material, showed that there was no increase in nearby air [11] . study on the safety of nanoparticles has started only recently, and no sufficiently systemized results have been obtained. what should be noted in particular is that the possibility of radial troubles caused by particulate matters are considered to increase by the decrease of particle diameter in nanoparticles. one typical example is the problem of dust explosion, caused by the high surface reactivity of fine particles. in other words, since nanoparticles are extremely fine particles, dust explosion is more likely to occur. explosion is more likely to occur because fine particles are different in their composition, in that low boiling point metal can be easily condensed, as described in section 7.2. however, of particular note here is that, since all particles do not necessarily exist independently in the form of a single particle, the possibility of dust explosion does not simply increase as particles become finer. fine particles with sizes of 1 m or smaller such as nanoparticles have an extremely high agglomeration propensity and secondary particles can be easily generated. therefore, in some cases they conversely behave like large particles. these are the points to be taken into consideration when studying the problems caused by nanoparticles. as shown in fig. 7 .3.1 [1] , the effect of the particle diameter on dust explosion tends to be that the smaller the particle diameter of the dust, the lower the minimum explosion concentration. in other words, explosion can be induced under conditions of lower concentration of particles in air as the particle diameter becomes smaller. due to the difficulty of conducting experiments to suspend particles with the same size in a uniform concentration, this result was obtained from particles far larger than nanoparticles; however, it has been clarified qualitatively that the smaller the particle diameter, the higher the possibility of dust explosion. from the perspective of composition, the possibility of explosion increase if materials that react easily with oxygen at low temperature are condensed into particles of small diameter. therefore, with regard to the effect of particle diameter on dust explosion, more careful attention needs to be paid in the case of combined materials than in the case of uniform materials, as assumed in fig. 7. 3.1. as described before, however, since nanoparticles are considered to exist often as agglomerates, it is necessary from the perspective of the particle diameter to take into consideration the diameter of not only primary particles but also of particles after agglomeration. to address the safety of nanoparticles, it will be important in the future to elucidate their behavior in detail including these factors. the terms 'nanoparticles' and 'nanomaterial' have been used for particles of which one representative dimension, for example, diameter of particles on cross-sectional diameter of fibers has at least 100 nm or less. some people hold that the majority of such fine particles are exhaled without depositing in the respirator tract, and that therefore the particles may not cause pulmonary diseases. however, the properties of nanoparticles are known to be different from the bulk material they are derived from. in cases where the biological effects of bulk materials have been reported, nanosized particles of these materials may be expected to have stronger dose response for the health effects. every effort must be made to clarify the uncertainty on the risks of these nanomaterials [1] . at the present time, there is no regulation or standard for assessing the biological effects of nanomaterials, and therefore there is a paucity of toxicological data concerning nanomaterials. much more systematic and strategic studies are needed to enable risk assessments for human health [2] [3] [4] [5] [6] . as regards risk assessment and risk management of nanomaterials, the characterization and identification of anticipated risks should be first determined for chemical substances or foods. conventional assessment methods are applicable for water-soluble particles. for insoluble nanoparticles, the assessment of potential health hazards should be made based on their properties or toxicity and dose-response relationship. the risk is a product of hazard and exposure; even if a nanoparticle has a hazard, the risk is lower when the possibility of exposure to the nanoparticle is small [2] . influence of particle diameter on lower limit of particle concentration for explosion. (1) exposure routes for nanoparticles nanoparticles can either be deliberately introduced into the body for medical purposes (drug delivery systems) or absorbed involuntarily from the environment (inhalation of nanoparticle-containing dust in the air). a distinction should also be drawn between nanoparticles manufactured for industrial application and those unintentionally generated and released in the environment, such as welding fumes or diesel exhaust particles (dep). in the fields of environmental science and toxicology, numerous studies on the potential health hazards caused by ultrafine particles have been conducted. practically, there are several definitions of nanoparticles or ultrafine particles, however, findings regarding biological effects of the ultrafine particles are useful as a starting point for estimating the effects of nanoparticles on human health. human and animals contact with nanoparticles through various routes: nanoparticles can be inhaled in the air, swallowed in the water, ingested in food, and absorbed via the skin in cosmetics. for successful risk assessment, it is important to determine how nanomaterials or nanoparticles are used, such as composites, surface coating, or powders. coatings or powders have the potential to release a part of their nanomaterials into the environment. workers who come into contact with nanomaterials have the possibility of exposure to nanoparticles at the workplace. consumers of products using nanotechnology can also be exposed to them. attention needs to be paid to the environments and ecosystems in which nanoparticles and nanomaterials are released. nanoparticles in the products may change their size, quantity, and composition during their life-cycle of manufacturing, use, transportation, and disposal. (2) respiratory uptake of nanoparticles inhalation is the main route of exposure to nanoparticles. particles inhaled with the air through the mouth and nose pass through the throat (nasopharynx and oropharynx) and tracheobronchial tree before reaching the alveolar region where oxygen moves from the alveoli to the blood and carbon dioxide moves from the blood to the alveoli. how deeply particles can penetrate and where they become deposited on each respiratory airway such as the nasal cavity, tracheobronchial tree, and the alveoli depend on their size under the various deposition mechanisms: inertial impaction, gravitational sedimentation and diffusion, etc. the respiratory airway includes the anterior nasal passage, posterior nasal passage, pharynx, larynx, trachea, main bronchi, bronchi, bronchioles, terminal bronchioles, alveolar duct, and alveoli, as shown in fig. 7. 3.2. in the human lungs, the trachea divides asymmetrically into the right main bronchus that enters the right lung where it divides into three lobes, that is, an upper, a middle, and a lower, and the left main bronchus that enters the left lung where it divides into two lobes, that is, an upper and a lower. the trachea divides into two branches, dividing progressively to the terminal alveolus. to quantitatively assess the pulmonary particle deposition needs human lung morphology models, respiratory physiology based models of the entire lung airway system and aerosol deposition models based on many experimental findings. in 1994, the icrp task group on lung dynamic (icrp: the international commission of radiological protection) published their revised lung model [7] . the deposition, clearance, and translocation of particles in each of the compartments were described. while the model has been widely used in the nuclear field, it is applicable to conventional aerosols as well as radioactive aerosol. in the nuclear field, aerosols including radon progeny that used to be nanoparticles have been studied. fig. 7.3.3 shows the deposition fractions of inhaled particles per adult nasal respiration of 1.2 m 3 /h in each region re-calculated for the nasopharynx, tracheobronchial, and the pulmonary (alveolar) region based on the model. inhaled aerosol particles deposit on different regions depending on their size; for example, nanoparticles larger than 10 nm deposit mostly in the alveoli and those less than 10 nm deposit in the nasal cavity. how deeply particles penetrate into the lung depends on their size. nanoparticles can reach pulmonary region in the lung and deposit more intensively and this, therefore, has become one of the reasons for concern about the effects of nanoparticles on human health. however, deposition of nanoparticle chain-like agglomeration and fibrous particles such as carbon nanotubes cannot be estimated by this model. most inhaled nanoparticles are deposited on the surface of the respiratory tract. generally, if insoluble particles are deposited in the ciliated airspaces which are lined with a mucous layer, they are transported to the digestive tract with the mucous flow by mucociliary movements. particles deposited on nonciliated bronchioles and alveoli are phagocytosed by macrophages, which is a kind of white blood cell. as a result, their residence time is longer, however, they are usually transported to the ciliated upper part of the respiratory tract. removal of deposited particles described above is called clearance. when the amount of deposited particles is below a certain value, no health effect is produced. in relation to a macrophage response to particles, crystalline silica is hazardous, whereas titanium dioxide is not. pneumoconiosis is a well-known lung disease that is caused by exposure to dust particles of several micrometers in diameter. silicosis is a typical form of pneumoconiosis resulting from exposure to crystalline silica dust and characterized by a progressive fibrosis of the lungs. the macrophage-mediated clearance (phagocytes) was effective for micron and submicron particles. it has been reported that only 20% of deposited nanoparticles were removed by the clearance mechanism [8] . it has been suggested that the remaining nanoparticles may pass through the alveolar walls, penetrate into the blood or lymphatic circulation, and be transported to other organs. many studies have shown that the smaller the particle size the greater the mobility or they pass easily through the alveolar wall and enter into the bloodstream. it is further presumed that the mechanism of health effects of nanoparticles on cardiovascular system other than the respiratory tract is similar to that in the airborne ultrafine particles from dep. in japan, two reference values, the 'administrative control levels' (acl) and the 'occupational exposure limits' (oels), are used for the regulation of hazardous chemicals as well as dust (particle matters). oels are recommended and revised every year by the japan society for occupational health. oel (oel-mean) for mean concentration of a chemical substance is defined as the reference value to the mean exposure concentration at or below which adverse health effects caused by the substance do not appear in most workers working for 8 h a day, 40 h a week under a moderate workload [9] . the 'threshold limit value' (tlv) has the same definition (but may not be the same values as oel of the same substance) provided by the american conference of governmental industrial hygienists (acgih). acl is an index to determine the control class to judge the propriety of the working environment control based on the results for working environment measurement, which have been implemented for the unit work area in accordance with the working environment measurement standard. the results of working environment measurement are evaluated by classifying the working environments concerned into three control classes (control class i, ii, and iii). these classes are used as the standards to classify the level of the working environment concerned. among those subject to working environment measurement, these standards apply to workplace where dust, lead, organic solvent, and specified chemical substances are used. article 65 of the industrial safety and health law stipulates that certain workplaces in which harmful substances are involved or harmful work operations are performed shall be the subject to working environment measurement. a 50% cut-off size of the dust particle is set at 4 m for both standards and is far larger than nanosized particles. oel or acl [10] for particulate matters are usually based on mass concentration, that is, milligram per cubic meter. therefore, if the particulate matters have broad size distribution, the contribution of nanoparticles is not large to in terms of mass of particles. evidence from a number of toxicological studies on insoluble particles indicates that the primary determinant of the health effect of particles depends on the surface area of particles deposited [11, 12] . on the basis of the results from a number of in vitro studies of insoluble nanoparticles, a hypothetical cellular interaction has been proposed [13] : inflammation and oxidative stress can be mediated by several primary pathways: (1) the particle surface causes oxidative stress resulting in increased intracellular calcium and gene activation; (2) transition metals released from particles result in oxidative stress, increased intracellular calcium, and gene activation; (3) cell surface receptors are activated by transition metals released from particles, resulting in subsequent gene activation; or (4) intracellular distribution of insoluble nanoparticles to mitochondria generates oxidative stress. in the workplace, the concentration of nanoparticles may be at a high level and most of the nanoparticles become agglomerates, while nanoparticles will form single nanoparticle at low levels in the general environment. it is a matter of debate whether agglomerates of nanoparticles react as a larger particle or a single nanoparticle in the lung or other organs. if insoluble particles are retained in the lung for a longer time without enough clearance mechanisms, they can cause pulmonary inflammation or pneumoconiosis. it is of interest that nanoparticles deposited in the lung can move into the blood vessel through alveolar epithelium and they can damage vessels or produce blood clots [14, 15] . in a recent study, nanoparticles deposited in the nose may move directly to the brain via the olfactory bulb [16] . (2) biological effects of fullerene the biological effects of fullerene have being investigated intensively. in rats dosed orally with radioisotope-labeled c 60 fullerenes, most were excreted in the feces and some were found in the urine. a small amount of them can be absorbed via the gastrointestinal tract. in contrast, in the same study, 90% of the same labeled fullerenes administered intravenously were retained after 1 week, with most found in the liver [17] . ld50s (acute toxicity) by intraperitoneal injection in mice and rats were 1.2 and 0.6 g/kg, respectively. the dose of 2.5 g/kg orally in rats did not result in death. the reproductive translocation of fullerenes was also observed in mice. fullerenes have shown mutagenic activity in ames tests. fullerenes have shown no skin irritation or allergic reactions [18] . on the other hand, fullerenes are being tested for possible medical use. fullerenes are basically hydrophobic but water-soluble derivatives have been synthesized to be used as drugs or its carrier. the derivative can be anticipated as drugs, for example, anti-aids drug. it has been stated that the toxicity of fullerenes changes due to slight structural changes including chemical modification [18] . (3) biological effects of carbon nanotubes carbon nanotubes are chemically stable and are similar in form and size to asbestos; these characteristics have given rise to concern that carbon nanotubes may have the potential to cause pulmonary diseases such as lung cancer and mesothelioma similar to asbestos. a few data are available concerning the biological effects of carbon nanotubes. the biological effects of carbon nanotubes are being researched. epithelioid granulomas and interstitial inflammation are induced in mice and rats following exposure to single-walled carbon nanotubes [18] [19] [20] . untreated carbon nanotubes contain the nanoparticles of transition metals such as iron and nickel, which are used as catalysts in forming carbon nanotubes. these nickel-containing carbon nanotubes have been reported to be toxic [19] . the concentration of airborne asbestos fibers is expressed as a number concentration, that is, fibres per cubic centimeter or fiber per liter. when fiber concentrations are determined by phase contrast light microscopy, the fibers with a diameter of less than 3 m, a length longer than 5 m, and a lengthto-diameter ratio (aspect ratio) greater than 3 are counted [21] . asbestos fibers having nanosized diameter were often observed in analyses of environmental samples using electron microscopy. international agency for research on cancer (iarc) rated asbestos as a known human carcinogen (group 1) [22] and the concentration of chrysotile asbestos is expressed as a risk level of 0.15 fiber/cm 3 [9] . health effects of vitreous fibers and other asbestos substitutes have been assessed to determine their oels or their carcinogenicity in humans. the health effects of carbon nanotubes are being intensively investigated now. the oel for carbon black respirable dust is 1 mg/m 3 and these for activated charcoal and graphite are 0.5 mg/m 3 in each [9] . in the ref. [23] , while rats and mice inhaled carbon black with a particle diameter of ϳ30 nm at a concentration of 5-13 mg/m 3 did not produce any specific changes, particles (agglomerate of small particles) of ϳ450 nm at a concentration of 2-6 mg/m 3 produced early pulmonary changes. micronsized titanium dioxide particles are thought to have almost no toxicity and often used as a negative control substance. the oel for titanium dioxide is 1 mg/m 3 for respirable fraction [9] . however, the results of a series of studies by oberdörster et al. [3, 11, 24, 25] on submicron-and nanosized titanium dioxide suggested that as size decreases, inflammatory effects are intensified, and normally nontoxic substances may assume hazardous characteristics. fig. 7.3.4 shows a part of the results by oberdörster et al. in which rats and mice were exposed to anatase titanium dioxide particles [25] . their results have been frequently cited in the discussion of whether the health effects of fine particles should be based on its mass or its surface area. in fig. 7.3.4 , percentages of neutrophils in lung lavage of rats are shown as indicators of inflammation after intratracheal instillation of different mass doses of 20 and 250 nm tio 2 particles. the steeper dose response of nanosized tio 2 particles is observed than for submicron tio 2 particles when the dose is expressed as mass (fig. 7.3.4a ). if the same dose-response relationship as in fig.7. 3.4a is indicated as particle surface area (fig. 7. 3.4b), the particle surface area seems to be a more appropriate dosimetric for comparing effects of different-sized particles of the same chemical structure. zinc oxide is a white powder and used in pigments. the oel is 1 mg/m 3 for its respirable fraction [9] , and the value for zinc oxide fume which causes metal fume fever is under consideration. nanoparticles of transition metals and rare earth elements and their oxides will be used widely. since many of these metals and their oxides have biological effects, particular attention should be given to them. nickel compounds are rated as a human carcinogen (group 1) by iarc. in particular, nickel oxide is particularly insoluble among the nickel compounds and remains longer in the lung. nanosized nickel oxide particles have greater toxicity to the lung than larger particles [26, 27] . pulmonary inflammatory responses induced by nanosized cobalt particles have been reported [28] [29] [30] . biological effects of nanosized particles of other transient metals such as iron and manganese have received attention [3] . rare earth elements are a general term of 17 chemical elements consisting of scandium (sc), yttrium (y), and a lanthanide series of 15 elements from lanthanum (la) to lutetium (lu). these elements have been used in magnetic alloy, fluorescent and hydrogen storage alloy. particularly cerium oxide nanoparticles are frequently used as a fuel additive and are incorporated in cosmetics formulation. the potential biological and environmental effects of these elements have not sufficiently been investigated. it has been demonstrated that ld50s for these elements in oral and intravenous administration are in a range from several dozens to several thousands milligrams per kilograms, indicating that none of these elements has high toxicity. in the results of studies in which the biopersistence and the distribution of rare earth compounds in the body were investigated, for example, the compounds deposited in bones and teeth, and organs including lung, liver, spleen and kidney following intratracheal, oral, intravenous, and intraperitoneal administration. although the compounds deposited predominantly in the liver other than bones, it has also been reported that the distribution of the compounds in the lung and spleen increased when the dose was increased [31] . the demand for indium compounds has been sharply increasing. the compounds have been used in the materials for transparent electrodes for flat panel displays. in japan, the cases of pulmonary interstitial pneumonia and pulmonary fibrosis have been reported in workers engaged in cutting and grinding of sintered indium-tin oxide (ito) and potentially having inhaled the dusts released from ito [32, 33] . the biological effects of indium arsenic compounds and indium phosphorus compounds also have been investigated. the acgih has proposed a value of 0.1 mg / m 3 for their tlv, and the value has been applied tentatively in japan. we have been experiencing amazing progress of the technology on the processing for the nanometer-sized materials. the applications cover even biomedical engineering in addition to information technology, material, environmental science, and energy production [1] [2] [3] [4] [5] . as the result, many kinds of new materials have been designed, fabricated, and discarded. from now on, this movement will be accelerated and even more new functional materials will be distributed in the world. here, we should not forget the safety of those materials in the process of the production, usage, and discard. without this safety assessment, we will go into the same problems as that of asbestos just we are facing now. first of all, we have to conduct experiments to reveal the minimal concentration for emergence of the toxicity. in other words, we have to fix the standard value for the threshold concentration for each material first of all. if we do not fix it, we should not use the material at any concentration, which means any engineering process could not be carried out. the applications in the various fields have started all over the world, and the safety assessment is urgently needed. in this article, we introduce the methods of the safety assessment of the semiconductor nanoparticles and describe the safety and the threshold depending on the surface treatment. the production process and the surface treatment play one of the most important roles for the safety of the nanoparticles. cd and se semiconductor nanoparticles coated with zns are one of the most widely used for the strong intensity of the fluorescent activity. cd oxide and se compounds once dispersed in the tri-octhil-phosphine oxide (topo) heated up to 300њc generate nanoparticles by self-assembly. then zns enhances the stability of the structure and raises up the fluorescent intensity than without the coated one. nanoparticles, thus manufactured, as materials for a novel memory in the field of intelligence technology (it), and as super-micro devices for laser in the field of optics, have been developed all over the world [1] [2] [3] [4] [5] . these nanoparticles cannot be dissolved into water, but dissolved into organic solvents like toluene. therefore, for biological and medical applications, various technologies for surface-conjugations to make them hydrophilic [6, 7] have been developed. for example, nanoparticles covered with topo are hydrophobic because an alkyl group on them is hydrophobic. therefore, a technology for replacing this alkyl group with hydrophilic carbonic acid (making the whole particles soluble in water) has been developed [6] . nanoparticles, thus surface-treated, can be dissolved into water, like sodium salt or potassium salt. with this method, various kinds of materials have been surface-conjugated. the mtt assay method is a way to evaluate the hazard assessment of nanoparticles, in which the activation metabolism in a mitochondrium in a cell is measured and the influence of nanoparticles on the prolification of the cell is qualitified. the mtt is a kind of tetrazolium, whose molecular formula is c 18 h 16 brn 5 s. taken into a cell, it is decomposed by a dehydrogenase enzyme in a mitochondrium into a pigment called 'hormazan'. the measurement of the fluorescence intensity of the pigment shows the prolification of the cell [8] [9] [10] . fig. 7 .3.5. shows the hazard assessment of vero cells and kidney cells of the african green monkey against cdse/zns nanoparticles. the horizontal axis shows the concentrations of the nanoparticles and the vertical axis the fluorescence intensity of the hormazan at 460 nm, that is, the metabolism of the cells. the figure indicates that cyto-toxicity is not observed for 10 5 cells when the concentration is less than 0.5 m. this result means that this concentration is the threshold of the cell toxicity. likewise, cyto-toxicity is not observed for the hela cells and for the human primary cells. further, in order to find out how the sizes of nanoparticles influence the cyto-toxicity, the cyto-toxicities were evaluated with three kinds of quantum dots; one whose fluorescence wavelength is 640 nm, red, one with 570 nm, yellow, and the other with 520 nm, green. the following results were obtained. the largest quantum dots whose fluorescence wavelength is 640 nm show a tendency to give cyto-toxicity. cyto-toxicity is observed at concentrations more than 1 m [11] . another method to evaluate cyto-toxicity is the flow cytometry [12] . the mtt assay alone cannot tell whether the toxicity observed is lethal to the cells or just restrains the prolification of them. in the flow cytometry, the nuclei of dead cells are dyed with propidium iodide (pi) after the nanoparticles are taken in and the ratios of the dead cells are measured. fig. 7.3.6 shows the lethal cyto-toxicity of mua conjugated nanoparticles (520 nm, green) against vero cells. the vertical axis indicates the numbers of the cells, and the horizontal axes show the fluorescence intensities and the cyto-toxicities. these experiments also show that dead cells cannot be observed at concentrations less than 0.5 m even though nanoparticles are taken in, as was shown in the mtt assay. however, at concentrations more than 2 m, the nanoparticles taken in cause damage to more than the half of the cells. that is, the cyto-toxicity of mua quantum dots against cells is lethal [11] . nanoparticles have been surface-conjugated for applications for various uses. some surface-conjugations cause more grave cyto-toxicity than others. therefore, relations between surface-conjugations and their safety for cells have to be considered. in order to find out the relations, the safety evaluations of nanoparticles surface-conjugated with two materials were made; one is with mua (quantum dots-cooh) and the other is with glycerol (quantum dots-oh), and their purified and unpurified particles. fig. 7.3.7 shows that the purification reduces the cyto-toxicity for the quantum dots-oh, and that the toxicity remains the same after the purification for the quantum dots-cooh. mua itself, a material with which particles are conjugated, has cyto-toxicity. this experiment shows that toxicity against cells is connected not only with particles themselves but also with kinds of surfaceconjugations and degrees of purification. it is shown in the above experiments that the toxicity of nanoparticles is lethal to cells. next, we found out by the comet assay method whether the toxicity is derived from damaged dna. the method is a way to evaluate quantitative damage of dna by electrophoresis. the fragmented dna seeps out of their cells by treating cells, whose dna has been fragmented, with agarose-gel to break their cell membrane, and then electrophoresing them. it looks like the "tales of comets". cells, whose dna has not been fragmented, have their nuclei keeping their spherical shape after electrophoresis, and the tales of comets cannot be observed [13] [14] [15] [16] . fig. 7 .3.8 shows the results of the experiments with quantum dots conjugated with cooh (both purified and unpurified) to wtk-1 cells, a human lymphoblast mutation strain [17] . the vertical axis shows the lengths of the tails, and the horizontal axis the concentrations of the quantum dots. the concentration of the nanoparticles is 2 m. the results are as follows: the unpurified quantum dots with cooh caused damage to dna. on the other hand, the electrophoresis with the purified quantum dots does not show dna damage. this is probably because the dna damage has been repaired during the longer cultivation time. conducted with quantum dots conjugated with mua, and quantum dots-nh 2 with an amino group. dna damage was not observed in either case. the results indicate that as far as particles themselves do not break down, the cyto-toxicity of quantum dots is derived from the chemical properties of the materials covering the quantum dots. the safety evaluation of nanoparticles has not been conducted sufficiently. as indicated above, the procedure for surface-conjugation could apply not only to cd/se nanoparticles but also to other nanoparticles. today, various kinds of techniques for surfaceconjugations have been available in academic papers, proceedings, and on the internet. some of them are widely known and others are patented. those techniques are all shared among the human race. even at this moment, the human beings are making breakthroughs in various fields and developing different kinds of technologies. sharing these technologies will lead to still more speedy developments of yet more advanced technologies. to achieve it, these technologies should be so structured that different fields, for example, bioimaging and biotechnology, structured on their own, can be linked to each other. such structured knowledge will play an essential part in merging different fields. in order to prevent nanoparticles release from a system so as to maintain environmental safety, the removal technique of nanoparticles must be established. in this section, separation techniques of particles from exhausted or suspended gas and liquid are described focusing on particles less than 100 nm. generally, as shown in fig. 7 .4.1, all particle separators for a dispersed system employ either one of three basic forms of particle separation. on the left hand side of the figure lie the separation methods in which particles are collected only by force field (electrostatic force, centrifugal force, gravity force, etc.), and the representative separator is electrostatic precipitator (esp). if some obstacles (collectors) are placed into the particle laden stream, particle separation is facilitated because particles are collected on obstacles with a smaller deviation from the fluid flow by the force exerting on the particles compared to the case without obstacles. typical collectors of this form are air filter, deep bed filter, etc. on the right hand side of the figure lie separators that collect particles utilizing only sieving effect of obstacles without any force field. in this case, geometrical size of channel between the obstacles must be smaller than that of particles. membrane filter, fabric filter, etc. belong to this group. when we apply the above collection forms to nanoparticles, the major collection mechanisms are brownian diffusion and electrostatic force for particles in gas, while sieving effect and interception/adhesion forces for those in liquid. as mentioned above, most airborne particles are collected by separators utilizing various kinds of forces such as gravity, centrifugal force, electrostatic forces, inertia, brownian diffusion force, and so on. therefore, the migration velocities or displacement of a particle per second due to the individual forces gives the basis for the comparison of removal efficiencies due to each force. in fig. 7 .4.2, migration velocities of particles due to various forces are depicted against particle diameter at normal temperature and pressure for particle density of 1 g/cm 3 [1] . as seen from the figure, the velocities due to gravity, centrifugal force, and inertia monotonically decrease with decreasing particle diameter, suggesting that the removal of nanoparticles with these forces is difficult. on the contrary, the velocities due to brownian diffusion and electrostatic forces increase with decreasing particle diameter for particles less than 100 nm. this suggests that brownian diffusion and electrostatic forces are most effective in collecting nanoparticles. fig. 7 .4.3 summarizes typical conventional dust collectors. among them, the effective collectors for nanoparticles are esp and fabric/air filter. however, for the case of esp, which relies on only electrostatic force, nanoparticles (ͻ 10 nm) fail to carry even one electron resulting in low collection efficiency. in this case electrically charged filters are effective because we can expect the combined effects of electrostatic forces and brownian diffusion. among charged filters, so-called electret filter, which consists of permanently polarized fibers, is the most favorable filter because of its charge stability. particle penetration data of electret filter are plotted against particle diameter in fig. 7 .4.4 and compared with that of uncharged filter with the same structure. for the three combinations of charged states of fiber and particle there exist the most penetrating particle diameters. for the uncharged fiber, collection efficiency of uncharged particle has a minimum at 100 nm and increases with decreasing particle diameter, showing that 100 nm is the most penetrating particle size. for the charged fiber, particle collection efficiency is very high even for uncharged particle, and the efficiency for charged nanoparticles is extremely high because of strong coulombic force between fiber and particle. the experimental data plotted in fig. 7 .4.4 are qualitatively in good agreement with the theoretical prediction following the particle size dependency on particle migration velocity (shown in fig. 7.4.2 ). however, as particle size becomes smaller and comparable with the size of a molecule, particles may rebound on a collector surface, and the adhesion probability of particles drops, resulting in a decrease in collection efficiency. fig. 7 .4.5 is an example of experimental data that confirm the particle rebound [2] . the figure shows the penetration of nanoparticles through a grounded circular tube. the solid curve is the theoretical line derived by assuming that particles are deposited from a laminar flow in a tube by brownian diffusion. it is evident that experimental penetration deviates from the theoretical line for particles less than 2 nm. this means that molecular behavior begins to appear when the particle size becomes as small as 2 nm, and as a result, the collection efficiency is reduced. it should be noted that considerable amounts of nanosized particles are contained in diesel exhaust particles (dep), possibly penetrating through the honeycomb type (tubular channel) diesel particulate filters (dpf). there are two types of methods that differ in the way the nanoparticles in liquid are collected. the first group, called membrane filtration, constrains the particles by a membrane, and the liquid is allowed to flow freely through the membrane. in the second group of ultracentrifugation, the liquid is constrained in a rotating vessel, and the particles move freely within the liquid by an external field of acceleration caused by ultracentrifugal field. these methods have been quite extensively used in separation of macromolecules and molecules from liquid, and they are recently becoming important also in separation of nanoparticles from liquid. in pressure-driven membrane filtration processes, the pressure gradient across the membrane would force solvent and smaller species through the pores of the membrane, while the larger molecules/particles would be retained. membrane filtration processes are usually classified into three general categories according to the size of separating components, as shown in fig. 7 .4.6. microfiltration (mf) is designed to retain suspended particles in the range of 50 nm-5 m. ultrafiltration (uf), on the other hand, retains macromolecules or nanoparticles in the range of 5-50 nm (nominal molecular weight cut-off (nmwco) ranging from 5,000 to 5,000,000 da). nanofiltration (nf) is a relatively new process that uses charged membranes, and it covers molecular sizes ranging from 0.1 to 5 nm (nmwco ranging from 200 to 1,000). it is useful in that it can separate dissociated forms of a compound from the undissociated form. one of the major factors limiting the use of membrane filtration is membrane fouling, resulting in a dramatic decline in flux with time of operation. to account for the membrane fouling, the resistancein-series model is frequently employed. the resistance model becomes (7.4.1) where u 1 is the permeate flux, v the filtrate volume per unit membrane area, the filtration time, p the applied transmembrane pressure, the viscosity of the permeate, r t the total resistance, r bm the resistance of the membrane per se plus the clogging of the membrane pores, r c the resistance of the filter cake, and r cp the resistance of the concentration polarization layer. significance of each resistance in membrane filtration is as follows. the membrane, even in the absence of any suspended particle, has a natural flow resistance. during membrane filtration, particles become attached to the pore channel of the membrane thereby reducing the flow channel dimension, or pores become blocked off completely. the last two effects lead to resistances that are due to adsorption and pore blocking. the blocking filtration model introduced by hermans and bredée [1] , and grace [2] is most commonly used as an interpretation of such phenomena. the clogging of the membrane pores is strongly influenced by such solution environment as ph and the ionic strength. the permeate flux of bovine serum albumin (bsa) (pi 5.1, molecular weight 67,000, stokes-einstein diameter 3.55 nm) solution by permeable mf membrane (nominal pore size 0.05 m) is lowest at around the isoelectric point [3] . as the bsa molecule carries no net charge at the isoelectric point, the molecule is in its most compact state at that point. the bsa molecules deposit themselves rather densely onto the pore walls of the membrane to form a compact configuration with a smaller lateral electrical interaction between the molecules. as a result of this, the flow resistance increases markedly at around the isoelectric point. in dead-end membrane filtration, which has a feed and permeate stream, each with the same mass flow rate, the resistance of the filter cake plays a major part in the filtration resistance. therefore, the cake filtration theory can be applied, and thus the permeate flux u 1 is described by (7.4.2) where m is the ratio of wet to dry cake masses, s the mass fraction of solids in the solution, av the average specific filtration resistance, the density of the permeate, and v m the fictitious filtrate volume per unit membrane area, equivalent to the flow resistance of the membrane [4] . for fine particle suspensions, colloidal forces which arise from interaction between the suspended particles control the nature of the filter cake. the average specific filtration resistance av and the average porosity av of the filter cake are strongly affected by the solution properties, including ph and electrolyte strength. for instance, in mf of suspensions of the titanium dioxide (pi 8.1, the original mean specific surface area size 470 nm), av goes through a minimum, and av is much larger near the isoelectric point [5] , as shown in fig. 7 .4.7. the titanium dioxide particles are destabilized around the isoelectric point where the van der waals attraction is more dominant. consequently, the particle tends to come together, that is, to flocculate, and the very porous flocs are then formed. thus, it is speculated that the filter cake formed from such porous flocs has often loose and wet structures. on the other hand, the filter cake becomes compact and dry when the particle carries the charge. since the most loose filter cake forms around the isoelectric ph, the filter cake is most permeable. it is interesting to note that the results in protein uf had a distinctly different behavior. in protein uf of bsa solution, the filter cake is in its most compact state around the isoelectric point [6] , as shown in fig. 7 .4.8. since the bsa molecules are hydrophilic colloids, their stability in the solution would appear to be influenced not only by the presence of a surface charge on the protein but also by hydration of the surface layers of the protein. the bsa molecules, because of hydrated layers surrounding them, are not destabilized by such consideration as depression of the electrical double layer. thus, the bsa molecules have water bound to them even around the isoelectric point. the hydrophilic bsa molecules maintain a dispersed state in the solution due to hydration of the surface layers of the protein even around the isoelectric point. when a bsa molecule acquires a charge, the filter cake becomes loose and wet due to electrostatic repulsion between the charged bsa molecules. this contrasts to the compact filter cake around the isoelectric point. the average specific filtration resistance av has a definite maximum around the isoelectric point since a compact filter cake provides a large hydraulic flow resistance. most membrane filtration processes are operated in the cross-flow mode, in which the feed is moved tangentially to the membrane surface so that the filter cake is continuously sheared off. during membrane filtration, particles in the feed are brought to the upstream surface of the membrane by convective transport, and this results in a higher local concentration of the rejected particles at the membrane surface as compared to the bulk solution which is referred to as concentration polarization [7] . the particle concentration in the solution adjacent to the membrane varies from the value at the membrane surface, c m , to that in the bulk feed solution, c b , over a distance equal to the concentration boundary layer thickness . the resulting concentration gradient causes the particles to be transported back into the bulk solution due to diffusional effects. at steady state, the rate of convective transport of particle toward the membrane is balanced by the rate of particle transport through the membrane plus the rate of the diffusive back transport of particle. thus, the permeate flux u 1 is given by (7.4.3) where cp is the particle concentration in the permeate, k (ϭd/ ) the mass transfer coefficient, and d the diffusion coefficient. the osmotic pressure model assumes that the deviation from pure water flux occurs solely due to the osmotic pressure difference across the membrane, and thus the permeate flux u 1 is given by (7.4.4) where is the osmotic pressure, which is a function of the concentration. equation (7.4.4) means that the effective driving force across the clean membrane is pϫ{ (c m )ϫ (c p )}. replacing pϫ{ (c m )ϫ (c p )} by the hydraulic pressure at the membrane surface, p m , equation (7.4.4) reduces to the cake filtration equation. to minimize the effects of cake buildup and concentration polarization, membrane filtration is usually conducted using the cross-flow geometry in which the feed flow is parallel to the membrane and perpendicular to the filtrate flow [8] . however, especially in mf the energy requirements associated with pumping the feed (plus any recirculation flow) along the membrane surface are typically very high. thus, there have been some innovations in recent years with cakeless filtration. the rotating disk module in which the membrane disk is stationary is suited for large-scale operation [9] . it is possible to enhance the permeate flux by using the vibrating modules [10, 11] . in the rotating cylinder device with the membrane on the inner rotating cylinder, counter-rotating taylor vortices within the annular gap are available [12, 13] . dean vortices that twist and spiral in the direction of flow inside a highly curved channel, similar to vortices in rotary modules can result in enhanced flux [14] . these vortices, or flow instabilities, induce turbulence into the system. periodic removal of the formed filter cake is also effective for enhancing the permeate flux. recently, several methods have been investigated: back washing using the filtrate or air pressurization [15] , periodic rotation of the cylindrical membrane [16] , pulsatile flow [17] , high-frequency transmembrane pressure pulsing with a frequency around 0.1-1 hz [18] . dead-end upward filtration, where the filtrate flow is in the opposite direction to gravity, and dead-end inclined filtration, where the membrane is inclined, can reduce the cake formation onto the membrane in uf of nanoparticulate suspension and protein solutions. in upward uf of silica sol (mean diameter 6.2 nm) and bsa solution, a sustained permeate flux is achieved, as shown in fig. 7 .4.9 [19, 20] , because the filter cake overlying the membrane is exfoliated continuously under the gravitational force acting on the particles comprising the filter cake. another approach for enhancing the permeate flux is to employ external force fields. electrofiltration, in which an applied electric field is used to drive charged particles away from the membrane surface, has been developed. in electrofiltration, the accumulation of the particles on the membrane surface is limited by the imposed electrophoretic force. in addition, the permeate flux through the filter cake is dramatically enhanced due to electroosmosis as a secondary electrokinetic phenomenon. this method can be applied to a broad combination ranging from mf of particulate suspension such as bentonite [21] to uf of protein solution. fig. 7 .4.10 shows the reciprocal permeate flux (d /dv) versus the permeate volume per unit membrane area, v, for various values of the strength of the dc electric field, e [22] . the steady permeate flux increases noticeably with the magnitude of the imposed field strength. also, a higher electric field strength causes the permeate flux to equilibrate more rapidly. a method has been developed for removing humic substances by hybrid uf combined with both flocculation and adsorption treatments, as shown in fig. 7 .4.11 [23] . flocculation by use of polyaluminum chloride (pacl) is particularly effective for the removal of humic acids, which constitute the relatively high molecular weight fractions of humic substances, whereas adsorption by use of powdered activated carbon (pac) is able to remove fulvic acids of relatively low molecular weight effectively, which cannot be fully flocculated by pacl. hybrid uf in combination with flocculation and adsorption treatments exhibits high permeate quality because the flocs and pac are easily retained by the uf membrane. in ultracentrifugal sedimentation, ultracentrifugal force field of several tens of thousands of revolutions per minute is applied to a rotor. in recent years, ultracentrifugal sedimentation is employed for concentrating dilute protein solutions and for separating proteins and other large biological molecules from low-molecular-weight solutes or from much larger particles. fig. 7.4.12 shows the results for ultracentrifugal sedimentation of an aqueous solution of the mixtures of bsa and egg white lysozyme (pi 11.0, mw 14,300) measured using schlieren optics in an analytical ultracentrifuge [24] . the angular acceleration of the rotor is 5,445 rad/s. the symbol r i and r i0 in the figure represent the distances from the center of rotation to the sedimentation boundary at time and 0 , respectively. the electrical nature of macromolecules plays a significant role in determining the sedimentation behavior in ultracentrifugation of binary protein mixtures. in the ph range where both protein molecules were electropositive, the molecules sediment independently due to the electrostatic repulsive force acting between bsa and lysozyme molecules. denpun kagaku filtr. sep institute for quality assurance and certification: basic criteria for the award of the environmental label (printer ral-uz 85) proc. air cleaning contam proc. air cleaning contam proc. air cleaning contam chemical contamination in semiconductor processing environments and its countermeasures the japan society of industrial machinery manufactures: report on behavior control of individual sort of contaminants -1993 report on introduction of advanced technologies to environmental equipment industry industrial application of nanomaterials -chance and risks. future technologies, division of vdi technologiezentrum nanoscience and nanotechnologies: isbn 0 85403 604 0 recommendation of occupational exposure limits who: environmental health series 4. world health organization summaries & evaluations -nickel and nickel compounds rapid colorimetric assay for cellular growth and survival ouyou earozorugaku crossflow filtration: theory and practice encyclopedia of fluid mechanics: slurry flow technology filtr. sep key: cord-310948-nt378esz authors: edwards, n. j.; widrick, r.; potember, r.; gerschefske, m. title: quantifying respiratory airborne particle dispersion control through improvised reusable masks date: 2020-07-14 journal: nan doi: 10.1101/2020.07.12.20152157 sha: doc_id: 310948 cord_uid: nt378esz objective: to determine the effectiveness of non-medical grade washable masks or face coverings in controlling airborne dispersion from exhalation (both droplet and aerosol), and to aid in establishing public health strategies on the wearing of masks to reduce covid-19 transmission. design: this comparative effectiveness study using an exhalation simulator to conduct 94 experiment runs with combinations of 8 different fabrics, 5 mask designs, and airflows for both talking and coughing. setting: non-airtight fume hood and multiple laser scattering particle sensors. participants: no human participants. exposure: 10% nacl nebulized solution delivered by an exhalation simulator through various masks and fabrics with exhalation airflows representative of "coughing" and "talking or singing." main outcomes and measures: the primary outcome was reduction in aerosol dispersion velocity, quantity of particles, and change in dispersion direction. measurements used in this study included peak expiratory flow (pef), aerosol velocity, concentration area under curve (auc), and two novel metrics of expiratory flow dispersion factor (edf) and filtration efficiency indicator (fei). results: three-way multivariate analysis of variance establishes that factors of fabric, mask design, and exhalation breath level have a statistically significant effect on changing direction, reducing velocity or concentration (fabric: p = < .001, wilks' {lambda} = .000; mask design: p = < .001, wilks' {lambda} = .000; breath level: p = < .001, wilks' {lambda} = .004). there were also statistically significant interaction effects between combinations of all primary factors. conclusions and relevance: the application of facial coverings or masks can significantly reduce the airborne dispersion of aerosolized particles from exhalation. the results show that wearing of non-medical grade washable masks or face coverings can help increase the effectiveness of non-pharmaceutical interventions (npi) especially where infectious contaminants may exist in shared air spaces. however, the effectiveness varies greatly between the specific fabrics and mask designs used.  the strength of this study is that it offers quantitative evidence on the effectiveness of wearing non-medical improvised masks in controlling airborne dispersion of particles from exhalation.  study can aid in establishing public health strategy that encourage the wearing of masks or face coverings for reducing airborne transmission of infectious disease in shared air spaces.  a limitation of this study is that is uses an exhalation simulator with the ppe industry standard nacl test solution for particle generation rather than a clinical study with exhalation of biomaterial particles.  the particle sensors used had a limited ability to detect fast moving aerosol clouds from coughing or talking with no-mask applied. in light of the current pandemic from rapid transmission of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2 or covid-19) and significant morbidity, there has been inconsistent medical guidance given to the public regarding the wearing of non-medical improvised fabric masks or face coverings to reduce the transmission of covid-19. if the sars-cov-2 aerosol is considered with an ability to infect for more than 3 hours with tcid50 of greater than 10 2 as noted in a recent laboratory study [1] then the understanding the effectiveness of non-medical masks and face coverings to control human exhalation aerosol dispersion has significant importance for broad public health infectious disease strategy, especially with asymptomatic or pre-symptomatic populations. of concern, recent studies show that bio-droplets of all sizes are generated from normal exhalation [2] [3] [4] [5] with 80-90% of droplets from human exhalation in the size range of 0.1-1µm, [6, 7] those from a cough can travel 23 to 27 feet (7-8 m) which is well beyond the recommended social distances of six feet or two meters, and smaller aerosols (≤5µm) stay aloft in the air and pose a greater risk for severe infection. [8, 9] in addition, social distancing is difficult to accomplish since many essential locations like grocery stores have aisles are narrow and result in the proximity of patrons being closer than 1 meter; reduced distance correlates to increased transmission of covid-19. [10, 11] a lack of definitive data on establishing the effectiveness of using non-medical masks or face coverings has resulted in medical practitioners giving broad public health guidance based on professional judgement only. existing guidance includes statements that facial coverings may offer minimal protection from small infectious particles, may only reduce large particulate matter, or only remind users to not touch their face considering infectious disease transmission from hand to face. a number of previous studies have been conducted to understand if wearing of masks reduce community infections of common diseases such as influenza, however most are inconclusive due to the application of masks post-exposure or lack of strict wearing compliance by study participants. [12] [13] [14] only a few well-executed studies conclude the prophylactic wearing of medical grade masks reduce community transmission of influenza or rsv. [15, 16] to make matters worse, the lack of definitive guidance has also led to social and political debates on the wearing of masks or face coverings [17] [18] [19] and deters the acceptance of any new public health strategy for reducing airborne transmission of infectious diseases. prior studies have established the filtration efficiency of a variety of fabrics but do not consider reducing covid-19 transmission by controlling airborne dispersion of human exhalation. [20] [21] [22] [23] [24] [25] [26] [27] other research investigates only forward dispersion of particles from coughing or sneezing by measuring a on a single optical plane, [8, 28] or on the aerodynamics of exhalation particles inside various rooms. [29] [30] [31] [32] several clinical studies showing reductions in virus shedding when wearing face masks,[2,3,5,33-35] but neither the clinical nor experimental studies have fully characterized the effectiveness of non-medical grade reusable masks in controlling aerosol dispersion of human exhalation particles in terms of three-dimensional direction, velocity, and particle concentration of various diameters in real world environments. the goal of this research is to determine the statistically significant factors and effectiveness of non-medical grade washable masks or face coverings in the control of aerosol dispersion of human exhalation, and to aid in establishing public health strategies or policies on the wearing of masks. [36] although broad clinical studies on the use of non-medical masks would offer results directly correlated to the community reduction of infectious diseases, this original research offers the experimental results that establish a basis for conducting such a study along with a more comprehensive set of effectiveness measurements for mask designs. we conducted a comparative effectiveness study using a randomized full factorial design of experiments with 10% nacl nebulized solution and an exhalation simulator to conduct 94 experiment runs with combinations of 8 different fabrics, 5 mask designs, no-mask as a control, and exhalation airflows (pef and fev1) that represent both talking and coughing. the experiment also included randomized runs of no-mask applied as the control and a preliminary comparison with the performance of a merv13 air filter media which has similar electrostatic filtration properties to the niosh n95 standard (95% filtration efficiency of 0.3µm particles). the exhalation simulator was constructed similar to previous research, [37, 38] but with some differences. the exhalation simulator was driven by a dry compressed air expansion chamber and timing-controlled relay, with a port for the small volume jet nebulizer, in-line spirometer, and a corrugated tube to emulate a trachea before exiting the mouth of the cpr manikin. details on the simulator equipment are in (online supplementary figure 1 ). the exhalation airflows were calibrated to simulate peak expiratory flow (pef) of coughing with a range 507l/min to 650l/min and pef for talking of approximately 120l/min as established by previous research. [37] [38] [39] [40] the typical air flows for talking are similar to that of singing. [40] four laser scattering particle concentration sensors (plantower pms5003) were placed at specific locations inside a non-airtight fume hood (online supplementary figure 2) to detect aerosol dispersion directly downward, laterally from the mid-line, and 1 meter forward of the mouth. preliminary testing of the configuration identified that the optimal position when used with various masks in this fume hood was 43 cm below the level of the mouth for all sensors. the frontal sensor represents an approximate halfway point of a 2 meter or 6 feet social distance. a nacl aqueous solution was selected as a polydisperse test aerosol which is also used as the exposure for niosh n95 respirator test methods. 10% nacl was used to generate a sufficient quantity of particles for the open-air fume hood environment and also to stay beneath the pms sensor maximum (65,535 particle count per 0.1l for any given size). the aerosol was produced by nebulizing the solution at 103 kpa (15 psi) for 5 seconds into the aerosol chamber of the exhalation simulator followed by a 3 ms delay before exhalation from the manikin through the applied masks. the simulated exhalations were driven by timing controlled compressed air at 827 kpa (120 psi) for "coughing" and 206 kpa (30 psi) for "talking". the intervention was provided by non-medical grade washable masks sourced from local materials that were available during the covid-19 supply chain interruptions. the fabrics were selected are shown in (online supplementary table 1) (fabric bolts were unavailable) which all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 14, 2020. . https://doi.org/10.1101/2020.07.12.20152157 doi: medrxiv preprint included natural fibers, polyesters, and other materials. the mask designs were selected from variety of community-based designs which included a bandana style, surgical mask style, folded no-sew, a simple mask with earloops, and a stylistic mask that had more coverage of the nose. microscopic images of the fabric weave and fibers were taken (keyence vhx-s660e) to further understand and explain the results. more details regarding the fabrics and masks designs and the basic test procedure are also included in the (online supplementary appendix). the primary outcome was to measure any significant reduction in aerosol dispersion velocity, quantity of particles, and change in dispersion direction. measurements used in this study included peak expiratory flow (pef), forced expiratory volume (fev1), as well as aerosol arrival time, time to peak concentration, aerosol velocity, area under curve (auc) for first minute and last minute as shown in figure 1 . a change in direction from sensor 5, reduction in velocity, or auc are considered a positive effect. two novel metrics of filtration efficiency indicator (fei) and expiratory flow dispersion factor (edf) are established in this study to present quantitative values that give relative indicators to the dispersion control performance of non-medical masks using simple and repeatable measurement techniques of the research. a description of all measurements and outcomes are presented in table 1 . indicates general filtration of mask design / fabric by comparing residual particle concentration with mask applied to nomask (control) after system has equalized. max value of the control is used to give ratio of particle conc. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 14, 2020. . https://doi.org/10.1101/2020.07.12.20152157 doi: medrxiv preprint worst case scenario. this is a unitless ratio. quantifies the overall reduction in aerosol velocity and direction from a mask application. this is a unitless ratio of the air velocity from exhalation and velocity of the first arriving aerosol coupled with direction. pef velocity is calculated from a standard volumetric flow formula. = 1 − , a since the data collected is a series of discrete measurements, the auc calculation is similar to a summation of trapezoidal areas but accounts for the ascending and descending edges. fei is a ratio of the particle concentration remaining after exhalation through a mask compared to no-mask and provides a quantitative indicator that aids in the filtration performance characterization. it is a ratio of remaining particle concentration (auc4 to 5 min) with a mask applied compared to the worst-case auc of no-mask applied. since this experiment does not lend itself to directly measure the particle concentration in the aerosol chamber prior to exhalation nor does it use the same calibrated equipment, test orifice and tube size, airflow dynamics, and other equipment from the niosh n95 test standard, [41] the filtration efficiency indicator values are relative to this experiment. similarly, many other recent studies seeking to establish the filtration efficiencies of non-medical grade masks or fabrics have the same constraint on relativity of results. however, while the actual values are relative to this study the fei measurement technique is broadly applicable to all exhalation dispersion studies. we also present edf as a measurement of the reduction in particle velocity and change in direction when a mask is applied. as shown in table 1 , it is the ratio of cloud velocity at the first arriving sensor to that of the exhalation airflow velocity derived from the pef measurement. the theory of edf is based on the airflow from exhalation simulator (bounded volume) and aerosol velocity in fume hood (unbounded turbulent airflow) which are correlated by bernoulli's ideal-gas law and further described in the field of kinetic theory of gases. full derivation of the volumetric flow formula is provided in literature; [42] in this study the airflow of pef equals the cross-sectional area of the spirometer multiplied by the average velocity of the air stream shown in equation (1) = ̅ (1) where: the inside diameter of the mir smartone spirometer was measured to be 28.67 mm which allows for an area calculation. using algebraic relationships, the formula for calculating the velocity of the exhalation using pef measurement is shown in equation (2) along with the unit conversion to meters per second. • . × (2) all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 14, 2020. . https://doi.org/10.1101/2020.07.12.20152157 doi: medrxiv preprint sample size: the overall sample size from the full factorial combination of 40 distinct masks, several randomly inserted test runs of no-mask as the control, and 2 exhalation levels resulted in 94 experiment runs. the overall experiment with four sensors and average sampling rate of 1 second, generated over 1.694 million time-series sensor data measurements for this study. the sample size of n=94 resulted in 2,496 measurements for each dependent response variable across all particle diameters. multivariable and multivariate analysis was conducted from the perspective of a null hypothesis that non-medical improvised masks do not affect the dispersion or offer source control. this study determines if the three independent variables (mask designs, fabrics and breathing levels) have a statistically significant effect on any of the dependent responses (direction, auc0 to 1 min, fei, and edf) at various sensor locations. three-way multivariate analysis of variance (manova) is used to simultaneously understand the significance of multiple effects from the independent variables and their correlations while minimizing type i statistical errors (false positives). details on the use of manova and validation against the assumptions of the data, including homogeneity of covariance, normality, independence of observations and multicollinearity [43] [44] [45] [46] [47] are provided in (online supplementary description of statistical methods). matlab version r2019a was used to import the raw data files, compute the response variable values, and calculate summary statistics. spss version 1.0.0.1327 was used to perform manova. to additionally validate the statistical results and measured outcomes, graphical analysis of the data was also performed to identify any anomalies that were not expected in the response variables. the mean (sd) pef for simulated coughing was 532.08 (75.65) l/min and fev1 of 5.92 (0.1) l. likewise, the mean (sd) pef for simulated talking was 148.35 (43.29) l/min and fev1 of 1.79 (0.07) l. both simulated exhalation levels are within range of previous studies. [37] [38] [39] [40] the aerosol particle concentration was measured at the one-meter frontal sensor during the last minute of all no-mask (control) runs and resulted in concentration levels and distribution that indicates good polydisperse particle generation (online supplementary figure 3 ). the mean (sd) concentrations for simulated talking generated peak concentrations of 32,199 (3,683) for 0.3µm particle diameters representing aerosols, and 201(42) for 10µm particle diameters representing droplets. likewise, simulated coughing generated peak concentrations of 27,731 (9,837) for 0.3µm particle diameters, and 131(27) for 10µm particle diameters. table 2 shows descriptive summary statistics of variables with respect to the primary dispersion direction and gives some insight into the generalized responses. the best overall performing mask is the surgical style with internal non-woven layers [auc0 to 1 min = 7.721x10 5 (6.606x10 5 ), fei = .468(.158), edf = .993(.005)]. the best overall fabric depends on a desired characteristic of reduced velocity and direction or increased filtration performance, a general comparison of edf across mask designs is shown in figure 2 . the velocity-ratio related performance for nomask applied, edf = .984(.009), indicates the overall slowdown of particles due to turbulence all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 14, 2020. . https://doi.org/10.1101/2020.07.12.20152157 doi: medrxiv preprint and aerodynamics in an open air system. the large standard deviations represent the divergence between the responses for each exhalation breath level (visible in (online supplementary figures 6 -13 ) and also indicate that the interactions between multiple factors and the multivariate responses. in addition, the mean velocities for no-mask are, in some cases, lower than certain masks or fabrics which is related to a pms sensor's sampling rate limitation. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 14, 2020. the results from manova on the complete data set are reported in table 3 table 3 shows that the results using pillai's trace are also significant (in case the manova assumptions of homogeneity of variance-covariance were violated). therefore, the null hypothesis that masks or face coverings have no effect on exhalation dispersion or source control is rejected. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 14, 2020. the statistics of wilks lambda and pillai's trace (table 3) converge at η 2 = .996 and indicate that 99.6% of the variance of dependent variables are associated with exhalation breath levels of talking or coughing. it should also be noted that there were statistically significant interaction effects between fabric, mask design, breath levels and the combination of all three independent variables also reported in table 3 (fabric*mask design, fabric*breath level, mask design*breath level, fabric*mask design*breath level). in some cases, the between-subject effects were marginally significant (p-value closer to .05), however the vast majority of individual between-subject effects 30/35 (85.7%) are significant. a full multivariate analysis of variance and multivariate tests of between-subject effects and interactions are provided in (online supplementary table 2 and 3) . conclusively, this quantitative comparative effectiveness study establishes that the application of improvised non-medical grade mask designs or fabric combinations were statistically significant in reducing airborne dispersion of particles from exhalation as defined by direction, velocity, auc0 to 1 min, fei and edf. the statistically significant interaction effects between combinations of all primary factors and partial η 2 values further establish the strong correlation of outcomes to fabrics used, mask design, and exhalation breath levels. this foundational research offers an orthogonal but complimentary result to previous research on respiratory protection and personal protective equipment which exclusively looks at inhalation filtration and airflow pressure gradients that support respiration. when considering airborne dispersion control (also known as source control in some literature) it is important to understand the primary mechanisms that affect the dispersion. the field of filtration theory offers significant understanding with the primary mechanisms for the respiratory use case: interception, brownian diffusion, inertial impaction, and sieving or blocking filtration. [48] [49] [50] [51] since the fibers and fabric meshes are typically larger than small aerosols or infectious particles at 5µm diameters or smaller, the first three filtration mechanisms are most applicable to this study and other studies in the field of personal protective equipment (ppe). special emphasis is placed on inertial impaction and brownian diffusion to disrupt the velocity and direction of airborne dispersion. further discussion on filtration mechanisms is provided in (online supplementary appendix). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 14, 2020. . https://doi.org/10.1101/2020.07.12.20152157 doi: medrxiv preprint one observation from this study is that the effectiveness in dispersion varies greatly between the specific fabrics and mask design combinations. for example, the factors of fabric, mask design, and the interaction of fabric and exhalation breath level that have significant effect on fei, while other factors and interactions are not significant (online supplementary table 3) . this suggests that a fabric's dynamic characteristics such as pliability (i.e. conforms to the face for fit and coverage) and dynamic response to airflow force (i.e. stretch characteristics) have an effect on the overall filtration of exhaled particles. ad hoc test data of a stretch fabric commercially available mask is consistent with this statement (online supplementary table 5). further materials analysis and characterization is justified to fully understand this observation however it does emphasize that proper wearing of masks [52] [53] [54] is important for ppe usage as well as dispersion control. characterization of fabric thickness, fiber density and weave, as well as layering will also aid in establishing accurate predictor coefficients of a dispersion control linear regression model for specific fabrics and masks. the strength of this study is that it offers quantitative evidence on the effectiveness of nonmedical improvised masks for helping to establishing public health strategies or policies that encourage the wearing of masks or face coverings. fundamentally the effectiveness of nonpharmaceutical interventions (npi) can be increased by reducing exhalation particle dispersion and is especially important where infectious contaminants may exist in shared air spaces. an overall public health strategy must consider the additive effect of wearing masks and face coverings for inhalation filtration (ppe) and that of dispersion and source control. however, the strategy would need to account for the non-ideal performance of various fabrics and masks, where the ideal particle dispersion performance would offer 95% filtration efficiencies and dispersion contained to the user's body. combining this research with recent community sir modeling [55] can help provide significant insights to the public health strategy. to summarize, it would be of most benefit for all people in community settings to wear masks and get full effect of controlling exhalation particle dispersion to reduce transmission of highly infectious respiratory diseases such as covid-19. one limitation of this study is that it provides approximations of human exhalation using polydisperse nacl solution rather than actual exhalation. real human exhalation adds additional compositions of particles that can be smaller than 0.3µm in diameters, moisture, proteins, gases, and other bio material[56] so the longer term effectiveness of masks for source or dispersion control cannot be directly established from this data, however this study utilizes industry and niosh accepted proxy for testing respiratory barriers of nacl. another limitation was the pms sensor performance: measurement minimum of 0.3µm particles and a slower intake fan speed limited its ability to accurately measure all characteristics of fast moving particle clouds from that of no-mask applied. regardless, the sensor data and experiment design were sufficient to determine statistical conclusions on the effects of wearing masks and face coverings of different fabrics and designs. future works should consider using a large test chamber and more sensors to result in more accurate measurement of airborne dispersion and turbulent airflows. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 14, 2020. . https://doi.org/10.1101/2020.07. 12.20152157 doi: medrxiv preprint the results show that the application of various non-medical grade mask designs or fabric combinations were statistically significant in reducing airborne dispersion of particles from exhalation during coughing and talking as well as singing. however, the effectiveness varies greatly between the specific fabrics and mask designs used. the best overall performing mask design is a surgical style with internal non-woven layers, while the best overall fabric depends on a desired characteristic of reduced velocity, change in direction, or increased filtration performance. conclusively this study can aid in establishing public health strategies or policies that encourage the wearing of masks or face coverings to increase the effectiveness of nonpharmaceutical interventions (npi) especially where infectious contaminants may exist in shared air spaces. mr. edwards was the principal investigator and primary author, ms. widrick created the design of experiment and conducted the mathematical analysis, dr. potember assisted in conducting background research and guided the approach to experimentation and rigor of analysis, mr. gerschefske assisted in the laboratory configuration and testing. we extend our appreciation to the american red cross of southeastern colorado and el paso county public health for allowing the use of cpr training equipment and manikin to rapidly conduct this study. we also acknowledge mr. asher edwards for his contributions to the early code development for the data acquisition system, and ms. lydia edwards for providing continuous awareness of open news research and public vetting of concepts. 56 popov ta. human exhaled breath analysis. ann allergy asthma immunol 2011;106:451-6. doi:10.1016/j.anai.2011.02.016 figure 1 : examples of the measurements performed on the time-series data from all experiment runs for each of the four sensors and particle sizes of 0.3µm, 0.5µm. 1µm, 2.5µm, 5µm, 10µm maskdesign all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 14, 2020. . https://doi.org/10.1101/2020.07.12.20152157 doi: medrxiv preprint aerosol emission and superemission during human speech increase with voice loudness the airborne lifetime of small speech droplets and their potential importance in sars-cov-2 transmission the size distribution of droplets in the exhaled breath of healthy human subjects size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities turbulent gas clouds and respiratory pathogen emissions: potential implications for reducing transmission of covid-19 collection, particle sizing and detection of airborne viruses physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov-2 and covid-19: a systematic review and meta-analysis natural ventilation for infection control in health-care settings face mask use and control of respiratory virus transmission in households review of economic evaluations of mask and respirator use for protection against respiratory infection transmission national academies of sciences e. rapid expert consultation on the effectiveness of fabric masks for the covid-19 pandemic mask use, hand hygiene, and seasonal influenza-like illness among young adults: a randomized intervention trial respiratory syncytial virus (rsv) infection rate in personnel caring for children with rsv infections: routine isolation procedure vs routine procedure supplemented by use of masks and goggles trump shares tweet that says masks represent 'slavery and social death' -business insider mandatory masks aren't about safety, they're about social control the mask wearing debate is dividing america. and the messaging isn't getting any clearer assessment of fabric masks as alternatives to standard surgical masks in terms of particle filtration efficiency multilayer nonwoven fabrics for filtration of micron and submicron particles simple respiratory protection--evaluation of the filtration performance of cloth masks and common fabric materials against 20-1000 nm size particles aerosol filtration efficiency of common fabrics used in respiratory cloth masks evaluating the efficacy of cloth facemasks in reducing particulate matter exposure the ultimate guide to homemade face masks for coronavirus. smart air filters effectiveness of common fabrics to block aqueous aerosols of virus-like nanoparticles visualizing the effectiveness of face masks in obstructing respiratory jets covid-19 outbreak associated with air conditioning in restaurant no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity novel coronavirus (covid-19) pandemic: built environment considerations to reduce transmission towards aerodynamically equivalent covid19 1.5 m social distancing for walking and running how sneeze particles travel inside an airplane | popular science testing the efficacy of homemade masks: would they protect in an influenza pandemic respiratory source control using a surgical mask: an in vitro study utility of substandard face mask options for health care workers during the covid-19 pandemic to mask or not to mask: modeling the potential for face mask use by the general public to curtail the covid-19 pandemic dispersion and exposure to a cough-generated aerosol in a simulated medical examination room a cough aerosol simulator for the study of disease transmission by human cough-generated aerosols exhaled air dispersion during coughing with and without wearing a surgical or n95 mask the control of air flow during loud soprano singing national institute for occupational safety and health. teb-apr-stp-0059: determination of particulate filter efficiency level for n95 series filters against solid particulates for non-powered a primer on multivariate analysis of variance (manova) for behavioral scientists 7 robustness of anova and manova test procedures handbook of applied multivariate statistics and mathematical modeling 7 -multivariate analysis of variance and covariance information point: wilks' lambda filtration theory for granular beds determining impaction efficiencies of mist collection equipment ullmann's encyclopedia of industrial chemistry masks and coronavirus disease 2019 (covid-19) recommendation regarding the use of cloth face coverings, especially in areas of significant community-based transmission use of cloth face coverings to help slow the spread of covid-19 a modelling framework to assess the likely effectiveness of facemasks in combination with 'lock-down' in managing the covid-19 pandemic key: cord-274355-6hiutrct authors: satheesan, manoj kumar; mui, kwok wai; wong, ling tim title: a numerical study of ventilation strategies for infection risk mitigation in general inpatient wards date: 2020-02-22 journal: build simul doi: 10.1007/s12273-020-0623-4 sha: doc_id: 274355 cord_uid: 6hiutrct aerial dispersion of human exhaled microbial contaminants and subsequent contamination of surfaces is a potential route for infection transmission in hospitals. most general hospital wards have ventilation systems that drive air and thus contaminants from the patient areas towards the corridors. this study investigates the transport mechanism and deposition patterns of middle east respiratory syndrome coronavirus (mers-cov) within a typical six bedded general inpatient ward cubicle through numerical simulation. it demonstrates that both air change and exhaust airflow rates have significant effects on not only the airflow but also the particle distribution within a mechanically ventilated space. moreover, the location of an infected patient within the ward cubicle is crucial in determining the extent of infection risk to other ward occupants. hence, it is recommended to provide exhaust grilles in close proximity to a patient, preferably above each patient’s bed. to achieve infection prevention and control, high exhaust airflow rate is also suggested. regardless of the ventilation design, all patients and any surfaces within a ward cubicle should be regularly and thoroughly cleaned and disinfected to remove microbial contamination. the outcome of this study can serve as a source of reference for hospital management to better ventilation design strategies for mitigating the risk of infection. hospitals are designed to accommodate a large number of patients with varying degrees of disease severity. as inpatient care facilities such as general medical and surgical hospitals are used by patients, healthcare workers and visitors simultaneously, the susceptibility of these people to hospitalacquired infections (hais) or nosocomial infections is reasonably high (giannini et al. 2009 ). the largest nosocomial outbreak of severe acute respiratory syndrome (sars) in hong kong, china and the recent outbreak of middle east respiratory syndrome (mers) in south korean hospitals have had substantial morbidity and mortality (oh et al. 2018; tang et al. 2015) . the three widely known transmission routes of sars coronavirus (sars-cov) and influenza viruses are close contact, long-range airborne and fomite (xiao et al. 2018) . although close contact is generally regarded as the possible transmission route of mers coronavirus (mers-cov) (zumla et al. 2015) , studies have indicated that airborne and fomite are other possible modes of mers-cov transmission (kim et al. 2016; van doremalen et al. 2013) . there is also sufficient evidence to support that an association exists between ventilation strategies and dissemination of nosocomial pathogens in indoor environments . hence, it is essential to revise and update current ventilation design strategies to contain potential outbreaks caused by novel emerging viruses in the future. infections are explicitly considered in the ventilation requirements for healthcare facilities (li et al. 2015) . most general hospital wards have ventilation systems that drive air from the patient areas to the circulation areas. airborne pathogens can thus spread from a ward cubicle to the rest of the ward and lead to a potential for nosocomial outbreaks. although it is widely assumed that increasing the air change rate (ach) can reduce infection risks, it was shown that the risk of exposure to pathogens could increase with an increased ventilation rate under certain circumstances build simul https://doi.org/10.1007/s12273-020-0623-4 satheesan et al. / building simulation 2 (bolashikov et al. 2012; pantelic and tham 2013) . several studies have also emphasized that apart from ach, the design of a ventilation system is a determinant controlling contaminant flow paths (ghia et al. 2012; licina et al. 2015; memarzadeh and xu 2012; pantelic and tham 2013) . as there is a paucity of guidelines or strategies for general ward ventilation (beggs et al. 2008; chaudhury et al. 2005; roy and milton 2004) , a review of present nosocomial infection control practices for presumably low risk zones and unprotected areas such as a general ward is all-important (humphreys 2006; wan et al. 2007) . to get meaningful estimations of the transport and deposition of pathogens in a mechanically ventilated space, accurate prediction of airflow pattern is essential. with the advent of improved turbulence modelling and computing power, the application of computational fluid dynamics (cfd) to indoor environment simulation has been on an increasing trend (nielsen 2015) . under varied operating conditions in an indoor environment with high temporal and spatial resolution, cfd numerical simulation techniques provide insights into the overall airflow and bioaerosol distribution (chao et al. 2008; . in fact, cfd techniques have been used to estimate ventilation effectiveness for contaminant removal in healthcare facilities. as the number of research studies on ventilation systems for general inpatient wards with respect to air change rate and exhaust airflow rate is limited, this study evaluates the combined impacts of these two parameters on the airflow as well as infection risk distributions of droplet nuclei of size 0.167 μm (i.e. mers-cov) within an air-conditioned general inpatient ward cubicle. moreover, a simple yet cost-effective ventilation system design that can minimize the risk of infection in an existing hospital ward is proposed. the risk of infection increases with increasing pathogen exposure. even a single pathogen can initiate the onset of an infectious disease in a susceptible person (nicas et al. 2004; wells 1955) . based on the bioaerosol transport and deposition mechanism, this study classified the types of infection transmission in a general inpatient ward environment into two categories: (1) cross infection within a ward cubicle; and (2) infection from a ward cubicle to the corridor. the deposition of particles in patients due to the exhalation of pathogens by other patients accommodated in the same ward can lead to cross infection. the severity of cross infection among patients depends on the location of each patient and the overall airflow distribution pattern within the ward environment. exposure to infection due to the inhalation or deposition of particles expelled by other patients through sneezing (infectors) can be estimated for a patient i (receptor exposure): where e i is the fractional exposure count for patient i, e j is the fractional emission from patient j and n is the total number of patients. based on this expression, the locations with the maximum and minimum risks of cross infection can be determined for the patients within the same ward cubicle. general inpatient wards in hospitals are recommended to have a relative humidity between 30% and 60% (ashrae 2013a). as mers-cov can survive for up to 72 hours on plastic or steel surfaces at a room temperature of 20 °c and a humidity of 40% (oh et al. 2018 ) and a portion of the exhaled particles will deposit to surfaces such as the ceiling, floor and walls in the ward, this study also took infection through surface contamination into account. based on the three deposition ratios expressed in eq. (2), namely wall deposition ratio r w , ceiling deposition ratio r c and floor deposition ratio r f , the infection transmission through surface contamination under all ventilation scenarios considered in this study can be estimated. where n s is the number of particles expelled by an individual patient through sneezing, and n w , n c and n f are the numbers of particles from the sneezing deposited onto the walls, ceiling and floor respectively. the dispersion of particles that were expelled by sneezing from an infected patient in a ward cubicle to the corridor was taken as the infection from a ward cubicle to the corridor. it was noted that these particles could also spread to adjacent ward spaces connected to the corridor (chen et al. 2011 ). the number of particles being exhausted to the corridor n e can be estimated based on the exhalation rates of individual supine patients in their respective beds. using the exhausted ratio r e for individual patients as expressed in eq. (3), locations of the patients who contribute the most and the least to the spread of infection to the corridor can be determined. e e s n r n = (3) a typical semi enclosed six bedded general inpatient ward cubicle with dimensions 7.5 m (l) × 6 m (w) × 2.7 m (h) and a between-bed spacing of 1 m as illustrated in fig. 1 was used in this study (li et al. 2005; yu et al. 2017 ). the cubicle was mechanically ventilated (with a positive pressure towards the corridor) and accommodated six supine patients. the supply air, i.e. mixed air estimated based on the room volume and air change rates (3 h −1 -13 h −1 ), was delivered to the cubicle through four ceiling mounted diffusers. in the base case as shown in fig. 1(a) , the supply air and ward air exhausted to the corridor were set to be equal for all air change rates. for exhausting 10% and 50% of supply air (i.e. ea = 10% and ea = 50%), local exhaust grilles (grille size 0.5 m × 0.2 m) were installed as depicted in fig. 1 after the extraction by exhaust grilles, the rest of the cubicle air was exhausted towards the corridor. the airflow distribution and the transport mechanisms of bioaerosols in the ward cubicle were investigated using a finite volume based cfd code (ansys fluent 13.0). the numerical simulation model consists of a continuum phase (air) and a discrete phase (droplet nuclei). in this study, the governing equations of continuity, momentum and energy for the continuum phase were based on an eulerian framework, and the discrete phase was modelled by a lagrangian framework (wong et al. 2010 ). the threedimensional airflow was modelled as a steady-state incompressible turbulent flow. cfd possess miscellaneous number of turbulence models, although it is very difficult to pinpoint one turbulence model that outperforms others for all class of problems. hence, the turbulence model selection is a compromise based on factors such as flow physics involved, established practice for predicting a particular set of problem, computational resources, accuracy level and simulation time (gao and niu 2005) . numerous studies on turbulent indoor airflow have been carried out through reynolds-averaged navier stokes (rans) simulations, whereas for some case studies large eddy scale simulations (les) have been preferred to provide accurate prediction of flow field variables. however, les demands much higher grid requirements as well as computational time compared to rans, which makes rans widely used (blocken 2018) . the reynolds-averaged navier stokes (rans) equation greatly reduces the complexity involved in the simulation of turbulent flows. the equations have time averaged flow field variables which would eliminate the turbulent fluctuations. although, this simplification leads to the creation of additional reynolds stress tensors which are unknowns within the equation and leads to closure issues. the eddy viscosity turbulence modelling provides closure to equations and the most preferred eddy viscosity turbulence model to simulate the indoor airflow distribution is the renormalization group (rng) k-ε. the rng k-ε model, was chosen to model the air turbulence in this study, as it offers better accuracy, stability and computing efficiency for low reynolds number as well as near wall flows (chen 1995; zhang and chen 2006; ). the diffuser inlets were defined as velocity-inlet, while corridor and exhaust grilles were treated as outflow boundary condition. ansys fluent treats outflow boundaries as having zero diffusion flux for all flow variables as well as it incorporates an overall mass balance correction. furthermore, outflow with flow rate weighting option enables the user to have multiple outflow boundaries with fractional flow rate through each boundary (ansys 2010). a second-order upwind scheme was applied to discretize the governing equations while the simple algorithm was used for the pressurevelocity coupling in the continuum phase. the metabolic rate of a reclining patient was assumed to be 0.8 met (ashrae 2013b) and half of the heat (23.3 w·m −2 ) from each patient is assumed to be transferred through convection (qian and li 2010; hang et al. 2014) . a constant heat flux thermal boundary condition was uniformly imposed on the whole surfaces of the supine patients (shen et al. 2013) . apart from walls of heat sources (patients), all other walls were treated as adiabatic. smooth no-slip condition is applied at all walls. in order to reduce modelling complexity arising from density differences due to temperature gradients, the boussinesq approximation was employed (zeytounian 2003) . the computational domain of inpatient ward was split in to multiple fluid zones. icem-cfd 13.0 was utilized to generate hexahedral mesh for these individual computational cell zones. the individual mesh files are then merged together using the tmerge filter functionality. in tmerge, before the individual meshes are combined together to one mesh file, the required scaling factor, translation distance and rotation information of the meshes is specified. with the existence of non-identical mesh node locations as shown in fig. 2 along the boundaries of the individual cell zones of the computational domain, non-conformal interfaces are established between individual cell zones. these interfaces connect each cell zones by transferring fluxes from one mesh to another (ansys 2010). the first cell height from wall is kept at a distance of 0.001 m and grid spacing of 1.2 is maintained throughout the domain. the near-wall mesh was made fine enough to resolve the viscous sublayer (y + < 5) and the near-wall modelling was done through the enhanced wall treatment approach. the three grid systems, namely 1002k (system1), 3202k (system 2) and 5110k (system 3), fig. 2 non-identical mesh nodes along the boundary of two cell zones were created for the grid convergence study and airflow simulations were performed on each grid. to analyse the convergence of the three grid systems, the grid convergence index (gci) concept was applied (roache 1998; wong et al. 2010) . the root mean square of the relative error (e rms ) for the fluid flow mean velocities (u) detected at 100 points along a vertical line in the centre of the ward cubicle was used to determine the gcis for the grid systems. the gci is calculated by: in the above equation, r is the grid refinement factor that is calculated as the ratio of the control volumes of fine and coarse grid systems, p is the order of the discretization method used, f s is the safety factor and e rms is determined by: using system 1 as a reference, the gcis for systems 2 and 3 were 3.11% and 3.40% respectively. as system 2 was adequate for studying the fluid flow characteristics, it was taken for further investigations by considering computational time as well as solution accuracy. the trajectories of individual particles were modelled using the lagrangian framework and the modelling assumptions made are listed below (tian et al. 2009; zhao et al. 2004 ):  the air-particle as well as particle-particle heat and mass transfers were neglected.  a particle would not rebound when hitting a surface such as wall, ceiling and floor.  particle coagulation was neglected in the deposition process.  all particles were modelled as spherical in shape. the droplets exhaled through exhalation activities such as sneezing will shrink in size due to evaporation within a short period of time (< 0.1 s) (xie et al. 2007 ). their dried out residuals, the droplet nuclei, may be carrying pathogens (wells 1955) . in this study, a small percentage (< 10%) of the total virus laden droplets from a vigorous sneeze was assumed. it was proved that with such a small percentage, virus particles will not form clusters (duguid 1946; gralton et al. 2011) . for simplicity's sake, droplet nuclei are referred to as particles in this article. the lagrangian particle tracking calculates the discrete trajectories of individual particles in the fluid flow separately by solving the following particle motion equation: where u a is the fluid velocity (m·s −1 ), u b is the particle velocity (m·s −1 ), μ is the molecular viscosity of air (kg·m −1 ·s −1 ), ρ a is the density of air (kg·m −3 ), ρ b is the particle density (kg·m −3 ), d b is the particle diameter (m), re is the particle reynolds number, c d is the drag coefficient, g a is the gravitational acceleration and f x is the auxiliary forces acting on the particles. the particle reynolds number is defined by, the drag coefficient c d for the bioaerosol particles is defined by, the drag constant k d for the bioaerosol particles as expressed in eq. (9) is given by, the above equations were solved in cfd simulations to determine the transport mechanisms of bioaerosol particles in a lagrangian scheme. the validity of eqs. (9) and (10) was established for a range of particles with equivalent bioaerosol diameters (d b ) from 0.69 μm to 6.9 μm and further examined for particles with d b as low as 0.054 μm (wong et al. 2015) . apart from the drag force, forces that can influence particle motions include basset force, magnus force, virtual mass force, brownian force and saffman lift force. although the magnitudes of these forces are greatly influenced by the fluid flow conditions and particle properties, a few of these forces are small enough to be neglected in some analyses (zhao et al. 2004 ). due to the particle size and non-isothermal flow conditions in this study, brownian, thermophorectic and saffman lift forces were taken into consideration for predicting the particle motion trajectories. the dispersion of particles as a consequence of turbulence in the flow field can be tracked through stochastic tracking methods. the discrete random walk (drw) model, a popular approach that takes velocity fluctuations into account, was employed in this study (lai et al. 2012) . further details for the cfd simulations are summarized in table 1 . the overall airflow distribution pattern based on ventilation strategies can have a great impact on the particle distribution within the space in a hospital ward. figure 3 depicts the air velocity distribution and velocity vector plot across a horizontal plane located at y = 1.0 m for the base case, i.e. a typical ward cubicle without exhaust grilles. in fig. 3(a) , stagnant air with velocity less than 0.05 m·s −1 is observed near wall 2 and fig. 3(b) depicts that the overall airflow pattern in the cubicle is directed towards the ward corridor. these results are consistent with those presented in our previous case study (yu et al. 2017 ). due to the existence of obstructions such as patients and beds, several eddies can be seen within the ward cubicle. figures 4 and 5 illustrate the temperature distribution as well as velocity vector plot across a vertical plane located at z = 1.625 m for an air change rate of 6 h −1 and 9 h −1 with ea = 50% respectively. as the thermal manikins cause thermal plumes, the effect of thermal plumes on the ward airflow pattern can be clearly seen from the vertical airflow distribution. there is an upward airflow (towards the ward ceiling) that returns to the floor level along the walls. recirculation zones, which may occur when the cold supply air from the diffusers interacts with the upward airflow caused by the thermal plumes, are observed. these zones can enhance the mixing of air within the ward space. moreover, the suction provided by the local exhaust grilles tends to alter the airflow pattern around a patient and thus can help to remove airborne contaminants in the immediate vicinity of the patient. in the base case, almost half of the virus particles exhaled from a patient's mouth deposited onto the patient's body and bed. according to fig. 6 , there were considerable amounts of virus particles on different cubicle surfaces including walls, floor and ceiling. a maximum r c (≈ 0.26), resulted from the supine patients and their exhaled air velocities, can be seen at 3 h −1 in fig. 6(a) . it can also be seen in the figure that r c decreases as the air change rate increases (e.g. r c ≈ 0.17 at 6 h −1 (a 34% decrease) and r c ≈ 0.11 at 13 h −1 (a 57% decrease)). in fact, the air supplied through the diffusers has a higher momentum with an increased air change rate and it moves the particles away from the ceiling to other spaces within the cubicle. as depicted in figs. 6(b) and (c), r w and r f were respectively greater than 0.07 and 0.03 in the base case for all air change rates. as shown in fig. 7(a) , patients in beds located at 1.625 m away from the corridor (i.e. patients 1 and 2) were most vulnerable to cross infection (with exposure risk (e) > 0.05) while those at 5.875 m away (i.e. patients 5 and 6) were least (with e < 0.025). with an increase in air change rate, a significant reduction in infection risk was observed for patients located farther away from the corridor. this can be explained by the general airflow patterns as illustrated in fig. 3 . on average, the infection risk level of patients 5 and 6 was only half of that of patients 1 and 2 for all air change rates. figures 4 and 5 demonstrate that the local exhaust grilles not only facilitated the removal of a portion of exhaled virus particles but also tended to increase the particle deposition in the source patient's body and thereby reduced the residual viral load present in the air. as exhibited in fig. 6(a) , the recorded values of r c at 3 h −1 were approximately 0.12 and 0.10 for ea = 10% and ea = 50% (i.e. decreases of 53% and 61%; comparing with the base case) respectively. for all other cases shown in the figure, similarly, r c decreased as the air change rate increased. according to figs. 6(b) and (c), wall and floor deposition ratios were also significantly reduced with ea = 10% and 50% (r w < 0.05 and r f < 0.03). however, deposition of particles was observed in all scenarios. as the deposition is random in nature and often happens irrespective of the ventilation system design, it highlights the importance of regular and proper ward housekeeping. furthermore, the randomness associated with particle deposition rates (r w , r c , and r f ) under different air change rate conditions can be attributed to the asymmetric airflow distribution patterns and locations of the infected patients. figures 7(b) and (c) indicate that the installation of exhaust grilles in close proximity to each patient can help prevent particle migration from an infected patient to different locations in the ward and significantly reduce individual patient exposures (e < 0.05). the results show that the location of an infected patient, exhaust airflow rates and air change rates within the cubicle work together influencing the mechanism of viral spread. the pathogen spatial distribution is dependent on the ward layout, ventilation strategy and location of the source patient. it can be observed in fig. 8(a) that exhausted ratio r e increases with air change rate in the base case. the maximum r e was recorded for patients 1 and 2. from r e < 0.05 at 3 h −1 to r e > 0.25 at 13 h −1 , an abrupt increase in risk (80%) was noted for these patients. at higher air change rates, specifically at 13 h −1 , the number of particles reaching the corridor due to the exhalation activities of patients 5 and 6 located at the rear end of the ward (i.e. 5.875 m away from corridor) was quite high (0.05 < r e < 0.1). this suggests that the use of a high air change rate such as 13 h −1 can put the users of the corridor and its connected amenities at high risk of exposure to pathogens and facilitate infectious disease outbreaks in the whole healthcare facility. as shown in figs. 8(b) and (c), the installation of exhaust grilles considerably reduced the risk of infection transmission. the exhaust grilles lowered the r e values of patients 1 and 2 at 13 h −1 to 0.15 with ea = 10% (i.e. a decrease of 40%; comparing with the base case) and < 0.03 with ea = 50% (i.e. a decrease of 88%; comparing with the base case). specifically, the supine patients at the rear end of the ward cubicle (i.e. patients 5 and 6) contributed the least to the spread of infection to the corridor (r e < 0.006). as the local exhaust airflow rate proved effective in reducing the risk of infection transmission, both the location and airflow rate of an exhaust grille are crucial factors for designing infection control strategies. this study investigated the transport mechanism and deposition patterns of mers-cov within an air-conditioned general inpatient ward cubicle. it demonstrated that both air change and exhaust airflow rates have significant effects on not only the airflow but also the particle distribution within a mechanically ventilated space. moreover, the location of an infected patient within the ward cubicle is crucial in determining the extent of infection risk to other ward occupants. hence, it is recommended to provide exhaust grilles in close proximity to a patient, preferably above each patient's bed. to achieve infection prevention and control, high exhaust airflow rate is also suggested. regardless of the ventilation design, all patients and any surfaces within a ward cubicle should be regularly and thoroughly cleaned and disinfected to remove microbial contamination. installation of uvgi lamps in the ward space is recommended to further enhance the risk mitigation strategies. the outcome of this study can serve as a source of reference for hospital management to better ventilation design strategies for mitigating the risk of infection. ansys fluent 13.0 documentation ashrae standard. ventilation for health care facilities ashrae fundamentals si handbook the ventilation of multiple-bed hospital wards: review and analysis les over rans in building simulation for outdoor and indoor applications: a foregone conclusion? building simulation exposure of health care workers and occupants to coughed airborne pathogens in a double-bed hospital patient room with overhead mixing ventilation transport and removal of expiratory droplets in hospital ward environment advantages and disadvantages of single-versus multiple-occupancy rooms in acute care environments: a review and analysis of the literature comparison of different k-ε models for indoor air flow computations role of two-way airflow owing to temperature difference in severe acute respiratory syndrome transmission: revisiting the largest nosocomial severe acute respiratory syndrome outbreak in hong kong the size and the duration of air-carriage of respiratory droplets and droplet-nuclei cfd study of the thermal environment around a human body: a review assessment of health-care worker exposure to pandemic flu in hospital rooms are toilet seats a vector for transmission of methicillin-resistant staphylococcus aureus? the role of particle size in aerosolised pathogen transmission: a review the influence of human walking on the flow and airborne transmission in a six-bed isolation room: tracer gas simulation overcrowding, understaffing and infection in hospitals extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards an experimental study of bioaerosol (1-10 μm) deposition in a ventilated chamber role of air distribution in sars transmission during the largest nosocomial outbreak in hong kong role of ventilation in airborne transmission of infectious agents in the built environment-a multidisciplinary systematic review engineering control of respiratory infection and low-energy design of healthcare facilities human convection flow in spaces with and without ventilation: personal exposure to floor-released particles and cough-released droplets role of air changes per hour (ach) in possible transmission of airborne infections the infectious dose of variola (smallpox) virus fifty years of cfd for room air distribution middle east respiratory syndrome: what we learned from the 2015 outbreak in the republic of korea adequacy of air change rate as the sole indicator of an air distribution system's effectiveness to mitigate airborne infectious disease transmission caused by a cough release in the room with overhead mixing ventilation: a case study removal of exhaled particles by ventilation and deposition in a multibed airborne infection isolation room verification of codes and calculations airborne transmission of communicable infection-the elusive pathway cfd study on the transmission of indoor pollutants under personalized ventilation aerosol-transmitted infections-a new consideration for public health and infection control teams numerical investigation of indoor aerosol particle dispersion under stratum ventilation and under displacement ventilation stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions dispersion of expiratory droplets in a general hospital ward with ceiling mixing type mechanical ventilation system airborne contagion and air hygiene. an ecological study of droplet infections an experimental and numerical study on deposition of bioaerosols in a scaled chamber drag constants for common indoor bioaerosols a study of the probable transmission routes of mers-cov during the first hospital outbreak in the republic of how far droplets can move in indoor environments-revisiting the wells evaporationfalling curve ventilation of general hospital wards for mitigating infection risks of three kinds of viruses including middle east respiratory syndrome coronavirus joseph boussinesq and his approximation: a contemporary view experimental measurements and numerical simulations of particle transport and distribution in ventilated rooms comparison of the eulerian and lagrangian methods for predicting particle transport in enclosed spaces evaluation of various turbulence models in predicting airflow and turbulence in enclosed environments by cfd: part 2-comparison with experimental data from literature comparison of indoor aerosol particle concentration and deposition in different ventilated rooms by numerical method middle east respiratory syndrome this work was partially supported by the research grants council of hksar and the hong kong polytechnic university (project no. 15208817e). key: cord-287708-0qvwjejv authors: gera, tamás; nagy, eszter; smausz, tamás; budai, judit; ajtai, tibor; kun-szabó, fruzsina; homik, zsolt; kopniczky, judit; bozóki, zoltán; szabó-révész, piroska; ambrus, rita; hopp, béla title: application of pulsed laser ablation (pla) for the size reduction of non-steroidal anti-inflammatory drugs (nsaids) date: 2020-09-25 journal: sci rep doi: 10.1038/s41598-020-72865-z sha: doc_id: 287708 cord_uid: 0qvwjejv we studied the application of pulsed laser ablation (pla) for particle size reduction in non-steroidal anti-inflammatory drugs (nsaids). grinding of the poorly water-soluble nsaid crystallites can considerably increase their solubility and bioavailability, thereby the necessary doses can be reduced significantly. we used tablets of ibuprofen, niflumic acid and meloxicam as targets. nanosecond laser pulses were applied at various wavelengths (krf excimer laser, λ = 248 nm, fwhm = 18 ns and nd:yag laser, λ(1) = 532 nm/λ(2) = 1064 nm, fwhm = 6 ns) and at various fluences. ftir and raman spectra showed that the chemical compositions of the drugs had not changed during ablation at 532 nm and 1064 nm laser wavelengths. the size distribution of the ablated products was established using two types of particle size analyzers (smps and opc) having complementary measuring ranges. the mean size of the drug crystallites decreased from the initial 30–80 µm to the submicron to nanometer range. for a better understanding of the ablation mechanism we made several investigations (sem, ellipsometry, fast photography) and some model calculations. we have established that pla offers a chemical-free and simple method for the size reduction of poorly water-soluble drugs and a possible new way for pharmaceutical drug preformulation for nasal administration. | (2020) 10:15806 | https://doi.org/10.1038/s41598-020-72865-z www.nature.com/scientificreports/ any chemical decomposition. in recent years, researchers have also started to use lasers for particle size reduction in drugs in liquid environment (plal) [18] [19] [20] [21] [22] [23] . in this work, three nsaids (ibuprofen, meloxicam and niflumic acid) with different chemical structures but similar solubility, dissociation constant, particle size and crystallinity were selected as targets to study the effect of a high-energy pulsed laser beam on the chemical degradation and particle size distribution of the ablated drug particles. being selective cyclooxygenase (cox-2) inhibitors, these model drugs are used in acute pain therapy where a basic requirement is rapid absorption through the gastric mucosa 24 . since these drugs have a weak acidic character, their solubility in gastric juice (ph = 1.2) is very poor. on the other hand, ablated particles dissolve faster and can produce rapid analgesic effect. our aim was to study the validity of the pla method for particle size reduction in different drugs with low dissolution rates, in ambient gas at normal pressure. ibuprofen, meloxicam and niflumic acid are frequently used, poorly water-soluble nsaids, representing three different molecular derivatives. we also wanted to explore the mechanism of pla when drug targets with different thermal and optical properties are ablated under various experimental conditions. to cover the uv-vis-ir regime with ablating laser wavelengths, a krf excimer laser (λ = 248 nm) and a nd:yag laser system (λ 1 = 1064 nm, λ 2 = 532 nm) were used. we adjusted laser fluences at each wavelength to the optical and mechanical properties of the target materials. ablated products were collected at normal ambient pressure using n 2 gas flow. we studied the chemical composition and particle size distribution of the ablated particles, the optical absorption of the target drugs and made fast photography measurements too. our motivation was to show that pla, a simple and chemical-free particle grinding method, has a great potential in pharmaceutics. laser ablation produced drug particles can be preferable during the preparation of medicines for per os, nasal and pulmonary drug administration. pulmonary administration of anti-inflammatory drugs like meloxicam or ibuprofen can be especially significant in the treatment of pneumonia or severe acute respiratory syndrome caused by viral infections (e.g. coronavirus). it has been shown that only sub-micrometer size particles can get to the lower lung area (alveolar region) 25 . therefore, size-reduced drug particles produced by pla may form the basis of a new type of pulmonary drug formulation for the fast and effective treatment of the hard-to-reach alveolar region of the lung. ibuprofen. ibuprofen (α-methyl-4-(isobutyl) phenylacetic acid) was obtained from sigma-aldrich ltd., (saint louis, missouri, usa). it is a white powder with a particle size of 15.3 μm (d(0.5)). nsaid classification: propionic acid derivatives. thermal properties: melting point t m = 75-77 °c; boiling point t b = 157 °c; degradation temperature t dec = 230 to 250 °c. meloxicam. meloxicam(4-hydroxy-2-methyl-n-(5-methyl-2-thiazolyl)-2h-benzothiazine-3-car-boxamide-1,1-dioxide) was obtained from egis ltd., (budapest, hungary). it is a yellow powder with a particle size of 3.78 μm (d(0.5)), 100% crystalline. nsaid classification: enolic acid (oxicam) derivatives. thermal properties: melting point and decomposition temperature are the same, t m = t dec = 254 °c. niflumic acid. conventional niflumic acid (nif) (2-[[3-(trifluoromethyl)phenyl] amino] -3-pyridinecarboxylic acid) was purchased from g. richter pharmaceutical factory, budapest, hungary. the particle size is 18.85 μm (d(0.5)). nsaid classification: anthranilic acid derivatives (fenamates). thermal properties: melting point and decomposition temperature are the same t m = t dec = 203 °c. pulsed laser ablation (pla) of drug tablets at different laser wavelengths. we applied three different laser wavelengths for pulsed laser ablation (pla) of various drug tablets. for uv irradiation a krf excimer laser (llg twinamp, fwhm = 18 ns, λ = 248 nm, f = 10 hz) was used, while for vis and ir ablation we applied the first and second harmonics of a nd:yag laser (quantel, fwhm = 6 ns, λ = 532 nm/1064 nm, f = 10 hz), respectively. we varied the number of laser pulses and adjusted the fluence from 1.5 to 12 j cm −2 . in order to achieve a significant ablation yield, we used fluences slightly above the ablation threshold. the highest applied fluence was limited by the mechanical fracture of the target tablets. the tablets were produced from commercially available drug powders using a hydraulic compactor at 175 mpa pressure. our experimental setup is shown in fig. 1 . the tablets were placed on a rotating sample holder at the bottom of the reaction chamber. the laser beam was focused on the target surface at a grazing angle of 45° by a fused silica (uv) and an n-bk7 (vis-ir) fused silica plano convex lens (focal length = 17.5 cm). to be able to direct and control the ablated particles, we created a flow field of n 2 gas along the chamber. a constant flow rate (0.3-2 l/ min) was maintained by a t-element at the entrance of the gas jet. the ablated particles were either collected for chemical analysis on a filter (pore size ~ 1 μm, merck millipore ltd. omnipore membrane filter), or they directly entered the particle size analyzer at the exit of the gas stream. theoretically, the pore size of the membrane filter is 1 μm, yet smaller elements can also be collected as the pores fill up with particles reaching the filter first. chemical composition characterization methods. fourier transformed infrared spectroscopy (ftir) . the preservation of the medical effect is an essential requirement during particle size reduction in pharmaceutical substances. to ensure that the chemical composition of the ablated products is identical with that of the initial drugs, the ablated particles were analyzed by ftir spectroscopy (thermo nicolet avatar 330, labx midland, on, canada). the particles were collected from the filters and mixed with kbr powder for pellet formation. ftir spectra were recorded in the 4000-400 cm −1 range, at a resolution of 4 cm −1 , using the average of 128 scans. particle size analysis. pla provides an efficient way for grinding; however, the size distribution of the ablated particles spans a wide range, wherefore we used two types of size analyzers, having complementary, slightly overlapping measuring ranges. scanning mobility particle sizer (smps). we investigated the particle size distribution in the 10-800 nm range with smps (grimm system, aerosol technik, germany, type smps + c). we set the scanning time to 7 min and repeated the measurements three times. the gas flow was 0.3 l/min during the scans. optical particle counter (opc). to establish if particles larger than 800 nm were also produced, we used an opc (grimm system, aerosol technik, germany, type 1.108). opc can reliably measure particles in the 400 nm to 10 μm size range. we set the scanning time to 4 min and took at least 4 scans during each measurement. the gas flow in the equipment was 1 l/min. to better understand the particle formation mechanism during ablation, we investigated the laser treated tablet surfaces, the morphology of the laser irradiated areas and the backscattered particles nearby using scanning electron microscopy (hitachi s-4700). prior to imaging, the samples were sputter-coated with gold (bio-rad sc 502). sem images were recorded at a magnification of 100 ×. ellipsometry. the optical absorbance of the target material plays a key role in laser ablation. since there is no relevant and reliable information about the optical absorbance ([α] = 1/nm) of the studied drugs in the 400-1100 nm wavelength range, we determined the relevant values from ellipsometric data. ellipsometric measurements were performed at different points on the tablets using a woollam m2000f rotating compensator ellipsometer. data were collected at 75° angle of incidence, applying focusing optics. the ellipsometric data measured in the 250-1000 nm wavelength range could be directly transformed to complex refractive index values, from which the absorption coefficients were determined according to the α = 4*π*κ/λ expression. fast photography of the ablation process. we also set up a commonly used pump and probe system ( fig. 2) for the visual observation of the ablation process. the pump laser was the same nd:yag laser (quantel, fwhm = 6 ns, λ = 532 nm/1064 nm) as for the ablation experiments. as probe lasers, we built two types of nitrogen laser induced dye lasers corresponding to the ablating wavelengths. in case of λ ablating = 1064 nm we used coumarin 153 as a laser active material in the dye laser (λ = 545 nm, fwhm = 1 ns), while in case of λ ablating = 532 nm we had to change the laser active dye to rhodamine 6g (λ = 590 nm, fwhm = 1 ns) in order to avoid an overlap between the pump and probe signals. the dye laser beams were transported to the experimental field by an optical fiber and collimated with an n-bk7 focusing lens (f = 15 cm). a ccd camera (the imaging infrared spectroscopy (ftir) of ablated drug particles. ftir spectra obtained from the collected particles are shown in fig. 3a . some contamination of the sample was inevitable while the collected particles were scraped or peeled off the filter. therefore, we subtracted the ftir spectrum of pure millipore filter as background spectrum. the two peaks between 2250 and 2500 cm −1 are related to the h 2 o and co 2 content of the samples and give no relevant information for this investigation. ftir spectra of ibuprofen particles generated at different wavelengths and fluences are shown in fig. 3 . it can be seen that all laser fluences applied at the uv wavelength (248 nm) caused significant chemical damage to the particles. however, the persisting characteristic peaks of ibuprofen [e.g. 1720 cm −1 (h-bonded c=o); 1231 cm −1 (h-bonded co-h); 780 cm −1 (c=o)] 26 in the ftir spectra indicate that no chemical changes occurred at vis (532 nm) and ir (1064 nm) wavelengths. we had similar observations in the case of niflumic acid [e.g. 1668 cm −1 (c-o); 1614 cm −1 (benzene ring); 1428 cm −1 (c=o/oh); 1110 cm −1 (c-f)] 27,28 and meloxicam [e.g. 1620 cm −1 (n-h); 1550 cm −1 (thiazole ring); 1346 and 1264 cm −1 (sulfone)] 29, 30 (fig. 4) . during ir ablation of niflumic acid, 4.5 j cm −2 and 6 j cm −2 laser fluences yielded a sufficient amount of particles for ftir analysis. since we wanted to avoid chemical modification of the pharmaceutical compounds, we used only vis and ir wavelengths for further investigations. www.nature.com/scientificreports/ raman spectroscopy of generated drug particles. we recorded several spectra at different places from each ablation product to obtain information about their homogeneity. for simplicity, in fig. 5 , only those spectra are compared with the original pharmaceuticals' spectra that were recorded from the particles ablated by the highest laser fluences (i.e., may have suffered the most severe chemical changes) at both wavelengths. we marked some characteristic bands of ibuprofen (477, 833 and 1608 cm −1 ) (fig. 5a) , niflumic acid (999, 1210 and 1605 cm −1 ) (fig. 5b) and meloxicam (1302, 1540 and 1595 cm −1 ) (fig. 5c) , and found no discrepancies in the spectra of the original drugs and their ablation products produced at 532 and 1064 nm. particle size analysis. using both smps and opc, we investigated the size distribution of the ablated particles in a very wide range, from 10 nm to 10 μm. for each fluence, the repetition rates of the laser shots were adjusted to prevent the suction tube from getting clogged by ablated particles. during evaluation, we normalized the particle numbers to 1 hz repetition rate for data comparison. size distribution (measured by smps) of ibuprofen particles produced by laser ablation at 532 nm and 1064 nm wavelengths can be seen in fig. 6 . the mode values in both distributions were below 100 nm. the ablation yield of niflumic acid was considerably higher than for ibuprofen, especially at 532 nm (fig. 7) . the broadest size distributions with the highest mode values were obtained for the ablated meloxicam particles (fig. 8) ; the full widths at half maximums (fwhms) of the distributions ranged from 300 to 400 nm and the mode values for higher fluences exceeded 200 nm. we fitted one or more lognormal curves to the measurements. in case of high ablation yields data could be fitted to a single curve, while we had to cumulate two or more curves for small amounts of ablated particles. www.nature.com/scientificreports/ all measurements above were also repeated with the opc system to see if particles larger than 800 nm were also produced. since opc uses a higher gas flow than smps, the number of particles had to be normalized to the flow rate of smps and 1 hz repetition rate. it must be noted that the two systems give different size values due to the different detection mechanisms (smps-by electrical mobility; opc-by optical scattering). hence the differences between them are minor and the values are mainly the same 31 . we did not find many particles larger than 800 nm in either case. figure 9 shows the particle size distributions obtained by both smps and opc for the ablation of niflumic acid. we chose this plot to demonstrate a common overlap of the distributions. the overlaps are the same for the other ablating parameters too. the main experimental results are summarized in table 1 . there is no sign of clear relationship between laser fluences and size distributions in the investigated fluence range. however, we can establish that the number of ablated drug particles grows with the applied fluence at any wavelength. the growth in ablated particles can clearly be seen at λ = 532 nm ablation. in case of ibuprofen and niflumic acid, the most abundant particles are smaller at ir ablation than at vis with equivalent fluences. in the case of meloxicam, there were no overlaps in the fluence values due to different ablating outcomes (thresholds, cracks). nevertheless, we can still claim that at higher laser fluences the modes of meloxicam particle size distributions belong to the same size range for ir and vis wavelengths. it can be clearly stated that in any case the efficiency of ablation (i.e. the number of ablated particles) is the highest for meloxicam, slightly lower for niflumic acid, while it is the lowest for ibuprofen. sem investigation of ablated surfaces. sem images taken from the intact and ablated drug tablet surfaces are shown in fig. 10 . laser irradiations at vis and ir wavelengths resulted in similar surface structures: ellipsometry. absorption spectra of the investigated drugs were recorded in a wide optical range, from ultraviolet to near infrared. the smallest absorption coefficients were obtained in the ir, while the biggest ones in the uv range (fig. 11) . the increased absorption in the uv is most noticeable for meloxicam and niflumic acid. this is in accordance with our observation that drug molecules degrade during ablation by uv light. 32 . this wavelength falls outside our measurement range (260-1000 nm), therefore we could not detect it. we estimated the laser induced temperature rise at different depths in the target drugs by simple model calculations in which we varied the laser wavelength and fluence for each drug. ignoring heat conduction, we applied the beer-lambert law (eq. 1) to calculate the temperature change at the surface and at the penetration depths: where α is the wavelength dependent absorption coefficient, x is the distance from the surface, f is the applied fluence of the laser beam, ρ is the density and c is the specific heat of the target material. we used the mean specific heat values between room temperature and the decomposition temperatures of the drugs in the absence of reliable data in the literature and since c is temperature dependent. we also calculated the reflexivity of the drugs for different incident angles and polarizations. since reflexivity was under 10% for all wavelengths, we neglected it in the calculations. the parameters and the calculated penetration depths (d = 1/α) are summarized in table 2 . in all three medicines the highest temperatures occur at the uv wavelength. the temperature values are close at vis and ir wavelengths, hence the differences between the absorption coefficient values are minor. at these two wavelengths, during ibuprofen ablation the estimated temperatures (depending on the fluences) exceed several thousand kelvins (2000-10,000 k) at the surface where the temperature values are the highest. this is followed in magnitude by meloxicam, where these values are higher and reach 10,000-20,000 k. surface temperatures are the highest in niflumic acid, where these values reach 30,000-100,000 k. the temperatures at the laser penetration depths always significantly exceed the decomposition temperatures of the materials. therefore, chemically preserved particles cannot originate from the volume element (v e )-defined by the laser spot size and penetration depth (v e = a spot *d)-and these particles must be produced by another mechanism. fast photography. successive stages of the ablation process were captured by fast photography. this information is valuable for us in representing and understanding the dynamics of particle generation. figure 12 shows fast photography images deemed essential for the important stages of particle generation. it can be clearly seen that the ablation mechanism is the same for all three studied drugs. the generated shock wave is closely followed by a cloud of non-condensed state of matter expanding at a slightly lower speed than the shock front. after that, solid particles increasing in size with time start to exit the surface, which means that bigger and heavier particles have lower velocities. it can be easily observed that particles of all sizes were generated during the ablation of drug tablets, although we could not detect a sufficient number of particles above 1 μm with the particle sizer methods. this can be explained with the mass dependence of the mean free path and the phenomenon that the magnitude of gas flow limits the sizes of transportable particles. (1) figure 11 . absorption coefficients of meloxicam (black), niflumic acid (red), and ibuprofen (blue). www.nature.com/scientificreports/ to estimate the significance of photomechanical effects during ablation, we calculated the pressures produced in the target material at the shock fronts 33 . first we calculated the propagation velocity of the shock front by measuring its travel distance as a function of time, and then the pressure was estimated according to eq. 2: where p is the pressure produced at the shock front, ρ is the density of the medium, v is the velocity of the shock front and γ is the proportion of specific heats (c p /c v ) of the medium. in fig. 13 some examples of the calculated pressures are shown. the recoil pressures-which are equivalent with the initial pressures at the shock front-are 70-350 atm. these relatively high pressure values confirm our assumption, that the photomechanical effects have a significant contribution to the ablation process. in the course of pla, two successive and significantly different material ejection mechanisms can be distinguished: (i) prompt evaporation of the target material directly from the center of the focused laser beam spot, where very high temperatures are created close to the surface. (ii) material ejection induced by the photomechanical effect due to pressure pulse and shock wave formation in the bulk material as a result of fast evaporation. according to our approximate calculations, temperatures as high as 10 3 k to 10 4 k can be created at the www.nature.com/scientificreports/ target surface. some of the imparted energy is transferred inside the target material by heat conduction, and in the volume element (v e ) the temperature exceeds a critical value and fast vaporization processes occur with the formation of high-pressure ionized plasma. in this nonequilibrium plasma micro-explosions take place, and the pressure pulse initiates the propagation of a high velocity shock wave outwards from the excited volume element. then vaporized and highly decomposed products burst out as shown in fig. 12a . however, a major part of the target material is ejected via photomechanical processes. the shock wave induced recoil forces and mechanical stresses result in the ejection of small and large fractures from the surface. the photomechanical ablation mechanism is more significant in case of nanosecond laser pulses 17 . the fractured particles leave the target at different velocities in accordance with their size and mass. this is in good agreement with our fast photography results (fig. 12) where submicron sized particle ejection occurs at nanosecond and few microsecond time scales. larger, micron-sized and irregularly shaped particles are ejected in the microsecond scale [16] [17] [18] [19] . the above observations apply mainly to homogeneous bulk materials. in case of drug tablets where the targets are highly porous and consist of micrometer-sized grains, the pla process is slightly different. the uneven surface of the ablation holes, which can be clearly seen in the sem images (fig. 10 ) and the pressure calculations in indicate strong photomechanical effects. thus, the subsequent laser pulses reach irregularly shaped target surfaces. the effect of target inhomogeneity is pictured in fig. 12 , as multiple shock waves are developing after one laser pulse. consequently, the ideal homogenous optical absorption mechanism does not hold for our porous drug tablet targets, where lower temperature changes and photomechanical effects occur. furthermore, since the drug grains are more loosely bound together in compacted tablets than in a bulk material, much higher ablation yields can be attained. occasionally the recoil forces rip out large pieces (several microns in diameter) from the tablets. these phenomena explain the random fluence dependency in the size distribution of the ejected particles (table 1 ). in bulk materials, the average size of the ablated particles and the fwhms of the particles' size distribution increase with the fluence 34, 35 . according to the ftir investigations, pla at uv (λ = 248 nm) laser wavelength is not an adequate method for creating chemically preserved particles. this conclusion is supported by the high absorption coefficients of drugs measured in the uv regime (see fig. 11 ). our calculations, which were based on the absorption coefficients, show that the uv laser beam creates the highest temperature in the excited volume element, and it has the lowest penetration depth in the target material (see table 2 ). therefore, the secondary photomechanical effects, which can lead to the ejection of chemically non-degraded particles, are less significant. moreover, the energy of the photons (e p (λ = 248 nm) = 5 ev) exceeds the energy of several chemical bonds in the drug molecules, enhancing the probability of direct photochemical reactions like photolysis or photo dissociation. we concluded that by pla at λ = 248 nm, the ejected particles consist mainly of degraded drug molecules, similarly to polymers seen before [36] [37] [38] . at higher wavelengths, i.e. at lower photon energies [e p (λ = 532 nm) = 2.33 ev; e p (λ = 1064 nm) = 1.16 ev], the photochemical effects are less significant, but at the same time more drastic photomechanical effects can be expected. due to the lower temperatures induced in the target material and the larger penetration depths of the vis/ir laser beams, the secondary material ejection mechanisms are more prominent at 532 nm and 1064 nm laser wavelengths. this allows for the production of chemically preserved drug particles 39 . with the use of a laser beam at 532 nm considerably higher ablation yields could be achieved even at lower fluences than at 1064 nm (table 1 ). this can be related to the higher (but still moderate) absorption coefficient at 532 nm. according to our observations, at 1064 nm laser wavelength the mechanical degradation (photo disruption) of the tablets was so severe that the tablets rapidly broke into pieces, greatly decreasing the ablation lifetime and ablation yield. a more elaborate evaluation of the ablation yields requires the consideration of the thermal and mechanical characteristics of the target materials too. it has been shown that there is a strong relationship between the mechanical properties (density, mechanical hardness) of the target and the ablation yield 40 . we suspect that the mechanical properties of the drug tablets influenced our experiments too. this can explain our observation that the ablation yields are higher for meloxicam than for niflumic acid although meloxicam's absorption coefficients are lower at both wavelengths. we must make two additional remarks. one concerns the absence of absorption bands in the ftir spectra, which can be related to the degraded molecules. we suppose that at temperatures of thousands of kelvins in the evaporation zone the material is practically atomized/ionized, and therefore cannot be filtered out from the gas jet. this means that if there are any chemically modified but not completely atomized molecules, they must represent a negligible proportion of the whole ablation mass. our other comment is that during the size distribution measurements (fig. 9 ) no particles above ~ 1 μm in diameter were detected, although the formation of bigger particles can evidently be seen in the fast photography pictures (fig. 12) . most probably, these bigger and heavier particles simply fell back to the surface, since even the highest adjustable gas flow was unable to transport them. conclusion pla can be applied for the size reduction of poorly water-soluble nsaids. in the case of meloxicam, ibuprofen and niflumic acid, submicron to nanometer size particles can be produced by careful selection of the laser parameters, reducing the initial mean average sizes by orders of magnitude. we have found that ablation without any chemical destruction of the initial pharmaceutical compounds can only be performed with laser beams having wavelengths in the vis and ir range. higher ablation yields are obtained at 532 nm than at 1064 nm laser wavelength, and the yields are generally increasing with the applied fluence for the studied drugs. without aiming to conduct a thorough investigation we could also establish the influence of the absorption coefficients and the structure of the target drug tablets on the ablation mechanism. the smps/opc measurements provided particle size data precisely from that fraction of the laser ablated aerosol which can be pumped through a nasal delivery device and inhaled by the patient. | (2020) 10:15806 | https://doi.org/10.1038/s41598-020-72865-z www.nature.com/scientificreports/ we concluded that pla offers a chemical-free, verifiably reproducible and simple method for the size reduction of poorly water-soluble drug crystallites. the achieved submicron size range can be especially advantageous for the development of new pulmonary drug formulations for the medication of the lower lung area in pneumonia or severe acute respiratory syndrome. the adjustable laser parameters and thereby the fine tuning of the grinding mechanism during pla can be advantageous over other commonly used methods when formulating drug delivery systems for sensitive compounds. drug solubility: importance and enhancement techniques a literary endorser solubility enhancement techniques-a review improvement in solubility of poor water-soluble drugs by solid dispersion production of ibuprofen in crystalline and amorphous forms by pulsed laser deposition (pld) enhanced solubility of non-steroidal anti-inflammatory drugs by hydroxyl terminated s-triazine based dendrimers nanocrystals: industrially feasible multifunctional formulation technology for poorly soluble actives particle size reduction of biomaterials using cryogenic milling process an investigation on the application of cryogenic ball milling to ibuprofen particle and its characteristics combinative particle size reduction technologies for the production of drug nanocrystals overview of milling techniques for improving the solubility of poorly water-soluble drugs methods of size reduction and factors affecting size reduction in pharmaceutics analysis of submicron-sized niflumic acid crystals prepared by electrospray crystallization laser ablation synthesis in solution and size manipulation of noble metal nanoparticles size reduction of gold nanoparticles by pulsed laser ablation and re-irradiation in water media laser ablation of inorganic and organic materials a study of particle generation during laser ablation with applications production of meloxicam suspension using pulsed laser ablation in liquid (plal) technique laser fragmentation as an efficient size-reduction method for pulmonary drug discovery: proof-of-concept study of beclomethasone dipropionate ultrafast laser processing of drug particles in water for pharmaceutical discovery nanoparticle formation by laser ablation of perylene microcrystals in an aqueous solution of triton x-100 reagglomeration mechanism of drug nanoparticles by pulsed laser deposition indomethacin nanoparticles directly deposited on the fluidized particulate excipient by pulsed laser deposition modifying the physicochemical properties of nsaids for nasal and pulmonary administration air quality criteria for particulate matter conformational stability of ibuprofen: assessed by dft calculations and optical vibrational spectroscopy spectral studies of niflumic acid aggregation in dissolved, solid and adsorbed states structural characterization of ambazone salt with niflumic acid mechanochemical preparation of organic-inorganic hybrid materials of drugs with inorganic oxides physicochemical characterization, the hirshfeld surface, and biological evaluation of two meloxicam compounding pharmacy samples a method for segregating the optical absorption properties and the mass concentration of winter time urban aerosol detection of ibuprofen and ciprofloxacin by solid-phase extraction and uv/vis spectroscopy approximate spherical blast theory including source mass permalloy nanoparticles generated by laser ablation particle generation by ultraviolet-laser ablation during surface decontamination laser application of polymers wavelength dependence of pulsed laser ablation of calcified tissue t.g. and e.n. carried out the ablation of drugs and ftir measurements. r.a. and p.sz.r. supported the pharmaceutical background of the article. t.s.k., t.g. and z.h. was responsible for fast photography measurements and calculations. t.a., f.k.s., and z.b. made the particle size measurements and prepared relevant figures. j.b. did the ellipsometry measurements. t.g., j.k. and b.h. wrote the main manuscript text. all authors reviewed the manuscript. the authors declare no competing interests. correspondence and requests for materials should be addressed to t.g.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-222700-yof5x4ct authors: slapik, aleksandra; spiechowicz, jakub title: tunable particle separation via deterministic absolute negative mobility date: 2020-08-31 journal: nan doi: nan sha: doc_id: 222700 cord_uid: yof5x4ct particle isolation techniques are in the spotlight of many areas of science and engineering. in food industry, a harmful bacterial activity can be prevented with the help of separation schemes. in health care, isolation techniques are used to distinguish cancer and healthy cells or in therapy for alzheimer's and parkinson's diseases. we consider a cloud of brownian particles of different sizes moving in a periodic potential and subjected to an unbiased driving as well as a constant force. we reveal an efficient separation strategy via the counterintuitive effect of negative mobility when particles of a given size are transported in a direction opposite to the applied constant force. we demonstrate a tunable separation solution in which size of the particle undergoing separation may be controlled by variation of the parameters of the external force applied to the system. this approach is an important step towards the development of point-of-care lab-on-a-chip devices. separation of (sub)micro sized particles is of paramount importance due to its vast applications including in particular medical diagnostics [1] . anomalies in a bioparticle size often indicate various illnesses. this is apparent, for instance, for alzheimer's, parkinson's [2] and huntington's disease [3] , needless to say that often cancer cells noticeably deviates in size from healthy ones [4] . a reliable and effective approach to separating bioparticles is therefore much in demand. they span a large size range covering several orders of magnitude from nanometers to micrometers [5, 6] what crucially complicates the development of such highly anticipated strategies. for instance, deadly viruses like hiv or covid-19 are approximately 0.1 micrometer in diameter [7] . on the other hand, the soma of a neuron can vary from 4 to 100 micrometers in diameter [8] . for such a broad (sub)micrometer scale, efficient isolation techniques are required to allow tunability of the particle size intended for separation. unluckily, the latter are rather scarce [9, 10] , however, recently some progress has been made in this direction [11] [12] [13] in this work we aspire to partially fill this significant know-how gap by demonstrating a nonintuitive, yet efficient separation strategy taking advantage of a paradoxical mechanism of negative mobility [14] [15] [16] [17] . we show that under the action of a static bias only particles of a given linear size move in the direction opposite to this net force whereas the others migrate concurrently towards it. this effect creates a possibility of steering different species of particles in opposite directions under identical experimental conditions thus facilitating their separation. a proof-of-principle experiment of a similar isolation scheme was performed with insulator dielectrophoresis in a nonlinear, symmetric microfluidic structure with electrokinetically activated transport [18, 19] . recently, such a setup allowed to induce the negative mobility not only for a colloidal particle but also for a biological compound in the form of mouse-liver mitochondria [20] . motivated by the large size range encountered in biochemical applications, a crucial result of this work is a demonstration of a tunable separation strategy, in which the size of the particle undergoing separation may be effectively controlled by variation of the parameters characterizing the external force applied to the particle, e.g. the magnitude of the static bias. the same setup can be applied to segregate particles with respect to their mass in a similar tunable manner [21] . this approach may provide selectivity required for individual isolation of nano and micro particles, proteins, organelles and cells and thus constitutes an important step towards the development of robust lab-on-a-chip devices exploited in both research and industrial applications, in particular point-of-care medical diagnostics. the paper is organized as follows. in sec. ii we outline the model of a brownian particle dwelling in a spatially periodic potential under the action of both an external harmonic driving as well as a constant bias. in the next section we exemplify the negative mobility phenomenon. sec. iv provides crucial results of the paper, namely, a tunable particle separation strategy. in sec. v we discuss the possibility of tailoring particle isolation in the considered system. finally, the last section is devoted to summary and conclusion. the system considered in this study is a classical inertial brownian particle of mass m which moves in a spatially periodic one-dimensional potential u (x) = u (x + l) of the period l, additionally subjected to an unbiased timeperiodic force a cos (ωt) of the amplitude a and the angular frequency ω, as well as an external static force f . dynamics of such a particle is described by the langevin equation [22] mẍ where the dot and the prime denote differentiation with respect to time t and the particle coordinate x, respectively. coupling of the particle with thermal bath of temperature t is modeled by gaussian white noise of zero mean and unity intensity, namely the noise intensity factor 2γk b t (where k b is the boltzmann constant) follows from the fluctuation-dissipation theorem [23] and ensures that the system reaches the equilibrium state when a = 0 and f = 0. the potential u (x) is spatially periodic with the period l and the barrier height 2∆u , at first glance the studied system looks simply, however, it exhibits peculiar transport behaviour including noiseenhanced transport efficiency [16] , anomalous diffusion [24, 25] , amplification of normal diffusion [26, 27] and a non-monotonic temperature dependence of normal diffusion [28] . as the first step of the analysis we transform the eq. (1) into its dimensionless form. this aim can be achieved in several ways. it often allows to simplify the setup description as after the rescaling procedure some parameters appearing in the corresponding dimensional version may be eliminated thus reducing the complexity of the problem. moreover, recasting into the dimensionless variables ensures that the results obtained later are independent of the setup which is essential to facilitate the choice in realizing the best setup for testing theoretical predictions in experiments. here, we propose the use of the following scales as the characteristic units of length and timê the langevin equation (1) transformed according to the above variables readŝ the rescaled dimensionless friction coefficient γ is the ratio of two characteristic time scales where τ 1 = m/γ is characteristic time for the velocity relaxation of the free brownian particle. the dimensionless mass is set to unity, m = 1. other rescaled parameters are as follows: a = (l/∆u )a, ω = τ 0 ω, f = (l/∆u )f . the dimensionless potentialû (x) = u (lx)/∆u = sin(2πx) has the periodl = 1. the dimensionless thermal noise ζ(t) assumes the same statistical properties as ξ(t), i.e. ζ(t) = 0 and ζ(t)ζ(ŝ) = δ(t −ŝ). the dimensionless noise intensity d = k b t /∆u is the ratio of thermal energy and half of the non-rescaled potential barrier. from now on, only the dimensionless variables will be used in this study and therefore, in order to simplify the notation, the ∧-symbol will be omitted in the equation (5) . the dimensionless friction coefficient γ is the most important parameter for later mentioned process of the particle separation with respect to its size. it is due to the fact that even for the simplest model of hydrodynamic interactions occurring in this setup it depends on the linear size r of the particle. for instance, the spherical particle diffusing in the surrounding medium is subjected to stokes drag −γẋ where γ = 6πηr and η is the viscosity of the environment [29] . we stress that (sub)micro sized particles typically possess small mass and therefore for them the dimensionless friction coefficient γ given by eq. (6) is expected to be either of the order or larger than the dimensionless mass m = 1. a. the directed velocity the particle mobility describes its ability to move through the medium as a response to the biased force acting on it. hence, the observable of foremost interest in this study is a directed velocity v of the particle which may be written as where · indicates averaging over all realizations of thermal noise as well as over initial conditions for the particle position x(0) and its velocityẋ(0). the latter is obligatory for the deterministic limit d ∝ t → 0 when the dynamics may be non-ergodic and results can be affected by the specific choice of initial conditions [30] . the fokker-planck equation corresponding to the langevin eq. (5) cannot be solved analytically in a closed form. therefore the system may be analyzed only by numerical simulations. equation (5) is characterized by a 5-dimensional parameter space {γ, a, ω, f, d} the detailed exploration of which is a very challenging task. all numerical calculations were performed using an innovative computational method which is based on employing graphics processing unit supercomputers. this procedure allowed us to speed up computations by about 10 3 times compared to traditional methods. for technical details we refer the reader to ref. [31] . in general, the directed velocity v is an increasing function of the static force f and the resultant particle transport follows the direction of the bias, i.e. v = µ(f )f with a positive nonlinear mobility µ(f ) > 0. however, in the parameter space there are also regimes for which the particle moves on average in the opposite way, namely v < 0 for f > 0. such anomalous transport behaviour is characterized by a negative mobility µ(f ) < 0 [15, 32, 33] . the key ingredient for the occurrence of the latter effect is that the system is driven away from thermal equilibrium into a time-dependent nonequilibrium state [15] . this fact provides a negation of the le chatelier-braun equilibrium principle [34] stating that the response of the perturbed system occurs into the direction of the applied bias towards a new equilibrium state. in our case this condition is guaranteed by the presence of the external harmonic driving a cos (ωt). it is known that there exist two fundamentally different mechanisms responsible for the emergence of negative mobility in the considered system. firstly, it may be generated solely by the deterministic dynamics given by eq. (5) with d ∝ t = 0 [15, 32] . secondly, it can be induced by an appropriate dose of thermal fluctuations [15] . among the first mentioned class we distinguish two completely distinct scenarios. for the deterministic counterpart of the system the negative mobility may be induced either by chaos-assisted dynamics [32] or regular, non-chaotic attractors transporting the particle in the direction opposite to the applied bias [35] . our numerical research reveals that the most common reason for the occurrence of the negative mobility is rooted in the complexity of the deterministic and chaotic dynamics [35, 36] . this observation is of great importance for uncovering the parameter regimes allowing the particle separation. in fig. 1 we illustrate the negative mobility effect. for γ = 1.29 the directed velocity v > 0 assumes the same sign as the force f > 0 leading to the normal particle transport regime with µ > 0. however, for γ = 1.23, the directed velocity v < 0 is opposite to the bias f > 0 and in consequence the negative mobility effect emerges µ < 0. if the value of f is positive and large enough then the sign of v coincides with the force f again. therefore this anomalous transport behaviour is observed only in the vicinity of the zero bias f = 0 and therefore often is termed as the absolute negative mobility [37] . as it is illustrated in the the panel (b) the presented parameter regime belongs to the class of deterministically induced negative mobility as for the limiting case of vanishing thermal noise intensity d → 0 the directed velocity is negative v < 0. we want to stress that this limit should be considered with utmost care as in such a case attractors transporting the particle in opposite directions may coexist and the dynamics may be non-ergodic. it means that depending on the initial conditions, a particle would either move in the direction of the bias force or opposite to it [32] . nevertheless, at any finite d > 0, possibly coexisting deterministic attractors turn metastable and due to thermally activated transitions between them the ergodicity of dynamics is restored. consequently, the directed velocity v is independent of the initial conditions. moreover, as it is shown in the panel (b) the deterministically induced negative mobility effect is generally quite robust with temperature change and usually survives up to moderate thermal noise intensities. we now want to harvest the negative mobility phenomenon to separate (sub)micrometer sized particles. this task may be achieved as follows. imagine that there is a mixture of several species of spherical particles each differing by its linear size r. the friction coefficient γ, via e.g. stokes formula, depends on the particle radius γ = γ(r). we can extract the given species of particles r * characterized by the friction coefficient γ * ≡ γ(r * ) if only for this particular group of particles the negative mobility effect arises, i.e. v < 0 for γ * and v > 0 for the rest. therefore this task translates to discovery in the complex four-dimensional parameter subspace {a, ω, f, d} regimes where in the characteristic v (γ) there exists only one interval δγ of the friction coefficient around the desired particle size γ * for which the negative mobility v < 0 emerges. in such a way only particles with the radius r * would be extracted from the mixture. motivated by the large size range typically encountered in biochemical applications, we aim to develop a tunable scheme that allows to control the particle size targeted for isolation by changing only one parameter of the system. in [21] it has been shown that the negative mobility effect can be harvested to separate the (sub)micrometer sized particles with respect to their mass. the particle mass targeted for isolation might be effectively controlled over a regime of nearly two orders of magnitude upon changing solely the frequency ω of the external harmonic driving. moreover, in [36] an efficient separation mechanism based on thermal fluctuation induced negative mobility phenomenon has been proposed. by tuning solely temperature of the system d ∝ t , one can extract from the mixture of particle species differing by size only those of a strictly defined radius. this scheme opens an opportunity to separate particles that carry no charge or dipole, however, it may be inconvenient since temperature variation typically takes too much time to offer a robust experimental implementation. therefore, in contrast, in this work we harvest the negative mobility effect to develop the particle separation strategy in which the particle size intended for isolation will be controlled by changing only the parameters characterizing the externally applied force, namely, the static bias f or the amplitude a or the frequency ω of the harmonic driving. unfortunately, there is no clear relationship between the presence of the negative mobility and the model parameter values. a tiny displacement in the parameter space may either cause a sudden emergence of the negative mobility or its rapid decay. therefore, extensive numerical simulations of eq. (5) were performed in order to systematically investigate the established parameter space. as the deterministically induced negative mobility is the most populated mechanism in the parameter space we set d = 0. then eq. overall, we considered nearly 10 9 different parameter sets. this exceptional precision was possible only because of our innovative simulation method [31] . the so collected data was transformed into two-dimensional maps presenting the directed velocity v versus two chosen model parameters to facilitate the further analysis. the results of foremost interest are those with γ dependence since the friction coefficient may be used as an indicator to differentiate particles by their size. we explored the data to discover any correlations between the presence of the negative mobility, the friction coefficient γ as well as the magnitude of the parameters a, ω and f . we exemplify such a situation in fig. 2 where we depict the directed velocity v as a function of the static bias f and the friction coefficient γ. the color bar in the plot represents the magnitude of the directed velocity v . the occurrence of the negative mobility is marked by blue areas for which v < 0. the reader can observe a linear trend between the friction coefficient γ, the static bias f and the emergence of the anomalous transport. the negative mobility effect occurs for progressively smaller values of γ as the force f increases. among many so discovered negative mobility regimes we distilled only those where the latter phenomenon is present solely for one indivisible interval δγ of the friction coefficient γ thus permitting the particle separation. we exemplify this procedure in fig. 3 where we depict the directed velocity v versus the friction coefficient γ which can be identified with the particle size. as it is illustrated in the panel (a), among many particle sizes corresponding to the friction coefficient γ ∈ [0. 2, 4] only those with the friction coefficient γ * ≈ 1.05 will move in the direction opposite v < 0 to the applied bias f > 0. other particles will travel concurrently towards it. as a result, only the particles with γ * ≈ 1.05 will be extracted from the heterogeneous mixture. panel (b) presents a magnification of the interval δγ where the effect of negative mobility emerges. it can be interpreted as a resolution capacity of this method. in this case it reads δγ ≈ 0.0678. the selectivity of the proposed separation scheme is impressive as δγ/γ * ≈ 0.06 . moreover, as it is illustrated, typically the velocity v is noticeably peaked in the interval δγ where the negative mobility occurs. the mechanism of the latter anomalous transport effect is rooted in the deterministic dynamics and as such thermal fluctuations generally have destructive impact on it [35, 36] . therefore the above outlined particle separation strategy is viable in low to moderate temperature regimes in which the size targeted for isolation γ * , i.e. the middle of the interval δγ, coincides well with the the friction coefficient γ min for which the directed velocity attains its minimal value v min ≡ v (γ min ). it means that not only the selectivity of this method is impressive but also the particle separation process is quick. such approach allowed us to distill parameter regimes which reveal a specific functional dependence between the particle size tailored for separation γ * and the parameters of the external force applied to the system thus allowing for tunable particle isolation. in fig. 4 we present the friction coefficient γ * versus the static bias f , the amplitude a and the frequency ω all depicted for different temperature of the system d ∝ t . the data were obtained from the characteristics v (γ) computed for many values of the control parameter, c.f. fig. 3 . each dot represents the friction coefficient γ * undergoing the separation process at the fixed f , a or ω, see panels (a)-(d). the bars indicate the friction coefficient interval δγ where the negative mobility emerges. here we note that the curves on the corresponding panels overlap with the one representing the deterministic solution d = 0. therefore to visualize the impact of temperature on the separation process in the corresponding insets we schematically show the solutions depicting subsequent d values. the reader may observe that using those tailored parameters read off from fig. 4 one is able to tune the negative mobility to the particle of a given size γ * by changing solely the static bias f or the amplitude a or the frequency ω. in this way it will be separated from the others possessing positive mobility and thus moving concurrently towards the applied bias f . from the experimental point of view the most convenient way to manipulate the particle separation is presumably by altering the static bias f . it is because in many realistic setups it is implemented via the constant external field, e.g. in the microfludic experiments in the form of a spatially uniform electric field which induces the particle electrophoresis [10] . in most cases its intensity can be changed relatively easily, as opposed to the frequency ω of the external harmonic driving which often requires complete rebuilding of the experimental setup. we note that the parameter sets reported in fig. 4 allow for the tunable separation of the particles in the regime of moderate-large friction coefficient which is characteristic for low reynolds numbers, being typical for (sub)micro sized particles immersed in a solution [38] . for example in the panels (a) and (b) corresponding to the isolation driven by the constant force f , the friction coefficient γ * ∈ [0.6, 2.5], whereas in (d) when the separation is controlled by the frequency ω the size γ * ∈ [0.25, 1.75]. the reader can observe that the friction coefficient γ * is a decreasing function of the force f and the frequency ω (panels (a), (b) and (d)) while for the amplitude a it depicts an increasing dependence (panel (c)). finally, we note that the dimensionless friction coefficient γ in eq. (6) is influenced not only by the actual friction γ but also by the parameters ∆u and l of the potential. therefore, experimentalists may exploit these characteristics of the periodic substrate to further adapt the particle size targeted for separation. last but not least, fig. 4 reveals the impact of temperature d ∝ t on the tunability of the separation process. as it was stated before, since the negative mobility effect derives from the deterministic dynamics of the system, thermal fluctuations have a destructive influence on it. when temperature increases the regions of negative mobility allowing for the controllable particle separation progressively shrink and eventually vanish completely. therefore the reported tunability is possible in the widest range of the friction coefficient γ * for low to moderate temperature regimes d ∝ t . however, for instance, the panel (c) illustrates an interesting effect of thermal fluctuations. an increase of temperature leads not only to shrinking of the range of the friction coefficient γ * for which the negative mobility is observed but also to significant decrease of the intervals where this phenomenon occurs, thereby optimizing the width δγ. it means that then the tunability of the method is limited but selectivity of the separation process increases. we remark that the particle isolation upon harvesting the negative mobility phenomenon is also present for different parameter regimes, however, the range of its tunability proves somewhat smaller. now we consider another, complementary issue. let us assume that we deal with particles of a given size γ * which we want to extract from the heterogeneous mixture. we address the following question: for how many different sizes γ * taken from the extended interval γ * ∈ [0.1, 10] it is possible to find a parameter set {a, ω, f, d} for which the negative mobility effect emerges in the small interval [γ * − δγ/2, γ * + δγ/2] around the targeted value γ * , therefore allowing its separation in a unique manner, c.f. fig. 3 ? we found that in most cases the magnitude δγ/γ * is equal to a few percent but frequently is even smaller. we note that for a single γ * there might be several parameter regimes fulfilling this condition thus facilitating the choice in realizing the best parameter set. we present the answer of this experimentally and practically relevant question in fig. 5 . the distribution of the size γ * targeted for separation is shown there in the parameter plane of the amplitude a and the frequency ω for different values of the static bias f . the color coded scale displays the friction coefficient γ * value. we observe that small particles can be isolated when the static bias f is likewise small and moderate to large values of the frequency ω. on the other hand, medium and large particles are separated for small frequencies ω. we note that the distribution of the particle size targeted for isolation γ * undergoes a stretching when the static bias f decreases. moreover, in such a case the range of the particles which might be segregated by harvesting the negative mobility effect is extended as well. finally, even though the considered panel depicts the deterministic d = 0 dynamics, we found that the distribution of γ * depicted there is quite robust with temperature change and survives up to moderate thermal noise intensity, see fig. 1 (b) . in this work we provide an efficient method for tunable separation of (sub)micro sized particles via the negative mobility phenomenon. the approach presented here requires only a spatially periodic nonlinear structure in combination with an unbiased external time-periodic driving. in this scheme the particle size intended for isolation can be effectively controlled by changing solely the parameters of the external force applied to the system, namely, the static bias or the amplitude or the frequency of the external harmonic driving. the approach can be further adapted to the needs by proper fabrication of the nonlinear potential landscape determined by its barrier height and period. it allows the possibility to not deflect the separated particles along the different angles but to steer them in the opposite direction making the isolation process robust. our theoretical predictions should be used as a guide towards physical reality indicating the direction for future theoretical and experimental research. in particular, one needs to carefully consider higher dimensional systems as well as geometrical constraints together with hydrodynamic interaction which in real experiments may play essential role. we expect that such research would potentially lead to implementation of the proposed scheme in a lab-on-chip device, as it has been recently demonstrated for a similar system [20] . we envision that current lithographic techniques with advantageous fabrication costs may be used to develop high throughput separation applications concerning in particular biophysical and biochemical problems. taking into account recent progress in 3d printing technologies allowing its scaling down to the nanometer range the proposed scheme may have even significant commercial potential in future [39] . we employed a weak 2nd order predictor-corrector method [40] to simulate stochastic dynamics given by eq. (5) . we integrated it with the time step scaled by the fundamental period t = 2π/ω of the external harmonic driving, namely h = 10 −2 ×t, with the exception of smallest ω < 1 values for which the step was chosen to be h = 10 −3 ×t. the initial positions x(0) and velocities v(0) were uniformly distributed over the intervals [0, 1] and [−2, 2], respectively. the directed velocity v was averaged over the ensemble of 2 10 = 1024 trajectories, each starting with a different initial condition according to the above distributions. the number of realisations of stochastic dynamics is not accidental and was chosen carefully to maximise the performance of the numerical simulation, see ref. [31] for more details. the time span of the simulations was set to [0, 10 4 t] to guarantee that the directed velocity v relaxed to its asymptotic long time stationary value. a. s. carried out numerical calculations. all authors contributed to the discussion and analysis of the results. j. s. wrote the manuscript. microfluidic diagnostic technologies for global public health cell-replacement and gene-therapy strategies for parkinson's and alzheimer's disease the early effects of ischemia upon skeletal muscle mitochondria biomechanics and biophysics of cancer cells microfluidics for cell separation size separation of biomolecules and bioparticles using micro/nanofabricated structures an overview of their replication and pathogenesis in coronaviruses principles of neural science particle separation and sorting in microfluidic devices: a review separation phenomena in tailored micro-and nanofluidic environments particle sorting by a structured microfluidic ratchet device with tunable 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of free diffusion by use of tilted periodic potentials josephson phase diffusion in the superconducting quantum interference device ratchet diffusion in a biased washboard potential revisited fluid mechanics (butterworth-heinemann transient anomalous diffusion in periodic systems: ergodicity, symmetry breaking and velocity relaxation gpu accelerated monte carlo simulation of brownian motors dynamics with cuda transient chaos induces anomalous transport properties of an underdamped brownian particle observation of negative absolute resistance in a josephson junction negative mobility of a brownian particle: strong damping regime temperature-induced tunable particle separation anomalous transport in biased ac-driven josephson junctions: negative conductances low reynolds number hydrodynamics small but perfectly formed? successes, challenges and opportunities for microfluidics in the chemical and biological sciences numerical solution of stochastic differential equations with jumps in finance in stochastic modelling and applied probability key: cord-322880-zfc9w5y6 authors: rohit, anusha; rajasekaran, shankar; karunasagar, indrani; karunasagar, iddya title: respiratory droplets get suspended longer and spread wider in temperate environments compared to tropics and implications for sars-cov-2 transmission date: 2020-06-09 journal: med hypotheses doi: 10.1016/j.mehy.2020.109958 sha: doc_id: 322880 cord_uid: zfc9w5y6 the new pandemic of sars-cov-2 has shown stark differences in number of affected patients between countries in the tropics and those with temperate environments. though there have been many theories on reasons for these differences, we hypothesise that this could be due to differences in the fate of respiratory droplets in the two environments. a simple understanding of the mechanics of droplet size, dispersion and displacement could help infection control and public health measures to minimize spread and mitigate the risk of people getting infected especially in hotspots like hospital environments or other closed spaces. this paper discusses the possibility of differences in number of infections and spread between different countries based on the spread of droplets. the situation with respect of covid-19 globally is very grim with 3,939,119 cases and 274,917 deaths as on may 9 th (1). interestingly, resource rich countries like us, germany, uk, italy, spain seem to have been much more affected than many of the tropical countries in south and south east asia and africa. for example, vietnam, which shares a long border with china has only 288 cases and no deaths though 2 cases were detected as early as 23 rd january. laos, sharing border with china had 19 cases with no deaths, myanmar, sharing border with china had 177 cases with 6 deaths. thailand had cases detected in january, but in april, the case count is only 3,004 with 56 deaths. in contrast, in us, cases started appearing only early march, but in a span of a month, the numbers have increased dramatically to 1,322,154 and 78,616 deaths. in italy, the cases started showing up during the last week of february, but escalated to 217,185 with 30,201 deaths. in spain, cases started appearing early march, but soon increased to 260,117with 26,299 deaths, while in germany, cases began appearing late february, but increased to 170,588 with 7,510 deaths. the explosive increase in cases in resource rich countries with well-established public health system compared to a very slow increase in countries in south east asia, which are resource limited and having weak public health system is perplexing. though inadequate testing contributes to lower numbers in some countries, the difference is indeed stark and calls for other explanations based on transmission of this virus. the predominant mode of virus spread is said to be through droplets generated by infected individuals during coughing, talking or sneezing and contact transmission (2) . the droplets may range in size from sub-micrometers to hundred of micrometers. from "stoke's drag force equation", the terminal settling velocity of a particle of known diameter and density can be calculated. the settling velocity of the particle would be directly proportional to the density of the particle and to the square of the particle diameter. owing to their size, larger particles settle down at a much faster speed than smaller particles. for instance, the settling velocity of a 10 micron water droplet will be approximately 88 times greater than a 1 micron water droplet. thus smaller particles will stay airborne for a longer time, while larger particles will settle down on surfaces more quickly. according to law of brownian motion, which is applicable to smaller particles, the diffusive forces would transport the particles in random directions. the diffusion coefficient of a particle as given by "stokes-einstein equation" is inversely proportional to the diameter of the particle. so, smaller the particle, higher the diffusivity. higher diffusivity helps smaller particles to travel through the room at a faster speed than larger particles. for instance, a 1 micron particle will have a net diffusion displacement that is 3 times more than that for a 10 micron particle (figure 1 ). thus a smaller particle will spread throughout the room at a much faster rate than a large particle. the relative humidity of the environment influences the density of the particle as well as the size of the particle (figure 2 ). upon release, the droplets may be subject to partial or total evaporation, depending on environmental conditions and would reduce in size. the environmental relative humidity (rh) will influence the extent of evaporation and final equilibrium size of the droplet. thus lower rh reduces the size of the particle as the particle desiccates. furthermore, relative humidity could affect the particle density too, as the particle desiccates. the combined effect of change in size of the droplet as well as the density amplifies the influence that environmental relative humidity would have on the particle settling velocity. marr et al (3) studied the effect of rh on droplet equilibrium using droplets containing 9mg/ml nacl, 3 or 76 mg/ml protein and 0.5mg/l surfactant. they noted that a droplet of 10μm shrinks to 2.8μm at 90%rh and to 1.9μm at less than 64%rh. the droplet size would greatly affect settling times, with 10μm droplet remaining suspended for only 8min at 100% rh while the 1.9 μm droplet at 64% rh remaining suspended for more than 3h. all the above apply to the dynamics of particles in still air, with no air currents. however, in real life scenario, air currents and turbulences are unavoidable. these would further affect the settling patterns of particles. therefore a significant portion of the particles are likely to be airborne indefinitely, drifting around freely. the expelled aerosol by a person on account of exhaling, talking, coughing, sneezing, etc would have a poly dispersed particle size distribution. the mean, median, mode and standard deviation of the distribution would vary based on expulsion characteristics. a significant portion of this cocktail of particles expelled by a person could hence stay airborne, influenced by environmental conditions and perhaps even get transported out of the source room, when provided with suitable conditions. our hypothesis suggests that mechanistically, in tropical conditions of high temperature and high humidity, droplets settle quickly, while in temperate conditions of low temperature and low humidity, droplets tend to remain suspended in air. in the case of respiratory droplets, these would dramatically affect airborne transmission of viruses in the droplets. it is possible that droplets discharged from infected individuals in places like new york city could shrink in size quickly and remain airborne for considerable period of time. a droplet size of 1 μm could be airborne for 8 hrs. the humidity in new york city which is experiencing a serious covid situation is around 64%, and in berlin, the humidity is around 60%. on the other hand, in hanoi, vietnam, the humidity is above 80%. under conditions of temperature and humidity in cities like hanoi and hochi minn city in vietnam, the droplets would settle quickly. this might explain higher transmission rates observed in temperate environments compared to tropical ones. the same argument holds good for air conditioned spaces, where the temperatures and relative humidity levels could be low. this may influence the measures needed to prevent infections. hand hygiene and social distancing could be the most important step in tropical settings, since transmission could be predominantly through droplets settling on various surfaces, while, use of masks, hand hygiene and social distancing would be important in temperate places, since virus could remain airborne for considerably longer period of time. for air conditioned environments, suitable modifications and adjustments would be required to the air conditioning system to control the spread of infections. in addition to differences in mode of dispersion, there could be differences in infectivity of virus due to survival under conditions of temperature and sensitivity. chan et al (4) studied survival of sars coronavirus on various surfaces at different temperature and humidity conditions. virus viability was retained up to 5 days on plastic surface at temperature of 22-25°c and humidity of 40-50% with only one log reduction in titre. temperature range of 28-33°c and humidity of >95% did not make much difference, but temperature of 38°c at 80-90% rh led to up to 2 log reduction in titres in 24hrs. studies with porcine coronavirus (transmissible gastroenteritis virus, tgev) showed that they remain viable longer at low relative humidity (rh) than at high humidity (5). tgev is also an enveloped virus like sars-cov-2. the persistence of influenza virus in the environment has been studied by a number of investigators. using systematic review methodology, irwin et al (6) surmised that increasing temperature (2-12°c vs >27°c) resulted in shorter virus half life. published studies indicate that virus half life was 16.5 times longer at temperatures between 7-12°c as compared to >27°c. harper (7) studied influenza survival at three temperatures (7-8°c, 20.5-24°c and 32°c) and five rh ranges (20-25%, 34-36%, 49-51%, 64-65% and 81.82%) and noted that virus survival was highest at lowest temperature and lowest rh studied. using enveloped virus phi6 in droplets as surrogate for influenza and coronaviruses, prussin ii et al (8) noted significant decrease in infectivity at rh range 60-85%. infectivity decreased two orders of magnitude between 19-25°c at humidity level of 75%. it can be expected that sars-cov-2 virus behaves on similar lines as sars-cov-1 and influenza virus. infectivity could be reduced under environmental conditions prevailing in cities like hanoi in vietnam compared with new york city in us. therefore, reduced infectivity and shorter airborne transmission could explain lower number of cases observed in south east asia. virology, epidemiology, pathogenesis and control of covid-19 mechanistic insights into the effect of humidity on airborne influenza survival, transmission and incidence effect of temperature and relative humidity on the viability of sars coronavirus fate of respiratory droplets in tropical vs temperate environments and implications for sars-cov-2 transmission the research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors the new pandemic of sars-cov-2 has shown stark differences in number of affected patients between countries in the tropics and those with temperate environments. though there have been many theories on reasons for these differences, we hypothesise that this could be due to differences in the fate of respiratory droplets in the two environments. a simple understanding of the mechanics of droplet size, dispersion and displacement could help infection control and public health measures to minimize spread and mitigate the risk of people getting infected especially in hotspots like hospital environments or other closed spaces. this paper discusses the possibility of differences in number of infections and spread between different countries based on the spread of droplets. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. all authors have no conflict of interest to declare. this work has not been published previously. key: cord-032188-y02b92pe authors: xu, zhonglin title: characteristics of air filters date: 2013-08-14 journal: fundamentals of air cleaning technology and its application in cleanrooms doi: 10.1007/978-3-642-39374-7_4 sha: doc_id: 32188 cord_uid: y02b92pe air filter is the main equipment in the field of air cleaning technology, and it is an indispensible equipment to create the clean air environment. so it is necessary to know the characteristic of air filters and its design principle so as to use it correctly and effectively. note: when the measured efficiency value meets the requirement for two types at the same time, the higher type is used for assessment sub-high-efficiency particulate air filter. it is used as final filter in the cleanroom to obtain a certain class of air cleanness (please refer to chap. 7), prefilter for hepa filter to further improve the cleanness of supply air, and final filter of fresh air system to improve the fresh air quality. it is mainly used to capture submicron particles with diameter less than 1 μm, which is similar as that of high-efficiency filter. hepa filter. it is mainly used as final filter in cleanroom. the purpose is to provide various cleanness classes corresponding to 0.5 μm, while its efficiency is usually tested with particle diameter 0.3 μm. if cleanness class corresponding to 0.1 μm is needed, its efficiency should be tested with particle diameter 0.1 μm and it is called ulpa filter. it is usually used as the final filter. there are several types of roughing air filters and medium-efficiency air filters, such as panel-type air filter, bag air filter, and folded media-type filter. it's better to choose air filters with larger filtration area. there are several types of high-efficiency filters, such as bag filter, cartridge air filter, and folded media-type filter. there are several types of sub-high-efficiency particulate air filters, such as cartridge air filter and folded media-type filter. the former is a type of low-pressure drop, which is a patent product of institute of hvac of china academy of building research. there are several types of hepa filters, such as folded media-type filter which could be classified as separator hepa filter and no-separator hepa filter. table 4 .9 presents the comparison between air filter standards home and abroad. this kind of comparison is only for information and may not match well, so care should be taken before selection of air filters. the foreign classification methods for general ventilation air filters are quite confusing, which will not be introduced here [1] . since 1993, iest classified hepa filters into two categories. one is hepa filter, and the other is ulpa filter. afterwards these terms are used frequently. the most important four indexes to evaluate the performance are face velocity (or filtration velocity), efficiency, pressure drop, and dust holding capacity. there are also other indexes, such as weight, energy consumption, and regeneration feature, which are mainly related to filter media. it is important to choose which kind of filter media is used to make air filters. except for the impacting factor of filter media, filter structure is also one of the important impacting determinants for the performance of air filters. for example, both the pressure drop and dust holding capacity are different, when the same filter media is used to make panel filter, bag filter, or wedge filter. so it is another important link to find reasonable optimal structure for air filter. these four performance indexes are introduced in the following section. both face velocity and filtration velocity can be used to describe the ability of airflow through the air filter. face velocity is defined as the airflow velocity passing the cross section of air filter (m/s), i.e., where q is the flow rate, m 3 /h; f is the cross-sectional area of air filter or frontal area, m 2 . so face velocity represents the passing capacity and installed area of air filter. the larger the face velocity is, the less the occupied area is. therefore, face velocity is an important parameter to reflect the structural characteristic of air filter. filtration velocity is defined as the airflow velocity passing the area of filter media, and it is expressed with the unit l/(cm 2 · min) or cm/s, i.e., v ¼ q â 10 3 f â 10 4 â 60 where f is the net area of filter media, i.e., the subtraction of binder area from the total area, m 2 . during the sample test on filter media, the unit of v is l/(cm 2 · min), while it is cm/s for the sample test on air filter. the multiplication of the former value with 16.6 equals with the latter value. 4 filtration velocity represents the ability of passing airflow of filter media, especially the filtration performance of filter media. generally speaking, the smaller the filtration velocity is, the higher the efficiency is. when the allowed filtration velocity of filter is smaller, the pressure drop of filter media is larger. for given structure of filter, the nominal flow rate can be used to reflect both face velocity and filtration velocity. with the same area of cross section, the larger allowed nominal flow rate is preferred. when the air filter is operated under lower flow rate, the efficiency increases and the pressure drop decreases. filtration performance of air filters can be described with efficiency, penetration, and decontamination factor. when weight concentration is used to describe the particle concentration in the airflow, performance is evaluated with arrestance. when particle counting concentration is used, performance is evaluated with particle counting efficiency. when other physical parameter is used, performance is evaluated with dust spot efficiency or turbidity efficiency. 1. to describe the efficiency with particle concentrations at both the inlet and outlet airflow, i.e., where g 1 , g 2 refer to particle mass or counting number at inlet and outlet airflow (mg/h or pc/h), respectively; n 1 , n 2 refer to particle concentration at inlet and outlet airflow (mg/m 3 or pc/l), respectively; q is the airflow rate passing through air filter (m 3 /h or l/h). this expression is valid for both arrestance and particle counting efficiency. 2. to describe the efficiency with particle concentrations upstream of air filter and particle mass captured on air filter, i.e., where g 3 is the particle mass captured on air filter, mg/h. this expression is only used for arrestance. the value of η calculated by this method is termed as dust removal efficiency in some countries. 3. to describe the efficiency with particle concentrations downstream of air filter and particle mass captured on air filter, i.e., this expression is also used to describe the arrestance. 4. to describe the efficiency with fractional efficiency corresponding to various particle size channels, i.e., where η 1 à η n is the fractional efficiency for various particle size, which is expressed in decimal; n 1 à n n is the percentage of particles for various particle size in the total particle group, which is expressed in decimal. it should be emphasized that which kind of method is used to obtain the efficiency when the efficiency value is mentioned. for example, when the arrestance with atmospheric dust is 98 %, it will bring misunderstanding or error when the efficiency is only said to be 98 % or the arrestance is 98 %. this will be explained in detail in chap. 17. in most cases, people care not only how many particles are captured on air filters but also how many have penetrated through air filters. the concept of penetration (or penetrating coefficient) can be used to represent the extent of the result, although the basic meanings are the same. in the exhaust cleaning system, penetration is used to replace filtration efficiency. it is customary to label penetration with k, i.e., for cases of η 1 ¼ 0.9999 and η 2 ¼ 0.9998, the difference between them is not substantial. when penetration is used, we get k 1 ¼ 0.01 % and k 2 ¼ 0.02 %, which means k 2 is two times of k 1 . when a filter with penetration k 2 is used, the number of particles penetrating through the filter is two times of the filter with penetration k 1 . this will attract people's attention. decontamination factor k c is defined as the reciprocal of penetration, i.e., it means the extent of the decrease of particle concentration when air passes through filters. when k ¼ 0.01 %, this means the difference between upstream and downstream of air filter is ten thousands. pressure drop of air filter is composed of two components: filter media and structure of air filter. pressure drop of airflow entering and exiting air filters is usually constant, which is about 5 pa and could be added as a fixed value. the following part will emphasize on the aforementioned two parts of pressure drop. in some literature and monograph, actually only the pressure drop of filter media layer is mentioned during the introduction of pressure drop of air filter, which will cause misconception to readers. for fibrous filter, pressure drop of filter media is caused by the frontal resistance during the airflows through fibrous layer. pressure drop depends on whether the airflow through fibrous layer is laminar or turbulent. generally speaking, extreme small fiber and low filtration velocity will result in extreme small re number, so airflow is laminar. for the isolated cylinder with unit length, when its long axis is perpendicular to the airflow, the force acting on its surface is a function of the cross section and dynamic pressure, i.e., where f is the drag force, n/m; c 0 is the drag coefficient; ρ a is the gas density, kg/m 3 ; v is the filtration velocity, m/s; d f is the fiber diameter, m. the drag force acting on all fibers inside filter media is fl where l is the total length of fibers. the drag force acting on all fibers inside filter media equals with the force that the filter media bears. when it is equally shared to the surface area, the pressure drop is obtained, which is expressed as δp and shown in eq. (3.40) . is the fiber length per unit volume; h is the thickness of filter layer; s is the area of filter media, i.e., filtration area. inserting eq. (4.10) into eq. (4.11), we could get this is the theoretical expression of pressure drop. the problem is how to determine the drag coefficient c 0 . because the value of c 0 is related to the arrangement of fibers, solid fraction and re number, it is impossible to obtain the relationship between δp and every parameter directly. therefore, experiment needs to be carried on. results from experiment on the five obvious factors show that [ it is known that δp / α m 2 , so replace α in the above equation with α m 2 and c 0 becomes c 0 m , i.e., with the experiment on fibers with different cross section and different re number, the relationship shown as the straight line in fig. 4 .1 could be obtained, i.e., where c 0 m is the correction factor of pressure drop when influencing factors such as cross-sectional shape are considered while the influence of α is ignored; k ¼ 60; inserting eq. (4.14) into eq. (4.13), we could get: in this equation, the relationship between δp and every parameter is consistent with the experimental results. for example, δp of filter media is linearly proportional to filtration velocity v, filter layer thickness h, and α m 2 , while it is inversely proportional to d 2 f . this means the equation is valid. according to eq. (4.15) which is the method of susumu and other equations by related literatures, pressure drops of three kinds of fibrous layer can be obtained, which is presented in table 4 .10. substantial difference exists between calculated results by various methods and actual experimental data. result given by norio method has the largest difference. there are many aspects for the difference, of which the accurate determination of every parameter is also important. it is comparatively easy to calculate by eq. (4.15), while it is complex to use other two methods which will not be introduced in detail here. for given air filter, the filter media is chosen, so h, α, d f, and φ are fixed. equation (4.15) can be simplified as this means for given particles, pressure drop is linearly proportional to filtration velocity in quite large range of filtration velocity, where a is a structural coefficient to reflect the structural characteristic of fibrous layer. give experimental results for nonwoven coarse filter media, medium-efficiency air filter media, and sub-high-efficiency particulate air filter media [6] . in these figures there are "front and rear." fibers near the front side are relatively large. inside the coarse filter media, fibers are inherently large and spaces between fibers are loose, so difference is small between front and rear. inside medium-efficiency air filter media, fibers near the rear are relatively dense and they will interference with the airflow. sub-high-efficiency particulate air filter media used here is not only polypropylene fiber filter paper, but it is composed of prefilter layer, main filter layer, and enhanced gauze. when the enhanced gauze is placed windward and leeward, the function of preventing filter media from stretching and deformation is different, so the resultant pressure drops are different. for common polypropylene fiber filter paper without enhanced gauze, the difference is not obvious. from above figures, we could see that for high-efficiency filter media, v is below 0.2 m/s; for sub-high-efficiency particulate air filter media, v is below 0.5 m/s; for medium-efficiency air filter media, v is below 0.8 m/s; and for coarse filter media, v is below 1.2 m/s. 2 pressure drops of various filter media: 1 foreign aec filter medium, 2 25 wire glass fibrous filter paper, 3 synthetic fibrous filter paper, 4 20 wire glass fiber filter paper, 5 φiiii-15 cloth, 6 synthetic fiber no. iv filter paper, 7 eight wire glass fibrous filter paper, 8 synthetic fiber no. 1 filter paper; 9 synthetic fiber no. ii filter paper; 10 five wire glass fibrous filter paper, 11 chemical microporous membrane for these four situations, the following approximated relationship is valid, even the filtration velocity is larger than the limit: except for the pressure drop of filter medium, structural pressure drop of air filter must be added to form the total pressure drop of air filter, where pressure drop of inlet and outlet of airflow occupies very small proportion. there is one view that except for the inherent structure of air filter, pressure drop of structure is also affected by filter media performance. the penetrating performance of filter media may influence the flow state passing through air filter; thus, the pressure drop of structure is affected. this view needs further experimental validation. experiments show that pressure drop of structure is no longer linearly proportional to the airflow velocity. the main reason to nonlinear relationship is that face velocity u is used to describe the airflows through filter frame, which has the magnitude of m/s and is much larger than the filtration velocity passing through filter media layer. the structural size of filter frame is much larger than that of fiber, so inertial force cannot be ignored at large re number flow (usually re > 1), and the flow is not laminar. in this situation, pressure drop is not linearly proportional to the velocity but is proportional to u n . therefore, pressure drop of air filter structure can be expressed as where b is the drag coefficient of air filter structure. the total pressure drop of air filter is: it is obvious that values of a and b are different for different air filters. taking a domestic-made gb-01 hepa filter for an example, experiment shows that n ¼ 1.37. when expressed with unified filtration velocity v, the total pressure drop can be written as: [7] , from which we can see that for hepa filter, the value of m is slightly larger than 1 when v ≯ 3 m/s, i.e., the flow rate is slightly larger than nominal flow rate. so the resultant error will not be big when the relationship between pressure drop and flow rate is considered to be linear. sub-high-efficiency particulate air filter has similar feature, which will be illustrated in fig. 4 .26. however, as for coarse filter and medium-efficiency air filter, since their structures differ a lot, the above characteristic is no longer common. dust holding capacity is an index directly related to the lifetime of air filter. when the final pressure drop of air filter at operation is about two times of the initial pressure drop (if two times is too low, other ratio can be set), or when the efficiency becomes less than 85 % of the initial efficiency, the dust weight deposited on air filter is called the standard dust holding capacity of this air filter, which is called dust holding capacity for short. when the flow rate is 1,000 m 3 /h, the dust holding capacity of common folded nonwoven air filter is about 100 g and that of glass fibrous air filter and hepa filter are 250-300 g and 400-500 g, respectively. even for the same kind of air filter, dust holding capacities are different for different size. it is shown that if the relationship is approximated with straight line, error is not large when it is below the standard dust holding capacity or the pressure drop increase is smaller than two times of initial pressure drop. the maximum difference of pressure drop for these three filters is within 10 pa. the less the upstream concentration is, the stronger the linear relationship is. so when prefilter especially one with high efficiency is usually placed before hepa filter, this characteristic appears. if the filtration velocity is larger than ordinary value or dust deposited weighs more than standard dust holding capacity, the pressure drop will increase sharply with the increase of the deposited dust. the increase of pressure drop is usually linearly proportional to the increase of deposited dust for medium-efficiency air filter. during the deposition of dust, the efficiency of filters with low efficiency will increase at first and then decrease. this is because the dust deposited is comparatively large for air filters with low efficiency and the filter medium is sparse, which will cause particles to penetrate when pressure drop increases and cause deposited particles to rebound and resuspend. during the operation of hepa filters, efficiency usually increases with the increase of deposited dust. classification of air cleanness at home and abroad is mainly evaluated with particle number of those with diameter !0.5 μm per unit volume air, while various kinds of air filters are evaluated with the fractional efficiency corresponding to certain particle size. therefore, during the design process in air cleaning technology field, efficiency of these certain particle size needs to be converted into these particles with diameter !0.5 μm. before delivery or during characterization, hepa filter is evaluated with monodisperse particles with diameter 0.3 μm, which has been introduced in the former chapter. in order to convert into efficiency with particle diameter !0.5 μm, the efficiency corresponding to 0.5 μm needs to be known. according to foreign experimental data, an empirical expression has been derived for the relationship between penetration of hepa filter and particle size [8] , i.e., where k 1 , k 2 refer to penetration of particles with diameter 0.3 μm and certain diameter which is larger than 0.3 μm, respectively; d 0.3 , d are the particle diameter 0.3 μm and certain diameter which is larger than 0.3 μm, respectively. the above equation was used to perform calculation on measurement data published abroad [9] , which is shown in table 4 .11. in the table, k 0 2 and k 1 are measurement data and k 2 is the calculated data with eq. (4.20) . from the comparison between the last two columns, we can see that except for the comparatively large difference for the data on the first row, differences of other data are extreme small. this empirical equation is only valid for hepa filter with 0.3 μm, while it is not useful for hepa filter with 0.1 μm. although the test methods for the efficiency of hepa filter are different at home and abroad, the results are almost consistent with that of particle counting efficiency with particle diameter 0.3 μm, which is shown in chap. 17. efficiency for hepa filter with particle diameter 0.3 μm is usually considered as the reference baseline, so eq. (4.20) is used to obtain the relationship of hepa filter between 0.3 μm and 0.5 μm, which is shown in fig. 4 .11 for reference. meanwhile, fig. 4 .12 shows the curve in ref. [10] , from which we can see that these results match well with each other. efficiency η in the figure is represented with decimal. according to the above curve and the particle diameter distribution of atmospheric dust introduced in chap. 2, the efficiency with particle diameter !0.5 μm can be derived, which is shown in table 4 .12. according to national standard "high-efficiency particulate air filter," filter with efficiency larger than 99.9 % is called hepa filter. for filter with efficiency equals with 99.9 %, efficiency with particle diameter larger than 0.5 μm becomes 99.9975 %, which is shown in table 4 .12. since efficiency of common hepa filter is actually larger than this limit value, it is reasonable to consider efficiency of particle diameter !0.5 μm to be 99.999 %. with the above conversion method, we compare the experimental data performed on hepa filter in the cleaning equipment with calculated data and find they are consistent. there is no literature abroad specially dealing with this problem. in ref. [11] , it is said that "for obtaining class 100 clean environment with all the fresh air when the particle concentration of outdoor air is about 3 â 10 5 relationship between efficiency of hepa filter and particle size pc/l (!0.5 μm), it's necessary to set filters with minimum efficiency 99.999 %. so hepa filter with efficiency larger than 99.95 % is recommended to use as the main filter." here hepa filter with efficiency 99.95 % (for 0.3 μm) is thought to have efficiency 99.999 % with particle diameter !0.5 μm. for comparison, according to the above conversion method, when particle efficiency is 99.95 % for particle diameter 0.3 μm, its corresponding efficiency for particle diameter 0.5 μm becomes 99.9992 %. domestic-made medium-efficiency air filters include glass fibrous mediumefficiency air filter and foam medium-efficiency air filter. at present the most commonly used is nonwoven medium-efficiency air filter, which is actually a kind of fiber felt air filter. particle counting efficiency of glass fibrous medium-efficiency air filter (d f ¼ 16 μm, h ¼ 20 mm, α ¼ 0.037, v ¼ 0.28 m/s) with atmospheric dust was performed at institute of hvac of china academy of building research, which is shown in table 4 .13. in the table, average efficiency corresponds with the arithmetic average diameter of atmospheric dust, and efficiency is obtained with the theoretical curve of fig. 4 .13 with this average diameter. this theoretical curve is obtained by the method of structural nonuniform coefficient. since the grouping range of particle diameter is comparatively large, difference between average particle diameter and actual value is large. but it is shown from the table that the efficiency calculated with average particle diameter is close to the actual measured data, fig. 4.12 relationship between efficiency of hepa filter and particle size [10] which is satisfactory. the calculation method has certain reference value. it is also shown from the figure that difference between calculated efficiency and experimental one is comparatively large when experimental coefficient method and the value of η 0 σ with eq. (3.21) are used. figure 4 .14 shows the experimental data of glass fibrous air filter and foam air filter at home and abroad. it is shown that the difference of efficiency between 0.5 and 0.3 μm is quite small, as well as the difference of efficiency between !0.5 and !0.3 μm. this is because filtration mechanism for medium-efficiency air filter for small particles has little difference. from fig. 4 .14, the following approximated relationship exists when η < 0.8: in actual air cleaning system, filters are usually placed in series. here the efficiency of air filters in series is emphasized. in filtration theory, for filtering polydisperse aerosol with the same kind of air filter (e.g., they are all fibrous medium-efficiency air filter or hepa paper filter), the penetration of second air filter should be larger than that of the first one, i.e., the efficiency of second air filter decreases. this is resulted from the selectivity of particles by filter medium, which has been introduced before. in short, mainly because the filtration mechanism for different particles is different, the dispersity of particles after the first air filter varies, which results in the change of total efficiency for the second air filter. from eq. (4.7) to derive the efficiency corresponding to various particle diameters, we can see that in order to calculate the efficiency of second air filter, the particle size distribution after the first filter and efficiency of various filers for different particle size must be known. these two problems have been solved, so detailed calculation can be made. table 4 .14 presents the calculation results for two hepa filters in series (when the atmospheric dust concentration m ¼ 10 6 pc/l) [12] . under normal conditions, atmospheric dust concentration is m < 10 6 pc/l. with the decrease of m, the absolute quantity of large particles decreases. so the number of large particles passing through the first air filter is close to zero, which makes the proportion of large particles upstream of the second filter smaller. efficiency of the second filter for particle diameter !d is approaching to that for particle diameter equals with d. this means the penetration of the second air filter is close to be two times of the first filter. therefore, when the third hepa filter is placed, its efficiency for particle diameter !d is much closer to that for particle diameter equals with d of the first filter. when d is 0.3 μm, efficiency decreases from 0.99955 to 0.9991, or the penetration increases two times from 0.045 to 0.09 % and then reaches stable. if monodisperse aerosol is filtered, the change of efficiency for various stages of air filter is small. reports on the problem of efficiency for air filters in series are rare [13] . both theoretical calculation and experimental data in field have proved that the efficiency of the second filter decreases a lot. there are two reasons: one is that particle concentration becomes extremely small after passing through the second air filter and so on, so data is not accurately measured because of the limit of measurement techniques, and even reverse conclusions are obtained, which has been clearly mentioned in the "nuclear air cleaning handbook" [14] ; the other is that during the field test, particle concentration will increase downstream of air filter in case of sealing problem during installation or even the trivial leakage. field test data from japan are listed below [13] : the increase of penetration between the third hepa filter and the second one is larger than the calculation result. tester have pointed out that this is caused by leakage. if there were no leakage made by improper installation, there would be no much difference of efficiency between the third and the second filter. for this aspect, the strict experimental data cited by "nuclear air cleaning handbook" denied the opinion that the efficiency of air filter in series will decrease. according to the data from "nuclear air cleaning handbook," the decontamination factor of the first hepa filter is 10 4 , and that of the second hepa filter remains the same, while that of the third hepa filter is 5 â 10 3 . it is known that so k 3 is two times of k 1 , which is consistent with the calculated result which increases from 0.045 to 0.09 %. therefore, it is suitable to choose hepa filters with these recommended penetrations for exhaust cleaning system: the first hepa filter k 1ð!dþ the second hepa filter k 2 ¼ 2k 1ð!dþ the third and later hepa filters k 3ðdþ 4.8 efficiency of air filters in series k 3(d ) means the penetration of the third and the following hepa filters for particle diameter !d equals with that for particle diameter d. in the inlet air cleaning project, efficiency for two hepa filters in series is quite large, so the influence of the decrease of the efficiency of the second air filter is too small to be neglected. the total efficiency can still be written as: there are two aspects of meaning to prove the small decrease of efficiency for hepa filters in series: 1. in the application field of exhaust cleaning system. as pointed out in nuclear air cleaning handbook, the emission permission for radioactive elements concentration (such as plutonium or other super uranium substance) is extreme low, so it is not enough to only install one hepa filter in the exhaust system. since the efficiency of air filter in series does not decrease, it is preferred to install two or more filters in series, which is easy to increase the efficiency of the first filter. 2. in the application field of cleanroom for cleanness higher than class 100. when a hepa filter is installed in series in the fresh air system, the influence of leakage is smaller. some cleanroom projects in china have adopted this method and the effect is satisfactory. for two medium-efficiency air filters in series, the efficiency of the second filter almost remains the same. if both filters are glass fibrous filters, in theory η !0.3 ¼ 0.4 and η !0.5 ¼ 0.54. we can obtain that the percentage of particles with diameter !0.5 μm decreases from 30 to 15 %. the efficiency for particle diameter !0.3 μm is 0.39 and that of particle diameter !0.5 μm is 0.54 which remains the same. so the total efficiency of coarse and medium-efficiency air filters in series can be written as: the weight of dust deposited on air filter can be expressed with the following equation: where p is the weight of deposited particles on air filter, g; t is the lifetime of air filter, d; n 1 is the particle concentration upstream of air filter, mg/m 3 ; q is the flow rate, m 3 /h; t is the operational time per day of air filter, h; η is the arrestance of air filter. when air filter is operated under the rated flow q 0 and the pressure drop increases to several times of initial pressure drop (usually it is two times), the air filter can no longer be used, and the weight of deposited particles is called standard dust holding capacity p 0 . the used time of air filter is called lifetime t 0 , i.e., where n 1 can be calculated with the method in chap. 10, i.e., where m is the atmospheric particle concentration, mg/m 3 ; s is the recirculation air ratio; n r is the return air concentration. for cleanroom with class 10 000, the concentration is between 0.001 and 0.01 mg/m 3 ; η n is the arrestance of air filter in the fresh air ventilation system; η r is the arrestance of air filter in the return air ventilation system. for different systems with different η n and η r , the detailed calculation method will be introduced in chap. 10. for example, p 0 ¼ 450 g for flow rate 1,000 m 3 /h, filter), q ¼ 1,000 m 3 /h, the service life of hepa filter can be calculated to be 660 day. if the operational time per day is 12 h, t can be prolonged to be 1,320 day, which is 3.5 years. since particles will also be deposited onto other surfaces, the lifetime of hepa filters is longer than that of calculation. figure 4 .15 shows the relationship between operational time and the increase of pressure drop of hepa filter [15] . in the figure, the dust spot efficiency of prefilter in curve b is 40-50 %, which is equivalent with the arrestance with atmospheric dust shown in chap. 17. the service time of air filter is close to the data in the above example. it should be mentioned that there is one opinion that the increase of pressure drop of hepa filter is faster than that of dust holding capacity, so it is unsafe to calculate the service time with dust holding capacity [16] . actually it is not clearly pointed out here. since the concept of dust holding capacity has included the increase of pressure drop, the service time or lifetime calculated with dust holding capacity equals with the operational time needed when the pressure drop becomes two times of the initial pressure drop (or other certain times). the following relationship is obtained from eq. (4.24): it should be noted that t 1 is not the lifetime of air filter under that flow rate q 1 but is the time needed for the weight of deposited particles on air filter to be p 0 under the flow rate q 1 . for example, if and vice versa. here t 1,0 is the lifetime under the flow rate q 1 . operational pressure drops are different when q 1 6 ¼ q 0 . experimental data were given which is given in fig. 4 .16 [17] . it is shown that when q 1 ¼ 1 2 q 0 , the lifetime is larger than 2 t 0 . when we denote q 1 q 2 ¼ k and the pressure drop h, tu guangbei obtained the following equations based on these curves [18] : the following comprehensive equation was obtained when log-log plot paper was used with k as the abscissa (fig. 4 when h ¼ h 0 , t becomes the lifetime t 0 . the value of t is only positive. however, the above expression was obtained from one test case, and it is inconvenient to use it for calculation, which is not obvious to obtain the characteristic at a glance. so it is still unsure whether the relationship between t and k is also universally valid. from another point of view, author proposed an approximation method for the theoretical analysis of relationship between t and k [19] . when both the operational and rated flow rates are known, the change trend of the lifetime can be calculated. for example, the increase of pressure drop is δh under the rated flow q 0 when the initial pressure drop is h 0 and standard dust holding capacity is p 0 , so the final when the flow rate becomes , what is the time t 1,0 when the pressure drop reaches h 0 + δh? simplification was made with the assumed condition δh % h 0 . from the aforementioned introduction, we know the increase of pressure drops of hepa filter and sub-hepa filter are linearly proportional to the weight of dust deposited under this condition. 1. from eq. (4.25), it is known that the operational time is reversely proportional to the flow rate, i.e., 2. after operation time t 0 k under flow rate q 1 , the weight of deposited particles becomes the standard dust holding capacity p 0 , but the finial pressure drop at this time is still far from h 0 + δh. from figs. 4.6, 4.7, 4.8, and 4.9, it is shown that h is approximately linearly proportional to q (when q is less than q 0 or slightly larger than q 0 ). since the final pressure drop is (1 à k ) times less, continuous dust loading process is needed to increase the pressure drop. it is known the pressure drop increase is approximately linearly proportional to the weight of deposited particles, and the weight of deposited particles is also linearly proportional to the time, so the time needed for continuous particle loading process or the increased time is: 3. according to the relationship between h and q, the initial pressure drop decreases to kh 0 under the flow rate q 1 , this is (1 à k) times less. if the operational flow rate is q 0 and (1 à k ) times of original pressure drop is added, the prolonged time needed is (1 à k )t 0 . now the operational flow rate is q 1 , so the time should be reversely proportional to q 0 , i.e., the actual prolonged time is 4. therefore, for the case k < 1, the time needed for the pressure drop to become the final pressure drop with k ¼ 1 should be . so the reciprocal of prolonged time q 0 /q 1 is obtained, which is the shortened time. with the above equations and principles, the relationship between k and t 1,0 for reaching the final pressure drop with rated airflow, which is shown in table 4 .15. in the table, k ¼ 1.25, which is equivalent with k ¼ 1 it is equivalent to the reciprocal of its multiple of t 0 , which is obtained with the value 0.8. taking fig. 4 .16 as an example, the lifetime t 0 of air filters for different k is calculated with the above analysis method, which is illustrated in table 4 .16, together with the measurement value and the calculated result with eq. (4.31). from the above comparison, results from three methods are almost consistent. so it is feasible to adopt the analysis estimate method which reflects the general laws. equation (4.31) is also thought to reflect the general relationship, although it was derived from single case. one important conclusion from the above analysis is obtained. the operational flow rate of air filter is suggested to be about 70 % of the rated flow, and the lifetime of air filter will be doubled, which is beneficial for both economic operation and energy saving. in practice, it is impossible to directly estimate how much particles have been deposited onto filters. it is usually to determine whether to change air filters according to the measured pressure drop or the outlet velocity of air filter. for air filters used in the radioactive exhaust system, except for the index of dust holding capacity or pressure drop, the index of surface contamination is also used to determine the service time. air filters must be replaced when each index reaches the specified value. the extent of surface contamination of air filter is determined according to the specific situation of usage. eq arrestance of air filter is used to calculate the service time of air filter in previous section. experimental data of arrestance can be used if it is available. but in the current national standard the fractional efficiency with atmospheric dust is used to assess the efficiency of air filters in general ventilation. therefore, the particle counting efficiency should be converted into arrestance. here an estimation method is introduced [20] . taking the data in table 2 .28 as an example, particle size distribution between 0.5 and 1 μm can be divided into the following parts according to the relationship shown in table 2 table 2 .28, when the particle counting efficiency for particles with diameter !0.5 μm is 100 %, at least 99 % of particles by weight are filtered. weight of particles with diameter less than 0.5 μm occupies 1 % of the total weight. since part of particles with diameter less than 0.5 μm will be captured, the penetration will be less than 1 %. this can be omitted since it is a small value. that is to say, when the particle counting efficiency for particles with diameter !0.5 μm is 100 %, the arrestance cannot reach 100 % in theory, but it can be still considered as 100 % because the error is less than 1 %. from the above analysis, we know when the particle counting efficiency for particles with diameter !1 μm is 100 %, at least 97 % of particles by weight and 18.51 % of particles with diameter larger than 0.5 μm will be captured. since part of particles with diameter between 0.5 and 1 μm will also be captured, the total arrestance will be slightly larger than 97 %. for the convenience of estimation, this excess value can be omitted. therefore, when the particle counting efficiency for particles with diameter !1 μm is 100 %, the corresponding arrestance can be estimated as 97 % or 100 %. when the particle counting efficiency for particles with diameter !1 μm is 80 %, 18.51 % â 0.8 ¼ 14.81 % of the total number with particle diameter !0.5 μm will be captured, so less particles with diameter between 0.5 μm and 1 μm will be filtered (because efficiency for particles with diameter !1 μm is less, the corresponding efficiency for smaller particles is much less, which can be omitted). therefore, the arrestance corresponding with the particle counting efficiency 14.81 % with particle diameter !0.5 μm can be used to express the particle counting efficiency 80 %, i.e., 96.5 %. but when only the particle counting efficiency with particle diameter !5 μm is known without any information about the efficiency with particles diameter between 1 and 5 μm, the above method to estimate the arrestance is not valid since the weight percentage of these particles cannot be omitted. according to the above analysis, the data in table 2 .28 are used to plot fig. 4 .18. the arrestance found in the figure is an estimation value or the minimum limit value. fig. 4.18 conversion from particle counting efficiency to arrestance: 1 100 % efficiency curve for with particle diameter !0.5 μm, 2 100 % efficiency curve for with particle diameter !1 μm, 3 100 % efficiency curve for with particle diameter !3 μm, 4 100 % efficiency curve for with particle diameter !5 μm 220 4 characteristics of air filters now the filter-paper hepa filter is one typical example of folded filter-paper filter, which was developed in the nuclear industry in the second world war to remove radioactive particles. the main characteristic is its extreme low resistance because the filter paper is thin and filtration area is dozen times of frontal area by the folded structure, which makes the practical use of filter-paper filter possible. in 1942 the folded filter-paper hepa filter was first manufactured in the usa and put into the market for sale in 1954. in 1956 hepa filter was imported into japan from the usa. later in 1958 japanese began to develop their own product, which appeared in the market in 1965 [21] . in 1960s, chinese began to develop hepa filter, which passed the identification and began the mass production. the first filter-paper material used in the air filter of nuclear industry was plant fiber together with blue asbestos fiber. blue asbestos fiber is very fine with diameter between 0.1 and 1 μm. the yield is very low. it is thought that asbestos fiber will cause cancer, so it is gradually replaced by popular ultrafine glass fiber and glass fiber filter paper, which promotes the application of high-efficiency filter-paper filter and the development of air cleaning technology. filter-paper hepa filters can be classified according to the type of filter media material, and they can also be divided based on whether separator is used; whether the diameter of filtered particle is 0.3 μm (it is called common hepa filter or 0.3 μm filter) or 0.1 μm (it is called ultrafine air filter or 0.1 μm filter); whether the frame material is board, laminate, plastic plate, aluminum alloy plate, steel plate, or stainless steel plate; whether the structural shape is flat or v type; and whether it is able to endure high pressure, endure high humidity, and endure acid and alkali, high resistance, low resistance, or sterilization. at present there are three types of structure in filter-paper filter, i.e., with separator, with inclined separator, and without separator. the product of air filters with inclined separator is rare, while the other two types are popular. structures of these three types are shown in figs. 4.19, 4 .20, and 4.21. inside hepa filters, separator is placed between two sides of folded filter paper to provide the airflow channel, which is the standard practice. so it is called as separator hepa filter. separator is also called as corrugated separator. after hot rolling stamp, high-quality kraft paper can be used to make the separator with different crests and pitches. in order to prevent the particle emission from the stretch of separator caused by the cold, hot, dry, or wet conditions, as well as to fix the separator shape, both sides of separator should be immersed into some kind of coating material, which has the disadvantage of abnormal odor. now chrome papers gluing at two sides are used to make separator. but some practical experience has shown that there is some hidden danger: particles will be released as the pollution source because of its stretch deformation with the variation of temperature and humidity. so aluminum and plastic can also be used to make separator. for the separator air filters, corrugation angle is one important parameter, which has great influence on the pressure drop. practice has shown that 90º corrugation angle is suitable. the influence of cross-sectional area on the pressure drop is not large. with the large cross section and same filtration velocity and thickness along the flow direction, the corrugation height has comparatively large influence on the pressure drop, which will be analyzed in detail later. the traditional practice to make the separator air filter is to glue at both edges of the corrugation at first, then the endsealing glue is added to the inner side at both two edges of wooden frame painting so as to make the glue sides in order and to prevent leakage. but practice has shown that the endsealing glue has little effect on the leakage prevention, and once there is leakage, it is more difficult to detect the leakage and make repair. taking the current gb-01 type air filter as an example, the cross-sectional area of filter cartridge is about 0.454 m â 0.454 m ¼0.206 m 2 and the width of endsealing glue is 1.5 cm. when it was considered as 1 cm, the corresponding cross-sectional area of filter cartridge is about 0.454 m â 0.434 m ¼0.197 m 2 , which means the cross-sectional area without endsealing glue is 5 % larger than that of with endsealing glue. when the net area of filter paper increases, the pressure drop will decrease to some extent. therefore, the practice to use the endsealing glue on air filter is canceled, which is replaced with potting glue method or inserting glue method. for traditional separator air filter, the cross-sectional areas of air channel formed by the corrugations on the separator are the same. when the concept of area variable cross sections is applied on air filter, inclined air filter is created, whose projection shape is a right-angled trapezoid when the separator is erected. the cross-sectional area is large when air enters into the channel. as air goes through the filter paper, the air volume near the channel terminal is the minimum, so is the cross-sectional area. in this way, both the length of filter paper around each separator and the number of corrugations increase. according to the product specification abroad, the filtration area will increase by 50 % for inclined separator air filter compared with vertical separator air filter, so the pressure drop under the same flow rate will be much less. another method of improvement on traditional hepa filter is to cancel the separator. it is beneficial for the mechanized production of mini-pleat filter. one case is to cancel the separator, and the filter paper is folded with corrugation matching corrugation and point matching point after corrugation and salient point are pressed on filter paper. the other case is to replace the separator plate with other separator, such as the streak formed by thermosol on filter paper, the dipping flame retardant silk thread, glass fiber thread, or filter strip pasted on filter paper. during the process of folding filter paper, filter strips are inserted from the two sides of corrugation and they are held with the friction force. it should also be mentioned that the cross-sectional size of domestic-made hepa filter includes 484 mm â 484 mm, 630 mm â 630 mm, etc., which are quite irregular. national standard issued in 2008 canceled the specification of maximum overall dimension. it also requires that (1), (2) separator plate is 5-8 mm lower than the frame edge, and (3) the filter element is 3-5 mm lower than the separator edge. the national standard also provides the following specification: (1) the frame width is 15 mm (when the side length is less than or equal to 600 mm) or 20 mm (when the side length is equal to or larger than 600 mm). this is a way to guarantee the quality of air filter, but some manufacturer does not pay attention to it. during the calculation related to air filter, these data should also be considered. in order to enlarge the filtration area, double folding structure is also adopted. the first folding structure is for filter paper itself, which means a piece of folded filter material is used. the second folding structure is the w type structure inside the frame. the structure is shown in fig. 4 .22. in early times, the former ussr made transversely placed large cylindrical filter with ϕii-15-1.5 filter fabric which is hard to be folded but can be pasted, which is shown in fig. 4 .23. strictly speaking, that is a kind of equipment, not a single air filter. ycg-type low-resistance sub-hepa filter is one typical example of filter-paper filter, which was one type of air filters with lower structural resistance innovated firstly in china [23] . hundreds of filter cylinders are hot welded with polypropylene fiber filter paper. filter cylinders are plugged onto the panel with the plastic cap stopper with wings. wings are meant to support the filter cylinders and separator the air channel into two parts. adhesive-free product, so secondary pollution caused by peculiar smell does not exist, which is suitable for the application with stringent environmental requirement. 5. light. the weight is only half of the hepa filter with the same size. structural calculation of this kind of air filter will be introduced in chap. 5. it usually means the filter paper made by plant cellulose. several domestic-made air filters with performance similar as sub-hepa filter are made of short cotton lint filter paper. the characteristic of this kind of filter paper is that its efficiency is between medium efficiency and sub-high efficiency. the efficiency is small for low filtration velocity. efficiency increases with the increase of filtration velocity. the performance differs for different kinds of particles. table 4 .17 shows the filtration efficiency of one cellulose fiber filter paper with psl [24] . it is shown that for particles with diameter smaller than 0.557 μm, the efficiency is the minimum for filtration velocity 9.5-17 cm/s. moreover, the minimum efficiency corresponds with smaller particle size with the increase of filtration velocity. the surface dust holding capacity of this kind of filter paper is slightly larger than that of glass fiber filter paper by 60-70 %, but it is smaller than that of membrane filter. this kind of filter paper has high efficiency and high resistance. since the surface dust holding capacity is also very high, even higher than that of glass fiber filter paper, it is usually applied in the exhaust treatment system for nuclear facility. this kind of filter paper also has high efficiency. efficiency changes little with the change of particle type and filtration velocity. the relationship between efficiency and composite proportion of glass fiber inside the filter paper is shown in table 4 .18 [25] . the pressure drop is smaller than that of cellulose-asbestos fiber filter paper. fiber diameters inside glass fibrous filter paper become smaller. in 1970s diameter of foreign made filter paper reduces to 0.3 μm, and the value of domestic made reduces to 0.5 μm. figure 4 .27 presents statistical analysis of the fiber diameter distribution inside domestic-made filter paper using sem graph, where all the average diameters are slightly smaller than 0.5 μm. in 1980s the diameter of domestic-made glass fiber was smaller than that of foreign made, which reduced to 0.04 μm. efficiency increases apparently and that of some filter paper was higher than that made in the usa. however, the most obvious shortcomings of domesticmade filter paper are fiber shedding and the amount of particles deposited by filter media itself is great, which is related to the inaccurate control of manufactory environment and production process. furthermore, pressure drop of common hepa filter paper is very high under the usual filtration velocity, and that of most ulpa filters is even much higher [9] . since the synthetic fiber has high resistivity and can bring large amount of electrostatic charge, it is an ideal material for making electrostatic material. perchloroethylene ϕii filter fabric produced in the former ussr in 1950s is one of the kinds of fibrous filter paper. penetration is 20 % when filtration velocity is 0.1 m/s, while it is 3.2 % when filtration velocity decreases to 0.01 m/s and the corresponding d max is 0.1 μm [22] . polypropylene fibrous filter paper developed in the late 1970s is another example. its performance is much better than ϕii filter fabric. polypropylene slice is used to make ultrafine fiber through the meltblown process. further filtration material is manufactured, which is a soft nonwoven felt. diameter of single fiber is 2-18 μm (usually it is 4 μm). the sodium flame efficiency with standard specific velocity is 99-99.999 %. it is quite difficult to make the fiber diameter smaller. fibers are not uniformly distributed inside the filter media. electrostatic charge on the filter media fades away gradually, so at present it cannot be used to replace glass fibrous hepa filter. but it is indeed a promising filter media. except for the features mentioned in the above chapter and sections in this chapter, polypropylene fibrous filter media has the following characteristics: 1. pressure drop. under the same efficiency range, the pressure drop is only 1/6 that of glass fiber. table 4 .19 presents the pressure drop of several domesticmade polypropylene filter paper when the filtration velocity is 1 cm/s. the reason for small pressure drop is that the fiber diameter is comparatively large and particles can penetrate deeper (several hundred micrometer). but particles can only penetrate tens of micrometers from the surface of glass fibrous filter paper. 2. stability of electrostatic charge. after passing through the corona discharge, filter media become the electrets which carry large amount of electrostatic charge, and the surface electrostatic potential can reach 1,000 v. with the electrostatic effect, the penetration decreases by 1-2 order of magnitude. after the filter media is immersed into the alcohol and then dried in the vacuum, the electrostatic charge is neutralized and the efficiency decreases a lot, which is shown in figs. 4.28 [26] and 4.29 [27] . at the same time, it is also found that the electrostatic potential increases by the friction effect when air flows through the filter media, which is shown in table 4 .20. the potential at smooth side is high, so the efficiency using this side as the frontal face is high, while the pressure drops using both sides facing upstream is almost the same ( but experiment was performed on the electrets air filters with polyolefin fiber which concluded that efficiency decreased from 99.9999 to 99.99 % after 2 years' operation without any prefilters. the reason is that the electrostatic effect is shielded by the deposited particles [29] . when it is placed in the environment with relative humidity 80 %, no obvious influence is made on both the efficiency and pressure drop [28] . 2. temperature characteristic. the operating temperature is à40 to +110 c. when it is baked under the temperature 50 c for 4 h, no obvious change appears in both efficiency and pressure drop [26] . but efficiency decreases when the operating temperature is above 120 c. the melting point is 164-174 c. 3. density. it is 0.91 g/cm 2 . the solid fraction is 0.12 with measurement [26] . 4. characteristic of acid and alkaline resistance. except chlorosulfuric acid, concentrated nitric acid, and some oxidants, it has good performance of resisting acid, alkaline, and organic solvent. strength. the transverse tensile strength is larger than 500 g/100 â 15 cm, and the longitudinal tensile strength is larger than 1,000 g/100 â 15 cm. the strength is more than two times of glass fibrous filter paper. it is fold resistant. 6. environmental property. nontoxic, odorless, no borer, and it can be disposed by combustion. 7. ability to absorb oil. it can absorb oil with weight equals with 14-15 times of self weight. 8. bonding characteristic. it is difficult to bond with the glue, but it's easy to bond with iron. the gel type microporous filter membrane is the main form, which is made of the nitrocellulose. the gel is the mixture of the ether alcohol with the fibers of nitrate ester, and it is also called celloidin. when the celloidin is diluted with the acetone and the pentanol, the gel used for make the membrane is formed. this kind of filtration membrane has very high efficiency and surface particle deposition rate, so it is usually used to act as a standard filter paper to measure the efficiency of other filter papers. it is also used to capture radioactive particles, but it is not convenient to use since the pressure drop is high and the tensile strength is low. pores on the surface of microporous membrane are irregular, which is similar as that of foam. their sem figures are shown in figs. 4.32 and 4.33 [30] , where the spheres are methylene blue particles. nuclear microporous membrane is also one kind of membrane filter paper. it is called nuclear track microporous membrane, which is developed in the late 1960s. thermal neutron during the nuclear reaction is used to bombard the heavy element such as 235 u. then the fission fragment from 235 u is used to bombard the plastic film such as polycarbonate film or polyester film, or the heavy element such as k r and x e accelerated by the accelerator is used to bombard these films, so track injury is left. afterwards, they are etched with chemical reagent, and pores appear on the surface. its strength is good. it is fold resistant and can bear high temperature 140-170 c. pore density can be controlled when both the bombardment intensity and time are monitored. pore size can be controlled when the reagent concentration, temperature, and etching time are controlled. the thickness of nuclear microporous membrane is usually between several micrometer and dozens of micrometer. the thickness of domestic-made nuclear microporous membrane is 11 μm. diameter of pore size is between 30 å and tens of micrometer, and it is usually about 1 μm. the porosity is about 20 %. the monodisperse of pore size is better than chemical microporous membrane. since the surface is quite smooth, it is suitable for qualitative analysis of aerosol sampling and study of bacteria filtration. the pressure drop of nuclear microporous membrane is large, so it is not suitable for common air filter, but it is very useful for special filtration (for the application field where particles with diameter larger than certain value are not allowed to penetrate). it is shown from fig. 4 .34 that almost no particles appear on the rear face when the frontal surface is already clogged. it is widely used in the medical applications. as for the filtration mechanisms of nuclear microporous membrane, domestic scholars have already made detailed investigation [31] , which will not be introduced here. except for the above five kinds of filter paper, there is also plastic fibrous filter paper, which is not illustrated here. special attention should be paid on the representative features during the selection of filter paper, which is presented in table 4 .21. it is hoped that the larger tensile strength and smaller fiber are preferred. while larger thickness and larger solid fraction will result in high efficiency, but the pressure drop will increase dramatically at the same time. the content of metal component is one important feature for filter paper. when the content of metal component in the captured particles is investigated, the background value in the filter paper itself is needed. there is little study in this aspect home and abroad. for reference purpose, table 4 .22 presents the contents of several metal components inside the common glass fibrous filter paper according to the literatures home and abroad. it is shown in table 4 .21 that the largest shortcoming of this kind of glass fibrous filter paper is the weak tensile strength. the ability to bear the shock press is extreme low. during the manufacturing process, it is too fragile to be damaged if it is not careful enough. experiment of the compressive strength of hepa filter was performed using the shock tube. protection device was developed to increase the ability to bear high shock force. common hepa filter with tensile strength of filter paper no less than 230 g and other air filters were used in the test. different conditions were compared when no baffle was placed and different baffles were set 5 cm upstream of the air filter. the test results for common hepa filters are shown in table 4 where the white part means the damaged part of filter paper which turned outwards [32] . moreover, the stiffness and rigidity of filter paper have decisive influence on the height of corrugation and pressure drop. from the test results shown above, the pressure for damaging domestic-made glass fibrous filter paper (or 6901 filter paper) used in common hepa filters is less than 0.16 kg/cm 2 . according to american air force design manual (ad295408, tdr-62-138 report), the pressure to cause damage on aec filter from us atomic energy commission is only 0.14 kg/cm 2 . therefore, when glass fibrous filter is installed on the pipeline with shock press, the protection device must be installed, and the most common measurement is to place the baffle plate. the tensile strength of glass fibrous filter paper is very small, and it decreases a lot under the high wet environment, so it is easy to be blown through. if special treatment is made on filter paper, its tensile strength can be improved. domestic researchers have proved that when the filter paper is treated by spraying with "soft no. 1" leather treatment agent, the tensile strength can be increased by ten times, while the pressure drop does not increase too much and the efficiency remains the same [33] . experimental results are summarized in table 4 .24. table 4 .24 is the experimental result of pressure drop with high filtration velocity 0.16 m/s. pressure drop data for low filtration velocity are not available. with low filtration velocity, the rise velocity of pressure drop is slower than that of high filtration velocity. the filter paper is still undamaged when it is treated with "soft no. 1" leather treatment agent and then with steam. this means it can bear high temperature environment of steam for sterilization, so it is useful for pharmaceutical and biological clean rooms. it is dependent on the application and performance to choose what kind of components including filter paper, frame, and separator (corrugation) for making air filter, especially hepa filters. for information, table 4 .25 shows the general performance of air filter made by components with different materials. because the filter paper is too fragile to be damaged during the manufacturing process, the efficiency of filter-paper air filter is usually smaller than that of small filter-paper sample by "half 9," and the poorest performance difference could be less than "one 9." therefore, for making hepa filter with efficiency for particle diameter 0.3 μm to be above "three 9" (i.e., 99.9 %), filter paper with efficiency "four 9" must be used. with the development of science, technology and manufacturing process, both the standard and test methods for hepa filters in many countries are developing, which will not be introduced here [34] . more stringent requirement for filter-paper air filter will be put forward. there are following requirements [35] : 1. efficiency for particle diameter 0.1 μm should get close to 99.99999 %, or for particle diameter 0.01 μm should get close to "eight 9," which is called ulpa filter and shown in fig. 4 .37. 2. it is more stringent to make requirement on the chemical pollution of filter paper. at present, most of hepa filter is composed of filter media with ultrafine glass fiber which is made from silicon boric acid. 58 % of its component is sio 2 . in 1980s the requirement of silicon pollution was proposed. silicon particles volatilize and emit from the hydrophobia material, which cause harmful effect on the production of hard disk drive. in 1990s the problem of phosphorus pollution was put forward. phosphorus pollution comes from the seal glue of air filter, which may cause pollution to the wafer. in the late twentieth century, the problem of boron pollution was put forward, since about 11 % of the component in the filter media is b 2 o 3 . except for the boron pollution of atmospheric air as the main source, air filter is also one important source. under high-humid environment, hydrofluoric acid will make corrosion on glass fiber and produce gaseous boric acid if hydrofluoric acid exits, which will pollute the wafer. 1. the pressure drop with rated flow is smaller than 50 pa. 2. the deposited particles are unlikely to reenter into the flow. 3. no crack exits inside the structure of air filter, so no seal material is needed and no leakage appears on it. leakage test before delivery is not needed. 4. the lifetime is more than 5 years. 5. it is easy to handle after usage. in the late of twentieth century, ptfe filter with filter media made by microporous ptfe membrane appeared in the market. the average diameter of the pores the time of filter paper with width 7.5 mm and length 25 mm having fractures during the repeated process of stretching and folding, when its one side is fixed and the other side is connected with an eccentric wheel which has an eccentricity 7 mm and speed 1,390 r/min the main characteristics of ptfe filter is: it has strong ability to anticorrosion including acid and alkali. the volatile amount of chemical substance is extremely low. for example, the volatile amount of boron and sodium is only 1/400 to 1/200 of that from glass fiber filter. the pressure drop is very small, which is less than common hepa filter by 2040 %. at present the price of ptfe filter is comparatively high and the dust holding capacity is a little small. as for the structure of nonleakage air filter with the requirement as item (5) mentioned above, it has been replaced by author's invention patent of zero leakage air supply outlet. when common air filter is installed in this air outlet, no leakage into the room can be realized. fibrous layer filter is mainly composed of a fibrous filling layer. the fiber used can be divided into three categories: one is natural fiber such as wool and cotton fiber, another is chemical fiber which is made with chemical method to modify the characteristic of raw material, and the third is artificial fiber which is separated from the raw material with fibrous shape by physical method or formed to be fibers from raw material. for the second type, the chemical feature of fiber is totally different from that of raw material. for the third type, the chemical feature before and after fiber formation remains the same, such as spinning after the melt of glass. the surface feature of fiber has great influence on the filtration effect. taking natural fiber including wool and cotton as an example, the particle capture efficiency is higher than that of smooth plastic fiber because of the scale shape and fibrous shape. the efficiency of glass fiber improves after treatment with hydrofluoric acid [37] . in order to prevent the fiber abscission during operation, binder is sprayed onto fibers. fibers with different diameter can be chosen to make packed bed with different solid fraction. particles with large diameter are captured by crude fiber layer. posterior fine fiber layer is used to filter small particles. in this way, the needed filtration efficiency and dust holding capacity are guaranteed, and the resultant pressure drop is not too high. fibrous layer made by nonwoven manufacturing process can also be used to make air filter. figure 4 .39 shows the nonwoven bag filter. the common techniques include needle injection sticking method and hot melt method. fibers with different diameters are used as raw material. it forms the web shape after loose carding and folding. movement of thousands of needles on the needle board of the needle machine makes fibers move along the perpendicular direction. after going forward and backwards, certain numbers of fibers entangle together on the fiber web because of this movement. the glue is sprayed on the surface and then dried. this is called the needle injection sticking method. if fibers with low melting point such as polypropylene are added into fibers such as polyester, the fiber web formed by folding is placed into the hot melt equipment and heated to certain temperature, then fibers with low melting point are melted and binding other fibers to form the filter media. this is called the hot melt method. nonwoven fabric made by this kind of method is in the range of coarse efficiency, which is suitable for making rolling filter material. in terms of nonwoven fabric shape, it is one kind of felt fibrous layer. the thickness of this fibrous layer is between less than 1 mm and dozens of micrometer. the efficiency range is so large to cover the coarse efficiency and sub-high-efficiency ranges. taking one kind of domestic-made polypropylene nonwoven fabric pp-k 2 for an example, its thickness is 1 mm and the pressure drop is about 8 pa with the filtration velocity 0.06 l/ (cm 2 · min). the particle counting efficiency with atmospheric dust is 94 % when particle counter is used. it is comparatively cheap and is only several yuan for each meter squared. table 4 .26 presents the characteristic of several fibers, which could be used for reference during selection of filter material [38] . fibrous layer air filter has small solid fraction, so its pressure drop is very low. it is especially suitable for the application of hvac air cleaning system as medium-efficiency air filter. there are two principles during the design and selection of fibrous layer air filter. one is based on a single index. it is the priority to consider the requirement of efficiency during the design of air filter. so with the same filtration efficiency, the optimum of certain index is expected. for example, the minimum pressure drop is expected, when requirements of other indexes should follow this requirement of pressure drop. this is a simple case. the other is based on the comprehensive index. with the same filtration velocity, the optimum comprehensive index is expected. when this comprehensive index is labeled with e, chen proposed to use the ratio of efficiency and pressure drop [3] , i.e., it is obvious that this index is only related to the technical performance of air filter, while its economic performance is not included. if the cost of filter material was considered, the comprehensive index reflecting the usage of filter material should be used [5] . when this index is denoted with j, we get, where w f is the usage amount of filter in the filter layer per each meter squared. it is shown from the above two expressions that e is larger when the efficiency is larger or the pressure drop is smaller. j is smaller when e is larger or the usage amount of filter material w f is more. therefore, the smaller value of j is preferred when the efficiency of air filter is given. moreover, comprehensive analysis with the fuzzy method was performed [6] . it is comprehensive, but it is not easy for visual understanding because of so many influencing factors. the weight of each factor is determined subjectively by the interest of referee. the comparability of this index is weakened. whatever evaluation method is adopted, the filtration efficiency requirement is of the priority, and then other comprehensive indexes are considered. the following items should be noted during the design of air filter: first of all, filter material with suitable fiber diameter should be selected. it is known from filtration theory that efficiency decreases with the increase of fiber diameter, but the decrease velocity is slower than the decrease velocity of pressure drop caused by the increase of fiber diameter. meanwhile, for the given filtration velocity and solid fraction of fibrous layer, thicker fibrous layer is needed for large fiber diameter in order to obtain the same efficiency. although the usage amount of filter material is obviously much for thicker fibrous layer, the pressure drop still decreases compared with that of small fiber diameter. therefore, suitable fiber diameter should be determined according to the design requirement. secondly, the fibrous layer thickness should be determined. this index depends on the structure and operation condition of air filter. thirdly, it should be remembered to keep the structure from being too close or too loose. it seems that the corrugation number could be increased, but this will cause two unreasonable consequences. one is that air does not flow thoroughly near the corrugation edge and stagnant airflow space is formed, so the effective filtration area reduces. at the same time, the corresponding support for filter material increases, so part of filtration area reduces by 7-20 % since the filter material is sheltered by the support. the other is that distance of filter material in each corrugation is very close when the corrugation number increases. under the airflow pressure, the soft filter material between two corrugations squeezed together, which increases the pressure drop. in order to obtain the maximum effective filtration area, comprehensive consideration of various factors including filter thickness, ridge distance, ridge angle, and filter material thickness. take bag-type air filter with frame of fixed volume, for example. when bag number increases from two to four, the filter material area increases and the pressure drop of filter material decreases. but the pressure drop of structure will increase because the flow channel is narrowed to increase the pressure drop of structure. when the number of bags is not large, the pressure drop decreases with the increase of the number of bags. on the contrary, when the number of bags is comparatively large, the pressure drop increases. the decrease of pressure drop of filter material cannot offset the increase of pressure drop of structure. meanwhile, when the number of bags is large or they are too long, the two walls of neighboring bags almost contact each other under the effect of airflow. so it is meant to increase the filtration area by increasing the bag number, but the result is different. it can be improved by setting formed line inside the bag or setting frame outside the bag. with the formed line, walls of bags will be tightened and bags will not be stretched outside. with the frame, walls are kept from stretching outside. for the filtration velocity between 0.2 and 0.5 m/s, the pressure drop can decrease by 30-50 %. there are similarities between folded air filters and the bag filter. if no frame is placed, with the same total filtration area, the more the number of bag is (or the smaller the bag is), the narrower the airflow channel is, and the larger the structural pressure drop is. since the pressure drop of filter material is the same, the total pressure drop increases. this is consistent between experiment and theoretical analysis [6] . foam air filter is a combination of individual pin hole. membrane between pin holes is melted with the chemical treatment, so that air can flow through it. it is then used as filter material. foam plastic filter is one example. it is composed of three dimensional net skeletal frames, whose cross section is not circular. the size of this skeletal frame differs a lot. when the skeletal frame is considered to be fibers inside the filling layer, the filtration efficiency can be derived with the application of fibrous filtration mechanism [39] . figure 4 .40 is the calculation plot for efficiency. in the figure, the number of pores is counted with microscopic after the surface of foam plastic is dyed. the usage of foam plastic filter is rare. in this section, the mechanism and device of electrostatic precipitation will not be introduced comprehensively. only one kind of popular electrostatic precipitation equipments -electrostatic cleaner is presented. in the turbulent cleanroom, eddy current forms near the four corners of rooms because of the limit of air supply mode, where the cleanness cannot be improved by air distribution. because of the eddy current and the dust source, the cleanness of the room will be greatly influenced. in order to reduce the particle concentration in these regions, local cleaning equipment can be used. air is cleaned when the local air goes through the air cleaner repeatedly. the pressure drop of electrostatic cleaner is very small, and it is usually only 10-20 pa. the axial fan can be used and the noise level is low. besides it has the advantage of flexibility and convenience to use. so it is especially suitable for self-purification of indoor air. in the past, the electrostatic cleaner was called electrostatic self-purifiers in china. if a layer of activated carbon filter is added into the electrostatic cleaner, it also has the effect of gas and carbon dioxide adsorption. now this kind of electrostatic cleaner can be used as self-purification in rooms where the air cleanness is required. for example, it can be applied in meeting room, guest room, and living room. test has shown that when an electrostatic cleaner with two-stage ionization and efficiency of 95 % operates in room with area 25 m 2 for 1.5 h, the dust concentration reduces to 1/8 of the original value, and the colony count becomes 1/6 of the original value. in cleanrooms, electrostatic cleaner should not be used as final filter, which has already specified in related standards. this is because the air delivery rate is very small. moreover, particle resuspension caused by power failure, shutdown of the device, and discharge will cause unexpected result. it efficiency is less than that of sub-hepa filter and hepa filter. it is mainly used in the air handling system of fresh air. it is shown in the expression that for given particle group, u e is proportional to n d p when other conditions remain the same. however, as mentioned before, for particles with diameter less than 1 μm, n d p is stable. so u e will approach stable and do not decrease. when it is noticed that the slip correction coefficient c will increase with the decrease of particle size (introduced in chap. 6), the separation velocity will be a little larger. that means the decrease of u e becomes stable. therefore, compared with other kinds of air filters, electrostatic cleaner is more suitable for capture of fine particles. when particle diameter is larger than 1 μm, u e is proportional to d p because n d p is proportional to d p (because n is proportional to d 2 p ). inside the electrostatic cleaner, the electric field usually has two forms: single zone and double zones, which are shown in fig. 4 .41. for the case of electric field with double zones, ionization electrode and dust collecting electrode are separate. in this way, the voltage of ionization electrode can be reduced from tens of thousands of voltage, which is used in single zone, to ten thousands voltage. several dust collectors can be used to increase the collecting area. the distance between collecting plates reduces so that the voltage can be as low as several thousands, which is much safer. therefore, the electrostatic cleaner with double zones is applied in the field of air-conditioning and cleaning system. the main difference between electrostatic cleaner used in air-conditioning and cleaning system and electrostatic precipitator used in industrial application is the discharge by positive corona instead of negative corona. for positive corona, it is easy to convert from corona discharge into spark discharge. so only lower charge voltage can be exerted, which reduces the generated ozone. for the occupied space, the concentration of ozone generated is limited. when positive corona is used, high enough dc positive voltage is exerted on the metal wire of ionization electrode, and two sides of polar plates are grounded. in this way, nonuniform electric field is formed near the ionization electrode. a few free electrons in the air obtain energy from the electric field. they collide with air molecules fiercely, which generates collision ionization, and incomplete discharge appears, which is called corona discharge. around the ionization electrode, a ring of light blue halo could be seen, which is termed as corona. so ionization electrode abounds with positive ions and electrons. electrons move towards metal wire and neutralize on it, while positive ions move regularly under the effect of electric field, and they attached onto neutral particles when they encounter each other. in this way, particles become positive, which is the first kind of charge mechanism, i.e., electric field charge. secondly, except for the movement under the effect of electric field, ions have thermal motion. ions attach onto particles during the process of thermal movement, which makes particles positive. this is called the second kind of charge mechanism, i.e., diffusion charge. according to the electrostatic theory, electric field charge mainly has influence on the particles with diameter larger than 1 μm. the maximum charge particles can obtain is where e 1 is the electric field intensity in the space of ionization electrode, e.s.u. (300 v/cm ¼ 1 e.s.u.); n is the number of charge; e is the unit charge, 4.8 â 10 à10 e.s.u.; d p is the particle diameter, cm; k is the coefficient, k ¼ 3ε εþ2 ; the average is between 1.5 and 1.8; ε is the dielectric constant; the average value is 2-3. diffusion charge has the main influence for particles with diameter less than 1 μm, especially less than 0.2 μm. however, no simplified expression has been obtained for the maximum diffusion charge so far. it is known from eq. (4.38) that the charge on particles with diameter larger than 1 μm is proportional to the square of particle diameter. but with the effect of diffusion charge, the charge on particles with diameter equal to or less than 1 μm is larger than that obtained by eq. (4.38) . so the ratio of charge and its particle size n d p keeps stable. it will not decrease inversely proportional to the square of particle size. charged particles enter into the space composed of parallel thin aluminum plates. since aluminum plates are placed by staggered rivets with one aluminum plate positive and the other grounded, an uniform electric field is formed in the space. with the coulomb force in the electric field, charged particles obtain repellent force from the positive plate, and they settle down onto the grounded plate. the repellent force can be expressed as where f e is the coulomb force; q is the charge on particle (e.s.u.). for the flow with small re (usually less than 1), the pressure drop of spherical particles is obtained by eq. (6.5). when the pressure drop is balanced with the coulomb force, i.e., 3πμd p v ¼ nee 2 , the motion velocity u e of particles in the electric field is obtained when the slip correction is considered, which is also called separation velocity or migration velocity. it can be expressed as where μ has the cgs unit (shown in chap. 6), and other symbols have already been explained. it is shown in the expression that for a given particle group, u e is proportional to n d p when other conditions remain the same. however, as mentioned before, for particles with diameter less than 1 μm, n d p is stable. so u e will approach stable and does not decrease. when it is noticed that the slip correction coefficient c will increase with the decrease of particle size (introduced in chap. 6), the separation velocity will be a little larger. that means the decrease of u e becomes stable. therefore, compared with other kinds of air filters, electrostatic cleaner is more suitable for capture of fine particles. when particle diameter is larger than 1 μm, u e is proportional to d p because n d p is proportional to d p (because n is proportional to d 2 p ). electrostatic cleaner is composed of box, power supply, fan, dust collecting electrode, ionization electrode, activated carbon filter, and prefilter. figure 4 .42 shows the structure of domestic jzq-ii electrostatic self-purifier [7] . the box is made by single layer of thin steel plate. with the requirement of air tightness, the box gates are connected with circlip, which is used for the convenience of maintenance. the ionization electrode is a nickel chrome silk with diameter 0.5 mm. dust collecting electrode is composed of rigid aluminum alloy plates with thickness 1 mm and inter-plate spacing 6 mm (between opposite plates). each polar is made of 46 pieces of plates with area of each plate 0.2 m â 0.23 m. the surface of these plates has been electropolished to get rid of the burr and sharp corner, so the phenomena of spark discharge will not occur and the voltage between plates will be decreased. figure 4 .43 presents the structure of jzq-1 electrostatic self-purifier with one time ionization method. the height of jzq electrostatic self-purifier is 0.8 m and the net cross-sectional area is 0.34 m â 0.34 m. during the design of structure, the leakage inside the structure is usually neglected, which will greatly reduce the dust capture efficiency. there are mainly two reasons: one is caused by the electric wire when it goes through holes (such as the separator between layers); the other is the leakage between frame of each layer and box. it should be noted during the design of structure that two grounded plates must be added at both sides of ionization electrode (metal wire) and dust collecting electrode (metal plate). if both the ionization wire and the electrode plate near the edge are only connected with the power and without being grounded, both the airflow and particles will not be easily ionized and particles will not deposit readily, which will lower the total efficiency. since the volume of electrostatic cleaner is small, silicon rectified circuit is used for the power supply. in order to reduce the output voltage of transmitter so as to insulate, four times voltage circuit is usually adopted. figure 4 .44 shows the circuit of jzq-ii electrostatic cleaner. when too much dust has been deposited on the dust collecting plate, the indicator light will turn dark. the plate should be taken out for clean in time so that the dust collecting efficiency will not be affected. the following steps can be used to derive the dust collecting efficiency of electrostatic cleaner when the dust collecting electrode is plate. suppose the concentration at x distance from the inlet of dust collecting plate is n x , the airflow velocity between plates is v, the total flow rate through the dust collecting plate is q, the total effective area of dust collecting plate is f, and the length of plate is l. during the time dt, the decrease of dust along the dust collecting plate (perpendicular to the flow) equals with the number of deposited particles at this section of dust collecting plate, i.e., when the concentration at x ¼ l , i.e., the outlet concentration, is n l , and the concentration at x ¼ 0, i.e., the inlet concentration, is n 0 , integration is performed on the above equation and the following expression is obtained: then the dust collecting efficiency is it is obvious that η increases with the increase of the separation velocity u e . for given particles, u e mainly depends on the voltage between the ionization electrode and the dust collecting plate. when the voltage of the dust collecting plate increases, the electric strength in the space between dust collecting plates also increases, which will increase the separation velocity. however, when the electric strength between dust collecting plates is too high, the phenomena of electrode discharge are likely to appear. even through the electropolishing, the surface of plates will inevitably unsmooth, especially burr exists near the edge. even through the surface is very smooth, discharge will occur when a large dust especially fiber deposits on the surface, which will decrease the electric strength rapidly. during the process of discharging, sounds with cracking will be heard. for common manufacturing level, the electric voltage of the dust collecting plate can be increased to 7,000-8,000 v, which is equivalent to the electric strength about 1 kv/mm. when the voltage of the ionization plate is elevated, particles will be charged more, which increases the separation velocity u e . but the extent of voltage increase is limited by the manufacturing precise. too much voltage will cause electric discharge. so the voltage is usually less than 15,000 v. the separation velocity u e calculated from eq. (4.40) is only a theoretic value. in practice there are many influencing factors which are not included in this equation. these influences include: distribution of air and airborne particles at the cross section between plates, movement characteristic of air in the channel, coagulation of particles, and re-entrainment of particles deposited on plates. therefore, the actual separation velocity is much less than theoretical value. research performed on industrial electrostatic cleaner shows that the actual velocity is half of the theoretical value. but the situation is better for electrostatic cleaner used in air cleaning system, because the distance between dust collector plates is small, and velocity is small so that the flow is laminar. the particle size distribution at inlet is comparatively uniform. so the disturbance extent on u e is small. therefore, the actual separation velocity of electrostatic cleaner is a little higher than that of industrial electrostatic cleaner. for the given height (width), the larger the effective area is, which corresponds to the larger length, the higher the efficiency derived by eq. (4.43) is. the effective area of plate mentioned in literature means the area which is effectively used in structure. according to the expression of efficiency, efficiency will reach 100 % when the area is large and the plate is lengthy. but in reality not all the area of the plate along its length can collect dust efficiently. test on jzq-i electrostatic cleaner with length 30 cm shows that only 2/3 of the area along the length collect dust. if all the particles are deposited on the plate along this 2/3 length, the efficiency of this electrostatic cleaner approaches 100 %, while in fact it is only 70-80 % which can been seen from the comparison table about efficiencies. this is not caused by the length of plate which is not long enough so that particles do not have time to deposit but by part of particles which is not charged or whose electric charge is not enough. for particles whose charge is not enough and u e is small, it is effective to prolong the length of dust collector plate. however, for particles which are not charged at all, they will not be deposited on plate even when its length is prolonged. since particles without charge do exist, a concept "effective length of dust collector plate" is proposed. it means that under certain electric field, only certain part of the plate has effect on dust collecting. when it is longer than this length, the efficiency of the left part of the plate cannot be described by eq. (4.43), which implies that no more particles can be captured or not all the particles can be collected. why do some particles carry very few charges or no charges? according to the theory of electric corona discharge, there are mainly two reasons: 1. since the electric ionization polar is a metal wire, the electric field with high electric strength only appears near the small distance around it, while the electric strength far from it is very weak. for the latter situation, the movement velocity of ions is very slow and the air in that region is not ionized (if all the air between plates is ionized, the electric field will be penetrated when spark discharge appears and short electric circuit is formed, thus the electrostatic cleaner stops). 2. as mentioned before, with certain voltage of electric ionization polar, the ionization strength for air is fixed and the charge amount is determined. if the dust concentration of air entering electrostatic cleaner is high, charge on every particle is not enough or some particle cannot be charged at all. it is obvious that the former reason is mainly for electrostatic cleaner. from the above analysis, if the effective length of dust collector is measured, the actual separation velocity can be derived from the dust collector efficiency. it is obvious that the less the flow rate of electrostatic cleaner, the higher the efficiency is. but for particles without charge, efficiency will become stable when the flow rate is less than a certain value. according to the above analysis, in order to increase the efficiency of electrostatic cleaner with one-stage ionization, the extent of particle charge must be increased. so author proposed a scheme "two-stage ionization" which put two electric fields in series. with this method, air molecules not ionized in the first electric field will be likely to be ionized in the second electric field. in the late of 1960s, institute of hvac at china academy of building science and the former tianjin medical equipment factory invented and manufactured jzq-ii electrostatic cleaner together, which adopted this method. it is meaningless if the height of the equipment with two-stage ionization is two times that of the original equipment. according to the above analysis, the effective dust collector length under the given conditions is about 0.2 m. so the length of dust collector of jzq-ii electrostatic cleaner is 0.2 m under the condition of compact structure, which is the same as that of the one-stage ionization. with the scheme of two-stage ionization, the predicted effect of electrostatic cleaner is realized. related experimental data are presented in the following tables. table 4 .27 is the experimental data about the relationship between efficiency and voltage of dust collector. table 4 .28 is the experimental data about the relationship between efficiency and velocity between plates of dust collector. table 4 .29 is the experimental data about the relationship between efficiency and capacity of capacitor in the rectifying circuit. the influence of capacity of capacitor in the rectifying circuit on efficiency is large. when the capacity is small, the decrease of voltage on each octave band pressure level will be large, which reduces the particle capture efficiency, while increasing the capacity will smooth the wave profile after rectifying and the effective voltage approaches the summit value. but it is not safe if the capacity of capacitor was too big. for jzq-ii electrostatic cleaner, it is feasible to choose 8,800μμf as the capacity of capacitor. according to eqs. (4.38) and (4.40) with the cgs unit and 1 e.s.u. of e 1 and e 2 , the separation velocity for the condition with c ¼ 1, k ¼ 2 (for oil mist from the transformer, k ¼ 1.5; for marble particles, k ¼ 2.4), and both e 1 and e 2 are 1 e.s.u. becomes u e ¼ 2d 2 e 1 e 2 12π â 1:8 â 10 à4 d % 0:03e 1 e 2 d â 10 4 cm=s when d ¼ 0.5 â 10 à4 cm, c ¼ 1.3, e 1 ¼ 14,000 v % 46.5 e.s.u., and e 2 ¼ 7,000 v % 23.3 e.s.u., the derived separation velocity is u c ¼ 21 cm/s (which is equivalent with the calculation result based on average particle size of atmospheric dust). according to eq. (4.40), the relationship between fu e q and η is where 46 is the number of dust collecting surfaces. each dust collecting plate has two surfaces. since the most outer two sides do not play a role, they are not included and the total number of dust collecting surfaces is 44. so the actual separation velocity of each section for given efficiency can be calculated, which is shown in table 4 .23. from table 4 .30, the average of actual separation velocity is 0.12 m/s, which is slightly higher than half of theoretical separation velocity. it is consistent with the aforementioned analysis. for jzq-ii electrostatic cleaner with two-stage ionization method, the comparison of its turbidity efficiency measured by photoelectric turbidimeter with the foreign similar products is shown in table 4 .31. in terms of efficiency, results obtained by the turbidimetry method are usually smaller than that of dust spot method and weighing method. so the performance of jzq-ii electrostatic cleaner is better than that presented in table 4 .31. for simplifying the structure, cylindrical electrostatic cleaner appears in the market. thin metal plate is used to make the cylinder with circular or hexagonal cross section. it acts as the grounding plate. circular electrode with cusp is placed in the center of cylinder, where high-voltage electrostatic is applied. it becomes the high-voltage discharging electrode in the electrostatic field, which is shown in fig. 4 .45. but the efficiency of this kind of cylindrical electrostatic cleaner is very low. table 4 .32 shows the test data from mao huaxiong [40] . more attention has been paid on the influence of chemical pollution inside cleanrooms (please refer to chap. 7), so people starts to care about activated carbon filter. activated carbon filter has functions of both physical adsorption and chemical adsorption, so in fact it is an adsorber. the adsorption ability of activated carbon has selectivity. for these chemical substances which cannot be removed by physical adsorption mechanism, different chemical agents must be used as adsorbent during the process of impregnation. with the chemical reaction between adsorbent and adsorbate, the property of adsorbate is modified and it becomes nontoxic and harmless. many monographs and literatures have introduced the general application of activated carbon filters, which will not be mentioned in this section. only several aspects are emphasized here: 1. at present there are three kinds of activated air filters. one is the activated carbon particulate filter where the size of particle varies from small to large. the second is activated carbon particles with diameter 0.5 mm pasted on multiply layers of porous polyurethane foam material. since the air permeability of foam material is good, its pressure drop is smaller than that of the former kind and the corresponding adsorption efficiency reduces. the third is activated carbon fibrous filter by carbonization of fibrous media. it is thin, and both the pressure drop and adsorption efficiency are comparatively small. 2. the problem of invalid layer exists in the activated carbon filters. invalid layer is meant to adsorb a certain amount of chemical pollutants. the larger the activated carbon particle is, the thicker this layer is. this problem is usually ignored. figure 4 .46 shows the theoretical relationship between the invalid layer thickness and the amount of pollutant adsorbed. when the amount does not reach a certain value, it is called the non-protective time. figure 4 .47 is the result performed with cyan chloride [41] . the existing of this invalid layer is related to the adsorption mechanism of chemical pollutant by activated carbon. when polluted airflows through activated carbon, the pollutant diffuses towards the whole surface of activated carbon particles. then it goes towards to the pore interior surface of the particles and the surface of pores. so chemical reactions occur inside the particle interior surface to decompose the pollutant by adsorption of pollutant molecules and between adsorbed chemical pollutant and chemical agent (catalyst) dipped with activated carbon or between adsorbed oxygen and water. if several adsorption mechanisms inside a layer with certain thickness do not have enough time to play a role, for example, pollutant only diffuses onto the particle surface while they do not have time to diffuse towards the interior surface of pores and then adsorbed and decomposed, but it has already penetrated this layer, the pollutant concentration cannot be reduced to allowable value or has no time to be reduced at all, this layer is called invalid layer. if the activated carbon is within the invalid layer, there is no effect of adsorption. with certain physical and chemical property of activated carbon and temperature/humidity conditions, the thickness of invalid layer is only related to the specific velocity and pollutant concentration. when both the specific velocity and concentration are fixed, the thickness is constant, which is not related to the thickness of whole activated carbon layer. 3. since the pressure drop of activated carbon filter filled with particulate activate carbon is very large, the allowable specific velocity cannot be very large. therefore, it is necessary to understand the specific velocity-pressure drop characteristic of this kind of activated carbon filter. 4. when the activated carbon filter is designed to be circular cylinder, it has been proved by the author that the performance is better when the polluted air flows from outer towards inside, which improves the amount of adsorption [40] . since the risk of microorganism becomes higher, antibacterial filters develop in the usa and japan. this kind of filter is made by adding bactericidal substance in the filter media. however, doubt about its effectiveness exists. one kind is only to spray the additive onto the surface of filter media, so not all the filter layer have the ability to kill bacteria. the second kind is only to add bacteriostatic agent, which cannot kill the bacteria and instead may cultivate the ability of drug resistance of the bacteria. the third kind may generate some gaseous substance or odor which is harmful for people. it should be emphasized, which will also be introduced in chap. 9, that it is difficult for the bacteria captured on the windward side of the hepa filter made by inorganic material to reproduce and even penetrate. only with the suitable conditions of temperature and humidity, they are likely to survive. so the final conclusion of the necessity to use the antibacterial filter has not been reached. ashrae has warned as for this issue and suggests using antibacterial product in hvac system cautiously, so as not to produce any chemical pollution and new harm to the indoor environment and people. application of air cleaning technology air filtration (3) filtration of aerosols by fibrous media particle capture performance of fibrous filling layer filter and influence of fiber cross sectional shape tu guangbei) (1980) fibrous filter media and air filter, science and technology information reference room at tianjin university study of influencing factors for the performance of air filters institute of hvac of china academy of building research (1973) assembly cleanroom calculation of cleanroom performance test of domestic air sampler and high efficiency filter media status of industrial cleanroom planning and design of cleanroom calculation and verification of in-series efficiency of hepa filters experimental study of the safety of hepa filter used in nuclear fuel facilities air cleaning handbook (trans: shi youren et al) japan refrigeration and air conditioning industry association (1971) handbook of refrigeration and air conditioning (application) air conditioning and air cleaning absolute filter of cambridge filter corporation discussion of standard flow rate for hepa filter influence of non rated flow volume on the life time of hepa filter conversion method between the particle counting efficiency and the arrestance with atmospheric dust the latest filter cleanroom technology (trans: yu zhaoji) tgg and ygf low resistance and sub-high efficiency air filter characteristic of filter paper used for collection of radioactive particles recent development of particulate capture system evaluation and performance study of air cleaner optimization design of corrugated sub-high efficiency air filter without separator performance of polypropylene fibrous media performance evaluation of electret hepa filter monodisperse aerosol generator, institute of hvac at china academy of building research study of nuclepore membrane filter structure and its filtration performance institute of hvac at china academy of building research (1975) two kinds of 300 type protective air filter soft leather treatment agent for treating with ultra-fine glass fibrous paper for air filtration introduction to chinese current standardization system on high efficiency particulate air filter performance and prospect of hepa filter used in cleanroom the mechanics of aerosols (trans: gu zhenchao) development status and trends in the filtration dust study on the filtration of ultrafine particles study of the application of electrostatic air cleaner for improving institute of hvac at china academy of building research (1974) two kinds of 300 type protective air filter key: cord-276323-0n4aws2q authors: dourmashkin, r. r; davies, h. a; smith, hillas; bird, r. g title: are coronavirus-like particles seen in diarrhœa stools really viruses? date: 1980-11-01 journal: the lancet doi: 10.1016/s0140-6736(80)92123-6 sha: doc_id: 276323 cord_uid: 0n4aws2q nan requisite for successful eradication of pyogenic streptococci with penicillin. we have shown that orally administered metronidazole achieves a statistically significant reduction in the anaerobic tonsillar flora. controlled studies are in progress to determine the therapeutic efficacy of metronidazole in tonsillitis. department sir,-workers in britain,' india,2 and australia3 have noted "coronavirus-like particles" in the stools of children with diarrhoea and also in presumably normal individuals. the particles carry a regular array of projections on their surface, as coronaviruses do, but the size and shape of the particles is highly pleomorphic. there are a number of distinct types of particle, the morphology of which will be reviewed elsewhere.4 we have investigated a case of diarrhoea in a 2z year-old asian child who had visited pakistan four months previously. campylobacter and cysts of entamceba histolytica were isolated from the stool, and the child was treated with two courses of metronidazole. urological investigations were negative. the diarrhoea remitted and the child was discharged without follow-up. examination of the differentially centrifuged ultracentrifugation pellet of the patient's stool by electrorimicroscopy with negative staining revealed large numbers of "coronavirus-1ike particles" type 34 ( fig. 1 ). the size and shape of the particles varied greatly; however, there was a regular array of projections around the periphery of the particles, approximately 23 nm in length. in close relationship to these particles were broad sheets of membraneous material, without distinctive morphology. an analogous pellet was fixed in buffered glutaraldehyde and osmic acid and embedded in spurr resin, and stained sections were examined. the material of the pellet consisted essentially of two components. one was a number of large, vesicular material is seen around periphery of envelope, and also some inside its cavity. (x70 000.) nm, and the thickness of this double layer was 14-0-30 nm (mean 23.3 nm). external to this there was another, single layered continuous membrane. the second component consisted of vesicles bounded by a double layered continuous membrane, 6.5-12.5 nm (mean 9.3 nm) in thickness. the diameter of the vesicles was 115-266 nm (mean 179nm). most of the vesicles appeared empty, although a few small vesicles contained dense material. the space between the vesicles was filled with a lightly staining amorphous substance. those vesicles that were separate from the rest showed projections from their surface that were similar to the projections seen in the coronavirus-like particles visualised by negative staining, (fig. 3 ). the vesicles were closely adherent to the surface of the large envelopes, their membranes sometimes appearing continuous. however, there were some areas in which vesicles alone were found. the interpretation of these findings is difficult. it seems likely that both types of structures represent one organism. an explanation may be that the large structures, which may be a yeast-like organism such as blastocystis, have lysed, releasing the smaller vesicles that are possibly derived from the endoplasmic reticulum. another explanation is that there are two separate entities-one, a virus (albeit different in appearance from sectioned coronaviruses) and the other a coincidental component derived from a yeast-like organism. we favour the first explanation but further study is required. beta-adrenergic blocking agents do not seem directly to intensify vasoconstriction, but their use may sometimes modify a situation where rest pain is occurring in the presence of a normal or rapid heart rate to one in which similar pain is associated with rates within the range 50-60/min, which you suggest to be desirable. we have used atrial or atrioventricular sequential pacing at a rate of 70-75/min to correct bradycardia in fifteen patients with angina. seven had moderate bradycardia apparently resulting entirely from administration of beta-blocking drugs. alternative forms of therapy had proved unsatisfactory; indeed, one patient with sinus node dysfunction had a myocardial infarction in association with a heart rate of 80/min when propranolol was withdrawn, despite intensive treatment with vasodilators. the immediate effect of pacing (combined with beta-blocking agents and vasodilators as required) has been salutary in all patients, with benefit persisting for up to 4 years and, so far, only one relapse to the previous severity of symptoms. recently, the technique has also been used temporarily to suppress pain in two patients awaiting cardiac surgery. it is evident that substitution by pacing for the chronotropic component of resting cardiac sympathetic activity may greatly extend the scope of the medical management of angina. it is tempting to speculate that the maintenance of heart rate (and resting cardiac output) may reduce alpha-sympathetic activity indirectly, resulting in maximum possible dilatation of critically stenosed coronary arteries, which are not adversely affected by simultaneous administration of beta-blockers. sir,&mdash;an endogenous opioid neurotransmitter system whose activation mimics exogenous opiate action had been postulated for many years but has only recently been describe.1,2 there is now compelling evidence that corticotrophin (acth) and (3-lipotropin/ (3-endorphin are formed from a larger recursor protein which has been called pro-acth/endorphin..5 acth and &bgr;-lhp/&bgr;endorphin are located and stored in the same cells and secretory granules within the pituitary, and under all stimulatory and inhibitory conditions all fragments are released together.3-5 in animals the major source of plasma &bgr;-lph/&bgr;-endorphin is the pituitary so that a reduced concentration in the pituitary causes reductions in circulating blood levels of p-lph, (3-endorphin, acth, and cortisol. 5 the effects of opiates (e.g., morphine and methadone) on neuroendocrine function can be used to provide evidence for the role of endorphins in the brain. 6,7 several neuroendocrine functions are influenced by exogenous opiates, endogenous opiates, and opiate antagonists (e.g., naltrexone).6-9 the infusion of the exogenous opiate methadone lowers plasma cortisol levels in man.l0 these and other data9,1 suggest that opiate receptor stimulation by exogenous opiates may, through a feedback mechanism, reduce the release and possibly the synthesis of pro-acth/endorphin, and therefore decrease acth and endogenous opiates or endorphins. opioid peptides (endorphins) in pituitary and brain endogenous opioid systems in brain corticotropin/&bgr;-endorphin precursor: concomitant storage of its fragments in the secretory granuyles of anterior pituitary corticotropin/ endorphin cells opioid peptides betaendorphin and adrenocorticotropin are secreted concomitantly by the pituitary gland the effects of opiate agonist and antagonist on serum prolactin in primates effects of naltrexone on mood and neuroendocrine function in normal adult males hormonal and other effects of naltrexone in normal men naloxone increases acth and conisol levels in man suppression of plasma cortisol in depressed patients by acute intravenous methadone infusion comparison of plasma hormonal levels between heroin addicts and normal subjects key: cord-006892-n2ncamqh authors: donaldson, braeden; lateef, zabeen; walker, greg f.; young, sarah l.; ward, vernon k. title: virus-like particle vaccines: immunology and formulation for clinical translation date: 2018-09-19 journal: expert rev vaccines doi: 10.1080/14760584.2018.1516552 sha: doc_id: 6892 cord_uid: n2ncamqh introduction: virus-like particle (vlp) vaccines face significant challenges in their translation from laboratory models, to routine clinical administration. while some vlp vaccines thrive and are readily adopted into the vaccination schedule, others are restrained by regulatory obstacles, proprietary limitations, or finding their niche amongst the crowded vaccine market. often the necessity to supplant an existing vaccination regimen possesses an immediate obstacle for the development of a vlp vaccine, despite any preclinical advantages identified over the competition. novelty, adaptability and formulation compatibility may prove invaluable in helping place vlp vaccines at the forefront of vaccination technology. areas covered: the purpose of this review is to outline the diversity of vlp vaccines, vlp-specific immune responses, and to explore how modern formulation and delivery techniques can enhance the clinical relevance and overall success of vlp vaccines. expert commentary: the role of formation science, with an emphasis on the diversity of immune responses induced by vlp, is underrepresented amongst clinical trials for vlp vaccines. harnessing such diversity, particularly through the use of combinations of select excipients and adjuvants, will be paramount in the development of vlp vaccines. virus-like particles (vlp) are a type of subunit vaccine based on virus-derived proteins, assembled to form a particle. vlp hold several advantages over other particulate subunit vaccines. these include a morphological resemblance to their parent virus, a highly repetitive immunogenic surface structure, and the retention of cell uptake and immune processing pathways associated with their parent virus [1] . vlp themselves are nonpathogenic, devoid of an intact virus genome, and are incapable of infection or replication. the noninfectious nature of vlp significantly improves their safety profile over live-attenuated vaccines, while also possessing advantages when compared to other forms of subunit, killed, or particulate vaccines. commercially successful vlp vaccines include hepatitis b virus (hbv) vlp, such as recombivax hb (merck), engerix-b (glaxosmithkline), elovac b (human biologicals institute), genevac b (serum institute) and shanvac b (shantha biotechnics), hepatitis e virus (hev) vlp, such as hecolin (innovax), and human papillomavirus (hpv) vlp, including gardasil (merck) and cervarix (glaxosmithkline). vlp vaccines currently under investigation in clinical trials include influenza a virus (iav) vlp (nct02768805, nct02233816) [2, 3] , chikungunya virus vlp (nct02562482) [4] , human cytomegalovirus (hcmv) vlp (nct02826798) [5] , and human norovirus (hunv) vlp (nct02669121, nct03039790, nct02661490) [6] [7] [8] . recent and current vlp clinical trials registered in the united states of america and the european union are summarized in table 1. vlp can be bulk expressed in bioreactor cultures, utilizing advanced in vitro protein expression systems for producing vaccine-grade vlp suitable for clinical administration. the manufacture of some vlp also includes additional processing steps, such as disassembly and reassembly of vlp. the manufacture of the hpv vlp utilizes disassembly and reassembly to improve vlp morphology and stability [9] . similarly, the manufacture of qβ vlp includes this method [10] . different vlp expression systems and the applications of vlp disassembly and reassembly was explored in a recent review [1] . compatibility with commercial upscaling technology, gmp production, and with minimal postproduction manipulation or modification supports large-scale use of vlp vaccines; however, some vlp vaccines struggle in their translation from laboratory research and development, to clinical trials and routine public access [11] [12] [13] . open accessibility to the molecular or genetic components of derivative constituents may place some limitations on the commercialization of specific vlp vaccines. this may be circumvented through tactical use of proprietary modification, or by utilizing the vaccine as a constituent within a composite formulation [14] . the development of some vlp vaccines can be challenged by issues with stability and longevity, which may be alleviated with formulation excipients, or other vaccine additives that facilitate vaccine distribution and storage [15, 16] . the purpose of this review is to explore some of the challenges in the translation [19] , and negative-sense rna viruses such as human parainfluenza virus type 3 [20] . variety can be observed in vlp size, ranging from ms2 bacteriophage vlp at around 27.5 nm in diameter [21] , to hpv vlp at around 60 nm [22] , and influenza vlp at around 100 nm [23, 24] . vlp also vary in structural complexity, as illustrated in figure 1 , including mono-layer vlp such as hbv vlp formed from hbv surface antigen (hbsag) or hbv core antigen (hbcag) [25] [26] [27] , and multi-layer vlp such as rotavirus vlp [28, 29] . vlp can be encapsulated within a phospholipid bilayer envelope to resemble their parent virus, such as hiv [30] or sendai virus vlp [31] . the envelope itself can also form the primary particle structure of some vlp, with recombinantly expressed virus envelope-stabilizing proteins embedded within the membrane, such as with iav virosomes [32] . some vlp are compatible with the formation of polyvalent or mosaic vlp, derived from multiple virus strains [33] . while this increases the diversity of vlp vaccines beyond the variety of parent viruses, the increased complexity of polyvalent or mosaic vlp may require post-production manipulation to facilitate stable particle formation [34, 35] . in addition to facilitating the development of complex vlp vaccine constructs, introduction of postproduction manipulation has also been demonstrated to improve the consistency and stability of some standard structure vlp vaccines [9, 36] . the diversity of vlp can also be characterized based on the variety of diseases these vaccines have been developed to prevent or treat. these include vlp vaccines developed for both human and veterinary pathologies, with some examples including an hbv hbcag core particle-based vaccine for her2 + cancer [37] , an adenovirus vlp-based vaccine for placental malaria [38] , and a qβ vlp-based vaccine for leishmania infection [39] . a selection of recently developed and investigated novel vlp vaccines are provided in table 2 . vlp can be produced using a variety of expression systems, usually involving vlp assembly by spontaneous polymerization upon the expression of virus protein constituents. vlp can be expressed in cells derived from bacteria, yeast, insects and mammals, in cell-free expression systems, and in live organisms such as silkworm pupae and various plants [40] [41] [42] . selection of an appropriate expression system is important, as each expression system can differ in their efficacy for expressing specific vlp, their post-translational modification (e.g. phosphorylation, glycosylation), and their potential for contamination with biologically compatible zoonotic viruses. protein expression for the production of vlp is highly amenable to modification, such as substituting strain-specific amino acid sequences [43] , or inserting foreign peptide sequences or proteins [44, 45] . manipulation of this process can facilitate the engineering of recombinant chimeric vlp containing xenogeneic antigens, inducing an immune response against targets other than the parent virus [46] . vlp can also present self-antigens in an immunogenic context, which can be particularly useful in immunotherapeutic vaccination for cancer [46] . a diverse range of applications for this type of modification have been investigated prophylactically and therapeutically for various conditions, including protection against infection from other organisms [47] , autoimmune inflammatory disease [48] , and as an antitumor immunotherapy [46, 49, 50] . chimeric vlp have been investigated as a potential therapy for conditions not usually associated with vaccination, such as atherosclerosis [51] , type ii diabetes mellitus [52] , and nicotine addiction [53] . some of these vlp vaccines utilize chemical conjugation for incorporation of antigenic sequences as opposed to recombinant insertion. vlp can serve as an effective immunogenic scaffold for chemical conjugation, compatible with a broad range of conjugation chemistries. the applications of chemical conjugation with vlp has been comprehensively reviewed in recent articles [1, [54] [55] [56] ; however, it is worth noting that introducing unique forms of chemical conjugation may provide some proprietary claim over methods involving vlp vaccines already in the public domain. vlp are a form of subunit vaccine, inducing a characteristic immune response shared amongst exogenous antigens. while vlp are taken up by cells through non-specific pathways such as phagocytosis and macropinocytosis, some vlp can also utilize specialized uptake pathways inherited from their parent virus. in general, protein-based subunit vaccines like vlp are internalized into antigen-presenting cells (apcs), digested within the phagolysosome, and the resulting antigen peptides are presented loaded on major histocompatibility complex (mhc) ii molecules to cd4 + t helper cells (t h cells). t h cells recognize epitopes presented on mhc-ii through their specific t cell receptor (tcr), and activate dependent on the strength of this interaction, in addition to the degree of signaling through costimulatory receptors and cytokines. these additional signals are usually supplied by the apc, which is activated to increase the presentation of costimulatory receptors and the release of cytokines in response to a variety of stimulatory signals. these stimulatory signals correspond with the activation of pattern recognition receptors (prrs), such as tolllike receptors (tlrs), nod-like receptors (nlrs), and rig1-like receptors (rlrs), which each recognize specific pathogenassociated molecular patterns (pamps) and damage-associated molecular patterns (damps). as vlp vaccines usually contain minimal or negligible amounts of these stimulatory molecules, these signals are instead induced by vaccine adjuvants. manipulation and selective targeting of specific apcs or other target cell populations may be an effective means of developing a novel proprietary vlp vaccine. chemical conjugation of receptor ligands onto the surface of compatible vlp should promote binding and uptake into cells expressing the corresponding receptor, while primary vlp uptake and processing pathways should continue unperturbed. this type of modification may be particular effective when targeting a specific apc population is desired for the induction of a specific type of immune response. for example, chemical conjugation of mannoside-based saccharides on the surface of rabbit hemorrhagic disease virus (rhdv) vlp selectively targets the mannose receptor expressed on the surface of apcs, inducing increased uptake and alteration of antigen cross-presentation in murine dendritic cells [57] . chemical conjugation of unmethylated cpg oligonucleotides on the surface of rhdv vlp has also been investigated for targeting uptake through the dec205 receptor [58] . similarly, coating of hiv vlp with phosphatidylserine-laced liposomes enhanced uptake into macrophages by mimicking apoptotic bodies [59] . although the immune response to vlp may be generalized due to similarities shared with other subunit vaccines, there are some unique components of a vlp-induced immune response. the pathways for uptake of vlp into apcs vary depending on the size and morphology of each vlp, including the potential retention of receptor binding motifs. particles smaller than 20 nm, such as norovirus p particles [60] , can drain directly from the vaccination site into the lymph through pores in the fenestrated lymphatic vessels [61] . trafficking of molecules within the lymphatics is likewise size dependent, with soluble proteins smaller than 70 kda or 5 nm transported by specialized small antigen conduits [62, 63] . vlp are larger than 5 nm, and many are instead transported passively to the lymph nodes bound on the surface of myeloid immune cells [64] [65] [66] . vlp larger than 200 nm cannot drain directly through the fenestration pores into the lymph, and instead require active transport following internalization by apcs at the vaccination site, such as in dendritic cells (dcs), macrophages and b cells [67] . uptake of virus antigens into antigen presenting cells can occur through multiple pathways, including phagocytosis, macropinocytosis, caveolae-mediated uptake, clathrinmediated uptake and clathrin noncaveolae-mediated uptake [68] . vlp have been identified to utilize a similar repertoire of uptake pathways, including phagocytosis [69] , size-dependent macropinocytosis [70] , and clathrin-dependent or independent forms of receptor-mediated endocytosis [71] [72] [73] . the retention of receptor-mediated endocytosis indicates that some vlp can inherit functional receptor binding domains, and are compatible with the corresponding uptake pathways derived from their parent virus. these mechanisms of receptor-mediated uptake can also be introduced to vlp through post-expression modification, such as the chemical conjugation of superantigen on hbv vlp for internalization through mhc-ii molecules [74] , or the chemical conjugation of transferrin on qβ vlp for internalization through the transferrin receptor, which is upregulated in some cancer cells [75] . non-specific uptake into apcs subjects vlp to standard components of exogenous antigen processing pathways, including lysosomal fusion, enzymatic digestion, mhc-ii loading and antigen presentation (figure 2(a) ). parent viruses of vlp internalized through these pathways engage additional mechanisms to progress toward virus replication, including capsid disassembly or uncoating, exportation of the virus genomic material, and disruption of vesicular fusion. while the retention of intracellular processing mechanisms resembling those utilized by parent viruses has been investigated for some vlp [76, 77] , it remains unclear whether vlp universally mimic the intracellular processing of their parent virus. vlp can retain access to cross-presentation pathways, facilitating presentation of antigens on mhc class i (mhc-i) for induction of a cd8 + t cell-mediated cytotoxic immune response. there are several known mechanisms of cross-presentation in apcs, including antigen escape from the early endosome, fusion of the endosome with the endoplastic vlp can be internalized and processed through a variety of intracellular pathways. (a) vlp internalized through non-specific pathways such as phagocytosis and macropinocytosis can be processed, with peptides presented on mhc-ii as exogenous antigen. (b) some vlp can also utilize cross-presentation pathways, facilitating the presentation of peptides on mhc-i [70] . (c) influenza vlp, in this example an influenza virosome, is internalized through receptor-mediated endocytosis prior to fusion of its envelope with the endosomal membrane [90, 92, 93] . (d) jcv vlp is thought to utilize the processing pathway of its parent virus to facilitate delivery of exogenous nucleic acids, including clathrin-dependent endocytosis, nuclear trafficking, and uncoating of the capsid within the nucleus [99, 100] . reticulum, and recycling of mhc-i receptors from the cell surface. rhdv vlp are known to utilize the mhc-i receptor recycling pathway of cross-presentation [70] (figure 2(b) ). additional examples of vlp known to induce cross-presentation of antigens include vlp derived from hbv [78, 79] , hepatitis c virus (hcv) [80] , hpv [73] , papaya mosaic virus [81] , and parvovirus [82] . the ability to induce cross-presentation is important when a cellmediated cytotoxic immune response is the primary desired outcome of a vlp vaccine. the retention of receptor-mediated internalization in vlp illustrates that these mechanisms of uptake in their parent virus are autonomous, independent of the integrity of the complete virion. vlp derived from influenza a or b viruses are prime examples of this process. influenza vlp can be produced in several forms, including enveloped vlp formed by expressing the influenza matrix 1 (m1) protein [24] , retroviral gag proteins [83] , or as independent virosome particles without a traditional protein core [84, 85] . each of these constructs includes the expression of influenza hemagglutinin (ha) with or without neuraminidase (na). these influenza proteins are crucial for the induction of an influenza-specific immune response, and they also play essential roles the budding of influenza virus-like particles [23, 86, 87] , and in receptor-mediated endocytosis. influenza vlp can bind to sialylated glycoproteins and glycolipids on the surface of cells from the inherent activity of ha, and are internalized through clathrin-mediated endocytosis [88, 89] (figure 2 (c)); however, alternative endocytic pathways have also been identified [90] [91] [92] [93] . as the early endosomal ph lowers, the envelope fuses with the endosome membrane. ha and na proteins distributed along the internal surface of the endosome membrane are enzymatically degraded, with peptides presented as exogenous antigens on mhc-ii (figure 2(c) ). specialized vlp vaccines designed to deliver a genetic payload add further complications to vlp uptake and processing. retention of the ability to deliver dna/rna requires conservation of intracellular processing pathways utilized by the parent virus for replication, or the induction of an alternative method of nucleic acid translocation. john cunningham virus (jcv) vlp is a polyomavirus vlp that has received significant attention over its ability to facilitate gene delivery and genetic manipulation [77, 94, 95] . jcv is internalized by clathrin-dependent endocytosis upon binding to an undefined plethora of receptors and molecules known to include the serotonin receptor and sialic acid [96, 97] . the virus then colocalizes to endosomes along with transferin, prior to cytosolic translocation, and subsequent trafficking through nuclear pores, mediated by the n-terminus of the vp1 capsid protein [98] . the virus then uncoats, exposing the genomic material within the capsid. jcv vlp are thought to mimic this process due to their ability to deliver dna plasmids or other nucleic acids to the nucleus [99, 100] (figure 2(d) ). vlp that demonstrate this ability to deliver nucleic acids likely utilize similar processing pathways to their parent virus [101, 102] , but vlp can also be modified to utilize alternative processing pathways [103, 104] . the primary desired outcome of most commercial vlp vaccines is the production of antibodies specific for the vlp parent virus. anti-vlp antibodies produced by these vaccines correspondingly neutralize the parent virus, protecting against infection. the production of anti-vlp antibodies involves the activation of a humoral immune response. b cells that specifically recognize vlp surface domains through their b cell receptor (bcr) bind and internalize vlp. binding of vlp to the bcr provides a stimulatory signal that primes the b cell for activation. most vlp consist of structurally identical capsid proteins arranged in a repetitive quasicrystalline pattern, which can crosslink multiple bcrs to provide a stimulatory advantage over other types of subunit vaccines. crosslinking of bcrs is important for inducing a robust humoral response, as b cells can ignore some monomeric soluble antigens [105] . potent stimulation through bcr crosslinking can override inherent tolerogenic mechanisms, and can activate unresponsive or anergic b cells [106] . highly repetitive structures also promote binding to low-affinity bcrs through multivalent, or highavidity interactions [107] . antigen primed b cells receive additional activation signaling induced by pamps delivered with the vlp vaccine. these signals are usually provided by vaccine adjuvants, but may also be induced by other virus-associated or expressionderived endogenous molecules associated with the vlp. vlpderived antigens are presented to t h cells loaded on mhc-ii, inducing the cytokine and costimulatory receptor signaling from the t h cell that continues b cell activation. activated b cells initially become plasmablasts, a transient extrafollicular activation state that can provide some immediate antibody production [108] . plasmablasts migrate to the follicular region of lymph nodes to form germinal centers, a specialized region where activated b cells proliferate. plasmablasts within germinal centers under affinity maturation and immunoglobulin class switching, guided by signaling from t follicular helper (t fh ) cells [109, 110] . this specialized t fh cell guidance results in the development of high-affinity antibody-producing plasma cells, and long-lived memory b cells. rapid t cellindependent activation can also be induced in marginal zone b cells and b1 cells, providing an alternative pathway for activation and antibody production over the predominant follicular b cell population [111] . some vlp have been identified utilizing this alternative form of b cell activation [112] . the production of anti-vlp antibodies has been investigated and characterized amongst a broad range of vlp. this process can be influenced by various vaccine components, constituents, and even unexpected molecules, such as those derived from the vlp expression system. for example, the presence of bacterial rna packed inside qβ vlp plays a role in the induction of igg2a/c antibodies in mice, and igg1 antibodies in humans [113, 114] . qβ vlp can also induce the production of iga class antibodies in immunocompetent mice when immunized through the intranasal route [115] , while t h cell-deficient mice required subcutaneous rather than intranasal vaccination to induce robust iga production through t-cell independent b cell activation [114] . tlr-7, simulated by singlestranded rna in endosomes, was also identified as crucial for the induction of both igg2c and iga antibodies against qβ vlp in mice. this further indicates the importance of endogenous bacterial rna present in these vlp. the induction of anti-hpv vlp antibodies has also been extensively studied. although the production of secretory iga antibodies is desired for protection against viral infection at mucosal surfaces, the predominant antibody class found in secretions from the female genital tract following vaccination with hpv vlp is igg [116] [117] [118] . while the presence of igg in these secretions may be due to transudation from the serum, some active transportation or local production in the mucosa may be possible [119, 120] . vaccination with hpv vlp induces the production of both igg and iga antibodies in the serum and cervical secretions of humans [121] . while serum titers of anti-hpv antibodies may initially decline post-vaccination, these levels plateau and remain stable to provide prolonged immunity [122] . while the induction of a potent humoral immune response and the subsequent production of anti-vlp antibodies is the primary desired outcome of most commercial vlp vaccines, these is increasing appreciation for the role of vaccine-induced cell-mediated immunity [123] [124] [125] . measurements of anti-vlp titers can provide an important indication of vaccine efficacy with respect to the neutralization of a virus challenge, a cellmediated immune response also plays an important role in antivirus immune defense [126, 127] . activation of a cellmediated immune response can also be the primary desired outcome of vlp vaccines, particularly for chimeric or other modified vlp vaccines that target non-virus pathologies. a potent cell-mediated response to vlp vaccines is dependent upon cross-presentation of vlp-derived antigens on mhc-i. cd8 + t cytotoxic (t c ) cells can recognize through mhc-i antigen complexes through their tcr, and become primed for activation. these antigen-primed t c cells require additional signaling for activation, including interaction with costimulatory receptors, and cytokines from t h cells. while many vlp may be capable of cross-presentation, some may be more effective at inducing cross-presentation due to uptake and processing pathways inherited from their parent virus [49, 128] . the induction of a potent cell-mediated immune response is particularly important for immunotherapeutic cancer vaccines. tumor cell-specific antibodies can enhance the inherent cytotoxic activity of natural killer cells through mechanisms such as antibody-dependent cell-mediated cytotoxicity, or directly through complement-dependent cytotoxicity; however these mechanisms tend to be restricted to passive vaccination with monoclonal antibodies, such as the her2-specific monoclonal trastuzumab [129] . a cell-mediated immune response combines target specificity with the in vivo expansion of effector cell populations, with the capacity to establish prolonged memory. rhdv vlp are particularly effective as a vector for cancer vaccines, capable of inducing the cross-presentation of vlp-derived antigens [70] , and compatible with recombinant insertion and chemical conjugation [49] . rhdv vlp vaccines have been investigated in models of hpvinfected cervical cancer [47] , melanoma [49] , lewis' lung carcinoma [130] , and colorectal cancer [46] . additional examples of vlp that can induce cross-presentation and a potent cellmediated immune response include qβ vlp [128] , ms2 vlp [131] , and alphavirus vlp [132] . preexisting immunity in the form of preformed anti-vlp antibodies can be an important consideration for some vlp vaccines [133, 134] . binding of anti-vlp antibodies may interfere with the normal uptake and presentation pathways of vlp vaccines. anti-vlp antibodies may be present in unvaccinated individuals due to environmental exposure to prevalent strains of the parent virus, or induced following vlp vaccination. while the presence of preexisting antibodies may decrease the efficacy or alter the immune recognition of some vlp vaccines [135, 136] , they can also be associated with measures of enhanced vaccine efficacy [133] . this may be due to accessing fc receptor-mediated uptake pathways, the formation of antibody-vlp complexes, or antigenic boosting of specific b cells. the presence of anti-vlp antibodies have also been found to have no observable effect on the intended vaccine outcome for several vlp vaccines, such as polyomavirus [137] , and rhdv vlp [138] . when anti-vlp antibodies present deleterious interference, alternating between different vlp vaccine vectors may be a suitable solution. alternating between rotavirus and adenovirus vlp has been found to enhance both the humoral and cell-mediated immune responses against target antigens in comparison to repeat delivery with the same vlp vector [139] . similarly, alternating between closely related vlp such as rhdv and human norovirus (hunv) vlp can be sufficient to enhance vaccine immunogenicity [140] . further complication can arise when vlp vaccines induce a phenomenon referred to as carrier-induced epitopic suppression (cies), in which the intended immune response is outcompeted by a strong anti-vlp response [135] . cies may be prevented by masking vlp surface antigens, or by avoiding recognition through recombinant modification. for example, recombinant insertion of the p18 domain of hiv into hev vlp was identified to avoid recognition by anti-hev antibodies [141] . the formulation of a vaccine refers to the constituents that make up the final administrable solution, including the vaccine vector, adjuvants, and excipients. for vlp vaccines, these excipients can include a variety of salts and compounds prepared as an aqueous solution or emulsion, which maintains the physical stability of vlp for enhanced shelf life of the vaccine. appropriate use of buffers can limit fluctuations in ph, while protection from fluctuations in temperature and desiccation may be provided by thermoprotectants and lyoprotectants, respectively. formulation science involves investigating the various components of a vaccine under different environmental conditions, with the intention of formulating a stable vaccine product suitable for the route of administration, and with maximized immunogenicity. the combination of formulation components, vlp vaccine preparation states and routes of administration is outlined in figure 3 . the majority of vlp vaccines currently on the market and under clinical evaluation are liquid suspensions, ready for administration. this places strict limitations on vlp vaccine storage and distribution for safe and compliant administration. for example, the commercial hpv vaccine gardasil (merck) must be refrigerated at 2-8°c, and protected from light. gardasil cannot be frozen, and guidelines for the use of gardasil advise that the vaccine must be used within 72 h when removed from refrigeration at temperatures below 25°c, or when stored at 0-2°c. these guidelines mirror those of many other commercial vlp vaccines, and outlines the primary stability, storage and distribution challenges for formulation science to investigate. maintaining the integrity and stability of vlp in solution is largely dependent on the combination of salts, and the buffering chemicals and compounds used. optimization of buffer ph, ionic strength, and other stabilizing components is imperative to developing a marketable, stable, liquid vlp vaccine [142] . an investigation into the stability of ev71 vlp identified that sodium phosphate-based buffers were superior to citrate or tris buffered solutions, with vlp stored in sodium phosphate buffer remaining stable for 1 month at both 25 and 37°c [142] . such prolonged stability at room and core body temperature suggests that this vlp may be suitable for importation into countries where maintaining cold-chain storage and distribution can be difficult. other important considerations for the selection of an appropriate buffer include compatibility with downstream applications, such as chemical conjugation, and the availability of scavengable nutrients that may promote the growth of potentially harmful organisms in an improperly stored vaccine. various additive molecules, particularly carbohydrates such as trehalose, sucrose and glycerol, have been investigated extensively in vlp vaccine formulations. the addition of these molecules to vaccine formulations demonstrated enhancement in vlp stability the role of formulation science in vlp vaccine manufacture includes the chemical composition of buffers, preservatives, additives and other stabilizing compounds for maintaining intact vlp. this includes protecting vlp from chemical or physical instability, and enzymatic degradation. formulations can also include targeted delivery compounds, such as muco-adhesives, and immunogenic components such as adjuvants. storage and distribution of vlp vaccines, and the subsequent route of administration are also important considerations in formulation science, critical in determining the efficacy and immunogenicity of the vaccine. within a liquid suspension of norwalk virus vlp [143] and rotavirus vlp [144] . other than sugar-based formulation additives, polyanionic solutions can also stabilize vlp that would otherwise be unstable at neutral ph, such as chikungunya virus vlp [145] . the majority of commercial vlp vaccines are distributed as a liquid suspension, requiring a cold chain maintaining 4-8°c throughout distribution and storage. even under stable cold chain conditions, the longevity of vlp can be limited. this can be prolonged by storage at temperatures at or below −20°c, stabilized with the addition of a cryopreservative such as glycerol or trehalose [146] ; however, this only further exacerbates the issue of delivering these vaccines intact where they are needed, such as in developing countries without reliable cold-chain delivery infrastructure. alternative storage methods such as freeze-drying, or lyophilization, can have variable effects on the stability of vlp. mechanical damage induced by ice crystallization, or exposure to varying salt concentrations and ph changes during the freezing process may adversely affect the integrity of vlp [147] . exposure to these factors can be limited through the addition of specific cryoprotectants and lyoprotectants. for example, red-spotted grouper nervous necrosis virus (rgnnv) vlp [148] reportedly retain stable particles and remain immunogenic when freezedried in the presence of sorbitol, but were adversely affected in the presence of mannitol. qβ vlp [147] , murine polyomavirus (mupyv) vlp [149, 150] , and hbv vlp [151] have likewise demonstrated some capacity to survive varying freeze-drying methodologies. spray-drying has also gained traction as an alternative vaccine storage mechanism, avoiding the potential mechanical damage induced by freeze-drying by instead forming a dry powder formulation through a combination of nebulization and dehumidification [152] ; however, spray-drying involves exposure to elevated temperatures, which can be similarly damaging to thermolabile vlp. hpv vlp suspended in a formulation containing mannitol, trehalose, dextran, l-leucine and inositol are capable of surviving this procedure [153] [154] [155] . spray-dried ms2-16l2 vlp stored at room temperature for 34 months were found to induce high titer anti-hpv l2 igg antibodies, and significantly protected vaccinated mice from challenge with hpv16 [155] . air or vacuum drying methods have also been reported for coating microneedles with influenza vlp suspended in a formulation containing carboxymethylcellulose (cmc). microneedle-based delivery of dried h1n1 (a/pr8) [156] and h3n2 (a/aichi) [157] were found to induce immune responses comparable to intramuscular injection, but with the advantage of prolonged longevity in storage. investigation of similar formulation and preparation strategies with different types of vlp may uncover some universality in their application, with the potential for these methods to eradicate the cold-chain limitation imposed on many vlp vaccines. as has been previously described, many vlp possess structural or molecular features that can confer some auto-immunostimulatory properties. these properties facilitate the induction of immune responses by vlp without the need for adjuvants; however, the use of adjuvants with vlp vaccines may enhance vaccine immunogenicity, and promote the activation of a specific type of immune response. currently licensed adjuvants for use with vaccines include aluminum sulfate salts (alum), proprietary combination adjuvants such as as03 (glaxosmithkline), as04 (glaxosmithkline) and mf59 (novartis), thermo-reversible oil-in-water immersions, and montanide isa51 [158] . while many standard vaccine adjuvants may be suitable for vlp vaccines where the generation of anti-vlp antibodies is the desired outcome, these adjuvants may fail to overcome immunotolerance and induce antitumor immunity in cancer vaccine formulations [159] ; however, where traditional adjuvants may fail, novel tlr agonist adjuvants have had success. the tlr7 agonist adjuvant imiquimod was recently approved for use with vaccines for melanoma, and another clinical trial is assessing its candidacy for treatment of bladder cancer [159] . tlr7/8 stimulate anti-viral interferon pathways in apcs, promoting the activation of cd8 + t cells and nk cells [160, 161] . the use of the adjuvant gardiquimod in combination with vaccination has been found to induce tumor regression in murine models of melanoma [162] , human hepatocellular carcinoma [163] and pancreatic cancer [164] . tlr9 agonist adjuvants such as unmethylated cpg oligonucleotides have drawn considerable interest due to their inherent ability to associate with some vlp [138, 165] . similarly, the presence of other nucleic acids can have downstream ramifications on the immune response induced by vlp. for example, the presence of rna inside qβ vlp can skew the humoral response induced in mice to produce igg2a antibodies, while the removal of this rna results in the production of igg1 antibodies [166] . qβ vlp can also contain additional adjuvant molecules derived from their expression system, encapsidated during particle formation. a recent study in mice investigated the use of various adjuvants in conjunction with a filovirus vlp vaccine, including the tlr3 agonist adjuvant poly-iclc (hiltonol), tlr4 agonist adjuvant monophospholipid a (mpla), cpg odn2395 and alhydrogel [167] . poly-iclc was found to induce a predominantly t h 1 response, with an igg2c subtype antibody production that correlated with protection from virus challenge. in comparison, administration of the vlp vaccine with alhydrogel elicited a strong t h 2 response with high antibody titers, but conferred no protection from virus challenge. this study highlighted the importance of carefully considering what kind of adjuvant is suitable to accompany a vlp vaccine, as the most immunogenic adjuvant may not necessarily provide the desired vaccine outcome. vlp vaccines approved for clinical use have utilized various administration modalities, including vaccination subcutaneously, intradermally, intramuscularly or at mucosal surfaces [168] . live attenuated vaccines are traditionally injected subcutaneously, while inactivated or subunit vaccines are often administered intramuscularly. in infants, intramuscular injection of vaccines tends to confer enhanced immunogenicity over subcutaneous administration [169] . modern vaccine delivery technologies, such as intradermal microneedle patches, have also been investigated for their potential applications with vlp vaccines [156, 157, 170] . while the majority of recent clinical trials involving the administration of vlp favor intramuscular injection, alternative routes of administration are being investigated based on differential immunogenicity at different delivery sites. for example, an investigation into the routes of administration of hunv vlp found that intranasal inoculation of mice induced the production of mucosal anti-hunv igg and iga antibodies, while intramuscular injection only induced igg production [171] . the study identified that only mucosal iga was suitable for neutralization of infectious hunv virions, rendering intramuscular injection unsuitable for administration of hunv vlp. a similar finding was identified regarding the administration of respiratory syncytial virus (rsv) vlp, with the desired t h 1 response, neutralizing mucosal antibodies and cd8 + t cell responses identified following intranasal inoculation, rather than intramuscular injection [172] . vaccination with vlp at mucosal surfaces also requires higher doses of vlp in comparison to parental administration. oral delivery of virus-like particles is also being investigated as an alternative, convenient route of administration. vlp expressed in plant-based systems represents a particularly efficient means of both production and delivery, with expression in an edible plant species potentially forgoing the need for vlp purification and vaccine formulation. examples of this concept include the expression of hbv and norwalk virus vlp in solanum tuberosum potato and lycopersicon esculentum tomato plants [173, 174] . nicotiana benthamiana tobacco are also used as an efficient plant expression system for production of vlp. hbv vlp purified from n. benthamiana are capable of withstanding a simulation of the acidic environment within the stomach [175] ; however, the hbcag polypeptide of hbv vlp was digested when exposed to porcine pepsin. the protection of protein-based vlp from enzymatic digestion is a significant hurdle for oral delivery, despite the promise of this vaccination route. the translation of vlp vaccines from preclinical research to routine clinical administration is a multifaceted task combining studies into stability, formulation science, immunogenicity, and clinical vaccine efficacy. these issues are not necessarily unique to the development of vlp vaccines, encompassing the breadth of research and development necessary for bringing any vaccine candidate to market. the expression of stable vlp is only the first step toward the development of a novel vlp vaccine, which may eventually include various excipients, adjuvants and other compounds in the administrable form. advancements in virology and vaccinology that have facilitated the development of novel vlp vaccines seemingly parallel regulatory and proprietary restrictions placed upon new vaccines; however, vlp vaccines also possess unparalleled potential due to their balance between clinical vaccine efficacy and safety, and their versatility as a vaccine vector. the field of vlp vaccines has continued to experience significant growth over the past decade, both in the diversity of vaccines and in the translation of vaccines toward routine clinical administration. our research focuses upon the development of rhdv vlp as a versatile vaccine scaffold, particularly for immunotherapeutic vaccination as an alternative treatment option for cancer. the translation of vlp vaccine constructs from proof of concept models toward clinical administration is no menial feat. we have observed a corresponding shift toward focusing on clinical translation amongst our colleagues in the field. focal points of discussion highlighted by the process include the relevance and translatability of research animal models, adaptation to the established dogma within the field of human vaccination, and the establishment of a developmental and commercial niche conducive to the advancement of novel vaccines throughout the rigors of clinical trials and regulatory approval. the immune response to vlp vaccines tends to strike a desirable balance between outcomes indicative of optimal vaccine efficacy, such as the induction of high-titer mucosal iga antibodies, while also maintaining an almost unparalleled vaccine safety profile. the inherent inability of vlp to infect or replicate alleviates potential vaccine risks, such as spontaneous reversion to pathogenesis, or incomplete inactivation. retention of the ability to facilitate cross-presentation of associated antigens, and the induction of a potent cell-mediated immune response can also have significant implications for vlp vaccines, diversifying the field to cover a broader range of disorders and diseases. the intracellular processing pathways remain largely unexplored for many vlp, and elucidation of the underlying mechanisms involved in some of the more advanced applications of vlp vaccines, such as gene delivery and immunomodulation. the role of formulation science appears underrepresented amongst clinical trials and clinical reports for vlp vaccines, with at times minimal elucidation of the vaccine administration route, the excipients included, and even exclusion of adjuvant delivered with the vaccine. the importance of formulation science and the relevance of excipients and adjuvants may be expectedly underappreciated during earlier phases of research and development, where establishment of vaccine efficacy may be paramount; however, early adoption of these vital constituents may ease clinical translation, potentially expanding the repertoire of marketable vlp vaccines. novelty, proprietary design, and versatility are almost as important in the development of vlp vaccines as the actual vaccine efficacy itself. maintaining compatibility with the upscaling of production and gmp manufacture pipelines is another important component in the development of a vlp vaccine for routine clinical administration. the benefit of such foresight is clear, and the promotion of clinical translation will be advantageous across the field of vlp vaccines. upon review across the field including current trends in new and emerging research, the progression of established vaccine research, and clinical trial schedules, vlp vaccine research is experiencing a period of rapid growth. this includes both the repertoire of organisms and conditions being targeted with vlp vaccines, and the advancement of established vaccines through clinical trials, translation, and clinical applications. over the next five years, we predict that there will be a significant increase in the diversity of vlp vaccines in active circulation. we expect that vlp will be used in more complex applications than as a vaccine against the cognate virus from which they were derived. we also predict the development of novel vlp vaccine constructs for human and zoonotic pathogenic organisms and conditions with or without current vaccination options, and the incorporation of advanced vaccination methodologies and techniques into routine vlp vaccine production, distribution and administration. in particular, we predict increased incorporation of alternative vaccine-storage techniques such as freeze-dried or spray-dried methods. the development of vlp vaccines is often focused on facilitating distribution amongst populations and communities where these vaccines are most needed. we predict that over the next five years, the capability to support vlp vaccine distribution will increase by limiting the current reliance on cold-chain delivery. these predictions outline an exciting future over the next five years for vlp vaccine research and development. • virus-like particles consist of virus-derived proteins and associated molecules that spontaneously form a particulate structure. • vlp vaccines have the safety profile of a subunit vaccine, but with efficacy and vaccination outcomes that can be comparable to killed or live attenuated vaccines, depending on the particular vlp vaccine. • while the predominant desired immune response amongst the majority of vlp vaccines is a potent humoral response producing high titer antibodies, some vlp vaccines can induce a potent cytotoxic immune response for selective elimination of cells currently infected by the target virus, or target tumor cells. • formulation science plays a major role in the development of vlp vaccines, facilitating the selection of appropriate combinations of excipients and adjuvants. excipients are used to increase vlp particle stability in the vaccine formulation, while adjuvants enhance vaccine efficacy, and help to select for a specific desired immune response and outcome. this research was funded by the university of otago. interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. peer reviewers on this manuscript have no relevant financial or other relationships to disclose. braeden donaldson http://orcid.org/0000-0003-2613-1669 vernon k. ward http://orcid.org/0000-0001-8692-5116 papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers antigen delivery by virus-like particles for 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microbial factories and cell-free systems as platforms for vaccine development chimeric gii. 4 norovirus virus-like-particle-based vaccines induce broadly blocking immune responses chimeric bivalent virus-like particle vaccine for h5n1 hpai and nd confers protection against a lethal challenge in chickens and allows a strategy of differentiating infected from vaccinated animals (diva) novel recombinant chimeric viruslike particle is immunogenic and protective against both enterovirus 71 and coxsackievirus a16 in mice multi-target chimaeric vlp as a therapeutic vaccine in a model of colorectal cancer an enhanced heterologous virus-like particle for human papillomavirus type 16 tumour immunotherapy protective effect of a germline, il-17-neutralizing antibody in murine models of autoimmune inflammatory disease virus-like particles from rabbit hemorrhagic disease virus can induce an anti-tumor response memory and effector cd8 t-cell responses after nanoparticle vaccination of melanoma patients a vlp-based vaccine against interleukin-1α protects mice from atherosclerosis development of an interleukin-1β vaccine in patients with type 2 diabetes a vaccine against nicotine for smoking cessation: a randomized controlled trial virus-like particles, a versatile subunit vaccine platform bioengineering virus-like particles as vaccines construction and characterization of virus-like particles: a review this review covers the breadth of expression systems available for vlp vaccine production, while also discussing vlp structural diversity and characterization mannosylation of virus-like particles enhances internalization by antigen presenting cells coupling the adjuvant cpg oligonucleotides to rhdv vlp. dunedin: university of otago αenv-decorated phosphatidylserine liposomes trigger phagocytosis of hiv-virus-like particles in macrophages norovirus p particle: a subviral nanoparticle for vaccine development against norovirus, rotavirus and influenza virus nanoparticles target distinct dendritic cell populations according to their size conduits mediate transport of low-molecular-weight antigen to lymph node follicles the humoral immune response is initiated in lymph nodes by b cells that acquire soluble antigen directly in the follicles virus-like particle (vlp) lymphatic trafficking and immune response generation after immunization by different routes follicular shuttling of marginal zone b cells facilitates antigen transport b cells acquire particulate antigen in a macrophage-rich area at the boundary between the follicle and the subcapsular sinus of the lymph node a molecular assembly system that renders antigens of choice highly repetitive for induction of protective b cell responses. vaccine dissecting virus entry via endocytosis despite differences between dendritic cells and langerhans cells in the mechanism of papillomavirus-like particle antigen uptake, both cells cross-prime t cells. virology cross-presentation of epitopes on virus-like particles via the mhc i receptor recycling pathway the length of vesicular stomatitis virus particles dictates a need for actin assembly during clathrin-dependent endocytosis single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes differential uptake and crosspresentation of human papillomavirus virus-like particles by dendritic cells and langerhans cells an engineered non-toxic superantigen increases cross presentation of hepatitis b virus nucleocapsids by human dendritic cells multivalent display and receptormediated endocytosis of transferrin on virus-like particles low ph-dependent endosomal processing of the incoming parvovirus minute virus of mice virion leads to externalization of the vp1 n-terminal sequence (n-vp1), n-vp2 cleavage, and uncoating of the full-length genome efficient gene transfer using the human jc virus-like particle that inhibits human colon adenocarcinoma growth in a nude mouse model cross-presentation of virus-like particles by skin-derived cd8-dendritic cells: a dispensable role for tap hepatitis b virus-like particles access major histocompatibility class i and ii antigen presentation pathways in primary dendritic cells uptake and presentation of hepatitis c virus-like particles by human dendritic cells proteasome-independent major histocompatibility complex class i cross-presentation mediated by papaya mosaic virus-like particles leads to expansion of specific human t cells in vivo, dendritic cells can crosspresent virus-like particles using an endosome-to-cytosol pathway influenza-pseudotyped gag virus-like particle vaccines provide broad protection against highly pathogenic avian influenza challenge eleven years of inflexal ® v-a virosomal adjuvanted influenza vaccine inflexal ® v a trivalent virosome subunit influenza vaccine: production influenza virus assembly and budding in raftderived microdomains: a quantitative analysis of the surface distribution of ha, na and m2 proteins influenza virus m2 ion channel protein is necessary for filamentous virion formation infectious entry pathway of influenza virus in a canine kidney cell line studies on the mechanism of influenza virus entry into cells epsin 1 is a cargo-specific adaptor for the clathrin-mediated endocytosis of the influenza virus dissection of the influenza a virus endocytic routes reveals macropinocytosis as an alternative entry pathway endocytosis of influenza viruses influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis gene therapy for human lung adenocarcinoma using a suicide gene driven by a lung-specific promoter delivered by jc virus-like particles in vivo sirna delivery using jc virus-like particles decreases the expression of rankl in rats the human polyomavirus, jcv, uses serotonin receptors to infect cells jc virus enters human glial cells by clathrin-dependent receptor-mediated endocytosis contrasting roles of endosomal ph and the cytoskeleton in infection of human glial cells by jc virus and simian virus 40 use of the baculovirus system to assemble polyomavirus capsid-like particles with different polyomavirus structural proteins: analysis of the recombinant assembled capsid-like particles human jc virus-like particles as a gene delivery vector encapsulation and delivery of plasmid dna by virus-like nanoparticles engineered from macrobrachium rosenbergii nodavirus a novel polyethyleneimine-coated adeno-associated virus-like particle formulation for efficient sirna delivery in breast cancer therapy: preparation and in vitro analysis gene transfer using recombinant rabbit hemorrhagic disease virus capsids with genetically modified dna encapsidation capacity by addition of packaging sequences from the l1 or l2 protein of human papillomavirus type 16 novel mir-122 delivery system based on ms2 virus like particle surface displaying cell-penetrating peptide tat for hepatocellular carcinoma neutralizing antiviral b cell responses virus-like display of a neo-self antigen reverses b cell anergy in a b cell receptor transgenic mouse model low-affinity b cells transport viral particles from the lung to the spleen to initiate antibody responses this research article investigates the transportation of vlp following intranasal delivery, mediated by low-affinity b cells. intranasal vaccination and the induction of mucosal iga anti-vlp production is an important vaccination outcome for vlp vaccines against viruses that infect through mucosal surfaces extrafollicular antibody responses germinal center b and follicular helper t cells: siblings, cousins or just good friends competence and competition: the challenge of becoming a long-lived plasma cell plasma cell development: from b-cell subsets to long-term survival niches b lymphocyte activation by human papillomavirus-like particles directly induces ig class switch recombination via tlr4-myd88 t cell-dependent and-independent iga responses: role of tlr signalling alveolar macrophages and lung dendritic cells sense rna and drive mucosal iga responses efficient induction of mucosal and systemic immune responses by virus-like particles administered intranasally: implications for vaccine design mucosal immunity in the female genital tract mucosal immunity in the female genital tract: relevance to vaccination efforts against the human immunodeficiency virus a comparison of antibody titres in mouse uterine fluid after immunization by several routes, and the effect of the uterus on antibody titres in vaginal fluid systemic and secretory humoral immunity in the normal human vaginal tract comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women specific antibody levels at the cervix during the menstrual cycle of women vaccinated with human papillomavirus 16 virus-like particles induction of immune memory following administration of a prophylactic quadrivalent human papillomavirus (hpv) types 6/11/16/18 l1 virus-like particle (vlp) vaccine. vaccine induction of virusspecific cytotoxic t lymphocytes as a basis for the development of broadly protective influenza vaccines influenza vaccines: t-cell responses deserve more attention this review article discusses the importance of inducing a potent cytotoxic immune response for vaccination against select virus infections t-cell-mediated and humoral approaches to universal influenza vaccines better influenza vaccines for older people: what will it take? influenza hemagglutinationinhibition antibody titer as a correlate of vaccine-induced protection efficient homologous prime-boost strategies for t cell vaccination based on virus-like particles use of chemotherapy plus a monoclonal antibody against her2 for metastatic breast cancer that overexpresses her2 antigen incorporated in virus-like particles is delivered to specific dendritic cell subsets that induce an effective antitumor immune response in vivo messenger rna vaccine based on recombinant ms2 virus-like particles against prostate cancer anti-tumor effect of the alphavirus-based virus-like particle vector expressing prostate-specific antigen in a hla-dr transgenic mouse model of prostate cancer universal influenza a m2e-hbc vaccine protects against disease even in the presence of preexisting anti-hbc antibodies segments of puumala hantavirus nucleocapsid protein inserted into chimeric polyomavirus-derived virus-like particles induce a strong immune response in mice carrier induced epitopic suppression of antibody responses induced by virus-like particles is a dynamic phenomenon caused by carrier-specific antibodies. vaccine innate immunity mediates follicular transport of particulate but not soluble protein antigen this research article investigates the transportation and delivery of qβ vlp to follicular dendritic cells within the lymph node, and the impairment of this delivery in the presence of anti-qβ vlp antibodies effects of preexisting anti-carrier immunity and antigenic element multiplicity on efficacy of a modular virus-like particle vaccine immunomodulation and vaccination with rhdv vlp: a thesis submitted for the degree of doctor of philosophy prime immunization with rotavirus vlp 2/6 followed by boosting with an adenovirus expressing vp6 induces protective immunization against rotavirus in mice can different virus-like particles be used for primeboost vaccination? dunedin: university of otago chimeric hepatitis e virus-like particle as a carrier for oral-delivery. vaccine evaluation of the stability of enterovirus 71 virus-like particle physical stabilization of norwalk virus-like particles downstream processing of triple layered rotavirus like particles development of a stable viruslike particle vaccine formulation against chikungunya virus and investigation of the effects of polyanions stability studies of hiv-1 pr55gag virus-like particles made in insect cells after storage in various formulation media rational design of a stable, freezedried virus-like particle-based vaccine formulation stability of virus-like particles of redspotted grouper nervous necrosis virus in the aqueous state, and the vaccine potential of lyophilized particles modular virus-like particles for sublingual vaccination against group a streptococcus. vaccine virus-like particle formulation optimization by miniaturized high-throughput screening freeze-drying of plant tissue containing hbv surface antigen for the oral vaccine against hepatitis b developments in the formulation and delivery of spray dried vaccines preclinical refinements of a broadly protective vlp-based hpv vaccine targeting the minor capsid protein, l2. vaccine optimized formulation of a thermostable spray-dried virus-like particle vaccine against human papillomavirus characterization of a spraydried candidate hpv l2-vlp vaccine stored for multiple years at room temperature. papillomavirus res this research article demonstrates the compatibility of a recombinant ms2-16l2 vlp vaccine with spray-drying, and the retention of vaccine immunogenicity after prolonged storage at room temperature immunization by vaccine-coated microneedle arrays protects against lethal influenza virus challenge transdermal influenza immunization with vaccine-coated microneedle arrays vaccine adjuvants: from 1920 to 2015 and beyond vaccine adjuvants as potential cancer immunotherapeutics distinct indirect pathways govern human nk-cell activation by tlr-7 and tlr-8 agonists tlr7/8-mediated activation of human nk cells results in accessory cell-dependent ifn-gamma production the tlr7 agonists imiquimod and gardiquimod improve dc-based immunotherapy for melanoma in mice tlr7/8 agonists promote nk-dc crosstalk to enhance nk cell anti-tumor effects in hepatocellular carcinoma activation of toll-like receptor 7 inhibits the proliferation and migration, and induces the apoptosis of pancreatic cancer cells immunotherapy of type-1 allergies with virus-like particles and cpg-motifs a universal virus-like particle-based vaccine for human papillomavirus: longevity of protection and role of endogenous and exogenous adjuvants. vaccine adjuvant-enhanced cd4 t cell responses are critical to durable vaccine immunity influence of parenteral administration routes and additional factors on vaccine safety and immunogenicity: a review of recent literature immunogenicity and safety of measles-mumps-rubella-varicella (mmrv) vaccine followed by one dose of varicella vaccine in children aged 15 months-2 years or 2-6 years primed with measles-mumps-rubella (mmr) vaccine long-term protective immunity from an influenza virus-like particle vaccine administered with a microneedle patch mucosal antibodies induced by intranasal but not intramuscular immunization block norovirus gii.4 virus-like particle receptor binding a single intranasal administration of virus-like particle vaccine induces an efficient protection for mice against human respiratory syncytial virus virus-like particle expression and assembly in plants: hepatitis b and norwalk viruses expression of norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice plant-expressed hepatitis b core antigen virus-like particles: characterization and investigation of their stability in simulated and pig gastro-intestinal fluids key: cord-340639-hdn85mik authors: uk lee, byung; yermakov, mikhail; grinshpun, sergey a. title: unipolar ion emission enhances respiratory protection against fine and ultrafine particles date: 2004-11-30 journal: journal of aerosol science doi: 10.1016/j.jaerosci.2004.05.006 sha: doc_id: 340639 cord_uid: hdn85mik abstract we developed a novel concept that allows to considerably improve the performance of conventionally used filtering-facepiece respirators against fine and ultrafine aerosols including airborne viral and bacterial agents. the concept is based on the continuous emission of unipolar ions. the effect was evaluated through the real-time monitoring of the concentration and size distribution of fine and ultrafine aerosol particles. the measurements were conducted inside and outside of a respiratory mask that was face sealed on a breathing manikin. a commonly used type n95 respirator and surgical mask were utilized for the tests. the manikin was placed in a 24.3-m3 indoor test chamber and exposed to polydisperse surrogate aerosols simulating viral and bacterial particles with respect to the aerodynamic size. the particle penetration through the mask was found to decrease by one-to-two orders of magnitude as a result of continuous unipolar ion emission in the chamber. the flux of air ions migrated to the breathing zone and imparted electrical charges of the same polarity to the aerosol particles and the respirator filter surface. this created an electrostatic shield along the external surface of the filter, thus enhancing the protection characteristics provided by the respirator. the above performance enhancement effect is crucial for minimizing the infectious risk in the cases when the conventional filtering-facepiece respirators are not able to provide an adequate protection against airborne viruses and bacteria. the outbreaks of emerging diseases (e.g., sars) and the threat of bioterrorism have triggered an urgent demand for adequate respiratory protection against bioaerosol agents, including airborne viruses and bacteria. particular interest has been directed towards increasing the e ciency of existing respiratory protection devices. the ÿltering-facepiece masks, including type n95 respirators, are frequently used in indoor air environments to prevent or considerably reduce inhalation of droplet nuclei that can potentially carry viable microorganisms. millions of workers, including health-care personnel, routinely use respirators in their workplaces (united states department of labor, 1995) . in case of bioterrorist attack in a major urban area, there may be a need in millions readily available respiratory protection devices. the existing respirators have been extensively evaluated against ÿne particles (e.g., brosseau, evans, ellenbecker, & feldstein, 1989; chen, ruuskanen, pilacinski, & willeke, 1990; chen & willeke, 1992; huang, willeke, qian, grinshpun, & ulevicius, 1998; johnston, myers, colton, birkner, & campbell, 2001; qian, willeke, grinshpun, donnelly, & co ey, 1998; halvorsen, 1998) and microorganisms (centers for disease control and prevention, 1994; lee, slavcev, & nicas, 2004a; qian, willeke, grinshpun, & donnelly, 1997; qian et al., 1998; reponen, wang, willeke, & grinshpun, 1999; willeke, qian, donnelly, grinshpun, & ulevicius, 1996) . at the same time, the protection e ciency of existing facepiece respirators have not been well characterized with respect to ultraÿne particles, i.e. those below 0:1 m (hinds, 1999) . the respirators di er from one another by their ÿltration e ciency, which is dependent on the ÿlter properties and the particle size. for example, a type n95 respirator may allow up to 5% penetration in "a worst case scenario," when most-penetrating sodium chloride particles of 0:3 m mass median aerodynamic diameter are drawn through the ÿlter at a ow rate of 85 l min −1 (federal register, 1995) . the penetration e ciency of larger mycobacterium tuberculosis bacteria (mtb, 0:8 m) through a face-sealed n95 respirator at strenuous workload is as low as about 0.5% . the face-sealed ÿlter of a conventional health-care mask, which ensures relatively low pressure drop and consequently good comfort level, allows approximately 15% of airborne mtb surrogate bacteria to penetrate, thus providing 85% protection against these bacteria (willeke et al., 1996) . if the bacterial concentration in the air is 1000 m −3 , an unprotected individual breathing at 30 l min −1 inhales 1800 microorganisms per hour, whereas the one wearing a perfectly ÿt n95 respirator inhales up to 90 microorganisms per hour. if the infectious dose of a bioaerosol agent of interest is less than 90, the n95 respirator may not provide an adequate respiratory protection once the exposure time exceeds one hour. the use of an improperly ÿt-tested tight-ÿtting respirator may further decrease the respiratory protection level because of the additional particle penetration that occurs through the face-seal leaks (chen et al., 1990; chen & willeke, 1992; oestenstad, dillion, & perkins, 1990a; oestenstad, perkins, & rose, 1990b) . based on the above considerations, it seems very useful if the ÿltration e ciency of existing respirators can be increased while the comfort level provided by these devices would remain the same. with respect to the respiratory exposure and protection, the particle aerodynamic diameter range of d a ∼ 0:04-2 m is of special public interest because of its health relevance. many bioaerosol agents, including viruses and bacteria that cause emerging diseases as well as those that can be used for biological warfare or in the event of bioterrorism, belong to this size range. for example, according to the national center for biotechnology information (http://www.ncbi.nlm.nih.gov), the dimension of the coronavirus virion (the etiological agent of the sars) are (60-120) × (160-200) nm, which corresponds to d a ∼ 0:1 m. for bacillus anthracis (bacteria causing anthrax), d a ∼ 1 m. as the above range is broad and includes both the ÿne and ultraÿne particle fractions (baron & willeke, 2001; hinds, 1999) , the particle penetration e ciency through the ÿlter media can be a ected by several mechanisms and is generally characterized by the particle aerodynamic size. this allows testing the performance of respirator ÿlters against pathogenic agents using non-pathogenic aerosol surrogates that simulate the aerodynamic characteristics of the particles of interest. in this study, we developed and evaluated a novel concept that drastically enhances the performance of conventional ÿltering-facepiece respirators against ÿne and ultraÿne aerosol particles. the concept is based on the continuous emission of unipolar ions into the air in the vicinity of the respirator. the aerosol particles are unipolarly charged by air ions primarily due to the di usion charging mechanism (adachi, kousaka, & okuyama, 1985; frank, cederfelt, & martinsson, 2004; hernandez-sierra, alguacil, & alonso, 2003; wiedensohler et al., 1994) . the ion-ÿlter interaction and the deposition of unipolarly charged particles on the external surface of the ÿlter impose signiÿcant unipolar charge on the ÿlter. this creates a shield for the incoming particles (as they carry charges of the same polarity), which decreases the penetration e ciency through the ÿlter. the new concept was experimentally evaluated in a non-occupied, unventilated indoor test chamber (l × w × h = 3:78 m × 2:44 m × 2:64 m = 24:3 m 3 ). this facility, developed in the center for health-related aerosol studies at the university of cincinnati, has been used in our previous studies (choe et al., 2000; grinshpun et al., 2002 grinshpun et al., , 2004 . the experimental setup is schematically shown in fig. 1 . a breathing manikin with a face-sealed respiratory mask was exposed to the airborne polydisperse surrogate aerosols that simulated viral and bacterial particles with respect to their aerodynamic size. the leakage tests were conducted between the mask and the face of the manikin with a bubble-producing liquid (trubble bubble, new jersey meter co., paterson, nj, usa). the manikin operated at a breathing ow rate, 30 l min −1 , representing human breathing during light workloads (mineral resources, 1994; johnson, weiss, & grove, 1992) . the electrical low pressure impactor (elpi, tsi inc./dekati ltd., st. paul, mn, usa) was used to determine the concentration and aerodynamic particle size distribution in real-time. this instrument utilizes the cascade impaction principle and-in addition-has a direct-reading capability. the aerosol particles are charged by the corona charger downstream of the elpi inlet and subsequently detected by the electrometers inside the cascade impactor. the particles were collected by the elpi inside and outside the respirator using identical sampling lines. for these measurements, a 10-mci kr 85 charge equilibrator (3m company, st. paul, mn, usa) was installed upstream of the elpi inlet to neutralize the particles to boltzmann charge equilibrium. this allowed us to avoid the in uence of high electric charges, imparted by the particles as a result of their interaction with air ions, on the elpi performance. the time resolution of the instrument was adjusted to 10 s. the data were recorded in 12 elpi channels (each channel = impaction stage), from 0.04 to 8:4 m. the latter sizes represent the midpoint diameters of the ÿrst and the 12th impaction stages (the midpoint = the geometric mean of the stage's boundaries). the natural aerosol concentration in the indoor test chamber was not su cient, particularly for the measurement inside the mask, because the ÿlter removed considerable number of ambient airborne particles. to increase the initial background aerosol concentration, we used a smoke generator. the smoke particles covered primarily the submicrometer aerodynamic size range (cheng, bechtold, yu, & hung, 1995) with a sharp decrease in the particle number at d a ¿ 1:5-2 m. thus, the data recorded in the ÿrst 8 measurement channels of the elpi (d a = 0:04-1:3 m) were used for the analysis. the measured aerosol concentrations inside (c in ) and outside (c out ) the mask were incorporated into the equation for the respirator penetration e ciency, e p : which was determined as a function of the particle aerodynamic diameter. e p is actually the inversed protection factor that is frequently used as a respirator performance index. initially, the background tests were conducted by measuring the penetration e ciency of the mask with no ion emission. then, a unipolar ion emitter was turned on at a distance of 20 cm from the mask, and e p (d a ) was determined in 3-min time intervals during 12 min. the continuous air ion emission in the chamber decreased c out as the particles, charged by ions to the same polarity, repelled and subsequently migrated toward the chamber's walls and deposited on these walls (grinshpun et al., 2004) . the change in the c out -value that occurred during each 3-min time interval due to ionic air puriÿcation in the chamber was taken into account through the linear interpolation of c out (t). in this study, we used a negative ion emitter (vi-2500, wein, inc., los angeles, ca, usa) producing an air ion concentration of n i ∼ 1:3 × 10 6 elementary charges per cm 3 as determined at a distance of 1 m from the source. the ion concentration was measured by the air ion counter (alphalab inc., salt lake city, ut, usa) that operates within the range of 10 -2×10 6 ions per cm 3 . two types of ÿltering-facepiece respiratory masks commercially available from a major manufacturer were tested in this study. one was the niosh (us national institute for occupational safety and health) certiÿed n95 respirator and the other one was a conventional disposable surgical mask. the n95 respirator consists of inner and outer cover webs made of rayon. its ÿlter made of polyester and polypropylene with the electrostatically charged microÿbers providing relatively high ÿltering e ciency. in the surgical mask, the polypropylene ÿlter is sandwiched between inner and outer webs made of rayon. the ÿlter of a surgical mask has lower ÿltration e ciency as compared to the one of an n95 respirator. thus, the e ect of ion emission on the respirator ÿlter e ciency was tested for the masks having two distinctly di erent original performance characteristics. the average values and the standard deviations of the penetration e ciency were calculated for each set of conditions as a result of at least three replicates. the data were statistically analyzed using the microsoft excel software package (microsoft co., redmond, wa, usa). fig. 2 shows the normalized initial aerosol concentration measured outside the respirator mask. each data point represents an average of six replicates. it is seen that the aerosol particles were primarily within a range of d a ≈ 0:04-0:5 m (the concentration n= log d a was between ¿ 10 4 and ¿ 10 5 cm −3 ), while fewer micron-size particles were detected ( n= log d a ∼ 10 3 cm −3 ). for each measured particle size, the initial aerosol concentration was reproducible with the variability (the coe cient of variation) not exceeding about 50% for six replicates. fig. 3 presents the data obtained with two types of face-sealed respirators: n95 respirator and a surgical mask. when no air ion emission was introduced, the e p -value, averaged over the test range of d a , was about 1.8% for the n95 respirator. the particle size did not considerably a ect the penetration through the n95 respirator ÿlter, although some decrease of e p with increasing d a was observed for d a ≈ 0:04-0:5 m. once the negative ion emission began, the penetration decreased to 0.27% during the ÿrst 3 min. at t = 12 min, it further decreased to about 0.11%, enhancing the n95 respirator ÿlter performance approximately by a factor of 17 (fig. 3a) . for the surgical mask, the initial penetration e ciency ranged from 18.7% (d a = 0:04 m) to 11.1% (d a = 1:3 m). resulting from the emission of negative ions, the average penetration e ciency through the surgical mask dropped from 15.4% (t = 0) to 0.19% (t = 12 min), demonstrating a 80-fold enhancement (fig. 3b) . the data in fig. 3 show that the most pronounced e ect occurred within the ÿrst 3-min interval. it was surprising to observe that the initial penetration e ciency (t=0) of ultraÿne particles through both masks slightly increased with decreasing particle size. in contrast, the available ÿltration models predict that the peak penetration is reached at d a between 0.1 and 0:3 m, and the particles below 0:1 m should be collected more e ciently as their size decreases (di usion regime) (halvorsen, 1998; hinds, 1999; lee & mukund, 2001) . the following considerations explain the results of our baseline test for the ultraÿne particles. these models (and the laboratory-generated experimental data that support them) characterize the particle penetration through a homogeneous perfectly sealed ÿbrous ÿlter material but not through a respirator mask. the design of a ÿltering-facepiece respirator does not assure a perfect peripheral connection of the assembly, so micro-leaks may be present between the core ÿlter material and the elastic peripheral support. these leaks can contribute to the penetration of the ultraÿne particles. in addition, although the mask was "glued" on the manikin, some very small, micrometer-or submicrometer-size leaks may still remain. most of soap-bubble-based air leak detection methods are capable to identify micro-leaks greater than 1 m. if d a is much lower than the characteristic size of the micro-leaks (1 m), the particles may penetrate through these remaining submicrometer micro-leaks, thus a ecting the overall aerosol penetration e ciency through the ÿltering mask. one more possible factor is associated with the spatial variations in ÿber diameter, orientation, packing density, as well as initial ÿber electrostatic charge level (for those masks utilizing electret ÿlter media). these variations have been shown to signiÿcantly a ect the respirator performance increasing the penetration e ciency of particles of ∼ 0:1 m (huang et al., 1998) . the enhancement of the respirator performance, observed almost immediately after the ion emitter started operating, can be attributed to the electrostatic e ect. the emitted negative ions as well as the particles charged by these ions in the air, impose signiÿcant negative charge on the respirator ÿlter. this forms the "electrostatic shield" against the particles moving toward the mask. the repelling forces decrease the number of particles that can approach the ÿlter. the above-described e ect works outside of the respirator, as opposite to the aerosol ÿltration by di usion, impaction, interception, and electrostatic deposition (lee & mukund, 2001 ) that takes place inside the ÿlter. therefore, the ion-induced decrease in the particle penetration e ciency does not cause the pressure drop increase through the ÿlter providing the enhanced performance with the same comfort level. to quantitatively characterize the e ect, we calculated the velocity of particle migration induced by the electrostatic interaction in the vicinity of the ÿlter. it was then compared to the velocity of the air ow through the ÿlter caused by inhalation. in this calculation, we used information about the airborne particle electric charges and the air ion density, obtained by the elpi and the air ion counter, respectively. the particle size of d a = 0:1 m was chosen, as it represents the dominant size range used in our experiments. furthermore, this value is at the boarder line between the ÿne and ultraÿne particle size ranges. in addition, many viral particles have an aerodynamic diameter of ∼ 0:1 m. two main assumptions were made. first, the aerosol particles and air ions that interacted with the respirator were assumed to give all their electric charges to the respirator ÿlter. second, the charged ÿlter was assumed to act as a point-charge located at the center of the respirator's surface. at the ion emission level produced in our experiment, a 0:1 m particle carries, on average, 10 elementary charges (lee, yermakov, & grinshpun, 2004b) . the calculation showed that the total electric charge acquired by the respirator ÿlter, as a result of a 3-min continued emission from the vi-2500 ion source, is about 1:5 × 10 13 elementary charges, if the breathing ow rate is 30 l min −1 . the ion concentration at the center of the respirator was determined to be approximately 1:6 × 10 8 cm −3 (this point located 20 cm from the ion emission source). thus, the particle migration velocity was found to exceed the air ow velocity, created by the inhalation in the breathing zone, approximately by a factor of 75. this suggests that the e ect of the repelling force between the unipolarly charged particles and ÿlter surface is much stronger than the aerodynamic force. therefore, although our assumptions may not be su ciently conservative, the above assessment demonstrates that the enhancement of the respirator performance by the unipolar ion emission is governed by the electrostatic "shield" mechanism. the drastic decrease of the particle penetration through the respirator ÿlter due to continuous unipolar ion emission may be critical in providing additional respiratory protection by existing masks against viral and bacterial particles. for example, an individual exposed to the in uenza virus concentration of 10; 000 m −3 inhales approximately 4500×0:18=810 viruses during 15 min when breathing through a conventional surgical mask at 30 l min −1 in the absence of ion emission (e p ≈ 18% for 0:1 m particles). the continuous emission of negative air ions (n i ∼ 10 6 e − cm −3 ) in a 25-m 3 room would reduce the indoor viral concentration by a factor of 9 during that 15-min interval (lee et al., 2004a, b) . in addition, it would enhance the surgical mask protection reducing e p from 18% to at least 0.19%. thus, only about (4500=9) × 0:0019 = 0:95 ≈ 1 virus would be inhaled in 15 min. given that the infectious dose of in uenza a2 is 790 viruses (lawrence berkeley national laboratory), the ion emission e ect would make an important di erence with respect to the health risk. while this study is limited to the negative ion emission, we anticipate that the respirator performance enhancement e ect can be achieved also by generating positive air ions, as long as the ion concentration in the vicinity of the mask (breathing zone) is su ciently high. future studies will address the e ects of polarity and the ion emission rate on the particle penetration e ciency through respirator ÿlters. generally, the mask protection factor depends not only on its ÿlter penetration e ciency but also on its face ÿt (in practice, the facepiece mask is not sealed to the human face allowing the particles to penetrate through the leak). this pathway may become especially apparent when the ÿlter material is highly e cient. therefore, future tests involving human subjects, di erent ÿt factors, and other experimental conditions ( ow rates and di erent masks) are needed to better characterize the enhancement e ect, discovered in this study, and link it to the exposure. unipolar and bipolar di usion charging of ultraÿne aerosol particles aerosol measurement: principles, techniques, and applications collection e ciency of respirator ÿlters challenged with monodisperse latex aerosols guidelines for preventing the transmission of mycobacterium tuberculosis in health-care facilities filter and leak penetration characteristics of a dust and mist ÿltering facepiece characteristics of face seal leakage in ÿltering facepieces incense smoke: characterization and dynamics in indoor environments particle settling after lead-based paint abatement work and clearance waiting period respiratory protective devices: final rules and notice characterisation of a unipolar charger for droplet aerosols of 0.1-20 m in diameter indoor air pollution control through ionization e ciency of ÿnal cleaning for lead-based paint abatement in indoor environments respirator fit testing application notes unipolar charging of nanometer aerosol particles in a corona ionizer aerosol technology: properties, behavior, and measurement of airborne particles method for measuring the spatial variability of aerosol penetration through respirator ÿlters review of respirator performance testing in the workplace: issues and concerns respirator performance rating table for mask design filter collection respiratory protection against mycobacterium tuberculosis: quantitative ÿt test outcomes for ÿve type n95 ÿltering-facepiece respirators removal of ÿne and ultraÿne particles from indoor air environments by the unipolar ion emission distribution of faceseal leak sites on a half-mask respirator and their association with facial dimensions identiÿcation of faceseal leak sites on a half-mask respirator performance of n95 respirators: reaerosolization of bacteria and solid particles performance of n95 respirators: filtration e ciency for airborne microbial and inert particles survival of mycobacteria on n95 personal respirators respirator use and practice a novel unipolar charger for ultraÿne aerosol particles with minimal particle losses penetration of airborne microorganisms through a surgical mask and a dust/mist respirator the experimental evaluation part of this investigation was supported by the wein products inc., los angeles, ca, usa. the participation of dr. lee in this study was partly due to the post-doctoral fellowship program and the advanced environmental monitoring research center (ademrc) program of the korea science & engineering foundation (kosef). the authors are thankful for this support. reference to any companies or speciÿc commercial products does not constitute or imply their endorsement, recommendation, or favouring by the university of cincinnati or the authors. key: cord-032174-qu5mm9r1 authors: xu, zhonglin title: sampling theory date: 2013-08-14 journal: fundamentals of air cleaning technology and its application in cleanrooms doi: 10.1007/978-3-642-39374-7_16 sha: doc_id: 32174 cord_uid: qu5mm9r1 in order to ensure the reliability of the measurement results of particle concentration, except for the reasonable detecting method and the sophisticated detection equipment, the correct sampling principle to minimize the sampling errors must be followed, which requires that people who carry out the measurement should master the correct sampling theory. in order to ensure the reliability of the measurement results of particle concentration, except for the reasonable detecting method and the sophisticated detection equipment, the correct sampling principle to minimize the sampling errors must be followed, which requires that people who carry out the measurement should master the correct sampling theory. sampling is generally divided into the planktonic method (the airborne state of sampled particles remains the same) and the capture method (airborne particles are captured). in the capture method sampling system, there are a few instruments: the sampler (as the clamp of a filter material, it is preferably made of stainless steel with the commonly effective diameter 25 mm, as shown in fig. 16 .1), the sampling tube (usually plastic pipe), flowmeter (the float flowmeter is commonly used; the flow rate is 0-30 l/min), vacuum pump (vacuum degree !300 mmhg; the flow rate !30 l/min), and valve (the needle valve or other fine-tuned valve). in the planktonic method sampling system, it is mainly composed of the dust analyzer (such as a particle counter) and the sampling tube. when the sampling system is designed, there are two problems should be paid attention to. one is the orientation of the sampling probe, and the other is the position of the flowmeter. onto the inner wall of the tube or the mouth edge, while some cannot enter the sampler or the sampling tube, so that the resultant collected particle number is less than the actual value. figure 16 .2 shows the curve about the effect of the inclination angle between the sampling mouth and the direction of the flow [1] . when the angle is less than 30 , for particles with diameter less than 10 mm, the sampling error is less than 5 %. figure 16 .3 provides a curve which can be used to calculate this kind of error more accurately [2] . one accurate method is to obtain the direction with the maximum dynamic pressure, i.e., the airflow direction, when the pitot tube is used. when sampling is performed outside, the filter material should be perpendicular to the ground to avoid the deposition of large particles or debris on the filter material. the essence of this problem is the influence of additional resistance on the flowmeter. the calibration of flowmeter is carried out in the environment with certain resistance (i.e., the additional resistance at the entrance of the flowmeter), pressure, and temperature, but in the actual application period, all these conditions changed, so it will certainly affect the original calibration value. in particular, the additional resistance caused by sampler, filter, and valve is often neglected, so it is necessary to emphasize this point. figure 16 .4 shows the experimental system to investigate the influence of additional resistance caused by different installation positions of the flowmeter [3] . for the completely same flowmeters 1 and 2 (after calibration), they are connected in series and k 1 is placed in between to create resistance. its value is indicated by 3. flowmeter 1 reflects the standard flow, and flowmeter 2 reflects the flow under different resistances. when the vacuum pump is turned on, it can be found that the flow rates showed by the flowmeter 1 and 2 are different. the former is less than the latter. the larger the resistance is, the larger the difference is. if k 1 and k 2 are adjusted in the way that the value indicated by the flowmeter 2 is fixed while the resistance of k 1 varies, the variation of the values indicated by flowmeter 1 is shown in fig. 16 .5. the main reason for this result is that air pressures inside the two flowmeters are different due to the additional resistance caused by spiral clamp k 1 . when air enters from the bottom of the flowmeter 1, there is almost no additional resistance (flowmeter calibration is often carried out in this case). when air goes through k 1 , the resultant air pressure in the flowmeter 2 decreases. according to the principle for orifice flowmeter and float flowmeter, when the same gas under different conditions is measured with a flowmeter, the following relationship about flow rates can be obtained: where q 1 and q 2 are the indicated flow rates under two kinds of working conditions; ρ 1 and ρ 2 are gas densities under two kinds of working conditions. it is known that the gas density ρ is proportional to the pressure p and is inversely proportional to temperature t. the above expression can be written as where p 1 and p 2 are the pressures upstream of the flowmeters under two different working conditions (the air pressure in calibration condition is generally one atmospheric pressure or 760 mmhg. the air pressure in measurement condition is the subtraction of the absolute value of the readings of the pressure gauge upstream of the flowmeter from the local atmospheric pressure b or the subtraction between b and the known additional total resistance before the flowmeter. only in the circumstance with the sampler, the total resistance becomes the resistance of the filter media under the sampling velocity.); t 1 and t 2 are air temperatures in two different working conditions, which is generally 20 c in calibration condition. there are two kinds of consideration methods for the application of this expression. the first consideration method. let the indication value q 2 represent the scale value in calibration condition, q 1 the indication value to be measured in the actual working condition, p 2 one atmospheric pressure in calibration conditions, and p 1 the pressure in the measurement condition; it is obvious that p 1 < p 2 , and q 1 > q 2 (for the convenience of discussion, it is assumed that there is no difference for the temperature), namely, the actual flow through the flowmeter is greater than the indication value. this is because of the reduced density and the decreased air pressure in the flowmeter. the second consideration method. let the indication value q 2 represent the actual measured indication value, p 2 the actual measured pressure, and q 1 the indication value with the calibration pressure p 1 ; it is obvious that p 2 < p 1 and q 1 < q 2 , i.e., the actual indication value q 2 in actual condition should be reduced to q 1 in the calibration condition, which is always easily neglected by people. the round dots in fig. 16 .5 are the indication values in the calibration condition after calculation (the temperature difference is ignored), and the value agrees well with the experimental data. if the above two calculation results should be converted to other conditions, the following equation can be used: where the superscript "'" in the equation represents the conversion state, such as the calibration state, standard state, sampling state, and special state in the flowmeter. the subscript "1" means the adopted symbol during the application of eq. (16.2) with one of the two consideration methods. as for what kind of the destination state of conversion, it depends on the need. for example, in inspection process of product quality or quality accident, the actual local particle concentration should be known, so it should be converted into the sampling state. for the general measurement, for the purpose of comparison, it should be converted to calibration state or standard state. so for the general measurement, the second method to correct the flow rate should be used, because after correction the result under calibration state is obtained, which no longer needs state conversion. according to the influence of the additional resistance on the flow rate, the following points in the sampling should be paid attention to: 1. when the particle concentration is measured with the filter method, under the premise of the particle collecting efficiency, the filter material with small resistance should be used, and the resistance of the pipeline should be reduced as much as possible. 2. when the flowmeter is installed at the suction end of a vacuum pump, the valve used to regulate the flow must be installed behind the flowmeter, as shown in fig. 16 .6. it should not be installed between the sampler and the flowmeter. if the flowmeter is installed at the exhaust end of the vacuum pump, there should be a buffer bottle between the flowmeter and the vacuum pump. particularly for removing large oil drops during the use of mechanical pump, apparatus such as valve and filter cannot be placed outside of the buffer bottle, as shown in fig. 16 .7. if sampling is performed in the cleanroom, the vacuum pump can only be placed indoors. because the exhaust of vacuum pump must pass hepa filter, it is not suitable to place the flowmeter at the exhaust end. 3. when filter paper is used as the media, because the resistance during sampling is not more than 30 mmhg, so its influence can be neglected. when microporous membrane filter is used for sampling, because its resistance during sampling is often more than 100 mmhg, the influence of this additional resistance on the indication value of flow rate will be more than 10 %. therefore the result must be corrected. now examples for calculation will be given as follows: example 16.1. during the indoor sampling in a cleanroom, the indication value of the flowmeter is 20 l/min, and the sampling time is 16 min. the resistance of sampling filter material (microporous filter membrane) under the sampling flow rate is 150 mmhg. after calculation, the total number of particles is 102,000 under the air temperature 300 c and the air pressure 750 mmhg during measurement. what kind of air cleanliness level can be achieved in the room? ans: 1. the flow without any corrections total sampling flow rate is q ¼ 20 â 15 ¼ 300 l the average particle concentration is n ¼ 102,000/300 ¼ 340 #/l so the air cleanliness in this room reaches class 7. with the first consideration method: (a). the actual flow rate through the flowmeter. the reading value of the flowmeter 20 l/min is the indication value in the calibration condition, so the actual flow rate q 1 through the flowmeter should be according to the actual collected particle concentration 18.31 l/min after correction, the calculated indoor particle concentration is 371.4#/l, so the indoor air cleanliness level does not reach class 7. from the above calculation result, it is found that the influence of additional resistance cannot be overlooked. when sampling is taken in the flowing air (especially when in the pipeline), the plane of the sampling mouth should be vertical to the direction of the air flow (namely, the axis of the sampling pipe and the direction of flow are the same), and the sampling velocity must be equal to the air flow velocity. otherwise, the sampled concentration (i.e., the measured concentration) will be greater than or less than the true concentration, which will cause an error. this is the principle of the isokinetic sampling. on the left of fig. 16 .8, the airflow velocity u 0 is greater than the sampling velocity u. in this case, the boundary limit of flow inhaled into the sampling tube is less than the diameter d of the sampling tube. if there are large particles outside the boundary limit of the inhaled flow or outside of the streamline with diameter d 0 , it will not follow the streamline to flow along the tube wall when it approaches to the tube mouth; instead it will enter the tube due to the inertial effect. small particles on this boundary limit will not enter the tube. in this way, in the same sampling air volume, particles outside this air volume will be carried to enter the tube, especially the large particles. so the sampled concentration of large particles increases, which means the whole sampled concentration will be higher than the actual concentration. on the right in fig. 16 .8, the airflow velocity u 0 is less than the sampling velocity u. in this case, the boundary limit of the flow inhaled into the sampling tube is bigger than the diameter d of the sampling tube. even if there are big particles within the boundary limit of the inhaled flow or within the streamline range with diameter d 0 , when it approaches the tube mouth, it will not enter into the tube but instead flow away along the tube wall, and only small particles in the streamline can enter into the tube. so in the same volume of airflow entering into the tube, particles especially big particles will be lost. so the total sampled concentration will be less than the actual one. therefore, factors that affect the sampling concentrations mainly include the difference between the air flow velocity u 0 and the sampling velocity u (i.e., the velocity at the sampling mouth), the dimensionless inertial parameter describing the inertial effect (stokes parameter) st. the form of the expression is where n is the sampled concentration; n 0 is the real concentration. as for the error caused by non-isokinetic sampling, the following semitheoretical formula [4] [5] [6] is now widely used: in refs. [4, 5] it provides: in ref. [6] it provides: it is shown that the relationship between n n 0 and u 0 u is more obviously demonstrated directly in eq. (16.6) (published in 1980) than eq. (16.7) (published in 1994), so eq. (16.6) was referred by 209e. the difference of the results with the two formulas is little, and the latter is slightly larger. from eq. (16.5) , it is shown that the general characteristic is: for the situation when particles are very big, and the flow rate is large, and the diameter of the sampling tube is very small); st ¼ const (the larger the difference between u 0 and u is, the greater the concentration difference is) u 0 /u ¼ const (the larger st is, the bigger the concentration difference is) when both eqs. (16.5) and (16.6 ) are plotted in fig. 16 .9 [7] , the above feature can be vividly demonstrated. it is shown that: 1. in the conditions of non-isokinetic sampling in the wide range u 0 / u ¼ 0.012-25.76, as long as st ≯ 0.001, or u 0 /u ¼ 0.15-3 and st ≯ 0.01, the sampling error≯ 5 %. because large particles are the sampled object in the field of dust removal technology, the requirement for the velocity deviation is less strict when error is ≯ 5 %. for example, in japanese standard jis z8808, the sampling error for non-isokinetic sampling is within ae5 % when the error of non-isokinetic velocity is between à5 %~+10 %. 2. the influence at the situation u 0 /u >1 which has slower sampling velocity (as on the right of fig. 16 .9) is much larger than that at the situation u 0 /u <1 which has faster sampling velocity (as on the left of fig. 16.9 ). this is also the reason why the negative deviation is less than the positive deviation specified in jis z88-8. at 20 c, μ ¼ 1.83 â 10 à5 pa · s. from table 6 .4, it is found that c ¼ 1.33 (0.5 μm) or 1.03(5 μm). the following results can be obtained with eq. it should be emphasized that, as pointed out in chap. 16 and relevant literatures [7] , the unit of μ during calculation of st must be paid attention to. if the engineering unit in the past is used, the value of μ decreases by 9.8 times, which will increase the particle loss rate by 9.8 times and will draw different conclusions. the above results show that the error with non-isokinetic sampling for 1 μm particles is less than 1 % even when particle counter with the sampling flow rate 28.3 l/min is used. but it reaches 10 % for !5 μm particles. if the requirement of sampling error is not only for particles with a certain size, but also for whole particles with diameter larger than a certain size such as !0.5 μm, it should be calculated according to the standard distribution of particle size. from chaps. 2, 3, and 7, when suppose particles with diameter ! 0.5 μm occupy 100 %, particles with diameter 0.5 μm occupy33%~38 %, so: it can be calculated that the error is 1-1.9 % for all the particles !0.5 μm with large sampling flow rate, and the average is 1.5 %. apparently the error is much smaller with the small sampling flow rate. this result is much smaller than the experiment data in the wind tunnel, but the error of the experiment data is 4.2 % on average [8] , which is also less than 5 %. for sampling of large particles in the flow field with large velocity fluctuation, such as the sampling in the field of dust removal technology, the error will be 5 % by the velocity deviation 10 %. the application of isokinetic sampling is mentioned in all the monographs about this aspect, where the allowable error is also mentioned to be more than 10 % [9] [10] [11] [12] . the velocity is required to be controlled strictly in these literatures. one method is the prediction of velocity. when a micromanometer is connected to both inside and outside of the sampling probe, the sampling velocity is adjusted so that the indication value is 0. of course, when the sampling flow rate changes, it is necessary to use the cumulative flowmeter, and the rotameter is only for the purpose of surveillance and control. in addition, there is another method called isodynamic pressure method. chinese academy of preventive medicine has developed a specialized isokinetic sampling probe. in cleanroom, it is obvious that the velocity field is stable. but it is not uncommon that the velocities at many positions are different from the average velocity by one time, or even much larger (e.g., in the blinder area below the shadowless lamp holder in the center of the operating table, where the air cleanliness must be measured). the transient change rate of velocity at each position can also be more than one time (as shown in fig. 8 .30). because of the time difference at each sampling position, the variation of velocity may be offset with each other. literatures have emphasized that the isokinetic sampling procedure must be followed. but the feasibility of isokinetic sampling at various measurement positions in large space is much less than that in the pipeline. in unidirectional flow cleanroom, the sampling velocity can be chosen to be the average velocity. but in turbulent flow cleanroom, it is meaningless to mention the average velocity. so for the measurement of the concentration field in space, the allowable error should be considered. all test results have errors. if the error is within the allowable range, the test results can be accepted. 209e proposed that the error less than 5 % is acceptable which is caused by non-isokinetic sampling. it also points out that when u:u 0 is between 0.3:1 and 7:1, the error is less than 5 %. the diagram to make correction was also given as appendix c in 209e, which is shown in fig. 16 . 10 . the example of the application of fig. 16 .10 is shown as follows: when the particle counter with small sampling volume 2.83 l/min is used to detect particles with diameter 5 μm (only for this particle size) from airflow with velocity 0.5 m/s, should any correction be made? ans: we know that u/u 0 ¼ 1.42/0.5 ¼ 2.84, and st ¼ 0.006 for 5 μm particles, so point a can be found on fig. 16 .10, which falls in the area where correction is not needed. so there is no need for correction. the specification of isokinetic sampling is listed below from the main standards, which is shown in table 16 it is shown that no matter what the particle size and the allowable error are, 209d required isokinetic sampling for unidirectional flow. but for 209e, isokinetic sampling is needed only when the error is >5 % for particles !5 μm, where the flow pattern is not specified. in fact it is turbulence flow. because particle counter with small sampling flow is used, the sampling error is smaller and influence exists only for large size particles. so 209e clearly pointed out: "although isokinetic sampling is good, if it is not feasible, the sampling deviation should be estimated with the method in appendix c," "it is only meaningful for particles with diameter equal to or larger than 5 μm in clean area by non-isokinetic sampling." in iso14644-1, it only kept the specification about the minimum sampling volume and the probe orientation which were given in 209e, while the requirement of isokinetic sampling is not explicitly mentioned. this is identical with the general opinion of 209e. although the sampling error is big for particles !5 μm with non-isokinetic sampling, a lot of practice shows that 10 μm particles do not exist in cleanroom, and 5 μm particles do not exist in high-level cleanroom either, and only a few exist in low-level cleanroom. so the influence on the particle number is small, which can be used for the understanding of iso specifications. the next chapter will illustrate that the sampling error including the non-isokinetic sampling by particle counter will generally not exceed the standard estimation range. based on the above analysis, we can suggest that (sampling error ≯ 5 % is used as the standard): 1. under the condition of u 0 /u ¼ 0.012-25.76, as long as st ≯ 0.001, isokinetic sampling is not required. 2. for sampling in cleanroom according to the current domestic and international standards, isokinetic sampling is not required for particles !5 μm. 3. if particle counter with large sampling flow rate is used in cleanroom, isokinetic sampling is required for particles !5 μm. (for particles less than 5 μm, calculation should be performed to determine whether isokinetic sampling is needed. generally isokinetic sampling is needed for 4 μm particles.) 4. when isokinetic sampling is needed but it is impossible, correction of the result should be made with the estimation error on fig. 16 .9. of course, it is absolutely impossible to obtain motionless air, and this is also the case even in some experimental device. here the so-called quiescent state means that the air velocity is very low, and it is completely in natural state. there are two kinds of errors when the sampling probe is facing upward in the still air: when the sampling rate is very low and the sampling mouth is upward, due to the settlement of particles, some particles outside the sampling volume will "fall" into the sampling probe. in the extreme case, the sampling flow rate is zero; sampling error will be infinite because of the natural sedimentation of particles. this is similar to the sampling in flowing air. with the larger sampling flow rate and the larger particle size, these particles may not be collected. in order to reduce the first kind of error, sampling with adequate sampling velocity must be performed. in order to reduce the second kind of error, sampling probe with large diameter should be used. davies provided a mathematical expression with the above two conditions [13] : for the first kind of condition μ ! 25ν s (16.8) where v s is the settling velocity. therefore the following expression can be obtained (where the slip boundary correction can be omitted) where d 0 is the diameter of the sampling probe; q is the sampling flow rate; d p is the particle diameter when ρ ¼ 1. for the second kind of condition (where the slip boundary correction can be omitted): with the mathematical expressions, figs. 16.11 and 16.12 can be obtained [14] , which facilitates the usage. the sampling conditions below the dotted line in fig. 16.12 show that the requirement for both the minimum and maximum allowable sizes of sampling probe cannot be simultaneously satisfied. when isokinetic sampling is required, it does not necessarily change the diameter of the whole sampling tube. it is fine as long as sampling probes with different diameters are placed at the head to the sampling tube, as shown in fig. 16 .13. if air velocity u 0 is expressed with "m/s," the sampling flow rate q with "l/min," and the sampling probe diameter d 0 with "mm," we can obtain: it is a matter of concern about the measurement error caused by particles loss in the sample tube. it is serious when the sampling loss of microbiology reaches 38.2 % for a 2.8 m long sampling tube [15] , which is not impossible. except for the electrostatic effect during sampling and filtration processes because of the electrostatic charge, there are also other reasons for the particle loss, which mainly include the deposition of particles in the tube caused by diffusion, deposition, collision, and coagulation effect. they will be introduced separately as follows. particles loss by diffusional deposition will occur since they will attach onto the tube wall by diffusional movement. expressions to calculate the particle loss by diffusional deposition are different according to the different flow states inside the tube. for the common particle counters with large, medium, and small sampling flow rates at home and abroad, reynolds numbers re in the sampling tube are shown in table 16 .3. it is laminar flow when re < 2,000. it is turbulent flow when re > 4,000. so for the particle counter with large sampling flow rate, it should be considered as the turbulent flow, while the laminar flow should be considered for the particle counter with medium or less sampling flow rate. according to the summary of the conclusions by fuchs in 1955 [16] , the following formula can be obtained. let when α > 0. 03 when α < 0. 03 where n/n 0 is the penetration of particles through the sampling tube; n 0 is the particle concentration at the entrance of the sampling tube, i.e., the sampling concentration, #/l; n is the particle concentration at the distance x from the entrance of the sampling tube, #/l; d is the diffusional coefficient of particles, m 2 /s; r is the radius of the sampling tube, m; x is the length of the sampling tube, m; u is the air velocity inside the sampling tube, m/s. with the above formula, the dotted line shown in fig. 16 .14 can be obtained [17] . the relationship can be found between particle loss rates, particle size, and flow rate. but it is inconvenient to determine the required length of the tube from the theoretical curve. fuchs gave a slightly different equation in the published monograph in new york in 1964 [18] (in the past this equation was referred by author in relevant publications). when α > 0.04, when α < 0. 04, the α value above may also be 0.02 in some literatures [19] . by comparison, the result from eq. (16.15) is slightly smaller than that from eq. (16.13) , and the result from eq. (16.16) is slightly larger than that from eq. (16.14) . but the differences for these two situations are very small. for particle counters with medium and low sampling flow rates, the value of α of the latter is greater than the former. the smaller the particles are, the larger the diffusion is, which is more disadvantageous. the most disadvantageous situation corresponds with d p ¼ 0.1 μm, when d ¼ 8 â 10 à10 m 2 /s. we can obtain so for calculating the diffusional loss of particles for particle counters with medium and small sampling flow rates, α < 0.04. eq. (16.16) can be used to obtain the relationship diagram between α and 1 à n/ n 0 , as shown in fig. 16 .15 [20] . in comparison of fig. 16 .14, this figure directly presents the relationship between α and the particle loss rate which is described in eq. (16.16) . it is convenient to calculate the particle loss rate and the pipe length with the known particle loss rate. it is shown in fig. 16 .15 that as long as α 0.0033, the diffusional loss rate will be 5 %. suppose the allowable maximum loss rate is 5 %, we know so the following requirement must be satisfied: for particle counter with medium and low sampling flow rates, calculation results are shown in table 16 .4. since the diffusional coefficient is independent of the particle density, the allowable tube length in table 16 .4 is also not related to the particle density. from the calculated results, the diffusional loss for !0.5 μm particles is extremely small, which can be completely ignored. in the literature from b.y.h. liu [19] , similar equation as eq. 16.16 was used to obtain the left part of fig. 16 .16 (only specified that α < 0.02). although it was pointed out that the formula was valid for laminar flow, it did not indicate in the figure that it is not suitable for the application in non-laminar flow such as the particle counter with large sampling flow rate. and example was given for this kind of particle counter (e.g., diameter d t ¼ 1 cm, and sampling velocity u ¼ 6 m/s). that means for a sampling tube with d t 1 cm, when pass-through time of the flow is 100 s and particle loss rate by diffusion is 5 %, the allowable tube length is 100 s â 6 m/s ¼ 600 m. if eq. (16.17) is used for calculation, when the particle loss rate is 5 %, we can obtain there is little difference between these two results. so it is acceptable to consider that results are consistent with fig. 16 . 16 and eq. (16.17) for calculation of the turbulent flow. but as mentioned before, the above formula and figure should not be used for the particle counter with large sampling flow rate where turbulent flow is inside. so it is incorrect to choose either 600 m or 618 m. it is difficult to obtain the particle loss in the particle counter with medium sampling flow rate from fig. 16 .16. but it is convenient to use eq. (16.17) . the allowable pass-through time t for the flow inside the tube can also be calculated with eq. (16.12). because because the particle loss by diffusion is known to be extremely small, the allowable tube length, i.e., the acceptable value t, is very big. the specific number of t will not be calculated. of course, the value of t can be obtained with the known tube length and flow velocity. the diffusional deposition onto the surface of tube wall by turbulent flow is much complex than that by laminar flow, and diffusional deposition onto the indoor wall which is introduced in sect. 6.5. as pointed out in sect. 6.5, there is a very thin diffusional boundary layer close to the wall. particle concentrations beyond the boundary layer will become uniform by turbulent flow. the boundary layer thickness is δ, which is difficult to determine. in hinds' monograph [21] , it is pointed out that davis had already discussed the problem, and fuchs provided the diffusional boundary layer thickness is δ inside the tube. so the whole penetration p through the tube with length x can be obtained with the following formula after the diffusional loss by the turbulent flow is considered: where v d is the diffusional velocity (see sect. 6.5) (m/s); where ρ is the air density. let log-log paper can be used to plot the linear relationship between the loss rate 1-p and y. so the value of y can be obtained from the figure according to the required loss rate, and then the value of x can be calculated. suppose it is required: substituting it into the eq. (16.20), we can obtain: substituting μ ¼ 1.83 â 10 à5 pa · s and ρ ¼ 1.2 kg/m 3 into the above expressions, we obtain: with these above conditions, the loss rate 5 %. if the value of y is obtained under a certain loss rate, we obtain: x 114yd 2 t u d for the particle counter with large sampling flow rate where turbulent flow exists inside the sampling tube, the allowable lengths are specified in table 16 .5. as shown in table 16 .5, the allowable tube length calculated according to the turbulent diffusion situation for 0.l μm is much less than that with laminar flow diffusion. it is obvious that it's inappropriate to calculate by the latter situation. and the allowable pass-through time t of the turbulent flow can also be obtained with eq. (16.23), which is: for the sampling in turbulent flow by particle counter with large sampling flow rate, the allowable pass-through time through the sampling tube is shown in table 16 .6. of course, the allowable pass-through time can also be calculated with the division between the allowable tube length and the airflow velocity. as mentioned before, b.y.h. liu just quoted the formula for diffusion loss in laminar flow [19] . he thought that the diffusional loss in the turbulent flow can be calculated according to the laminar flow at first, and then an additional item considering the vortex deposition is added. but the calculation method is very inconvenient for use, so it will not be introduced here. for laminar flow, fuchs quoted the same formula of г. натансол in two versions of his monographs [16, 18] : for turbulence flow, fuchs gave the following expression in his latter version [13] : it has been pointed out that the difference is small between the calculation results when n/n 0 ! 0.9 in two conditions. here calculation for 5 μm particles is taken as an example: the allowable tube length is as follows when n/n 0 ¼ 0.90: deviations for these two kinds of flow regime are both within 10 %, so they can be calculated by laminar flow. the theoretical curve made by eq. (16.26) has been shown in fig. 16 .14 [17] , which is the solid line. it is also not convenient to determine the tube length by this curve. the relationship diagram between β and 1 à n/n 0 is shown in fig. 16 .15, which is very convenient for use to calculate the loss rate. [19] . for the sampling tube with d t ¼ 1 cm, taking 1 μm, for example, when the particle penetration is 0.95, we obtain t d t % 13 s 1 cm the allowable tube length for the particle counter with large sampling flow rate u ¼ 6 m/s is the deviation is 8 % with the value 85.4 m in table 16 .8. since interpolation is needed during the application of the figure, it is inconvenient to calculate specifically. in addition to vertical and horizontal tubes, the sampling tube generally has a certain degree of bending. figure 16 .17 shows a real photo. it forms a 90 bend in the extreme case, as shown in fig. 16 .18 [19] . the minimum bending radius is the calculation results of collision loss are listed in table 16 .9. it is shown from the table that the larger the particle is, the greater the collisional loss is; the bigger the value st is, the bigger the collisional loss is; the smaller the value r 0 is, the greater the collisional loss is. for particles with diameter equal to and less than 5 μm (this is the maximum controlling particle size in various recent standards), the collisional loss can be ignored. but for l0 μm particles, it should be taken into consideration. equation (6.49) has given the method to calculate the coagulation concentration of monodisperse particles. if it is used to estimate the coagulation concentration of polydisperse particles, separate calculation should be performed with different particle size. and further correction can be made according to the geometric standard deviation σ g (see chap. 1) of the group particles. the correction values from literature [22] are listed in table 16 .10. take the unfavorable 0.1 μm particles as an example: when t ¼ 10s, and n 0 ¼ 10 3 #/cm 3 , the coagulation coefficient is found from table 6.13 that k 0 ¼ 8.6 â 10 à10 cm 3 /s. so even when polydispersity is taken into consideration with σ g ¼ 2, k 0 increases by 1.94 times. the result shows that the coagulation loss is very small, so it can be completely ignored. from the above theoretical calculation, the particle loss by collision, eddy, and coagulation for particles with diameter equal to and smaller than 5 μm inside the sampling tube is much smaller than that by diffusion and deposition, which generally can be ignored. it should be considered only for particles with diameter more than 5 μm and in special sampling conditions. for the particle loss by diffusion and deposition, the deposition loss is much important. the diffusional loss of large particles can also be negligible. comparison is performed between three experimental data and theoretical calculation results: 1. for the horizontal sampling tube with diameter 0.4 cm and length 15 cm (a) and 500 cm (b), respectively, particle counter with small sampling flow rate was used to measure the concentration of standard particles after steady state reached. the sampling velocities were both 27 cm/s. the test result is shown in table 16 .11 [23] . for tube a with length only 15 cm, even for 1 μm particles, the theoretical deposition loss rate is much lower than 1 %, and the diffusional loss is even smaller. so the average concentration in tube a can be considered as the standard value without any particle loss, which can be used to compare with that in tube b (table 16 .11). 2. for a horizontal sampling tube with diameter d t 1.09 cm and length 500 cm, measurement was performed to test the concentration of the standard particles when the sampling velocity was 0.44 m/s. the result is shown in fig. 16 .19 [24] . by comparison of the particle loss rate in the figure with the theoretical particle loss rate, results are shown in table 16 .12. in the two experiments, small spherical psl particles were used as the standard particles, whose density is 1.06 g/cm 3 . from comparison, the actual particle loss rate is close to the theoretical value for particles with diameter less than 2 μm. but the actual loss rate is 70 % of the calculated value for particles with diameter larger than 2 μm. this is because the resuspension phenomenon is not considered in the formula after particles deposit. however, it cannot be ignored for big particles. because the smaller the particles are, the larger the molecular force is. the corresponding suspension velocity is bigger, which needs bigger force to blow them up, and it is hard to resuspend again. as shown in fig. 16 .13, the suspension velocity suddenly decreases from about 2-25 μm. it showed that it is hard for particles with diameter more than 2 μm to resuspend, while it is quite easy for particles with diameter more than 2 μm to resuspend again. it shows that the deposition loss rate is smaller than that of small particles. 3. for the horizontal sampling tube with diameter 0.5 cm and length 400 cm, when the sampling velocity is 0.28 m-1.41 m/s, the actual loss rate for polydisperse dop particles in the tube is shown in fig. 16 .20 [17] . taking u ¼ 0.42 m/s, for example, comparison with theoretical results is shown in table 16 .13 (the particle density is 0.981 g/cm 3 ). in the experimental results with six grades of velocity channels, most of the actual tested results are closed to the theoretical value, and both have the same trend. for example, the loss rate becomes bigger when the sampling velocity is small, and the loss rate increases quickly with the increase of the particle size. with the same sampling velocity, for most particle sizes, the particle loss rate in pipes with small diameter is bigger than that in big pipes. through the comparison of three examples, it is reasonable to consider that the theoretical calculation is closer to the reality. correct on the actual test data will be performed according to the theoretical calculation if necessary. what should be emphasized is that, the theoretical calculation results are all obtained from the formula and the given parameters in this section, instead of directly in the fig. 16 .14. the values from this figure are a little smaller. since the values of some parameters are unknown during the plot of the figure, the reason why it is smaller cannot be confirmed (table 16 .14). through the above analysis of particle loss inside the sampling tube, the following conclusions can be obtained: 1. for particles with diameter less than 5 μm, the length of sampling tube should be considered just according to the particle loss by diffusion and deposition. 2. for 0.5-5 μm particles, the deposition loss rate is gradually bigger than that by diffusion, so the length of the sampling tube should be considered according to the deposition loss. 3. for particles with diameter less than 0.5 μm, the length of the sampling tube should be considered according to the diffusional loss when only particle counter with large flow rate is used. when particle counters with medium and small sampling flows, the length should be considered according to the deposition loss. in the appendix b 40.2 of fs209e, the sampling tube in the "air sampling system" for particle counters is required that "the size of the sampling tube should be determined in such a way that the flow time in the tube should not be more than 10s." and in the b40.2.1, further suggestion was given in the section about "the consideration for particle delivery." "for 0.1-1 μm particles, the maximum sampling tube could be 30 m. for 2-10 μm particles, the length of the sampling tube should not be more than 3 m. in this condition, the particle loss rate can be less than 5 % for small particles by diffusion and large particles by deposition and inertia (see appendix c)." in the above references, the reynolds number of sampling flow is required to be 500-2,500, and the diameter of the sampling tube is not specified. so in this re conditions, when calculation is performed with the turbulent formula, if the diameter is bigger than that the diameter 1 cm for the particle counter with large sampling flow rate by 50 %, the allowable length could be 30 m for 0.1-1 μm particles. if the diameter is 1 cm, the maximum length could not be more than 17 m according to table 16 .5. the second channel should not be 2-10 μm, but instead it should be 2-5 μm as the interval, because 5 μm is the prescribed maximum control particle size in 209e. therefore from table 16 .8, it is suitable that the length of the horizontal sampling tube is not more than 3 m (209e only mentioned it as the sampling tube, which is not accurate). if the diameter of control particles is 2-10 μm, it cannot be more than 1 m. for the special needs with the 5-10 μm particles as the control object, it should not be more than 1 m for the horizontal sampling tube. so for 209e, it is reconcilable that 2-10 μm is changed to 2-5 μm. for the particle counter with so-called medium sampling flow rate 2.83 l/min in china, the control particle size generally begins from 0.5 μm. so the lower limit 0.1-0.5 μm can be classified as one channel (corresponding to 0.1-1 μm in 209e). based on the results from table 15 .2 and the calculated results from ref. [19] cited in 209e, the length of the allowable sampling tube can be long in terms of the diffusional loss. therefore only the deposition loss should be considered. from table 16 .8, when the atmospheric particle concentration is large (2000), it is safe to set the length of the sampling tube within 30 m in theory. for cleanroom where the maximum control size is 5 μm commonly, it is more likely that only the deposition loss is considered. from table 16 .8, it is known that the horizontal sampling tube should be less than 0.5 m, which means that in order to ensure the loss rate less than 5 % for 5 μm particles, it is better that the length of the horizontal sampling tube is less than 0.5 m. as shown in fig. 16.17 , it is allowable that the length of the horizontal sampling tube is not more than the instrument length, and it is not suitable if the tube is longer. ref. [20] gives the suggested length value of the sampling tube for domestic situation, as listed in table 16 .15. for the environment with different air cleanliness levels, the sampling flow rates should be different. during the measurement with the balance meter, it is relevant to the sensitivity of the balance. during the dust sampling with membrane filter, the sampling flow rate is related to the background value of the membrane filter. an appropriate proportion must be kept between the captured particle number and the background value of the membrane filter. for both cases, appropriate sampling flow rates are needed. the minimum sampling volume put forward here is for the particle counter [25] . in the 1970s, particle counters with medium and big sampling flow rates had appeared. due to the difference of the measurement results between large sampling flow rate and small sampling flow rate, this problem is naturally proposed. for measurement in cleanroom with air cleanliness level higher than class 100, what is the requirement for the sampling flow rate of the particle counter? if requested, how to determine the minimum sampling volume? this is the new problem that is put forward by the development of air cleaning technology. in us standards before 209c in 1987, including that from federal government, nasa, and air pollution control association, and in related standards from britain and germany, the problem of the minimum sampling volume, the minimum sampling times, and the necessary number of measuring points were not provided. only in 1973, 209b generally put forward that for turbulent flow cleanroom, it is suitable to set the sampling flow rates 0.01, 0.1, and 0.25 ft 3 /min, and for laminar (now unidirectional flow) cleanroom, it is suitable to set the sampling flow rates 0.25, 1.0, and 5.0 ft 3 /min. in 1983, german standard vdi-2083-iii only put forward that it is better to use particle counter with sampling flow rate 1 ft 3 /min for cleanroom with air cleanliness level above class 100. in 1984 the standard ies-rp-cc-006 made by institute of environmental sciences and technology in usa pointed out the number of measuring points for the first time, and the method to determine the number of measuring points by the area of the cleanroom was proposed. for turbulence and laminar flow cleanroom, the area (ft 3 ) corresponding to each measuring point is ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi air cleanliness level p , but the minimum sampling volume was also not mentioned. in october of 1977, sato [26] proposed to perform short-period sampling on several positions with small sampling volume particle counter for the first time. for class 100 cleanroom, it is reasonable to doubt the accuracy of the measured results. but how to determine the sampling volume was not discussed. it was the first time in china that the concept of the minimum sampling volume was officially put forward also in october of 1977. the nonzero principle to determine this minimum sampling volume was given, and the calculation method was also provided [25] . in 1980, the concept of necessary number of measuring points and the corresponding calculation method were put forward [27] . the minimum sampling volumes obtained by this method and by data (data from japan jis standard in 1987 adopted from us standard) from 209c later (in 1987) belong to the same class, which will be described in detail later. particle counter can only display numbers which are positive integers such as 0, 1, 2, and 3. if the particle concentration of the measured space is extremely low, and the sampled air volume for each reading number every time, namely, the sampling volume is very small, the average concentration in the sampling volume may be a number that is less than 1. in this case, each reading may appear many times with the value of "0," or the majority is "0." for the occasion that the particle concentration is extremely low, when the average reading at each sampling point is "1" and there are few sampling points such as only three points, the average concentration should be "1." if there are errors, for example, there are two times of "1" and one time of "0," the average concentration becomes "0.7," so the difference between two concentrations reaches 1.5 times. in this case, the real particle concentration cannot be really reflected. if the sampling volume is big, each reading from every sampling volume may be more than 1, such as 2 and 3, then the frequency with number "0" in each sampling volume is few. even if there are some particles, the average concentration is only affected in the magnitude of 0.01-0.1, so the difference from true concentration will be smaller. the above situation mainly appears in the place with high air cleanliness requirement (such as higher than class 1000). therefore, the minimum sampling volume of the particle counter should have such characteristics. the possibility that particles fall into the minimum sampling volume is large, which means there is no possibility for the appearance of zero particle inside the sampling volume. this method can be called "nonzero inspection principle." as pointed out in chap. 1, if the average concentration in the sampling volume is set as λ, the particle distribution in the space can be described by the poisson distribution when λ 10. eq. (1.16) has given the probability p for the appearance of k particles at most in the sampling volume. it is rewritten here: so the probability for "0" particle is: the curve in fig. 16 .21 can be made with the two above formulas. the "0 particle" curve corresponds to the probability for the appearance of "0 particle." the " 1 particle" corresponds with the sum of appearance of "0" and "1" particle. the probability of appearance "1" particle is the difference between " 1" and "0," which is analogous for other cases. from fig. 16 .21, if the probability of nonzero reading is required to reach 95 %, namely, the probability of the appearance of "0" particle is only 5 %, λ must reach 3. the sampling volume obtained is the minimum sampling volume. the calculation example of the minimum sampling volume will be given below. now the lower limit of the particle concentration for each air cleanliness level is used to represent the concentration for this level (which is safer than that with the upper limit of the concentration). we can obtain: the minimum sampling volume ¼ 3 particles the lower limit of concentration at this level (16.32) the calculated results are shown in table 16 . 16 . from table 16 .16, we can obtain: 1. in china, the legal unit system is used, while in air cleaning technology, "l" is used as the basic unit. so it is appropriate to take 0.1 l. 2. for the environment with the particle concentration less than 35#/l, when ordinary particle counter with small sampling flow rate is used, the sampling time should be extended to ensure adequate sampling volume and larger possibility of the appearance of nonzero particle. if the sampling time is not prolonged and the sampling volume is not increased, most of the measured results will be zero, which will reduce the reliability of the measurement results. 3. for the measurement in the space with high air cleanliness requirement with ordinary small sampling flow rate particle counter, if the extension of the required time is not long, the extension of time is feasible. if the extension of the time is too long, it is not only uneconomic, but also likely to cause other errors. so it is necessary to use the particle counter with large sampling flow rate in this case. but the effect of large sampling volume on the concentration field must be considered, which may be also likely to produce false phenomena. so in the above circumstances, it may not be necessary to use large sampling flow rate particle counter. as already mentioned before, the poisson distribution may not be suitable to describe the particle concentration distribution in the cleanroom. although this aspect has been explained in chap. 1, which believed that the poisson distribution can still be used as the basic means to predict the particle distribution in space with high air cleanliness, if local serious leakage occurs in unidirectional flow cleanroom, it will have effects. but this kind of influence also exists in the turbulent flow cleanroom, especially when the sampling points are close to the leakage airstream. so code for construction and acceptance of cleanroom (gb50591-2010) has required that air filters installed in the cleanroom must undergo the leakage detection one by one. after installation, leakage scanning must be performed along the frame of the air supply surface. when all the situations are confirmed, the statistical result is effective. otherwise, if the frequency of a particle size is more than their due value, it is apparent that there are abnormal reasons, which means the data is not believable or the possible reason must be found. it does not mean that the poisson distribution cannot be used to predict the particle distribution. for example, during the measurement with 20 times (the sampling volume is 1 l), there are 19 times with "0" particle and one time with "1" particle. please judge if this is a normal situation. with eq. (16.30), we can calculate λ ¼ 1/20 ¼ 0.05, which means the probability of the appearance of "0" particle should be 95 %, namely, it should be 19 times, which is consistent with the actual condition. so although the sampling volume is not enough according to the value of λ, the result can be considered as normal. when the first measured value is "3" in the above example, please judge if this is a normal situation. after calculation, we obtain λ ¼ 0.15, so the frequency of the appearance of "0" particle should be 17 times. there are obvious differences. from eq. (16.30) we know: where k is the total times of measurement; x is the times of the appearance with "0" particle. if there are 19 times of the appearance of "0" particle in total 20 times, λ ¼ 0.051, which means the average particle concentration in theory should be 0.051#/l, instead of 3/20 ¼ 0.15#/l. since the average concentration in the sampling volume 1 l is 0.051, the probability for the appearance of three particles is determined by eq. (1.13) in this sampling volume, which is: so the probability of the appearance of three particles is extremely small, which is impossible. if it appears, it is an illusion, and it cannot be used to calculate the average concentration. it is very likely that the occurrence of three particles is caused by leakage. but as illustrated in 209e, the statistics method is not aimed to changing multipoint leakage positions during sampling (the possibility of leakage at one place is very small), which means the application of statistical methods or the minimum sampling volume cannot modify the random abnormal error caused by the leakage. in other words, the abnormal situation of measurement results should not deny the particle distribution characteristic and the application of statistical methods. instrument performance may also become one of the abnormal reasons. table 16 .17 listed foreign measurement data (the data at each point is the average value of many sampling times), where air cleanliness class 1 was realized in the cleanroom when instrument 1 was used, while the conclusion was opposite when instrument 2 was used [28] . the performances of these two instruments were compared, and it was found that the function to reset zero cannot be performed in instrument 2. so it is inappropriate to conclude that the above particle distribution characteristic is not correct and the minimum sampling volume is not applicable when two instruments with different performances were used for measurement and the measured results do not follow the above distribution characteristics. in table 1 .8, an example was presented where two instruments with identical performance after calibration were used to determine the particle distribution in three rooms, and the results are completely consistent. when statistical analysis is made on these data, it is more likely to conform to the distribution characteristic law. the monograph air cleaning technical measures was published before the concept of "nonzero inspection principle." so the data of table 16 .15 were not adopted, but the empirical data were used. at that time the concept of the minimum sampling volume was not established, and instead the concept of the sampling volume at every time was used. that is: when particle concentration < 30#/l (equivalent to class 1000), sampling volume !0.9 l; when particle concentration 30-300#/l (equivalent to class 10000), sampling volume !0.3 l; when particle concentration !300#/l (equivalent to class 100000), sampling volume !0.1 l. the code for design of clean room approved in 1984 and implemented in 1985 also adopted the similar data as the monograph "air cleaning technical measures," which was later than the "nonzero inspection principle." the data are: the concept of the minimum sampling volume was also not given. it did not mention that particle distribution in cleanroom belongs to poisson distribution, so the method with "nonzero inspection principle" was not cited to obtain the minimum sampling volume. as for the sampling volume specified in code for construction and acceptance of cleanroom (jgj 71-90) published in 1991 and code for design of clean room (gb50073-2001) published in 2002, it is consistent with related international standards (209d, iso14644-1). the exact data will be presented later. the above discussion is aimed to measure accurately, so the concept of the minimum sampling volume must be used. but it is still not enough only to use the minimum sampling volume; there must be enough sampling points, which have already pointed out in the past [27] . but the specification in this aspect in related foreign standards is not enough. the problem will be discussed in the next chapter. number [29] [30] [31] because sampling process is the collection of the counting data at every sampling point, it can be treated as the poisson process. according to the poisson distribution, the interval estimation of the average value for the test data can be made. from eq. (1.15), if the total number n obtained from the actual measurement is used to replace k, the distribution function of the estimation value λ 0 for λ is where λ 0 0 is the upper confidence limit of λ 0 , as shown in fig. 16 .22. when ζ is the probability (i.e., the confidence level) for the given value of þand integration can be performed for the right expression of the above equation, the upper limit of λ 0 0 of the estimation parameter λ 0 can be obtained with the value of n for different ζ. results are shown in table 16 .18. the random absolute error of this sampling is: the random relative error is: 10 l' 20 l 0 l 10 as shown in fig. 16 .22, for the given confidence level ζ, the more the sampled total particle number is, the bigger the upper confidence limit λ 0 0 is. from eq. (16.36), the relative error r decreases. as for the minimum sampling volume in us 209c standard, it is based on the principle that the total sampled particle number is 20 for the corresponding air cleanliness level according to the explanation of 209b revision attendant. with this principle, when the confidence level is 95 %, the maximum concentration is 31 particles and the minimum is 12 particles. that means there is 95 % probability that the total particle number sampled is between 12 and 31. for example, when the upper concentration limit is calculated, it could be 30.89 % 31 when table 15 .15 is used with the confidence level 97.5 %, which is consistent with the above explanation by 209b revision proposals. from the table, the sampling error is r ¼ 54.5 %. if the confidence level reduces to 95 %, λ 0 0 ¼ 29 particles. although the increased total sampled particle number can reduce error, the sampling time will be prolonged. it is completely artificial to set 20 particles. so we obtain: minimum sampling volume ¼ 20 upper concentration limit with the air cleanliness (16.37) for class 100, the upper concentration limit is 100#/ft 3 , 20/100 ¼ 0.2 ft 3 . for class 1000, the upper concentration limit is 1,000#/ft 3 , 20/1,000 ¼ 0.02 ft 3 . according to the same principle, iso14644-1 uses "l" to represent the sampling volume, so: minimum sampling volume ¼ 20 upper concentration limit with the air cleanliness=m 3 â 1;000 l (16.38) in code for design of clean room (gb50073-2001), the value 1,000 in the above equation was mistaken as 100. the above results are shown in table 16 . 19 . the mantissa of l in 209e and iso are different slightly. at the same time, iso specified that the minimum value should not be less than 2 l. from the comparison of tables 16.16 and 16.19, we can know: 1. the above two principles to determine the minimum sampling volume are based on the collection of the point data. so all of them applied the poisson distribution, but with different methods. the method with λ ¼ 3 is aimed to guarantee that the probability of nonzero data is not less than 95 %. the method with total particle number ¼ 20 is slightly optional. so although eq. (16.32) and eq. (16.37) have similar shapes, the nature is different. only american literature [29] has not pointed out that the test statistics is based on the counting points' data when it comes to poisson distribution. so when the statistical analysis about the average concentration (it will be introduced in detail in next chapter) was mentioned in this literature, it concluded that "both experience and theory have proved that poisson distribution is seldom applicable for the actual concentration in the cleanroom," and t distribution was recommended, which will cause misunderstanding. it seems that t distribution may fit the particle concentration in the cleanroom. in fact, different principles were used to treat the statistical problems with two different features between the counting points' data and the average value. from statistics theory, it is known that this is the problem with two different natures, and it is not a problem that which distribution is more suitable to describe the particle distribution in cleanroom. one is the density distribution, where the poisson distribution is applicable. the other is the particle number distribution according to the particle size, which is aimed to calculate the error of the average value, namely, the so-called treatment of "actual measured concentration data from the cleanroom," so t distribution method reflects the average distribution characteristics from small subsamples. the method will be discussed in the next chapter. 2. the us federal standard 209c adopted the english unit, which chooses ft 3 as the basic unit. it is understandable that 0.1 of this unit, namely, 0.1 ft 3 , is taken. 3. the minimum sampling volume specified in 209c is calculated according to the upper concentration limit. although the concentration increased by 10 times, λ only improves 6.7 times. so in contrast, the minimum sampling volume of each air cleanliness level should be less than that in table 16 .15. because the difference of the specific value represented by 0.1 unit for air cleanliness level lower than class 100, the minimum sampling volume (l) is bigger than that in table 16 . 16 . but in all, the two values of the minimum sampling volume are very close. like the measurement of particles, there is also the problem of the minimum sampling volume for airborne bacteria sampling. now the minimum sampling volumes are calculated with "nonzero inspection principle" and the airborne bacterial concentration, which is shown in table 16 .20 [32] . as pointed out in chap. 1, deposition of particles on surface follows the poisson distribution. therefore like the minimum sampling volume mentioned in the above section, there is also the minimum sampling volume problem during the measurement of particle settlement. if the deposition area is too small, it will reduce the reliability of the whole measurement because the probability of the appearance of "0" particle is extremely large. this problem is especially important during measuring the colony in biological cleanroom with the deposition method. as the minimum sampling volume analyzed in the previous section, in order to ensure 95 % of readings are nonzero, the particle deposition density must also reach or exceed 3. from table 9 .11, the allowable colony in the specified period in each petri dish is known, which is the deposition density (take one petri dish as the base). so the minimum deposition area needed for measuring the bacterial concentration with the deposition method in the environment with different air cleanliness levels can be obtained, which corresponds with the minimum number of petri dishes. for example, when it is 3.5#/l from table 9 .11, the probable maximum settlement in each petri dish is 0.239 cfu. then in order to obtain three cfu, it needs 12.55 dishes, approximately 13 dishes. results are listed in table 16 .21. if it is 3#/l, the number of petri dishes is 14 and 4. that is the upper concentration limit corresponding with the air cleanliness level in fig. 16 .23 or the value used by author in the past. the minimum number of petri dishes should be adapt to the necessary number of sampling points or the number of sampling points specified in the regulation, which will be introduced later. if the minimum number of petri dishes is bigger than the number of sampling points, the former value is adopted, and both the requirement of the number of sampling points and the minimum deposition area should be satisfied. if the minimum number of petri dishes is less than the number of sampling points, the number of petri dishes should choose the number of measurement points. if the sampling space is small and petri dishes cannot be placed, the deposition period could be appropriately prolonged (not more than 1 h), and the number of the petri dishes can decrease proportionally. measurement method of particle concentration in exhaust air aerosol technology (trans: sun yufeng). heilongjiang science and technology press influence of additional resistance on the flowrate calibration of the flow meter aerosol technology (trans: sun yufeng). heilongjiang science and technology press aerosol technology: properties, behavior and measurement of airborne particles handbook of air cleaning on the sampling error due to non-isokinetic sampling analysis of sampling problem in laminar flow cleanroom theory and practice of dust removal and collection (trans: chan wenchang). scientific and technical documents publishing house dust removal in the workshop of crushing and screening industry ventilation dust removing technology heilongjiang science and technology press aerosol technology (trans: sun yufeng). heilongjiang science and technology press the fifth institute of military medical college, development of jwl-1 type sampler the mechanics of aerosols (trans: gu zhenchao) particle loss in sampler the mechanice of aerosols electrostatic effects in aerosol sampling and filtration discussion of the sampling tube length of the particle counter aerosol technology (trans: sun yufeng). heilongjiang science and technology press heilongjiang science and technology press study on property of atmospheric aerosol (the 1st report) dust meter -light scattering particle counter calculation of the minimum detection volume of the particle counter with different air cleanliness levels status of industrial cleanroom calculation of the necessary sampling points in cleanroom certifying a class 10 cleanroom using federal standard 209c. (trans: zhang yaping, checked by feng peiming) rationale for proposed revisions to federal standard 209b (cleanrooms) (trans: liu xianzhe, zhan yingmin) interval estimation of particle concentration -process of measured data measurement of low concentration particles and evaluation of air cleanliness level in clean area measurement of bacterial concentration. handouts for the seminar on the specification of construction and acceptance inspection of cleanroom key: cord-252147-bvtchcbt authors: domingo-espín, joan; unzueta, ugutz; saccardo, paolo; rodríguez-carmona, escarlata; corchero, josé luís; vázquez, esther; ferrer-miralles, neus title: engineered biological entities for drug delivery and gene therapy: protein nanoparticles date: 2011-11-15 journal: prog mol biol transl sci doi: 10.1016/b978-0-12-416020-0.00006-1 sha: doc_id: 252147 cord_uid: bvtchcbt the development of genetic engineering techniques has speeded up the growth of the biotechnological industry, resulting in a significant increase in the number of recombinant protein products on the market. the deep knowledge of protein function, structure, biological interactions, and the possibility to design new polypeptides with desired biological activities have been the main factors involved in the increase of intensive research and preclinical and clinical approaches. consequently, new biological entities with added value for innovative medicines such as increased stability, improved targeting, and reduced toxicity, among others have been obtained. proteins are complex nanoparticles with sizes ranging from a few nanometers to a few hundred nanometers when complex supramolecular interactions occur, as for example, in viral capsids. however, even though protein production is a delicate process that imposes the use of sophisticated analytical methods and negative secondary effects have been detected in some cases as immune and inflammatory reactions, the great potential of biodegradable and tunable protein nanoparticles indicates that protein-based biotechnological products are expected to increase in the years to come. the development of genetic engineering techniques has speeded up the growth of the biotechnological industry, resulting in a significant increase in the number of recombinant protein products on the market. the deep knowledge of protein function, structure, biological interactions, and the possibility to design new polypeptides with desired biological activities have been the main factors involved in the increase of intensive research and preclinical and clinical approaches. consequently, new biological entities with added value for innovative medicines such as increased stability, improved targeting, and reduced toxicity, among others have been obtained. proteins are complex nanoparticles with sizes ranging from a few nanometers to a few hundred nanometers when complex supramolecular interactions occur, as for example, in viral capsids. however, even though protein production is a delicate process that imposes the use of sophisticated analytical methods and negative secondary effects have been detected in some cases as immune and inflammatory reactions, the great potential of biodegradable and tunable protein nanoparticles indicates that protein-based biotechnological products are expected to increase in the years to come. the design of new chemical entities (nce) for diagnosis and treatment of human diseases has relied on the discovery of active chemical drugs from a diverse library of compounds or from naturally occurring molecules. 1, 2 further chemical modifications improve pharmacokinetic properties to obtain a final product with a known mechanism of action and decreased toxicity. 3 nonetheless, using such approaches, the final products present low specificity for their target molecules, interacting with many other molecules and accumulating in some tissues, disturbing the correct homeostasis of the system. in some cases, the adverse effects of drug administration exceed pharmacological effect and despite the concise mechanism of action of the drug over the target molecule representing an improvement in the patient's state, the treatment has to be prevented or discontinued. 4 in fact, although a maintained steady increase in the number of launched nce has been observed in the last years, the question arises whether this classical approach has already exhausted the discovery of innovative molecules. 5 on the other hand, macromolecular new biological entities (nbe) have been used to supplement cellular deficiencies or to inhibit cellular pathways exploiting their relatively specific mode of action. proteins and peptides have been obtained first from their natural source or produced as recombinant 248 versions after the development of genetic engineering techniques in the late 1970s. however, the delivery of biological entities is sometimes hampered by its low half-life in the bloodstream by unspecific degradation, resulting in an expensive and ineffective process. nevertheless, some solutions have already been explored for biopharmaceuticals to increase solubility and stability and to reduce immunogenicity including postranslational modifications such as glycosylation and covalent conjugation of polyethylene glycol. 6 thus, one of the main objectives in the use of drugs (for either nce or nbe) is the need to optimize the delivery system to reduce the pharmacological dose which would consequently represent a concomitant reduction in toxicity and cost. in that scenario, new delivery approaches have been implemented using biological interactions such as antigen-antibody binding (immunoliposomes) 7 or more sophisticated interactions including the binding between nutrient concentrator sparc (secreted protein acidic and rich in cysteine) and albumin in the treatment of some types of cancer (abraxane ). 8, 9 proteins can be then used for their targeting qualities as molecular delivery vehicles both for the specific delivery of drugs or nucleic acids in gene therapy approaches and by themselves as therapeutic molecules. one of the interesting characteristics of proteins is their ability to form intermolecular driven complexes as sophisticated and structurally perfect as in the case of viral capsids. in addition, through the use of genetic engineering, recombinant proteins can be tuned to include additional properties to optimize drug delivery and nucleic acid delivery in gene therapy. in this chapter, the main available strategies to develop protein-based nanovehicles or biopharmaceuticals will be described. in this context, several parameters will be defined such as proper formulation, stability, immunogenicity, and delivery to the correct cell type and cell compartment. modular protein engineering, virus-like particles (vlps), and other self-assembling entities are envisioned as modulatable novel protein nanoparticles able to include many desirable properties in the correct delivery of drugs and nucleic acids. finally, some successful examples of protein nanoparticles on the market will be described in addition to protein products currently in clinical trials and under preclinical research in order to envision which type of protein nanoparticles will be available soon on the market. with the therapeutic molecule to generate a vehicle capable of being transported in the blood if a systemic administration is needed and retaining a significant stability before reaching the target cell. 10, 11 in addition, the biological system poses specific barriers that have to be overcome such as membranes (cytoplasmic, endocytic, and nuclear), degradation (protease degradation induced by acid denaturalization in lysosomes, cytosolic proteosomes, and nucleases), cytosolic transport, and nuclear entry if necessary. 12, 13 for central nervous system therapies, the blood-brain barrier (bbb) represents the main bottleneck, and for that, a specific strategy has to be designed. 14 furthermore, the therapeutic complex has to be flexible enough in order to release the therapeutic molecule in the specific cell compartment. thus, several protein motifs have been described to overcome each and every process described earlier so that a modular multifunctional protein can be generated including those modules that are necessary to achieve its goal. in order to get a rational construction of the multifunctional vector, each step has to be carefully taken into account so as to overcome every step which is needed to achieve its final goal (table i) . the dna/rna condensation or drug interaction with the protein vector is a critical step in the formulation of protein nanoparticles for gene therapy. they have to remain attached to the vector during the whole transport process through the body and the cell until it can be released in the desired localization within the target cell. highly positively charged peptides containing a large number of arginines or polylysines have been used to promote electrostatic interactions since nucleic acids are highly negatively charged molecules. [15] [16] [17] [18] [19] [20] [21] [22] natural dna-condensing proteins as nuclear histidines or protamines can also be used to bind nucleic acids. [22] [23] [24] [25] protamine, which is the protein that replaces histidines during the spermatogenesis process, is a sperm chromatin component and just as the histidines do, it has very high dna condensation ability to protect nucleic acids form cytosolic endonucleases. 23, 26 in addition, as soon as the complex reaches the cellular nucleus, protamine is degraded by chromatinremodeling proteins, releasing the transported dna allowing its expression. 15, 23 in contrast, polycationic dna condensation modules such as polylysines and polyarginines-even they can present higher dna condensation ability depending on the polycationic chain length-usually present lower dna-releasing ability, interfering negatively with the accessibility of cellular transcription factors and dna expression capacity. 15 all these dna condensation modules described above interact with any dna that is incubated in an unspecific way. however, there are proteins such as gal4 that are able to recognize specific dna sequences [27] [28] [29] and that permit to bind and condensate specific dna sequences in the final vector. 30 in many cases, the multifunctional protein vector is in vivo administrated by the systemic route in order to travel in the blood and reach the target cells. that exposes the vector to all blood components, making it susceptible to be degraded. thus, it is completely necessary that the vector remain in the blood long enough to be able to reach the target cells. it has also been described that naked dna has an estimated half-life in blood of minutes 10 ; so protein nanovehicles in gene therapy, among other properties, are intended to protect nucleic acids from degradation. one important factor when the vector is exposed to the blood is that it can be recognized by the immune system components and produces an immune response against the vector. thus, it is also very important to try to make the vector as less antigenic as possible in order to avoid being degraded or even being toxic to the organism. 32 peptide uptake or internalization involves a step before the protein binding to the cell surface. this attachment can be either specific or unspecific but in all cases the promotion of its internalization is required. 33 positively charged peptides usually bind the cellular surface by unspecific electrostatic interactions with the negatively charged cell surface proteoglicans. this kind of peptides can be used in the multifunctional protein if specific targeting is not required. 33 cell-penetrating peptides (cpps) have been widely described as unspecific cell-binding and internalization peptides [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] (see also the chapter ''peptide nanoparticles for oligonucleotide delivery'' by lehto et al. in this volume). however, specific interactions can be obtained by incorporating cell receptor ligands if cell or tissue targeting is required for the therapeutic action. moreover, some of those ligand-receptor interactions promote the ligand-receptor complex internalization. many peptides have been described in the literature as receptor-specific ligands so any of them can be added to the multifunctional proteins in order to confer them cell specificity. [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] the most natural specific ligands that can also be used for cell targeting are monoclonal antibodies. 32, [63] [64] [65] in addition, if no specific peptides are available for an intended target, new specific binding peptides can be found by using phage display 66 or combinatorial chemistry. 67 2. endosomal escape several internalization pathways are possible depending on the vector properties, 27, 33 including endocytosis (clathrin/caveolae-mediated, clathrin/ caveolae-independent), macropinocytosis, and non-endocytic pathways. 254 it is known that more than one internalization pathway can be performed at the same time but usually the peptide-based vector uses endocytic pathways. 68 moreover, it seems that proteins that interact with a specific cellular receptor are internalized by the clathrin-mediated endocytic pathway. 33 most of the generated endosomal vesicles will converge to late endosomes that eventually will fuse with cellular lysosomes. 15, 33 remaining in the cellular endosomes, the multifunctional protein will be degraded, so it is strictly necessary that the internalized multifunctional proteins be released into the cellular cytoplasm escaping from degradation. several peptides have been described that are able to promote endosomal escape and can be classified into two types depending on their escape mechanism: fusiogenic peptides and histidine-rich peptides. 36 the fusiogenic peptides are small peptides that have hydrophobic amino acids (aa-s) interspersed at constant intervals with negatively charged aa-s. 12, 19, 39, 40, 45, 46, [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] thus, when early endosomes become late endosomes, their low ph induces a conformational change in the peptide, which adopts a alpha-helix structure, in an amphipathic structure able to fuse with the endosomal membrane, leading to pore formation and releasing all the endosomal content into the cell cytoplasm. 36 the histidine-rich peptides are small peptides with a high histidine content whose endosmolytic activity is mediated by a mechanism called ''proton sponge''. 12, 22, [80] [81] [82] [83] when the endosomal ph becomes low in late stages, the imidazole groups of the histidines are protonated and attract endosomal cl à ions, buffering against the proton pump. thus, the endosomes collapse by an osmolytic swelling process and the endosomal content is released to the cell cytoplasm. 36 further details are given in the chapter ''peptide nanoparticles for oligonucleotide delivery'' by lehto et al. in this volume. once the protein has achieved the cellular cytosol, it can be degraded by cellular proteases or by the cellular proteosome system. 84 it is important to avoid this process, especially if the protein has to reach the cellular nucleus. if the final target of the nanoparticle is the cellular cytoplasm, it is necessary that it remain there at least long enough to perform its therapeutical action. several peptide proteosome inhibitors have been described that are able to avoid this type of protein degradation. by adding these peptides to the final protein vector it is possible to protect it and enhance cytoplasmatic stability. epstein-barr virus nuclear antigen 1 (ebna1) contains a proteosome inhibitor consisting of glycine-alanine repeats able to prevent proteosomal proteolysis. it has been shown that a minimum of 4 aa-s gly-ala repeats are necessary to achieve such protective activity. [85] [86] [87] if the protein vector is carrying nucleic acids (dna or rna), degradation by the cytosolic endonucleases has to be taken into account, so it is also very important to protect this nucleic acid in order to maintain its integrity. some dna/rna condensing peptides as protamines also protect the dna against cytoplasmic endonucleases and enhance its stability as has been described above. 15 the cellular cytoplasm is a very crowded and compartmentalized environment where cellular organelles and cytoskeleton make the free diffusion of macromolecules such as protein vectors difficult. however, cytoskeleton elements such as microtubules are used by endosomes and other cytosolic macromolecules for intracytosolic mobility. 33 dyneins have been described as being capable of carrying those macromolecules and endosomes along the microtubules in a retrograde transport toward the nucleus. some small peptides that are able to bind dyneins have been identified. they can be added to the multifunctional protein vector in order to mediate an intracytosolic mobility toward the cellular nucleus. 36 several dynein-binding proteins have been identified in viruses that are able to use this transport system. comparing those protein sequences, a consensus peptide sequence (kstqt) that is able to bind to the dynein lc8 light chain has been identified. 88 molecules lower than 45 kda/10-30 nm are able to enter in the cellular nucleus by passive diffusion. however, macromolecules higher than 45 kda/ 10-30 nm generally require an active transport system through the nuclear pore system. this transport mechanism generally requires a specific targeting signal peptide named nuclear localization signal (nls). these signaling peptides are usually rich in basic aa-s, which are recognized by the cellular importines and actively transported through the nuclear pore. 15, 89 monopartite or bipartite nls sequences which are nls peptides that have one or two nls recognized sequences respectively have been described. 12 thus, these peptidic sequences can be added into the final multifunctional protein if nuclear localization is required in order to express a carried dna. it has been reported that a single nls sequence is sufficient to transport the vector to the nucleus and that a large number of nls sequences can result in inhibition of its activity. 90 one of the most used nls signal peptides are fragments derived from the 111-135 aa-s of the simian virus sv40 large tumor antigen (t-ag). other nls sequences can be found in gal4, protamines, or tat. 23, 36, 37, 77, [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] it is important that when the transported dna reaches the cellular nucleus, it has to be released in order to be accessible to the nuclear transcription factors and achieve the desired expression level. thus, while designing the multifunctional protein vector, this aspect has to be taken into account. once the dna has been released in the cell nucleus, it will be necessary to control its expression level depending on which therapeutic action is being promoted. when the goal is to kill a cell as in cancer therapies, the uncontrolled dna expression levels would not be a problem. however, when a specific protein expression level is required, achieving good control is very important. 13 some expression systems have been developed that can be pharmacologically regulated by oral drug formulation. 103 cell-specific promoters and enhancers can be also used in order to confer high cell specificity to the therapy. 104, 105 d. ways to get over the bbb the bbb is a hermetic barrier that only allows nonlipophilic molecules smaller than 400 da to cross it. however, some human proteins such as insulin, transferrin, insulin-like growth factor, or leptins are able to go across it by receptor-mediated transporters. thus, the most important factor limiting central nervous system-targeting therapeutics is the presence of the bbb. 106 finding the way to cross it will be the main challenge. some peptides have been described that are able to reach the brain crossing the bbb. moreover, it has been seen that they can be associated with another molecule and transported through the barrier. thus, they could be interesting candidates to be included in the multifunctional vectors if central nervous system targeting is required. 14, 56, 107, 108 antibodies have also been described that bind transferrin and insulin receptors and that are able to cross the bbb efficiently. they can be conjugated with large molecules, allowing its translocation to the central nervous system. 63, 64, [109] [110] [111] synthesis, and rational design the development of genetic engineering techniques has increased the natural repertoire of proteins for the design of useful and/or valuable proteins with the aim to obtain new proteins with desired functions. there are three main strategies leading to the construction of engineered proteins: (a) direct evolution, (b) de novo protein design, and (c) rational design. directed evolution has developed quickly to become a method of choice for protein engineers in order to create enzymes having desired properties for all kind of processes. over the past decade, this technique has become a daily part of the molecular toolbox of every biochemist. this is emphasized by the increasing number of publications about the subject. 112 in nature, evolution and creation of new functionalities is achieved by mutagenesis, recombination, and survival of the fittest. directed evolution mimics this and is a process of iterative cycles of producing mutants and finding the mutant with the desired properties. mutations can be introduced at specific places using site-directed mutagenesis or throughout the gene by random mutagenesis. several mutagenesis techniques have been developed in order to avoid codon bias. 113, 114 the first technique used to mimic evolution was dna shuffling. 115 this method is based on the mixing and subsequent joining of different related small dna fragments in order to form a complete new gene. in the process of shuffling, the recombination frequency is dependent on the degree of homology. a high level of recombination is important to get all possible combinations of mutations. since recombination can be biased, several methods to overcome problems arising from the use of shuffling in the early years were tackled by novel strategies, all having their own advantages and disadvantages. 112 the products obtained by these methods have to be screened for desired qualities and not all of them can be easily screened. de novo protein design offers the broadest possibility for new structures. it is based on searches for amino acid sequences that are compatible with a three-dimensional protein backbone template using in silico techniques. several research groups in the field have applied in silico methods to design the hydrophobic cores of proteins, with the novel sequences being validated with experimental data. 116 in silico protein design has allowed novel functions on templates originally lacking those properties, modifying existing functions, and increasing protein stability or specificity. beyond any doubt, intense research activities are ongoing in the field, the potential of which is simply enormous. 117 so far there have been numerous examples of full sequences designed ''from scratch'' that were confirmed to fold into the target three-dimensional structures by experimental data. 118 the zinc-finger protein designed by dahiyat and mayo 119 was the first one to appear by this method. rational design of proteins is based on the modification or insertion of selected amino acids or domains in a polypeptide chain backbone to obtain proteins with new or altered biological functions. when using that strategy, a detailed knowledge of the structure and function of the backbone protein is needed to make desired changes. this generally has the advantage of being inexpensive and technically feasible. however, a major drawback of this approach is that detailed structural knowledge of a protein is often unavailable or it can be extremely difficult to predict the effects of various mutations. modular engineering enables, by using simple dna recombinant techniques, the construction of chimerical polypeptides in which selected domains, potentially from different origins, provide the required activities. an equilibrate combination and spatial distribution of such partner elements has generated promising 258 prototypes, able to deliver expressible dna or molecules to tissue culture but also to specific cell types in whole organisms. 120 modular fusion proteins that combine distinct functions required for cell type-specific uptake and intracellular delivery of dna or drugs present an attractive approach for the development of self-assembling vectors for targeted gene or drug delivery. 121 one of the first examples was described by the group of uherek et al. they combined a cell-specific target module (antibody fragment specific for the tumor-associated erbb2 antigen), a dna-binding domain (gal4), and a translocation domain for endosomal escape. 121 in this context, many strategies for the construction of safer vehicles are being explored and the number of nonviral prototype vectors for gene and drug delivery is noticeably increasing. here, the common steps that an approach like this might explore are presented ( fig. 1) . when designing a new protein for drug or gene delivery there are many critical aspects, namely (a) design of the vehicle itself, required functions, stability, etc.; (b) production of the protein, suitable expression system, purification procedure, scaling up process, etc.; (c) characterization of the vehicle by physicochemical and functional tests; and finally (d) the administration route and regulatory guidance for biological products. although all these aspects belong to different disciplines, they have to be overviewed together. here, the major needs of a modular protein for gene and drug delivery are presented. to enhance the physicochemical stability of the cargo molecules and their resistance to nuclease/protease-mediated degradation, protein vehicles should ideally exhibit, like their natural counterparts (viruses), nucleic-acid binding and condensing properties. 27 such abilities are, in general, conferred by cationic segments of the main scaffold molecules that interact with nucleic acids, mainly through electrostatic interactions. in addition, such complexes need to efficiently release the nucleic acid in the nucleus (if the cargo is a therapeutic gene), for which endosomal escape is required. such functions have been found in some peptides in many natural molecules and they are suitable for functionalizing protein vehicles. the ability to bind a particular cell type with high specificity is especially significant in a systemic delivery in which appropriate biodistribution and tissue targeting are essential. 122 for nuclear targeting, only naked short nucleic acids can freely enter the nucleus of nondividing cells via free diffusion through the nuclear pore. large molecules require active transport mediated by nlss that are often found in viral proteins. because the molecular mass of plasmidic dna varies from to 2 to 10 mda, dna that is to be expressed, and essentially any macromolecular complex for nucleic acid delivery, requires nlss. 123 the role and types of functional modules peptides used for all these purposes will be discussed in depth in the following sections. in vivo experiments finally, which protein or peptide is better for a given cargo is to be determined empirically and only few rules can be taken literally. 38, 124 c. production of protein nanoparticles some steps in the production of a protein-based vehicle after molecular cloning such as protein production and protein purification 125 might be experimentally labor intense with a variable success rate. for that reason, when small proteins are needed, solid-phase peptide synthesis 44 guarantees the process. however, the classical procedure of biological production allows scaling up the process in most of the cases and the production of larger polypeptides and fulllength proteins. generally, in protein nanoparticle approaches, the protein is composed by different modules of natural sources such as the cell-penetrating peptide transactivator of transcription (tat) derived from the tat of the human immunodeficiency virus (hiv) 126 or artificial sequences not present in any organism such as the polylysine dna-condensing sequence. 127 once it has been defined which modules will be part of the protein, it is important to define the order they will have in the final construct. it has been demonstrated by boekle and coworkers using melittin conjugated to polyethylenimine (pei) that depending on the side of the linkage (c-or n-terminus), the lytic activity could be changed. some other modules have the need to be in a determined position for its correct function. 128 when producing a protein for gene or drug delivery, it is important to know the origin of its domains to choose the most suitable expression system for its production. for instance, if any module naturally carries a posttranslational modification that is essential for its biological function, the expression system chosen will have to be able to reproduce the same crucial modification. the main biological production systems for protein drugs are described below. escherichia coli is the most widely used prokaryotic organism for the expression of recombinant proteins. 129 the use of this host is relatively simple and inexpensive. 130 added advantages include its short duplication time, growth to high cell densities, ease of cultivation, and high yields of the recombinant product. however, since it lacks fundamental prerequisites for efficient secretion, recombinant proteins manufactured by e. coli systems are mainly produced as inclusion bodies. 125, 131 moreover, posttranscriptional modifications are not achieved with this system. there are many examples of proteins for gene delivery produced in e. coli with probed efficiency. 132, 133 like e. coli, yeasts can be grown cheaply and rapidly and are amenable to high-cell-density fermentations. besides possessing complex posttranslational modification pathways, they offer the advantage of being neither pyrogenic nor pathogenic and are able to secrete more efficiently. species established in industrial production procedures are saccharomyces cerevisiae, kluyveromyces lactis, pichia pastoris, and hansenulapolymorpha. s. cerevisiae is the best genetically characterized eukaryotic organism among them all and is still the prevalent yeast species in pharmaceutical production processes. 131 in spite of their physiological advantageous properties and natively high expression and secretion capacity, the employability of yeasts in some cases, however, might reach a limit, particularly when the pharmacological activity of the product is impaired by the glycosylation pattern. in such cases, either a postsynthetic chemical modification has to be considered or the employment of more highly developed organisms. most examples of nanoparticles produced in yeast are for vlps. 134 animal cell expression systems show the highest similarity to human cells regarding the pattern and capacity of posttranslational modifications and the codon bias. however, their culture is more complicated and costlier and usually yields lower product titers. among the known systems, insect cells infected by baculovirus vectors have reached popularity since they are considered to be more stress-resistant, easier to handle, and more productive compared with mammalian systems and are thus frequently employed for high-throughput protein expression. for commercial application, scale-up related questions have to be solved. [135] [136] [137] preferably applied in pharmaceutical production processes are mammalian systems like chinese hamster ovary (cho) cells and baby hamster kidney (bhk) cells. these systems are genetically more stable and easier to transform and handle in scale-up processes, to grow faster in adherent and submerged cultures, and to be more similar to human cells and more consistent in their complete spectrum of modification. 138 in some cases, mammalian cell systems can be the only choice for the preparation of correctly modified proteins. peptides, being complex and unique complex molecules with regard to its chemical and physical properties, can be produced synthetically by the solidphase method. 139, 140 this technology can be used to avoid problems related to biological production. general advantages of synthetic peptides are that they are very stable compounds, solid-phase chemistry produces highly standardized peptides, and the crucial polycation component is provided by a ''natural'' polycation, thus minimizing toxicity. 141 however, some disadvantages related to synthetic peptides have been reported such as the difficulty to synthesize long and well-folded oligopeptides, peptides with multiple cysteine, methionine, arginine, and tryptophan residues due to technical limitations or production cost. 141 when working with protein nanoparticles, it is very important to characterize them physically and functionally in order to understand their behavior. the size and charge of protein/cargo particles are crucial properties which influence rates of diffusion, binding to polyanionic components of connective tissues, transversal of anatomical barriers, binding of serum proteins, attachment to cells, and mechanisms of endocytosis, among other factors. stability in physiological salt solutions is a key issue for in vivo delivery, as salt is found everywhere in the body. 141 mixing a multivalent polycation and dna results in electrostatic binding of both molecules, with charge neutralization of dna and a particle formation named conjugate. charge neutralization can be easily seen by retardation gel assays and particle formation by dynamic light scattering (dls). dls is a good method to see particle formation but not to quantify relative number of particles of different sizes. 142 to visualize particles, many groups have used transmission electron microscopy (tem) 15, 143 with good results while others have used fluid particle image analyzer (fpia) to photograph individual particles in physiological solutions. 58 the net charge of protein/cargo particles is an important variable. generally, optimal gene delivery for cell lines requires a net positive charge but, as stated previously, it has to be determined empirically. one of the best techniques to determine the net charge is by calculating the zeta potential that measures the electrophoretic mobility of particles. 144 despite the fact that physical characterization is a key element, understanding and testing the functionality and pharmacokinetics of a gene or drug is the most important part of its development process. most of the initial tests are done using cell lines in in vitro experiments using reporter genes, rna, or drugs. 145, 146 quantifying the percentage of transfected cells or drug-induced changes is a very valuable tool to evaluate nanoparticle performance in both nuclear and cytoplasmic delivery, respectively. in addition, in vitro experiments may be designed to select a candidate for the in vivo experiments from a group of possible therapy vectors. the quantitative kinetics of particle binding, the molecular basis of particle interactions with target cell membranes, the efficiency of particle internalization, and endosomal escape are all poorly understood. 141 interaction of particles with plasma membranes prior to protein internalization can be either unspecific or specific. untargeted delivery normally is the consequence of electrostatic interactions between anionic ligands in the cell surface and cationic components of the vehicle. on the other hand, targeted delivery to specific membrane molecules is a more sophisticated approach. it aims to improve cell specificity and efficiency, by directing to molecules, only expressed or overexpressed in a particular cell type, that initiate internalization by endocytosis. targeting moieties include many types of molecules and is discussed afterwards. internalization of particles, its mechanisms, and kinetics are not well known and most studies about nanoparticle delivery do not focus on this aspect. there are several endocytic pathways each initiated by different ligands. 147 enhancing the delivery by addition of chloroquine, a synthetic molecule used primarily for the prophylaxis and treatment of malaria that disrupts endosomes, 148 is an accepted parameter to demonstrate endosomal localization of particles. endosomal escape is the area most intensively investigated but is poorly understood. an important practical point to note is that some reagents that are used can be toxic. 141 to enhance this step, anionic fusogenic peptides can be used. these peptides fuse to membranes in an acidic-dependent manner causing its disruption. 149 in gene delivery approaches, translocation of dna expression plasmids into the cell nucleus involves an active, energy-dependent process through the nuclear pore complex. 150 directly injected dna into the cytosol is usually, but not always, poorly transferred to the nucleus 150, 151 and because of that, the use of proteins carrying cationic nuclear-localizing sequences (such as that of sv40 large t antigen) has been widely used to overcome this step. 143 iv. natural self-assembling protein nanoparticles: vlps ideal drug delivery and gene therapy vehicles must accomplish some desired features such as appropriate packaging size for its cargo, target cellspecificity, safe and efficient cargo delivery, and protection against immune recognition, or capability to escape immune recognition. moreover, these vehicles must avoid inflammatory toxicity and rapid clearance. 152 in this context, viral vectors have been exploited as one of the vehicles of choice. viruses are nano-sized (15-400 nm) supramolecular nucleoproteinbased entities, covered or not with a lipid bilayer (enveloped/nonenveloped viruses) that satisfy, into relatively simple structures, outstanding properties and functions that are relevant to drug and gene delivery. viruses are able to recognize and interact specifically with cells by receptor-mediated binding, internalize, escape from endosomes, and uncoat and release nucleic acids in different cellular compartments. they are also capable of transcribing and translating their viral proteins to self-assemble into new infectious virus particles and exit the host cell. 120, [153] [154] [155] despite all these relevant properties of viral vectors or some other rising vehicles in drug and gene delivery such as cationic liposomes, their therapeutic use presents some limitations and risks because of the complexity of production, limited packaging capacity, insertional mutagenesis and gene inactivation, low probability of integration, reduced efficacy of repeat administration or reduced expression overtime, unfavorable immunological recognition or strong 264 immune response against vehicle and transgene, inflammatory toxicity, and rapid clearance. 120, 152 in this context, virus capsids or vlps, produced by recombinant capsid proteins but lacking the viral genome, have noticeably emerged as a safer alternative to viral vectors. a. structure of protein self-assembled nanovehicles vlps are classically described as self-assembling, nonreplicative and nonpathogenic, highly organized supramolecular multiprotein nanoparticles (coats) (ranging from 20 to 100 nm) that can be formed from the minimal spontaneous self-assembling of one or more viral structural capsid proteins. it has been described that the self-assembling process of the structural viral proteins for vlp formation involves both spontaneous assembly, under favorable experimental conditions, and the requirement of scaffold proteins as catalysts. 156, 157 therefore, vlps are considered protein ''coats'', ''shells'', or ''boxes'' that lack the viral genome, still conserve the structure, morphology, and some properties of viruses. some of these properties such as cellular tropism and uptake, intracellular trafficking, membrane translocation, and transfer of nucleic acids or molecules across the cytoplasmic, endosomal, and nuclear membranes are important for drug delivery and gene therapy. 120, 153, 155, [158] [159] [160] usually, the degree of similarity of vlps and their viruses depends on the number of proteins incorporated into the constructs. 161, 162 since the first description in 1983 of the viral dna packaging into mouse polyomavirus (mpyv) vlps and its transduction in vitro, 163 vlps of different viruses such as papillomaviruses, [164] [165] [166] hepatitis b, c, and e viruses, [167] [168] [169] polyomaviruses, 163 vlps offer some structure, dynamics, characteristic features, and functions that make them appealing bionanomaterials to be exploited in the biomedicine arena as drug and gene delivery vehicles and are discussed in detail afterward. on the one hand, viral coat proteins have the ability to spontaneously selfassemble, which ensures the formation of highly organized, regular, repetitive structurally stable, and very low morphological polydisperse particles that provide useful properties to be used as scaffolds for bioimaging, synthesis of bionanomaterials, and as nanocarriers in drug and gene therapy. 186 in addition, homogeneity of particle size and composition is a desired production factor when developing therapeutic molecules. the overexpression of structural viral proteins in a convenient expression system renders recombinant proteins capable of being folded and assembled in discrete organized nanoparticles with a defined size corresponding to the natural capsid geometry. [187] [188] [189] moreover, even though vlps are structurally stable particles, some biochemical and structural studies have observed that viral capsids and bacteriophages may show some structurally dynamic properties varying in shape, size, or rearrangements of the coat proteins, in response to different factors such as ph. [190] [191] [192] [193] on the other hand, vlps are considered biologically safe nanostructures since they are not infectious (lack of viral genome) and do not replicate, representing a safer alternative to viral vectors. 160, [194] [195] [196] [197] however, they can elicit immune and inflammatory responses, especially when repeated administration is needed. 152 it has to be also noted that when used in vaccination, vlps could show excellent adjuvant properties and the majority of vlps stimulate strong cellular and humoral immune responses as direct immunogens. 198 it has been suggested that recombinant vlps derived from infection of insect cells with baculovirus or even those derived from prokaryotic systems could be contaminated with different residual components of these host cells, contributing those impurities to the adjuvant properties. 153 one interesting property of vlps is that coat viral proteins present an enormous elasticity and adaptability to be modified chemically and/or by protein genetic engineering 154, 160, 199 to incorporate multiple directed functionalities, in order to be addressed in biomedical applications such as drug delivery or gene therapy. it has been recently reviewed that chemically and/or genetically modified vlps, including cpmv, ccmv, ms2, m13 bacteriophages, and other virus-based nanoparticles, 155,186 could maintain their structural integrity and improve their physical stability 154 and, moreover, these modifications could also confer desired cell-targeting properties to the nanovehicle. [153] [154] [155] 186, 200, 201 vlps can be successfully engineered with spatial precision to incorporate (attached or genetically displayed on the surface) targeting tissue-specific ligands such as epidermal growth factor (egfr) and antibodies, or other molecules such as oligonucleotides, peptides, gold, and other metals, target proteins, carbohydrates, polymers, fluorophores, quantum dots, drugs, or small molecules. 152, 154, 155 moreover, one of the potential benefits of such modifications is that the specific geometric rearrangement confers precise recognition patterns. 200, 201 furthermore, accessibility of the materials carried within the particle and the ability of inclusion and separation of nucleic acids, small molecules, and unusual cargoes with appropriate charge is another outstanding feature and key advantage of vlps that has also made them excellent vessels for gene and drug delivery. 152, 195 as described above, vlps can be used as empty nanocarriers to transport molecules chemically attached on their surface or can be loaded ex vivo with therapeutic small molecules such as drugs, dnas, mrnas, sirnas, oligonucleotides, quantum dots, magnetic nanoparticles, or proteins. 155, 157, 160 vlps of different papillomavirus and polyomavirus have been widely characterized and used for directed delivery in biomedical applications. 132, 165, 173, 174, 194, 202 osmotic shock and in vitro self-assembling of vlp subunits in the presence of 266 the cargo have been the two main strategies used to packaged nucleic acid or other small molecules. it has to be taken into account that some attachment of the cargo on the vlp surface can occur. 195 besides, diversity of natural tropism including liver for hepatitis b vlps, spleen for some papillomavirus and polyomavirus vlps, antigen-presenting cells for certain papillomavirus vlps, and glial cells for human polyomavirus jc (jcv) vlps, among others 152 is one of the key advantages offered by vlps providing a wide spectrum of specific targeting and distribution profiles depending on the directed application. although each vlp has its own characteristic receptors, entry pathway, and intracellular trafficking, it has been demonstrated that tropism of vlps could be customized, modifying the residues identified as ligands of the cellular receptor on vlps' surface or even varying the delivery routes. 155, 189, 203 another key advantage of vlps is that they can be easily produced by using a wide range of hosts and expression systems, each of them with its own conditionings. 162 in the past years, there has been an increasing need to improve and optimize efficient large-scale production systems, process control and monitoring, and up-and down-streaming processes. 153, 157, 159, 204 production of vlps usually involves transfection of the cell host expression system of choice with a plasmid encoding one or more viral structural proteins, further and rigorous purification for the removal of immunogenic cellular contaminants, and quality control of the produced vlp and encapsulation of the cargo ex vivo before administration. 152, 158 the most frequent and convenient expression systems, adaptable to large-scale processes are (1) yeast cells 176 205, 206 , (4) bacteria 204,207 , (5) green plants infected with modified viruses 208, 209 , and (6) cell-free systems. 163, 204 the preparative and large-scale manufacture of vlps in some of these hosts has been reviewed by pattenden et al. and can be classified into two main methods of bioprocessing: in vivo and in vitro systems. 157 in addition, the capability of in vitro dissociation and reassociation of vlps contribute to the application of easy and more accurate purification methods than those of viral vectors. 152, 157 furthermore, depending on the expression system, the resulting vlp might be significantly different even though expressing the same viral proteins. thus, a broad spectrum of vlps could be customized depending on the vlp type, the number of proteins needed for vlp assembling, and the targeted final application. 158, 210 as described above, vlps have great potential as nanocarriers in drug and gene delivery. at the same time, although there is an increasing flow of developments in this area, these vehicles also present some limitations that should be addressed and taken into account, such as residual cellular components, variable yield of functional vlps after disassembly/reassembly process, immunostimulation and unsuitability for repeated administration, tolerance to the transgene, ineffective therapeutic molecule loading, and low transfection rates. 152 protein nanoparticles engineered for drug delivery and gene therapy 267 due to their versatile nanoparticulate structure and morphology, and nonreplicative and noninfecting nature combined with their natural immunogenic properties and ease production, vlps have principally emerged as an excellent alternative tool to attenuate viruses for vaccination. 152 182 and ebola virus. 215, 216 although vlp-based vaccines have been primarily developed for their use against the corresponding virus, in the last decades genetic engineering or chemical modifications have been applied in order to generate chimeric vlps. thus, on the one hand, commonly short heterologous peptide epitopes or full proteins that are unable to form vlps or that are unsafe for vaccination have been presented on surface-exposed loops or fused to n-or c-exposed termini of structural viral capsid proteins on vlps. 154, 161, 210 different hpv, 217-219 hbv, 220,221 parvovirus, 222, 223 and chimeric polyoma vlps have been engineered 170, 175 and tested for different applications including vaccination against viral or bacterial diseases, against virus-induced tumors, and more recently, for immunotherapy of nonviral cancer. 161, 210 on the other hand, chemical bioconjugation for covalent coupling of protein epitopes and small molecules to lysines, cysteines, or tyrosine residues of vlp surfaces has been applied in viral or cancer vaccines. 200 chackerian et al. have demonstrated the efficient induction of protective autoantibodies using self-antigen conjugation to hpv vlps. 224 it is important to point out that vlps can also be engineered to incorporate heterologous cell-specific ligands to cell receptors, thus altering their cellular tropism. 154, 155, 186, 201 this great convertibility and flexibility of vlps to be modified (chemically and/or genetically), their high stability, natural and diverse tropism, their nanocontainer properties, and their ability to enter in the cell and incorporate, bind, and deliver nucleic acids and small molecules have positioned vlps as appealing entities not only for vaccination applications but also for a broad spectrum of other diverse and emerging applications in nanomedicine and nanotechnology such as immunotherapy against cancer, 210,225 gene therapy delivery of therapeutic genes into specific cells, 161, 165, 171, 184, 226, 227 and targeted delivery of drugs and small molecules using vlps as nanocarriers. 174, 196 268 domingo-espín et al. although there is no commercial vlp as vector in gene therapy, since the initial work in 1970 of uncoating polyoma pseudovirus in mouse embryo cells as gene delivery vector 228 and the establishment in 1983 of the viral dna packaging into mpyv vlps and its transduction in vitro, 163 different vlps such as hbv and hepatitis e virus, 229 hpv and polyomavirus nanoparticles 172, 178, 229 have been modified toward the specific delivery of therapeutic genes and proteins in different target cells, organs, and tissues in vitro and in vivo by systemic injection 229 or oral administration. 230 for example, recombinant vp1-based polyomavirus vlps can encapsulate in vitro exogenous dna, and deliver it by cell surface sialic acid residues to human brain cells and fetal kidney epithelial cells. 178 furthermore, vlps have recently emerged as novel nanocarriers or nanocontainers to store unnatural cargos, deliver modified oligonucleotides, 154 synthetic small interfering rnas, and plasmids expressing short hairpin rnas as therapy to downregulate gene expression. 171, 231 in this context, chou et al. have recently described the use of jcv vlps as an efficient vector for delivering rnai in vitro using murine macrophage raw 264.7 cells and in vivo using balb/c mice in silencing the cytokine gene of il-10 without significant cytotoxicity for systemic lupus erythematosus gene therapy. 171 one of the key aspects in targeted gene and drug delivery is cell-specific delivery. it is important to point out that vlps are tunable nanoparticles that can also be chemically or genetically engineered to modify their natural cellular tropism in order to diversify the range of therapeutic applications in targeted gene or drug delivery. 154, 201 some effective approaches to modify the natural cellular tropism include: (1) genetic engineering of vlp chimeras incorporating heterologous cellspecific short peptides that contain recognition sites of target cell receptors. 232 in this context, polyoma and papillomavirus, with solved atomic structures of their major structural capsid proteins, have been extensively used to obtain chimeric vlps as delivery vector systems. 165, 233 however, this approach has some bioprocessing limitations such as low production levels as a consequence of vlp modification, alterations of size and properties of the vlps that could affect the structural interactions and conformations for vlp assembly, disassembly and packaging, and low transduction efficiencies. 157 (2) chemical bioconjugation of purified vlps with epitope-containing peptides 234, 235 or a wide range of small molecules conferring cell-specific targeting such as transferrins, folic acid, or other targeting molecules. as an example, cmpv vlps have been successfully conjugated with tfn using ''click'' chemistry 236 and with nhs-ester-derivatized folic acid, demonstrating both as internalized into hela cells and kb cells, respectively. 183, 184 (3) high-throughput library and directed evolution method is a rational approach that has been recently used to engineer viral vectors with the desired tropism properties. 237 (4) pseudotyping, which consists of replacing the envelope protein of one virus species by the envelope protein of another virus species. 238 (5) modification of the delivery route of the vlps. it has been shown that the levels of expression of b-galactosidase in heart, lung, kidney, spleen, liver, and brain are different depending on the delivery route of polyomavirus vp1 vlps. 203 the great accessibility and reactivity showed by vlps, as well as their ability to serve as nanocarriers, which made them suitable to be exploited in gene therapy, have also been applied to targeted drug delivery. 195 genetic modification and/or chemical functionalization of exposed amino acid residues on the capsid surface in order to attach small molecules, such as markers or bioactives molecules, is one of the most common approaches applied to target drug delivery. 174, 239 as an example, canine parvovirus (cpv) vlps produced in a baculovirus expression system and exhibiting natural tropism to transferrin receptors (tfrs) were chemically modified on accessible lysines of the capsid surface with fluorescent dye molecules and delivered to tumor cells. derivatization of cpv-vlps did not interfere with the binding and internalization into tumor cells. 183, 184 one limitation of vlps in gene therapy is the low efficiency of gene transduction due to inefficient dna packaging. however, a recent study presented a novel in vivo dna packaging of jcv vlps in e. coli that effectively reduced human colon carcinoma volume in a nude mouse model. in this study, the exogenous plasmid dna was transformed into the jcv vp1 expressing e. coli. the packaging of the second plasmid occurs simultaneously as the in vivo assembly of the jcv vlp. even though it is still not clear how the plasmid dna molecules are encapsidated in the vlp, the authors showed that gene transduction efficiency by their in vivo package system was about 80% in contrast to the 1-2% of gene transduction efficiency achieved by the in vitro osmotic shock system. 226 in addition, the administration of exogenous proteins may induce the immune system response, reducing therapy effectiveness or causing undesirable secondary effects, albeit immunological response of protein nanoparticles can be modulated. 240 spontaneous protein self-assembly to form ordered oligomers is a common event in biology. it can prove advantageous in terms of genome-size minimization, formation of large structures, stabilization of complexes, and inclusion of 270 functional features. 241 it has been widely documented that cellular oligomer proteins as well as viral capsids are stabilized by several weak noncovalent interactions as hydrophobic interaction, electrostatic energy, and van der waals forces. [242] [243] [244] these interactions result in a complex quaternary structure described by three symmetry point groups named cyclic (cn), dihedral (dm), and cubic (t, o, i). 245, 246 the development of computational techniques to predict protein-protein interactions using solved 3d protein structures makes it possible to predict and/or strengthen experimental data performing in in silico approaches. 247 furthermore, its use opens up the possibility to design proteins not only displaying specific biological functions but also interesting intermolecular interactions to obtain increased multivalency in the resulting complexes. moreover, it should be considered that not only whole proteins can self-assemble in smart nanoparticles; oligopeptides are also capable of forming organized structures. many applications are possible due to the enormous quantity of different combinations and features that can be exploited with peptides. 248, 249 furthermore, protein-protein interactions are not the unique parameters involved in particle formation, nucleic acid-peptide interactions, salt concentration, order of mix, and ratio between nucleic acid and protein can also strongly influence the condensation process. 250, 251 due to their natural tendency to self-assemble forming highly ordered structures, viruses provide a wide variety of scaffold proteins which are used as gene/drug carriers. among them, vlps have been reviewed in the previous section. however, simple bacterial proteins can be also utilized as carriers for gene delivery. for example, heat shock proteins (hsp) from hyperthermophilic archeaon methanococcus jannaschii can assemble in a small structure of 24 subunits having an octahedral symmetry. these 12 nm structures are stable at high temperature, up to 70 c, and wide range of ph. residue modifications are allowed to elicit specific attachment of small molecules. 186, 252 in bacteria, bacterial microcompartments (bmc) which are intracellular organelles consisting of enzymes encapsulated within polyhedral, protein-only shells, somewhat similar to viral capsids, have been described. bmcs are composed of a few thousand copies of a few repeated protein species (including one or more enzymes involved in specific metabolic pathways), and with sizes of around 100-150 nm in cross section. the general role of bmcs is to confine toxic or volatile metabolic intermediates, while allowing enzyme substrates, products, and cofactors to pass. the first described bmc, the carboxysome, was isolated in the early 1970s 253, 254 and has been found to contain both co 2 -fixing ribulose bisphosphate carboxylase/oxygenase (rubisco) 253, 254 and carbonic anhydrase [255] [256] [257] enzymes. carboxysomes' function is to enhance autotrophic co 2 fixation at low co 2 levels. other bmcs were later identified in cyanobacteria and some chemoautotroph bacteria. among them, bmc proteins have been later found to be encoded in the propanediol utilization operon (pdu) of the heterotroph salmonella 258 and by an operon for metabolizing ethanolamine (eut) in enteric bacterial species, including salmonella and escherichia. 259 salmonella enterica forms a polyhedral organelle during growth on 1,2-propanediol (1,2-pd) as a sole carbon and energy source, but not during growth on other carbon sources. 260, 261 the pdu organelles' function is to minimize the harmful effects of a toxic intermediate of 1,2-pd degradation (propionaldehyde). [261] [262] [263] other studies have shown that a polyhedral organelle is involved in ethanolamine utilization (eut) by s. enterica. 259 the function of the eut microcompartment is to metabolize ethanolamine without allowing the release of acetaldehyde into the cytosol, therefore minimizing the potentially toxic effects of excess aldehyde in the bacterial cytosol [264] [265] [266] and also preventing volatile acetaldehyde from diffusing across cell membrane. 267 so far, about 1700 proteins containing bmc domains have been identified, covering at least 10 different bacterial phyla. the typical bmc protein consists of approximately 90 amino acids, with an alpha/beta fold pattern. 268, 269 some individual bmc proteins self-assemble to form hexamers, which further assemble side by side to form the flat facets of the shell. 268, 270, 271 the formation of icosahedral, closed shells from such flat layers was elucidated in part by structural studies in carboxysomes: some bmc proteins assemble to form pentamers, which are located at and form the vertices of the icosahedral shell. 270 mechanisms directing enzyme encapsulation within protein-based bmcs have been studied during the last years. it has been described that, in some carboxysomes, protein ccmm is used as a scaffold to form interactions between both shell proteins and enzymes, 272,273 through a ccmm c-terminal region with homology to the small subunit of rubisco. 274 other studies revealed that pdu shells can self-assemble without needing interior enzymes 275 and that carboxysomes can self-assemble in vivo when rubisco has been deleted. 276 regarding properties of the encapsulated enzymes, in the pdu bmc some of the internal enzymes are encapsulated by specific n-terminal targeting sequences. 275, 277 in this line, sutter and colleagues 278 described a conserved c-terminal amino acid sequence that mediates the physical interaction of an iron-dependent peroxidase (dyp) or a protein closely related to ferritin (flp) with a specific type of bmc (encapsulins). in another example, an icosahedral enzyme complex, lumazine synthase (aals) from bacillus subtilis and aquifex aeolicus, was engineered to encapsulate target molecules by means of charge complementarity and can also be modified to give different characteristics to the assembled structure. 279 moreover, enzymatic subunits, like e2 of pyruvate dehydrogenase from bacillus stearotermophilus, can be modified to be used in gene delivery. e2 peptides naturally form a dodecahedron of 60 subunits of 24 nm in diameter allowing modification for drug-like accommodation. the assembling/disassembling of these structures can be modulated by changing the operative ph in the experimental environment. these nanoparticles can also be functionalized with antigens for vaccine development. 281, 282 according to these results, specific targeting sequences could be of use in biotechnological applications to package proteins inside the stable selfassembled icosahedral shell of bmcs, offering appealing opportunities to manipulate in the laboratory such nanocages to fill them with therapeutic molecules. the simplicity of this system makes it very attractive for engineering studies to design, mimicking nature, new applications in biotechnology, providing a new, intriguing platform of microbial origin for drug delivery. bovine serum albumin (bsa) is able to form microspheres after sonochemical treatment in aqueous medium. chemical effects of ultrasound radiation and coupling with an anticancer drug such as taxol (paclitaxel) led to the assembling of a spherical carrier with an average diameter of 120 nm. bsa particles resulting from s-s bonds, due to ho 2 radical formation, are able to release the encapsulated taxol in cancer tissue with best results if compared with mere taxol treatment. this drug for breast cancer treatment is commercially available. 283, 284 also little cationic peptides can lead to self-assembling particles. among others, arginine-rich cationic peptides are widely known as good tools for gene delivery. for example, purified r9-tailored gfp in solution is described to form nanodisk particles 20 nm in diameter. this structure is proved to be induced by the 9 arg tails and is able to bind and condense dna. these nanodisks are also able to deliver dna toward the nucleus where the reporter gene is expressed. 285 on the other hand, the expression of recombinant proteins over physiological rates can cause a bad functioning of cellular quality control system, leading to self-organizing, pseudo-spherical, protein aggregates known as inclusion bodies. these mechanically stable nanoparticles, ranging from 50 to 500 nm in diameter, were considered for a long time as undesired bio-products. recently, it became clearer that they are suitable for medical approaches when utilized as scaffold surface to promote cellular proliferation. [286] [287] [288] one of the most difficult goals for a foreign gene delivery is to reach the nucleus. an approach to overpass this obstacle is by fusing an nls in a nonessential position of a dna-binding protein. such type of modification has been described for a tetracycline repressor protein (tetr) fused with an sv40 nls. the tetr-nls affinity and specificity to teto dna sequence is exploited to form spontaneous protein-dna complexes which allow an enhancing of dna transportation into the nucleus and subsequent expression of foreign genes, combining the two peculiar characteristics of each fusion component. 289 there is still a tremendous gap between progresses made in protein-based nanoparticle research for drug delivery and clinical reality. hundreds of publications in basic research describe the combination of two or more functional elements in a single protein nanoparticle, by which the delivery of a carried drug is enhanced. these agents act by improving critical steps in the drug delivery process, such as increasing the systemic stability or tissue specificity, favoring internalization, endosomal escape, and entry into the nucleus, or transporting therapeutic material through the bbb, in in vitro and in vivo studies. besides the human recombinant therapeutic proteins currently on the market (or functional segments of them), there are also some fusion proteins approved for clinical use (most by incorporating an antibody fragment or a ligand to enhance cell specificity). sadly no gene therapy trials have so far used full protein carriers in vivo, but rather peptide-functionalized vehicles. bottlenecking the gap between research and clinical application, the us fda/european medicines agency (emea) only approves human proteins, to avoid the risk of an immune response that could affect not only the effectiveness of the nanoparticle but also challenge patients' health. another critical factor is the administration route, where the protein is degraded before arriving at the target; this problem could be solved or minimized by the use of protein d-isomers, pegylation, or the design of protecting groups for labile sites. despite the current situation mentioned above, there are many good examples of multifunctional modular proteins that, when carrying therapeutic material, can improve the prognosis in vivo in animal models for different diseases. these examples are reviewed below, along with those few protein nanoparticles that are currently on the market or in clinical trials. albumin is a natural protein transporter of hydrophobic molecules throughout plasma that has been approved by the fda to reversibly bind water-insoluble anticancer agents, as is the case of albumin-bound (nab) paclitaxel, abraxane . this albumin-nab technology-based drug is in use in patients with metastatic breast cancer who have failed combination therapy, and it is the first protein nanoparticle approved by the fda. albumin potentiates paclitaxel 274 concentration within the tumor by increasing paclytaxel endothelial transcytosis through caveolae formation. it also contributes to the fact that tumors secrete an albumin-binding protein sparc (also called bm-40) to attract and keep albumin-bound nutrients inside the tumor cell. 290 the albuminpaclitaxel complex was not formally considered a nanoparticle in the united states (due to an average size of 130 nm) but only so in europe. apart from whole recombinant therapeutic proteins being currently commercialized, there are also some examples of vehicles formed by chimerical proteins with target ligands already in the market. dab389il-2 (denileukin diftitox or ontak) is a fusion of diphtheria toxin catalytic and translocation domains for lethal effect and interleukin-2 (il-2) to gain cell specificity in the treatment of persistent or recurrent t-cell lymphoma. belatacept (bms-224818) is a ctla4-ig fusion protein formed by the cytotoxic t-lymphocyteassociated antigen 4 joined to an immunoglobulin g1 fc fragment fusion protein, developed by bristol-miers-squibb. etanercept (enbrel) fusion tumor necrosis factor receptor (tnfr), which binds and inhibits specifically tnf activity, to an immune globulin g1 fc, to prevent inflammation mediated by tnf in autoimmune diseases like arthritis and psoriasis. on the other hand, fusion proteins which include an antihuman epidermal growth factor receptor 2 (her2) monoclonal antibody that binds tumor cell surfaces, among them the so-called ''trastuzumab'' (commercialized as herceptin by roche), associated to dm-1, an antimitotic drug, aimed at improving the treatment of breast cancer. finally, vlps, that is, empty viral entities formed by the self-assembly of a viral capsid protein, are the only truly protein nanoparticles (architectonically speaking) which are currently used in clinical practice. hbsag recombinant protein of hbv expressed in yeast and the capsid l1 recombinant protein of hpv (types 6, 11, 16, and 18) administered currently as vaccines tend to form spontaneously vlps that elicit t and b immune response. recently, there have been preclinical and clinical trials to test the security and efficacy of vlp vaccines against chikungunya 291 and seasonal influenza virus (http://www. medpagetoday.com/meetingcoverage//icaac/22129), respectively. influenza vlp vaccines have proven to provide complete protection against h1n1 2009 flu pandemics, 292 within a record preparation time when compared to 9 months for traditional vaccines. the use of vlps as a delivery system for drugs or nucleic acids in gene therapy is still under investigation. 194 drugs and proteins may be transformed through pegylation, a process that can assist them in overcoming some of the potential problems that delay the adoption of protein nanoparticles for clinical use. the covalent attachment of peg can reduce immunogenicity and antigenicity by hiding the particle from the immune system, can increase the circulating time by reducing renal clearance, and can also improve the water solubility of a hydrophobic particle. the use of pegylation has been approved for commercial use by the fda and emea, and some examples of pegylated protein products are adagen (peg-bovine adenosine deaminase), the first pegylated protein approved by the fda in 1990, pegasys (peg-interferon alpha), and oncaspar (peg-l-asparaginase). the majority of protein nanoparticles studied in clinical trials (http://clinicaltrias.gov) are fusion proteins composed of a therapeutic protein/peptide and a target cell-specific ligand. an example is alt-801, a biologic compound composed of il-2 genetically fused to a humanized soluble t-cell receptor directed against the p53-derived antigen. the clinical trials evaluated whether directing il-2 activity using alt-801 to the patient's tumor sites that overexpress p53 results in clinical benefits (nct01029873, nct00496860). another ligand joined to il-2 is l19, a tumor-targeted immunocytokine constituted of a single chain fragment variable (scfv) directed against the ed-b domain of fibronectin, one of the most important markers for neoangiogenesis. l19-il-2 is in a phase i/ii study for patients with solid tumors and renal cell carcinoma (rcc) (nct01058538). l19 has also been fused to tnfa with the intention to target tnfa directly to tumor tissues resulting in high and sustained intralesional bioactive tnfa concentrations. the l19tnfa is under clinical trial using isolated inferior limb perfusion (ilp) with the standard treatment with melphalan 10 mg/l limb volume in subjects affected by stage iii/iv limb melanoma (nct01213732). ngr-htnf is another bifunctional protein which combines a tumor-homing peptide (ngr) that selectively binds to amino peptidase n/cd13 highly expressed on tumor blood vessels, thus affecting tumor vascular permeability, and htnf, with direct anticancer activity. ngr-htnf is undergoing 14 clinical trials as a single agent to treat different cancers, as well as in combination with chemotherapy agents. another strategy to direct a therapeutic protein to the target cell is through fusion to a growth factor receptor ligand. an example is tp-38, a recombinant chimerical protein composed of the egfr binding ligand (tgf-a) and a genetically engineered form of the pseudomonas exotoxin, pe-38, to treat recurrent grade iv malignant brain tumors (nct00071539). many clinical trials are based on a therapeutic protein fused to a targeting antibody, as is the case of apc8015. this drug stimulates the immune system and stops cancer cells from growing by the combination of biological therapies with bevacizumab , an already approved monoclonal antibody that locates tumor cells and kills them in a specific way (nct00849290). there are also many putative protein drugs against cancer which include antibodies antiintegrins (e.g., cilengitide and imgn388), sometimes in combination with 276 classical therapies. a recently developed tool, the nanobodies or single domain antibodies, 293 have several advantages: small size (only 12-15 kda), which lowers the possibility of triggering immune response, safety in clinical trials (nct01020383), and is easy to be joined to different kinds of compounds. all these features make nanobodies competent drugs against different diseases, and have been tested in vivo as bifunctional proteins associated to a prodrug, very efficient in mice cancer xenografts. 294 even though cpps are very useful tools to deliver drugs and in gene therapy (see the chapter ''peptide nanoparticles for oligonucleotide delivery'' by lehto et al. in this volume), their toxicity and endosomal entrapment slows their inclusion for systemic delivery in clinical trials. nevertheless, there are a few examples of use to prevent undesirable cell proliferation in coronary artery bypass grafts, as is the case of a cpp (r-ahx-r) 4 ahxb-pmo conjugate targeted to human c-myc to be applied ex vivo. the trial, in phase ii, has been completed in 2009 (nct00451256). another case is psorban , a product patented for the treatment of psoriasis based on a cyclosporine-polyarginine conjugate of local application, which circumvents the specificity problem of intravenous (i.v.) application. it is in clinical trial phase iii, but not yet in the market. finally, kai-9803, a pkcd inhibitor peptide conjugated to tat to function as an intravenous drug for the treatment of acute myocardial infarction, is currently in phase 2b clinical trial (nct00785954, kai pharmaceuticals). there are many proteins, often organized as nanoparticles, that when associated to a drug, therapeutic protein, peptide, or nucleic acid increase the therapeutic efficacy of a cargo alone in the treatment of various diseases. some of them proved effective in animal models, which are discussed in more detail in this section, with relevant examples listed in table ii . these nanoparticles may simply be (a) a cpp to promote nonspecific internalization, 295-300 (b) a peptide to confer cargo specificity by joining a receptor distinctive of a cell type, including scfvs or peptides obtained by phage display, 301 and (c) a mixture of both, 302 since as observed in several studies the cpp does not reduce ligand specificity and increases nanoparticle potency. [303] [304] [305] complex and multifunctional vehicles including endosomal escape peptides enhance the therapeutic potency of the complex, or other domains that allow their selective activation in certain contexts. 306, 307 apart from the cases listed in table ii , the spectrum of additional examples of multidomain protein nanoparticles tested in vivo is wide, and a considerable proportion of them include cpps, mainly tat and polyarginines. a classical tat fusion protein is the transducible d-isomer ri-tatp53c 0 ' fusion protein that activates p53 protein in cancer cells, but not in normal cells. ri-tatp53c 0 treatment in terminal peritoneal carcinomatosis and peritoneal lymphoma preclinical models results in significant increases in life span (higher than sixfold) and full recovery from the disease. 308 there are also several studies in vivo using tat-fused therapeutic proteins which have proven effective in treating tumors [309] [310] [311] and cerebral ischemia 312,313 when applied intraperitoneally (i.p.). regarding polyarginines, kumar and colleagues have presented two different models in which a bifunctional peptide formed by nine arginines (9r) and a specific ligand constitute an effective sirna vehicle. in the first model, a chimerical peptide derived from rabies virus glycoprotein (to confer neuronal specificity) fused to 9d-arginines (rvg-9r), was able to transport si-rna across the bbb and silence specific gene expression in the brain when applied intravenously. 56 in the second model, a cd7-specific single-chain antibody was conjugated to oligo-9-arginine peptide (scfvcd7-9r) for t cell-specific antiviral sirna delivery in humanized mice reconstituted with human lymphocytes. in hiv-infected humanized mice, this treatment controlled viral replication and prevented the disease-associated cd4 t cell loss. moreover, it effectively suppressed viremia in infected mice. 314 some other examples of polyarginines in tumor models are 9-d-arginines fused to a tumor-suppressor peptide, which stopped tumor growth in hepatocellular carcinoma-bearing mice when applied intraperitoneally, and also colesteryl oligoarginines carrying vegf sirna, which inhibited tumor growth in colon adenocarcinoma after local application. 315 another bbb-crossing peptide is g7, which is able to transport nanoparticles loaded with loperamide. 107 in general, the partner fusion peptide can confer specificity instead of penetrability, as is the case of egfr fab fragment associated to liposomes that contain anticancer drug, which increases efficiency of anticancer effect in egf overexpressing xenograft tumors 316 ; in addition, rgd-4 c-doxorubicin in human breast xenografts increases efficacy and diminishes toxicity. 317 in many conjugates, the therapeutic peptide of the chimerical proteins is a toxin. anthrax lethal toxin has been modified to be activated by methaloproteases, and it has probed to be effective for human xenografted tumors such as melanoma, lung, and colorectal cancer. 318 anthrax toxin has also been associated to antibodies or growth factors for lethal effects specifically on cancer cells. 319 the specific cytotoxicity desired to treat a tumor might derive from a tissue factor, which promotes clotting to restrict blood supply in tumor vessels, fused to peptides that provide specificity, like v-cam antibodies, fibronectin, and integrin ligands. 320 eventually, drug activity may decrease when conjugated to a carrier protein, although if the entry of the drug is favored, the overall balance of activity can be much more efficient. 321 on the other hand, the use of noncovalent bond drug carrier could avoid interfering with the activity of the drug. an important issue in a preclinical study to be considered for a clinical trial is the administration route. in in vivo experiments, most of the protein nanoparticles are administered by local or intraperitoneal injection, avoiding systemic spreading and clearance in the vascular system, in a way very similar to in vitro experiments. the fda and emea, on the other hand, will preferentially approve i.v. and oral administrations rather than intraperitoneal or local injections except for very accessible tissues. another relevant issue is the number of active domains to be included in a therapeutic protein carrier, an issue that seems to be relevant for the functionality of the construct. for example, the cpp neutralization of a ligand may depend on the cpp/ligand ratio that is in the vehicle. 322 it has also been observed that the integrin binding power of rgd-containing motives increases with the number of rgd domains over the monomer until a maxim of four moieties. 323 another example is tat activity empowerment when attached to molecules that form tetramers, such as beta-galactosidase 108 and p-53. 324 some multidomain protein carriers allow the drug entrance only in selected target cells by tailored smart selective mechanisms. 325 for instance, cpps neutralized by polyanions are activated and enter the cells when they are released by metalloproteases 326 or by lowering the ph, 327 both situations being very common in tumors. cpp-morpholino oligomer (pmo) nanoparticles have also shown their effectiveness in treating viral infections by inhibiting viral replication, as demonstrated with the carrier (r-ahx-r) 4ahxb-pmo administered i.v. in animal models infected with picornaviruses, i.p. in mice infected with coronaviruses and flaviviruses, and the carrier r9f2c-pmo administered also i.p. in mice infected with ebola virus. furthermore, it has also been shown in some of these studies that the efficacy of the treatment is dependent on the incorporation of arginine-rich peptides in the nanoparticle. 328 a good example of how a cpp can improve the internalization of a therapeutic protein is the case of insulin. the instability and low absorption in the digestive tract of insulin prevents its oral administration, even though it would be very convenient for a daily administrated drug. in recent studies, noncovalent conjugation of insulin to different cpps enhances its absorption without toxic intestinal effect, l-penetratin being the most efficient as insulin carrier. 329 among the protein nanoparticles tested in vivo, it is worth making special mention of trojan horses generated in pardridge's laboratory to cross the bbb, through a strategy of fusing within a chimerical peptide the therapeutic protein which has to reach the cns to a monoclonal antibody against the human insulin receptor (hirmab). this trojan horse is very potent for humans and primates, and has proven effective to transport b-glucuronidase, a-l-iduronidase, gdnf, abeta amyloid peptides, paroxonase, etc., with potential benefits in diseases like mucopolysaccharidosis type vii, hurler syndrome, parkinson, alzheimer, and organophosphates toxicity, respectively. 330 there are also promising results when protein nanoparticles have been tested as carriers for gene therapy in vivo, some examples being listed in table ii . in this regard, the use of modular proteins generated by insertional mutagenesis of b-galactosidase condensing the sod gene are able to protect neurons against ischemic injury 133 ; a bifunctional galactosylated polylysine is able to conjugate plasmid dna and to differentially promote expression in hepatocytes that display asialoglycoprotein receptor 331 ; a suicide multidomain protein particle formed by herpes simplex virus thymidine kinase (hsv-tk) conjugated to transferrin (tf) by a biotin-streptavidin bridging, which, administered i.v. in k562 massively metastasized nude mice, was able to reduce tumor size and to increase mouse survival. 332 in this chapter, proteins and peptides have been envisioned as potent biotechnological tools for the development of new biocompatible biological entities that can be used as therapeutic agents by themselves or as nanovehicles for the delivery of associated drugs. proteins are nanostructures that can form complex high-order entities such as vlps, resulting in appropriate cages for the internalization of therapeutic molecules. in addition, the design of modular proteins displaying selected functions has been possible by using in silico approximations to the feasibility of recombinant protein production. this approach has demonstrated the versatility of such molecules in the generation of novel delivery nanovehicles opening up the possibility of new functional combinations to enhance the specific interaction with the target tissue. such tunable specificity in the delivery of drugs, nucleic acids, or other proteins is one of the main properties that make multifunctional proteins appealing as more rational delivery vehicles. the presence on the market of such complex entities, which started with the approval of insulin for the treatment of diabetes, has been increasing over the past years, and this tendency is expected to continue. in fact, there are some products in clinical trials that will probably end up being approved and some more are being explored in preclinical experiments which might enter in clinical trials. identifying actives from hts data sets: practical approaches for the selection of an appropriate hts data-processing method and quality control review natural products in the process of finding new drug candidates when analoging is not enough: scaffold discovery in medicinal chemistry dose-toxicity models in oncology is declining innovation in the pharmaceutical industry a myth? the impact of pegylation on biological therapies anticancer activity of celastrol in combination with erbb2-targeted therapeutics for treatment of erbb2-overexpressing breast cancers soon-shiong p. sparc expression correlates with tumor response to albumin-bound paclitaxel in head and neck cancer patients improved effectiveness of nanoparticle albumin-bound (nab) paclitaxel versus polysorbate-based docetaxel in multiple xenografts as a function of her2 and sparc 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specifically stabilized and activated in hypoxic tumor cells the 104-123 amino acid sequence of the beta-domain of von hippel-lindau gene product is sufficient to inhibit renal tumor growth and invasion dendritic cells transduced with protein antigens induce cytotoxic lymphocytes and elicit antitumor immunity protein kinase c delta mediates cerebral reperfusion injury in vivo in vivo delivery of a bcl-xl fusion protein containing the tat protein transduction domain protects against ischemic brain injury and neuronal apoptosis t cell-specific sirna delivery suppresses hiv-1 infection in humanized mice cholesteryl oligoarginine delivering vascular endothelial growth factor sirna effectively inhibits tumor growth in colon adenocarcinoma epidermal growth factor receptor-targeted immunoliposomes significantly enhance the efficacy of multiple anticancer drugs in vivo cancer treatment by targeted drug delivery to tumor vasculature in a mouse model matrix metalloproteinase-activated anthrax lethal toxin demonstrates high potency in targeting tumor vasculature anthrax fusion protein therapy of cancer comparison of three different targeted tissue factor fusion proteins for inducing tumor vessel thrombosis overcoming methotrexate resistance in breast cancer tumour cells by the use of a new cellpenetrating peptide tumor cell retention of antibody fab fragments is enhanced by an attached hiv tat protein-derived peptide improved targeting of the alpha(v)beta (3) integrin by multimerisation of rgd peptides probing the impact of valency on the routing of arginine-rich peptides into eukaryotic cells cell-penetrating and cell-targeting peptides in drug delivery tumor imaging by means of proteolytic activation of cell-penetrating peptides tat peptide-based micelle system for potential active targeting of anti-cancer agents to acidic solid tumors cell penetrating peptide conjugates of steric block oligonucleotides usefulness of cell-penetrating peptides to improve intestinal insulin absorption biopharmaceutical drug targeting to the brain gene transfer in vivo: sustained expression and regulation of genes introduced into the liver by receptor-targeted uptake in vivo gene delivery to tumor cells by transferrin-streptavidin-dna conjugate the authors appreciate the financial support received through grants bfu2010-17450 from micinn, ps0900165 from fiss, and 2009sgr-108 from agaur. the authors also acknowledge the support of the ciber de bioingeniería, biomateriales y nanomedicina (ciber-bbn), an initiative funded by the vi national r&d&i plan 2008-2011, iniciativa ingenio 2010, consolider program, ciber actions and financed by the instituto de salud carlos iii with assistance from the european regional development fund. protein nanoparticles engineered for drug delivery and gene therapy 281 key: cord-006947-nrzjedhi authors: dasgupta, s; auth, t; gompper, g title: nanoand microparticles at fluid and biological interfaces date: 2017-09-20 journal: j phys condens matter doi: 10.1088/1361-648x/aa7933 sha: doc_id: 6947 cord_uid: nrzjedhi systems with interfaces are abundant in both technological applications and biology. while a fluid interface separates two fluids, membranes separate the inside of vesicles from the outside, the interior of biological cells from the environment, and compartmentalize cells into organelles. the physical properties of interfaces are characterized by interface tension, those of membranes are characterized by bending and stretching elasticity. amphiphilic molecules like surfactants that are added to a system with two immiscible fluids decrease the interface tension and induce a bending rigidity. lipid bilayer membranes of vesicles can be stretched or compressed by osmotic pressure; in biological cells, also the presence of a cytoskeleton can induce membrane tension. if the thickness of the interface or the membrane is small compared with its lateral extension, both can be described using two-dimensional mathematical surfaces embedded in three-dimensional space. we review recent work on the interaction of particles with interfaces and membranes. this can be micrometer-sized particles at interfaces that stabilise emulsions or form colloidosomes, as well as typically nanometer-sized particles at membranes, such as viruses, parasites, and engineered drug delivery systems. in both cases, we first discuss the interaction of single particles with interfaces and membranes, e.g. particles in external fields, non-spherical particles, and particles at curved interfaces, followed by interface-mediated interaction between two particles, many-particle interactions, interface and membrane curvature-induced phenomena, and applications. systems with interfaces are abundant in both technological applications and biology. while a fluid interface separates two fluids, membranes separate the inside of vesicles from the outside, the interior of biological cells from the environment, and compartmentalize cells into organelles. the physical properties of interfaces are characterized by interface tension, those of membranes are characterized by bending and stretching elasticity. amphiphilic molecules like surfactants that are added to a system with two immiscible fluids decrease the interface tension and induce a bending rigidity. lipid bilayer membranes of vesicles can be stretched or compressed by osmotic pressure; in biological cells, also the presence of a cytoskeleton can induce membrane tension. if the thickness of the interface or the membrane is small compared with its lateral extension, both can be described using two-dimensional mathematical surfaces embedded in three-dimensional space. we review recent work on the interaction of particles with interfaces and membranes. this can be micrometer-sized particles at interfaces that stabilise emulsions or form colloidosomes, as well as typically nanometer-sized particles at membranes, such as viruses, parasites, and engineered drug delivery systems. in both cases, we first discuss the interaction of single particles with interfaces and membranes, e.g. particles in external fields, non-spherical particles, and particles at curved interfaces, followed by interface-mediated interaction between two particles, many-particle interactions, interface and membrane curvature-induced phenomena, and applications. keywords: membranes, nanoparticles, capillary interactions, lipid bilayers, emulsions, viruses, interfaces (some figures may appear in colour only in the online journal) original content from this work may be used under the terms of the creative commons attribution 3.0 licence. any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and doi. also compartmentalize cells and thereby define organelles. transmembrane transport is essential for the communication both inside a cell as well as of cells with their environment [6, 7] . the interaction of particles and pathogens with biological membranes-and therefore also their cellular uptake and intracellular transport-crucially depends on the particle size, shape, softness, and surface functionalization. nowadays a whole zoo of micro-and nanoparticles can be fabricated from various materials, with engineered shapes and surface functionalizations. the particles can be used for applications in food science [8] [9] [10] , cosmetics [8, 9, 11] , as antimicrobials [12, 13] , and in nanomedicine [14] [15] [16] ; therefore systematic studies and a careful consideration of potentially toxic effects are required [17] [18] [19] [20] [21] . figure 1 shows examples for oblate, bullet-shaped, pill-shaped, and dumbbell-shaped microparticles that are made from a polymeric material. figure 2 shows cube-like, rod-like, irregularly-shaped, and spindle-like metal and metal-oxide nanoparticles. all these particles can also be considered as model systems for 'particles' found in nature. for example, the malaria parasite is micrometer-sized and has an egg-like shape [22, 23] . milk contains casein micelles with sizes below 100 nm that stabilize fat globules [24] . viruses resemble roundish, filamentlike, and bullet shapes with sizes below 100 nm [25] [26] [27] ; in particular, the filamentous ebola and marburg viruses are of much interest due to their enhanced virulence that leads to high mortality rates [28, 29] . fluid interfaces are rough on the molecular scale and can be analytically well described by a hyperbolic tangent-shaped density profile [31, 33] , see figure 3 . however, the typical interface width is much smaller than the sizes of the particles that we consider. in order to study the interaction of particles with interfaces, the interface can therefore be thought of as mathematical surface with its physical properties characterized by an interface tension. lipid-bilayer membranes often consist of many components, e.g. different lipids and cholesterol, and biological membranes usually also contain membrane proteins. this can lead to phase separation within the membrane, as shown in figure 4 . the typical thickness of a lipid bilayer membrane is ≈ d 4 nm and cannot be neglected for small nanoparticles with sizes of few nanometers. for nanoparticles with radii of 10 nm and above, the membrane can be described as mathematical surface with curvature-elastic properties. the possiblity to model both fluid interfaces and biological interfaces (membranes) using mathematical surfaces is our motivation for discussing particles at interfaces and at membranes in a single review article. for biological interfaces, we will focus on sufficiently large particles for which a continuum model is not only feasible, but also more appropriate than an atomistic model. the deformation energy of the surface can then be calculated using the helfrich hamiltonian [34] , here, the interface conformation is characterized by the two principal curvatures at each point of the interface, c 1 and c 2 , that enter the hamiltonian via the mean curvature = + h c c 2 1 2 ( )/ and the gaussian curvature = k c c 1 2 . the total deformation energy is obtained by integration over the entire interface area s. tension γ, bending rigidity κ, spontaneous curvature c 0 , and gaussian saddle splay modulus κ describe the mechanical and elastic properties of the interface. equation (1) applies to biological interfaces if the bending energy contribution dominates, and to fluid interfaces if the energy is given only by the tension term. for spherical particles at planar fluid interfaces, the particle size, the interface tensions between the particles and the two phases, as well as the interface tension between both phases characterize the system. if the interface tensions between the particles and both phases are identical, the particles attach to the interface because their presence reduces direct contact between both phases without any additional costs for the contact of the particles with the fluids. for high-tension interfaces and micrometer-sized particles, such as for silica particles at oil-water interfaces, the attachment energy gain can be as high as k t 10 7 b [41] . therefore, such particles are irreversibly adsorbed to the interface. if the interface tensions of the particles with both fluids differ, the attachment energies of the particles can be strongly reduced. for particles attached to membranes, the membrane elastic properties in equation (1) and the adhesion energy between particles and membranes characterize the system. here, s ad is the membrane area adhered to the particle, and the adhesion strength w for the contact interaction can be mediated by van der waals forces, by electrostatic interactions, and by specific adhesion (receptor-ligand bonds). for small spherical particles attached to nearly planar membranes and weak adhesion strengths, wrapping may not be energetically favourable, while for spherical particles with a radius of 50 nm and for a high adhesion strength = − w k t 0.2 nm b 2 the energy gain through wrapping can be as high as k t 5000 b . particle shape can strongly alter the attachment energy. table 1 provides an overview of typical attachment energies of spherical particles to interfaces and membranes. for particles at interfaces, we assume that they have equal interface tensions with both fluid phases and that they are therefore half immersed in each phase. for particles at membranes, we assume that they are attached to a membrane with half of their surface area and that there is no deformation energy cost for the membrane surrounding the particles 5 . interface deformations induced by particles lead to interface-mediated interactions and self-assembly; many-particle systems minimize the deformation energies also with respect to the particle positions. for example, capillary forces between micrometer-sized ellipsoidal particles increase with increasing aspect ratio and the energy gain at particle contact can be as high as 10 4k t 10 5 b already for ellipsoid aspect ratios of 2-3 [43, 45] . membrane-mediated binding energies of few k t b have been calculated for spherical particles at membranes [44, 46, 47] 6 . table 2 provides an overview of interface-mediated and membrane-mediated bond energies for particles at contact. structures on the micrometer scale can readily be observed using light microscopy. in order to access the nanometer scale, more sophisticated techniques, such as electron microscopy or super-resolution microscopy, have to be employed [49] [50] [51] [52] [53] [54] . whereas ramsden and pickering have reported on particle-stabilised emulsions already in the early 20th century [1, 3] , images of nanoparticles, such as bacteriophages, have only been reported in 1940s, soon after electron microscopy has become available [55] . since the very early studies of particles at fluid and biological interfaces, observation techniques and abilities to engineer particles have continuously advanced. besides these experimental developments, also mesoscopic and atomistic modeling and computer simulation techniques and speed have rapidly developed. both allows the characterization of particles at interfaces with increasing accuracy; this review article provides an overview of recent achievements. for both fluid and biological interfaces, we will discuss similar aspects: attachment to the interface, mechanisms for deforming the interface, particle orientation at the interface for nonspherical particles, long-range and short-range interactions, many-particle interactions, and applications. many concepts can easily be transferred between systems with fluid and with biological interfaces. we stress this parallelity by the analogous structure of the sections for both systems. section 2 is devoted to particles at fluid interfaces, section 3 to particles at biological membranes. the overview of the experimental studies of selfassembly of microparticles at fluid interfaces may not only benefit scientists investigating such systems, but also those interested in nanoparticles at biological membranes, where systems are less accessible experimentally and fewer studies are therefore available. particles adsorb at fluid interfaces because they reduce the interface area and thereby also the total interface energy. here, we have to account for the interface energies between the three phases, liquid , vapor (or a second liquid) v, and solid s. the system can be characterized by the three interface tensions γ sv , γ v , and γ s , see figure 5 . the force balance at the contact line, where the liquid-vapor interface is located at the particle, is given by the young-dupré equation, which defines the contact angle θ c , see figure 5. the energy difference to bring a spherical particle with radius a from the bulk phase to the interface is the trapping energy [56] [26] . copyright © 2010 american chemical society. where τ is an effective line tension and l c is the length of the contact line. for micrometer-sized particles, the contrib ution of the line tension is negligible; the trapping energy is proportional to the liquid-vapor interface tension γ v and decreases with decreasing contact angle, see figure 6 . for small particles with sizes below one micrometer line tension becomes relevant [57] , for large particles with sizes of several micrometers gravity has to be taken into account [58] [59] [60] . the eötvös or bond number compares the contributions of interface tension and gravity to the total energy, where ρ ∆ is the density difference of the two phases, g is the gravitational acceleration, and l is a characteristic length of the particle. similarly, the energies due to line tension and surface tension are compared using the dimensionless number ( ) . (a) a vertical slice of width σ 3.5 and height σ 40 is shown with the interface region highlighted. (b) corresponding mean density profile z ( ) . reproduced with permission from [31] . © epla. all rights reserved. for micrometer-sized particles and typical density differences between, for example, silica and water, ≈ − bo 10 8 [61] ; for line tensions and the corresponding surface tensions measured for polystyrene and poly(methyl methacrylate) particles at oil-water interfaces, = li 0.01-0.1 [57] . in the following sections, we mainly discuss systems with bo 1 and li 1, where both gravity as well as line tension can be neglected. the estimate for li indicates that line tension may not always be entirely negligible for small micrometer-sized and large nanometer-sized particles. line tension may qualitatively alter the behaviour of non-spherical particles at interfaces: for example, ellipsoidal and cylinderical particles may undergo orientational changes in order to minimise the length of the contact line. in [57] , contact angles have been measured for spherical particles and for prolate ellipsoidal particles with various aspect ratios obtained by deforming initially spherical particles. here, an apparent decrease of the contact angle with increasing aspect ratio of the particles has been measured. this can be attributed to a line tension and a contact angle that is independent of particle shape [57] . the experiment predicts an effective line tension τ ≈ 1 nn that also includes experimentally-observed heterogeneities in the contact line. other values for line tensions reported in the literature are in the pn to µn range [62] . one of the key reasons for the stability of colloidal assemblies at interfaces and of pickering emulsions are the very high trapping energies for the particles [56, 63] , see table 1. calculations for non-spherical particles show that the trapping energies depend only weakly on particle shape and increase for both oblate and prolate deformations of spherical particles, see figure 6 . at curved interfaces, the trapping energies of spherical particles have been shown to depend on the interface curvature and therefore on the laplace pressure difference at the interface [64] . for spherical particles and a contact angle θ = 90 c , the trapping energy decreases with increasing interface curvature. for contact angles θ ≠ 90 c , the trapping energy either increases or decreases depending on the sign of the contact angle and on whether the interface is curved towards or away from the particles. the white arrow points to a cholesterol oriented in between the monolayer leaflets. reprinted with permission from [32] . copyright © 2008 national academy of sciences. [36, 37] . based on [43] and [44] . to satisfy the young-dupré equation locally at every point on the three-phase contact line, see figure 5 , the contact lines and therefore also the interfaces are often not planar for non-spherical particles. also particle-surface inhomogeneities, either due to roughness or chemical surface patterning, e.g. for janus particles, may induce contact-line undulations. furthermore, imposed fields, such as gravity for large mm-sized particles or electromagnetic fields for electrically-charged or for magnetic particles, may lead to interface deformations. the capillary forces due to the system's tendency to reduce the total interface area between liquid and vapor is the origin of the capillary forces between particles, see table 2; very strong bond energies of k t 10 4 b for micrometer-sized particles correspond to interface height perturbations of only 50nm [43] . the importance of hydrodynamic interactions for the dynamics of particles at interfaces is characterized by the capillary number with fluid viscosity η and typical particle velocity v. for typical velocities of µ − 100 m s 1 for particles with sizes of up to few micrometers in water [65] , we find < − ca 10 5 . therefore, for the systems discussed in this review article ca 1, such that hydrodynamic interactions can be neglected. particle velocities can therefore be directly related to the forces acting on the particles and the friction by the fluid. self-assembly of colloidal particles at fluid interfaces is governed by both direct interactions, such as van der waals and electrostatic interactions, and indirect interactions, such as forces due to overlap of interface distortions, popularly coined as capillary interactions [62, 66] . in 1980, pieranski reported a two-dimensional colloidal crystal of spherical polystyrene colloids with a triangular lattice structure at an air-water interface induced by an asymmetric charge distribution [63] , forced into two dimensions by the interface. a large number of more recent studies on colloids at interfaces that assemble due to interface-mediated and direct interactions show a rich variety of two-dimensional structures. an overview of both single-particle and many-particle systems at interfaces is provided in the remainder of this section. in sections 2.1-2.4, we focus on various aspects of the interaction of single particles with interfaces, while in sections 2.5 and 2.6, we discuss two-particle and many-particle interactions, respectively. section 2.7 focuses on the special case of particles at interfaces that are half immersed into an ordered fluid. we finally discuss applications in section 2.8. an undulating contact line at a particle distorts a surrounding planar fluid interface. for a cylindrically-symmetric system, such as a spherical particle at an interface, the young-laplace equation that describes the interface deformation is best expressed in cylindrical coordinates [67] , the height profile φ h r, ( ) of the interface can then be expressed as product of a function of the radial coordinate r and a function of the angular coordinate φ around the sphere, the general solutions, are characterized by the contact line at the particle, described by φ m,0 , φ m,0 , and r m,0 . the interface deformation decays faster with increasing distance from the particle the larger m, i.e. the larger the number of undulations along a circle around the particle. the contact-line undulations can be expressed as multipole expansion, where the contact radius r c is often similar to the particle radius, h m are the expansion coefficients, and φ m,0 the phase spherical particles at a fluid-fluid or fluid-gas interface in a system with a solid spherical particle (s), a fluid (l), and a gas/ second fluid (g). the contact angle θ c for the green particle is 90 , while the contact angle for the red particle is smaller than 90 . the three interface tensions γ sv , γ s , γ v are sketched for each particle using arrows. they indicate the force balance given by the young-dupré equation that holds for all points along the three-phase contact lines. angles. the first two terms of the expansion vanish, because without additional external forces the particles are at their optimal height and orientation at the interface. the lowest multipole contribution and the most important contribution for long-ranged interactions is the quadrupolar deformation with = m 2 [67] , where h 2 characterizes the undulation amplitude of the contact line. the long-ranged interactions between two particles in the far field therefore decay with − d 4 , where d is the interparticle distance. experimentally, imprinted contact-line shapes have to be distinguished from mere surface roughness. whereas surface roughness often leads to temporary and random pinning of the contact line, engineered contact-line deformations are stable and well controlled. for instance, undulating plates allow the engineering of the interface deformation and the tuning of the capillary interaction between particles from attractive to repulsive [68, 69] . however, also nanometric roughness on microspheres or disc-like particles leads to strong capillary interactions due to contact-line pinning [67, [70] [71] [72] . the temporary nature of contact-line pinning can be observed for instance for metastable orientations of dumbbell-shaped particles at interfaces; the distribution of particle orientations relaxes towards the globally stable particle orientation when the system ages [73] . in 1992, lucassen first suggested that particles with complex shapes can induce interface distortions in absence of gravity [69] . he systematically calculated the interaction between sinusoidal interface deformations. however, the most common interface distortion due to non-spherical particles is the quadrupolar deformation, which is found for all elongated particles with homogeneous surface functionalization at planar interfaces in the far field [65, 74, 75] . for ellipsoidal particles, quadrupolar interface deformations can also be found in the near field [43, 75, 76] . for contact angles θ < 90 c , the interface is pulled down at the tips and pulled up near the long sides of the particle-and reverse for the inverse-wetting condition with θ > 90 c , see figure 7 (a). for ellipsoidal particles and θ = 90 c -and for spherical particles-a planar interface remains undeformed. strength and nature of the interface distortions can be characterized using different quantifications: contour maps of the interface distortion, see figures 7(a) and (b), shapes of contact lines, see figure 7(c), differential heights ∆u of the interface between the crest and trough along the contact lines, see figure 7(d), or height profiles along cross-sections, see figure 9 (e). for well-defined particle shapes, in particular for ellipsoidal particles and quadrupolar interface deformations, the characterization of the interface deformation fields is uniquely determined by ∆ = − u z z max m in , where z max is for both particles. the spherical particle approaches the ellipsoidal particle at the side. reprinted with permission from [43] . copyright (2014) american chemical society. (d) maximal height difference ∆u of the contact line for ellipsoidal particles with several aspect ratios b a 1 1 0 ⩽ / ⩽ . the numerical data are normalized by the half the length of the minor axis a and plotted as function of the contact angle θ c . the maximum value of ∆u, depicted by ×, shifts to smaller contact angles with increasing particle aspect ratio, as indicated by the grey lines that serve as guides to the eye. reprinted with permission from [43] . copyright (2014) american chemical society. the maximum and z min the minimum height along the contact line. the height difference ∆u for ellipsoidal particles vanishes for θ = 0 c and for θ = 90 c and assumes a maximal value ∆u max for θ < < 43 5 4 c for ellipsoids with aspect ratios b a 1. 1 1 0 ⩽ / ⩽ [43] , see figure 7 (d). the position of the peak shifts from larger to smaller contact angles with increasing aspect ratios of the particles [77] , while the height of the peak increases from ∆ ≈ u a 0.013 . studies are available for ellipsoidal particles [43, 74, 75] , for cylindrical [65] , for cuboidal particles [43] , and for rounded box-like particles [70] . the height difference along the contact line of elongated particles can be thought of as a measure for their capillary interaction strength. systematic theoretical and experimental calculations thereby provide routes to tailor capillary assembly of multiple particles. the experimental determination of contact angles using light microcopy is difficult, because the same interface deformation can be derived for two different contact angles. this 'contact-angle mystery' arises because the differential interface height distortion, i.e. ∆u a / , does not show a monotonic increase with the contact angle θ c , see figure 7 (d). due to the non-monotonic profile for ellipsoidal particles, for each value of ∆u a / there are two possible values for θ c , see figures 8(a) and (b). however, the 'mystery' can be resolved by measuring a second quantity, such as the excess area s s 0 / plotted in figure 8 (c), i.e. the ratio of the projected area by the contact line and the projected area at θ = 90 c [74, 77] . in figure 8 , results from theoretical contact-angle calculations for ellipsoidal particles with various aspect ratios are plotted together with corresponding experimental measurements of ∆u a / and with s s 0 / as function of the contact angle. the lower branch for smaller θ c has already been reported in [74] , the upper branch more recently in [77] . only with the knowledge of both, experimental values for ∆u a / and for s s 0 / , the correct branch of contact angle solutions can be singled out. a new technique using electron microscopy provides means to measure contact angles more directly than optical microscopy. this so-called freeze-fracture shadow-casting cryo-sem (fresca) has been used to measure contact angles for wetting of spherical and ellipsoidal micro-and nanoparticles at liquid-liquid interfaces [57] . for more complex particle shapes, the connection between interface deformation and particle aspect ratio can be different from the case of 'simple' elongated particles. for example, figures 9(a)-(e) shows deformations around rounded box-like particles, where rises and dips depend on local particle shape rather than aspect ratio. cuboidal particles can produce octupolar distortion fields with eight lobes (rises and dips), see figure 9 (f). for rounded cylinders, there can even be multiple branches of possible contact angles due to the multiple peaks in the ∆u a / variation for certain aspect ratios (unpublished results). table 3 provides an overview of the dominant multipole contributions for interface deformations and corresponding systems, several of them are discussed in more detail in the following. we discuss here gravity, buyoancy, and thin films in part 1 and non-planar interfaces in part 2. 2.3.1. gravity, buyoancy, and thin films. the presence of graviational forces leads to floatation forces that cannot be neglected in the regime of large absolute values of the bond number [59, [83] [84] [85] [86] [87] [88] 89] . particles either sink into the interface / , for ellipsoidal particles with aspect ratios in the range b a 1. 25 7 ⩽ / ⩽ . the data from [43] is plotted together with numerical data taken from [77] . for a spherical particle, s s 0 / varies as θ sin c 2 , as shown by the solid line above. for all contact angles between 0 and 90 , s s 0 / attains higher values for ellipsoidal particles in comparison to the analytical estimate obtained for a spherical particle. adapted with permission from [43] . copyright (2014) american chemical society. if they are heavier than the fluid or float up if they are lighter than the fluid (buyoancy). gravitational forces therefore induce dominant monopole interface deformations, so-called 'capillary charges', see equation (23) [90] . some fascinating outcomes of the interplay of capillarity and gravity are water striders [99] , meniscus-climbing insects [83, 84] , and the cheerios effect in cereal bowls [59] . monopole interface deformations are obtained as well for particles in thin films, where instead of gravitation the small thickness of the film compared with the particle size induces interface deformations [79, 100] , see figure 10 . the ideal interface positions on the particle are incompatible with the film thickness, which leads to immersion forces. for a thin film of fluid at a solid interface, the horizontal projection of the immersion force is [79] πγ with the radius r c of the three-phase contact lines at the particles, the mean meniscus slope angle ψ c at the contact line, and the distance d cc between the particles. this expression holds ( /( )) / , where ρ ∆ is the density difference between the fluid and the gas. figure 11 compares the floatation forces between particles with immersion forces for polystyrene latex particles in a water film on a glass substrate; in this example, the floatation forces decrease faster with increasing interparticle distance than the immersion forces. as an instructive example, we discuss froth floatation as an application in more detail [101] . here, grains of one solid are carried away with the froth, while grains of a different solid sink to the bottom of the floatation system. the selective adsorption of a collector chemical onto a mineral in a flotation plant determines-among others-the attachment of the mineral to bubbles used for separation. the stability of the froth with the particles depends mainly on the properties and the amounts of particles [80] . the so-called capillary pressure due to the liquid that drains from the film because of gravity measures the pressure when the film ruptures. this critical pressure increases if particles stabilise the film, e.g. spherical particles with contact angles far below 90 , and decreases if particles destabilise the film, e.g. particles with sharp edges. / . the heights are scaled by the length scale r. reprinted with permission from [78] copyright of the royal society of chemistry (f) deformation profiles of an interface around a particle with hauser's cube shape at contact angle θ = 56.28 whether a particle stabilizes or destabilizes a film furthermore depends on the orientation that the particle assumes at the interface [102] , see section 2.4. figure 12 shows how a thin film ruptures due to the presence of a non-spherical sharpedged particle. particles at curved interfaces experience forces not only due to the presence of the interface, but also because of the laplace pressure. curvature gradients of the interface lead to lateral forces, and curved interfaces modify interface-mediated interactions between particles [61, 64, 71, 72, 103, 104] , see figure 13 ; vice versa, particles at high densities can induce spontaneous interface curvature [105] . mean curvature h and deviatoric curvature δ = − c c c 1 2 determine the trapping energy [104] where e f is the trapping energy at the flat interface, see equation (4). for a spherical colloid of radius a on a spherical drop, which is the interface curvature-induced contribution. this term is evaluated by considering contributions due to both the interface energies and the work done against the laplace pressure. a similar estimate for the trapping energies attempted previously in [106] without including the work done by the laplace pressure resulted in the correction term to be . this result is consistent with equation (15) when the contribution due to the pressure term, , is added to it [104] . for a minimal surface with mean curvature = h 0, such as for a catenoidal interface shape, the correction term compared with the flat interface depends only on the deviatoric curvatue δc. the trapping energy of a colloidal particle on a catenoidal minimal surface is thus . on an interface with varying curvature, the lateral force on the trapped particle is given by [ thus, spherical particles on interfaces with curvature gradients experience forces depending on both the absolute values and the gradients of the mean and deviatoric curvature to move towards regions of high mean and deviatoric curvatures (and therefore also high gaussian curvatures because . recent experiments of microspheres on interfaces with different curvatures have evaluated these forces to be of the order = − ± [35] . theoretical estimates of capillarity at curved interfaceswhich neglect pinning-suggest that capillary forces for particles that are smaller than the capillary length where ρ is the density of the fluid, are proportional to both a 4 and the gradient of the gaussian curvature, ∇k [35] . this has been verified in experiments where large microposts induce a curvature gradient and cause smaller particles to migrate along the deformed interface to assemble in regions of high gaussian curvature, see figure 14 . experimental evidence further suggests that capillary migration on curved interfaces can be enhanced by contact-line pinning. contact-line undulations, which lead to finite local interface curvatures, induce capillary forces of the order ⋅ 6 10 3 -⋅ 5 10 4 k t b , which are much larger compared with the weak forces due to capillarity for perfectly smooth spherical particles. the experiments demonstrate that rough microspheres, microdisks, and rod-like particles move along deterministic trajectories to regions of maximum deviatoric curvature [71, 72, 103] . non-spherical particles do not only migrate figure 11 . comparison between immersion and floatation capillary forces between two spherical latex particles in a water film on a glass substrate: dependence of the capillary interaction energy, ∆w, on the particle radius, r. the distance between the particles is = l s 2 , the contact angle θ α = c , and the liquid-vapor interface tension γ γ = v . see figure 10 for a sketch of the system. reprinted with permission from [79] . copyright (1992) american chemical society. translationally by sensing the background interface curvature, but also orient themselves to align their long axes, such that the excess area is minimized. for example, cylindrical particles reorient either parallel or perpendicular to the groove as the interface curvature changes from concave to convex [107] . particle orientations for non-spherical particles, the orientation of the particles at interfaces, as well as particle shape, size, and surface properties have to be taken into account to determine adsorption energies and interface deformations. cube-like particles can be oriented in a corner-top or face-top orientation depending on the contact angle. elongated particles in a magnetic field or elongated janus particles in their stable orientations can be oriented with their long axes tilted with respect to the interface. one way to estimate the adsorption energy of a particle at an interface is to calculate the area of the fluid interface that is 'cut out'. this estimate can be exact if the interface tensions between particle and both phases are equal. for a particle that is symmetric with the interface acting as a mirror plane, the surrounding interface remains planar. however, in general particle-induced deformations of the surrounding interface can change adsorption energies and stable particle orientations even qualitatively. figure 15 (a) sketches the various angles by which a cube-like particle can be rotated. figure 15 for θ = 90 c the flatinterface approximation predicts the edge-top orientation to be globally stable, while accounting for the deformation of the surrounding interface the corner-top orientation is found to be globally stable. figure 15 (c) shows specific orientations for a cube-like particle at an interface. for a contact angle θ = 72.5 c , the globally stable state is the face-top orientation, while for θ = 90 c the globally stable state is the corner-top orientation. figure 15 (d) shows the predicted interface deformations for both cases using the same scale: hexapolar for the corner-top orientation and almost planar for the face-top orientation. capillary interactions between the cubes are therefore expected to be much stronger in the corner-top orientation compared with the facetop orientation. for particles with complex shapes, such as cube-like particles, a multitude of kinetically stabilised orientations and corresponding multipolar interface deformations is expected to be observed in experiments. a simple non-spherical particle with a stable tilted state is a janus dumbbell, which consists of two spherical particles made from different materials, see figures 16 and 17. these particles do not deform planar interfaces, because the contact line around each of the spherical particles is a circle. figure 16 (a) shows the experimentally observed orientations for dumbbell particles that consist of an hydrophobic spherical particle of radius r a and a hydrophilic spherical particle of radius r p . in their lowest-energy orientation, the spherical particles intersect the interface at different heights. the tilt angle of the dumbbell in the lowest energy state is [73] see figure 16 (b). here θ p is the contact angle at the polar particle, θ a is the contact angle at the apolar particle, α = r r a p / is the relative size of the two lobes, and with decreasing aspect ratio, the tilt angle of the janus dumbbell increases. for aspect ratio = b a 2 / , only one of the spherical particles is in contact with the interface. however, also for cases where the tilted orientation of the dumbbell is predicted to have the lowest energy, a substantial fraction of particles are found experimentally in one of the kinetically stable orientations, where only one of the spherical particles intersects the interface [73] . the lowest-energy orientation, where both spheres are at the interface, coexists with two kinetically stable orientations where only one of the spheres is at the interface. because the energy for an orientation where only one spherical particle is in contact with an interface does not depend on the tilt angle, an arrest in such a state could be caused by surface roughness. in these trapped states, the particles can therefore reorient to the tilted orientation only by diffusion. unlike janus dumbbell particles, janus ellipsoidal particles that are half hydrophobic and half hydrophilic do not show a continuous transition of their the orientation between a tilted long axis and a perpendicular long axis to the interface, see figures 17 and 18. energy landscapes for various orientations of an ellipsoidal and a dumbbell particle are shown in figure 17. the lowest energy states for different aspect ratios are indicated in the figure using green symbols; the discontinuous transition in the particle orientation is clearly visible. the tilted orientation is only stable above a threshold aspect ratio and the orientation of the particle orientation jumps to the perpendicular orientation below this aspect ratio [108] . these energies for various particle orientations have been calculated numerically, under the assumption of a flat interface surrounding the particles. whereas for ellipsoidal particles with homogeneous surface properties the deformations of the surrounding interface are quadrupolar with a dip at the tips and a rise at the long sides for contact angles below 90 , see section 2.2, the deformations of the interface around janus ellipsoids can show both a dip and a rise along their long sides [93] , see figure 18 . similar interface deformations have been observed for double-hydrophilic janus cylinders with aspect ratios 0.9, 1.2, and reprinted with permission from [97] . copyright (2016) by the american physical society. 2.4 [94] , see figure 19 . the cylinders are found in kinetically trapped end-on orientations as well as globally stable tilted orientations. the cylinders have asymmetric hydrophilicity unlike most studies where the janus particles have amphiphilic properties [94] . in their tilted orientations, they induce hexapolar interface deformations that lead to capillary interaction. complex particle assemblies have been observed due to the multi-lobed deformations and the non-determininistic assembly. fields. an external magnetic field can be used to tune the orientation of prolate ellipsoidal magnetic particles at fluid interfaces [41, 92, 109] , see figure 20. the field tends to align the magnetic dipole with the direction of the applied magnetic field, where µ and h are the dipole and the field, respectively. the angle φ indicates the particle orientation and µ = | || | b h represents the field-dipole strength. the total free energy of the particle at the interface, neglecting particle-induced interface deformations, can therefore be written as where s is the area of the interface 'cut out' by the particle, and a sv is the area of the particle in contact with the vapor/the second liquid. if the field is oriented normal to the interface and if the direction of the permanent magnetic dipole coincides with the major axis of the ellipsoid, the analytical estimation of the tilt energy predicts that at a critical field strength the particle 'jumps' from a tilted orientation to a vertical orientation. here, φ = 0 and φ = 90 correspond to vertical and horizontal particle orientations, respectively. in figure 21 , the energies of magnetic prolate ellipsoidal particles are shown as function of their orientation angle for various aspect ratios and field strengths. for particles with aspect ratio = b a 1.67 / and length of the minor axis a = 30 nm , the energy barrier between vertical and tilted states can be several hundred k t b , such that the particles are trapped in one of both orientations. lattice-boltzmann simulations have been used to investigate the orientations of magnetic ellipsoidal particles in external fields including the deformations of the surrounding interface [91] . the simulations show that the interface deformations around the particles significantly affect the tilt angles for a given dipole strengths, altering the properties of the reorientation transition. figure 22 shows a simulation snapshot of the deformed interface and a plot of the tilt angle for various field strengths together with the analytical approximation discussed above. the simulation snapshot shows that the interface deformations remove more interface than in the planar-interface approximation, which lowers the free energy. deviations from the approximate result are found mainly for high aspect ratios. the discontinuous transitions allows the switching of the particle orientations using external magnetic fields, which could find applications for instance using its dynamically-tunable optical properties for electronic readers [110] . furthermore, also the dipolar interface deformations can be switched on and off and thus lead to switchable capillary interactions between particles adsorbed at fluid interfaces. overlaps of interface deformations around particles lead to interface-mediated interactions, also called (lateral) capillary forces. capillary interaction energies between two particles, see figure 23 , can be calculated based on the difference between the changes of the fluid-vapor interface area for placing two particles at given center-of-mass separation d cc and for placing two isolated particles at a planar interface. the corresponding capillary interaction energy is [67] where ∆s ab is the change of the fluid-vapor interface area around the interacting particles, and ∆s a and ∆s b are the changes of the interface areas around the single particles. interface-mediated interactions follow power laws in the far field and are thus long-ranged. in the near field, the interactions strongly depend on the particle shapes. because the dynamics occurs in the stokes regime for small capillary numbers, figure 16 . amphiphilic dumbbell particles at an oil-water interface. (a) in experiments, three dumbbell orientations are found: only the hydrophilic sperical particle intersects the interface, only the hydrophobic spherical particle intersects the interface, and both spherical particles intersect the interface. the circles and arrows indicate the size of the particle crossection at the interface and the shadow length obtained by freeze-fracture, shadow-casting (fresca) cryo-scanning electron microscopy, that can be used to measure the contact angle. (b) schematic diagram highlighting the geometry of the dumbbells at a liquid-liquid interface. reprinted with permission from [73] . copyright (2014) american chemical society. see equation (7), the particle velocities are proportional to the interaction forces and can therefore be extracted for example from videos. analytical solutions for the interaction forces are available in the far field, while numerical calculations have to be used in the near field. for large distances between particles, the deformation field of particle a can be assumed to be small at the position of particle b and vice versa. the capillary interaction energy can then be calculated using the superposition of the interface deformations around the single particles [58, 67, 111 ] where h a and h b are the interface height deformation fields due to particle a and b, respectively. the lowest multipole contribution for a given system, see table 3, dominates the interaction. for sufficiently large particles for that gravity or buyoancy have to be considered, the capillary forces act between two monopole interface deformations [58] πγ with the capillary length defined in equation (17), the mass density ρ ∆ between the upper and the lower phase, and the capillary charges the angle ψ i is the slope of the interface at radius r i , the radius of the contact line. for )/( ), particle mass density ρ i , fluid mass density ρ , and upper fluid/gas mass density ρ g . stamou et al first presented an analytical result for the pair potential between two quadrupolar deformation fields in polar coordinates [67] . in this quadrupolar approximation, the energy between two ellipsoidal particles is figure 17 . janus ellipsoid and janus dumbbell at an interface with corresponding energy landscapes for particle orientations. while the dumbbell smoothly transitions to the perpendicular orientation with increasing aspect ratio, the ellipsoid jumps from a small tilt angle to the perpendicular orientation beyond a critical aspect ratio. reprinted with permission from [108] . copyright (2012) american chemical society. interestingly, the magnitudes of the interaction potentials are equal in s-s, t-t, and t-s orientation for equal center-of-mass distances. kralchevsky et al extended the multipole approach to multipoles of arbitrary orders [105] . whereas for weak deviations from circular contact lines a polar multipole expansion is most appropriate, for ellipsoidal particles with higher aspect ratios the appropriate choice are elliptical multipoles [75] . ( )/ ( / ) , are observed for both cylindrical and ellipsoidal particles [65, 75] . deviations are observed for small interparticle distances, which is discussed in section 2.5.2. elongated particles that touch each other have lower bond energies than predicted by the quadrupolar approximation in side-by-side orientation and higher bond energies in end-to-end orientation, see figure 24 . capillary self-assembly is determined by the contact interactions between the particles, therefore near-field interactions are important for all many-particle systems. uncharged ellipsoidal particles at low bond numbers preferentially align in side-byside orientation [45] . the higher stability of the side-by-side orientation over the tip-to-tip orientation is obvious from equation (24) and is qualitatively unchanged by the deviation from the ideal quadrupolar approximation in the near field. using triangulated surfaces and energy minimisation, numerical calculations of deformation energies have been applied for various particle shapes, orientations, and contact angles. figure 25 shows snapshots and interaction potentials for pairs of identical ellipsoidal particles with aspect ratios = b a 2 / and = b a 3 / [43] . the bond energies increase quadratically as function of the bond angle φ | | that quantifies deviations from the side-by-side orientation with φ = 0 . figure 26 shows the bond-bending energies for two ellipsoids with shows a linear elastic regime with φ = t g , where γ = g a 0.0015 2 [76] ; the torques may reach values up to 10 3k t 10 4 b . a bending modulus for the polymer-like assemblies of particles at the interface can be extracted from many-particle calculations. if two ellipsoids with different aspect ratios interact, the stable assembly is an arrow [112] , see figure 27 . unlike ellipsoidal particles, cylindrical particles preferentially assemble in end-to-end orientation, see figure 28 . because of the flat ends of cylindrical particles, the contact figure 19 . configurations of double-hydrophilic janus cylinders at the air-water interface. the first and second columns are sem images of janus cylinders embedded in pdms slabs prepared by the gel trapping method. arrows indicate the location of the wettability separation line. the schematic representations show the side view of particle configurations at the air-water interface where four colors represent four different particle-fluid surfaces: weakly polar surface in air (black), weakly polar in water (red), strongly polar in air (blue, rarely shown), and strongly polar in water (cyan). the third column shows the corresponding optical microscopy images. the scales bars are µm 50 . reprinted with permission from [94] . copyright (2013) american chemical society. figure 20 . sketch of an ellipsoidal particle adsorbed at a liquidliquid interface in (a) vertical, (b) horizontal, and (c) tilted orientation. the variables defining the geometry and orientation of the particle are discussed in the text. the direction of the magnetic field is indicated by b. reprinted from [41] . © iop publishing ltd. all rights reserved. line can adjust its height rather freely if the height of the surrounding interface changes because the contact angle remains unchanged. when two cylindrical particles approach each other, the energy gain for wetting the surfaces overcomes the costs for the deformation of the surrounding interface, thus the interface forms a capillary bridge between the two ends. the bond-bending energies for cylindrical particles around their preferred tip-to-tip state are very high, with restoring torques up to = t k t 10 6 b [76] . therefore, such chains are usually not distorted. two cylindrical particles may switch to the metastable side-by-side orientation only for bond angles larger than φ ≈ 120 c -140 , which can be achieved for particulate monolayers under compression or shear stress. the height of the capillary bridge that forms between two cylindrical particles decreases when the distance between them is increased, compare snapshots for = d a 0.9 / with = d a 0.3 / in figure 28 ; it changes also if the particles are tilted with respect to each other, compare snapshots for = d a 0.3 / and φ = 0 , and for = d a 0.3 / and φ = 20 . for cylindrical particles, bond bending is not elastic as for ellipsoidal particles, but shows non-elastic hinging behaviour. therefore, both bond energies and bond-bending energies are significantly affected by deformations of the surrounding interface and by changes of the wetting of the planar faces of the particles. this importance of wetting energy is a qualitative difference for the capillary interactions between ellipsoidal and between cylindrical particles. the crossover between elastic bond-bending and nonelastic hinging can be further explored using superegg-shaped particles with variable edge curvatures, as discussed in [43] . . reprinted from [41] . © iop publishing ltd. all rights reserved. / . (b) comparison of the tilt angle of the particle obtained from the analytical, planarinterface approximation in [41] and the numerical data in [91] . although the numerical data qualitatively confirms the prediction of a discontinuos transition, there are quantitative deviations in particular at higher aspect ratio. reprinted from [89] with permission from the royal society of chemistry. and ω 2 indicate the orientation of the particle 1 or 2, respectively, with respect to the vector joining the centers of the two particles. the center-to-center distance is given by d cc for a particle of aspect ratio b a / . reprinted with permission from [43] . copyright (2014) american chemical society. in general, the presence of one particle changes the contactline position on the other particle. figure 7 (c) shows the deformation of the initially circular contact line around a spherical particle by a nearby ellipsoidal particle. figure 29 shows that although the spherical particle at a planar interface by itself does not deform the interface and does not induce interfacemediated attraction, an ellipsoidal and a spherical particle mutually attract each other. the attractive interaction between spherical and ellipsoidal particles is larger for side-on configurations of the spherical particles than for tip-on configurations. in both configurations, however, the attraction between spherical and ellipsoidal particles is significantly weaker than the attraction between two ellipsoidal particles with similar sizes that have equal aspect ratios and surface properties. attractive energies for capillary bonds and energy barriers between locally stable configurations are often high, therefore computer simulations show self-assembly in kinetically-trapped dendritic and raft-like structures [114] . numerical calculations for two-particle, three-particle, and four-particle interactions predict a variety of stable and metastable configurations [77, 115] . many-particle studies of particles at interfaces can also be extended to polydisperse particle mixtures [116] . , and ellipsoidal particles with aspect ratio = b a 4 / that have been obtained by deformation of spherical particles with µ = a m 5 [77] . for prolate ellipsoidal particles mostly the side-by-side orientation is observed in experiments, hexagonal networks can be stable if an additional repulsive electrostatic repulsion contributes to the particle-particle interaction. figure 31(a) shows self-assembly of slightly charged micrometer-sized ellipsoidal particles. the structure initially shows many tip-to-tip contacts; after slow relaxation it compactifies slightly and shows less triangular and more chain-like aggregates in side-by-side orientation after 12 h [113] , see figure 31 (b). the chains of particles in side-by-side orientation can be thought of as 'colloidal polymers': worm-like chains or rings with bending elasticity. for non-spherical particles at planar interfaces, particleparticle interactions may affect the orientations of particles and vice versa. for example, cube-like particles at lower interfacial densities assemble on hexagonal and honeycomb lattices in corner-top orientation [97] , and at higher interfacial densities on a square lattice in face-top orientation [117] . figure 32 shows how particle orientations affect interparticle interactions: chains form for prolate ellipsoidal particles that are oriented with their long axes parallel to the interface, while particles disperse homogeneously if they are oriented with their long axes perpendicular to the interface. particle orientation can be controlled using an external magnetic field [110] , see section 2.4.3. a similar tunable (dipolar) interface deformation and therefore tunable capillary interaction can be achieved using magnetic spherical janus particles [118] . interface curvature alters particle-induced interface deformations and therefore many-particle self-assembly, see section 2.3.2. in order to systematically understand the interaction of many particles at non-planar interfaces, it is important to study model systems with controlled curvatures [61, [118] [119] [120] [121] . for example, spherical particles at interfaces with saddle-like shapes induce quadrupolar interface deformations and therefore at low densities assemble on a square lattice [61] , see figure 33(a); at high densities, the optimal packing on a hexagonal lattice is observed. the local lattice structure can be investigated using bond-orientational order parameters, where n j indicates the number of neighbors of a particle. the angle θ j is the angle between the bond with a neighboring particle j and an arbitrary reference axis. figure 33 (b) shows that the lattice order switches continuously from square to hexagonal with increasing particle density. particles embedded within a nematic liquid crystal (lc) interact with the nematic directors in the vicinity of the colloid and therefore impose boundary conditions on the nematic order parameter at the colloid-lc boundary. this leads to longranged anisotropic elastic interactions for spherical and ellipsoidal particles dispersed in nematogenic fluids [122, 123] . due to varying nature of nematic defects at the colloid-lc boundary, typically elastic multipoles, such as dipoles and quadrupoles, are observed. large pair interaction strengths render the self-assembled aggregates insensitive to thermal or hydrodynamic fluctuations [124] . capillary interactions dominate the elastic energies [125] . for microspheres in nematic films, strength and nature of the elastocapillary interactions depend on film thickness and particle size [126] . for small thicknesses of films, giant elastic dipoles occur due to interface distortions. defects appear around particles, elastic dipoles and nematic elasticity counterbalance the strongly attractive capillary interactions and stabilize particles pairs at finite distance. figure 34 shows how cylindrical nanoparticles first assemble at a nematic interface in their energetically favourable tip-to-tip orientation and then align with the nematic director [125] . the weak elastic interactions are able to manifest themselves at flat nematic interfaces, but under curvature gradients again capillarity dominates [125] . particle anisotropy shall also play a significant role for defect formation at colloid-lc boundaries and shall thus also control nature and strength of pair-particle interactions. future experiments and theoretical investigations to elucidate these elusive interplay between capillarity and elasticity are warranted. applications for (engineered) particles at interfaces range from emulsion stabilisation and colloidosomes to suppression of the coffee-ring effect and froth floatation. in addition to capillary forces, also for example marangoni flows, surfactants, electric charges, or gravity have to be taken into account. a classical application is the stabilisation of emulsions. here, the interactions of particles at interfaces on the mesoscale can be expected to determine macroscopic rheologial properties. in general, the strong adsorption of particles to interfaces leads to thermodynamically stable droplets of oil (water) suspended in water (oil) [1, 3, [127] [128] [129] . at low particle concentrations, the mechanism of limited coalescence generates narrow and reproducible droplet-size distributions [117] . phase inversion of droplet emulsions is obtained by increasing the volume fraction of oil (water). for mixtures for small hydrophilic and hydrophobic particles also sponge-like, bicontinuous phases and non-spherical colloidosomes have been observed [4, 130] . particles with anisotropic surface functionalization, such as janus particles, can be used as amphiphilic colloidal surfactants [131, 132] . particle-stabilised emulsions are used for example in food and cosmetic industries [8] [9] [10] [11] . colloidosomes are solid capsules engineered with controlled permeability and mechanical strength that can have sizes from sub-micrometers to millimeters [133] . the capsules are prepared by self-assembly of colloidal particles to emulsion droplets that are then locked together for example using sintering or electrostatic binding of an oppositely-charged polyelectrolyte. this ensures that the shells remain intact when they are transferred to a different fluid. with the help of centrifugation, the particles are typically transferred into a solvent that is identical to the internal phase. colloidosomes can be used for encapsulation of drugs, proteins, vitamins, flavors, gas bubbles, and even living cells. particles that are homogeneously dispersed over an entire drop often form ring-like deposits when the drop evaporates, so-called 'coffee rings' [134] . addition of ellipsoidal particles with aspect ratio 3.5, obtained by deforming spherical particles with µ = a 0.65 m , to droplets that contain the spherical particles has been found to lead to more homogeneous deposition [135] . for droplets that contain ellipsoidal particles only, a strong suppression of the coffee ring effect is already observed for very moderate aspect ratios ≈ b a 1.5 / . numerical calculations predict capillary attraction between spherical and ellipsoidal particles [43] ; the uniform deposition may therefore be caused by capillarity-induced cluster formation. applications for that a more uniform deposition of the material instead of 'coffee rings' may be desired are inkjet printing, fabrication of microand nanostructures, and coating. froth floatation is used to separate minerals from gangue. the separation efficiency depends on both the particle-bubble attachment and the froth stability [80, 136] . while the particle-bubble attachment can be adjusted using collector chemicals that modify the particle's surface properties, the film stability is affected by particle shape. one complication that decreases froth stability can be particles with sharp edges [79] . therefore, a systematic understanding of the interaction of non-spherical particles with thin films and potentially also of immersion forces may help to improve froth floatation [80, 98, 102, 136] . particles adsorb at biological interfaces, often called membranes, because of an adhesion strength w between the particles and the membranes. in addition to the adhesion strength, the minimal ingredients required to study spherical particles are the bending ridigity κ and the tension γ of the membranes, and the radius a of the particles. for small particles, molecular interactions are important. coarse-grained or even atomistic computer simulations are used to investigate theoretically translocation through and incorporation within membranes [142] [143] [144] [145] . experimentally, the interaction of small particles with membranes can be investigated using a combination of microfluidic devices, fluorescence microscopy, and electrophysiological measurements [146] , by scattering techniques [147] , and by quartz crystal microbalance and afm [148] . for larger particles with radii > a 20 nm , wrapping is the dominant mechanism of the interaction between particles and membranes, as will be discussed in detail in this section. particles that interact with membranes can be engineered and are found in biological systems with a wide variety of sizes, shapes, and surface functionalizations, see figure 35 . comparing the different energetic contributions, large spherical nanoparticle adhesion and wrapping is determined by two characteristic crossover radii, the radius κγ * a that compares the bending energy with the membrane tension characterizes the crossover between the bending-dominated regime for particles with < κγ * a a and the tension-dominated regime for larger particles. while for nanoparticles bending energy is thus the main player, for micrometer-sized particles tension plays the dominant role. within the bending-dominated regime, small particles with < κ * a a w remain unwrapped, while larger particles get wrapped, determined by the subtle balance of bending and adhesion energy [139, [149] [150] [151] . for given particle radius, the threshold adhesion strength κ = * w a 2 2 / therefore marks the transition between the unwrapped and the complete-wrapped regime. adhesion may be mediated by van der waals interaction [152] , hydrophobic interaction [152] , electrostatic interaction [153] , and specific adhesion via receptor-ligand bonds [154] [155] [156] [157] [158] [159] [160] . in biological media, nanoparticles can be surrounded with a corona of proteins that effectively changes their surface properties [161] [162] [163] [164] [165] [166] . throughout this section, we assume that the adhesion can be modeled by a continuous and mostly homogeneous adhesion strength, which is appropriate for many systems, but not always sufficient for / . an overview of both single-particle and many-particle systems at membranes is provided in this section. in sections 3.1-3.4 we focus on various aspects of the interaction of single particles with membranes, while in sections 3.5 and 3.6 we discuss two-particle and many-particle interactions, respectively. section 3.7 focuses on particles at membranes with spontaneous curvature. we finally discuss applications in section 3.8. for a spherical particle and an infinitely large tensionless membrane, the membrane surrounding the particle assumes a catenoidal shape, see figure 36 (a). this shape is found around all cylindrically-symmetric, 'conical' membrane inclusions, figure 29 . interaction of a spherical particle with an ellipsoidal particle of aspect ratio = b a 5 / . the contact angle θ = 48.47 c has been used for both particles. the energies are plotted for spherical particles with radii = r a 3 and = r a 4 that approach the ellipsoidal particle (a) at the side (b) and at the tip, here shown for a spherical particle with = r a 3 . (c) interaction energies γ ∆e a 2 / as function of d a cc / between the centers of mass of the particles. inset: doublelogarithmic plot and the fit of the numerical data to a power-law decay. reprinted with permission from [43] . copyright (2014) american chemical society. which includes curved proteins [140, 169, 170] and attached polymers [171] [172] [173] [174] . a catenoid is a minimal surface with vanishing mean curvature at every point and therefore with vanishing bending energy. the deformation energy cost for the membrane adhered to the particle and the adhesion energy gain for contact between particle and membrane can therefore be directly compared with each other in order to predict whether a particle gets wrapped or not. the wrapping transition is fully characterized using the critical particle radius κ * a w for wrapping in equation (26) . however, in general the deformation energy of the membrane surrounding a particle will contribute to the total deformation energy of the membrane. a convenient way to characterize wrapping states of particles for various parameters are wrapping diagrams based on energy minimisation, analogous to phase diagrams in thermodynamics [139, 149, 175] , see figure 36 (b). spherical particles remain unwrapped below a threshold adhesion strength and get partialwrapped or complete-wrapped for higher adhesion strengths. particles at membranes with finite tension can be found in stable partial-wrapped states for adhesion strengths just above those for the binding transitions, see figure 36 (b). the stable partial-wrapped states are separated by continuous transitions from the unwrapped states and by discontinuous transitions from the complete-wrapped states. the latter trans itions with energy barriers are associated with two spinodals that indicate those values for the adhesion strength beyond which particles transition spontaneously from partial-wrapped to completewrapped states and below which particles transition spontaneously from complete-wrapped to unwrapped states. membrane deformations induced by non-spherical particles depend on both particle shape, discussed in this section, and particle orientation, discussed in section 3.4. in general, inhomogeneous surface curvature of particles corresponds to energy barriers for wrapping [139, 176] . for non-spherical particles, membrane tension is therefore not required to stabilize partial-wrapped states; such particles are ideal membrane markers for imaging. thus not only size and aspect ratio, but also local particle surface curvature matters for wrapping of non-spherical particles [176] . the importance of local particle surface curvature can be demonstrated well for hauser's cube-shaped particles. the phase diagram in figure 37 shows that no membrane deformation energy costs arise if cube-like particles attach with a flat face to a membrane. the particles therefore adhere already for very small adhesion strengths and are found in shallow-wrapped states. the first energy barriers are encountered between shallow-wrapped and deep-wrapped states when the membrane has to bend around the highly-curved edges to increase wrapping from one face to five faces. the energy barriers between deepwrapped and complete-wrapped states correspond to the last four edges to be wrapped. both energy barriers shift to higher adhesion strengths for higher values of the membrane tension. we discuss here particles within the hydrophobic tail region of lipid bilayers in part 1 and particles at curved membranes in part 2. particles with sizes of few nanometers may incorporate themselves in membranes and locally distort both lipid order and membrane thickness [142, 145, 177] , see figure 38 (a). while lipid order can only be investigated on the molecular scale, thickness variations can also be investigated using continuum models. here the headgroup positions of the monolayers can be modeled by mathematical surfaces with bending rigidity and monolayer tension, and a confinement potential which maintains the lipid bilayer thickness. analogous to integral membrane proteins with hydrophobic mismatch [178] [179] [180] [181] , particles that locally induce membrane-thickness deformations experience short-ranged deformation-mediated interactions. while the interactions may be repulsive at long distances as predicted by the continuum models, both continuum models and molecular dynamics simulations predict short-ranged attractive interactions and therefore clustering of nanoparticles within membranes. for high membrane tensions, the energy barrier for a third particle approaching a pair of particles is considerably decreased, such that particle aggregation may occur spontaneously [145] , see figures 38(b) and (c). not only particle shapes, also membrane curvature prior to wrapping has to be taken into account for the interaction of particles with membranes. this can be demonstrated by calculating entry of particles into and exit of particles out of vesicles with different curvatures, see figure 39 . the vesicles are assumed to freely adjust volume but keep a fixed membrane area. vesicles without particles thus adopt an overall spherical shape that is then locally distorted by binding a nanoparticle. the adhesion strength w and / , for vesicles with radius r v , are the relevant parameters to characterize this system. they are a subset of the full parameter space, determined in addition by membrane spontaneous curvatures and by osmotic pressures [183] . for particles that enter vesicles from the outside, > c 0 r , wrapping is hindered by an energy barrier. for particles that exit vesicles from the inside, < c 0 r , upon increasing the adhesion strength the particles continuously transit from the free state via stable partial-wrapped states with increasing wrapping fractions to the complete-wrapped state. partial-wrapped particles can be used as probes for the local membrane curvature [184] . a direct and barrierless transition between the free and the complete-wrapped state is only present for particles at infinitely large planar membranes. with decreasing absolute relative curvature c r , both the energy barriers for particle entry as well as the stability of partially-wrapped states for particle exit decrease. the adhesion strengths for the wrapping trans itions can be well approximated by the instability relations [42, 183] for non-spherical particles, the orientation of particles at membranes-in addition to particle shapes, sizes, and surface properties-has to be taken into account to determine / and membrane tensions γ γ κ = a 2 / . (a) the membrane deforms in a cylindrically symmetric way around the symmetry axis and the shape can be described by a radial deformation profile. for an infinitely large tensionless membrane, the shape of the free membrane is catenoidal. (b) the wrapping phase diagram shows that the nanoparticles are unwrapped below a critical adhesion strength = w 4 , and completely enveloped for adhesion strengths beyond the thick solid line. the thin dotted line is the envelopment transition calculated neglecting the deformation energy of the free membrane. the thin dashed lines are the spinodals for spontaneous unwrapping from the complete-wrapped to the free/non-wrapped state and for spontaneous envelopment from the partially wrapped to the fully wrapped state. adapted with permission from [168] . © epla. all rights reserved. wrapping states and membrane deformations. elongated particles can be oriented in rocket and in submarine orientation, with their long axes perpendicular and parallel to the membrane, respectively. janus particles preferably bind with their most adhesive side to the membrane. changing the orientation of particles in magnetic fields can be used to probe the elastic properties of membranes and cells. the orientations of elongated particles at membranes crucially depend on particle shapes, membrane elastic properties, and membrane-particle interactions, see figure 40 . table 4 summarizes wrapping states and transitions for various particles shapes and membrane elastic parameters based on the results of [139, 149, 176, 185] . for prolate ellipsoidal particles, the particles bind to membranes with their points of lowest curvature at the sides of the particles. in these shallow-wrapped states, they are in the so-called 'submarine orientation'. for fast wrapping, if reorientation does not occur, the particles remain in submarine orientation until they reach the complete-wrapped state [139] . for for two corresponding states at the w 2 phase boundary: (a) a shallowwrapped state with approximately 10% of particle wrapped, and (b) a deep-wrapped state with approximately 80% of the particle wrapped. (c) phase diagram for wrapping of hauser's cube for reduced membrane tension γ γ κ = a 2 / and reduced adhesion strength πκ = w wa 2 /( ), where a is the particle surface area. we find a shallow-wrapped (sw), a deep-wrapped (dw), and a completewrapped (cw) state, separated by two discontinuous wrapping transitions, labeled as w 2 and w 3 . adapted with permission from [176] . copyright (2014) american chemical society. morphology diagram of stable states of a particle adhering to a vesicle. the wrapping state is plotted for various values of rescaled adhesion energies κ = u wa 2 / and relative curvatures c r of the vesicle membrane and the particle surface. the three black lines divide the diagram into three regions in which the particle is either partially wrapped, unwrapped, or fully wrapped. in the grey shaded region, the transitions between the unwrapped state and the wrapped state of a particle outside the vesicle require the crossing of an energy barrier. the red dashed lines are analytical instability lines derived from the stability relations in [42] . reproduced from [183] .-published by the royal society of chemistry. oa cc by 3.0. slow wrapping, the particles are able to reorient to their minimal-energy states at every time, they transit from submarine orientation to the so-called 'rocket orientation' at deep wrapping. the rocket state is energetically preferable at deep wrapping, because only one of the pointed tips needs to be wrapped. from a stable deep-wrapped state, ellipsoidal particles then continuously transition to the complete-wrapped state [176] . for rod-like particles, energy minimisation predicts that the particles readily bind with their blunt tips in rocket orientation at very small adhesion strengths. if the edges of the particles are sharp and their aspect ratios are small, the particles remain in rocket orientation until they reach complete wrapping; the transitions between shallow-wrapped and deep-wrapped, as well as between deep-wrapped and complete-wrapped states are discontinuous. for high aspect ratios or round edges of the particles, rod-like particles bind with their blunt tips in rocket orientation only with a very small fraction of their surface area. they then rotate to submarine orientation in the shallow-wrapped state, and back to rocket orientation in the deep-wrapped state [176, 186] , see figure 40. in particular, the theoretical prediction for the rotation from submarine orientation to rocket orientation is in agreement with experimental observations for budding of filamentous viruses [28, 29] . modes of entry for nanoparticle uptake by membrane wrapping: (i) submarine mode with the long axis of the particles oriented parallel to the membrane, (ii) rocket mode with the long axis oriented perpendicular to the membrane, and (iii) competition between submarine and rocket mode as observed for rod-like particles with high aspect ratios. the complete-wrapped particle is connected by an infinitely small catenoidal neck to the membrane; the particle orientation in this state is irrelevant. reprinted with permission from [176] . copyright (2014) american chemical society. table 4 . shape dependence of particle wrapping, based on the results of [139, 149, 176, 185] . the membrane can be characterized by bending rigidity only, 'κ', or by bending rigidity and membrane tension, 'κ and γ'; the binding transition can occur at finite or vanishing adhesion strength w; the particle can be in submarine or rocket orientation; transitions can be continuous (cont.) or discontinuous (discont.) and may involve reorientation (reorient.). the binding transition for ellipsoids is independent of the membrane tension and is given in [139, 185] . binding transition a fast wrapping at high adhesion strength, such that a bound ellipsoid cannot reorient to rocket orientation. b rocket mode for supereggs with blunt tips and small aspect ratio (e.g. = n 4 and = b a 1.5 / ). reprinted with permission from [176] . copyright (2014) american chemical society. the dynamics of wrapping of spherical nanoparticles is determined by the typical time scales for the relevant processes, such as membrane deformation [170] , receptor or protein diffusion that may be hindered by a cortical cytoskeleton [187] [188] [189] [190] [191] , and potential metabolic remodeling of the cytoskeleton [192] [193] [194] . after initial contact between nanoparticles and membranes, the deformed area of the membrane surrounding the particles increases until half wrapping and then again decreases. while the membrane deformation is catenoid-like for small and for large wrapping fractions of particles, for finite membrane size the highest deformation energy costs are expected for about half-wrapped particles. formation of a defect in the neck towards the end of the wrapping process can induce the separation of bent and flat membrane and completes wrapping [186] . the reorientation dynamics for elongated particles can be calculated using molecular dynamics simulations for nanoparticles that interact with initially flat lipid bilayer membranes [186, 195] . also local free-energy analysis and incremental changes of the nanoparticle orientation in the direction of lowest energy at each time step allow to predict the wrapping pathway [186] . in figure 41 , the corresponding curvature-energy landscapes are displayed. a spherocylindrical nanoparticle that is initially oriented in the unfavourable rocket orientation first reorients towards submarine orientation. however, although energetically most favourable until half wrapping the particle may never actually reach submarine orientation. beyond half wrapping, the particle turns back to the then favourable rocket orientation, in agreement with the energetics discussed at the beginning of this section. we are not aware of systematic experimental studies for the interaction of lipid-bilayer membranes with nanoparticles that have anisotropic surface functionalization. however, micrometer-sized spherical particles that are half coated with ligands preferably orientate with the ligandcoated side towards the membrane during phagocytosis [196] . furthermore, viruses have been modeled as partially adhesive particles [140] . the reorientation of the malaria parasite in tipfirst orientation at the beginning of the invasion process may also be due to an adhesive gradient on the parasite surface [22] . analytical calculations for partial-wrapped particles, e.g. janus particles, at non-spherical vesicles show that membrane curvature-induced forces pull the particles to regions with preferred membrane curvature [197] . in analogy to magnetic particles with switchable orientation at fluid interfaces, micrometer-sized magnetic particles can also be attached to cell membranes with an underlying cytoskeleton [198] . such microrheological measurements reveal glassy behaviour for various cell types [198] [199] [200] [201] . for red blood cells with their cortical spectrin cytoskeleton responsible for the shear elasticity of the complex cell membrane [202] , the values of the elastic parameters obtained using magnetic particle microrheology agree well with those used for computer simulations for cell stretching and blood flow [37] . membrane deformations induced by partial-wrapped particles lead to membrane-mediated interactions that minimise the sum of both membrane deformation energy and particlemembrane attachment energy [203] . several recent studies are discussed in this section. however, much more is knownand this knowledge may partially be transferred to nanoparticles-about the related systems of lipid bilayer-mediated interactions between curved inclusions, both for membranedeformation mediated interactions [48, 140, 169, 170, 204] , as well as for membrane-fluctuation mediated casimir interactions [205] [206] . membrane-mediated interaction between two parallel and long cylindrical particles at distance d attached to the same side of membranes under lateral tension is repulsive [208] , / predicted by local energetics. the nanoparticles take a general laying-down-thenstanding-up sequence during endocytosis. the heat maps show the curvature energy for various particle orientation angles and wrapping fractions. nanoparticle wrapping at the turning points (i-v) along the endocytic pathways predicted by local free-energy analysis is schematically shown on the right: green-shaded areas are wrapped, while yellow-shaded areas are naked. reprinted with permission from [186] . copyright (2013) american chemical society. where ξ γ κ = / is a characteristic reciprocal length, and a cyl is the cylinder radius. the interaction between two cylindrical particles attached to opposite sides of the membrane is attractive, here, the length of the cylinders is contained in the param eters for the bending rigidity, adhesion strength, and membrane tension for these effectively one-dimensional calculations. results for stronger membrane deformations can be found in [209] . only few studies are available for far-field interactions between spherical nanoparticles that are partially attached to lipid bilayer membranes. figure 42 shows both numerical calcul ations and experimental data. membrane-mediated particle attraction is found for distances below a 5 , with an attractive energy well of κ ≈ − e 0.17 a for an interparticle distance of about a 2.8 [44] . for typical lipid bilayer bending rigidities κ < < k t k t 20 100 b b , this corresponds to binding energies the figure shows larger attraction strengths for membrane tensions γ < 10 nn m / compared with membrane tensions γ µ > 1 n m / . many computer simulation studies for membrane-mediated interactions between partial-wrapped nanoparticles, as shown in figure 42 , rely on finite-element calculations. here, the bending energy is discretized on triangulated surfaces [210] [211] [212] [213] . large systems that require at the same time fine discretization in high-curvature regions around particles are computationally expensive, therefore membrane-mediated interactions between the particles have so far mostly been studied in the near field. however, long-ranged membrane-mediated interactions between partial-wrapped nanoparticles might be similar to long-ranged interactions between curved inclusions. for example, for two spherical-cap inclusions attached to the same side of planar membranes, the repulsive membranemediated interaction is [169] where α 1 and α 2 are polar angles that determine the sizes of the spherical caps, a is their radius, and d is the distance between the centers of the caps. strongly-curved inclusions at very close distances experience an attractive interaction [48] . interactions between two (spherical) nanoparticles have been shown to be attractive, see e.g. figure 42 . this is surprising on the first view, because catenoid-like membrane deformations around partial-wrapped nanoparticles at large distances require only very small deformation-energy costs. if two spherical-cap inclusions or two particles approach each other on a planar membrane the ideal catenoid-like deformations cannot form any more because of the boundary conditions. therefore, the membrane-mediated interactions because of deformation-energy are expected to be repulsive [169, 204] . however, for spherical particles the attractive membrane-mediated interactions because of membrane-particle adhesion energy dominate and lead to an overall attraction [203] . for two partial-wrapped particles in the near field, not only the interparticle distance, but also the orientation of the pair of particles with respect to the membrane has to be taken into account. for example, in [46] the connecting line between two particles adsorbed to a vesicle is parallel to the membrane at large distances and reorients to perpendicular orientation at small distances. the attractive interaction is significantly higher for this particularly stable tubular arrangement [214] . many-particle interactions are important for membrane-mediated interactions, which has been demonstrated in several cases for integral membrane proteins with hydrophobic mismatch and for spherical-cap inclusions [140, 170, 182, 215] . it is therefore essential to not only consider pair interactions between particles. we start our discussion with the 'inclusion case', i.e. with membrane deformation-mediated interactions only, where the membrane area attached to each particle is fixed. while two weakly-curved spherical-cap inclusions that are attached to the same side of the membrane repell each other as discussed in section 3.5.1, many inclusions have been observed to aggregate and to induce bud formation [140, 170, [215] [216] [217] . a membrane that is curved prior to adhesion of inclusions or that gets curved by cooperative budding screens the repulsive interaction and can therefore lead to effective attraction. figure 43 highlights catenoidal membrane deformations around inclusions on vesicles; the size of the catenoidal 'halo' shrinks with increasing background curvature, thereby screening the repulsive interaction between the inclusions. these catenoidal patches reduce the total bending energies of the vesicles, which vanish at optimal inclusion density. in comparison, on planar membranes curved inclusions always increase the deformation energy. a particle dimer switches from a linear aggregate on the membrane to a tubular aggregate for increasing wrapping fraction [46, 47] , compare also section 3.4 for the orientation of elongated particles. on a vesicle, the wrapping fraction can be tuned by adjusting the reduced volume of the vesicle, where v is its actual volume and v sph is the volume of a spherical vesicle with the same membrane area. a small reduced volume also allows more than two particles to join the tube. the energy gain of such tubular assemblies compared with single, complete-wrapped particles strongly depends on the ratio of the range ρ of the particle-membrane interaction potential and the particle radius a. the finite potential range leads to a higher adhesion energy gain for tubular arrangements compared with single-particle buds, because some fraction of the tubular necks between particles are inside the interaction range; for example the energy gain per particle is about κ 5 for ρ = a 0.2 / [214] . simulation snapshots and energies for several configurations of three particles attached to membranes are shown in figure 44 . if the particles are all located in the plane of the membrane, a third particle that attaches to form a linear aggregate gains membrane-mediated binding energy of few k t b and attaches without an energy barrier. in contrast, a particle that attaches from the side to the existing particle aggregate experiences an energy barrier and has a few k t b higher energy in the (metastable) bound state compared with the unbound state. bending energy has been shown to favour compact aggregation, . here, φ = 0 corresponds to a linear arrangement of the particles. the four snapshots depict minimum-energy conformations at the angles φ = 0 , 45 , 90 , and 120 . reprinted with permission from [46] . copyright (2012) by the american physical society. adhesion energy linear aggregation [203] . also for tubular aggregates, the linear-tube configuration is preferred over the more compact, triangular configuration [46] , see figure 44 (b). linear aggregates in the plane of the membrane have been experimentally observed for colloidal particles bound to giant unilamellar vesicles (guvs) [218] , tubular aggregates for the interaction of viruses with cells and guvs [219] . figure 45 (a) shows a phase diagram for many-particle systems in terms of bending rigidity κ and particle-membrane binding energy d 0 [203] . in the limit of small bending rigidities, partial-wrapped particles form a hexagonal cluster phase where the membrane penetrates in-between the particles. in the limit of high bending rigidities, the particles are barely attached to the membrane. they deform the membrane only weakly, therefore they also interact only weakly and are found in loose, mostly hexagonal aggregates. linear aggregates are observed inbetween both limites for biologically relevant bending rigidities κ < < k t k t 10 100 b b . figure 45 (b) shows a phase diagram in terms of particle diameter and particlemembrane binding energy. free unbound nanoparticles are found at low adhesion strengths and for small particle radii, then linear aggregates, tubular aggregates, and single-particle buds are observed with increasing adhesion strength [47] . this sequence of configurations is consistent with the reorientation reported for elongated nanoparticles in [176] , see section 3.4. biological membranes are often not symmetric, the two monolayers usually consist of different lipids. this asymmetry can be modeled with the help of a spontaneous curvature c 0 of the membrane. if the directions of spontaneous curvature and the curvature of the particle surface coincide, the spontaneous curvature facilitates complete wrapping compared with a symmetric membrane [42] . at the same time, the direct transition between free and complete-wrapped states is replaced by a discontinuous transition. for a spontaneous curvature opposite to the curvature of the particle surface, the complete-wrapped state is shifted to higher adhesion strengths. furthermore, a new regime with partial-wrapped states is found. with increasing spontaneous curvature, also the regime of adhesion strengths where partial-wrapped states are stable increases. for fixed adhesion strengths, finite spontaneous curvatures lead to size selectivity for wrapping of nanoparticles, see figure 46 . for vanishing or small spontaneous curvatures, all particles with radii beyond a threshold radius get figure 45 . membrane-mediated interactions between many particles. (a) phase diagram for nanoparticle self-assembly in terms of membrane bending rigidity κ and particle-membrane binding energy d 0 , which replaces the adhesion strength w in the simulations. the snapshots show typical aggregates in the h1, the l, and the h2 phase (top to bottom). adapted with permission from [203] . copyright (2012) by the american physical society. (b) phase diagram in terms of nanoparticle diameter a 2 and d 0 . with increasing d 0 , the gaseous phase g with non-attached particles is followed by the linear aggregation phase l, the tubeformation phase t, and the single-particle bud phase b. the radius of the vesicle is σ = r 30 v and the particle surface fraction is 0.15. adapted with permission from [47] . copyright (2012) by the american physical society. wrapped, see equation (26) . with decreasing spontaneous curvatures, in addition to the lower threshold radius for complete wrapping, also an upper threshold radius beyond the that particles remain unwrapped is found [42] . the regime with stable complete-wrapped states thus narrows to a small window in the particle radius for high negative values of the spontaneous curvature. such a preferred radius for particle wrapping has been observed in experiments [220, 221] . size selectivity by spontaneous membrane curvature is therefore an alternative to receptor-based models for cellular uptake that are usually used to motivate an upper limit for the particle radius to achieve complete wrapping [154] . on the one hand, particle-membrane systems can be used for applications in therapeutics and diagnostics. on the other hand, for numerous other applications in industry, potential toxic effects have to be considered. furthermore, nanostructured surfaces can be rationalised as nanoparticles bound to a substrate, which opens an entire new field for applications. for basic research, particle-membrane systems can serve as model systems to understand biological processes, such as viral budding, malaria invasion, and phagocytosis. 3.8.1. drug delivery. nanoparticles are potential drug-delivery vectors and tools for diagnosis [222] [223] [224] [225] [226] . for diagnosis, in particular non-spherical nanoparticles with enhanced stability of partial-wrapped states can serve as membrane markers for imaging [141] . for drug delivery, rough hydrophobic particles have been proposed to have a high drug-carrying capacity and high loading efficiency [227] . such particles can prolong the time of release of a drug payload, which can be therapeutically advantageous. nanoparticles may even cross the blood-brain barrier via transcytosis and may therefore be applied for drug delivery in the brain [228, 229] . recently hollow shell-shell nanocontainers with stimuli-responsive properties that could allow for controlled drug release have been suggested for drug delivery [230] . for studies of potential toxic effects, not only single-particle properties, such as composition and size, but also particle concentrations play an important role [19] . toxicity can be quantified by ec 50 values that refer to the concentration of particles that induce a response of cells or organisms halfway between the base line and the maximum after 72 h exposure time to nanoparticles. while agno 3 leads to growth inhibition for algae already at concentrations of − − 10 mg l 3 1 , ag nanoparticles lead to growth inhibition only at concentrations between − 0.1 mg l 1 and − 1 mg l 1 , see figure 47 . most other organisms are less sensitive to nanoparticles and growth inhibition occurs only at higher particle concentrations compared with algae. in general, the ec 50 values increase significantly with particle size, which means that the toxic effect is reduced. for mammalian cells they range from 3.8.3. nanostructured surfaces. multi-electrode arrays allow to electrically couple excitable cells to electronic devices, see figure 48 . the challenge is to achieve an optimal coupling, which requires to minimize the cleft between the cell and the electrode and to achieve tight attachment of the cell to the substrate in general. while thin electrodes pierce cell membranes [232] , micro-electrodes get wrapped [231, 233] . a systematic study of different micro-electrode shapes reveals that mushroom-shaped pillars are engulfed more than cylindrical pillars without caps [231] . furthermore, cells have been found to preferably engulf pillars in their center compared with their edge. quantitative evaluation of focused ion-beam cuts allows to extract normal cytoskeletal stresses between few pa for cylinders with caps and several hundred pa for cylinders without caps. sis. passive endocytosis of particles with a homogeneous adhesion strength between particles and membranes is often modified by specific adhesion and active processes for biological systems. figure 49 shows viruses with different shapes and a malaria parasite that have similar sizes as the particles discussed earlier. one specific example for active wrapping is the invasion of the malaria parasite into an erythrocyte, see figure 50 . reorientation of the parasite, possibly because of a gradient of adhesive molecules, is followed by invasion. during invasion, a tight junction forms and reseales the membrane, which completes the formation of the parasitophorous vacuole. for the egg-like shape of the malaria parasite, line tension at the tight junction, and anisotropic adhesion of the parasite leads to a complex phase diagram for passive endocytosis with stable non-wrapped, shallow-wrapped, deep-wrapped, and complete-wrapped states [22] , see figure 51. as indicated in figure 51 , secretion of unstructured membrane from the parasite and favourable spontaneous curvature of the erythrocyte membrane may help the parasite to overcome energy barriers for wrapping and to finally invade the cell. in addition, also motor forces have been proposed to assist invasion [22] . another active biological 'wrapping' process is phagocytotic uptake, see figure 52 . here, the growth of the phagocytotic cup with its actin cytoskeleton is the major dynamic process [238] . quantification using microscopy and image analysis shows fast uptake at the beginning, followed by a plateau with weakly increasing wrapping fraction, and again fast uptake of the particle towards the end of the process [237] . various mechanisms for phagocytotic uptake have been suggested, among them receptor diffusion and directed motion [239] , a zipper-like mechanism based on membrane fluctuations and membrane adhesion to the particle [240] , and hindered uptake by increase and relaxation of membrane tension [241] . multi-phase fluid systems and cellular biological systems abound with interfaces. nano-and microparticles naturally collect at such interfaces, because their localization at the interfaces lowers their interaction energy with the environment. we have considered here two types of interfaces: fluid-fluid interfaces governed by interfacial tensions, and biological interfaces controlled by curvature elasticity. particles at both types of interfaces show many common behaviours. the interface attraction depends on particle size, shape, and surface properties. particles can orient in different ways at interfaces, they deform the interfaces around them, and these interface deformations lead to interface-mediated interactions and collective behaviour. although systems and concepts for particles at fluid and biological interfaces are very similar, and for instance sds stabilisation of droplets can drive the system from fluid interfaces towards membranes [242] , there is a major difference. in many cases, particle sizes at fluid interfaces are in the micrometer range, while particle sizes at lipid bilayer membranes are in the nanometer range. this implies that different experimental techniques are required in both cases, where the nanometer scale makes particles at membranes more difficult to observe and more difficult to manipulate. therefore, we have a better knowledge about particles at fluid interfaces, and there is an urgent need for systematic and well-controlled experiments for particles at membranes. nevertheless, a unified numerical and theoretical description of both systems is possible-at least for particles above a threshold diameter of about − 15 20 nm -on the basis of continuum models for two-dimensional surfaces and their deformations embedded in three-dimensional space. from the technological point of view, micrometer-sized particles at fluid interfaces can be used in many ways to control and tailor interface properties. such applications range for both the upper and lower lobes, after engulfment begins at ≈ t 10 s there is an initial slow stage (light gray) followed by a much quicker second stage (dark gray). engulfment is complete by ≈ t 46 s . reprinted from [237] , copyright (2014), with permission from elsevier. from emulsion stabilisation through an effective reduction of the interface tension to the design of solid shells and surfaces with controlled optical properties. from a biological point of view, nanoparticles are interesting in nanomedicine as biomarkes and drug carriers, but their effect on cells also has to be assessed because of their potential nanotoxicity. in addition, there is a large range of biological particles, such as viruses and parasites, the interaction of which with cells is also highly desirable to be controlled. the interaction of single hard particles with interfaces is by now reasonably well understood. therefore, we believe that future research should move towards particle-mediated interactions and collective properties for many particles at both fluid and biological interfaces, as well as towards soft particles. for example, size and concentration effects for the interaction or nanoparticles with liposomes and polymersomes have been studied using electron microscopy and scattering [49] [50] [51] . using force measurements, nanoparticlemediated adhesion between elastic gels has been measured [243] . the hard particles can serve as 'glue' between soft interfaces. soft particles, e.g. microgel and polymeric particles, add further complexity by allowing the particle shape to adjust in response to the interaction with a fluid or biological interface [160, [244] [245] [246] [247] [248] . finally, dynamical behaviour of particles at fluid and biological interfaces awaits further characterization. for instance, dynamics of wrapping of non-spherical particles by biological interfaces has been discussed in section 3.4.1, but calculations that include brownian motion are missing. colloids adsorbed to stabilised oil-water interfaces show different diffusion properties than colloids in bulk [242] , systematic studies could be used to measure the viscosity of lipid bilayers. furthermore, nanoparticles in combination with superresolution microscopy can be used as probes to study dynamics in biological cells [249] [250] [251] . to conclude, particles are already widely used for applications, but a systematic understanding of the interactions of engineered nano-and microparticles with soft and biological matter is often lacking. furthermore, our understanding of basic biological processes on the cellular scale, such as phagocytosis and blood-stage malaria, will benefit from a 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microgel monolayers from fluid interfaces: particle size effects on interface microstructure and nanolithography cellular uptake of elastic nanoparticles size and dynamics of caveolae studied using nanoparticles in living endothelial cells protein-based fluorescent nanoparticles for super-resolution sted imaging of live cells super resolution imaging of nanoparticles cellular uptake and trafficking our research on the interaction of particles with biological membranes has been supported by the eu fp7 nmp collaborative project prenanotox (309666). key: cord-336901-q6kgzuob authors: choi, jeongan; kang, miran; jung, jae hee title: integrated micro-optofluidic platform for real-time detection of airborne microorganisms date: 2015-11-02 journal: sci rep doi: 10.1038/srep15983 sha: doc_id: 336901 cord_uid: q6kgzuob we demonstrate an integrated micro-optofluidic platform for real-time, continuous detection and quantification of airborne microorganisms. measurements of the fluorescence and light scattering from single particles in a microfluidic channel are used to determine the total particle number concentration and the microorganism number concentration in real-time. the system performance is examined by evaluating standard particle measurements with various sample flow rates and the ratios of fluorescent to non-fluorescent particles. to apply this method to real-time detection of airborne microorganisms, airborne escherichia coli, bacillus subtilis, and staphylococcus epidermidis cells were introduced into the micro-optofluidic platform via bioaerosol generation, and a liquid-type particle collection setup was used. we demonstrate successful discrimination of syto82-dyed fluorescent bacterial cells from other residue particles in a continuous and real-time manner. in comparison with traditional microscopy cell counting and colony culture methods, this micro-optofluidic platform is not only more accurate in terms of the detection efficiency for airborne microorganisms but it also provides additional information on the total particle number concentration. may not satisfy the specific growth requirements of all species of interest, and also requires prolonged incubation (i.e., > 24 hours) 16 . recently, detection and enumeration of airborne microorganisms has been accomplished using techniques based on polymerase chain reaction (pcr) and enzyme-linked immunosorbent assays (elisa), which are highly sensitive and quantitative techniques [17] [18] [19] [20] [21] . however, additional pretreatment processes (such as particle condensation/purification), and elaborate sample handling by well-trained operators in a clean environment are required 22 . although these pcr and elisa techniques have been applied to microfluidic chips to simplify the treatment processes, [23] [24] [25] most such microfluidic systems have been developed as disposable chips for point-of-care diagnosis of target microorganisms, which often leads to a limited number of samples or poor accuracy or rates of detection 26 . novel real-time methods for the rapid detection and continuous monitoring of airborne microorganisms have the potential to address the public health problems associated with bioaerosols. one of the most frequently used real-time detection techniques for detection of airborne microorganisms exploits auto-fluorescence following illumination with ultraviolet (uv) light 27, 28 . auto-fluorescence is caused by metabolites and structural components of living cells 29 . although this technique allows continuous real-time monitoring and detection of bioaerosols directly in the air stream, problems remain due to low fluorescence intensity, which leads to poor limits of detection, and requires precise optical systems for the measurements. for these reasons, such devices are typically not portable, and cannot provide an integrated "micro-total analysis system". to overcome the limitations discussed above, cell and particle detection methods based on microfluidic flow-cytometry have been reported [30] [31] [32] [33] . microfluidics benefits from miniaturization, low power consumption and high sensitivity. microfluidic methods of counting water-borne microorganisms include tracking the population of marine algae and quantification of bacterial cells using pre-staining [34] [35] [36] . chung et al. (2014) developed a real-time single-cell detection system using target aptamer-conjugated fluorescent nanoparticles in a microfluidic flow-cytometry platform for microbial diagnostic applications 37 . however, there have been no reports of micro-optofluidic platforms for real-time continuous detection and monitoring of airborne microorganisms. here, we demonstrate a micro-optofluidic platform for real-time detection and quantitative analysis of airborne microorganisms. our optofluidic system involves the following steps: (1) sampling of airborne microorganisms; (2) mixing and reacting in a microchannel for staining; and (3) real-time detection and analysis of the particle by means of light scattering (sc) and bacterial fluorescence (fl). from these optical signals, we may discriminate and quantify airborne microorganisms, enabling simultaneous measurement of the total particle concentration. the performance in terms of particle detection is evaluated using standard particles and three bacterial taxa, and is compared with that of conventional microscopy cell counting and colony counting methods. design and operation of the micro-optofluidic platform. figure 1 shows a schematic diagram of the micro-optofluidic platform. the 150-μ m-deep polydimethylsiloxane (pdms) microfluidic channel was fabricated using conventional soft-lithography processes 38 . the micro-optofluidic platform is based on single-layer pdms channel and has four inlets (one sample inlet, one dye inlet, and two hydraulic focusing sheath inlets), and one outlet for waste. it consists of four main components: sample and dye inlets, a mixing region, a hydraulic focusing zone, and a sample optofluidic detection component (see fig. s1 ). we used syto82 dye (molecular probes inc., eugene, or, usa) with an excitation wavelength of 541 nm and an emission wavelength of 560 nm for nuclear staining of live cells in the microfluidic mixing zone. syto82 provides cell-permeant nucleic acid staining and exhibits low intrinsic fluorescence in cell-free systems, with marked enhancement upon binding to deoxyribonucleic acid (dna) or ribonucleic acid (rna) 39, 40 . following injection of the sample and dye using a syringe pump (kds200; kd scientific inc., holliston, ma, usa), the two fluids mixed rapidly at the entrance to the mixing zone. numerical analysis was carried out using the commercial computational fluid dynamics (cfd) software package cfd-ace (esi us r&d inc., huntsville, al, usa) to identify the optimal design and operating conditions. the navier-stokes equations were solved with fick's law of diffusion to describe the dynamics and homogenization of the mixing process in the channel. figure 1 (b-1,b-2) show the results of the cfd simulations and optical micrographs of the mixing of the sample and dye in the micro-mixer part. the numerical simulations show that the mixing of the two fluids was complete approximately 0.1-0.4 mm from the entrance of mixing zone with flow rate conditions for each fluid in the range 2.5-25 μ l/h (simulation duration, ~72-180 ms) (fig. s2 ). the mixing zone was 136.8 mm long and 50 μ m wide. the residence time for mixing (and hence for the reaction between the sample and dye) was in the range 25-246 s for total flow rates in the range 5-50 μ l/h. after passing through the mixing zone, the mixed sample fluid was focused horizontally using a dual sheath flow with sterilized distilled water (sdw) in the hydraulic focusing component (see fig. 1 (a) ). the focused core flow was 7.5 μ m wide, which is one-twentieth the width of the channel. this minimized variation in the optical signals among locations in the particle detection zone 30 . figure 1 (c-1) shows a schematic diagram of real-time detection of target particles in the core flow, as well as the optical setup for continuous detection and signal quantification. a diode-pumped solid-state (dpss) laser with a wavelength of 532 nm and an output power of 2.0 mw (cni laser, changchun, china) was coupled with a single-mode optical fiber (core diameter: 3.5 μ m, cladding diameter: 125 μ m). the laser beam was focused on the core fluid using an objective lens (rms20x-pf; 20x/0.5; thorlabs) located beneath the microfluidic channel (~7 μ m beam spot size at full-width half-maximum). a highly sensitive multi-pixel photon counter (mppc) (c10507-11-50u; hamamatsu photonics, hamamatsu city, japan) module was used to detect the sc and fl signals from the target particles. the particle fl was collected using the objective lens, and entered the mppc via the optical long-pass filter and focusing lens. to bring the sc light from the particles, multi-mode optical fiber with a core diameter of 105 μ m and a cladding diameter of 125 μ m (thorlabs) was carefully inserted into the channel with the axis of the fiber at 90° to the direction of flow, and linked to the mppc. a data acquisition (daq) unit (ni usb-6356; national instruments, austin, tx, usa) was employed to collect the analog data from the mppc modules, which were recorded using a desktop computer. an oscilloscope (dpo 2012b; tektronix, beaverton, usa) was used to monitor the signal in real-time, as shown in fig. 1 performance evaluation of the micro-optofluidic platform using standard psl particles. the particle detection performance of the optofluidic platform was evaluated under various microfluidic and sample conditions, such that the sample flow rate and the mixing ratios of fl and non-fl particles were varied. we used standard spherical polystyrene latex (psl) and fluorescence psl (flpsl) particles that were 1 μ m in diameter. figure 2 shows the particle fl and sc signals with flow velocities in the range 1.42-14.3 mm/s. the flpsl particle suspension (~1.45 × 10 3 /μ l) was injected into sample inlet. the ratio of the flow rate of the flpsl particle suspension to that of the sdw was 1:19 in the narrow core region (which had dimensions of 7.5 × 150 μ m). as the flow velocity increased from 1.42 to 14.3 mm/s, the frequency of the particle sc and fl signals increased in proportion to the particle flow velocity, and the average intensity of the measured sc and fl signals (sc/fl) decreased from ~140 arbitrary unit (a.u.)/~60 a.u. to ~46 a.u./~30 a.u., respectively ( fig. 2 (a) ). this is attributed to the decreased photon integration time of the photodetector with decreasing residence time of the particles in the detection zone of microchannel 41 . figure 2 (b) shows that the event rate (i.e., number of counts per second) of the particle sc and fl signals followed a linear relationship with the particle flow velocity; for fl we have y = 1.04x + 0.37, with r 2 = 0.996, and for sc we have y = 1.07x + 0.52, with r 2 = 0.998. the index of coincidence between the total frequencies of particle sc and fl signals was ~96.5% over the entire range of particle flow velocities. mcclain et al. reported that only ~5% of the particle sc signals were unaccompanied by coincident fl signals, which was attributed to unresolved fl peaks due to the relatively low signal intensity 36 . when the number of counts of the particle sc and fl signals are plotted as histograms as a function of the signal intensity, both sets of signals form gaussian-like distributions, as shown in fig. s3 . the variation in the intensity results from variations in the particle size and particle position in the detection zone. as expected, the particle sc signal was significantly stronger than the fl signal. we evaluated the particle detection performance of the micro-optofluidic platform with various mixing ratios of flpsl and psl particles. the total particle concentration was fixed at ~1.45 × 10 3 /μ l, and the total flow rate of the mixed suspension was 25 μ l/h, giving a flow velocity of ~7.1 mm/s. the mixing ratio of flpsl and psl particle suspensions was varied in the range 0 -100%. as shown in fig. 3 (a) , the ratio of the count rate of fl signals to sc signals increased in proportion to the mixing ratio of flpsl particles. figure 3 (b) shows fluorescence microscopy images of the flpsl particles in the mixing suspension. when the mixing ratio of flpsl particles was 0%, the flpsl particle number concentration in the micrographs was 0/μ l; at a mixing ratio of 50%, the flpsl particle number concentration was 688/μ l (~95% of the initial flpsl particle concentration); and at a mixing ratio of 100%, the flpsl particle number concentration was 1406/μ l (~97% of the initial flpsl particle concentration). psl particles could not be enumerated using normal bright-field microscopy because of the limit of resolution and small field of view. from this performance evaluation (figs 2 and 3) , we may conclude that the micro-optofluidic platform demonstrated not only real-time and continuous particle detection, with suitable performance for standard psl particles, but also quantitative and accurate discrimination between fl and non-fl particles. real-time detection of airborne microorganisms. to simulate a hazardous airborne bacteria-contaminated environment, we prepared a 1 × 1 × 1 m test chamber containing live, airborne escherichia coli, bacillus subtilis and staphylococcus epidermidis bioaerosols separately. details of the experimental setup can be found in the methods and supplemental information (fig. s4) . figure 4 shows the size and morphology of the bacterial bioaerosols in the test chamber. real-time particle size distribution data of the bacterial bioaerosols were obtained using an aerodynamic particle sizer (aps; 3321; tsi inc., shoreview, mn, usa). as shown in fig. 4 (a) , the bacterial bioaerosols exhibited mono-modal curves with a specific geometric mean diameter (gmd), peak diameter, and geometric standard deviation (gsd). table 1 lists these data for the three bioaerosols; the gmds of e. coli and b. subtilis were similar at ~1 μ m, and that of s. epidermidis was somewhat smaller, at ~0.8 μ m. airborne bacterial bioaerosols in the test chamber were collected using a biosampler (skc inc., eighty four, pa, usa), which is a highly efficient collection device that traps airborne microorganisms in a swirling liquid for subsequent analysis 42 . the airborne particle collection efficiency of the biosampler was ~94.3% for the 1-μ m-diameter psl particles (fig. s5) . figure 4 samples containing the bacterial suspension were injected into the micro-optofluidic platform using a syringe pump to evaluate the device performance for the real-time detection of airborne bacterial particles. figure 5 (a) shows the acquired sc and fl signals of the test bacterial bioaerosols. the average sc/fl signal intensity ratios were ~555/240 for e. coli, ~565/227 for b. subtilis, and ~433/142 for s. epidermidis. because of the size of these bacteria (i.e., 0.5 -1.6 μ m), they may be considered to lie within the mie scattering regime, and the intensity of the sc signals may be assumed to be proportional to the cross-sectional area of particles 36 . in the same manner, the fl signal intensity increased proportionally with the size of the bacteria because larger particles have a higher concentration of nucleic-acid fluorescent dye 33, 36 . the particle number concentration was determined based on the frequency data for the sc and fl signals. table 2 lists the sc signal concentration, which corresponds to the total particle number concentration. this was higher than the fl signal concentration, which corresponds to the total concentration of microorganisms. the difference between the total particle count (sc signals) and the total bacterial concentration (fl signals) was ~7% for e. coli, ~16% for b. subtilis, and ~23% for s. epidermidis. it follows that the sample included other particles; i.e., non-microorganism particles or impurities, such as non-dyed particles or small debris. to evaluate the particle detection efficiency for airborne microorganisms, the total concentration of bacteria obtained from the fl signal of the micro-optofluidic platform was compared with the conventional fluorescence microscopy cell counting and colony counting methods. figure 5 (b) shows a comparison of the concentration of bacteria measured using fluorescence microscopy and colony counting, both of which were normalized to that measured using the micro-optofluidic platform. for e. coli the normalized cell concentration by microscopic cell counting was 88 ± 4.6%, and was 67 ± 8.5% using colony counting; for b. subtilis, the normalized cell concentration using microscopic cell counting was 88 ± 4.7%, and was 77 ± 6.6% using colony counting; and for s. epidermidis the normalized cell concentration using microscopic cell counting was 85 ± 11.1%, and was 73 ± 12.4% using colony counting. the concentration of bacteria measured using both conventional cell-counting methods was lower than that using the micro-optofluidic platform. the micro-optofluidic platform exhibited the highest cell counts and the lowest standard deviation, which is indicative of superior performance. photobleaching of fluorophores may occur during cell counting via conventional fluorescence microscopy, which results in a reduction in the fluorescence intensity. furthermore, the low signal-to-noise ratio in the imaging process may lead to underestimation of the number of cells. an inappropriate image threshold intensity setting is required to discriminate fluorescence particles from the background, and this may exclude particles that either have a low fluorescence intensity or are slightly out of focus 43 . furthermore, quantitative analysis of the total concentration of particles with sizes of less than 1 μ m is limited by the resolution and the field of view of bright-field microscopy. as shown in figure 5 (b), the colony counting method resulted in the lowest cell concentration of the three methods. the colony counting method measures only culturable cells in the medium. therefore, it is difficult to apply the colony counting method for the analysis of viable but non-culturable (vbnc) microorganisms, or microorganisms that require a specific growth environment and/or specific nutrients 44 . the micro-optofluidic platform with integrated sample preparation and detection simplifies the system, can significantly reduce the measurement time, and can yield more accurate quantitative results 48 . the number concentration of the particles in air was recorded for each aps channel size, divided by the logarithmic interval of the corresponding particle size range and plotted as a function of the aerodynamic diameter 48 table 1 . size characteristics of test airborne bacterial particles. the geometric standard deviation (gsd) is defined as exp( where d j is the diameter of an individual particle, n j is the number of particles in the j th group, n is the total number of particles, and ln d g is the natural logarithm of the geometric mean diameter (gmd) of the particles, defined as ∑ / n d n ln compared with the abovementioned conventional methods. furthermore, our system provides additional information on the total particle number concentration of aerosols, which is difficult to obtain using conventional methods. we have demonstrated continuous, rapid and real-time detection of bioaerosols using a micro-optofluidic platform. the performance of our device was investigated using standard psl particles with various flow speeds and mixing ratios with flpsl particles. accurate quantitative flpsl particle discrimination with high efficiency was achieved. this first application of this integrated micro-optofluidic platform for the analysis of airborne microorganisms showed that our device could reduce the time for sample preparation and manual analysis compared with conventional microorganism-counting methods. the micro-optofluidic platform has potential applications in aerosol analysis, and can enable portable, highly sensitive, continuous real-time detection of airborne particles and microorganisms. in future, we plan to include a 3d focusing stream to optimize the optical signals, which is expected to enhance the resolution and efficiency of the measurements, and to reduce the noise due to spatial deviations of the particles. accurate particle sizing using the current micro-optofluidic platform is possible 30 ; furthermore, a micro-pump between the biosampler and microfluidic channel could be included for continuous real-time analysis of airborne microorganisms. various species of dye or marker for specific target materials, such as target-specific antibodies or aptamers, could be used to create an analysis system that provides data on the physical, chemical and biological properties of the airborne particles 37 . note that the purpose of this work was to quantitate airborne microorganisms rapidly and in real-time using a fully integrated micro-optofluidic platform. sample preparation. two types of standard uniform particles and three types of bacteria were used in this study. fluorescence polystyrene latex (flpsl) particles (f8820; fluorescent microsphere; 1.0-μ m diameter; orange fluorescent (540/560); invitrogen) and polystyrene latex (psl) particles (4010a; monosized microsphere; 1.0-μ m diameter; refractive index of 1.59; density of 1.06 g/cm 3 ; duke scientific corporation) were used as standard particles for the performance evaluation of particle detection and quantification. as test airborne microorganisms, gram-negative escherichia coli (korean collection of type cultures (kctc) 1039, biological resource center, republic of korea), gram-positive bacillus subtilis (kctc 1022), and gram-positive staphylococcus epidermidis (atcc 12228), were used 45 . the bacteria were incubated in nutrient broth (becton dickinson, franklin lakes, usa) at 37 °c, and harvested using a centrifuge (mini, gyrozen, south korea) at 6000 rpm for 10 minutes. bacterial pellets were washed three times using sdw with a centrifuge to remove the residual medium. to create the bacteria suspensions, 30-ml aliquots were placed in a six-jet collision nebulizer (bgi corp., usa) to aerosolize the bacteria. microchannel design and fabrication. the microchannel was a single-layer pdms channel fabricated using conventional soft lithography. su-8 (2025; microchem corp.), which is a negative photoresist, was spin-coated onto a 4.2-inch si wafer. the photoresist was soft baked on a hotplate at 65 °c for 5 min, followed by 95 °c for 10 min. a chrome photomask pattern was installed using a uv aligner, and the photoresist was exposed to uv irradiation. following this exposure, the resist was baked at 65 °c for 2 min and then at 95 °c for 10 min. the su-8 photoresist pattern was developed using 1-methoxy-2-propyl acetate (microchem corp.) for 7 min. after completion of the developing process, the pattern was rinsed using isopropyl alcohol (ipa) and deionized (di) water to yield a master mold. pdms was poured over the developed wafer with the pattern, and cured in an oven at 65°c. the patterned pdms channel was then removed from the wafer, and bonded onto glass using o 2 plasma to seal the microchannel. fluorescence microscopy cell counting. to enumerate flpsl particles and bacterial cells using the fluorescence microscopy method, 10-20 μ l of sample (following enrichment via centrifugation in the case of low particle concentrations) was loaded into the sample injection area of a disposable hemocytometer (dhc-n01; incyto, republic of korea), and visualized using a fluorescence microscope (b x 51; olympus, tokyo, japan) with a u-mwg2 filter set; the excitation wavelength was in the range 510-550 nm, and the emission wavelength was > 590 nm. for each sample, images of at least 16 microscopic fields were captured using a ccd array camera. microscopy cell counting was carried out using the imagej software package (http://imagej.nih.gov/ij/). colony counting. bacterial suspensions were serially diluted and spread onto the surface of nutrient agar (becton dickinson) in a petri dish, followed by incubation at 37°c for 24 hours. the resulting colonies were counted manually. scanning electron microscopy. the morphology of airborne bacterial particles was investigated using sem (nova nano sem 200; fei co., hillsboro, or, usa). the modified karnovsky's fixation protocol was used 46, 47 , and bacterial samples were fixed using 2% paraformaldehyde (15713; electron scientific reports | 5:15983 | doi: 10.1038/srep15983 microscopy sciences (ems), hatfield, pa, usa) and 2% glutaraldehyde (16020; ems) in a 0.05 m sodium cacodylate buffer (scb; ph 7.2) (11652; ems) at 4°c for 2 hours, followed by washing with 0.05 m scb at 4°c for 2 hours. post-fixation, the samples were treated with 1% osmium tetroxide (19152; ems) in 0.05 m scb at 4°c for 2 hours, and then washed with distilled water at room temperature. the samples were dehydrated at room temperature in a 30-100% ethanol series with 10-minute exposure to each concentration. the ethanol was then replaced with hexamethyldisilazane (16700; ems). following dehydration, each sample was spread onto polycarbonate membrane filters with a pore size of 0.4 μ m (isopore membrane filters http01300; millipore, billerica, ma, usa) and coated with osmium by chemical vapor deposition (hpc-1sw; vacuum device inc., ibaraki, japan). real-time detection of an airborne microorganism using inertial impaction and mini-fluorescent microscopy the role of particle size in aerosolised pathogen transmission: a review bioaerosol health effects and exposure assessment: progress and prospects measurement of airborne influenza virus in a hospital emergency department rapid quantification of bioaerosols containing l. pneumophila by coriolis ® μ air sampler and chemiluminescence antibody microarrays microbiological quality of indoor air in university rooms exposure to organic dusts, endotoxins, and microorganisms in the municipal waste industry upper airway inflammation assessed by nasal lavage in compost workers: a 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toxins using a microflow cytometer three dimensional, sheathless, and high-throughput microparticle inertial focusing through geometry-induced secondary flows disposable flow cytometer with high efficiency in particle counting and sizing using an optofluidic lens rapid, semiautomated quantification of bacterial cells in freshwater by using a microfluidic device for on-chip staining and counting optofluidic characterization of marine algae using a microflow cytometer flow cytometry of escherichia coli on microfluidic devices fast and continuous microorganism detection using aptamerconjugated fluorescent nanoparticles on an optofluidic platform soft lithography analysis of apoptosis by laser scanning cytometry syto probes in the cytometry of tumor cell death single molecule fluorescence under conditions of fast flow improved aerosol collection by combined impaction and centrifugal motion optimization of an automatic counting system for the quantification of staphylococcus epidermidis cells in biofilms accuracy of plate counts the complete genome sequence of the gram-positive bacterium bacillus subtilis optimization of fixation methods for observation of bacterial cell morphology and surface ultrastructures by atomic force microscopy adhesion forces between e. coli bacteria and biomaterial surfaces aerosol technology: properties, behavior, and measurement of airborne particles this research was supported by the kist institutional program (2e25290). supplementary information accompanies this paper at http://www.nature.com/srep competing financial interests: the authors declare no competing financial interests. key: cord-339475-okw6la2b authors: nan title: chapter 11 health effects date: 2005-12-31 journal: interface science and technology doi: 10.1016/s1573-4285(05)80015-8 sha: doc_id: 339475 cord_uid: okw6la2b abstract there are beneficial as well as harmful aerosols. according to their nature, harmful particles can be classified into three categories: chemically toxic, infectious, and radioactive. in general, there is a relationship between the response and the dose received. a biochemically active particle may contain only a small amount of active agents. in this respect, an inhaled particle simply acts as a carrier that facilitates delivery of deleterious or beneficial components to specific surface areas of lung airways. in view of the tortuous narrow passageways and sharp turns they have to go through, aerosol particles are an effective carrier. as an indication of their effectiveness, about one half of all 3-m particles inhaled through the mouth deposit in the alveolar region. there are beneficial as well as harmful aerosols. according to their nature, harmful particles can be classified into three categories: chemically toxic, infectious, and radioactive. in general, there is a relationship between the response and the dose received. a biochemically active particle may contain only a small amount of active agents. in this respect, an inhaled particle simply acts as a carrier that facilitates delivery of deleterious or beneficial components to specific surface areas of lung airways. in view of the tortuous narrow passageways and sharp turns they have to go through, aerosol particles are an effective carrier. as an indication of their effectiveness, about one half of all 3-~tm particles inhaled through the mouth deposit in the alveolar region. in addition to respiratory disease, some types of deposited particles can cause systemic diseases following their dissolution and absorption. the period between first exposure to an agent and the appearance of symptoms varies depending on the characteristics of the agent and disease. some symptoms appear within hours from an acute exposure, whereas others may have latency periods as long as 20 years. synergistic or antagonistic effects play an important role. synergism represents the interaction of two or more agents that produces an adverse effect greater than the sum of the effects resulting from exposure to each agent separately. antagonism is an interaction between two or more agents that results in a reduction in adverse effect of each agent. host factors have a strong influence on biological responses to harmful particles. the main host factors include state of health, genetic trait, immunological status, and psychological state such as anxiety and stress. inhalation of particulate toxicants can lead to various types of responses including fibrotic, systemic, irritant, allergic, mutagenic, teratogenic, and carcinogenic responses. injury of tissues may result from the inhaled compound or its metabolic products. effects may differ considerably between direct exposure of epithelial cells to toxicants and exposure to the toxicants after phagocytosis. toxicity also depends on particle size and physicochemical properties of the agent. concentration of the agent and duration of exposure are two important factors influencing response. acute and chronic exposure may result in different adverse effects. pneumoconioses, characterized by scars due to increase in interstitial fibrous tissue, are diseases resulting from retention of certain types of particles in the alveolar region. different types of dusts may produce different patterns of fibrotic lesions. for example, crystalline free silica found in mines, foundries, blasting operations, and glass manufacturing can give rise to a nodular type of fibrosis (silicosis). on the other hand, asbestos fibers can cause a diffuse fibrosis (asbestosis) if they exceed 2 ~tm in length and 0.1 ~tm in width (lippmann, 1990) . coal workers' pneumoconiosis has a complicated form. most pneumoconioses result in shortness of breath and chronic cough, but pneumoconioses from certain types of dust, such as iron oxide, may have no evidence of functional impairment. systemic response arises from chemical toxicants that affect certain organs or tissues. target sites include respiratory tract, gastrointestinal tract, hematopoietic system, liver, kidney, bones, endocrine system, and nervous system. herbicides, pesticides and heavy elements such as mercury, lead, cadmium, arsenic, molybdenum, and selenium are known systemic toxicants. an example of systemic response is metal fume fever, an acute flu-like illness with symptoms of throat irritation and dry cough. the symptoms appear within hours following heavy exposure to fumes of metal oxides. irritant response generally results from inhalation of particulate sulfates, pesticides, and droplets of sulfuric acid or other strong acids. they cause inflammation in affected regions such as rhinitis, pharyngitis, laryngitis, bronchitis, pulmonary edema, and pneumonia. allergic response involves sensitization by initial exposure and reaction to subsequent exposure. such response can lead to bronchial asthma. examples of allergens include nickel, cobalt, arsenic, chromium, organic dusts, herbicides, and insecticides. inhalation is a route of entry for airborne mutagens (agents that produce changes in dna), teratogens (agents that cause developmental malformations), and carcinogens. among known or suspected chemical mutagens are ddt, sodium arsenate, cadmium sulfate, and some lead salts. examples of known or suspected chemical teratogens include dioxin, organic mercury, cadmium sulfate, and sodium arsenate. there is a broad variety of chemical carcinogens including cigarette smoke, soot, asbestos, arsenic, chromates, and certain petroleum products. changes in lung tissues can begin when an individual is exposed to a cancer-causing substance. a few abnormal cells may appear in the lining of bronchi. if exposure to the carcinogen continues, more abnormal cells can appear and lead to formation of a tumor. in addition to asbestosis, asbestos fibers can cause bronchial cancer and mesothelioma. the latency period is 20 years or longer. to have chronic toxicity, inhaled asbestos fibers must exceed the following critical length limits" 5 ~tm for mesothelioma and 10 ~tm for lung cancer. in addition, fiber width must be less than 0.1 ~tm to induce mesothelioma and larger than this limit to cause lung cancer (lippmann, 1990) . cigarette smoke, containing particles and vapors, has been identified to be the cause of a plethora of diseases. in addition to cancers of the lungs, larynx, esophagus, bladder, cervix, kidney, pancreas, and stomach, smoking can give rise to emphysema, chronic bronchitis, pneumonia, chronic heart and cardiovascular diseases, abdominal aortic aneurysms, acute myeloid leukemia, cataracts, and gum disease. central bronchial airways are the most common sites for lung cancer in cigarette smokers (schlesinger and lippmann, 1978) . the diseases resulting from exposure to ambient aerosols include pulmonary emphysema, bronchitis, and, perhaps, lung cancer. asthmatics are particularly susceptible to the adverse effects of aerosol acidity in ambient air. emphysema is a condition of over-inflation of structures in the alveolar region. the over-inflation arises from a breakdown of alveolar walls. early symptoms of emphysema include shortness of breath and cough. the primary cause of emphysema is cigarette smoking. there is also an inherited form of emphysema due to deficiency of a protein called alpha 1-antitrypsin. bronchitis is an inflammation of the lining of bronchial airways. when bronchial airways are inflamed, less air is able to flow to and from the alveolar region and a heavy mucus or phlegm is coughed up. cigarette smoking is the number one cause of chronic bronchitis. dusts and fumes in ambient air and workplaces are other common causes of this disease. higher rates of chronic bronchitis are found among workers exposed to particles of high concentrations, such as coal miners, grain handlers, and metal molders. emphysema and chronic bronchitis in combination comprise chronic obstructive pulmonary disease (copd). a copd patient has difficulty breathing because (1) the airways lose their elasticity and therefore cannot keep open properly; (2) some alveolar walls are destroyed; (3) airway walls are inflamed; and (4) cells in airways make more mucus than usual, which tends to clog the airways. ambient aerosol is a mixture containing numerous chemical species of natural and anthropogenic origins. these particles are either directly emitted into the atmosphere from many different types of sources or formed in air by gas-to-particle conversion processes. harmful components identified in highly polluted air include acid sulfate species, heavy metals, reactive organic compounds, and peroxides. at sufficiently high doses these components can produce local respiratory diseases or systemic disorders. however, none of the harmful components mentioned above exists in ambient particles at sufficiently high concentration levels to cause a specific disease. ambient particles are harmful when their concentration exceeds a certain limit; epidemiological studies have indicated a strong association of increases in human morbidity and mortality rates with increased concentrations of ambient particles in a certain size fraction (wilson and spengler, 1996; vedal, 1997) . complexity in chemical characteristics of ambient particles has led to considerable difficulty in identifying the components responsible for adverse health effects. thus, it remains largely unclear which specific components are responsible. concentration alone cannot explain the causal relationship. according to the threshold limit values recommended by american conference of governmental industrial hygienists, a worker can be exposed to nuisance or inert dust at the concentration of 5 mg/m 3 in the respirable fraction without measurable injury. questions have been raised whether ambient particles are a carrier of shortlived, difficult-to-quantify, but harmful compounds. friedlander and yeh (1998) have provided supporting evidence for the involvement of peroxides in the adverse health effects of particulate pollutants. reactive oxygen species (ros) in particles also have been shown to play an important role. highly reactive oxygen-containing species, such as hydroxyl radical and hydrogen peroxide, are collectively described as ros. in ambient air, photochemical reactions involving reactive organic gases can produce ros, which are distributed in the gas and particulate phases. combustion aerosols, such as vehicular exhaust, also contain relatively high concentrations of ros. hung and wang (2001) have reported that the concentrations of particulate ros in ambient air are strongly associated with photochemical activities. there is a good correlation between the concentrations of ambient ozone and ros in submicron particles, especially in the ultrafine fraction that are freshly produced by either photochemical reactions in ambient air or combustion processes in vehicular engines. aerosol particles can serve as an effective carrier for ambient peroxides and reactive oxygen species to reach the alveolar region. in the absence of particles, inhaled peroxides and ros mainly deposit in tracheobronchial airways because of their high solubility and diffusivity. when these reactive species are adsorbed on particle surfaces, they can easily reach the alveolar region and thereby lead to an adverse effect greater than in tracheobronchial airways. because of their small sizes and surface characteristics, ultrafine particles appear to be more toxic than larger particles. respiratory effects are shown to be associated with the number of ultrafine particles (peters et al., 1997) . a recent study indicates that ultrafine particulate pollutants are capable of inducing oxidative stress and mitochondrial damage (li et al., 2003) . pulmonary effects of ultrafine particles have been reviewed by oberd6rster (2001) . studies on rodents indicate that ultrafine particles administered to the lungs cause a greater inflammatory response than larger particles of the same total particle mass do. surface chemistry appears to be an important contributing factor of their high toxicity. furthermore, it appears that deposited ultrafine particles largely escape capture by alveolar macrophage and therefore have higher probabilities to enter the pulmonary interstitium. these results lend support to the hypothesis that ultrafine particles are causally involved in adverse responses seen in susceptible groups of the population. the need for further studies on ultrafine particles has been stressed in a couple of recent reports (national research council, 1998; friedlander and pui, 2003) . inhalation is an important route of entry for bacterial and viral pathogens. single bacterial particles can remain airborne for a long time and therefore have high probabilities to be transmitted through air. viruses in droplets generated by sneezing and coughing of a patient can survive for hours and gain access to another host while they are airborne. droplet size plays an important role in determining the atmospheric spread of infectious diseases. sneezing and coughing can produce droplets of various sizes. the distance to which a droplet can be transported depends on the rate at which the droplet evaporates. large droplets settle out of air quickly and, even if they are inhaled, may not reach the pulmonary region. for many pulmonary diseases, the susceptible part is the alveolar region of the respiratory tract. however, smaller droplets can evaporate rapidly to less than 10 ~m in diameter. it is mainly these residual particles, named droplet nuclei (wells, 1934) , that can spread the diseases. tuberculosis and bacterial pneumonia are two common respiratory diseases caused by bacteria. symptoms of tuberculosis include feeling tired, loss of appetite, fever, coughing up blood, and night sweats. pneumonia is a serious infection of the lungs. when the lungs are infected, the alveoli fill with pus and other liquid, thereby greatly reducing the ability to transfer oxygen to the bloodstream. legionnaires' disease is a form of bacterial pneumonia. it has acquired its name, because the first known outbreak occurred in the bellevue stratford hotel in philadelphia where a convention of the pennsylvania department of the american legion was held. in that outbreak, over 200 people contracted this previously unknown type of pneumonia as a result of exposure to mist from comaminated water that was used to cool the air in the hotel's air conditioning system. the disease can also be contracted by inhaling mist from water sources such as whirlpool baths and showers that are contaminated with legionella bacteria. anthrax is a disease caused by bacillus anthracis, a spore-forming bacterium. its symptoms are similar to a common cold initially, but progress to severe breathing problems and shock in a few hours to a few days. to contract the pulmonary form of anthrax, it requires inhalation of 8 to 20 thousand spores into the alveolar region of the lungs. droplets that deposit in the nose are not likely to cause the disease. examples of diseases due to viruses include influenza, common cold, viral pneumonia, and severe acute respiratory syndrome (sars). when a person is infected by influenza virus, the tissues of respiratory tract become swollen and inflamed. prominent symptoms of flu are high fever, muscle aches, severe headache, dry cough, and sore throat. patients with common colds have milder fever, stuffy nose, sneezing, coughing, and also sore throat. flu and common colds mostly spread through transfer of virus-containing droplets by inhalation or hand-to-hand contact. sars is a respiratory disease due to a type of coronavirus. first reported in asia in february 2003, its symptoms include high fever (temperature higher than 38.0~ shortness of breath, headache, coughing, diarrhea, and malaise. most sars patients develop pneumonia. according to the world health organization, a total of 8,098 people worldwide were infected during the 2003 outbreak. of these, 774 died. a probable transmission route of the sars virus is the spread of droplets produced by the cough or sneeze of a patient. infection can occur if these droplets deposit on the mucous membranes of the mouth, nose, or eyes of persons who are nearby. the most common radioactive particles in ambient air are radon daughters. after release from soil, radon gas continues to decay and generate a series of radioactive products. these radon daughters easily attach to ambient particles and become a source of radiation exposure for the general population. occupational exposure to radioactive particles mainly occurs in uranium, tin, and hematite mines. the primary health effect from inhalation of radioactive particles is cancer. damage caused by ionizing radiation at the cellular or molecular level can give rise to uncontrolled growth of cells. this is a stochastic effect re:~ulting from long-term, low-level exposure to radiation. increased doses lead to increase in probability of occurrence. all regions of the respiratory tract are susceptible, but the frequency of incidence differs from region to region. the types of radiogenic tumors and target tissues have been reviewed by icrp (1994) . soluble radionuclides in deposited particles can be absorbed into bloodstreams. depending on their metabolic behavior, these radionuclides can be retained in various organs for different periods of time. a broad variety of pharmaceutical agents can be effectively delivered through inhalation to treat respiratory and systemic diseases. examples of aerosolized agents for treating respiratory disorders include bronchodilators and mucolytics. antibacterial and antiviral agents have been used in aerosol form for treating pulmonary infections. for aerosol therapy of systemic illnesses, various degrees of success have been achieved with hormones, vaccines, antibiotics, and cardiovascular drugs. among the agents tested for systemic medication, aerosolized insulin and heparin appear to be very promising. two proven classes of aerosolized drugs for treatment of asthma and chronic obstructive pulmonary disease are 132-adrenergic agonists and glucocorticoids. inhalation of 132-adrenergic agonists can relax bronchial smooth muscle, stimulate skeletal muscle, and inhibit release of inflammatory mediators. short-acting 132-adrenergic agonists can provide bronchodilation within minutes of inhalation. glucocorticoids are useful for treatment of inflammation. anticholinergics are among other common agents for treatment of asthma and chronic obstructive pulmonary disease. as bronchodilators, anticholinergics agent have a slower onset but a comparable duration of action as 132-adrenergic agonists. aerosolized formulations of corticosteroids have become commonly used agents in treating respiratory diseases such as asthma, cystic fibrosis, and obstructive lung disorders. these formulations are able to influence existing inflammation. mucolytics such as n-acetylcysteine can give rise to rapid liquefaction of viscid mucus. when administered in aerosol form, they can help maintain adequate mucus viscosity in patients with chronic obstructive airway disease. 11.1 a review of the sars epidemic in 2003 indicates that, in some countries, a majority of cases were caused by nosocomial infection. discuss possible transmission routes of the sars coronavirus in hospitals. 11.2 a potential terrorist weapon is the release of plant and animal pathogens in aerosol form. these pathogens can have devastating effects on agriculture if they are released at points where they can spread out rapidly. what strategies can be used to protect agriculture from such terrorist attacks? the submicron atmospheric aerosol as a carrier of reactive chemical species: case of peroxides emerging issues in nanoparticle aerosol science and technology (nast) experimental determination of reactive oxygen species in taipei aerosols human respiratory tract model for radiological protection ultrafine particulate pollutants induce oxidative stress and mitochondrial damage effects of fiber characteristics on lung deposition, retention, and disease research priorities for airborne particulate matter. i. immediate priorities and a long-range research portfolio pulmonary effects of inhaled ultrafine particles respiratory effects are associated with the number of ultrafine particles selective particle deposition and bronchogenic carcinoma ambient particles and health: lines that divide on air-borne infection; ii. droplets and droplet nuclei particles in our air: concentrations and health effects key: cord-313825-bbjxd86y authors: xia, tian; zhu, yifang; mu, lina; zhang, zuo-feng; liu, sijin title: pulmonary diseases induced by ambient ultrafine and engineered nanoparticles in twenty-first century date: 2016-10-08 journal: natl sci rev doi: 10.1093/nsr/nww064 sha: doc_id: 313825 cord_uid: bbjxd86y air pollution is a severe threat to public health globally, affecting everyone in developed and developing countries alike. among different air pollutants, particulate matter (pm), particularly combustion-produced fine pm (pm(2.5)) has been shown to play a major role in inducing various adverse health effects. strong associations have been demonstrated by epidemiological and toxicological studies between increases in pm(2.5) concentrations and premature mortality, cardiopulmonary diseases, asthma and allergic sensitization, and lung cancer. the mechanisms of pm-induced toxicological effects are related to their size, chemical composition, lung clearance and retention, cellular oxidative stress responses and pro-inflammatory effects locally and systemically. particles in the ultrafine range (<100 nm), although they have the highest number counts, surface area and organic chemical content, are often overlooked due to insufficient monitoring and risk assessment. yet, ample studies have demonstrated that ambient ultrafine particles have higher toxic potential compared with pm(2.5). in addition, the rapid development of nanotechnology, bringing ever-increasing production of nanomaterials, has raised concerns about the potential human exposure and health impacts. all these add to the complexity of pm-induced health effects that largely remains to be determined, and mechanistic understanding on the toxicological effects of ambient ultrafine particles and nanomaterials will be the focus of studies in the near future. air pollution is a major global problem associated with human health in both developing and developed countries [1] [2] [3] [4] [5] [6] [7] [8] . the most common sources of air pollution include particulate matter (pm), ozone, nitrogen dioxide and sulfur dioxide [1, 3, 5, 6] . pm can be defined by their different size ranges, including coarse (<10 μm), fine (<2.5 μm) and ultrafine particles (ufps, <100 nm) [4] [5] [6] 9] . because numerous studies have shown that fine pm 2.5 particle levels can be linked to premature mortality and adverse health effects, pm 2.5 levels are often used as a major surrogate for air pollution [1, [3] [4] [5] [6] 9, 10] . according to the world health organization (who) ambient air pollution database that comprises urban air-quality data for about 1600 cities from 91 countries for the years 2008-13, almost 90% of the urban population endured a pm 2.5 concentration exceeding the annual mean values of 10 μg/m 3 in the who air-quality guidelines [6] . in addition, who reported that, in 2012, around 7 million people died-one in eight of the total global deaths-as a result of air pollution exposure [5] . the new data revealed a stronger link between both indoor and outdoor air pollution exposure and the development of respiratory diseases, including acute respiratory infections, chronic obstructive pulmonary disease (copd) and lung cancer, as well as cardiovascular diseases such as strokes and ischemic heart disease [5, 6, 11] . this finding further confirmed that air pollution is now the world's largest single environmental health risk factor and reducing air pollution could save millions of lives. globally, we are witnessing two opposing trends in terms of changes in the air pollution levels in different countries. using new high-resolution global review xia et al. 417 satellite maps of air quality indicators, nasa scientists tracked air pollution trends from 2005 to 2014 in various regions and 195 cities globally [12] . according to the research findings, the usa, europe and japan have improved air quality owing to strict emission-control regulations. as a result, in the usa, reductions in pm 2.5 were associated with lower premature mortality and improvement in life expectancy. long-term improvements in air quality were associated with statistically and clinically significant positive effects on lung-function growth in children [13] . on the contrary, china, india and the middle east countries, with their rapid population growth, fast-growing economies and vastly expanding industrialization, have shown alarming increases in air pollution, along with increased mortality and disease burden in those countries [14] . on a positive note, in recent years, china's overall air quality has seen early signs of improvement due to a decrease in coal consumption, increase in renewable energy sources and stricter emission control. it is worth noting that, although air pollution mostly is considered a local or regional problem, studies have shown that air pollution could be transported over long distances, even across continents, in the troposphere [15] . thus, mitigation of local air pollution could have far-reaching health benefits and a collective effort between every country involved would be important. despite the progress in our understanding of air pollution-induced diseases, much research remains to be done [1, 8, 9, 14, 16] . for instance, among the pm fractions, ufps possess the highest particle number and surface area, carrying higher chemical contents than pm 2.5 [4, [17] [18] [19] [20] . studies have shown that ufps have detrimental effects on both the respiratory and cardiovascular systems, and exacerbation of asthma [2] [3] [4] 17, 21, 22] . however, our understanding of ufps is still incomplete because of a deficiency in extensive ufp-monitoring networks, rapid physicochemical characterization techniques, and limited epidemiological and toxicological studies. in addition, the rapid advances in nanotechnology lead to production of high volumes of nanomaterials and increasing use in commercial products [18, 23, 24] . the use of nanomaterials increases the potential for human exposure to nanomaterials that could generate adverse health effects. for instance, ceria (cerium dioxide) are used in diesel as a catalyst to promote the combustion process, which are released in the diesel exhaust, potentially leading to lung exposure. the addition of ceria to diesel fuel resulted in ceria-concentration-dependent emission reductions of co 2 , co, total particulate mass, formaldehyde, acetaldehyde, acrolein and several polycyclic aromatic hydrocarbons; however, it also led to decreases in the size of emitted particles and a substantial increase in the number of ufps (+32%), together with increases in certain other air pollutants, specifically nox (+9.3%) and the particle-phase benzo[a]pyrene toxic equivalence quotient (+35%) [25] . this shows the complexity of the potential impacts introduced by nanomaterials. given the health concerns related to ufps and increasingly produced and used nanoparticles, further research is needed to evaluate the health risks associated with these tiny particles. evidence that links ambient pm to a decrease in life expectancy and increase in premature mortality came from epidemiological studies that have been extensively reviewed before [1, 7, 8, 24, [26] [27] [28] . in a recent study, chen et al. presented findings that implicated long-term exposure to air pollution particles contributed to enormous loss of life expectancy in china [26] . these results were based on an experimental design making use of a chinese policy that provided free coal for heating in cities located north of huai river, but not in the south, which produced an arbitrary discontinuity for pm air pollution, where the major difference was coal combustion. as a result, mean life expectancy is about 5.5 years (95% conficence interval (ci): 0.8, 10.2) lower in northern compared with southern china due to an increased incidence of cardiorespiratory mortality [26] . this finding correlated well with a study by pope et al. that used a temporal difference in pm levels observed since the 1990s, when air quality across cities in the usa improved substantially. they found that there was an association between reductions in pm 2.5 and an increase in life expectancy; a reduction of 10 μg/m 3 was associated with an increase of 0.61 years in life expectancy [8] . there was also a strong evidence base for morbidity and mortality associated with both short-term (days to weeks) and long-term (years to decades) pm exposures. early evidence linking ambient pm to mortality came from welldocumented short-term extreme air pollution episodes (that lasted for days) in the 1930s to 1950s [28] . more recently, numerous daily time-series and case-crossover studies have observed a small but statistically robust relationship between daily mortality and short-term (days to weeks) elevation in pm [28] . in addition, short-term air pollution exposure could also increase the mortality rate of patients with respiratory diseases. for example, cui et al. evaluated air pollution using the air pollution review figure 1 . the contribution of outdoor air pollution sources to premature mortality on a global scale. the data were derived from a global atmospheric chemistry model that links premature mortality to outdoor air pollution, mostly by pm 2.5 . study found that, in 2010, outdoor air pollution, mostly by pm 2.5 , leads to 3.3 million premature deaths per year worldwide, predominantly in asia. unit of mortality was expressed as deaths per area of 100 km × 100 km (color-coded). in the white areas, annual mean pm 2.5 and o 3 are below the concentration-response thresholds where no excess mortality is expected. the figure was originally published by [14] and has been approved for reuse by nature publishing group. index (api) derived from the concentrations of particulate matter, sulfur dioxide, nitrogen dioxide, carbon monoxide and ground-level ozone and their relationship with the case fatality of severe acute respiratory syndrome (sars) in china [29] . case fatalities of patients from regions with high apis (api > 100) and moderate apis (75-100) were compared with that of patients from regions with low apis (api < 75). the study showed that the case-fatality rate increased with the increment of api (case fatality = -0.063 + 0.001 × api). the correlation coefficient between api and sars fatality was 0.8568 (p = 0.0636) [29] . short-term exposure demonstrated that sars patients from regions with moderate apis had an 84% increased risk of dying from sars compared with those from regions with low apis (relative risk (rr) = 1.84, 95% ci: 1.41-2.40). similarly, sars patients from regions with high apis were twice as likely to die from sars compared with those from regions with low apis (rr = 2.18, 95% ci: 1.31-3.65). for long-term studies, two large-scale prospective cohort studies in the usa showed that there were statistically robust associations between mortality risk and pm 2.5 exposure even after smoking and other risk factors were controlled for [8, 27] . long-term air pollution exposure could also increase the mortality rate of patients with respiratory diseases such as sars [29] . although ecologic fallacy and uncontrolled confounding effects might have biased the results, the possibility of an effect of air pollution on the prognosis of sars patients was indicated [29] . in a recent study, lelieveld et al. used a global atmospheric chemistry model to investigate the link between premature mortality and ambient pm 2.5 concentrations [14] . the authors found that more than 3.2 million deaths per year could be attributed to outdoor pm 2.5 exposure. the majority of the mortality happened in asia, which strongly influenced the global mortality rate. the highest number of deaths was in the western pacific, where china was the main contributor (1.36 million per year). southeast asia had the second highest premature mortality, where india was the main contributor (0.65 million per year) ( fig. 1) [14] . this was in addition to the estimated 3.54 million deaths per year caused by indoor air pollution resulting from biomass or coal combustion for cooking and heating [14] . the cause of premature mortality includes lung diseases, such as chronic obstructive pulmonary disease (copd), lung infection and cancer (described in detail below), as well as cardiovascular diseases and cerebrovascular diseases, etc. [14] . in addition, many birth cohort studies have linked pm 2.5 exposure to asthma and allergic diseases [3, 30] . altogether, a large amount figure 2 . general toxicological pathways linking pm lung exposure to cardiovascular and cerebrovascular diseases that cause morbidity and mortality. the first line of defense against pm is the lung, where pm can induce or exacerbate lung diseases including copd, asthma, lung infection disease and lung cancer. furthermore, ufps could translocate out of the lung into the blood stream and can cause systemic inflammation and oxidative stress that negatively impact blood and blood vessel, heart function and brain. of literature provided evidence that breathing combustion-related pm 2.5 , even at exposure levels common to urban populations worldwide, contributed to cardiorespiratory disease mortality and diminished the lifespan [1, 7, 8, 14, 28] . there was also encouraging evidence showing that improvement in air quality benefited human health and increased the lifespan [8, 27] . for example, long-term improvements in air quality were associated with a significant improvement in lung function in children [13] . however, there are no conclusive data yet available for the human health impacts of ambient ultrafine particles and engineered nanoparticles, which warrant further studies. the lung is the first target organ for air pollution and pm exposure is associated with reduced lung function, increased lung inflammation, asthma, respiratory infections, lung cancer and exacerbation of copd, which lead to systemic inflammation and oxidative stress affecting blood, vasculature, heart and brain, ultimately contribute to the premature mortality ( fig. 2) [3, 8, 14, 16] . it is notable that the acute adverse health effects of pm are most often found in susceptible populations, including children, elderly people and those with chronic diseases [3, 7] , while not obvious for short-term exposure in normal healthy people except at very high concentrations [17] . however, air pollution exposure could induce adverse health effects to normal healthy people [17] . a panel study conducted among chinese healthy adults during the beijing olympics found that peak expiratory flow levels increased in 78% of the participants when compared with the during-and pre-olympics time points, while peak expiratory flow levels decreased in 80% of participants for the post-and during-olympic periods comparison [31] . children are more susceptible to air pollution because they have smaller airways, higher breathing rates per body mass, immature detoxification and metabolic systems, and they are more frequently exposed to outdoor air [3, 10] . the elderly population and people with chronic diseases are susceptible likely because of less efficient particle clearance in the lung or impairment of immune functions. the adverse effects induced by air pollution are not limited to review the lung, pm could induce systemic inflammation and oxidative stress, and nano-sized particles could even translocate out of the lung to other tissues and organs, which induces pathological changes in blood, vasculature, heart and brain ( fig. 4) [17, 23] . the main pulmonary diseases that are associated with pms are the following. copd is not only associated with smoking, but also has high incidence among non-smokers [3, 32, 33] . the risk factors for copd among non-smokers include indoor air pollution from biomass combustion and second-hand tobacco smoke, as well as occupational exposures and outdoor air pollution [32] [33] [34] . epidemiological studies from both developing and developed countries have shown the association between outdoor pm exposure and copd. in addition to pm, ozone and no 2 could also exacerbate copd. an increase in ambient pm 2.5 could induce acute exacerbations and mortality from copd, while improvement in air quality decreases the incidence of copd [32] [33] [34] . abundant evidence has shown that air pollution could induce and exacerbate asthma [35] . in a recent meta-analysis of several birth cohort studies, the authors suggested that early childhood exposure (1-12 years old) to traffic-related air pollution (trap) containing ufps and pm 2.5 were associated with increased incidence of asthma and risk of sensitization to common allergens [30] . furthermore, a 10-year longitudinal study of 23,704 adults from eight european countries showed an association between trap exposure and increased incidence of asthma [36] . the authors found that adult asthma incidence was positively associated with exposure metrics, including pm 10 , pm 2.5 , nitrogen oxides, traffic load and intensity. further research with improved personal-level exposure assessment (vs. residential exposure assessment only) and phenotypic characterization was recommended by the authors [36] . in october 2013, the international agency for research on cancer (iarc) of who announced that outdoor air pollution and pm were classified as a group i carcinogen, which was determined based on the most recent data from human, animal and mechanistic studies [11] . in one of the first studies of lung cancer among female non-smokers in china with measured indoor exposure to pm 1 , pm 2.5 , pm 7 , pm 10 and total suspended particulate (tsp), mu et al. reported that the pm 1 levels in cases were more than three times higher than those in the controls. every 10 μg/m 3 increase in pm 1 is associated with a 45% increased risk of lung cancer [37] . multiple studies in recent years have shown a correlation between air pollutants including pm 2.5 and no 2 and lung cancer risk [3, [38] [39] [40] . in 2010 alone, 223,000 deaths from lung cancer worldwide were associated with air pollution [11] . recent data showed that air pollution was associated with respiratory infections, due to the impaired immune functions of the lung and the susceptible population including children, elderly people and those with chronic diseases [41, 42] . two recent epidemiological studies demonstrated associations between air pollution (trap and ozone) and acute respiratory infections as well as increased emergency department visits in young children [41, 42] . the most common sources of air pollution include pm, ozone, nitrogen dioxide and sulfur dioxide [3, 7, 14] . among these components, pm has been shown to play a major role in human morbidity and mortality [3, 7, 14] ; thus, we will focus our discussion on pm. pm comes from natural and anthropogenic activities and processes [3, 4, 7, 14, 24] . anthropogenic sources of outdoor pm are hightemperature processes (e.g., welding, smelting), combustion (e.g., power generation, land traffic, residential and commercial heating and cooking), industrial and other processes, including agriculture, dust and biomass burning. indoor pm sources are mostly biomass and coal combustion for cooking and heating purposes [3, 4, 7, 14, 24] . although the physiochemical properties (chemical composition, metal content, etc.) of pm are different worldwide, they are all associated with adverse human health effects although at different potency levels [14] . pm 10 is referred to the mass of particles collected with 50% efficiency for particles with an aerodynamic diameter equal to or less than 10 μm; it should be noted that all particles down to the ultrafine size range were collected [17] . at this size range, according to the international standards organization thoracic convention, the mass fraction of inhaled particles could penetrate beyond the larynx to the airways [23] . pm 2.5 refers to the respirable fraction . tem images of ambient ultrafine particles and engineered nanoparticle as well as particle characteristics that promote or contribute to ros generation. (a) photoactivation effects of nps, such as the formation of electron-hole pairs during ultraviolet exposure of tio 2 ; this effect has been associated with the generation of oxidative stress and inflammation by tio 2 ; (b) discontinuous crystal planes and material defects of nps that lead to oxygen radical generation due to the active electronic state of the material surface; (c) redox cycling contributes to ros production. this can occur due to the presence of transition metals or redox-cycling organic chemicals on the pm, ufp and np surfaces. pms and ufps, for example, contain organic compounds such as quinones, which can generate ros through redox cycling. moreover, transition metals can generate hydroxyl radicals through the fenton reaction. the fenton reaction is one of the mechanisms by which metal impurities on the cnt surface can induce ros production. finally, (d) particle dissolution (e.g. zno, cdse, cu) can produce free ions that are capable of inducing ros generation and oxidative stress in cells. metal fume fever is a real-world example of this toxicity, commonly for welders. that also contains the ultrafine component, which penetrates to the unciliated regions of the lung and is now being considered worldwide as the standard [23] . ultrafine particles are found to a large extent in urban air as both singlet and aggregated particles (fig. 3) , and indeed are the predominant particle type by number in urban pm 10 and pm 2.5 , although they contribute insignificantly to mass [4, 18, 23, 24] . pm from combustion processes characteristically has an elemental or organic carbon core carrying trace metals, sulfate, ammonium, and volatile and semi-volatile components [43, 44] . the composition of combustion-generated pm usually depends on fuel type, burn conditions and atmospheric conditions. compared with pm 10 and pm 2.5 , traffic-derived ufps are challenging to characterize geographically or spatiotemporally, as their concentrations decrease sharply downwind from sources, and ufps shift in size from nucleation mode to accumulation mode with time and distance from their emission point due to agglomeration and condensation [45] [46] [47] . for combustion sources, the fuel, combustion conditions and pollution controls will alter the particle numbers and size distribution of the pm emitted [43, 44] . however, studies have shown that ufps carried more organic chemicals due to their significantly larger surface area; many of these compounds were redox-active and had the ability to generate reactive oxygen species (ros) [43, 44] . studies have shown that ufps were more toxic than their larger counterparts; however, more research is needed to clarify the role of ufps [17, 21, 22] . nanoparticles (nps) are intentionally created with specific size, shape, surface characteristics and functionality that are required for their applications [18, 23] . there are similarities between nps and ufps; however, there are also major differences (table 1) . nps and pm including ufps could both generate ros or release toxic ions through dissolution, through similar or different mechanisms (fig. 3) [18, 24, 48] . although currently there is no definitive evidence to link np exposure to any human disease, much experimental data indicate that some nps with certain physicochemical properties may be potentially hazardous [19, [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . obviously, more research is needed on this front. the potency of pm in causing an adverse health impact is dependent, in part, on its deposition in the airways and chemical composition [17, 23] . the main deposition mechanisms in the respiratory tract for inhaled pm include impaction, sedimentation, interception and diffusion [23] . pm in different size ranges has drastic differences in distribution and deposition in the lung [23] . this is clearly demonstrated in fig. 4 , which shows the differential deposition of inhaled particles with different sizes in the nasopharyngeal, tracheobronchial and alveolar regions of the human respiratory tract [23] . for ufps and nps that are in the size range of 1-100 nm, the particles could penetrate deeper into the alveolar region and deposit there at high percentages. for larger particles including pm 10 and pm 2.5 , they generally deposit in the nasopharyngeal and laryngeal region and poorly deposit in the alveolar region (fig. 4) [23]. the different deposition profiles among particles with diverse sizes could at least partially explain their different health effects [17, 23] . for example, the deposition site determines the clearance rate of particles [17, 23] . for large particles including pm 10 and pm 2.5 , the majority deposited in the upper airways could be removed with the mucus via the mucociliary escalator (fig. 5) . the small frac-tion of particles that could pass through upper airways into the alveolar region (fig. 4) , such as pm 2.5 fine particles, could be easily endocytosed and removed by alveolar macrophages (fig. 5) . however, for ufps and nps, the majority of these particles could pass through the airways into the alveolar region. although they also deposit in the larger airways, this only accounts for a small percentage of review xia et al. 423 figure 5 . scheme for physicological and toxicological events occurring after pm 2.5 and ufp/np exposure. pm 2.5 mostly sticks to the airway, where they are cleared by mucociliary escalator. the remaining pm 2.5 particles that get inside the alveoli will be phagocytosed by resident macrophages. however, for ufps and nps, the majority of the particles will get inside the alveoli, where the cellular uptake of nano-sized particles by macrophages is impaired, leading to delayed clearance of particles and generating oxidative stress and pro-inflammatory effects by macrophages and epithelial cells because of prolonged exposure to particles. the prolonged interaction between particles and the epithelium also lead to extrapulmonary translocation to the lung interstitium and even into the circulation. adapted from [17] . the total number of particles in the lung. furthermore, the particles are so tiny and in such vast numbers that macrophages could not take them up effectively (fig. 5 ) [17, 23] . the interactions among particles, epithelial cells and macrophages could generate oxidative stress and pro-inflammatory effects in the lung, and there are reports showing that nano-sized ufps could be more toxic than their larger counterparts (which will be discussed in later sections) [2, 17, 21, 23] . in addition, extrapulmonary translocation across the epithelium could occur because of the reduced removal rate and longer retention of ufps and nps that allows transcytosis of these nano-sized particles [17, 23] . this could lead to secondary deposition of these particles in other tissues and organs, which may contribute to further adverse health effects (fig. 5 ) [17, 23] . emerging evidence has shown that, among different particles, ufps are potentially the most dangerous owing to their small size, deep penetration, large surface area/volume ratio, high content of redox-cycling organic chemicals, alveolar deposition and high rates of retention in the lung [4, 21, 22] . compared with pm 10 and pm 2.5 , traffic-derived ufps are capable of carrying more organic chem-icals on their significantly larger surface; many compounds (polycyclic aromatic hydrocarbons (pahs) and quinones) are redox-active and have the ability to generate ros [4, [20] [21] [22] 24, 44, 60] . while pm 10 and pm 2.5 can be easily removed by phagocytosis, the extremely small size of ufps enables them to evade such a process and ufps could have more interactions with other cell types in the lung (fig. 5 ) [17, 23] . these specific features of ufps can significantly contribute to the adverse effects through ros over-production by the redox-active organic chemicals and metals on particle surface, resulting in cellular oxidative stress [18, 19, 21, 44, 48] . oxidative stress has been identified as a major mechanism for pm 10 -, pm 2.5and ufp-associated health effects, including exacerbation of asthma and copd, and promotion of atherosclerosis [18, 19, 21, 22, 44, 48] . some nps, including carbon nanotubes, silver nanoparticles, zno, cuo, etc., have also been shown to be able to generate ros and oxidative stress [19, 55, 56, 58, 59, 61] . at the cellular level, particleinduced oxidative stress can activate a cascade of signaling pathways that mediate the production of pro-inflammatory cytokines/chemokines and induce apoptosis (figs 6 and 7 ) [18, 48, 53] , resulting in inflammation and tissue injury in the respiratory and cardiovascular systems [17, 33, 38] . pm-induced ros production in biological systems and targeted cells originates from a variety of sources ( fig. 6 ) [18] [19] [20] [21] 24, 44, 60] . these include: (i) carbon core of pm and ufps could induce ros generation and oxidative stress; (ii) catalytic conversion of pahs to quinones by cytochrome p450 in the endoplasmic reticulum; (iii) quinone redox cycling by nadph-dependent p450 reductase in microsomes; (iv) mitochondrial perturbation leading to electron leakage in the inner membrane; and (v) nadph oxidase activity in the macrophage surface membrane and associated phagosomes. both the carbon cores as well the chemicals coated on their surface play a role in these biological events [2, 17, 44] . this also includes the involvement of transition metals in generating ros by a fenton reaction [2, 17, 44] . the pm backbone is formed by chain-like carbon-based nanoparticles. to see whether the particles themselves could induce ros and adverse effects, ultrafine carbon black was selected as the model particle. studies found that ultrafine review figure 6 . sources of pm-and ufp-induced ros production and their cellular effects. quinones, under the catalytic influence of nadph-cytochrome p450 reductase, can redox cycle to produce ros in the endoplasmic reticulum. phagocytosis can induce the assembly and activation of nadph oxidase to produce superoxide. ufps can interfere in electron transduction in the mitochondrial inner membrane as well as perturb the pt pore to generate ros. ros induce lipid peroxidation in the cell membrane, cross-linking of protein thiol (sh) groups and dna damage. ros can also deplete glutathione (gsh), resulting in oxidative stress in the cell. depending on the levels of oxidative stress, the response could range from induction of nrf2 release to the nucleus, activation of mapk and nf-kb signaling cascades, or cytotoxicity. according to the hierarchical oxidative stress hypothesis, nrf2 interaction with the antioxidant response element (are) leads to heme oxygenase 1 and other phase ii enzyme expression at lower levels of oxidative stress (tier 1), while, at a intermediary level of oxidative stress, activation of the mapk and nf-kb signaling cascades can induce pro-inflammatory responses (e.g. cytokine and chemokine production) (tier 2). at the highest oxidative stress level (tier 3), ros can induce the opening of the mitochondrial pt pore, followed by cytochrome c release, caspase-3 activation and induction of programmed cell death. carbon black could generate ros in cell-free systems and cause oxidative stress to cultured cells [2, 17, 23] . in addition, ultrafine carbon black caused systemic pro-inflammatory responses in the lung including modest neutrophil influx and protein leak [17, 23] . there was evidence of systemic oxidative stress in the plasma and increased production of plasma factor vii, which is an independent risk factor for cardiovascular disease [17, 23] . redox-cycling organic chemicals on pm surfaces, such as quinones, are capable of generating ros in cellular targets such as bronchial epithelial cells, macrophages and endothelial cells [24, 44, 60] . quinones are byproducts of fossil fuel combustion, as well as the enzymatic conversion of pah in the lung. redox-cycling quinones undergo one-electron reductions mediated by nadph-cytochrome p450 reductase to form semiquinones. these semiquinones are metastable and donate electrons to o 2 , leading to the formation of o 2 •− . due to their high content of organic chemicals, ambient ufps contribute proportionally more redox-cycling chemicals than larger particles [4, 24, 44, 60] . catalytically active metals adsorbed on the pm surface have been shown to contribute to oxidative stress in vitro and in vivo [4, 24, 60] . pm contains a number of transition metals (coarse > fine > ufp) that contribute to ros production. among the transition metals, fe, al, cu, ni, mn, zn, cr, ba and sr are the most abundant [44] . in the presence of hydrogen peroxide, some of these metals, including fe 2+ , have the ability to generate the hydroxyl radical ( • oh) through catalysis of a fenton reaction: the • oh radical is more reactive than o 2 •− and hydrogen peroxide by several orders of magnitude. besides ros generation, transition metals also have the ability to directly perturb the function of the mitochondrial permeability transition (pt) pore [24, 44, 60] . transition metals may also act synergistically with other pm components in impacting mitochondrial function, ros generation, atp production and cell viability [24, 44, 60] . pm components could perturb mitochondrial function, leading to generation of ros and cell death [21, 60] . mitochondria have been shown to be a direct subcellular target for ambient ufps. for example, ambient ufps have been found to lodge in the mitochondria of target cells and cause mitochondrial damage [21] . although the mechanism of mitochondrial localization is still elusive, we hypothesize that particle size, hydrophobicity and presence of organic chemicals may play a role in the process. in addition to direct effects, studies have found that organic pm chemicals are capable of generating ros by their ability to interfere in these electron transfer events. this includes the effect of polar chemicals such as redox-cycling quinones to disrupt electron transfer in the inner membrane [44] . this disruption in electron flow could favor the formation of ubisemiquinones, thereby contributing to mitochondrial o 2 •− production. in addition, organic chemicals such as quinones and pahs also have the capability to perturb the mitochondrial pt pore (fig. 6) [44, 60] . the pt pore is a redox-, ph-, calcium-and m -dependent protein complex, which plays a pivotal role in regulating mitochondrial function and controlling cellular apoptosis. opening of the pt pore could lead to mitochondrial swelling and rupture of the mitochondrial outer membrane, by which various proapoptotic proteins such as cytochrome c, smac, apoptosis-inducing factor (aif), etc., are released . potential mechanisms of pm-, ufp-and np-induced nlrp3 inflammasome activation and il-1β production. activation of nlrp3 inflammasome complex can be induced by various stimuli including pm and np particles, and the pro-il-1β is processed by the inflammasomes to produce mature il-1β, while il-1β plays a major role in inducing the pro-inflammatory and pro-fibrogenic effects in the lung. nlrp3 inflammasome activation requires two signals in vitro. for signal 1, pathogen-associated molecular patterns (pamps) such as lps is recognized by toll-like receptor 4 (tlr4) residing on the cell membrane, which further leads to nf-κb activation and the production of pro-il-1β and nlrp3 proteins. nlrp3 inflammasome activation mechanisms include ros generation, potassium efflux, lysosomal damage and cathepsin b release. after phagocytosis of pm, ufps and nps, nadph oxidase is activated to generate ros. the over-production of ros may cause the destabilization and permeabilization of lysosomes and cathepsin b release, which will initiate the inflammasome activation cascade. mitochondrion is another important source of ros in cells. over-production of mitochondrial ros could lead to release of mtdna and cardiolipin that could induce nlrp3 inflammasome activation. in addition, potassium efflux induced by particles could also induce mitochondrial and cellular ros production leading to nlrp3 inflammasome activation. levels of activated nlrp3 inflammasomes are tightly regulated by autophagy. into the cytosol where they activate a number of apoptotic pathways, ultimately leading to programmed cell death [18, 24, 44, 48, 60, 62] . pm-, ufp-and np-induced oxygen radical generation can result in cellular and tissue injury responses such as inflammation, apoptosis, necrosis, fibrosis and carcinogenesis [18, 20, 48, 63] . cellular oxidative stress can generate a wide range of responses that can be experimentally detected, including damaged dna-mainly dna single-strand breaks or generating 8-oxo-2 -deoxyguanosine (8oxo-dg) that promote cell turnover and proliferation [63] . at the lowest level of oxidative stress (tier 1), the induction of antioxidant and protective responses is mediated by the transcription factor nrf2, which regulates the activation of the antioxidant response element in the promoters of phase ii genes [18, 64] . as the levels of oxidative stress increases (tier 2), this protective re-sponse may yield to pro-inflammatory responses because ros induces redox-sensitive signaling pathways such as the mitogen-activated protein kinase (mapk) and nuclear factor-kappa b (nf-κb) cascades [18, 20, 24, 53, 60] . at the highest level of oxidative stress (tier 3), a perturbation of mitochondrial inner membrane electron transfer and the open/close status of the pt pore can trigger cellular apoptosis and cytotoxicity. this outcome is also known as toxic oxidative stress (fig. 6 ) [18, 20, 24, 53, 60] . using this three-tier screening platform, we have also shown for ambient airpollution particles that one can link the hierarchical oxidative stress paradigm to the in vivo outcomes in animal disease models [18, 20, 24, 53, 60] . oxidative stress is also involved in mutagenesis [65] . future screening could also include dna damage and mutagenesis tests to assess their carcinogenic potential. nps are intentionally created with specific size, shape, surface characteristics and functionality that are required for their applications [18, 23, 24, 48, 53, 66] . although currently there is no definitive evidence to link np exposure to any human disease, experimental data indicate that many nps may be potentially hazardous [19, [49] [50] [51] [52] [54] [55] [56] 58, 59, 61, 67, 68] . the physicochemical characteristics of nps that may have health implications include particle size, shape, aspect ratio, composition, surface reactivity, solubility and ability to generate ros [18, 48, 53] . similar to ufps, the nano-scale size can enhance np translocation and deposition by interfering with their clearance [17, 23] . np composition or modification may affect particle surface reactivity such as the ability to generate ros, which is an important mediator involved in various human diseases. however, ufps and nps are inherently different in many aspects, such as source, morphology, organic chemical content, homogeneity, ros production, possible exposure route and the potential adverse health effects (table 1) . extensive in vitro and in vivo studies have been performed to elucidate the toxic effect of engineered nanomaterials (enms) and physicochemical properties of enms, including morphology, size, dissolution, aspect ratio, surface coating, surface reactivity, bandgap and aggregation, have been suggested to determine their toxicity potentials [17, 18, 23, 48, 53] . for example, ceo 2 has excellent antioxidant properties that are enabled by the duality of the cerium ion to easily cycle review between ce 4+ and ce 3+ ; it is widely used as a catalyst or fuel additive in diesel to facilitate the combustion process and is released into the air as exhaust. there are reports over the past few years that utilize the antioxidant property of ceo 2 in treating diseases such as cancer, alzheimer's, cardiac arrest, radiation-induced cell death and aging [69] . however, a recent study found that ceo 2 nps could lead to a decrease in cell viability and the treated cells exhibited characteristic hallmarks of apoptosis [70] . in this case, ceo 2 np toxicity is caused by mitochondrial damage leading to aif release, but not caspase activation or ros production. moreover, ceo 2 np exposure leads to autophagy and inhibition of autophagy partially reverses cell death by ceo 2 nps [70] . obviously, more research should be done to clarify the effects of ceo 2 under in vitro and in vivo conditions. tio 2 is the most widely produced nanomaterial and is found in cosmetics, sunscreen, paint, vitamins, toothpaste, food colorants, nutritional supplements, etc. however, study has shown that tio 2 nanoparticles could cause oxidative stress-mediated acute lung inflammation. oberdorster et al. have shown that, on a mass-dose basis, ultrafine tio 2 is more toxic than fine tio 2 [23] . however, when the doses of particles were expressed in forms of particle surface area, the responses of ultrafine and fine tio 2 particles fell on the same dose-response curve. this suggests that surface area is an important property for nps when considering their toxic potential. also, there is a study showing that tio 2 in anatase form was more potent in ros generation than in rutile form, revealing the important role of crystal structure [23] . under low levels of ultraviolet light, tio 2 nanoparticles could generate ros and induce cytotoxicity because of their photoactivation property [71] . another example is zno nanoparticles, which have received significant attention due to their wide use in sunscreens, electronics, optics and photonics. however, pulmonary exposure of zno nanoparticles could lead to transient increases in acute lung inflammation, similarly to a disease called metal fume fever. it has been shown that the toxicity of zno is dependent on the particle dissolution property and shedding of toxic zn ions, which induce oxidative stress and pro-inflammatory effects in vitro and in vivo [58, 68, 71] . in addition to oxidative stress paradigm, recently studies have found nod-like receptor protein 3 (nlrp3) inflammasome activation plays a major role in pm-and np-induced chronic effects such as lung fibrosis [49] [50] [51] 55, 56, 61, [72] [73] [74] [75] . nanomaterials including rare earth oxides (reos), high-aspectratio materials including carbon nanotube (singlewalled and multi-walled), tio 2 nanobelts and ceo 2 nanorods, 2d materials including graphene and graphene oxide could induce nlrp3 inflammasome activation and a series of events leading to epithelial mesenchymal transition and lung fibrosis. carbon nanotubes (cnts) are a type of long-aspectratio nanomaterial which is drawing wide interest because of their potential applications in electronics, optics, drug delivery and cancer therapy. however, animal studies have shown that they could promote the production of pro-fibrogenic cytokines and growth factors (il-1β, tgf-β and pdgf-aa, etc.) that may lead to lung fibrosis [72] [73] [74] [75] . the mechanism involves cellular uptake of cnts, overproduced ros by nadph oxidase activation, lysosomal damage induced by the surface reactivity of cnts and the release of lysosomal protein, cathepsin b, which activates nlrp3 inflammasome and produces il-1β. il-1β plays a major role in inflammation and fibrosis by activating epithelial mesenchymal transition in the lung (fig. 7) [56, [72] [73] [74] [75] . wang et al. showed that the dispersal state, hydrophobicity and purity of multi-walled carbon nanotubes (mwcnts) could affect the pro-fibrogenic cellular responses that also correlate with the extent of pulmonary fibrosis [72] [73] [74] [75] . furthermore, other long-aspect-ratio enms also showed similar effects. ji et al. demonstrated the toxicological effects of long-aspect-ratio nanomaterials using a library of inhouse-made ceo 2 nanoparticles [76] . in vitro toxicity study demonstrated that, at lengths ≥200 nm and aspect ratios ≥22, ceo 2 nanorods induced progressive pro-inflammatory effects and cytotoxicity [76] . the relatively low 'critical' length and aspect ratio were associated with small nanorod/nanowire diameters (6-10 nm), which facilitates the formation of stacking bundles that could pierce through cell membrane, causing the release of cathepsin b and further the activation of nlrp3 inflammasome. the same ceo 2 nanorods could also induce significantly higher lung fibrosis than spherical particles in vivo [77] . our recent studies show that rare earth oxide nanoparticles (reos), which are widely used in electronics and upconversion nanoparticles for bioimaging, are unstable in the acidic physiological environment, including the lysosomal compartment of the cells [49] [50] [51] . these reos can dissolve and the dissolved ions could bind to phosphates strongly; as a result, the particles transform from spheres to 'sea-urchin'-shaped or mesh-like structures with composition changes to the repo 4 . the sources of phosphates are not limited to free phosphate ions in the lysosomal compartment; they also include phosphate groups on intracellular proteins and membranes. on the one hand, biotransformation of reos could lead to dephosphorylation of phospholipids on the lysosomal membrane, which destabilizes the membrane, leading to lysosomal damage. released cathepsin b from lysosomes to the cytosol review xia et al. 427 will activate nlrp3 inflammasomes to produce il-1β, which initiate a cascade of events that culminate in pulmonary fibrosis [24, 26, 27] . on the other hand, stripping of phosphate groups from lysosomal proteins will lead to loss of enzymatic activities of the proteins, resulting in lysosome dysfunction, which leads to lysosome and autophagosome fusion inhibition and compromised autophagosome degradation in the autophagy flux. this leads to accumulation of activated nlrp3 inflammasomes because autophagy is the major homeostatic mechanism to remove activated inflammasomes [51] . the above examples show that the physicochemical properties of nps determine their toxic potential to human health, and more research is needed to understand these interactions between numerous other types of nps and biological systems occurring at the nanobio interface [18, 48, 53, 66, 78] . studies have shown that exposure to ufps and nps has the potential to cause adverse health effects in humans [18, 21, 22, 24, 48, 53] . in order to assess the toxic effects of these nano-sized particles, we advocate a predictive toxicological approach with the goal of linking particle physicochemical properties to their toxic effects [18, 48, 53] . to establish this predictive toxicological paradigm, there are five major requirements. the first is to comprehensively characterize their physicochemical properties that may lead to biological injury. it also requires the establishment of nanomaterial libraries with property variations, which allows building the link between material property and the biological/toxicological activities. the second requirement is to develop in vitro cellular screening assays that reveal particleinduced injury mechanisms and pathways such as oxidative stress and nlrp3 inflammasome activation. third, where possible, it is important to develop high-throughput screening platforms to assess the large number of material physicochemical properties, dosage and time points that are likely to lead to biological injury. fourth, the in vitro data could be used for in silico modeling to establish hazard ranking and structural-activity relationships that can be used to predict enm toxicity. finally, the in vitro hazard ranking and structural-activity relationships will then need to be validated by limited in vivo animal experiments to establish the 'predictiveness' of this approach. we have successfully demonstrated the usefulness of the predictive toxicological approach for the hazard assessment of over 200 different nano-materials and development of major structure activity relationships for groups of nanomaterials. we are confident that this approach would facilitate research on the toxicological effects induced by pms, ufps and nps [53] . convincing evidence has established the association between pm and many pulmonary diseases that contribute to early mortality and reduced life expectancy. however, for ambient ufps and nps, although much progress has been made in understanding their toxicological effects and mechanisms of toxicity, there are still many knowledge gaps on their impact on human health. in this review, we have summarized recent major findings on cellular, animal and human research with a focus on the respiratory effects and mechanisms of toxicity induced by pm, ufps and nps. available evidence strongly suggests that ufps and nps may be more potent in causing adverse health effects in humans because of their high deposition rate in the alveolar region, impaired clearance by alveolar macrophages and higher surface reactivity, pro-oxidative and proinflammatory effects than their larger counterparts. thus, it is imperative to focus future research on the health effects of nano-scale pollutants so that preventive strategies and regulatory guidelines can be developed to reduce exposure and improve human health. an association between air pollution and mortality in six u.s. cities ambient particle inhalation and the cardiovascular system: potential mechanisms pulmonary health effects of air pollution the role of oxidative stress in ambient particulate matter-induced lung diseases and its implications in the toxicity of engineered nanoparticles million premature deaths annually linked to air pollution, geneva, world health organization health effects of fine particulate air pollution: lines that connect fine-particulate air pollution and life expectancy in the united states cardiopulmonary mortality and air pollution influence of air pollution on respiratory health during perinatal development outdoor air pollution: a leading environmental cause of cancer deaths images show human fingerprint on global air quality-release materials association of improved air quality with lung development in children the contribution of outdoor air pollution sources to premature mortality on a global scale rapid increases in tropospheric ozone production and export from china lung cancer, cardiopulmonary mortality, and long-term exposure to fine particulate air pollution ultrafine particles toxic potential of materials at the nanolevel comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm the role of reactive oxygen species and oxidative stress in mediating particulate matter injury ultrafine particulate pollutants induce oxidative stress 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particulate matter air pollution during the beijing olympics: a panel study association of ambient air pollution with the prevalence and incidence of copd ambient air pollution: a cause of copd? the global contribution of outdoor air pollution to the incidence, prevalence, mortality and hospital admission for chronic obstructive pulmonary disease: a systematic review and meta-analysis ambient air pollution, lung function, and airway responsiveness in asthmatic children ambient air pollution and adult asthma incidence in six european cohorts (escape) indoor air pollution and risk of lung cancer among chinese female non-smokers outdoor particulate matter exposure and lung cancer: a systematic review and meta-analysis the international agency for research on cancer (iarc) evaluation of the carcinogenicity of outdoor air pollution: focus on china particulate matter air pollution exposure, distance to road, and incident lung cancer in the nurses' health study cohort air pollution and acute 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of upconversion nanoparticles through phosphonate coating interference in autophagosome fusion by rare earth nanoparticles disrupts autophagic flux and regulation of an interleukin-1 beta producing inflammasome crucial role of lateral size for graphene oxide in activating macrophages and stimulating pro-inflammatory responses in cells and animals nanomaterial toxicity testing in the 21 st century: use of a predictive toxicological approach and high-throughput screening graphene oxide induces toll-like receptor 4 (tlr4)-dependent necrosis in macrophages reduction of acute inflammatory effects of fumed silica nanoparticles in the lung by adjusting silanol display through calcination and metal doping nadph oxidase-dependent nlrp3 inflammasome activation and its important role in lung fibrosis by multiwalled carbon nanotubes silver nanoparticles induced rna polymerasesilver binding and rna transcription inhibition in erythroid progenitor cells comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties cationic polystyrene nanosphere toxicity depends on cell-specific endocytic and mitochondrial injury pathways impairment of mitochondrial function by particulate matter (pm) and their toxic components: implications for pm-induced cardiovascular and lung disease engineering an effective immune adjuvant by designed control of shape and crystallinity of aluminum oxyhydroxide nanoparticles a study on permeability transition pore opening and cytochrome c release from mitochondria, induced by caspase-3 in vitro air pollution and cancer by international agency for research on cancer role of the nrf2-mediated signaling pathway as a negative regulator of inflammation: implications for the impact of particulate pollutants on asthma oxidative stress-induced mutagenesis in single-strand dna occurs primarily at cytosines and is dna polymerase zeta-dependent only for adenines and guanines where are we heading in nanotechnology environmental health and safety and materials characterization surface charge and cellular processing of covalently functionalized multiwall carbon nanotubes determine pulmonary toxicity decreased dissolution of zno by iron doping yields nanoparticles with reduced toxicity in the rodent lung and zebrafish embryos antioxidant properties of cerium oxide nanocrystals as a function of nanocrystal diameter and surface coating cerium dioxide nanoparticles induce apoptosis and autophagy in human peripheral blood monocytes role of fe doping in tuning the band gap of tio 2 for the photo-oxidation-induced cytotoxicity paradigm use of a pro-fibrogenic mechanismbased predictive toxicological approach for tiered testing and decision analysis of carbonaceous nanomaterials pluronic f108 coating decreases the lung fibrosis potential of multiwall carbon nanotubes by reducing lysosomal injury quantitative techniques for assessing and controlling the dispersion and biological effects of multiwalled carbon nanotubes in mammalian tissue culture cells dispersal state of multiwalled carbon nanotubes elicits profibrogenic cellular responses that correlate with fibrogenesis biomarkers and fibrosis in the murine lung designed synthesis of ceo 2 nanorods and nanowires for studying toxicological effects of high aspect ratio nanomaterials aspect ratio plays a role in the hazard potential of ceo 2 nanoparticles in mouse lung and zebrafish gastrointestinal tract mechanisms of nanosilver-induced toxicological effects: more attention should be paid to its sublethal effects key: cord-008454-8brxpotx authors: field, anne m. title: diagnostic virology using electron microscopic techniques date: 2008-04-09 journal: adv virus res doi: 10.1016/s0065-3527(08)60432-7 sha: doc_id: 8454 cord_uid: 8brxpotx this chapter illustrates the development of the use of electron microscopy in viral diagnosis. the field covered is confined to medical viral diagnosis, but parallel developments have taken place in both veterinary and botanical fields and techniques derived from both these sources are also included where relevant. it is reported that the scanning transmission mode of operation, which can induce image contrast changes electronically, may enhance studies with unstained sections and perhaps facilitate thin section immune electron microscopy (iem). the application of negative stain iem has been particularly useful for the study of the antigenic nature of some of the newly discovered noncultivable viruses. viral antigens can also be detected in thin sections of infected cells by iem with suitably labeled specific antibodies. confirmation of viral infection by electron microscopy on tissues originally processed for light microscopy is also frequently useful. diagnosis of viral infections by observation of virus particles in thin sections of infected tissues has been a continuing but perhaps rather underused technique for the last 30 years. observation of virus particles in suspension in metal-shadowed preparations had some diagnostic applications, but when the negative staining technique was introduced in 1959 and, as a result, virus particles were more readily recognizable, diagnostic use of the electron microscope became extremely practical. the presence in the world of smallpox infection and the consequent necessity for rapid differentiation between the smallpox virus and other viruses established the electron microscope as a n essential tool in a few selected laboratories. naturally these instruments were utilized for other virus diagnostic problems and gradually experience accumulated. confirmation of electron microscopy as a good diagnostic technique for samples direct from patients came in the late 1960s with hepatitis b serum testing. in the 1970s the essentially noncultivable fecal viruses of hepatitis a and various diarrheal conditions were discovered and in these studies electron microscopy played an indispensable role. the purpose of this article is to review the development of the use of electron microscopy in viral diagnosis in the last 20 years and to place it in context with other laboratory techniques. the field covered is confined to medical viral diagnosis, but parallel developments have taken place in both veterinary and botanical fields and techniques derived from both these sources are included where relevant. viral diagnostic electron microscopy in the medical field has been reviewed by doane (19741, by doane and anderson (19771, by donelli et al. (19791, and by hsiung et al. (1979) . virus particles have characteristic morphologies (i.e., shape, substructure, and size) which, because they are fundamental properties, are important in viral classification. viral structure is thus the basis for the use of the electron microscope for diagnosis and viruses having the same structure constitute a morphologic group, which may be a family of viruses or a genus. within a group individual viruses cannot be differentiated on appearance and more sophisticated methods of antigenic analysis must be used, but it is often sufficient for diagnostic purposes merely to place a virus within such a group. many diagnoses are made by recognition of the characteristic viral structure in the image displayed on the microscope screen and need not await confirmatory micrographs; for this an appreciation of the full range of viral morphology by the operator is essential. the salient features of viral morphology as observed by negative staining and thin sectioning methods (section 111) are here briefly reviewed and illustrated diagrammatically in fig. 1 , and selected examfig. 1 ples are illustrated photographically (figs. 2 to 12). reviews of viral structure have recently been published (rabin and jenson, 1967; madeley, 1972; dalton and haguenau, 1973; cheville, 1975) . this review is not intended to be comprehensive but stresses the features with diagnostic significance and is largely based on personal observations and the reviews quoted above. terminology is according to recent viral classification (report, 1979) . poxviruses are the largest and most complex virus particles. the orthopoxvirus genus includes vaccinia, variola, cowpox, and monkeypox viruses, all of which can infect man. negatively stained particles are brick-shaped, 220 to 270 nm long, and 180 to 220 nm wide. when penetrated by stain the particles appear larger than when unpenetrated. the latter display a random arrangement of 9-nm-wide surface filaments. members of the parapoxvirus genus causing human infections are orf virus and milker's nodule virus. the negatively stained particles are ovoid, 220 to 300 nm long, and 150 to 180 nm wide. the single surface filament is narrower than on orthopoxvirus particles and is arranged in a regular spiral which usually gives a criss-cross appearance because both sides of the particle are imaged. stain-penetrated particles are identical with orthopoxviruses except in the ovoid shape and generally greater length and lesser width. a probable member of the poxvirus family is molluscum contagiosum virus which has brick-shaped particles resembling orthopoxviruses but with more rounded corners. the slightly narrower surface filaments are arranged rather more regularly than on orthopoxviruses. stainpenetrated particles closely resemble the orthopoxviruses except in the rounded corners. in thin sections of infected cells it has been shown that all poxviruses mature within a cytoplasmic matrix. in mature particles a n outer coat encloses two lateral bodies which lie on each side of a dense core. particles are released by cell lysis. because of their large size and complex structure the poxviruses cannot be confused with other viruses. some of the larger icosahedral viruses, although of different families, can resemble each other under some circumstances, particularly if damaged. the families concerned are the herpesviridae, adenoviridae, reoviridae (including reoviruses, rotaviruses, and orbiviruses), and papovaviridae, arranged in descending size order. negatively stained herpesvirus particles have icosahedral 100-to 110-nm-diameter capsids bearing 162 capsomeres which are hexagonal when seen end-on and tubular in profile. capsids are often surrounded by a membranous envelope bearing irregular short projections. this gives the virus a total diameter of 120 to 150 nm. varying degrees of stain penetration of both capsid and envelope have been observed (watson and wildy, 1963; tyrrell and almeida, 1967) . in thin sections of infected cells it has been shown that capsids are assembled in the nucleus where they acquire cores, which have variable morphology, and where paracrystalline arrays of capsids may be seen. envelopes are formed around capsids at several sites: the nuclear membrane, cytoplasmic membranes, and cell membrane. members of the group infecting humans are herpes simplex virus, cytomegalovirus, epstein-barr (eb) virus, and varicella zoster virus. adenoviruses negatively stained have icosahedral 70to 80-nmdiameter capsids with a rigid angular appearance, becoming spherical when damaged. there are 252 capsomeres which are 7 to 9 nm in diameter. twelve vertex capsomeres bear fibers (valentine and pereira, 19651, but these are rarely observed in the conditions used to prepare diagnostic specimens. in thin sections of infected cells it can be seen that capsids assemble and mature in the nucleus where they often form paracrystalline arrays. at least 33 antigenic types of adenovirus can infect humans. the reoviridae have particles with icosahedral symmetry and two concentric protein coats both bearing capsomeres. members of the reovirus genus, which includes three types capable of infecting humans, have particles which are 70 to 80 nm in diameter when negatively stained. the outer layer of capsomeres is rarely spontaneously lost, but when it is a n inner, 50-to 55-nm-diameter particle, it remains ( fig. 2) . rotaviruses occur in feces and negatively stained particles are 65 to 75 nm in diameter when complete and 55 to 60 nm without the outer coat which strips off readily in natural conditions. complete rotavirus particles have a smooth circular outline but removal of the outer layer leaves a particle coated with capsomeres having the appearance of spokes of a wheel (fig. 3) . colorado tick fever virus is an orbiuirus genus member capable of infecting humans. the virus has a substructure resembling the reoviruses but with less clearly defined capsomeres on the surface of a 65-to 75-nm-diameter capsid (palmer et al., 1977) . thin sections show that all the reoviridae replicate in the cytoplasm and usually mature from a matrix of viroplasm. reoviruses frequently associate with the mitotic apparatus whereas rotaviruses replicate in close association with the endoplasmic reticulum. viral release is by cell lysis except for orbiviruses which bud from cell membr anes. papovaviruses when negatively stained show icosahedral symmetry, but with a skew arrangement, and the capsomeres are more prominent around one side of the particle than on the opposite side. particles of the papillomavirus genus are 50 to 55 nm in diameter. this genus includes human wart virus. particles of the polyomavirus genus have identical morphology but are smaller, 40 to 45 nm in diameter. this genus includes two human viruses, bk virus and jc virus. filamentous forms and several isometric forms smaller than true virus particles can occur. in thin sections of infected cells capsids are seen t o be assembled in the nucleus where they often form paracrystalline arrays. particles may also be seen in cytoplasm, wrapped around by cellular membranes on entry to cells, while progeny virus particles are usually aligned on cytoplasmic membranes. papillomaviruses tend to be more confined to the nucleus than polyomaviruses. another collection of morphologically similar particles is the range of smaller sized (20to 40-nm-diameter) isometric viruses, most of which do not have clearly recognizable surface subunits. this group includes caliciviridae, picornaviridae, parvoviridae, the hepatitis viruses, norwalk agent, and astroviruses. members of the proposed family caliciviridae negatively stained have roughly spherical particles, 29 t o 40 nm in diameter, bearing 32 cup-shaped surface depressions arranged in icosahedral symmetry. in suitably oriented particles these give rise to a distinctive surface pattern of a hollow-centered star recently compared with the "star of david" (madeley, 1979) . in thin sections the viruses are seen to mature in the cytoplasm in the cisternae of the endoplasmic reticulum and may form crystalline arrays (love and sabine, 1975) . human fecal calicivirus appears t o be a member of this group (fig. 4) . negatively stained picornaviruses are spherical particles with a smooth surface and outline measuring 22 to 30 nm in diameter (fig. 5 ) . in infected cells they mature in the ground substance of the cytoplasm, sometimes forming paracrystalline arrays. because they are only slightly larger than ribosomes individual particles may be difficult t o recognize. the many antigenic types of poliovirus, coxsackievirus, echovirus, and rhinovirus all infect humans. rhinoviruses are more fragile than enteroviruses in negatively stained preparations and empty shells and partial shells are frequently observed. enteroviruses have a peak buoyant density of 1.33 to 1.34 gm/ml in cesium chloride; the rhinovirus density is 1.38 to 1.41 gm/ml. members of the parvoviridae when negatively stained closely resemble picornaviruses. they are spherical with a smooth surface and outline and the diameter ranges from 18 to 26 nm. in both families some particles may appear to have hexagonal outlines. empty shells and partial shells are, as with rhinoviruses, commonly seen in parvovirus preparations (fig. 6) . thin sections of infected cells reveal empty and complete parvovirus capsids in the nucleus and at later stages progeny virus particles are found in the cytoplasm embedded in matrix material or in membrane-bounded spaces. the peak buoyant density in cesium chloride is 1.39 to 1.42 gm/ml. possible human members of this family have been observed in feces and sera. negatively stained hepatitis a virus particles are 27 nm in diameter, spherical, smooth surfaced, and smooth outlined (fig. 7) . in thin sections they are found in the cytoplasm. possession of ribonucleic acid and other properties indicate that this virus is a member of the picornaviridae. hepatitis b virus is pleomorphic. in negatively stained preparations of serum three particle types are seen: small round particles, 19 to 23 nm in diameter with a smooth surface and edge; long filamentous forms, 19 to 23 nm wide and varying lengths with a smooth surface or small isometric viruses. with cross striations; dane particles which are 42 nm in diameter and have a smooth outer layer enclosing a 27-nm spherical core (fig. 8) . the cores (hbcag) can be extracted from the dane particles and are antigenically different from the other pleomorphic particles (hbsag) which are all antigenically similar (almeida et az., 1971) . in thin sections of infected liver the hbcag particles are seen in the nucleus and resemble parvoviruses. the three forms of hbsag are seen in cisternae of endoplasmic reticulum in the cytoplasm (huang and neurath, 1979) . norwalk agent and other morphologically similar viruses are at present unclassified. the particles bear some resemblance to caliciviruses but do not show the star of david substructure. the major structural protein has properties similar to that of caliciviruses (greenberg et al., 1981) . the outline of the particle is structured indicating the presence of some substructure. the particles are difficult to measure accurately because of the ragged outline but are approximately 27 to 34 nm in diameter. no thin section studies have been done. the agents are found in human feces (kapikian et al., 197213) . astroviruses are also unclassified at present. negatively stained, they are 26-to 30-nm-diameter round particles with a smooth edge. a proportion of the particles bears a solid five-or six-pointed star on the surface (madeley, 1979) . no thin section studies of a full replicative cycle of the human virus are available but abortive cycles in cell culture show the virus located in cytoplasm (kurtz et al., 1979) . sheep astrovirus particles are cytoplasmic in vivo (e. gray et al., 1980) . the particles are found in human feces ( fig. 9 ). viruses which are notable for bearing a fringed outer membrane include orthomyxoviridae, paramyxoviridae, coronaviridae, rhabdoviridae, and marburg/ebola viruses. the orthomyxovirus particles are 80 to 120 nm in diameter and are pleomorphic in negatively stained preparations. spherical and filamentous forms are the commonest. spherical forms may show small blebs but these are probably preparation artifacts (nermut, 1972) . the surface of the limiting membrane is covered with 10-nm-long spikelike projections spaced 7 to 8 nm apart. this fringe has a regular appearance around the periphery of negatively stained particles (fig. 10) . end-on views of the projections give a regularly dotted appearance to the surface of particles. some influenza c virus particles have a reticulate surface pattern (apostolov and flewett, 1969) . the orthomyxovirus outer membrane is rarely penetrated by stain to reveal the internal component, which is a ribonucleoprotein helix, 9 nm in width, arranged in a tight coil (murti et al., 1980) (fig. 11) . nuclei of sectioned cells show various granular and fibrillar structures in the early stages of infection but virus particles are recognized only in the cytoplasm where they mature by budding at the plasma membrane where it covers the already assembled nucleocapsid. this group includes the influenza viruses. paramyxoviruses are also pleomorphic, membrane-bound particles, with diameters 120 to 450 nm. surface projections are approximately 8 nm long and are spaced at 8-to 10-nm intervals. in contrast with orthomyxoviruses these particles frequently fracture revealing the enclosed ribonucleoprotein which is a loosely arranged helix with a pitch of 5 nm and a width of 17 to 18 nm in the paramyxouirus genus (parainfluenza viruses and mumps virus) and in the morbilliuirus genus [measles and subacute sclerosing panencephalitis (sspe) viruses] (fig. 12) . in the pneumouirus genus [respiratory syncytial virus (rsv)] the helix width is only 12 to 15 nm (joncas et norrby etal., 1970; bachi and howe, 1973; berthiaume et al., 1974) . in sections the tubular nucleocapsid is seen to accumulate in the cytoplasm and to be aligned beneath the cellular membrane which acquires projections in those regions. finally mature virus particles bud through the membrane. the morbilliviruses also accumulate nucleocapsid in the nucleus. sspe virus has lost the budding maturation stage in the natural host. coronaviruses have pleomorphic membrane-bound particles with diameters ranging from 75 to 160 nm. they are surrounded by clubshaped surface projections approximately 20 nm long which however are easily lost. in thin sections of infected cells particles are seen to assemble in the cytoplasm and mature by budding into vesicles, accumulating there before being released by cell lysis. the particles in thin sections consist of a translucent core surrounded by a 20-nm-thick membrane bearing 15to 20-nm-long projections. the human coronavirus affects the respiratory tract. another possible member of the family is the human enteric coronavirus which, in common with some other animal enteric coronaviruses, has narrow surface spikes rather than club-shaped projections. these spikes are 20 nm long and frequently have knobs and extra t pieces a t the distal ends (caul et al., 1977) . rhabdoviruses are bullet-shaped particles 130 to 250 nm long and 70 to 80 nm wide. the group includes rabies and vesicular stomatitis virus. rabies virus is very fragile and in negatively stained preparations most particles are pleomorphic and have variable lengths. particles penetrated by the stain exhibit internal cross-striations at 4.5to 5-nm intervals which is the coiled ribonucleoprotein helical component. some particles exhibit a reticular surface structure but all have surface projections which are 8 to 10 nm long. in thin sections viral matrix material can be seen in the cytoplasm. the site of virus maturation depends on the virus and the cells used; rabies particles mostly bud from intracytoplasmic membranes but can bud in the viral matrix from de novo membranes. marburg and ebola viruses, so far unclassified, have structural similarities to rhabdoviruses. i n effect they are very long bullet-shaped particles which are frequently curved into hooked and circular forms and are 80 nm wide and 130 to 2500 nm long or even longer. surface projections are 10 nm long and a helical inner structure with 40 to 50 nm diameter has 5 to 6 nm perodicity. in sections nucleocapsids are formed in the cytoplasm and particles mature by budding at the plasma membrane (murphy et al., 1978) . a collection of viruses which are not often encountered in diagnostic electron microscopy, largely because the negatively stained morphology is not distinctive, includes the togaviridae, arenaviridae, bunyaviridae, and retroviridae. togaviruses infecting man include sindbis and chikungunya in the alphavirus genus, dengue, yellow fever, and japanese b encephalitis in the flavivirus genus, and rubella in the rubivirus genus. with negative staining the viruses are difficult to recognize but they consist of a 40-to 70-nm-diameter envelope closely applied to a 25to 35-nm nucleocapsid which probably has icosahedral symmetry. most particles bear surface projections with little regularity. viruses are more clearly seen in thin sections of infected cells where they multiply in the cytoplasm and mature by budding a t the plasma membrane (alphaviruses), the endoplasmic reticulum and golgi zone membranes (flaviviruses), and into vacuoles and at the plasma membrane (rubiviruses). particles in thin sections exhibit a n electron-dense core surrounded by a translucent zone bordered by the viral envelope (holmes et al., 1969; matsumara et al., 1971) . arenaviruses are spherical or pleomorphic, 110-to 130-nm-diameter particles with club-shaped 10-nm-long surface projections. the most distinctive morphology is shown in thin sections where it can be seen that the limiting membrane encloses a varying number of 20-to 25nm-diameter, ribosome-like particles. in the infected cells a mass of ribosomes accumulates in a dense cytoplasmic matrix and the virus particles mature by budding through the cell membrane . viruses of the tacaribe complex, lassa fever virus and lymphocytic choriomeningitis virus are human pathogens. bunyaviruses are spherical or oval, 90-to 100-nm-diameter particles with a n envelope bearing surface projections. the envelope encloses helical ribonucleoprotein 2 to 2.5 nm wide. the particles assemble i n the cytoplasm and mature by budding through smooth surfaced vesicles in the golgi region. human agents are bunyamwera and california encephalitis virus. the oncovirus subgroup of the retroviridae were originally described morphologically as a, b, c, and d type virus particles by bernhard (1960) . when negatively stained the particles are spherical, enveloped, 80 to 100 nm in diameter, and have surface projections which are seen best in the type b oncovirus genus. often particles have surface blebs or tails but these are probably preparation artifacts. beneath the envelope the core is probably icosahedral and contains the ribonucleoprotein which may be helical. in thin sections the particle outer envelope encloses a nucleoid which resembles a n a type particle and is eccentrically placed in mature type b oncoviruses and is central in type c oncoviruses. mature type b particles are 90 to 200 nm in diameter and type c particles are 85 to 110 nm diameter (dalton, 1972; de harven, 1974) . although viruses of this family are found in mam-mals, the morphological evidence for human oncoviruses is very slight. the other subfamily, the spumaviridae, includes the foamy viruses and a possible human member. negatively stained particles are enveloped, about 90 nm in diameter, with 12-to 15-nm-long surface projections. in thin sections of infected cells virus particles are observed in the cytoplasm budding into cytoplasmic vacuoles and through the plasma membrane. the moderately electron-dense core is 40 to 60 nm in diameter and is separated from the 70-nm envelope by empty space bridged by striations; spikes on the surface increase the total diameter to 90 nm (clarke et al., 1969) . although size is a n important aid to identification it is unwise to rely on the accuracy of size estimates; the sizes quoted for virus particles in the preceding section are only approximate as measurement is subject to many variables. in practice it is useful to have a size marker in the instrument so that a rough estimate of virus particle size can be made using the image on the fluorescent screen. the microscope itself must be calibrated to ensure the accuracy of any given magnification as manufacturing tolerance is generally only within 5%. at high magnifications, such as those used for virus work, calibration based on crossgrating replicas is inaccurate and calibration using measurements of the lattice spacing in negatively stained catalase crystals is considered more reliable (wrigley, 1968). even small height changes of the specimen in the microscope affect magnification (agar e t al., 1974) and this creates problems as it is almost impossible to ensure that specimen grids will stay perfectly flat and will be positioned at exactly the height of the calibration grid. another cause of possible error is that lens currents vary over a period and this too can affect magnification. in the photographic darkroom careless enlarger setting affects the magnification of prints and photographic paper dimensions can change. thus measurements are best made on the negatives except when particle images are so small that this in itself induces errors. the thickness of stain surrounding particles is variable in negatively stained preparations, consequently particles may be flattened and so appear to be larger (nermut, 1977) ; in addition excessive photographic contrast of the image can affect apparent particle size by obscuring the edge of the particle. in thin sections virus particles are usually smaller than in negatively stained preparations as a result of the processing used (glauert, 1975) . a comparison of sizes obtained for bacteriophage particles by negative staining, thin sectioning, and freeze drying accompanied by metal shadowing, showed that negative staining gave the best correlation with the size derived from x-ray analysis (earnshaw et al., 1978) . the preceding descriptions of negatively stained virus particles refer to viruses untreated as far as possible. the shape of the more delicate viruses can be altered by high-speed centrifugation (polson and stannard, 1970) , and on the grid by stretching of the support film (ronald et al., 1977) . myxovirus pleomorphism is probably a preparation and storage artifact (nermut, 1972) . chemicals used in purification and concentration of viruses may affect morphology . even the negative stain can alter viral morphology: phosphotungstic acid is probably the least damaging in this respect but even this can disrupt the helical paramyxovirus ribonucleoprotein into short lengths (hosaka, 1968) while uranyl acetate shrinks bacteriophage heads and causes the tails to swell (ackermann et al., 1974b) and potassium borotungstate splits myxovirus surface projections (flewett and apostolov, 1967) . fixation of foot and mouth disease virus with glutaraldehyde before negative staining produced empty particles and increased their diameter by 25% (sangar et al., 1973) . the internal details of enveloped herpesviruses were also obscured by either glutaraldehyde or osmium tetroxide fixation, probably because negative stain no longer penetrated the fked envelope (vernon et al., 1976; field, unpublished observations) . murphy et al. (1970) found that glutaraldehyde or osmium tetroxide fixation obscured the envelope detail of arenaviruses; glutaraldehyde-fixed coronaviruses lost the clarity of the surface projections in negatively stained preparations (caul et al., 1977) . on the other hand rotavirus structure was stabilized by formaldehyde fixation (woode et al., 1976) and retroviruses were less pleomorphic if glutaraldehyde fixed and critical point dried than if unfixed and air dried (gonda et al., 1978) . influenza virus particles fixed in osmium tetroxide were less pleomorphic than when unfixed but the detailed surface structure was not so clear (reuss et al., 1967) . the intentional disruption of virus particles on the grid with detergent has been used to study their internal structure (almeida and brand, 1975) . the basis of negative staining is that the electron-dense stain surrounds the virus particles and flows into surface crevices, giving a clear image of the outside surface of the particles. sometimes stain penetrates to the interior of the capsids and an image of the outer shell in profile results (horne, 1975) . the technique was first described in 1959 (brenner and horne, 1959) and was soon applied successfully to viral diagnostic specimens. the usual method for viral diagnosis is to mix virus suspension and negative stain in equal volumes, place a drop of mixture onto a formvar-carbon-coated grid, remove excess fluid by touching the edge of the grid to filter paper, and allow the grid to air-dry. alternatively virus suspension can be applied to the grid and dried before stain is added. the stain used most commonly is phosphotungstic acid. samples must be rich in virus to overcome the limitations of the technique. some samples, even diagnostic specimens from patients, contain sufficient virus to be examined without concentration; however, others need concentration and a simple method is to pellet virus by ultracentrifugation of a clarified sample (almeida et al., 196713) . a further refinement is purification by density gradient centrifugation. virus may be concentrated from samples by adding lyphogel, which is a polyacrylamide hydrogel capable of absorbing water, salts, and small molecules to leave virus particles in a greatly reduced volume (ashcavai and peters, 1971; whitby and rodgers, 1980) . virus can be concentrated from very large volumes by membrane filtration (torrella and morita, 1979) or can be adsorbed to a polyelectrolyte and eluted to a smaller volume (chaudhary and westwood, 1972) . virus particles can be released from infected cells of solid tissues, cell cultures, and organ cultures by lysing the concentrated cells in a small volume of distilled water (almeida et al., 1967c; almeida and tyrrell, 1967) and, if necessary, further concentration of virus in such cell lysates can be effected by one of the techniques mentioned above. various methods have been used to remove contaminating salts from samples. an agar diffusion technique devised by kellenberger and arber (1957) was developed by kelen et al. (1971) and by anderson and doane (1972a) . it utilizes an agar substrate and either the grid with a microdrop (approximately 0.01 ml) of virus suspension on it is placed on the agar or the microdrop is applied to the agar surface and the grid is floated on the microdrop. when the drop has dried the fluid, salts, and low-molecular-weight substances have diffused into the agar leaving virus particles and larger debris on the grid ready for negative staining. salts may also be removed by careful washing of grids after airdrying the viral sample onto them (cartwright et bond and hall, 1972) . sucrose can be dialyzed from viral samples already applied to grids by using a wick of filter paper to run a buffer solution continuously across the grid (webb, 1973) . the pseudoreplica method concentrates virus from samples and removes salts simultaneously (smith and melnick, 1962) . the viral sample is applied to the surface of a small block of agar and allowed to dry. the agar surface is covered with formvar solution, and the resultant virus-coated film is floated onto negative stain and mounted on a grid. the technique has been used successfully with diagnostic samples (burtonboy et al., 1978; lee et al., 1978) but is perhaps unnecessarily complicated. although excellent viral morphology results from spraying virus particles onto grids, it is not to be recommended for diagnostic samples since the technique is relatively insensitive, needing very high concentrations of virus, and the resultant aerosol could be dangerous (horne, 1967; england and reed, 1980) . it is generally accepted that the threshold concentration of virus necessary for detection in negatively stained preparations is lo5 to lo6 particles per milliliter (galasso, 1967; monroe and brandt, 1970; ball and harris, 1972; chaudhary and westwood, 1972) . if viral samples are too well purified it may be difficult to obtain adherence to grids, but adding a wetting agent such as bovine serum albumin or bacitracin may solve this problem (horne, 1967; gregory and pirie, 1972) . of the many different negative stains available phosphotungstic acid at ph 6.4 has been found to be the most reliable for general diagnostic work. silicotungstate gives a less granular background and preserves paramyxovirus structure better than phosphotungstate (bloth and norrby, 1967) . phosphotungstate often gave better definition of the viruses encountered in veterinary diagnosis when used a t ph 6 as opposed to ph 7 (spadbrow and francis, 1969). caul et al. (1977) found phosphotungstate superior to ammonium molybdate or uranyl acetate for examination of fecal coronaviruses. flewett and apostolov (1967) found potassium borotungstate damaged myxovirus surface projections. the morphology of negatively stained virus particles is sufficient for grouping purposes but it is necessary to use immune electron microscopy (iem) to differentiate morphologically identical but antigenically distinct viruses. when specific antiserum is added to a suspension of virus particles molecules of antibody attach to the particles and can be seen after negative staining. when optimal proportions of virus and antiserum are used the virus particles are agglutinated by the antibody into immune complexes. methods and applications have been reviewed by waterson (1969a1, doane (1974) , and doane and anderson (1977) . the simplest method for negative stain iem is to mix small volumes (i.e., 0.1 ml) of viral suspension and antiserum, incubate at 37â°c or room temperature for 1 hour, dilute to a reasonable volume, and ultracentrifuge to pellet the immune complexes for negative staining (almeida and waterson, 1969a) . when reagents are scarce smaller volumes (0.01 to 0.02 ml) can be mixed and incubated, then microdrops are removed to a n agar surface and grids are floated on the drops. when the fluid has dried the grids are treated with negative stain (kelen et al., 1971) . alternatively, microdrops of virus-serum mixture may be applied directly to the grids, but before negative staining repeated washing is necessary to remove salts and low-molecular-weight substances which would otherwise obscure the immune reaction (milne and luisoni, 1977) . unfortunately it is necessary to use salt-containing fluids for immune electron microscopy to ensure optimal combination of virus with antibody (ball and brakke, 1968) . for routine virus identification specific antibodies can be incorporated into agar in microtiter plate wells and the plates can be stored with a grid on top of the agar in each well. when required, microdrop samples of the virus to be identified are added on top of the grids in the wells and allowed to dry into the agar. grids are removed and treated with negative stain (doane, 1974) . gel immunodiffusion test precipitin lines can be cut out, homogenized, and negatively stained to show virus-antibody complexes (beale and mason, 1968) . a method of specific attraction of virus particles to antiserum-treated grids was developed for plant viruses (derrick, 1973) and further refined by shukla and gough (1979) who used staphylococcal protein a to enhance antibody coating of the grid. the technique has recently been used successfully in rotavirus diagnosis (nicolaieff et al., 1980) but it was noted that coronaviruses and small round viruses also present in the samples were not seen on the rotavirus-antiserum-treated grids. virus particles specifically adsorbed onto antiserum-coated grids may be treated further with antisera to investigate their antigenic nature (milne and luisoni, 1977) . complement causes immune lysis of some viruses and may change the appearance of immune complexes so sera are best heat inactivated before use to eliminate these effects (almeida and waterson, 1969a) . addition of a second antiserum specific for the immunoglobulin in the original immune complex increases the total antibody layer around the virus particles making detection of complexes easier (saif et al., 1977) . antibody may be conjugated to ferritin molecules which makes identification of antibody easier (brzosko et patterson, 19751 , although antibody molecules can be seen readily without a marker when negatively stained. ferritin labeling has been found useful when a second layer of antibody is used to identify the species of immunoglobulin involved in immune complexes (locarnini et al., 1977) . the basic necessities for satisfactory negative stain iem are virus particles in large numbers, free from cell debris and free of antibody, and a n antibody preparation which is also free of immune complexes. if the viral antigen is a sample from a patient some antibody may be present. although it is possible to use such antigens by careful grading of the amount of antibody seen in immune complexes interpretation is more difficult. false clumping of virus particles occurs particularly during abrupt ph changes (floyd, 1979) . rheumatoid factor can induce mixed clumping of nonidentical viruses to give misleading results in typing tests by iem (stannard et al., 1980) . it is best to use viruses unfixed because some lose antigenicity when fixed (narayan et al., 19731 , although others retain activity (woode et al., 1976; chaudhary et al., 1979) . the application of negative stain iem has been particularly useful for the study of the antigenic nature of some of the newly discovered noncultivable viruses. for example human parvovirus-like particles have been compared antigenically by this technique as have rotaviruses from humans and from veterinary samples (woode et al., 1976) . the system can be reversed and, using known viral antigens, specific viral antibodies can be detected in human sera. again the technique is most useful when the antigen is a noncultivable virus such as hepatitis a virus (dienstag et al., 1976a) or norwalk agent (parrino et az., 1977) . negative stain iem can be used to detect viruses in clinical samples or after culture in uitro since with certain viruses it increases the sensitivity of negative stain visualization some 100 times (doane, 1974) . such enhanced sensitivity depends upon the titer of the antiserum used (lamontagne et al., 1980) and zissis et al. (1978) found negative stain iem did not in fact increase sensitivity of rotavirus detection. when single virus particles are readily recognizable, such as rotaviruses, it is probably more sensitive to have many scattered single particles in an ordinary negative stain rather than a much smaller number of virus aggregates in immune preparations. perhaps the main advantage of iem in virus detection is the specific aggregation of virus particles of unremarkable morphology so that their viral nature can be appreciated. this was the method used to identify rubella virus (best et al., 1967) and the small round virus particles such as hepatitis b antigen (almeida et al., 1969b) , rhinoviruses (kapikian et al., 1972a) , and hepatitis a virus (feinstone et al., 1973) . incorporation of atypical forms along with typical virus particles in the same specific immune complex demonstrates their common viral antigenicity. this was shown for the tubular forms of human polyomaviruses (albert and zu rhein, 1974) and for filamentous forms of rotaviruses (holmes et al., 1975) . conversely negative stain iem demonstrated that hbcag particles differed antigenically from the three forms of hbsag because they were not associated in the same immune complexes . amorphous material possessing viral antigenicity can also be identified using negative stain iem methods (almeida et al., 1981) . while tissues can be homogenized to extract virus particles for negative staining it is often preferable to search for viruses in situ in thin sections. when particles are scanty the thin section technique may be more sensitive than negative staining. some viruses have a more distinctive morphology in thin sections than when negatively stained. thin sectioning allows observation of the pathogenesis of the infection as well as identification of the viral cause. there are many standard schedules for tissue fixation and embedding and methods will not be reviewed here. examination of thin sections for viruses entails the use of relatively high magnifications in the electron microscope and methods should be chosen which will satisfy this condition. for samples where results are required urgently there are rapid embedding methods (doane and anderson, 1977) . examination of thick sections of resin-embedded material stained with toluidine blue for light microscopy may reduce sampling error and so reduce time spent examining thin sections. even with such selection the thin sectioning technique has very limited potential in rapid viral diagnosis. confirmation of viral infection by electron microscopy on tissues originally processed for light microscopy is frequently useful. for example, virus particles have been seen in thin sections of tissues which had been stored in formalin over a long period (zu rhein and chou, 1965; hashida and yunis, 1970) . paraffin sections can be marked to indicate cells with inclusions and the appropriate areas of the section reembedded for electron microscopic examination of the same cells (blank et al., 1970; rossi et pinkerton and carroll, 1971; bhatnagar et al., 1977) . cytologic preparations have been reembedded in a similar fashion and virus particles seen in cells (takeda, 1969; coleman et al., 1977b) . cells positive for viral antigens by immunofluorescence have also been reembedded and the same cells have been found to contain the expected virus particles (epstein and achong, 1968 ). tissue and cell structure is generally adversely affected in reprocessed samples, because the original processing for light microscopy is not suitable for electron microscopy, but viral structures rarely disintegrate so far as to be unrecognizable. however, initial use of bouin's fixative did destroy herpesvirus structure (cockson and holmes, 1977) . viral antigens can be detected in thin sections of infected cells by iem with suitably labeled specific antibodies. certain factors limit the technique for diagnostic virology: prolonged fixation and the use of standard concentrations of fixatives reduce antigenicity and also limit the penetration of reagents into fixed cells, thus making it difficult to investigate intracellular antigens (smit et al., 1974; brown and thormar, 1976) . the average diagnostic schedule is not oriented toward light fixation of specimens immediately followed by intricate processing. various methods are available to process already heavily fixed tissues but the preservation of fine structure is generally poor (miyamoto et al., 1971; hadler and dourmashkin, 1975; mohanty, 1975; wendelschafer-crabb et al., 1976; bohn, 1980; sisson and vernier, 1980) . reactions can be attempted on already thin-sectioned material but nonspecific staining is a problem (thomson et al., 1967; hansen et al., 1979; takamiya et al., 1979) . trypsin digestion has restored viral antigenicity to formalin-fixed material for immunofluorescence and might be useful for thin section iem (huang et al., 1976; swoveland and johnson, 1979; johnson et al., 1980) . the antibody markers used in thin section iem are usually ferritin or peroxidase and the methods have been reviewed by howe et al. (1974) and by kurstak and kurstak (1974) . cytochrome c has also been used to label antibodies (singer, 1974) and peroxidase-antiperoxidase methods based on those used in light microscopy have recently been developed (hsu and ree, 1980) . reprocessing immune light microscopy preparations for thin sections or thin section iem has confirmed reaction specificities (epstein and achong, 1968; chapman, 1970; kumanishi and hirano, 1978) . virus particles are sometimes present in such large numbers in clinical specimens that they can be detected directly by electron microscopy and negative staining methods in particular can be used to provide a rapid diagnosis. there are of course limitations i n sensitivity and in the fact that the technique gives only a morphological grouping in the first instance. however, iem can be used in certain circumstances to give further identification. viral skin lesions may contain a large number of virus particles and samples are thus highly suitable for electron microscopy. before the advent of negative staining techniques viruses extracted from such samples were examined, with variable success, after metal shadowing (nagler and rake, 1948; van rooyen and scott, 1948; melnick et al., 1952) and tissue biopsies have been thin sectioned to demonstrate virus particles (cf. sutton and burnett, 1969; kimura et al., 1972) . however with negative staining a rapid diagnosis can be made. vesicular fluid is a suitable starting material for this technique; equally good are dried smears of lesion scrapings, rehydrated in a minimal quantity of distilled water. crude extracts of solid tissue in distilled water may also be utilized (macraeet al., 1969) . because of the high viral content of the lesions it is usually unnecessary to concentrate virus before negative staining. samples are best unfixed as this facilitates extraction of virus from the cells and fixation before negative staining may hinder virus recognition, especially of herpesviruses. because of variation of viral content in different samples it is advisable to prepare specimens from more than one lesion (cruickshank et al., 1966; harkness et al., 1977) . application of negative staining techniques to diagnosis of viral skin lesions, particularly to the differential diagnosis of smallpox (a poxvirus as opposed to the herpesvirus of varicella zoster), was a major factor in establishing electron microscopy in diagnostic virology (peters et williams et al., 1962) . although smallpox has been eradicated other diagnostic problems remain for solution by electron microscopy. human monkeypox infections were diagnosed by electron microscopy and in some cases other laboratory tests were negative (breman et al., 1980) . orf virus grows only with difficulty and electron microscopy is the only practical diagnostic method; similarly, laboratory diagnosis of molluscum contagiosum is possible only by electron microscopy. compared with virus isolation and gel precipitin tests electron microscopy was conspicuously effective in diagnosis of varicella zoster virus infections (cruickshank et macrae et al., 1969) . although poxviruses and herpesviruses are morphologically distinguishable, individual orthopoxviruses (vaccinia, variola, and cowpox) cannot be differentiated morphologically nor can the herpesviruses (herpes simplex and varicella zoster). negative stain iem is not useful in this diagnostic situation because of the small sample size and because other laboratory tests can be more simply used to give precise identification (macrae et al., 1969) . the viral etiology of skin warts has been repeatedly demonstrated by electron microscopy. in thin sections paracrystalline arrays of papillomavirus particles have been observed (strauss et al., 1949; bunting, 1953) and these thin section appearances have been correlated with intranuclear inclusions seen by light microscopy . the virus particles reacted with wart virus antiserum in thin section iem preparations (viac et al., 1978) . negative staining was successfully applied to homogenates of skin warts and negative stain iem was used to study antigenicity of extracted virus . some skin warts contain only a small number of virus particles in thin sections (maciejewski et al., 1973) and genital warts usually have a low viral content. oriel and almeida (1970) suggested that the best method to extract virus from biopsies of genital warts was by light grinding to disrupt only the surface layers rather than complete homogenization of the tissue. a recent study of the wart virus lesions of epidermodysplasia verruciformis showed that negative staining was slightly more sensitive than thin sections to detect virus in the early malignant lesions where viral content is very low (yabe and sadakane, 1975 al., 19771 , but as yet there has only been one report of a successful negative stain diagnosis (goodfellow and calvert, 1979) . laryngeal wart thin sections have shown scanty papillomavirus-like particles some of which were not entirely convincing in the published micrographs (dmochowski et al., 1964; boyle et al., 1971; spoendlin and kistler, 1978) , but these findings were confirmed when papillomavirus antigen was found in laryngeal papillomas by light microscopy peroxidase-antiperoxidase techniques (lack et al., 1980) . papillomavirus particles have also been observed in thin sections of atypical genital warts resembling early dysplasia in the cervix and vagina (laverty et morin and meisels, 1980) . particles resembling paramyxovirus nucleocapsid have been reported in thin sections of cells in measles skin rash biopsies (kimura et al., 19751 , in skin lesions of discoid lupus (hashimoto and thompson, 1970) , in behqet skin lesion biopsies (tawara et al., 1976) , and in warts, bowen tumours, and basal cell carcinomas (maciejewski et al., 1973) . these findings must be viewed with caution as artifacts which resemble nucleocapsid are not uncommon. this will be discussed in section vi,b. nasopharyngeal secretions have been examined for virus particles a>fter dilution in distilled water and negative staining. in samples revealing paramyxovirus particles studied by doane et al. (1967) all yielded parainfluenza virus type 1 in cell culture . joncas et al. (196913) examined lysates of cells in nasopharyngeal secretions by negative staining and also used doane's method and found that several samples contained paramyxoviruses. these were differentiated into respiratory syncytial virus (rsv) and other paramyxoviruses according to the width of the nucleocapsid. isolation studies confirmed all the rsv identifications. however adenoviruses and picornaviruses isolated from these samples were never detected by electron microscopy. in a large survey of routine specimens examined by electron microscopy and virus isolation it was shown that electron microscopy was comparatively insensitive for the detection of myxoviruses and paramyxoviruses (pavilanis et al., 1971 ) although valters et al. (1975) found that negative stain iem increased the sensitivity of electron microscopy for detection of viruses in throat swabs. the difficulty of differentiating the paramyxoviruses seen has made it necessary to continue either routine virus isolation or immunofluorescence, which has proved the most useful rapid and specific diagnostic method providing suitable samples are available (gardner and mcquillin, 1980; minnich and ray, 1980) . partially enveloped herpesvirus particles observed in negatively stained preparations of concentrated throat washings from a patient excreting virus were identified as eb virus by culture methods (lipman et al., 1975) and lee et al. (1978) detected herpesvirus particles by the pseudoreplica method in throat swabs of five infants which were identified as cytomegalovirus in cultures from all samples. many patients excreting rotavirus in their feces have respiratory as well as gastrointestinal symptoms, but a study of nine such patients using negative staining methods failed to detect rotaviruses in throat swabs or nasopharyngeal secretions (lewis et al., 1979) . saliva has often been subjected to negative staining and negative stain iem studies for hepatitis b particles. occasionally the search has been successful (kistler et bancroft et al., 1977) though the photographic evidence is not always completely convincing, probably because so few particles are present. a recent report correlated the presence of dane particles in negatively stained density gradient fractions of saliva with the presence of dna polymerase. small round particles resembling those of hbsag were also present but negative stain iem was not done to prove their identity (macaya et al., 1979) . norwalk agent particles have been found in a sample of vomit concentrated 100-fold by ultracentrifugation before negative staining. however, three more vomit samples positive for norwalk agent by radioimmunoassay were negative by electron microscopy (greenberg et al., 1979) . the transmission of hepatitis b in blood was known for many years before the first electron micrographs of negatively stained small round and long particles of hbsag in serum were published (bayer et al., 1968) . shortly after this negative stain iem was used to aggregate the particles so rendering them more recognizable (almeida et al., 1969b; hirschman et al., 1969) . this was immediately useful in diagnosis and sera began to be widely examined by negative stain iem after concentration. circulating immune complexes were observed in some sera which were examined without adding antibody (almeida and waterson, 1969b) . sera with such complexes frequently gave false-negative results in the other relatively crude detection tests then available (krohn et al., 1970; cossart et al., 1971) . it has been suggested that electron microscopy is more sensitive than the modern radioimmune and hemagglutination assays for detection of immune complexes (trepo et al., 1974) . dane described the larger particles bearing his name in 1970 (dane et al., 1970) and almeida et al. (1971) showed that the cores of the dane particles could be extracted and, by negative stain iem, were antigenically distinct from the other particles. positive sera always contain the small round particles; long particles are next in frequency and dane particles are the least common (dane et al., 1970; stannard et al., 1973) . the extra sensitivity of modern tests has rendered electron microscopy less useful in routine detection of hepatitis b antigens but it is a relatively easy way to identify sera containing dane particles and their presence can be correlated with high risk of hepatitis transmission. gel precipitin techniques are still used in routine testing and confirmation of the specificity of these tests by negative staining of extracted gel lines is useful. hepatitis a infection is not blood transmitted but there is one report of virus-like particles of variable size in human sera in the acute stages (zanen-lim, 1976) . the samples examined were gel precipitin lines formed between patients' sera and animal sera raised against them, a system susceptible to nonspecific reactions. some particles illustrated resembled common artifacts found in human sera which will be discussed in section vi, a. it is uncertain if non-a, non-b hepatitis has one etiologic agent or many. studies are underway to examine sera by negative staining for possible particles but the results which have been obtained are inconsistent with each other. in one study seemingly specific gel precipitin lines between sera were examined by a n unusual method: the gel containing the line was fixed in osmium and embedded in araldite blocks before negative staining. no significant particles were seen (tabor et al., 1979) . in another study particles 60 nm in diameter with 40-nmdiameter cores were seen in some sera (coursaget et al., 1979) . hantz et al. (1980) reported the presence of particles closely resembling those of hbsag in size and shape; however these were antigenically distinct from hbsag, were present in very low concentration, and were not reactive in negative stain iem with antisera which gave gel precipitin lines with the same sera. the gel lines were not examined . yoshizawa et al. (1980) reported the presence in very low concentrations of viruslike particles approximately 27 nm in size detectable only by negative stain iem using large volumes of sera from patients with non-a, non-b hepatitis. similar particles were observed in chimpanzee sera after experimental infection. mori et al. (1980) , in a negative staining study of density gradient fractions of non-a, non-b sera, showed hexagonal 32-nm enveloped particles with 22-nm hexagonal cores as well as free 22-to 24-nm particles, but no iem was done. theoretically, any virus causing massive viremia could be detected in serum by electron microscopy. ebola virus was seen in human serum in the course of one infection (bowen et al., 1978) . when testing sera by negative stain iem for hbsag we occasionally observed parvovirus-like particles with a diameter of approximately 23 nm. the human hbsag detector serum routinely used had antibodies to the parvovirus-like particles and so formed them into immune complexes closely resembling those of hbsag small round particles (cossart et al., 1975) . these parvovirus-like agents are antigenically distinct from those found in feces and no diseases, except possibly a short febrile illness (shneerson et al., 1980) and onset of hypoplastic crisis in sickle cell anemia (pattison et al., 1981) , have yet been associated with them. particles resembling coronaviruses have also been seen in sera during routine hbsag testing by negative staining techniques stannard et al., 19731 , but these particles are probably forms of serum lipoproteins (ackermann et al., 1974a) . negative staining examination of urine after suitable concentration has revealed herpesvirus particles in patients excreting cytomegalovirus (paradis et al., 1969) . montplaisir et al. (1972) found electron microscopy was more successful if large volumes, 30 to 50 ml, were used. they also noted that cytomegalovirus isolation in suitable cell cultures, although slower than electron microscopy, was much more sensitive. this was confirmed, comparing isolation with pseudoreplica methods, by lee et al. (19781, who observed however that electron microscopy was most sensitive in tests on children younger than 6 months; presumably in these congenital infections large numbers of cytomegalovirus particles are excreted. in contrast henry et al. (1978) found that negative staining of ultracentrifugation pellets from 5-ml samples of urine was more sensitive than virus isolation, however, they were using a detection level of only four or five virus particles to a grid indicating prolonged examination in the electron microscope. the same authors examined thin sections of cells excreted in urj ne but saw no herpesvirus particles. immune electron microscopy has not been used to differentiate between herpesviruses seen in urine. we have seen herpesvirus particles in urine from which herpes simplex virus has been grown; without the culture results the particles might have been assumed to be cytomegalovirus (field and gardner, unpub-lished observations) . our experience with urine of immunosuppressed adults has been that cytomegalovirus is frequently cultured but rarely seen (coleman et al., 1973) . we first observed papovaviruses, of the polyomavirus subgroup, in concentrated urine samples from a n immunosuppressed renal transplant recipient in 1971 (gardner et al., 1971) . since then we and other groups of workers have seen similar particles in urine of various categories of patients: renal transplant recipients (coleman et al., 1973; lecatsas et al., 1973; dougherty and di stefano, 1974; hogan et al., 1980a) , patients under treatment for malignancy (reese et gardner, 19771, and pregnant women (coleman et al., 1977a, 1980; lecatsas et al., 1978) . sometimes the virus particles are coated with a n antibody-like substance and specific antiviral antibodies can be detected in urine by negative stain iem and other tests (gardner et al., 1971; reese et al., 1975) . when sufficient virus is present in urine it is possible to identify the precise type of virus by negative stain iem provided the particles are not already coated with antibody . all our attempts to detect polyomavirus particles in urine by negative stain electron microscopy have been accompanied by virus culture and cytologic studies. to confirm cytologic detection of virus the urine cells with viral inclusions were embedded for thin sectioning. although ultrastructural preservation of the cells was poor, polyomavirus particles were clearly identified (coleman et al., 1977b) . cytology is often more sensitive than either virus isolation or negative stain electron microscopy and reprocessing in this way has increased the diagnostic potential of the electron microscope (coleman et al., 1980). as well as typical polyomavirus particles lecatsas and prozesky (1975) observed filamentous and minispherical forms in one urine. recently papillomavirus particles were seen in urine from pregnant women (lecatsas and boes, 1979) . urine samples can be contaminated by feces and this may be the origin of rotavirus particles which were seen in one sample of urine of nine examined from babies excreting rotaviruses in feces (chrystie et cl., 1975) . urine of two patients in the acute phase of non-a, non-b hepatitis contained virus-like particles 60 nm in diameter with 40-nm cores (coursaget et al., 1979) . in 1972 anderson and doane described a n agar filtration technique to rid samples of undesirable salts and to concentrate virus particles onto the grids. a sample chosen to illustrate the technique was a fecal extract and the micrograph showed particles resembling rotaviruses though the identification made was reovirus. this may be the fist picture published of human rotavirus (anderson and doane, 1972a) though it is not clearly stated in the text that the feces were human. in the same year the detection by negative stain iem of norwalk virus particles in fecal extracts from volunteers in gastroenteritis experiments was reported (kapikian et al., 1972b) . the observation in 1973 of rotaviruses in thin sections of duodenal biopsies of infants with diarrhea (bishop et al., 1973) was closely followed by the observation of rotaviruses in fecal extracts by negative staining methods (flewett et al., 1973; middleton et al., 1974) . all these illustrated the suitability of electron microscopy for the diagnosis of viral agents in diarrhea and opened up a new field of fecal virology which continues to expand (flewett, 1979; holmes, 1979) . fecal extracts may be examined by negative staining without concentration but this is successful only if virus content is very high. it is more usual to employ some method, usually ultracentrifugation, to concentrate virus . portnoy et al. (1977) compared three methods: direct examination of uncentrifuged fecal extracts; a pseudoreplica method using clarified fecal extracts; and virus concentration by ultracentrifugation. they found that the last two techniques were more sensitive than direct examination and that the small viruses were more readily detected by ultracentrifugation than by the pseudoreplica method. concentration of enteroviruses from fecal extracts by adsorption onto a polyelectrolyte followed by elution into a smaller volume for negative staining was successful for detection of lo6 poliovirus particles per milliliter (chaudhary and westwood, 1972) . concentration of virus in fecal extracts by selective removal of water, salts, and low-molecular-weight substances into lyphogel had a sensitivity equal to or greater than ultracentrifugation and the viral morphology was not affected (whitby and rodgers, 1980) . ammonium sulfate precipitation of viruses from fecal extracts has been found useful and the morphology of coronaviruses was particularly well preserved (caul et al., 1978) . narang and codd (1980a) found that low-speed centrifugation of lightly clarified fecal extracts onto grids placed at the base of specially shaped tubes was sufficient for most fecal diagnostic work although smaller virus particles (20 to 35 nm) did not appear on the grids unless present in large aggregates (narang and codd, 1980b) . comparing narang and codd's method with other techniques, roberts et al. (1980) found that leaving the prepared centrifuge tubes on the bench gave the same results as the low-speed centrifugation and they suggested the aggregates of virus seen were released from infected cells which had settled on the grid to be lysed in the subsequent staining. narang and codd's experiments show that the comparatively high centrifugal forces used in most laboratories to clarify fecal extracts might rid the preparation of much of the desired virus particularly if it is aggregated. juneau (1979) suggested incorporating normal human immunoglobulin into agar used in the agar-filtration techniques thus making virus particles in fecal extracts more easily recognized by coating them with antibody. however, if there is too much antibody on the particles viral structure may be obscured and identification becomes difficult. it has also been shown that human antibodies are capable of indiscriminate coating of viruses, virus-like particles, and bacteriophages locarnini et al., 1974) . the diagnosis of rotavirus infection has considerable clinical value. tests other than electron microscopy to detect rotaviruses are available (who scientific working group, 1980) but are all limited by a requirement for high quality viral antisera. rotaviruses have a typespecific antigen on the outer layer of capsomeres and a group-specific antigen on the inner layer. loss of the outer layer is common and immune detection methods are imprecise a s a result. rotaviruses may already be coated with antibody when excreted (watanabe and holmes, 1977) which also renders detection by immune methods more difficult. this has ensured a continued role for electron microscopy in rotavirus diagnosis. in addition electron microscopy is a nonselective method and in the search for rotaviruses other agents may be rev e a 1 e d. aberrant rotavirus capsids in tubular form are sometimes observed. negative stain iem studies showed these were antigenically identical to the spherical capsids (holmes et al., 1975) . human rotavirus subtypes have been identified by various techniques including negative stain iem . adenovirus particles have been observed by negative staining in feces of patients with gastroenteritis and frequently are noncultivable (bruce white and stancliffe, 1975; bryden et al., 1975) . it has recently been demonstrated that they belong to a new adenovirus serotype (johansson et al., 1980) . coronavirus-like particles have been seen in human feces but their presence often cannot be correlated with illness mathan et al., 1975; schnagl et al., 1978; clarke et al., 1979) . morphologically they differ from the classical coronavirus by having greater pleomorphism and narrower surface projections (caul and egglestone, 1977; caul et al., 1977) . the viral nature of these particles has not yet been established conclusively. although coronavirus-like particles were seen in the abortive replication cycle in cell culture the thin sections failed to show typical coronavirus maturation stages (caul and egglestone, 1977) . the viral nature of the fecal particles has been questioned by dourmashkin et al. (1980) who sectioned a fecal extract pellet which by negative staining contained typical pleomorphic coronavirus-like particles. no typical coronaviruses were seen in the thin sections although large numbers of fringed membrane-bound objects were present. however, it is possible that the pleomorphism of the negatively stained particles reflects some abnormality, such as lack of core material, which would give rise to this appearance in thin sections. norwalk agent was first described in feces from volunteers with experimental gastroenteritis (kapikianet al., 1972b) . unlike the smoothsurfaced parvoviruses and picornaviruses, negatively stained norwalk particles appear to have a structured surface and edge. particles are 27 to 34 nm in diameter with a density of 1.36 to 1.41 gm/ml. particles similar in appearance and antigenically related were observed in feces in a gastroenteritis outbreak in montgomery county and other morphologically similar but antigenically unrelated particles were reported from hawaii (thornhill et al., 1977) . particles resembling norwalk morphologically and antigenically, 27 to 30 nm in diameter and with density 1.38 gm/ml, were excreted by patients with gastroenteritis following the consumption of oysters in australia (cross et al., 1979; murphy et al., 1979) . two further agents morphologically identical to norwalk, with densities 1.35 to 1.37 and 1.37 to 1.4 mg/ml, but both antigenically distinct from norwalk were found in feces of gastroenteritis patients in japan (taniguchi et al., 1979; kogasaka et al., 1980) . morphologically similar particles have also been seen in the united kingdom and, based on the examination of these particles and of norwalk agent, it has been suggested that there are morphological similarities with caliciviruses . viruses morphologically indistinguishable from classical caliciviruses were observed in human feces (madeley and cosgrove, 1976) but were not associated with illness until more recently when they were implicated in winter vomiting disease and gastroenteritis (mc-swiggan et al., 1978; chiba et al., 1979 chiba et al., , 1980 cubitt et al., , 1980 suzuki et al., 1979) . negative stain iem has been used in these studies to detect the appearance of antibodies to the agents seen and to investigate the antigenic nature of the caliciviruses. particles measuring 26 to 30 nm with a solid star-shaped pattern on their surface have been termed astroviruses. the surface pattern dif-fers from the hollow star of david pattern on the 29-to 33-nmdiameter calicivirus. madeley (1979) has described the differential morphology in detail. astroviruses were first observed in negative staining studies of feces of babies in maternity ward outbreaks of gastroenteritis and in other infants with gastroenteritis (madeley and cosgrove, 1975) . parvovirus-like particles with a diameter of 22 nm were first observed in human feces by paver et a2. (1973) . for their detection it was found necessary to use negative stain iem with postinfection human sera t o agglutinate the particles. identification of parvoviruses depends upon the smooth-surfaced morphology; the size, which is slightly smaller than the morphologically similar enteroviruses; and the density, which is higher than enterovirus density. because of overlap in both size and density ranges between these two groups precise identification is often impossible. fecal parvovirus-like agents are noncultivable whereas enteroviruses can usually be grown in cell cultures. appleton et al. (1977) described 26-nm parvovirus-like particles with density 1.38 to 1.40 gm/ml in feces from a school outbreak of winter vomiting disease (the ditchling agent) and other 25-to 26-nm parvovirus-like particles with density 1.40 gm/ml (appleton and pereira, 1977) in feces of patients with gastroenteritis after eating cockles (cockle agent). negative stain iem showed that these two agents differed antigenically, both were unrelated to norwalk but ditchling was related to the w agent, an earlier reported parvovirus-like particle (paver et al., 1973) . another school outbreak of gastroenteritis was associated with a 23-to 26-nm parvovirus-like particle but no density estimations or cross-reactions with other parvovirus-like agents were described for the paramatta agent (christopher et al., 1978) . although norwalk was assumed to be the cause of the australian oyster-associated gastroenteritis, parvovirus-like particles, 22 to 25 nm in diameter, were also seen in many of the fecal samples and similar particles were seen in one oyster sample . negative staining examination of feces of hepatitis patients, originally in a search for hbsag and latterly for hepatitis a virus, has revealed interesting particles . cross et al. (1971) found 15-to 25-nmdiameter small round particles and 35-to 45-nm-diameter particles resembling dane particles. by gel diffusion and negative stain iem there was no antigenic similarity between the smaller particles and the small round particles of serum hbsag but slight cross-reactivity between the larger particles and the dane particles of hbsag was observed. moodie et al. (1974) observed that gut digestive enzymes would degrade all hepatitis b antigen with the exception of the dane particle cores and in fact there have been no convincing electron microscopy reports of hepatitis b particles in feces. the causative agent of hepatitis a was revealed as a 27-nm-diameter small, round, smooth-surfaced virus particle by feinstone et al. (1973) who used volunteers' fecal samples and negative stain iem with convalescent sera to detect the virus. excretion of the particles was time related to symptoms. these findings have been confirmed by others (locarnini et al., 1974; gravelle et al., 1975) and particles seen in different outbreaks have been found to be antigenically identical. coulepis et al. (1980) showed that maximal virus excretion occurred just before the onset of symptoms, fell slightly in the 5 days after onset while patients had dark urine, and then reduced steadily until by 2 weeks after onset virus was only just detectable by electron micros-copy * breast milk has been tested for hbsag by negative stain iem after concentration of the samples by ultracentrifugation and small round particles have been seen (boxall et al., 1974) , but no strict tests were done to exclude the presence of occult blood. following the analogy of the mouse mammary tumor virus human breast milk has been surveyed for retroviruses. particles resembling type b oncoviruses were observed by using thin sections and negative staining in parallel with biochemical studies on fractionated milk. some samples of human breast milk degraded the structure of true type b oncovirus particles added to them (sarkar and moore, 1972) rendering the particles unrecognizable. chopra et al. (1973) found type d oncovirus-like particles in breast milk using negative staining but could not correlate electron microscopy and biochemical findings. it is possible to demonstrate viral antigens by immunofluorescence in cells in cerebrospinal fluid (csf) in viral encephalitis or meningitis (dayan and stokes, 1973; taber et al., 1973) . a paramyxovirus has been seen in csf by negative staining and mumps virus was isolated from the sample (doane et al., 1967) . thin sections of csf cells in presumed mumps virus meningitis showed cytoplasmic collections of tubules resembling paramyxovirus nucleocapsid (herndon et al., 1974) . most human tissues have been examined by electron microscopic methods for virus particles at some time. however, it is comparatively rare for such investigations to be included in routine viral diagnosis. sample selection is a considerable problem for both thin-sectioned and negatively stained preparations of tissues. immune electron microscopy on sectioned material presents considerable technical difficulties and viral content of tissue homogenates may be too low for negative stain iem so a virus seen in the tissue cannot always be sufficiently well identified for diagnostic purposes. immunofluorescence and other light microscopy immune methods have greater diagnostic potential because they are simpler, larger samples are used, and precise virus identification is possible. nevertheless, electron microscopy has been important in revealing viral etiology, often for the first time, and it has frequently been the impetus for development of the more convenient light microscopy techniques. of recent years examination of tissues for viruses by electron microscopy has concentrated upon three major areas: the brain, the liver, and tumors. these applications illustrate well the techniques, problems, and achievements i n viral diagnosis by electron microscopy of tissues. ultrastructural studies of virus infections of the human brain have been reviewed recently (mirra and takei, 1976) . the light microscopy neuropathology of brain affected by the rare condition progressive multifocal leukoencephalopathy (pml) was first described by astrom et al. (1958) . lesions were later shown by thin section electron microscopy to contain papovavirus particles by zu rhein and chou (1965) . extracts of formalin-fixed brain were negatively stained and typical papovavirus particles were observed which were 41 nm in diameter and were clearly members of the polyomavirus subgroup (howatson et al., 1965) . virus particles were also demonstrated in unfixed pml brain homogenates by negative staining (schwerdt et al., 1966) . it was not until 1971 that a new polyomavirus, jc virus, was cultivated from pml brain (padgett et al., 1971) . the following year two isolates of a polyomavirus antigenically similar to simian virus 40 (sv40) were reported from pml brains (weiner et al., 1972) but all subsequent strains isolated from such material have been identified as j c virus (padgett et ul., 1976) . in thin sections of brain typical spherical polyomavirus particles are found in the nuclei and cytoplasm of oligodendrocytes and filamentous forms of the virus are also frequently present (fig. 13) . brain homogenates may be so rich in virus that concentration by ultracentrifugation before negative staining is unnecessary. filamentous particles have not been seen in negatively stained brain extracts even when they were plentiful in the thin sections (field, unpublished observations) . when jc virus had been grown in uitro and specific antisera were prepared, virus particles extracted from infected brains were identified by negative stain iem and parallel immunofluorescence studies were performed on brain sections (narayan et al., 1973) . for successful negative stain iem virus content of the brain sample must be high and virus particles must be free of cell debris. because j c virus is relatively difficult to cultivate this technique has great potential. although diagnosis of the polyomavirus infection by recognition of the typical particles in thin sections and negative stains of formalin-fixed brain is straightforward, it has proved impossible to identify the virus antigenically by negative stain iem after formalin fixation (narayan et al., 1973; padgett et al., 1976; field and gardner, unpublished observations) . laboratory diagnosis of infection with the measles-like virus of subacute sclerosing panencephalitis is generally based upon detection of measles antibodies in serum and in cerebrospinal fluid. in rare fig. 13 . thin section of pml brain showing both spherical and filamentous polyomavirus particles within the nucleus. ~25,000. bar = 1 pm. cases when brain biopsy is performed the usual laboratory diagnosis is by immunofluorescence with specific measles virus antiserum or, less desirably, measles convalescent human serum. confirmation by electron microscopy may be sought because virus culture, although possible (chen et horta-barbosa et al., 19691 , is technically difficult. thin sections of sspe brain have been found to contain collections of 17-to 19-nm-diameter tubular paramyxovirus nucleocapsids in nuclei and cytoplasm of oligodendrocytes and neurons (herndon and rubinstein, 1968) (fig. 14) . the nuclear particles are clearly tubular and have no surface coating but cytoplasmic nucleocapsid is usually coated with a granular substance. there have been only two reports of clearly recognizable paramyxovirus nucleocapsid in negatively stained sspe brain homogenates (dayan and cumings, 1969; dayan and almeida, 1975) . we have examined concentrated homogenates from nine sspe brains, of which four contained typical sspe viral tubules in thin sections and two contained measles antigen by immunofluorescence, but in none have we seen any paramyxovirus nucleocapsid by negative staining (richmond and field, unpublished observations). antigenic identification of the virus seen in brain is thus dependent richmond.) upon thin section iem with all its attendant technical difficulties (jenis et al., 1973) . sspe brain cells cultured in uitro have been shown to contain paramyxovirus nucleocapsids in nuclei and cytoplasm (chen et al., 1969; katz et al., 1969) and typical negatively stained paramyxovirus helix has been seen in extracts of these cultured cells . in cases of suspected herpes encephalitis laboratory confirmation of herpes simplex is sometimes urgently required and the most sensitive and rapid method for this is immunofluorescence on brain biopsy material. herpesvirus particles are found in thin sections (fig. 15 ) in nuclei and cytoplasm of neurons and glial cells (harland et al., 1967; roy and wolman, 1969 ; baringer and swoveland, 1972; viloria and garcia, 1976) . the thin section technique is too slow for rapid diagnosis and as virus-infected cells are distributed unevenly sample selection is a major problem. virus can be seen in negatively stained brain homogenates but this procedure is comparatively insensitive (flewett, 1973; ross, 1973; joncas et al., 1975) . in our experience virus particles are extremely sparse even after ultracentrifugation of brain homogenates (field, porter, and richmond, unpublished observations al. (1969) saw herpesvirus particles in glial cells in thin sections of the brain from a patient with encephalomyelitis and cultured varicella zoster virus. in the absence of virus culture complete identification of an observed herpesvirus is difficult. thin section iem is technically difficult although possible (kumanishi and hirano, 1978) ; negative stain iem is unsatisfactory because there is insufficient virus and even by immunofluorescence there are problems with antigenic crossreactions with other herpesviruses (emmons and riggs, 1977) . electron microscopy has not on the whole proved useful for the laboratory diagnosis of rabies infection of human brain. the methods more generally used are histology, immunofluorescence, and virus isolation. the negri bodies in histological sections are sometimes but not always identical with the cytoplasmic viral factory areas of thin sections (morecki and zimmerman, 1969; lemercier et al., 1970; vallat et al., 1977) and rabies virus particles may bud from factory sites or from diverse cytoplasmic membranes of neurons. factory sites and virus particles are easily recognized in thin sections. cell culture-derived rabies virus is difficult to identify in negatively stained preparations because of its fragility, indicating that attempts at rapid diagnosis by this technique on brain samples would be unsatisfactory. adenovirus encephalitis was investigated by chou et al. (1973) who found inclusion bodies in histological sections and typical, mostly intranuclear, adenovirus particles in neurons and glial cells in thin sections. adenovirus particles were also seen in negatively stained brain homogenate, from which adenovirus type 32 was later isolated. investigating eastern equine encephalomyelitis, bastian et al. (1975) isolated the virus, found typical histology, and for the first time demonstrated typical togavirus particles in thin sections of human brain. particles resembling paramyxovirus nucleocapsids have been observed in thin sections of multiple sclerosis brain lesions, mostly sited in nuclei with probable leakage t o the cytoplasm (prineas, 1972; lhermitte et watanabe and okazaki, 1973) and sometimes exclusively in the cytoplasm (narang and field, 1973; pathak and webb, 1976) . however, similar particles have been seen in unrelated conditions of the brain, in normal brain, and in other organs; immunofluorescence and iem studies have failed to identify the particles as viral. suggestions have been made that abnormal condensation of nuclear chromatin and multiple invaginations of cytoplasmic membranes might be the cause of these virus-like structures (baringer and swoveland, 1972; dubois-dalcq et al., 1973; tanaka et al., 1974 tanaka et al., , 1975b hayano et al., 1976; kirk and hutchinson, 1978; lehrich and arnason, 1978) . although a paramyxovirus (parainfluenza virus type 1) was re-trieved from multiple sclerosis brains by cell fusion techniques, no virus particles were seen in thin sections of the original brains, although some cytoplasmic nucleocapsid was seen at the eleventh pass of brain cells in culture (ter meulen et al., 1972) . not surprisingly, in view of the experience with sspe brain, no paramyxovirus particles have been reported in negatively stained extracts of multiple sclerosis brain. particles identified by bastian (1971) as papovaviruses were seen in cytoplasm and extracellularly in a human choroid papilloma, but the particles were not morphologically characteristic and the lack of intranuclear particles was unusual. similar particles in other human choroid papillomas were shown to be glycogen (carter et al., 1972) . early reports claimed ultrastructural evidence for association of a papovavirus with the sspe paramyxovirus (koprowski et oyanagi et al., 1970) but none of the published micrographs has convincingly demonstrated papovavirus and the particles were never intranuclear and were seen only in cultured brain cells and not in the original tissue. a large number of 45-nm-diameter granules with irregular edges were observed in astrocyte cell processes in a creutzfeldt-jakob diseased brain and were identified as papovaviruses although the morphology was not convincing (de reuck et al., 1976) . kirk and hutchinson (1978) believe that these cytoplasmic papovavirus-like structures are probably reticulosomes and related structures normally present in cells. creutzfeldt-jakob disease has some characteristics of a virus infection and brain tissue has been examined by electron microscopy for a possible etiologic agent. the histology, characteristic of a spongioform encephalopathy, is the usual means of laboratory diagnosis. in thin sections tubular particles and variably sized round and hexagonal particles with and without cores have been reported (vernon et bots et al., 1971; narang, 19751 , but none has been demonstrated to be viral. recently objects resembling spiroplasmas have been observed in thin sections of creutzfeldt-jakob brains (bastian, 1979; a. . infection of the liver with hepatitis b virus is accompanied by ultrastructural changes. parvovirus-like particles were fist described in hepatocyte nuclei in thin sections by nowoslawski et al. (1970) and these observations were soon confirmed (nelson et al., 1970; scotto et az., 1970) (fig. 16) . for some time it was assumed that these particles were identical to the small round particles of hbsag in negatively stained serum and immunofluorescence and thin section iem using human antisera tended to confirm this impression (gerber et al., 1972; huang et al., 1972) . meanwhile cytoplasmic particles resembling the pleomorphic forms of serum hbsag were seen in liver cells in thin sections (huang, 1971; stein et al., 1971) . by immunofluorescence, sera specific for hbsag revealed antigen in cytoplasm and sera specific for hbcag traced a nuclear antigen and so it was suggested that the nuclear, parvovirus-like particles must be hbcag and the pleomorphic cytoplasmic particles hbsag (gerber et al., 1974; gyorkey et al., 1974) . thin section iem has confirmed this and has also shown hbcag in cytoplasmic maturing dane particles (huang and neurath, 1979) . examination of liver homogenates with negative staining had already shown the three morphologic forms of hbsag and when 25-to 27-nm round particles were also seen it was suggested that these might be the intranuclear, parvovirus-like particles since they were certainly antigenically different from the particles of hbsag huang and groh, 1973a) . extracts of heavily infected human liver are a good source of hbcag for detection of antibodies by negative stain iem (cohen and cossart, 1977; cohen, 1978) . recent reports have described intranuclear, roughly spherical, 27nm-diameter particles in human liver thin sections in non-a, non-b hepatitis (gmelinet al., 1980; grimaudet al., 1980) . these particles are however somewhat unconvincing as virus because of their size range and irregular outlines. particles with similar morphology to the hbcag have been extracted from non-a, non-b liver but they are antigenically distinct (hantz et al., 1980) . herpesvirus particles have been seen in the nuclei and cytoplasm of liver cells of a patient who died with infectious mononucleosis and extensive hepatic necrosis (chang and campbell, 1975) . arenavirus particles were described in liver thin sections of a patient with lassa fever (winn et al., 1975) and of another patient with argentine hemorrhagic fever (junin virus) (maiztegui et al., 1975) . ebola virus particles have been observed in large numbers in hepatic cells and bile canaliculi in human infection (ellis et al., 1978) . the benign human tumors molluscum contagiosum and warts have been shown by electron microscopy to have poxvirus and papovavirus etiology, respectively, and have already been described in section iv,a. papovavirus-like particles measuring 45-50 nm were seen in a cell line derived from a nephroblastoma (wilms' tumor) a t the fortieth and subsequent pass levels. the particles were recognized in nuclei in thin sections but were less convincing in negatively stained preparations. attempts to confirm the findings on other wilms' tumor cell lines were fruitless and no virus particles were seen in the original tumor . although herpesvirus particles of eb virus have frequently been observed in cultured burkitt lymphoma cells by thin section electron microscopy since they were first described by epstein et al. (1964) , there has been no report of such particles in the original tumor. eb virus particles have also been seen in lymphoblastoid cell lines derived from nasopharyngeal carcinoma (vuillaume and de the, 19731, in leukemic buffy coat cell cultures (zeve et al., 19661 , and in lymphoid cell lines derived from lymph nodes from patients with various cancers (jensen et al., 1967) , and in one of these cases herpesvirus particles were seen in a few cells of the original lymph node biopsy. epstein-barr virus particles have also been seen in lymphoid cell lines from patients with infectious mononucleosis (moses et al., 1968; steel and edmond, 19711, with hepatitis (douglas et al., 19691 , and from a n apparently normal patient (moore et al., 1967) . generally thin section methods have been used to detect the virus. negative staining has been used (hummeler et al., 1966) but appears to be less sensitive (moses et al., 1968; hillman et al., 1977) . the sophisticated immunofluorescence tests for the various eb virus antigens have supplanted electron microscopy in diagnosis particularly as even uncultured burkitt tumor cells carry one of these antigens. katayama et al. (1974) suggested the morphology of burkitt tumor cells in thin sections was so characteristic, even though no virus was seen, that electron microscopic diagnosis was superior to histology. full identification of particles seen i n cultured lymphoid cell lines is not always attempted, but herpesvirus particles seen in cell lines from patients with kaposi's sarcomas were associated with cytomegalovirus in some instances (giraldo et al., 1972) . the search for retroviruses in human tumors, cell cultures of tumors, and in placentas by electron microscopy was reviewed by de harven (1974) who reluctantly concluded that the morphologic evidence for human types a, b, and c oncoviruses at that time was extremely slight. since 1974 the situation has not changed markedly. tests for biochemical markers for the presence of a retrovirus are now frequently performed in parallel with electron microscopy. such parallel studies have utilized thin sectioning (birkmayer et al., 1974; warnaar et al., 1976) and negative staining (mak et al., 1974) of fractionated cell extracts. retrovirus-like particles were seen in cultured myeloid cells from a patient with acute myelogenous leukemia but not in the original tumor, although there was biochemical evidence for retrovirus in the uncultured cells (gallagher and gallo, 1975) . this virus was later shown to be closely related to simian sarcoma-associated virus isolated from a woolly monkey fibrosarcoma. the placenta has provided somewhat better morphological evidence for human retroviruses (dalton et al., 1974; imamura et al., 1976) but the viral morphology was not absolutely comparable with other mammalian c-type particles (dalton et al., 1974; dirksen and levy, 1977) . similar particles were detected in cultured testicular tumor cells but parallel biochemical studies were negative (bronson et al., 1979) . human embryonic cells in culture have been studied and particles resembling type c oncoviruses have been observed (chandra et al., 1970; panem et al., 1975) . types a, b, and c oncoviruses have somewhat variable structure in both thin sections and negatively stained preparations (sarkar and moore, 1972; sarkar et al., 1975) which makes morphological diagnosis difficult. the budding stage of maturation, which is perhaps the most convincing evidence of the viral nature of these particles, has rarely been observed in human tissues. the negatively stained morphology, particularly of type c oncoviruses, is so variable that this method is unsuitable for diagnosis although lo and howatson (1978) have suggested detergent treatment before negative staining to standardize particle shape. the other retrovirus subgroup, the spumaviridae, have more distinctive morphology. although not seen in the original tumor a spumavirus was identified in thin sections of cultured nasopharyngeal carcinoma cells (achong et al., 1971 ). electron microscopy alone rarely gives a sufficiently specific diagnosis of viral infection on samples taken directly from the patient and other techniques are generally employed to reach a more conclusive result. in the course of laboratory tests on such samples electron microscopy can be used to detect the presence of virus in inoculated cell cultures, in the precipitin lines of gel immunodiffusion tests, and following passage of an agent in laboratory animals. morphology can be very helpful in the preliminary identification of the virus, and, provided suitable controls are examined, electron microscopy can also detect contamination with endogenous viruses from cell cultures. density gradient studies to characterize a virus are often monitored by electron microscopy, particularly if the virus is noncultivable. simple negative staining techniques are most useful and negative stain iem can be used for more specific identification of viruses seen. thin sections are useful to detect viruses which do not have clear negatively stained morphology, such as rubella virus, and thin sections are also useful in examination of tissues from inoculated laboratory animals. viruses inoculated into cell cultures usually take some days to grow and produce typical cytopathic effects. negative staining can give early confirmation of the presence of a virus and preliminary identification by its morphology. this is a useful diagnostic aid in most circumstances and particularly so when the diagnosis is urgent as in dangerous infections such as ebola johnson et al., 1977) . but early electron microscopic examination of cultures, before cytopathic effects are well advanced, is often fruitless (field, unpublished observations) . however, the use of negative stain iem in-creased the sensitivity of virus detection so that cultures examined only 24 hours after inoculation showed virus particles (edwards et al., 1975) . rhinoviruses are particularly difficult to detect in cell cultures by negative staining and kapikian et al. (1972a) found negative stain iem was better able to detect these viruses in crude cell culture extracts. in practice diagnostic use of iem is seldom feasible because of the large number of possible serotypes. we have found that examination of inoculated cell cultures by negative staining is useful for rapid differentiation of viruses when initial biological observations cannot group them. examples are differentiation of myxoviruses from paramyxoviruses, adenoviruses from herpesviruses, and vaccinia virus (an orthopoxvirus) from enteroviruses. electron microscopy of viruses which grew in cell cultures without clearly discernible cytopathic effects was used in initial work with human coronaviruses mcintosh et al., 1967; tyrrell and almeida, 1967) and human polyomaviruses (gardner et al., 1971) . this procedure has remained useful in our laboratory to monitor growth of the human polyomaviruses both on isolation and further passage in cell cultures. viruses which are endogenous in cell cultures are a particular hazard in diagnostic laboratories and electron microscopy by negative staining and thin sectioning is useful for the detection of these agents (anderson and doane, 197213) . frequently endogenous viruses cause no cytopathic effects to arouse suspicion of their presence. for those who work with monkey kidney cell cultures the simian paramyxovirus sv5 and the simian polyomavirus sv40 have been particular problems, but these agents can be easily detected in negatively stained preparations. on the other hand simian foamy virus, a retrovirus, can be easily detected by its cytopathic effect but by electron microscopy is more difficult, thin sections being more sensitive than negative staining (anderson and doane, 1972b) . polyomaviruses have been detected by electron microscopy in vero cell lines (waldeck and sauer, 1977; gardner and field, unpublished observations) and in pig kidney cell lines (newman and smith, 1972; tischer et al., 1974) . bhk-21 hamster cell lines carry the hamster r virus detectable in thin sections (shipman et al., 1969) . parvoviruses were detected in many continuous cell lines by techniques which included electron microscopy (hallauer et al., 1971) . it was assumed that many of these originated from trypsin used when subculturing, but recently parvoviruses detected in cultured cells had as their source the calf serum used in culture media (nettleton and rweyemamu, 1980) . virus particles may be purified by gel electrophoresis. the particles are concentrated into certain regions of the gel which can then be extracted for electron microscopy. weintraub et al. (1962) purified plant viruses from plant sap by this technique and examined the gel fractions by metal shadowing to prove the purity of the harvests. ahmad-zadeh et al. (1968) used negative staining techniques on gel electrophoresis eluates to prove separation of adenovirus soluble antigens into hexons, fibers, and pentons. gel immunoprecipitin lines formed between viral antigens and homologous antibodies can be treated in the same way and the specificity of the lines becomes evident when the expected virus particleantibody complexes are seen. beale and mason (1968) investigated the antigenic nature of full and empty poliovirus particles this way. huang and groh (1973b) applied the technique to hbsag-antibody and hbcag-antibody reactions. however, almeida et al. (1974) found that immunoprecipitin lines between fecal extracts and human sera were frequently devoid of recognizable virus particles. when gel precipitin lines were examined for herpesviruses in thin sections by konn et al. (19691, of the three lines formed between eb virus and a rabbit antiserum, herpes particles, which were surrounded by antibody, were seen in only one line. fractions from density gradient centrifugation can be examined in the electron microscope for virus particles to determine their buoyant density. the cesium or sucrose must be removed before examination by application of the methods for eliminating salts described in section ii1,a. use of the technique depends upon the presence in the fractions of enough virus for particles to be detectable either by negative staining or by negative stain iem. a typical study was that of torikai et al. (1970) on a parvovirus using negative stain on the gradient fractions: particles banding at a density of 1.43 gm/ml were complete and were not penetrated by stain; particles in the 1.34 gm/ml fraction were partially penetrated by stain but were intact; at 1.30 gm/ml only shells completely penetrated by stain were seen. parallel studies showed the particles with 1.43 gm/ml density contained normal nucleic acid while those at 1.34 gm/ml had little if any nucleic acid. cultivable virus can be located in density gradient harvests by its infectivity but for noncultivable viruses electron microscopy is a prac-tical detection method which has been widely applied in density determination of agents such as the fecal parvovirus-like particles (paver et al., 1974; appleton and pereira, 19771, norwalk agent (kapikian et al., 19731 , other fecal agents resembling norwalk (kogasaka et al., 1980) , and hbsag (bond and hall, 1972) . the interpretation of results in the absence of infectivity experiments can be difficult as shown by the discrepancy in original estimates of the density of hepatitis a virus. some estimates gave a peak density around 1.4 gm/ml, consistent with parvoviruses (feinstone et al., 19741 , while other estimates were 1.34 gm/ml, consistent with enterovirus density (provost et al., 1975b; maynard et al., 19751. eventually, general agreement was reached that the latter estimates were correct (moritsugu et al., 1976; schulman et az., 1976) . the confusion arose because both enteroviruses and parvoviruses display multiple peaks in density gradients and the ranges overlap significantly. laboratory animals are sometimes inoculated with known viruses in order to develop model systems for the study of human disease. examples are picornavirus-induced hepatitis in mice (burch et al., 19731, adenovirus infection of mouse adrenal glands as a model for allison's disease (hoenig et al., 19741 , and parainfluenza type 1 infection of mouse brain as a model for multiple sclerosis (tanaka et al., 1975a) . electron microscopy of affected tissues in these experimental infections can ultimately be useful in providing examples of what might be expected in the human diagnostic situation. laboratory animals infected with viruses which do not grow in cell cultures may generate sufficient virus for use as reagents in diagnostic tests. production of hbcag from chimpanzee livers for use in detecting antibodies by complement fixation (hoofnagle et al., 1973) was suggested by thin sectioning electron microscopy studies which demonstrated intranuclear hbcag particles in liver cells, the antigenicity being confirmed by immunofluorescence . similarly, thin section studies on livers of marmosets infected with hepatitis a virus showed cytoplasmic picornavirus-like particles which could be extracted for use as antigen to detect antibodies by negative stain iem (provost et al., 197513) and by complement fixation (provost et al., 1975a) . specimens from animals infected with virus-containing material may be examined by electron microscopy, using all the same techniques as for human samples. the gb hepatitis agent passaged in marmosets has been examined using serum, liver, and feces in negative staining and negative stain iem studies (almeida et al., 1976; dienstag et al., 197613; appleton, 19771 , but no firm conclusions resulted. current descriptions of non-a, non-b hepatitis agents in chimpanzees are similarly conflicting (bradley et al., 1979; 'biquaye et al., 1980; yoshizawa et al., 1980) . since any sample examined by negative staining in diagnostic virology is likely to contain some cellular debris it is important to appreciate that this can give rise to artifacts which can be confused with virus particles. cellular membranes may bear projections comparable in size with the surface projections of orthomyxoviruses, paramyxoviruses, and rhabdoviruses (cunningham and crane, 1966; berg et al., 1969) . most cellular membrane projections tend to be globular rather than spike-like, particularly those on mitochondria1 internal membranes, and with practice it is easy to differentiate such artifacts from virus particles. particles with 5to 10-nm projections are occasionally seen in sera and have been tentatively identified as coronaviruses, but ackermann et al. (1974a1, by enzyme digestion experiments, proved they were composed of lipoprotein. it is worth noting that negatively stained cell debris is indistinguishable from mycoplasmas (wolanski and maramorosch, 19701, thus the negative staining technique should not be used for the detection of mycoplasma contamination of cell cultures and thin sectioning and even scanning electron microscopy are the methods of choice (boatman et al., 1976) . small lipoprotein particles in some sera have diameters about 20 to 23 nm and closely resemble the small round particles of hbsag (solaas, 1978) . while individual particles are difficult to distinguish from hbsag they tend to cluster together into a palisade which differs from hbsag immune complexes because the edges of adjacent particles tend to flatten against one another (fig. 17) . virus-like artifacts in feces are common and are a serious problem since confirmation of the viral nature of the particles seen by culture is rarely possible. bacterial cell walls often display substructure similar to arrays of small virus particles (dalen, 1978) . the smaller isometric bacteriophages and viruses from edible plants which might be expected to make a n occasional appearance in feces are often comparable in size, fig . 17 . artifact in human serum resembling hepatitis b antigen. negative stain. x 180,000. bar = 100 nm. appearance, and buoyant density with the small fecal viruses (tikhonenko, 1970; brown and hull, 1973) . even the development of antibodies to an agent is not proof that it is a human virus as human convalescent sera do contain antibodies to fecal bacteriophage particles locarnini et al., 1974) . brief consideration has already been given to papovavirus-like and paramyxovirus-like particles in thin sections resulting from artifacts (section iv,h,l). kirk and hutchinson (1978) have explained cytoplasmic particles in both these categories as normal cellular components. cytoplasmic paramyxovirus-like tubules in dilated cisternae of endoplasmic reticulum have often been described. in high-quality micrographs schurch and fukuda (1974) demonstrated continuity between the tubules and the cisternal membranes, proving that the tubules arose as invaginations of the membrane and were cellular rather than viral. eady and odland (1975) confirmed this in wounded tissue and suggested the phenomenon occurred in regenerating cells. intranuclear filaments which sometimes appear to be tubular have also been identified as paramyxovirus nucleocapsid, but the interpretation generally favored is that this is a postmortem chromatin change (blinrichmond. ) zinger et hayano et al., 1976) (fig. 18 ). in addition, tangentially sectioned nuclear pores can resemble herpesviruses (fig. 19) . densely stained spherical cytoplasmic particles approximately 20 nm in diameter have been observed in muscle cells and variously interpreted as picornaviruses (gyorkey et al., 1978) or, with histochemical proof, as glycogen (collins and gilbert, 1977; green et al., 1979) or, when histochemistry disproved glycogen, as arrays of ribosomes (oshiro et al., 1976) . dalton (1975) described 30-to 60-nm-diameter structures which resembled viruses and could be observed in thin sections of cultured cells, both intra-and extracellularly. he demonstrated that these originated from the fetal bovine serum used in the media and showed that none of the particles was viral. electron microscopy is a relatively good test in virus diagnostic work because positive results are convincing, there is photographic evidence, and results obtained from samples direct from the patient are not complicated by possibilities of cross-contamination. but it does suffer from a low sensitivity and from a lack of automation for large-scale studies compared with some of the other available tests. it is not the method of choice in the diagnosis of herpes simplex encephalitis because, although rapid, electron microscopy is insensitive compared with immunofluorescence on brain tissue (flewett, 1973; ross, 1973) . it is, however, the method of choice for varicella zoster diagnosis because of the difficulty of culturing virus from the generally unsuitable specimens provided and the lack of sensitivity of other available tests (macrae et al., 1969) . electron microscopy is useful in cytomegalovirus detection only in very young children and the need for parallel virus isolation procedures is apparent, for even cytology is less sensitive than virus culture (montplaisir et al., 1972; henry et al., 1978; lee et al., 1978) . when virus particles are antibody coated and unable to multiply in cell culture, electron microscopy has the advantage as a detection method. this can be seen in studying polyomaviruses in urine where virus isolation and negative stain electron microscopy otherwise have roughly equivalent sensitivities (gardner et al., 1971; coleman et al., 1973) . cytology is more sensitive in polyomavirus detection than either of the other two techniques and reprocessed cytologically positive cells can be thin-sectioned to confirm the presence of polyomavirus (coleman et al., 1980) . a new immunofluorescence test promises greater sensitivity for the detection of urine polyomavirus (hogan et al., 1980b ) and a peroxidase-antiperoxidase light microscopy technique for polyomaviruses in tissues should also prove useful (gerber et al., 1980) . electron microscopy was initially sufficiently sensitive to confirm positive results by complement fixation and gel immunodiffusion tests for hbsag (cossart et al., 1971) . when the more sensitive radioimmunoassay (ria) and hemagglutination techniques were developed as routine tests for hepatitis b, the use of electron microscopy was largely discontinued except for specialized applications such as the assessment of dane particle content of positive sera. immunofluorescence and immunoperoxidase light microscopic detection of hepatitis b antigens in liver are more sensitive than thin section electron microscopy (roos et al., 19761 , but false-positive reactions can be a problem (omata et al., 1980) . for the detection of hepatitis a virus ria and enzyme-linked immunosorbent assay (elisa) seem to be equally as good as negative stain iem (hollinger et purcell et al., 1976; locarnini et al., 1978; mathiesen et al., 1978) . electron microscopy has been of major diagnostic importance in the detection of noncultivable fecal viruses, but for large-scale investigations of rotavirus-associated nonbacterial gastroenteritis more suitable tests have been developed. an early report indicated that gel immunoelectrophoresis was not very sensitive for detecting rotaviruses but complement fixation was almost as sensitive as electron microscopy (spence et al., 1975) . the immunofluorescence test on cells after centrifugation with rotavirus-containing fecal extracts was not as sensitive as electron microscopy, perhaps because particles lacking the outer capsomere layer were not taken into the cells bryden et al., 1977) . direct immunofluorescence on the fecal extract, however, was as specific as electron microscopy and slightly more sensitive (yolken et al., 1977) . ria and elisa tests have greater sensitivity than electron microscopy (middleton et al., 1977; birch et al., 1979; sarkkinen et al., 1979; seigneurin et al., 1979) and usually specificity is good, though elisa false positives have been observed (yolken and stopa, 1979) . similarly, the ria for nonvalk is specific, at least as sensitive as negative stain iem, and more sensitive than immune adherence assay (iaha) (greenberg et al., 1978 (greenberg et al., , 1979 . but in a recent series of tests negative stain iem proved more sensitive than ria in detecting australian strains of norwalk agent (grohmann et al., 1980) . an elisa to detect noncultivable adenoviruses has recently been described and is almost as sensitive as electron microscopy but might not detect antibody-coated particles (johansson et al., 1980) . adenovirus in tonsils was detected by the presence of viral nucleic acid in the tissue with greater sensitivity than by virus isolation (lord et al., 19801 , and similar studies with wart virus have been reported (krzyzek et al., 1980) . the great advantage of electron microscopy not possessed by ria, elisa, and other such immune tests is that detection of a range of agents is possible in a single test. this advantage is inevitably lost if specific iem methods are used to concentrate virus from specimens. negative stain iem can be used to serotype viruses but it is scarcely justifiable to use this difficult technique for viruses which can be cultivated and thus serotyped more easily by conventional means. despite this, iem typing has been described for adenoviruses (luton, 19731, picornaviruses (chaudhary et al., 1971; hughes et al., 19771, and orthoand paramyxoviruses (kelen and mcleod, 1974) . papovaviruses were also serotyped by negative stain iem because, although some grew well in cell culture and were easy to type by conventional means, others were not as amenable field et al., 1974) . negative stain iem has been a standard method of comparing strains of the noncultivable fecal viruses such as hepatitis a viruses (locarnini et al., 1974; gravelle et al., 1975) , rotaviruses (woode et al., 1976; zissis and lambert, 1978) , parvovirus-like particles appleton and pereira, 19771 , and norwalk group agents (thornhill et al., 1977; kogasaka et al., 1980) . the development of alternative tests for the detection of these agents has also facilitated serotyping and, for example, norwalk-like agents found in a n australian outbreak of oyster-associated gastroenteritis were confirmed as norwalk by ria . negative stain iem utilizing known viral particle antigens has been used in recent years to assess the antibody response. certain precautions are necessary and ideally the antigen should consist of virus particles which are well separated and clear of all attached cell debris. if particles are already antibody coated interpretation of results becomes difficult. these tests have been used to detect antibody response to eb virus (henle et al., 19661, rabies virus (chaudhary et al., 1979) , papovaviruses (ogilvie, 1970; gardner et al., 19711, hepatitis a virus (feinstone et al., 1973; gravelle et al., 1975; locarnini et al., 1977; coulepis et al., 1980) , hepatitis b virus (almeida et al., 1971; cohen, 1978) , rotavirus (kapikian et al., 19741, norwalk agent (kapikian et al., 1972b; parrino et al., 1977; thornhill et al., 1977; murphy et al., 1979) , fecal calicivirus (chiba et al., 1979; suzuki et al., 1979) , and astrovirus (kurtz et al., 1977) . the interpretation of the norwalk negative stain iem for antibody detection has been most difficult because sera taken in both the acute and the convalescent phases of infection contain antibody and careful grading of the amount of antibody coating the particles has been necessary to demonstrate rising titers. other tests originally developed to detect these agents, such as ria and elisa, can be reversed if suitable amounts of antigen are available to detect antibodies as, for example, in hepatitis b (cohen, 1978) , in hepatitis a (purcell et al., 1976; mathiesen et al., 19781, in norwalk (greenberg et al., 19781 and, by immunofluorescence, in astrovirus infections (kurtz and lee, 1978) . generally these tests are a t least as sensitive for antibody detection as negative stain iem and have advantages for mass screening. because of the limited excretion period of hepatitis a virus the detection of antibody, particularly immunoglobulin m, is more likely to be used to establish the diagnosis. specificity of some of these tests may be a problem and negative stain iem can be used to monitor this aspect. immunoglobulin m (igm) molecules have a distinctive shape and can be differentiated from immunoglobulin g (igg) in negatively stained preparations of immune complexes between virus particles and serum immunoglobulin fractions (almeida et al., 1967a; svehag and bloth, 1967; green, 1969) . it is necessary to use fractionated serum; otherwise, the igm structure would be obscured by any igg present. thus negative stain iem can be used to detect specific antiviral igm response, a useful criterion for recent infection. this test has been used successfully with papillomavirus (goffe et al., 1966) , polyomavirus (flower et al., 19771, and hepatitis a virus (locarnini et al., 1977; coulepis et al., 1980) . used in conjunction with other tests for igm directed toward hbcag, cohen (1978) noted that igm was undetectable in iaha and complement fixation tests. gibson et al. (1981) the emergence of diagnostic tests which may be more sensitive than electron microscopy and more suitable for large-scale screening of specimens has still demanded the continuing use of electron microscopy to monitor the performance of these tests by confirmation of results on selected specimens. electron microscopy also plays a role in quality control of the viral reagents utilized in these tests. there is now a general awareness of the need for greater safety in the laboratory, not only for the laboratory worker but also for engineers servicing equipment, and all potentially infectious material must be inactivated before examination by electron microscopy. the electron beam may kill viruses, but only in the area of the specimen grid irradiated, and the high vacuum conditions will not inactivate virus. chemical fixation prior to embedding and thin sectioning does kill viruses but some agents are not inactivated, negative staining procedures do not inactivate viruses (horne and wildy, 1963) . the whole aim of electron microscopy in viral diagnosis is to examine recognizable virus particles and some of the most effective ways of sterilizing the starting material have an adverse effect on negatively stained viral morphology, as discussed in section i1,c. exposure to ultraviolet irradiation inactivates most viruses (cameron et hughes et al., 1979) , but papovaviruses are comparatively resistant (shah et al., 1976; cameron et al., 1979) . negatively stained viral morphology is usually unaffected by ultraviolet irradiation although poliovirus particles were penetrated by stain after prolonged exposure (katagiri et d., 1967) . our standard virus inactivation procedure is to expose negatively stained grids to a high intensity, short wavelength, ultraviolet lamp at a distance of 6.5 cm from the source where the emission registers 700 to 800 pwlcm2. the grid is irradiated for 5 minutes on each side. the lamp has been tested for its capacity to kill vaccinia virus and human polyomaviruses under these conditions (unpublished observations). inactivating viruses in tissues for embedding and thin sectioning must be done with a fixative which also preserves viral and cellular structure. glutaraldehyde and formalin are most commonly used and are quite effective virucidal agents a t the usual fixative concentrations, although the markedly lower concentrations needed for thin section iem studies may not be active (borick, 1968; graham and jaeger, 1968; bowen et al., 1969; sabel et al., 1969; saitanu and lund, 1975) . papovaviruses tend to be more resistant (tevethia and tevethia, 1976) and viruses in tissues may resist fixative action longer than when tested in suspension (cunliffe et al., 1979) . the agents of slow virus cns diseases such as creutzfeldt-jakob disease are highly resistant to formalin (gajdusek and gibbs, 1976) and special care must be exercised when handling any material from such cases. care must also be taken when handling the various toxic chemicals which are routinely used in electron microscopy (drury, 1980) . the supreme advantage of electron microscopy in virus diagnosis is that any virus, if present in the sample in sufficient quantity, will be recognized. the technique is flexible because it is nonselective and this applies to both negative staining and thin sectioning. the disadvantages are the high cost of the electron microscope, the need for highly trained operators, the comparative lack of sensitivity for virus detection, and the relatively small number of specimens which can be examined in a given time. as a means of opening up new fields of diagnostic virology electron microscopy has been preeminent, but after this initial stage it tends to be replaced by techniques based on newer more biochemical concepts, especially for large-scale diagnostic work. the slowly developing techniques of electron microscopy itself seem at present to have little to offer diagnostic virology. even highresolution scanning electron microscopy is incapable of revealing virus particle-cell interactions in a way which can be utilized in diagnostic work. the scanning transmission mode of operation, which can induce image contrast changes electronically, may enhance studies with unstained sections and perhaps facilitate thin section iem. it might even alter ways of examining virus particles in suspension, but early results are not particularly encouraging. the immense contribution of electron microscopy to diagnostic virology in the last 10 years should not be underestimated. the whole concept of a virus diagnostic laboratory has changed from one in which most diagnoses are serological but with a n occasional virus being isolated from a large number of samples tested, to a laboratory in which some virus isolation work continues together with a great deal of useful diagnostic work on infections with viruses which are never cultured in the laboratory. this change of outlook has been largely brought about by discoveries made with the electron microscope. for the future, electron microscopes already heavily used in virus diagnostic work will continue to be used in this field and new discoveries will be made from time to time as in the past. the newly established laboratory, especially in financially less well endowed parts of the world, will probably utilize diagnostic tests in kit form rather than electron microscopy to search for clinically important noncultivable viruses as a first priority. however, the electron microscope has now become an established and essential part of any large virus diagnostic laboratory. practical methods in electron gesarnte virusforsch microbios 1 a m lancet 2, 821 a m arch. gesamte virusforsch. 45, best proc. int. colloq. ebola virus infect. other haemorrh. fevers lancet 2, 703 proc. soc cancer (philadelphia) 30, 1130 cytopathology in viral diseases lancet 2, 92 ultrastructure of animal viruses and bacteriophages: an atlas i n "viral diseases of the central nervous system viral immunodiagnosis comparative diagnosis of viral diseases. human and related viruses proc. soc virus diseases lancet 2, 1497. flower, a proc. soc. exp. b~o l rapid virus diagnosis: application of proc. soc a m a m practical methods in electron microscopy proc. eur. congr. electron microsc., 5th n. 11972). oral surg. oral med. oral a m a m viral immunodiagnosis a m a m a m a m a m viral immunodiagnosis viral immunodiagnosis lancet 2, 1007 der virus morphology lancet 2, 124. madeley a m a m lancet 2, 318 proc. int. colloq. ebola virus infect. other haemorrh. feuers lancet 2, 703 principles and techniques of electron microscopy lancet 2, 311 proc. soc classification and nomenclature of viruses: third report of the international committee on taxonomy of viruses proc. soc a m proc. soc. exp. b i d . med viral immunodiagnosis a m a m lancet tevethia ultrastructure of bacterial viruses acknowledgments i should like to thank dr. m key: cord-324585-2rx84imv authors: konda, abhiteja; prakash, abhinav; moss, gregory a.; schmoldt, michael; grant, gregory d.; guha, supratik title: aerosol filtration efficiency of common fabrics used in respiratory cloth masks date: 2020-04-24 journal: acs nano doi: 10.1021/acsnano.0c03252 sha: doc_id: 324585 cord_uid: 2rx84imv [image: see text] the emergence of a pandemic affecting the respiratory system can result in a significant demand for face masks. this includes the use of cloth masks by large sections of the public, as can be seen during the current global spread of covid-19. however, there is limited knowledge available on the performance of various commonly available fabrics used in cloth masks. importantly, there is a need to evaluate filtration efficiencies as a function of aerosol particulate sizes in the 10 nm to 10 μm range, which is particularly relevant for respiratory virus transmission. we have carried out these studies for several common fabrics including cotton, silk, chiffon, flannel, various synthetics, and their combinations. although the filtration efficiencies for various fabrics when a single layer was used ranged from 5 to 80% and 5 to 95% for particle sizes of <300 nm and >300 nm, respectively, the efficiencies improved when multiple layers were used and when using a specific combination of different fabrics. filtration efficiencies of the hybrids (such as cotton–silk, cotton–chiffon, cotton–flannel) was >80% (for particles <300 nm) and >90% (for particles >300 nm). we speculate that the enhanced performance of the hybrids is likely due to the combined effect of mechanical and electrostatic-based filtration. cotton, the most widely used material for cloth masks performs better at higher weave densities (i.e., thread count) and can make a significant difference in filtration efficiencies. our studies also imply that gaps (as caused by an improper fit of the mask) can result in over a 60% decrease in the filtration efficiency, implying the need for future cloth mask design studies to take into account issues of “fit” and leakage, while allowing the exhaled air to vent efficiently. overall, we find that combinations of various commonly available fabrics used in cloth masks can potentially provide significant protection against the transmission of aerosol particles. t he use of cloth masks, many of them homemade, 1,2 has become widely prevalent in response to the 2019− 2020 sars-cov-2 outbreak, where the virus can be transmitted via respiratory droplets. 3−6 the use of such masks is also an anticipated response of the public in the face of future pandemics related to the respiratory tract. however, there is limited data available today on the performance of common cloth materials used in such cloth masks, 7−12 particularly their filtration efficiencies as a function of different aerosol sizes ranging from ∼10 nm to ∼10 μm scale sizes. this is also of current significance as the relative effectiveness of different droplet sizes in transmitting the sars-cov-2 virus is not clear, and understanding the filtration response across a large bracketed size distribution is therefore important. 13 −16 in this paper, we report the results of experiments where we measure the filtration efficiencies of a number of common fabrics, as well as selective combinations for use as hybrid cloth masks, as a function of aerosol sizes ranging from ∼10 nm to 6 μm. these include cotton, the most widely used fabric in cloth masks, as well as fabric fibers that can be electrostatically charged, such as natural silk. respiratory droplets can be of various sizes 17, 18 and are commonly classified as aerosols (made of droplets that are <5 μm) and droplets that are greater than 5 μm. 3 although the fate of these droplets largely depends on environmental factors such as humidity, temperature, etc., in general, the larger droplets settle due to gravity and do not travel distances more than 1−2 m. 19 however, aerosols remain suspended in the air for longer durations due to their small size and play a key role in spreading infection. 14−16 the use of physical barriers such as respiratory masks can be highly effective in mitigating this spread via respiratory droplets. 20−22 filtration of aerosols follows five basic mechanisms: gravity sedimentation, inertial impaction, interception, diffusion, and electrostatic attraction. 23, 24 for aerosols larger than ∼1 μm to 10 μm, the first two mechanisms play a role, where ballistic energy or gravity forces are the primary influence on the large exhaled droplets. as the aerosol size decreases, diffusion by brownian motion and mechanical interception of particles by the filter fibers is a predominant mechanism in the 100 nm to 1 μm range. for nanometer-sized particles, which can easily slip between the openings in the network of filter fibers, electrostatic attraction predominates the removal of low mass particles which are attracted to and bind to the fibers. electrostatic filters are generally most efficient at low velocities such as the velocity encountered by breathing through a face mask. 25 there have been a few studies reported on the use of cloth face masks mainly during or after the influenza pandemic in 2009; [8] [9] [10] [11] [12] 26 however, there is still a lack of information that includes (i) the performance of various fabrics as a function of particle size from the nanoscale to the micron sized (particularly important because this covers the ∼10 nm to ∼5 μm size scale for aerosols) and (ii) the effect of hybrid multilayer approaches for masks that can combine the benefits of different filtering mechanisms across different aerosol size ranges. 9, 26 these have been the objectives of the experimental work described in this paper. in addition, we also point out the importance of fit (that leads to gaps) while using the face mask. 27, 28 the experimental apparatus (see figure 1 ) consists of an aerosol generation and mixing chamber and a downstream collection chamber. the air flows from the generation chamber to the collection chamber through the cloth sample that is mounted on a tube connecting the two chambers. the aerosol particles are generated using a commercial sodium chloride (nacl) aerosol generator (tsi particle generator, model #8026), producing particles in the range of a few tens of nanometers to approximately 10 μm. the nacl aerosol based testing is widely used for testing face respirators in compliance with the niosh 42 cfr part 84 test protocol. 29,30 two different particle analyzers are used to determine particle size dimensions and concentrations: a tsi nanoscan smps nanoparticle sizer (nanoscan, model #3910) and a tsi optical particle sizer (ops, model #3330) for measurements in the range of 10 to 300 nm and 300 nm to 6 μm, respectively. particles are generated upstream of the cloth sample, whose filtration properties are to be tested, and the air is drawn through the cloth using a blower fan which can be controlled in order to vary the airflow rate. effective area of the cloth sample during the tests was ∼59 cm 2 . measurements of particle size and distribution were made by sampling air at a distance of 7.5 cm upstream and 15 cm downstream of the cloth sample. the differential pressures and air velocities were measured using a tsi digital manometer (model #axd620) and a tsi hot wire anemometer (model #avm410). the differential pressure (δp) across the sample material is an indicator of the comfort and breathability of the material when used as a face mask. 31 tests were carried out at two different airflows: 1.2 and 3.2 cfm, representative of respiration rates at rest (∼35 l/min) and during moderate exertion (∼90 l/min), respectively. 32 the effect of gaps between the contour of the face and the mask as caused by an improper fit will affect the efficiency of any face mask. 21, 27, 28, 33 this is of particular relevance to cloth and surgical masks that are used by the public and which are generally not "fitted", unlike n95 masks or elastomeric respirators. a preliminary study of this effect was explored by drilling holes (symmetrically) in the connecting tube onto which the fabric (or a n95 or surgical mask) is mounted. the holes, in proximity to the sample (figure 1 ), resulted in openings of area ∼0.5−2% of the active sample area. this, therefore, represented "leakage" of the air around the mask. although the detailed transmission specifics of sars-cov-2 virus are not well understood yet, droplets that are below 5 μm are considered the primary source of transmission in a respiratory infection, 13,15,34 and droplets that are smaller than figure 1 . schematic of the experimental setup. a polydisperse nacl aerosol is introduced into the mixing chamber, where it is mixed and passed through the material being tested ("test specimen"). the test specimen is held in place using a clamp for a better seal. the aerosol is sampled before (upstream, c u ) and after (downstream, c d ) it passes through the specimen. the pressure difference is measured using a manometer, and the aerosol flow velocity is measured using a velocity meter. we use two circular holes with a diameter of 0.635 cm to simulate the effect of gaps on the filtration efficiency. the sampled aerosols are analyzed using particle analyzers (ops and nanoscan), and the resultant particle concentrations are used to determine filter efficiencies. www.acsnano.org article 1 μm tend to stay in the environment as aerosols for longer durations of up to 8 h. 19 aerosol droplets containing the sars-cov-2 virus have been shown to remain suspended in air for ∼3 h. 13, 35 we have therefore targeted our experimental measurements in the important particle size range between ∼10 nm and 6 μm. we tested the performance of over 15 natural and synthetic fabrics that included materials such as cotton with different thread counts, silk, flannel, and chiffon. the complete list is provided in the materials and methods section. for comparison, we also tested a n95 respirator and surgical masks. additionally, as appropriate, we tested the efficiency of multiple layers of a single fabric or a combination of multiple fabrics for hybrid cloth masks in order to explore combinations of physical filtering as well as electrostatic filtering. we determine the filtration efficiency of a particular cloth as a function of particle size ( figure 2 ) by measuring the concentration of the particles upstream, c u (figure 2a ,b) and the concentration of the particle downstream, c d (figure 2c ,d). concentrations were measured in the size ranges of 10−178 nm (using the nanoscan tool) and 300 nm to 6 μm (using the optical particle sizer tool). the representative example in figure 2 shows the case for a single layer of silk fabric, where the measurements of c u and c d were carried out at a flow rate of 1.2 cfm. following the procedure detailed in the materials and methods section, we then estimated the filtration efficiency of a cloth from c u and c d as a function of aerosol particle size. the results plotted in figure 3a are the filtration efficiencies for cotton (the most common material used in cloth masks) with different thread counts (rated in threads per inchtpi and representative of the coarseness or fineness of the fabric). we compare a moderate (80 tpi) thread count quilter's cotton (often used in do-it-yourself masks) with a high (600 tpi) cotton fabric sample. additionally, we also measured the transmission through a traditional cotton quilt where two 120 tpi quilter's cotton sheets sandwich a ∼0.5 cm batting (90% cotton−5% polyester−5% other fibers). comparing the two figure 2 . particle concentration as a function of particle size at a flow rate of 1.2 cfm. plots showing the particle concentration (in arbitrary units) upstream and downstream through a single layer of natural silk for particle sizes <300 nm (a,c) and between 300 nm and 6 μm (b,d). each bin shows the particle concentration for at least six trials. the particle concentrations in panels (b) and (d) are given in log scale for better representation of the data. the y-axis scales are the same for panels "a" and "c"; and for panels "b" and "d". acs nano www.acsnano.org article cotton sheets with different thread counts, the 600 tpi cotton is clearly superior with >65% efficiency at <300 nm and >90% efficiency at >300 nm, which implies a tighter woven cotton fabric may be preferable. in comparison, the single-layer 80 tpi cotton does not perform as well, with efficiencies varying from ∼5 to ∼55% depending on the particle size across the entire range. the quilt, a commonly available household material, with a fibrous cotton batting also provided excellent filtration across the range of particle sizes (>80% for <300 nm and >90% for >300 nm). electrostatic interactions are commonly observed in various natural and synthetic fabrics. 36, 37 for instance, polyester woven fabrics can retain more static charge compared to natural fibers or cotton due to their lower water adsorption properties. 36 the electrostatic filtering of aerosols have been well studied. 38 as a result, we investigated three fabrics expected to possess moderate electrostatic discharge value: natural silk, chiffon (polyester−spandex), and flannel (cotton−polyester). 36 the results for these are shown in figure 3b . in the case of silk, we made measurements through one, two, and four layers of the fabric as silk scarves are often wrapped in multiple layers around the face (the results for two layers of silk are presented in figure s1 (supporting information) and omitted from this figure) . in all of these cases, the performance in filtering nanosized particles <300 nm is superior to performance in the 300 nm to 6 μm range and particularly effective below ∼30 nm, consistent with the expectations from the electrostatic effects of these materials. increasing the number of layers (as the error bars on the <300 nm measurements are higher, particularly for samples with high filtration efficiencies because of the small number of particles generated in this size range, the relatively poorer counting efficiency of the detector at <300 nm particle size, and the very small counts downstream of the sample. the sizes of the error bars for some of the data points (>300 nm) are smaller than the symbol size and hence not clearly visible. shown for silk in figure 3b ), as expected, improves the performance. we performed additional experiments to validate this using the 600 tpi cotton and chiffon ( figure s1 ). we note that the performance of a four-layer silk composite offers >80% filtration efficiency across the entire range, from 10 nm to 6 μm. in figure 4a , we combine the nanometer-sized aerosol effectiveness (for silk, chiffon, and flannel) and wearability (of silk and chiffon because of their sheer nature) with the overall high performance of the 600 tpi cotton to examine the filtration performance of hybrid approaches. we made measurements for three variations: combining one layer 600 tpi cotton with two layers of silk, two layers of chiffon, and one layer of flannel. the results are also compared with the performance of a standard n95 mask. all three hybrid combinations performed well, exceeding 80% efficiency in the <300 nm range, and >90% in the >300 nm range. these cloth hybrids are slightly inferior to the n95 mask above 300 nm, but superior for particles smaller than 300 nm. the n95 respirators are designed and engineered to capture more than 95% of the particles that are above 300 nm, 39, 40 and therefore, their underperformance in filtering particles below 300 nm is not surprising. it is important to note that in the realistic situation of masks worn on the face without elastomeric gasket fittings (such as the commonly available cloth and surgical masks), the showing the filtration efficiencies of a surgical mask and cotton/silk with (dashed) and without a gap (solid). the gap used is ∼1% of the active mask surface area. the error bars on the <300 nm measurements are higher, particularly for samples with high filtration efficiencies because of the small number of particles generated in this size range, the relatively poorer counting efficiency of the detector at <300 nm particle size, and the very small counts downstream of the sample. the sizes of the error bars for some of the data points (>300 nm) are smaller than the symbol size and hence not clearly visible. www.acsnano.org article presence of gaps between the mask and the facial contours will result in "leakage" reducing the effectiveness of the masks. it is well recognized that the "fit" is a critical aspect of a highperformance mask. 27, 28, 33, 41 earlier researchers have attempted to examine this qualitatively in cloth and other masks through feedback on "fit" from human trials. 11, 12 in our case, we have made a preliminary examination of this effect via the use of cross-drilled holes on the tube holding the mask material (see figure 1 ) that represents leakage of air. for example, in figure 4b , we compare the performance of the surgical mask and the cotton/silk hybrid sample with and without a hole that represents about ∼1% of the mask area. whereas the surgical mask provides moderate (>60%) and excellent (close to 100%) particle exclusion below and above 300 nm, respectively, the tests carried out with the 1% opening surprisingly resulted in significant drops in the mask efficiencies across the entire size range (60% drop in the >300 nm range). in this case, the two holes were ∼0.635 cm in diameter and the mask area was ∼59 cm 2 . similar trends in efficiency drops are seen in the cotton/ silk hybrid sample, as well. hole size also had an influence on the filtration efficiency. in the case of an n95 mask, increasing hole size from 0.5 to 2% of the cloth sample area reduced the weighted average filtration efficiency from ∼60 to 50% for a particle of size <300 nm. it is unclear at this point whether specific aerodynamic effects exacerbate the "leakage" effects when simulated by holes. its determination is outside the scope of this paper. however, our measurements at both the high flow (table s1 ). the filtration efficiencies of all of the samples that we measured at both 1.2 cfm and 3.2 cfm are detailed in the supporting information (figures s2−s4) . in table 1 , we summarize the key findings from the various fabrics and approaches that we find promising. average filtration efficiencies (see materials and methods section for further detail) in the 10−178 nm and 300 nm to 6 μm range are presented along with the differential pressures measured across the cloths, which represents the breathability and degree of comfort of the masks. the average differential pressure across all of the fabrics at a flow rate of 1.2 cfm was found to be 2.5 ± 0.4 pa, indicating a low resistance and represent conditions for good breathability (table 1) . 31 as expected, we observed an increase in the average differential pressures for the higher flow rate (3.2 cfm) case (table s1) . guidance. we highlight a few observations from our studies for cloth mask design: fabric with tight weaves and low porosity, such as those found in cotton sheets with high thread count, are preferable. for instance, a 600 tpi cotton performed better than an 80 tpi cotton. fabrics that are porous should be avoided. materials such as natural silk, a chiffon weave (we tested a 90% polyester−10% spandex fabric), and flannel (we tested a 65% cotton−35% polyester blend) can likely provide good electrostatic filtering of particles. we found that four layers of silk (as maybe the case for a wrapped scarf) provided good protection across the 10 nm to 6 μm range of particulates. combining layers to form hybrid masks, leveraging mechanical and electrostatic filtering may be an effective approach. this could include high thread count cotton combined with two layers of natural silk or chiffon, for instance. a quilt consisting of two layers of cotton sandwiching a cotton−polyester batting also worked well. in all of these cases, the filtration efficiency was >80% for <300 nm and >90% for >300 nm sized particles. the filtration properties noted in (i) through (iii) pertain to the intrinsic properties of the mask material and do not take into account the effect of air leaks that arise due to improper the filtration efficiencies are the weighted averages for each size rangeless than 300 nm and more than 300 nm. www.acsnano.org article "fit" of a mask on the user's face. it is critically important that cloth mask designs also take into account the quality of this "fit" to minimize leakage of air between the mask and the contours of the face, while still allowing the exhaled air to be vented effectively. such leakage can significantly reduce mask effectiveness and are a reason why properly worn n95 masks and masks with elastomeric fittings work so well. in conclusion, we have measured the filtration efficiencies of various commonly available fabrics for use as cloth masks in filtering particles in the significant (for aerosol-based virus transmission) size range of ∼10 nm to ∼6 μm and have presented filtration efficiency data as a function of aerosol particle size. we find that cotton, natural silk, and chiffon can provide good protection, typically above 50% in the entire 10 nm to 6.0 μm range, provided they have a tight weave. higher threads per inch cotton with tighter weaves resulted in better filtration efficiencies. for instance, a 600 tpi cotton sheet can provide average filtration efficiencies of 79 ± 23% (in the 10 nm to 300 nm range) and 98.4 ± 0.2% (in the 300 nm to 6 μm range). a cotton quilt with batting provides 96 ± 2% (10 nm to 300 nm) and 96.1 ± 0.3% (300 nm to 6 μm). likely the highly tangled fibrous nature of the batting aids in the superior performance at small particle sizes. materials such as silk and chiffon are particularly effective (considering their sheerness) at excluding particles in the nanoscale regime (<∼100 nm), likely due to electrostatic effects that result in charge transfer with nanoscale aerosol particles. a four-layer silk (used, for instance, as a scarf) was surprisingly effective with an average efficiency of >85% across the 10 nm −6 μm particle size range. as a result, we found that hybrid combinations of cloths such as high threads-per-inch cotton along with silk, chiffon, or flannel can provide broad filtration coverage across both the nanoscale (<300 nm) and micron scale (300 nm to 6 μm) range, likely due to the combined effects of electrostatic and physical filtering. finally, it is important to note that openings and gaps (such as those between the mask edge and the facial contours) can degrade the performance. our findings indicate that leakages around the mask area can degrade efficiencies by ∼50% or more, pointing out the importance of "fit". opportunities for future studies include cloth mask design for better "fit" and the role of factors such as humidity (arising from exhalation) and the role of repeated use and washing of cloth masks. in summary, we find that the use of cloth masks can potentially provide significant protection against the transmission of particles in the aerosol size range. materials. all of the fabrics used as well as the surgical masks and n95 respirators tested are commercially available. we used 15 different types of fabrics. this included different types of cotton (80 and 600 threads per inch), cotton quilt, flannel (65% cotton and 35% polyester), synthetic silk (100% polyester), natural silk, spandex (52% nylon, 39% polyester, and 9% spandex), satin (97% polyester and 3% spandex), chiffon (90% polyester and 10% spandex), and different polyester and polyester−cotton blends. specific information on the composition, microstructure, and other parameters can be found in the supporting information (table s2) . polydisperse aerosol generation. a polydisperse, nontoxic nacl aerosol was generated using a particle generator and introduced into the mixing chamber along with an inlet for air. the aerosol is then mixed in the mixing chamber with the help of a portable fan. the particle generator produces particles sizes in the ranges of 10 nm to 10 μm. detection of aerosol particles. the particles were sampled both upstream (c u , before the aerosol passes through the test specimen) and downstream (c d , after the aerosol passes through the test specimen) for 1 min. the samples collected from the upstream and downstream are separately sent to the two particle sizers to determine a particle concentration (pt/cc). each sample is tested seven times following the minimum sample size recommended by the american industrial hygiene association exposure assessment sampling guidelines. 42 we observed a significantly lower particle count in the upper size distribution for both of the data sets, that is, for particles greater than 178 nm for the data from the tsi nanoscan analyzer and greater than 6 μm for the data from tsi ops analyzer. we exclude the data above these thresholds for all of the studies reported due to the extremely low counts. we categorize our data based on these two particle analyzersindividually the two plots (figure 2a,b) show two size distributionsparticles smaller than 300 nm and particles larger than 300 nm. two different flow rates of 1.2 cfm (a face velocity of 0.1 m/s) and 3.2 cfm (a face velocity of 0.26 m/s) were used that corresponded to rates observed at rest to moderate activity, respectively. the velocity of the aerosol stream was measured at ∼5 cm behind where the test specimen would be mounted using a velocity meter. differential pressure. the differential pressure (δp) across the test specimen was measured ∼7.5 cm away on either side of the material being tested using a micromanometer. the δp value is an estimate of the breathability of the fabric. data analysis. the particle concentrations from seven consecutive measurements were recorded and divided into multiple bins 10 for nanoparticle sizer (dimensions in nm: 10−13, 13−18, 18−24, 24−32, 32−42, 42−56, 56−75, 75−100, 100−133, 133−178) and 6 for optical particle sizer (dimensions in μm: 0.3−0.6, 0.6−1.0, 1.0− 2.0, 2.0−3.0, 3.0−4.0, 4.0−6.0). the seven measurements for each bin were subjected to one iteration of the grubbs' test with a 95% confidence interval to remove at most one outlier per bin. this improves the statistical viability of the data. following grubbs' test, average concentrations were used to calculate the filtration efficiencies as described below. filtration efficiency. the filtration efficiency (fe) of different masks was calculated using the following formula: where c u and c d are the mean particle concentrations per bin upstream and downstream, respectively. to account for any possible drifts in the aerosol generation, we measured upstream concentrations before and after the downstream measurement and used the average of these two upstream values to calculate c u (for runs that did not include a gap). we do not measure upstream concentration twice when the run included a gap. the error in fe was calculated using the quadrature rule of error propagation. due to noise in the measurements, some fe values were below 0, which is unrealistic. as such, negative fe values were removed from consideration in figures and further calculations. in addition to the fe curves, we computed an aggregate filter efficiency for each test specimen. to do this, we took a weighted average of fe values weighted by the bin width for the two particle size ranges (<300 nm and >300 nm). these values are reported in table 1 and table s1 . the supporting information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.0c03252. filtration efficiencies for various fabrics tested at two different flow rates and the effect of layering on the filtration efficiencies of chiffon, silk, and 600 tpi cotton; detailed information on various fabrics used (pdf) acs nano www.acsnano.org article how to sew a fabric face mask transmission routes of respiratory viruses among humans exhalation of respiratory viruses by breathing, coughing, and talking ) national academies of sciences. medicine. rapid expert consultation on the possibility of bioaerosol spread of sars-cov-2 for the covid-19 pandemic a cluster randomised trial of cloth masks compared with medical masks in healthcare workers evaluating the efficacy of cloth facemasks in reducing particulate matter exposure simple respiratory protection-evaluation of the filtration performance of cloth masks and common fabric materials against 20−1000 nm size particles testing the efficacy of homemade masks: would they protect in an influenza pandemic? professional and home-made face masks reduce exposure to respiratory infections among the general population airborne transmission of sars-cov-2: the world should face the reality transmission potential of sars-cov-2 in viral shedding observed at the university of nebraska documentary research of human respiratory droplet characteristics. procedia eng world health organization. annex c -respiratory droplets. in natural ventilation for infection control in health-care settings droplet fate in indoor environments, or can we prevent the spread of infection? indoor air reducing risk of airborne transmitted infection in hospitals by use of hospital curtains effectiveness of facemasks to reduce exposure hazards for airborne infections among general populations respiratory virus shedding in exhaled breath and efficacy of face masks aerosol technology: properties, behavior, and measurement of airborne particles 21 -aerosol sample applications and field studies 5 -filtration mechanisms comparison of filtration efficiency and pressure drop in anti-yellow sand masks, quarantine masks particle size-dependent leakage and losses of aerosols in respirators nanoparticle penetration through filter media and leakage through face seal interface of n95 a comparison of total inward leakage measured using sodium chloride (nacl) and corn oil aerosol methods for air-purifying respirators title 42: public health, part 84approval of respiratory protective devices. code of federal regulations 35the determination of the air permeability of fabrics air flow measurements on human subjects with and without respiratory resistance at several work rates performance of an n95 filtering facepiece particulate respirator and a surgical mask during human breathing: two pathways for particle penetration airborne contagion and air hygiene: an ecological study of droplet infections covid-19: a call for physical scientists and engineers 37) frederick, e. r. fibers, filtration and electrostatics -a review of the new technology experimental study of electrostatic aerosol filtration at moderate filter face velocity do n95 respirators provide 95% protection level against airborne viruses, and how adequate are surgical masks? manikin-based performance evaluation of n95 filtering-facepiece respirators challenged with nanoparticles reusable elastomeric respirators in health care: considerations for routine and surge use a strategy for assessing and managing occupational exposures key: cord-258118-j26v354r authors: karuppal, raju; surendran, sibin; patinharayil, gopinathan; muhammed fazil, v.v.; marthya, anwar title: it is time for a more cautious approach to surgical diathermy, especially in covid-19 outbreak: a schematic review date: 2020-05-16 journal: j orthop doi: 10.1016/j.jor.2020.05.013 sha: doc_id: 258118 cord_uid: j26v354r background: many surgeons are unaware of the risks posed by the surgical diathermy. apart from the numerous chemicals, surgical smoke had been shown to harbour intact bacterial and virus particles especially covid-19 in the current time. objective: to identify the inhalational, infectious, chemical, and mutagenic risks of surgical smoke and suggest evidence-based hazard reduction strategies. also to cogitate on the very high risk of viral spread spread by the use of surgical diathermy in covid-19 outbreak. methods: a review of articles indexed for medline on pubmed using the keywords surgical smoke, diathermy, electrocautery, surgical smoke hazards, smoke evacuator, and guidelines for surgical smoke safety was performed. the review included evidences from 50 articles from the dermatology, surgery, infectious disease, obstetrics, and cancer biology literature. results: there are risks associated with surgical smoke. although some surgeons were aware, majority were not keen in the hazard reduction strategies. conclusion: many chemical and biological particles have been found in surgical smoke. it is highly recommended to follow the standardised guidelines for surgical smoke safety. surgical smoke carries full virus particle(such as covid-19 virus), it is strongly recommended to minimise or avoid electrocautery during the covid-19 outbreak. surgical diathermy, also known as electrocautery is a useful and common surgical technique for tissue ablation. here, an alternating current is passing through a resistant metal wire electrode, generating heat which is then applying to living tissue to achieve the hemostasis or varying degrees of tissue destruction (1) . heating of tissues causes vaporisation of protein and fat which results surgical smoke(2) which contain particles from combustion and numerous chemicals like hydrocarbons, acrylonitrile, phenols and fatty acids and biological particles, viruses, and bacteria which are known to be potentially hazardous. applications of high-frequency electrocautery in cutting and coagulation modes produce odorous smoke from tissue pyrolysis. thus surgical smoke is a by-product of the heat produced by electrosurgical tools. most orthopaedic surgeons are often unaware of the risks posed by the surgical diathermy in their daily work environment. this issue is potentially harmful to cause cancer risk on long-term exposure among surgical personnel (3) . surgical smoke had been shown to harbour intact bacterial and virus particles (4, 5) . in covid-19 outbreak scenario, the use of surgical diathermy has to be minimised or avoided due to the very high risk of viral spread among operating room personals. 2. how does surgical smoke become dangerous?: information on the hazardous nature of surgical smoke is not new. now, a multiplicity of chemical and biological hazards have been identified in surgical smoke. it is reported that surgical smoke has mutagenic effects as well (6) . surgical smoke contains contaminants such as hydrocarbons, acrylonitrile, phenols, fatty acids, nitriles and carbon monoxide and viable cellular elements (7, 8) . the main chemical in surgical smoke is polycyclic aromatic hydrocarbons(pahs). amongst these pahs, benzopyrene(bap) is the most significant carcinogenic compound (9) . benzopyrene is often used as an indicator of cancer risk for target populations. studies have shown that higher cauterisation temperatures produce more harmful chemical components while lower temperatures produce more cell particles (5) . it is also found that different electrosurgical techniques produce different amounts of surgical smoke (10) . the chemical composition of surgical smoke also varies by the type of tissue dissected. muscular tissue produces aldehyde and ketone; liver and fatty tissue liberates carbon monoxide and hydrocyanic acid; epidermal tissue produces xylene, toluene, and ethyl benzene(5). studies have been showing various pulmonary changes when exposed to smoke plumes. (11, 12) . the size of particulates and concentration of chemical or cellular particulate in surgical smoke is a critical issue. particulates can range from 10 nm to 100 µm. smaller the particles, the deeper inhaled into, particles 7 µm or smaller can be deposited in the alveoli (10) . electrocautery produces more surgical smoke than laser or ultrasonic scalpel. electrocautery led to the formation of smaller particles (0.07 µm), where as lasers and ultrasonic devices lead to the formation of larger particles (0.31 µm-6.6 µm). smaller particles are chemical health hazards and larger particles are acting as biological hazards (5) . studies have shown similarities between surgical smoke and the adverse effects of air pollution and passive cigarette smoke (5, 13) . surgical smoke also contains a variety of cellular particles which harbour contagious, viable malignant cells, and even to contain live bacteria and viruses. different studies have clearly mentioned many viruses like hiv, infectious polio virus, human papilloma virus(hpv) and hepatitis b virus (hbv) are present in surgical smoke (3, 4, 14, 15) , this knowledge is relevant in covid-19 outbreak.. there for, it is possible to have viruses like covid-19 in cellular particles. surgical smoke is a visible suspension of water vapour, volatile organic compounds and other organic matter of biological origin released when the instruments raises intracellular temperatures >100°c (16) . the cutting mode can create a higher tissue temperature in a shorter period of time, resulting in rapid expansion and explosive vaporisation of intracellular contents (17) . while coagulation is performed using waveforms with lower average power, heat generating is insufficient for explosive vaporisation. so the risk of surgical smoke production is more with cutting mode and will be absent or negligible with coagulation mode. surgical smoke is a nuisance because it has a repulsive odour and can obstruct the surgeon's view of the surgical site. individuals in the operating room, including surgeons, surgical technicians, nurses, anaesthesiologists, and others, are exposed to surgical smoke annually (18) . transmission has been well demonstrated in animal studies (7, 19, 20, 21, 22, 23) . smaller particles in surgical smoke are chemical health hazards and larger particles are acting as biological hazards (5). hence, viral dna content seems to be higher in laser and ultrasonic smoke than in electrocoagulation smoke (25) . since surgical smoke contains many viruses(4,14,15) many of the surgeons acquired their infection through it (21) . in this context, we cannot ignore the possibility of spreading covid-19 through cellular particles in surgical smoke. i. direct physical injury: direct physical injuries like alveolar congestion, interstitial pneumonia, and emphysematous changes by surgical smoke have been demonstrated in animals. the chemicals contained in the surgical smoke are pulmonary irritants which have resulted to cause coughs, burning throats, sneezing, and rhinitis in surgeons and or personal (11) . this may have a significant impact on the health and safety of everyone in the operating room, because these compounds pose a potential health risk (11) . animal studies have shown that the use of 0.1-m ultra-low permeability air filters does not cause substantial parenchymal damage (24) . ii. chemical and mutagenic effects: surgical smoke produced by electrocautery will produce more harmful chemical components. some of the chemical substances, such as benzene, formaldehyde, acrolein, co and hydrogen cyanide, are also present in the smoke released by laser tissue ablation (30) . benzene can induce headache, dizziness, nausea, and irritation of the mucous membranes. (26) . what's more, it also contained carcinogens and teratogens (11) . toxicity equivalency factors (tefs) were recommended for calculating the relative toxicity of individual pahs to bap for the purpose of simplifying risk assessment (27) . acute deleterious effects of exposure to surgical smoke includes eye, nose and throat irritation, headache, cough, nasal congestion, asthma and asthma-like symptoms (2, 18, 28, 29) . chemicals present in surgical smoke may cause cancer in humans (5) . although there have been no human studies on the carcinogenic effects of smoke, surgical smoke has been shown to be carcinogenic in vitro (30) . it was calculated that the total mutagenicity from surgical smoke condensates generated for 60 seconds from 1 g of tissue was equivalent to that of 3 (in the case of co2 lasere induced smoke) to 6 (in the case of electrocautery-induced smoke) cigarettes (26) . although direct physical injury and carcinogenesis have been well demonstrated in in vitro and animal models, it is difficult to describe the long-term effects on humans due to the inherent time lag and the inability to prove causality (30) . some of the risks are theoretical, until there is evidence that disproves these risks to humans. 4 . deleterious effects to patients: electrical shocks and burns are possible from electrocautery arise from a faulty grounding pad or from an outbreak of a fire (31) . this issue happens if the surface of the contact pad is small and the pad is disconnected from the radio frequency generator or through a metal implant (32) .modern electrocautery systems have sensors that prevent similar injuries. there have also been reports of flash fires, especially in the case of increased oxygen concentrations of anesthetics (33) . the toxicity of surgical smoke may cause harm to patients.there have been no reports that surgical smoke exposure to patients can cause any obvious chronic diseases such as cancer or chronic obstructive pulmonary disease (cold). such diseases might be caused by accumulation processes. diathermy can also cause tissue damage through energy transfer to the implanted spinal cord stimulation system, resulting in serious injury or death. diathermy incision has many advantages compared with the scalpel because of reduced incision time, less blood loss, and reduced early postoperative pain (34) , but there are reports which show increased tissue damage and a significant reduction in the tensile strength of healing wounds (35). although a reasonable body of research exists on the aspects of surgical smoke, little substantive data exist about the actual exposure patterns. traditionally, the removal of surgical smoke through conventional ventilation and continuous hourly ventilation(air changes per hour-ach). the capability of 20 ach can be able to remove hazardous substances below those of exposure standards (36) . it was described that conventional ventilation will work effectively if exhaust grills must be free of obstruction by equipment or furniture (37) . the use of specific suction or extraction measures for surgical smoke has been recommended by many regardless of exposure duration or concentration level (38, 39) . smoke evacuation in the operating room has been highly recommended to minimise exposure(3). high efficiency particulate air filters (hepa) is another recommendation to reduce the hazards of surgical smoke. in hepa contaminated air first passes through the operating room corner vents, and is then filtered and re-circulated in the operating room. hepa efficiency and the air exchange rate determine operating room air quality. the surgeon's, exposure to an average of 16 typical pahs in the gaseous and particle phases were 1,279 and 37 ng/m3, respectively. though gaseous pah concentrations were 30 times higher than those associated with particles, it should be noted that high molecular weight pahs often result in higher carcinogenic effects (40) . though the hepa can remove particles in recirculated air, gaseous pollutants may penetrate through filters and accumulate in the or air. so the effectivity of removing surgical smoke in operating room should be emphasised to minimise the potential adverse health effects. hazards and risks are identified and risks are managed based on control measures from best to least effective by methods like elimination, substitution, isolation, engineering controls, administrative controls, and personal protective equipment (41) . multiple precautions like use of a standard surgical mask, laser or high filtration mask, masks coated with nanoparticles, operating room ventilation guidelines, and use of wall suction have been using to reduce the health hazards, but each one has its own limitations. therefore, it is strongly recommended to use smoke extraction devices. in view of surgical smoke has been shown to harbour intact virus particles, it can very well carry covid-19 like viruses. so it is mandatory to minimise or avoided electrocautery especially in covid-19 outbreak, as it was recommended in middle east respiratory syndrome outbreak(mers) (42) . a negative-pressure operating room is the optimal environment to prevent all airborne viruses including covid-19, bacteria, fungi, yeasts, gases, volatile organic compounds, small particles and chemicals spreading to adjacent areas (43) . surgical masks do not work well at filtering submicrometer-size particles, and a poorly fitted mask greatly compromises the performance. the bacterial filtering efficiency of n95 respirator is superior to that of the surgical mask, and this fact is especially true in environments with high concentrations of airborne bacteria. although n95 respirators have higher filtration efficiency in a laboratory environment, there is insufficient data to determine whether n95 respirators are superior to masks in protecting medical staff from infectious infections (44, 45, 46) . the national institute of occupational safety and health (niosh) conducted a detailed study and said that little is known about the health effects of long-term exposure to surgical smoke (28) . niosh is the federal agency responsible for conducting research and making recommendations for preventing work-related illness and injuries. a. employees have to use local exhaust ventilation (lev) for all procedures where surgical smoke is generated. smoke evacuators should be used in situations where considerable plume is generated and room wall suction systems should be used for controlling small amounts of smoke when there is adequate room air ventilation (28, 29, 47) . b. train employees on the hazards of surgical smoke and methods to minimise exposure prior to working in areas where surgical smoke is generated (48) . c. ensure that procedures that address the hazards of surgical smoke are available (47) . d. use a properly fitted, filtering face piece respirator like n95 mask rather than a ordinary surgical or laser mask, especially in situations where lev is lacking or not functioning properly. respiratory protection should be at least as protective as a fit-tested n95 filtering face piece respirator when working with known disease transmissible cases (viral infections like hpv, and/or during aerosol-generating procedures or with aerosol transmissible diseases (e.g., tb) (47, 49) . it is interesting to noticed in a survey by niosh that only half (47%) of respondents reported that lev was always used during laser surgery and even fewer (14%) reported that lev was always used during electrosurgery (28) . surgical smoke safety guidelines will establish a safe environment for surgeons, individuals or patients." though the concern about respiratory exposure is warranted, little evidence is available to determine actual exposure levels. this lack of evidence is compounded by a lack of research comparing electrosurgical techniques, type of surgery, and ach. future researches have to focus on surgical smoke exposure levels in operating room while performing a specific type of surgery, using a specific electrosurgical technique and the ability of air changes per hour(ach) and other administrative measures to reduce surgical smoke exposure to acceptable levels (36) . cold steel scalpel has been the instrument of choice for surgical incisions because of accuracy, and predictable tissue damage (50) . however, the use of cold steel scalpel must be accompanied by electrocautery to maintain hemostasis and a clear surgical field. furthermore, research is needed on a equally or better alternative to electrocautery, which can can also reduce incision time, bleeding, and thermal tissue damage might be beneficial as well. the ace blade and mega power generator (ace electrosurgical system) is a next-generation electrosurgery system that is intended for use in a broad range of surgical procedures requiring the use of electrosurgery (50) . many chemical and biological particles have been found in surgical smoke. they have potentially serious occupational hazards, especially the viruses like covid-19. the surgical smoke exposure for surgeons and anaesthetist have been fully investigated by measuring the pah concentrations in both the gaseous and particle phases in their breathing zones. efficient smoke extractors in the operating room or high-efficiency masks are suggested to minimise potential health hazards. insufficient knowledge on these regards demands the need for further investigation and research. so further studies are needed for its effects on duration of exposure, the composition of surgical smoke produced by different electrosurgical techniques and the impact of ach. we need to receive education about the hazards of smoke plume to raise awareness of the health risks of surgical smoke. each of the many precautions currently available has its own limitations, so smoke extraction devices are very much recommended than any other. it is highly recommended to follow the standardised guidelines for surgical smoke safety. since it has been proven that surgical smoke carries full virus particle(such as covid-19 virus), it is strongly recommended to minimise or avoid electrocautery during the covid-19 outbreak. surgical smoke poses numerous risks to the surgeon, including the transmission of infectious diseases, mutagenicity, and direct physical injury. smoke safety guidelines would establish a safe environment for the surgeon, or personal, and patient. the usage of surgical diathermy is mainly for the ease and comfort of the surgeons and not for any direct benefit of the patient. we hope to invent an equivalent or more effective alternative to electrocautery in the near future, which will not causes any of the above harmful effects. till then we have to reduce the it's usage judiciously. the hazards of surgical smoke surgical smoke and infection control detecting hepatitis b virus in surgical smoke emitted during laparoscopic surgery. occupational and environmental medicine 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medicine masks against airborne bacteria in different working environments evaluation of single-use masks and respirators for protection of health care workers against mycobacterial aerosols effectiveness of n95 respirators versus surgical masks in protecting health care workers from acute respiratory infection: a systematic review and meta-analysis understanding and controlling the hazards of surgical smoke occupational safety and health administration. nd osha hazard communication standards orthopaedic patient workflow in covid-19 pandemic in italy advanced cutting effect system versus cold steel scalpel: comparative wound healing and scar formation in targeted surgical applications. plastic and reconstructive surgery global open the editorjournal of orthopaedics sir,we the authors hereby declare that we do not have any financial and other conflicts of interest that might bias the work. we have not published or submitted this article elsewhere. we also declare that it is not under consideration for publication with any other journal. dr. raju karuppal key: cord-289521-zun6tp2o authors: leonard, scott; strasser, wayne; whittle, jessica s.; volakis, leonithas i; debellis, ronald j.; prichard, reid; atwood, charles w.; dungan, george c. title: reducing aerosol dispersion by high flow therapy in covid‐19: high resolution computational fluid dynamics simulations of particle behavior during high velocity nasal insufflation with a simple surgical mask date: 2020-05-29 journal: j am coll emerg physicians open doi: 10.1002/emp2.12158 sha: doc_id: 289521 cord_uid: zun6tp2o objective: all respiratory care represents some risk of becoming an aerosol generating procedure (agp) during covid‐19 patient management. personal protective equipment (ppe) and environmental control/engineering is advised. high velocity nasal insufflation (hvni) and high flow nasal cannula (hfnc) deliver high flow oxygen (hfo) therapy, established as a competent means of supporting oxygenation for acute respiratory distress patients, including that precipitated by covid‐19. although unlikely to present a disproportionate particle dispersal risk, agp from hfo continues to be a concern. previously, we published a preliminary model. here, we present a subsequent high‐resolution simulation (higher complexity/reliability) to provide a more accurate and precise particle characterization on the effect of surgical masks on patients during hvni, low‐flow oxygen therapy (lfo2), and tidal breathing. methods: this in‐silico modeling study of hvni, lfo2, and tidal breathing presents ansys fluent computational fluid dynamics simulations that evaluate the effect of type i surgical mask use over patient face on particle/droplet behavior. results: this in‐silico modeling simulation study of hvni (40l∙min(‐1)) with a simulated surgical mask suggests 88.8% capture of exhaled particulate mass in the mask, compared to 77.4% in lfo2 (6l∙min(‐1)) capture, with particle distribution escaping to the room (>1m from face) lower for hvni+mask versus lfo2+mask (8.23% versus 17.2%). the overwhelming proportion of particulate escape was associated with mask‐fit designed model gaps. particle dispersion was associated with lower velocity. conclusions: these simulations suggest employing a surgical mask over the hvni interface may be useful in reduction of particulate mass distribution associated with agps. this article is protected by copyright. all rights reserved covid-19, the clinical disease related to infection with the sars-cov-2 coronavirus (covid19) represents a major world-wide health risk. it is associated with varying degrees of respiratory distress, hypoxemia, and failure. management of the oxygenation of these patients has been the topic of much discussion, receiving current guidance from numerous international and national agencies and organizations. 1,2,3,4 these guidance documents all include the use of high flow oxygen (hfo) therapy in the list of possible interventions. these guidance documents also caution regarding the potential aerosol generation from the use of respiratory support. according to the who, covid-19 is transmitted via respiratory droplets and fomites during close unprotected contact between people. 5 based on current evidence, classically defined airborne spread has not been reported for covid-19 as of this writing and it is not believed to be a major driver of transmission. caution is recommended due to the unclear nature of particle dispersion during aerosol generation. 6 all guidelines are clear and reinforce the requirement for strict adherence to personal protective equipment (ppe) guidelines and use of environmental controls, including negative pressure rooms, when available. agps can produce airborne particles which can remain suspended in the air, travel over a distance and may cause infection if they are inhaled/contacted, therefore, agps create the potential for airborne transmission of infections that may otherwise only be transmissible by the droplet route. 7 respiratory therapies that are known agp's include intubation, extubation and related procedures such as manual ventilation and open suctioning, bronchoscopy, non-invasive ventilation (niv) such as non-invasive positive pressure ventilation (nippv) and continuous positive airway pressure (cpap), high-frequency oscillating ventilation (hfov), high flow oxygen (hfo, hfnc, and hvni), induction of sputum, and any procedure that induces coughing. 8, 9 particle sizes of droplets or aerosolized infectious pathogens can directly have bearing on transmission distance. other factors such as room ventilation, people walking nearby, environmental factors, and air flows of any variety can influence particle dispersion distance from the host/origin. [10] [11] [12] [13] [14] the transmission risk of hfo was brought into question, noting that (1) all agps, including hfnc, are high risk for covid-19 infection transmission; and (2) due to non-universal interfaces across multiple device technologies, that hfnc and niv (specifically nippv), with a potential for poor application to the patient, is not recommended for use without an isolation room. 15 prior research found there was no significant increase in risk for hfo therapy, with lower risk compared to manipulation of oxygen mask, endotracheal aspiration, bronchoscopy, and nebulizer treatment. 8 studies have shown that good interface practice should be in place for niv and hfnc. 6, 16 these studies determined that hfnc aerosol dispersion distance is lower than found in nippv and cpap. good interface practice for hfnc is a simple and practical consideration of cannula placement into the patient nares, whereas a more significant challenge exists in the case of placing an nippv mask-toskin interface. 6, 16 although mechanistically different for ventilatory effect, hvni shares characteristics with hfnc/hfo therapy, in that both deliver heated humidified gas through an open nasal interface, and both able to deliver oxygen-rich gas at supraphysiologic flows, allowing a more precise fio 2 delivery in management of hypoxic respiratory failure. hvni and hfnc have been shown to effectively manage hypoxic respiratory failure in adult patients presenting in the emergency department. 17, 18 high flow therapy in general has demonstrated broad capability to manage acute respiratory failure, wherein a meta-analysis demonstrated that hfnc provided superior outcomes regarding avoidance of endotracheal intubation as compared to conventional oxygen therapy, and comparable rates of intubation compared to nippv. 19 computational fluid dynamics (cfd) is an in-silico simulation method used to evaluate fluid-flow problems. complex geometry is broken down into a mesh of discreet elements. boundary conditions, such a flow inlets, outlets, and surface conditions are applied to the surfaces of some of the elements. an algorithm then iteratively solves the flow in each element based on the boundary conditions and flow conditions in the elements surrounding it. smaller elements increase the accuracy, but also the total number of mesh elements (mesh count) required to model the geometry, thus increasing the computation time for the simulation. there are several advantages in comparing in-silico cfd with in-vitro and in-vivo testing. cfd allows measurements of any fluid property simultaneously at all points in the flow, where other methods only allow measurement at specific points at which sensors are placed. for particle studies, cfd allows precise tracking of particle sizes and location that is generally not possible with other methods. most importantly, cfd allows testing of complex problems without the need to fabricate a physical experiment. there are also disadvantages to cfd, such as the need for careful design of the model and assumptions to avoid obtaining inaccurate results. the addition of a simple surgical mask over a high flow therapy interface has been proposed as a mitigation to particle dispersal. 3 this study evaluated this recommendation in the case of tidal breathing, low flow oxygen therapy (lfo2), and hvni. this study expands upon previous work, and by employing high-resolution cfd. the in-silico model included a room simulation to evaluate the fluid dynamic behavior of the effect of a surgical mask on particles which may be generated in the airway while receiving hvni therapy. a preliminary study was published with lower mesh-count and lower resolution. 20 this report provides a subsequent high-mesh-count simulation with improved fidelity, performed in ansys fluent cfd, of the noted models, thereby generating results with higher reliability/acuity than initially published. computational fluid dynamics (cfd) allows for the simulation of complex flow fields, tracking of particles through those fields, interactions of those particles with the carrier fluid, and the differential capturing of particles by a porous media. evaluation of the use of a type i surgical mask with hvni, low flow oxygen, and tidal breathing was performed in ansys fluent cfd (ansys, inc, canonsburg, pa, usa). simulations were performed on control cases modeling a patient on hvni (40l•min -1 ), low flow oxygen (lfo2) therapy (6l•min -1 via nasal cannula), and simulated breathing (tidal breathing, no therapy) without a surgical mask. summary of all the evaluated cases are shown in table 1 . the patient is modelled to be breathing at 32 breaths per minute with a tidal volume of 500ml with a sinusoidal 1:1 ratio (inspiratory:expiratory) breath curve, without a pause (no interbreath interval) amongst the inspiratory/expiratory phases. the peak expiratory flow rate is 49.0l•min -1 . detailed and expanded description of of the cfd modelling methods, validation, and assumptions are provided in the appendix. a 3d modeled human head, positioned 736mm above the floor with a 30° incline, was placed on a bed in a room. the head model includes a simplified airway structure, an adult small / pediatric cannula (vapotherm® inc, exeter, nh, usa) and a surgical mask (figure 1 ).there are two inlet vents and two outlet vents located on the ceiling and wall near the floor across the room from the patient. there are 6 air changes per hour (ach) implemented to simulate a conservative (non-negative pressure) room ventilation flow. a type i surgical mask, appropriate for use on patients to prevent the spread of droplet particles carrying infectious diseases, is modeled for the simulation (figure 2 , left). the model employs a mask fitted to a simulated head. to imitate clinical practice, gaps between skin and mask were modeled in eight discrete locations (figure 2 , right). simulated gaps were modelled as a 'poor-fitting' mask at the nose (e.g., failure to 'pinch the nose' at the bridge of the nose/face interface). each side of the face has one gap which simulates a cannula tube passing through the edge of the mask. the vapotherm adult-small/pediatric cannula nose piece is modeled in position with its prongs in the simulated nares. therapeutic gas flow is defined to emit from the prongs of this cannula. the mask properties were obtained from the standard governing surgical masks, en14683 21 , and from data in chen et al., aerosol penetration through surgical masks. 22 the mask is modelled as a porous medium which allows flow to pass through with a pre-defined resistance. particle penetration is obtained from chen et al data for a mask with a filtration layer at 100l•min -1 . as the simulated flow rate through the modeled mask does not exceed 80l•min -1 , this is conservative. as the chen et al data does not include efficiency for particle sizes above 4µm, the data was extrapolated with the assumption that for every additional 5µm, the percentage of particles passing through is halved. the distribution of particle sizes emitted by a patient breathing, talking, coughing, and sneezing has been investigated in several studies. the specific distributions vary, however, the range of particle sizes that are meaningful to the study is generally from 0.1 to 100µm. particles larger than 100µm are highly unlikely to penetrate a mask or travel far without a very high velocity flow. particles smaller than 0.1µm account for a very small fraction of the total particles are likely to escape regardless of a mask and will remain airborne regardless of the velocity. the particle distribution (table 3) , for the simulation is taken from exhaled droplets due to talking and coughing. 13 particles smaller than 0.1µm and larger than 100µm were not included virtual particles are introduced in this simulation at just above the larynx, and material properties (mass) of the particles/droplets were defined as water in the ansys model. this allows the model to account for both particle size, as denoted in table 2 , and particle mass using water as a correlate. the standard clinical room simulation is transient, accounting for variations of the flow with time caused by cyclic breathing. the simulation was allowed to run until the flow in the room reached a steady state. streamlines of the fully developed room ventilation flow are shown in figure 4 . the simulations then run for six breath cycles (9.8sec). particles are injected at five timesteps near peakexpiratory (pe); pe -0.2sec, pe -0.1sec, pe +0.1sec, and pe +0.2sec. the particles are tracked along their trajectories (lagrangian) and their final position is calculated. the particle final positions are grouped into one of the following categories, with the results averaged across the five timesteps: (1) caught in the mask, (2) trapped in the vicinity of the patient, and (3) escaped. particles caught in the mask are absorbed in the filter and become trapped, and as such are removed from consideration in the additional airflow dynamics. particles trapped in the vicinity of the patient are deposited on the patient head, upper torso, bed, or pillow with path lengths less than 1m, normally considered a likely risk area. escaped particles are considered to have traveled further than 1m and could remain in an airflow for longer time within the room. sensitivity studies were performed to ascertain the effects of various computational methods used in the fluid flow simulation. based on the sensitivity studies, the final simulations were all run using the 16.2.1 solver, sst turbulence model, 0.001sec time step, and without advanced numerics (to be more conservative). particle modelling was performed in post-processing using 1-way coupling, so the feedback effects of momentum and turbulence augmentation or suppression by the particles on the fluid flow are not considered. a sensitivity study of mass flow rate of particles on the results confirmed that the mass flow does not affect the percentage of particles caught in the mask or in the vicinity of the patient. larger particles may remain suspended for a longer time in a flow which has a higher velocity. this demonstrates that in all cases the velocity of the expiratory flow is substantially reduced by the presence of a mask. the mask resists high velocity flow through the media and distributes the flow through a larger area. this diffusing effect results in a much lower velocity in the area near the patient's face and does not allow jetting of flow over long distances, shown graphically by the velocity contours, along the sagittal plane, for all tested cases ( figure 5 ). even with the loosely fitted mask modelled in this simulation, most of the flow does not pass through the simulated mask, but rather exits through the designed gaps between skin and mask. near peak-expiratory flow was defined as expiratory flow that is within 10% of peak-expiratory flow ( table 3) . although the percentage of flow loss near peak-expiratory flow is very high, ranging from 67.9% (hvni) to 76.2% (tidal breathing, no therapy), the exiting flow velocity is similarly reduced as compared to flow that passes through the mask, demonstrated by evaluating the isosurfaces for all cases at peak expiratory flow ( figure 6 ). all points on the isosurfaces have a velocity of 0.5m•sec -1 . in the cases where a mask is present, the velocity is both reduced and is redirected back toward the patient rather than out into the room. images of results without a mask in place are not shown. with an actual mask in place, flow passes through the mask and is filtered to remove approximately 96.5% of particles by mass when weighted by the distribution of particles and the efficiency of the filter for those particle sizes. this efficiency would be the upper limit of filtration for a perfectly sealed mask. the flow demonstrated in this simulation, which does escape through the gaps followed a tortuous path, which tended to cause the larger particles to impact the surface of the mask, face, and cannula. these particles remain trapped in the areas around the model's head. relative pressure in the airway and inside the mask for each of the three cases modeling the mask show increasing therapy flow (from 6 to 40l•min -1 ) resulting in an increase in relative pressure in the upper airway and mask (figure 7 ). in the case of hvni, a high-pressure region is also present at the surface of the mask where the high velocity flow from the mouth impacts the mask. this localized high pressure gradient across the mask forces flow through the mask in the localized area. this can also be observed as a region of increased velocity is present outside of the mask ( figure 5 ), directly opposite the high-pressure region. for hvni at 40l•min -1 with a mask, 88.8% of the total particle mass is captured and terminated/deposited in the mask (table 4, figure 8 top), as compared to 77.4% of the total particle mass captured in the mask whilst on lfo2 at 6l•min -1 , and 73.4% for tidal breathing with a mask. the low proportion of total particles which escape the mask during hvni have a longer travel length, with 2.97% of particles settling within 1m, compared to 5.47% for lfo2, and 6.81% for tidal breathing. when the patient simulation is tidal breathing in the room, without therapy and without a surgical mask, 52.3% of total particle mass leaving the nose and mouth will deposit greater than one meter from the face. the proportion of particles >5μm, which are captured in the simulated mask over hvni therapy is 93.4%, as compared to 85.1% whilst receiving lfo2, and 83.9% during tidal breathing. table 5 provides the disposition distribution into two categories: particles sized ≤5μm and >5μm (figure 8 , middle and bottom). this study adds information for current clinical practice decision-making. particle-mass dispersion is reduced in this simulated model with the addition of a surgical mask analogue. the simulation showed that the greatest particle loss was associated through the gaps between the skin and mask , but those locations imparted lower velocities of escaping gas, limiting the overall virtual particle mass dispersion. the amount of particle mass captured by the mask was actually greater for the hvni+mask scenario as compared to the lfo2+mask or tidal breathing+mask scenarios. this is likely due to the greatly increased velocity of gas outflow into the mask matrix seen in this model, promoting capture at the mask and diffusion/deflection of the gas stream. the increased particle capture in the model by hvni+mask as compared to lfo2+mask or tidal breathing+mask is unexpected, given the degree of flow loss by hvni+mask was expected to be higher with higher flow. this suggests that the flow loss occurs 'after' deposition of the particle mass within the mask matrix or on the face. although seemingly counterintuitive, the high-pressure region where the flow impacts the mask offers a probable explanation -the velocity of the flow exiting the mouth during hvni is significantly higher than the other cases, causing greater momentum of the gas and particles through the mask. as much of the flow is redirected toward the designed gaps, the particles' momentum resists directional change. the momentum propels particle trapping/deposition into the mask. while a greater proportion of larger particles (>5μm) are trapped/deposited in the mask, the effect of increasing the velocity of the flow (lfo2 to hvni) promulgates a greater difference in the capture of smaller particles (≤5μm). for particles >5µm during hvni, the mask captures 9.75% more particles than lfo2, and 11.32% more particles than no therapy (tidal breathing). for particles ≤5µm during hvni, the mask captures 130.72% more particles than lfo2, and 223.45% more particles than no therapy (tidal breathing). in all cases, the importance of using a mask to reduce the particle dispersion is evident (figure 8 ). without a mask, most particles disperse beyond the immediate area of the patient. increased velocity translates to increased particle spread into the room (77.0%, 63.7%, 52.3%) for hvni, lfo2 and tidal breathing, respectively. this difference is primarily due to the behavior of larger particles/droplets escaping into the room (73.3%, 58.1, 44.0%), respectively. there is less disparity between therapies (100.0%, 98.0%, 94.5%, respectively), for the smaller particles (<5μm), as these may remain suspended in the room with less velocity. adding to previous simulations, these ansys results are considered more robust due to the higher mesh density (3.0•10 6 vs 8.8•10 5 elements), finite mask thickness, improved mesh design, finer timesteps, better numerics, inclusion of lift effects, and more robust particle tracking. 20, 23 this model suggests that the addition of a surgical mask, placed over the mouth and nose of the patient, may significantly reduce the spread of these particles by reducing the flow's velocity through the gaps, thereby decreasing the particles escaping into the room. an important finding was that the overwhelming majority of particles escaping the mask were associated with the model design of the simulation. while this model is intended to simulate a 'worst-case' real-world clinical application, the findings suggest attention must be paid to securing the mask to the face. the who has suggested the primary mode of transmission was droplet. 5 this has been brought into question with more recent findings suggesting the maintenance of viral activity in smaller (aerosol) particles. 24 the mask also captured a greater proportion of smaller particles (≤5μm) than were captured while receiving lfo2 or tidal breathing alone. these findings support the notion that a mask over the nasal interface during hvni may substantially reduce the particulate burden in the room around the patient. clinicians and healthcare workers must always wear personal protective equipment (ppe) as well as practice droplet precautions during patient interactions with suspected or confirmed covid-19, as the risk of agp in the care of the patients may remain an issue. newer information suggests important physiological phenotypes which may be better suited to noninvasive support than to invasive mechanical ventilation. 25 the role of invasive mechanical ventilation as the 'first line' therapy after simple oxygen management fails has been brought into question. 26 as such, the ability to limit the overall environmental exposure becomes important. although modeling of liquid and bacterial pathogen dispersal demonstrated that high flow therapy limited the dispersion to the area proximate to the face and cannula, the issue of partial contamination remains concerning. 27 particle sizes of droplets or aerosolized infectious pathogens can directly have bearing on transmission distance. although sars-cov-2 associated with covid-19 is reported as 0.06-0.14μm in size, these viral/infectious particles may be carried in droplets and aerosols when you talk/cough/sneeze. 28, 29 a 2018 study measuring influenza virus presence in droplets from a coughing patient noted that the influenza virus was present in 42%, 23%, and 35% of droplets/particles sized <1μm, 1-4μm, and >4μm, respectively. 30 this cfd simulation suggests that there may be capture of the majority of aerosol particles (≤5μm) and almost all larger particles (>5μm). the area immediately proximate to the patient's face may remain the likely 'hot zone' for higher contact likelihood even when a mask is employed in clinical practice. the strength of the study was built upon the following: inclusion of real-life variables outlining details such as an average fitting type i surgical mask with appropriate gaps as a result of fitting. the quality and type of mesh in the mask was designed per type i mask specifications. a room demonstrating particle dispersion via a fixed patient space, over time, under appropriate hospitalgrade airflow conditions. the particle sizes were engineered to simulate a range of human particle sizes that are emitted during breathing and while on multiple varieties of oxygen support. the quality and high fidelity of analysis using ansys in this simulation which increased the specificity of the test and carried out the particle behavior in the room over time compared to the previous simulation trial that utilized solidworks for simulation. in addition, the range and modality of comparators used, dispel bias measured with and without a mask in place, tidal breathing, lfo2 at 6l•min -1 , and hvni at 40l•min -1 . this method of testing provided extremely specific measurable qualities. this simulation contained aerosolized particles that were engineered to have a wide range of size. these results may be applicable to other operations characterized as agps. limitations of the simulation include the fact this is an in-silico model. in-vivo testing to determine particle distribution would be methodologically difficult, with limited ability to further quantify the nature and result of the droplets/particles. such testing may be an important follow-on study once adequate methodologies have been identified. the current simulation model is high-fidelity and takes both a 'functioning room' geometry and dynamics into account, including air currents, as well as plausible particle mass distributions in a breathing model, and includes an accurate rendering of the mask behavior. as such it is informative for clinical decision making but has not been tested in vivo. such testing will no doubt further refine the clinician decision making in the management of these patients. the designed limitation of mask-fit served the simulation well in highlighting the importance of mask-fit for any real-world utilization of a similar model. another limitation may be the studied particle size thresholds in the findings and applying these to situations in which there are predominantly smaller particles. however, smaller particle masses were studied as part of this evaluation and an advantage of using the mask is demonstrated in this subgroup. finally, the actual dynamics were tested at a single flowrate for hvni and for lfo2, although the anticipated behavior of the mask capture simulated in that pair of scenarios would suggest 'worst case'. the fact that tidal breathing and breathing with lfo2 in this simulation were both associated with potential particulate dispersal, these findings may suggest a role for use of a simple surgical mask in the care of any covid-19 patient in the acute care environment. the use of masks are likely to help address issues of cough and sneeze, which have been associated with high velocity impulses know to transit particulate mass great distances. 31 these surgical mask simulations would not change current recommendations to use negative pressure rooms, when available. hvni with a mask may be the safest option in an overwhelmed system (insufficient negative pressure rooms), where patients are treated in simple rooms (as modeled in this study). regarding disinfection/cleaning implications, these findings suggest no substantive change to current practices because particle escape remains dependent on mask security and due to the fact that respiratory-therapy related transmission is only one means of dispersal of particulate contaminants. however, practically, the results for this particular configuration and simulation infer that additional focus could be placed on areas behind the patient head. cleaning procedures should be completed correctly and consistently to prevent excess viral load accumulation on fomites, thereby decreasing risk of infectious particle/droplet transmission. non-invasive therapies such as high flow oxygen are widely used in management of acute critically ill patients with respiratory distress. such patients present a potential droplet-transmissive risk during care. personal protective equipment and environmental control/engineering should be a primary concern/consideration when managing patients with covid-19. this model corresponds with prior work indicating that even tidal breathing disperses particles some distance. this model also suggests that when making decisions regarding limitation of potentially infectious droplets/particles, the application of a simple surgical mask, which is well-fit to the patient's face, may reduce the velocity of escaping gas and capture particles. this adjunct must be balanced against the totality of the patient care situation, as the addition of a surgical mask adds, albeit very slightly, to the complexity of management of the patient. clinicians will have to decide on the cadence for changing such masks, if required, based on the particular circumstances in management of that patient. no recommendations regarding that aspect of care can be made from these data. these insilico findings should be evaluated in-vivo with appropriately constructed clinical trials. this appendix is intended to provide additional cfd model specificity for reproducible study results. the room model used in the simulation measures 4.87m x 3.65m x 2.44m with a total volume of 43m 3 . two inlet vents are positioned on the ceiling (dimensions 0.305m x 0.305m), and two outlet vents (0.305m x 0.305m) located on the centerline of the wall across from the patient, with one near the floor and the other near the ceiling. a 3d model of a human head is positioned on a bed 736mm above the floor with a 30° incline. the head model includes a simplified airway structure. the mouth opening is rectangular with rounded corners (5mm x 20mm with 2.5mm radius corners). the nasal openings each measure 8mm x 9mm with 2.5mm rounded corners. an adult small / pediatric cannula (vapotherm® inc., exeter, nh, usa) is positioned with its prongs in the center of the nasal openings. the flow opening of the nasal cannula measured 2.62mm in diameter. the airway model extends to a point just above the larynx, where the fluid passage is terminated with a planar surface. this surface is used as both a flow opening for the breathing flow and the source of the particles introduced into the flow. a model of a type i surgical mask is fitted to simulated head. the mask is modelled with a thickness of 4mm to allow a mesh with 4 hexahedral elements across the thickness without requiring prohibitively small mesh elements. the mask properties are adjusted to account for the modelled thickness. to imitate clinical practice, the designed emission-openings were modeled in eight discrete locations (figure 2 , right). the total cross section of the emission-openings is 679mm 2 , thereby modeling a 'poor fitting' of the mask at the nose (e.g., failure to 'pinch the nose' at the bridge of the nose/face interface). each side of the face has one emission-opening which simulates a cannula tube passing through the edge of the mask. the discrete emission-openings have the same cross section as a 1.3mm gap around the entire perimeter. discrete emission-openings were used to improve cfd performance. modelling the emission-opening as a continuous narrow channel would have required a finer mesh to accurately simulate the emissions. the mask pressure-drop properties were obtained from the standard governing surgical masks, en14683 21 , and from data in chen et al., aerosol penetration through surgical masks. 22 the mask is modelled as a porous medium which allows flow to pass through with a pre-defined resistance. the pressure drop of the mask is modelled to be in accordance with en14683 and chen et al, providing a pressure drop of 29.4 pa•cm -2 for a test area of 4.9cm 2 . the velocity and pressure drop data used to model the porous media properties is given in table a1 . the porous media properties were validated by modelling the standard test defined in en14683. using the 4mm thick mask, a test area of 4.9 cm 2 and the defined porous media properties a pressure drop of 29.4 pa•cm -2 was obtained in the validation model. particle penetration is obtained from chen et al. data for a mask with a filtration layer at 100l•min -1 . as the simulated flow rate through the modeled mask does not exceed 80l•min -1 , this is conservative. as the chen et al. data does not include efficiency for particle sizes above 4µm, the data was extrapolated with the assumption that for every additional 5µm, the percentage of particles passing through is halved. above 20µm the filtration efficiency is assumed to be 100%. the filtration efficiency, summarized in table 2 , was used to implement a user defined function (udf) in ansys fluent to remove particles that pass through the filter. when a particle trace enters the porous media volume, the mass travelling along that particle trace is reduced by the filtration efficiency for that size particle. cfd allows for the simulation of complex flow fields, tracking of particles through those fields, interactions of those particles with the carrier fluid, and the differential capturing of particles by a porous media. evaluation of the use of a surgical mask with hvni, low flow oxygen, and tidal breathing was performed in ansys fluent cfd (ansys, inc, canonsburg, pa, usa). simulations were performed on control cases modeling a patient on hvni (40 l•min -1 ), lfo2 therapy (6 l•min -1 via nasal cannula), and simulated breathing (tidal breathing, no therapy) without a surgical mask. summary of all the evaluated cases are shown in table 1 . the patient is modelled to be breathing at 32 breaths per minute with a tidal volume of 500ml with a sinusoidal 1:1 ratio (inspiratory:expiratory) breath curve, without a pause (no inter-breath interval) amongst the inspiratory/expiratory phases. the peak expiratory flow rate is 49.0 l•min -1 . the distribution of assumed spherical particle sizes emitted by a patient breathing, talking, coughing, and sneezing has been investigated in several studies. the specific distributions vary, however, the range of particle sizes that are meaningful to the study is generally from 0.1 to 100µm. particles larger than 100µm are highly unlikely to penetrate a mask or travel far without a very high velocity flow. particles smaller than 0.1µm account for a very small fraction of the total particles are likely to escape regardless of a mask and will remain airborne even in very low velocity flows. the particle distribution (table a2) , for the simulation is taken from exhaled droplets due to talking and coughing. 13 a rosin-rammler diameter distribution method is used during the simulation to generate particles that approximate this particle distribution. particles smaller than 0.1µm and larger than 100µm were not included. the rosin-rammler parameters, ̅ and n = 0.990 are used for the simulation, with the parameters' curve fit shown ( figure a1 ). the particles are released at five time-steps near peak expiratory flow and traced through the flow field at that time step. particles are introduced at just above the larynx, representative as water for material properties. note that all surfaces inside the airway are defined to have ideal reflection for particles that contact those surfaces, such that particles are not absorbed until particles exit the nasal-oral opening of the human airway. table a2 . mass fractions of the particle distribution used in the room simulation (left) and for the curve fit (left and right) of the rosin-rammler diameter distribution ( figure a1 ). figure a1 . rossin-rammler diameter distribution of the particles in the room simulation. mesh geometry of 3 million elements used in the simulation is shown in figure 3 . a hex-dominant mesh is implemented in the mask and the majority of the room volume. tetrahedral elements converted to polyhedron elements are implemented in the regions near the head, where hexdominant meshing was unachievable. as the mask is a critical component for an accurate simulation, the mesh density within the mask is set to achieve a thickness of at least 4 hexahedral elements. hexahedral elements were used in this instance since validation testing in ansys reveals that hexahedra are required for accurate pressure drop results and are preferred for atomization studies. 32 mesh refinement is also applied to the regions around the patient to best achieve accurate particle movement trajectories both near the patient and where particles settle. the patient breathing is modelled as a sinusoidal flow with a frequency of 32 breaths per minute. the breath curve is defined by the equation ̇ where ̇ is the mass flow rate in kg/s. therapy flow is modelled as a constant inlet with a mass flow rate of 8.02•10 -4 kg/sec (40 l/min) or 1.204•10 -4 kg/sec (6 l/min). room ventilation is modelled as an inlet with a constant flow of 0.066 m 3 /sec the simulations are transient, accounting for variation of the flow with time caused by cyclic breathing. the simulation is allowed to run until the flow in the room reaches a quasi-steady state. figure 3 shows the streamlines of the fully developed room ventilation flow. a fixed time step of 0.001sec is used for the simulation. the solver (16.2.1 v 2020r1), turbulence model (rsm v sst), and timestep (0.001s v 0.0001s) had negligible effect on the results (data not shown). use of the advanced numerics option showed a non-negligible increase in particle capture in the mask. this is possibly due to the numerics affecting how the porous media resistance is computed at the interface between the porous zone and the bordering fluid zone. the particle model uses a specified mass flow rate for normalization purposes only. all results are reported as a percentage of the total mass flow rate. a sensitivity study of mass flow rate of particles on the results confirmed that the mass flow does not affect the percentage of particles caught in the mask or in the vicinity of the patient. saffman lift effects are included in the particle simulation. a sensitivity study showed saffman lift increases capture in the mask as compared to neglecting this effect (data not shown). the preliminary simulation and model was published in a chest research letter. 20 upon review of this preliminary work, the authors designed a subsequent model, with the aim to provide a more reliable and accurate simulation. there are several differences between the preliminary study and the current study, which improve the simulation, and thereby provide more reliable findings. first, the mesh across the surgical mask is now hexahedral element instead of tetrahedral element, as tetrahedral element mesh may constrain/influence the properties of the porous media and subsequently may affect the differential particle filtration. second, the hexahedral elements replaced the bulk room geometry mesh, and an increased mesh density was now applied to the area of the room immediately surrounding the patient. third, the current study involves a significantly higher computational cell count and resolution/quality on and around the patient mouth, nose, and internal airway, which provides a lower growth rate as element size transitions more gradually from smaller to larger elements. finally, the emission-openings between the face and mask were previously artificially restricted by use of tetrahedral elements that were converted to polyhedral elements, thus reducing the mesh cell count (resolution) through the emission-openings. this study, with hexahedral elements, does not artificially constrict these emission-openings by allowing the boundary conditions to be fully realized. the effect of this change was to invert the preliminary model results for the proportion of flow that passes through the emission-openings. table 5 . ansys results for the percentage of particle mass by disposition and particle size (≤5µm or >5µm) for all tested cases, both with and without a surgical mask. . ansys results for the percentage of (top) total particle mass disposition, (middle) particle mass disposition for particles ≤5µm, and (bottom) particle mass disposition for particles >5µm for all tested cases with a surgical mask. for particles >5µm during hvni, the mask captures 9.75% more particles than lfo2, and 11.32% more particles than no therapy (tidal breathing). for particles ≤5µm during hvni, the mask captures 130.72% more particles than lfo2, and 223.45% more particles than no therapy (tidal breathing). clinical management of severe acute respiratory infection when novel coronavirus (2019-ncov) infection is suspected: interim guidance. who reference number: who/ncov/clinical surviving sepsis campaign: guidelines on the management of critically ill adults with coronavirus disease 2019 (covid-19) respiratory care committee of chinese thoracic s. [expert consensus on preventing nosocomial transmission during respiratory care for critically ill patients infected by zhonghua jie he he hu xi za zhi = zhonghua jiehe he huxi zazhi = chinese journal of tuberculosis and respiratory diseases report of the who-china joint mission on coronavirus disease 2019 (covid-19) exhaled air dispersion during high-flow nasal cannula therapy versus cpap via different masks. the european respiratory journal infection prevention and control of epidemic and pandemic prone acute respiratory infections in health care aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review department of health and social care -public health wales -public health agency northern ireland -health protection scotland -public health england guidance for infection prevention and control in healthcare settings: adapted from pandemic influenza guidance for infection prevention and control in healthcare settings the role of particle size in aerosolised pathogen transmission: a review factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises the size and concentration of droplets generated by coughing in human subjects exhaled droplets due to talking and coughing study on the initial velocity distribution of exhaled air from coughing and speaking staff safety during emergency airway management for covid-19 in hong kong exhaled air dispersion during noninvasive ventilation via helmets and a total facemask high-velocity nasal insufflation in the treatment of respiratory failure: a randomized clinical trial high-flow oxygen through nasal cannula in acute hypoxemic respiratory failure. the new england journal of medicine can high-flow nasal cannula reduce the rate of endotracheal intubation in adult patients with acute respiratory failure compared with conventional oxygen therapy and noninvasive positive pressure ventilation?: a systematic review and meta-analysis preliminary findings of control of dispersion of aerosols and droplets during high velocity nasal insufflation therapy using a simple surgical mask: implications for high flow nasal cannula aerosol penetration through surgical masks discrete particle study of turbulence coupling in a confined jet gas-liquid separator aerosol and surface stability of sars-cov-2 as compared with sars-cov-1. the new england journal of medicine covid-19 pneumonia: different respiratory treatments for different phenotypes? intensive care medicine basing respiratory management of coronavirus on physiological principles assessment of the potential for pathogen dispersal during high-flow nasal therapy a novel coronavirus from patients with pneumonia in china transmission potential of sars-cov-2 in viral shedding observed at the university of airborne spread of expiratory droplet nuclei between the occupants of indoor environments: a review turbulent gas clouds and respiratory pathogen emissions: potential implications for reducing transmission of covid-19 the war on liquids: disintegration and reaction by enhanced pulsed blasting phd, is a research scientist at vapotherm key: cord-337957-z9ssp0rs authors: berrouk, abdallah s.; lai, alvin c.k.; cheung, albert c.t.; wong, s.l. title: experimental measurements and large eddy simulation of expiratory droplet dispersion in a mechanically ventilated enclosure with thermal effects date: 2009-06-26 journal: build environ doi: 10.1016/j.buildenv.2009.06.016 sha: doc_id: 337957 cord_uid: z9ssp0rs understanding of droplet transport in indoor environments with thermal effects is very important to comprehend the airborne pathogen infection through expiratory droplets. in this work, a well-resolved large eddy simulation (les) was performed to compute the concentration profiles of monodisperse aerosols in non-isothermal low-reynolds turbulent flow taking place in an enclosed environment. good care was taken to ensure that the main dynamical features of the continuous phase were captured by the present les. the particle phase was studied in both lagrangian and eulerian frameworks. steady temperature and velocity were measured prior to droplet emission. evolution of aerosol concentration was measured by a particle counter. results of the present les were to compare reasonably well with the experimental findings for both phases. indoor air quality (iaq) issues have been receiving much research attention from various disciplines over the last couple of decades [1] [2] [3] [4] [5] [6] . this has been mainly triggered by the need of promoting more comfortable and healthy indoor environment on one hand, and by the necessity of protecting indoor environment against the intentional release of biological and/or chemical agents on the other hand. after the epidemic outburst of severe acute respiratory syndrome (sars) and avian influenza in east and southeast asia, there has been a growing research interest in studying the transport and control of airborne bacteria and viruses indoors and in confined environments such as aircraft cabin [7, 8] . dispersion of microorganism-laden aerosols exhaled from infected patients was recognised as a potential airborne transmission pathway. thus, proper understanding of aerosol transport is required to improve exposure assessment tools and models and adopt better ventilation strategies that can substantially reduce indoor particle concentrations and improve the indoor air quality. depending on the original and final size, droplet nucleus can remain suspended in air for several hours and thus distribute widely throughout indoors. this also depends on the ventilation scheme used. indeed, the ventilation system determines the airflow pattern in the room which in turn decides the droplet nucleus fate. displacement ventilation has been acknowledged to provide better indoor air quality than mixing ventilation. some existing studies that used passive gaseous and small inertial particles as contaminant sources stated that in the presence of heat sources, displacement ventilation is more contributive to pollutants removal without much mixing to the whole indoor environments and this compared to other types of ventilation [9, 10] . these studies also showed that in order to design an effective ventilation system, it is crucial to have a reliable tool that is capable to predict airflow pattern and particle distribution and dispersion indoors. this can be achieved through the use of computational fluid dynamics (cfd) which has the capacity to provide microscopic information on the indoor air environment like the air velocity, pressure, temperature, and pollutant's concentration distribution which are useful to obtain pertinent macroscopic parameters for engineering goals. among these cfd tools, reynolds averaged navier-stokes (rans) simulation has been extensively used for simulation of airflow indoors to predict the averaged velocity or temperature. large eddy simulation (les) solves directly for the transient behavior of the large-scale turbulent motion which tends to have the greatest influence on the turbulent transport but less adjustable constants are required [11] . les approach to model turbulent flows has been recognised as a powerful tool that is able to satisfy a continuing desire for higher fidelity of predictive capabilities. its use has been invigorated by the extraordinary development of computational facilities. since les solves time-dependant turbulent flows, it can provide detailed description of the turbulence phenomenon, such as three-dimensional instantaneous velocity field. thus, les properly accounts for the history and transport effects of turbulence on aerosol dispersion and deposition. the very informative picture of the turbulent transport provided by les has usually been associated with a large computational expense that has prevented its use in the past. however, many recent works have shown that les computations carried out on current computer workstations have been successfully applied to several indoor airflow problems with reasonable computation overheads [3] . among these studies, we can mention the work of emmerich and mcgrattan [12] and musser and mcgrattan [13] who applied les to investigate isothermal room airflow. jiang and chen [14] who explored the potential of les to study natural ventilation in buildings. zhang et al. [15] evaluated various turbulence models, including les, in predicting airflow and turbulence in enclosed environments and zhang and chen [11] proposed a filtered dynamic subgrid scale model to be used in conjunction with les of indoor airflow. all these investigations have shown the great potential of les in predicting turbulent airflow in enclosed environments but none of them have considered thermal effects generated by human. for les of two-phase turbulent flows, the numerical simulation of the particulate trajectories and dispersion pattern in airflows are treated along two streams namely the lagrangian and the eulerian methods. the eulerian methods consider the particle phase as another continuum. transport equation for the mass concentration is derived from the mass (species) conservation conditions and solved to give details of the particle concentration field. when the particle phase is considered as scalar species, the gravitational settling and the deposition rate should be taken properly into account to reflect the aerosol's inertial character [1, 6] . for the lagrangian methodology, the motion of the discrete matters are described by the force balance including those induced by the interactions with the carrier phase. in indoor particle studies, each method gains its own reputation depending on the research goals. the eulerian method is widely used to predict particle concentration distributions in rooms. generally these simulations agree well with experimental data although remarkable discrepancies exist in some studies [4] . if the particle motion and dispersion history is of interest, the lagrangian method is more suitable. indeed, the lagrangian approach appears to provide a more natural description of the actual physical phenomena compared to the eulerian approach. investigations using two-phase les to study particle transport in enclosed environments are rare in literature. we can mention the works of tian et al. [16] , bouilly et al. [17] and chang et al. [18] . almost all of the previous studies did not consider thermal effects. for conventional ceiling schemes the influence of high momentum airflow overwhelms the buoyancy effect and the pollutant transport is mainly governed by the bulk airflow [5] . nevertheless the working principle of displacement ventilations relies completely on the existence of internal heat sources, i.e. occupancy. hence it is very important to study aerosol transport by displacement ventilation with internal heat sources. in the present work, measurements and numerical simulations of contaminant particle concentration evolution in a mechanically ventilated room with heated sources have been carried out. this investigation aims to provide a realistic simulation of the time-dependant flow field in a chamber populated with two heated manikins using the wellresolved les approach. the potential of both the lagrangian and the eulerian approaches coupled to les of non-isothermal airflow to study dispersion characteristics of expiratory aerosols has been explored. it is important to mention that by conducting this study, there was no intention to treat the experimental and numerical findings as portable results and hence draw practical conclusions about the effectiveness of the displacement ventilation scheme for the contaminant removal in full-size model. this is because the scaling rules were not respected, in particular for the thermal aspects due to the physical restrictions imposed by the type of chamber used for the experiment. however, the aim of this work is twofold: (i) to test the capabilities of the in-house code code_saturne to investigate aerosol transport under displacement ventilation with two heated human models using les, (ii) to compare the aerosol concentration fields computed by both the lagrangian and the drift-flux eulerian approaches in the framework of les, and (iii) to compare the experimental results with model predictions. both measurements and numerical simulations of airflow field and particle concentration were carried out in a downscaled chamber with two identical model occupants as depicted in fig. 1 . the chamber is mechanically ventilated using the displacement ventilation scheme. table 1 shows the details of the room and occupants geometries and the boundary conditions. the center point of each object is indicated in the table. the geometry of the human occupant used in this study is the one originally proposed by brohus and nielsen [9] . an opening (0.004 m â 0.004 m), measured at 0.33 m above floor, located at the centerline of the head was added to simulate the mouth of the occupant. table 2 shows the details of the geometry of the model occupant used for this study. two planes are defined in the geometry; a plane (x-y) crossing through the human model near at the inlet and a midplane (y-z) at x ¼ 0.25 m. the wall temperature is set to 297 k while the occupant temperature is set to 317 k. one of the occupant is emitting droplets (source) and faces directly the second occupant (receptor). the source emits water spherical droplets with initial velocity of 10 m/s lasting for 0.1 s. similar duration period has been adopted in the literature [19] . in this work, some well-justified assumptions were made. first, the evaporation period of the emitted droplets was ignored and only the droplet nuclei is directly modelled. two recent reviews have demonstrated the time scale of evaporation. nicaset et al. [20] and morawska [21] estimated that the shrinkage time from the original droplet to droplet nuclei is rapid and is in the order of 0.5 s. this time scale is at least an order of magnitude shorter than the residence time of the droplet nuclei suspended in the room. also, the droplets are assumed trapped once they touch any surfaces and do not re-suspend or break up. these assumptions are valid for the present low air velocity environment. coagulation effect has been examined by applying a simple estimation [22] . the results revealed that the coagulation effect can be neglected. the filtered spatial and temporal evolution of non-isothermal incompressible newtonian fluid flow is governed by the following equations: where u i is the component of the filtered fluid velocity in the x i direction, p i is the fluid pressure, t is the filtered temperature, r is the fluid density, n is the fluid kinematic viscosity, g j is the gravitational acceleration, b is the thermal expansion coefficient, pr is the molecular prandtl number and d ij is the kronecker symbol. s ij and q j are the sub-grid scale (sgs) stress tensor and heat flux, respectively. the sub-grid scale (sgs) stress tensor s ij is modelled using the algebraic eddy-viscosity model proposed by smagorinsky [23] : table 1 geometry and boundary conditions. where n sgs is the sub-grid scale viscosity: here c s is the smagorinsky constant, its value is taken equal to 0.12. of-strain tensor. the length scale d is equal to the grid spacing, is the van driest damping function that accounts for the reduction of the sub-grid length near solid walls [24] . it is based on the dimensionless distance from the wall y þ ¼ yu s /n. u s is the friction velocity and a þ is taken equal to 25. in the same way the heat flux q j is modelled: the subgrid scale prandtl number pr sgs is taken equal to 0.5 [25] . an unstructured grid (non-conforming embedded refinement) consisting of 1,360,000 cells was used for the computational domain discretisation. the first grid point near the chamber walls and manikins at which the velocity is computed is located at y þ z 1. two grid points are placed within the viscous sublayer, the depth of which equals 5 wall units. a non-uniform grid is employed in the normal-to-the-wall and normal-to-manikins directions. this is done in order to locate more gridpoints where they are most needed. care should be taken when non-conforming embedded refinement is used. it is well known that the near-solid region is characterised by steep gradients and very small energy containing eddies that should be well captured. these near-solid coherent structures contain most of the turbulence and are responsible for the correct distribution of the turbulent energy from the streamwise into the other directions. moreover, the near-solid turbulence has a significant impact on the deposition of inertial particles and therefore it should be properly resolved. the different non-conforming layers were designed such that the aspect ratios between the different directions for all the cells do not exceed 5. this is done to avoid numerical instabilities that arise when using very flat cells in the les context. thus the use of non-conforming layers should be efficient since it allows a cell distribution that responds to the requirement of the flow dynamics without introducing further numerical errors. speziale et al. [26] pointed out that a reliable les is the one that becomes a dns when the grid resolution is as small as the kolmogorov scales. in this numerical study, the reynolds number is 1300 based on the length of the inlet opening and the expected kolmogorov microscale is 70 microns. consequently, one cannot seek a grid independent les, as we usually do for rans. this is because a grid independent les is essentially dns, and the philosophy of les that is based on grid dependency, looses its meaning. celik et al. [27] developed a method to assess the quality of les results. it consists of estimating an index of quality which is a measure of the percentage of the resolved turbulent kinetic energy. they stated that if more than 75% of the kinetic energy is captured, then the les is considered adequate. pope [28] has also stated that the amount of turbulent kinetic energy that is carried by the sgs scales should not exceed 25% for the les to be well resolved. a simple way of estimating the sgs kinetic energy can be made based on the assumption of equilibrium at the cutoff. then the dissipation rate and the sgs kinetic energy can be evaluated as the following: typically c e z 1 [29] . for non-equilibrium turbulent flows characterised for instance by zones of strong recirculation and/or boundary layers detachment, the assumption of equilibrium at the cutoff is not valid and it is necessary to solve a transport equation for the sgs kinetic energy to get an accurate estimation of the filtered out kinetic energy [30] . in this work, eqs. (9) and (10) were used to give such an estimation. a time step, dt ¼ 0.01t * was used to advance calculations. t * is the integral time scale defined as the ratio of the inlet height to the inlet velocity. the size of the time step was dictated by the numerical stability. the les computations are initiated from a randomly generated instantaneous inlet velocity with mean velocity and turbulent kinetic energy profiles fitted to analytical formulae [31] . the time advancement was carried out until t ¼ 150t * to achieve a flow field independent of the initial conditions. at t ¼ 150t * , residuals of eqs. (1)-(3) became smaller than the set convergence tests indicating that the computations had reached a nearly statistically steady state. from t ¼ 150t * , the calculations were continued until t ¼ 200t * . in this interval, the final statistical data was accumulated. a flow solver from the r&d section of electricite de france named code_saturne (http://www.code-saturne.org) was used as starting point of the present work. the discretisation in code_saturne is based on the collocated finite-volume approach. it allows solving navier-stokes and scalar equations on hybrid and nonconform unstructured grids. velocity and pressure coupling is ensured by the prediction/correction method with a simplec algorithm. the collocated discretisation requires the rhie and chow interpolation in the correction step to avoid oscillatory solutions. a second order centred scheme (in space and time) is used. the flow solver has been extensively tested for les of single-phase flows [32] . aerosols are released and tracked in the turbulent flow that is described in the previous section. the physical properties of these inertial particles are summarized in table 3 . as a result of the high density ratio between particle and fluid densities, the equation describing particle motion is reasonably simple and only the drag and gravity forces will be retained since other forces are in this case negligible [33] . since the dispersion of very small particles is investigated in this work, the brownian force should be taken into account. thus, the tracking of the inertial particles within the turbulent flow obeys the following system of equations: dx p;i ¼ u p;i dt; du p;i ¼ u p;i à u s;i s p dt þ n i ðtþdt à g i dt; here x p and u p are the particle position and velocity, u s is the fluid velocity seen by an inertial particle along its trajectory, g is the gravity force per unit of mass, d p and r p are the diameter and the material density of inertial particles, s p is the particle response time, c d is the drag coefficient and re p is the particle reynolds number, re p ¼ d p ju s à u p j=v with n is the kinematic fluid viscosity. c n is cunningham slip correction factor. it is considered herein to correct the drag coefficient in order to take into account the free-slip boundary conditions that occur at the surface of the particles. here l is the molecular mean free path. n i (t) is the brownian force per unit of mass. here g i is zero-mean, unit variance-independent gaussian random number, dt is the time step and s 0 is spectral intensity which is computed using: the number of particles injected was set to 500 per time step for a period of time lasting 0.1 s which corresponds to a number of iteration equal to 500 iterations. both particle and fluid seen velocities were set equal to the fluid velocity at the secondary inlet (occupant's mouth); i.e. 10 m/s. calculations were repeated using 1000 particles and only slight differences in the concentration fields were noticed (around 2%). because of the high velocity at the secondary inlet, the time step of the simulation was decreased to dt ¼ 0.001t * to keep the courant-friedrichs-lewy number around 1. for the eulerian approach, the concept of a particulate phase consisting of individual, distinguishable droplets is abandoned. the particulate matter phase is considered as continuum that can be described using a set of generalized equations similar to the equations used to solve the gas phase. this approach is known in the literature as the two-fluid approach. if only the instantaneous mass concentration of the particulate phase c is of interest as it is the case of this study, a transport equation by turbulent motion for this property can be derived: where g is the particulate matter mass diffusivity and s c is the rate of creation or destruction of the mass concentration per unit volume. in the context of les, eq. (16) should be spatially filtered giving rise to the filtered transport equation for particulate phase mass concentration: the term vf j =vx j is considered as a supplementary turbulent diffusional process occurring at the sub-grid scales. this additional concentration flux is approximated following: where sc sgs is taken equal to 1. eq. (18) the sink term s c is computed as the mass wall flux per unit of volume. it is computed for all the cells that have at least one wall face. this sink terms account for a decrease in mass concentration due to loss of mass to the walls. this sink term corresponds to particle deposition in the lagrangian approach. where a is the area of the wall face linked to its corresponding cell. v d is the deposition velocity estimated for the different wall orientation using the three-layer model developed by lai and nazaroff [34] . this was done by integrating the particle loss across the boundary layer. this afformentioned approach is not a new approach and its use in the framework of rans has been discussed by holmberg and li [1] and chen et al. [6] . residual charge. the airflow was induced by means of a dc fan and regulated by a power supply. the inlet duct length was sized at least 60 times of hydraulic diameter of the size of the duct to ensure that the flow is fully developed at the chamber inlet. monodisperse particles of 10 mm were generated by atomization of diluted standard polystyrene microsphere suspensions (thermo fisher scientific). the diluted suspension was pre-filled to the cup container attached to a spray gun. the spray gun was fixed at the centreline (x ¼ 0.25 m, y ¼ 0.33 m, z ¼ 0.4 m) of the head of the source. a flow regulating valve was used to adjust the emission velocity to match with the simulation boundary conditions. the expiratory process was mimicked by a short release of particles through a spray gun connected to a compressor. the spraying duration was controlled by a timer circuit and it can be adjusted by a labview program. in this work 0.1 s was selected. hepa filters were installed at inlet and outlet to minimize the background particle count inside the chamber and to prevent crosscontamination. type t thermocouples were selected as it has better accuracy than the other types of thermocouples. prior to the temperature measurement, the thermocouples were all calibrated in-situ by 5-points measurement. the two manikins that are made of aluminuim were heated by wrapping heating wire around the body. thirteen thermocouples were used to measure inlet, manikins' surface temperature and different pre-fixed locations inside the chamber. the air inlet temperature is the room temperature and during the entire experimental period the inlet air temperature was maintained at approximate 24 c. the temperature difference between the inlet air and the surface temperature of the manikins was kept at 20 c. all the temperatures were monitored and controlled through the same labview program. the emission velocity was measured by a thermal anemometer (tsi, 9555). the particle concentration was measured by an optical particle counter (tsi, 3775). conductive sampling tube was used to sample the particles to minimize the electrostatic loss. since there was one counter available, only a single point measurement made at a time. background concentration was measured 5 min prior to the emission starting. the measured concentration was subtracted from the background. figs. 2 and 3 show the predicted steady state averaged velocity contour at the mid-plane of section y-z and at the plane crossing the manikin closer to the air inlet respectively. a cooled-jet can be easily identified near the low-level of the floor and a strong vertical buoyancy-plume is formed above the heated manikin. one key feature of displacement ventilations is the low inlet velocity. as the cold air flows around the manikin and picks up the heat, the velocity increases rapidly. it is noticed that the low momentum cooled air (0.2 m/s) near the floor level absorbs heat from the two occupants and creates a dominant vertical thermal plume in the boundary layer around each occupant. the airflow velocity is fairly weak in all regions except inside the buoyancy plume [35] and the bulk velocity is less than 0.1 m/s. this low velocity will influence significantly the convective transport of aerosol. fig. 4 shows the predicted averaged temperature contour along the mid-plane of y-z direction. this shows you a clearer picture on how the temperature varied from the cooled-inlet to the exhaust. fig. 5 shows an instantaneous velocity at the same plane. two similar but unequal strengths of vertical plumes can be observed. inferring carefully from both figures, it can be seem that the buoyancy plume of the heated manikin near the inlet is warmer and stronger than another one. it is due to the coolest air contacting that heated manikin while the air contacting the other manikin has been warmed. it is not straightforward to compare the experimental data with those modeling results directly. the particle counter output is number (or mass) concentration expressed in particle number (mass) per cm 3 . the native output for the eulerian and lagrangian models are concentration or dimensionless concentration. to compare with both eulerian and lagrangian modeling results, the (concentration) data obtained by the counter was transformed to a dimensionless concentration and is defined as where c(x, t) is the temporal concentration at the measuring point x and c vol ðt ¼ 0þ is the initial volume-averaged concentration for the entire chamber. it was evaluated by calculating the particle injected during the emission period (0.1 s) and divided by the volume of the chamber. figs. 6-8 show the experimental measured steady state airflow pattern and temperature distribution prior to the emission of aerosols. the results were compared with those predicted by the les. all the results were measured at mid-plane along y-z. the velocity profile along the centerline of the inlet duct (i.e. y ¼ 0.075 m) is shown in fig. 6 . excellent agreement between the modeling and experiment can be observed. due to the very low velocity and the sensitivity of the velocity probe used, there was no measurement data for z greater than 0.6 m. it is interesting to note that a small recirculation zone is existed at z ¼ 0.7-0.75 m. fig. 7 shows the temperature measurement along a central vertical line (i.e. y from 0 to 0.5 m). the computational prediction experimental data consistently overestimates the measured temperature for most of the locations. it is interesting to notice that the discrepancy decreases with the y-direction. the simulated results were fluctuated temperature while the experimental results were averaged value. besides, the discrepancy may also be attributed to the uncertainty of the thermocouples. fig. 8 depicts the temperature profile along the z-direction at the vertical height of the spray gun. since the separation of the two manikins was less than 0.2 m, seven thermocouples were placed. it can be seen that the measurements and modeling results agree well as it shows the increasing trend when it approaches the heated manikin. concentration evolutions were measured in three streamwise locations for two different transverse points. figs. 9-12 show the experimental results for four locations and the results are compared with the two modeling predictions. the x-axis is the elapsed time after the aerosol emission and the y-axis is the dimensionless concentration. it should be noted that unlike reynolds averaging methods les gives instantaneous concentrations of particles that are convected by a turbulent flow. all the reported results were measured downstream of the emission point. hence it can be anticipated that the concentration will decay with elapsed time. regardless of the modeling approaches or experimental results, first three figures show clear decay profiles. for fig. 12 where the location is at the exhaust, the result is more complex as both lagrangian and experimental results depict non-decay profiles. it can be seen that the variation of the experimental measurement at point f is very small which is ranging from 0.01% to 0.07%. further investigation is required to explain the observation. it is also interested to compare the ''decay'' rate for different locations as the decay magnitude should be correlated to the ''local'' air velocity. this issue will be addressed in future work. the experimental results were best fitted by an exponential function using the least square method and the corresponding decay rates are shown (figs. 9 and 10). inferring from the results figs. 9 and 10, the decay rates are comparable to each. by taking into account of the uncertainly of the particle counter used which is ae12%, the agreement between the experimental results and the computational modeling predictions is acceptable [36] . in this work observable discrepancy between the modeling results by lagrangian and eulerian approaches is seen. more experimental results are required to validate the accuracy of the model. in this work particle dispersion in a scaled chamber was studied numerically and experimentally. a well-resolved les was performed to study aerosol dispersion in a turbulent scaled chamber populated by two manikins. numerical predictions were compared to the experimental observations. due to the complexity of the turbulent flow in such an enclosed environment, a good deal of care has been taken to ensure that the simulation of the carrier phase is reasonably accurate. efforts were made to adapt the mesh to the dynamical features of the flow. the numerical predictions showed a good quantitative agreement with the experimental findings for both phases though some discrepancies were noticed in some regions of the chamber. both steady state and transient parameters were measured experimentally. the measured steady state temperature and velocity agreed well with the simulation. particle concentration was measured a cpc. the measured concentration was lower than that predicted by lagrangian and eulerian models. by taking into account of the measurement uncertainty of the instrument and some limitations of the set-up, the result comparison is acceptable. modelling of the indoor environment-particle dispersion and deposition a study of the dispersion of expiratory aerosols in unidirectional downward and ceiling-return type airflows using a multiphase approach numerical studies of indoor airflow and particle dispersion by large eddy simulation experimental measurements and numerical simulations of particle transport and distribution in ventilated rooms study of expiratory droplet dispersion and transport using a new eulerian modeling approach modeling particle distribution and deposition in indoor environments with a new drift-flux model role of ventilation in airborne transmission of infectious agents in the built environmenta multidisciplinary systematic review numerical simulation of airflow and airborne pathogen transport in aircraft cabins -part i: numerical simulation of the flow field personal exposure in displacement ventilated rooms removal of contaminants released from room surfaces by displacement and mixing ventilation: modelling and ventilation large eddy simulation of indoor airflow with a filtered dynamic subgrid scale model application of a large eddy simulation model to study room airflow evaluation of a fast large-eddy simulation model for indoor airflows study of natural ventilation in buildings by large eddy simulation evaluation of various turbulence models in predicting airflow and turbulence in enclosed environments by cfd: part 2 -comparison with experimental data from literature on the numerical study of contaminant particle concentration in indoor airflow effect of ventilation strategies on particle decay rates indoors: an experiment and modelling study transport mechanisms of airborne particulate matters in partitioned indoor environment comparison of the eulerian and lagrangian methods for predicting particle transport in enclosed spaces toward understanding the risk of secondary airborne infection: emission of respirable pathogens droplet fate in indoor environments, or can we prevent the spread of infection? aerosol technology general circulation experiments with the primitive equations on the turbulent flow near a wall large eddy simulation of non-isothermal room inflow, comparison between standard and dynamic type of smagorinsky model modelling the pressure-strain correlation of turbulence: an invariant dynamical system approach index of quality for large-eddy simulations ten questions concerning the large-eddy simulation of turbulent flows velocity filtered density function for large eddy simulation of turbulent flows subgrid scale model for finite difference simulations of turbulent flows in plane channels and annuli lengthscales and correlations for rans-les coupling internal report d3.3-15 for desider project code_saturne: a finite-volume code for the computation of turbulent incompressible flows -industrial applications equation of motion for a small rigid sphere in a nonuniform flow modeling indoor particle deposition from turbulent flow onto smooth surfaces numerical modeling of exhaled droplet nuclei dispersion and mixing in indoor environments model 3775 condensation particle counter operation and service manual key: cord-271616-h6iauddk authors: block, karin a; trusiak, adrianna; katz, al; gottlieb, paul; alimova, alexandra; wei, hui; morales, jorge; rice, william j; steiner, jeffrey c title: disassembly of the cystovirus ϕ6 envelope by montmorillonite clay date: 2013-12-19 journal: microbiologyopen doi: 10.1002/mbo3.148 sha: doc_id: 271616 cord_uid: h6iauddk prior studies of clay–virus interactions have focused on the stability and infectivity of nonenveloped viruses, yielding contradictory results. we hypothesize that the surface charge distribution of the clay and virus envelope dictates how the components react and affect aggregation, viral stability, and infectivity. the bacteriophage cystoviridae species φ6 used in this study is a good model for enveloped pathogens. the interaction between φ6 and montmorillonite (mmt) clay (the primary component of bentonite) is explored by transmission electron microscopy. the analyses show that mmt–φ6 mixtures undergo heteroaggregation, forming structures in which virtually all the virions are either sequestered between mmt platelet layers or attached to platelet edges. the virions swell and undergo disassembly resulting in partial or total envelope loss. edge-attached viral envelopes distort to increase contact area with the positively charged platelet edges indicating that the virion surface is negatively charged. the nucleocapsid (ncs) remaining after envelope removal also exhibit distortion, in contrast to detergent-produced ncs which exhibit no distortion. this visually discernible disassembly is a mechanism for loss of infectivity previously unreported by studies of nonenveloped viruses. the mmt-mediated sequestration and disassembly result in reduced infectivity, suggesting that clays may reduce infectivity of enveloped pathogenic viruses in soils and sediments. it is estimated that there are 10 31 virus particles on earth making viruses more prevalent in number than prokary-otes, the vast majority of viruses being bacteriophages (weinbauer 2004; breitbart and rohwer 2005) . clays are a primary nonorganic component of soils and aquatic sediments, therefore the dynamic interactions between clay minerals and phage are expected to affect soil-bacteria activity (ostle and holt 1979; vettori et al. 1999; weinbauer and rassoulzadegan 2004; syngouna and chrysikopoulos 2010) . montmorillonite (mmt) is a highly reactive, expandable, hydrous aluminum smectite clay. smectite platelets are positively charged at the edges and negatively charged along the faces to produce an overall negative charge at ph > 3. clays readily form colloidal suspensions that have the potential to interact with dispersed viral particles which influences the aggregate structure. clay speciation is important in considering the fate of viral particles in the environment. numerous studies on the interaction between viruses and clays have yielded conflicting information regarding the effect of clay minerals on virus survival and infectivity; see jin and flury (2002) or kimura et al. (2008) for reviews. the contradictory findings in the literature imply that virus morphology plays a role in the interaction with clays. to our knowledge, published studies on the interactions between clays and viruses have only investigated nonenveloped viruses, although a number of pathogenic viruses found in the environment are enveloped (e.g., avian influenza, coronavirus). enveloped viruses differ from nonenveloped viruses in that they possess a lipid-protein layer surrounding the nucleocapsid (nc). cystoviridae, a bacteriophage family that infects the plant pathogen, pseudomonas syringae pv. phaseolicola, is one of the few phage families with an external lipid envelope. cystoviridae species are often used as a model for enveloped animal and human pathogens (mindich 1988 (mindich , 2004 . many studies have focused on clay influence on virus survivability, but only several have investigated factors that can affect the virus-clay interaction. roper and marshall (1974) found that at low salinity, mmt particles surrounded escherichia coli bacteria resulting in greater protection from phage than under higher salinity conditions. roper and marshall (1978) determined that mmt inhibited infection of e. coli and that finer suspended clay particles provided the e. coli with greater protection from phage by forming a barrier around the bacteria. however, they found that particles greater than 0.6 lm in diameter offered no protection. lipson and stotzky (1984) observed that several proteins (lysozyme, chymotrypsin, and ovalbumin) reduced the adsorption of reoviruses to mmt, likely a result of the proteins competing for adsorption sites on the clay. in contrast, kaolinite and mmt enhanced φx174 infection of e. coli (lipson and alsmadi 1989) . zeph et al. (1988) determined that mmt protected phage p1 from inactivation, although the presence of mmt had no effect on transduction of e. coli. vettori et al. (2000) found that clay minerals protect phage pbs1 from inactivation and loss of ability to transduce bacillus subtilis by uv light. animal pathogenic viruses similarly interact with clays. vilker et al. (1983a,b) investigated the interaction of poliovirus with nonaggregated mmt, and through scanning electron microscope (sem) analysis determined that the negatively charged virions adhere to the positively charged mmt edges. they also suggested that clay aggregation accompanies adsorption of poliovirus and that the mmt enhances poliovirus survival (vilker et al. 1983a,b) . stotzky (1983, 1986) showed that viruses (e.g., poliovirus, coxsackie virus, reovirus) are adsorbed onto clays with enhanced survivability. the adsorption of reovirus onto mmt or kaolinite was almost immediate and correlated with the cation exchange capacity of the clays (lipson and stotzky 1983) . this indicates that reovirus adsorbs onto negatively charged regions of the clays. lipson and stotzky (1985) found that coliphage t1 and reovirus type 3 adsorb onto different sites on kaolinite and mmt. they also investigated mixtures of kaolinite and reovirus and determined that these were more infectious than equivalent concentrations of virus alone and attributed the increased infectivity to improved viral transport in the presence of kaolinite. the seemingly contradictory effects of clays on virus survivability are likely a result of differences in the mechanisms behind the clay-virus interactions. although the literature has focused on the effect of environmental conditions (clay type, clay charge distribution [lipson and stotzky 1985; christian et al. 2006; templeton et al. 2008] , ph [zhuang and jin 2008; walshe et al. 2010 ], ionic strength [tong et al. 2012] , buffer composition [zhuang and jin 2008; gutierrez et al. 2010] , and cation exchange capacity [lipson and stotzky 1983; vettori et al. 1999] ), the surface morphology of viruses has been largely ignored. the attachment of phages t1, t7, t2, pbs1, or φx174 to cation exchanged clay is related to positively charged sites on clay edges (schiffenbauer and stotzky 1982; chattopadhyay and puls 1999; vettori et al. 2000) . however, lipson and stotzky (1983) found that reovirus adsorption occurs at negatively charged sites on cation exchanged kaolinite and mmt, consistent with derjaguin, landau, verwey, overbeek (dlvo) (derjaguin and landau 1941; verwey and overbeek 1948) theory of colloidal aggregation. rossi and aragno (1999) found that colloidal aggregation of phage t7 and mmt results in reversible binding and protection from inactivation. phages ms2 and φx174 attach to kaolinite and mmt by hydrophobic interactions (chrysikopoulos and syngouna 2012). chattopadhyay and puls (2000) studied the different forces involved in phage (t2, ms2, φx174) sorption on soil particles (hectorite, kaolinite, and norman clay) and found that van der waals attraction dominated over electrostatic repulsion. in this work, the objective is to explore the interaction between mmt clay and an enveloped virus to better understand the mechanisms of mmt-induced virus deactivation in clay-rich environment, such as earth's critical zone. in being the first study of the interaction between clays and enveloped viruses, the aim is to provide insight into the methods of viral inactivation, and the application to many enveloped human pathogens. a high-purity na-mmt (smectite) clay (commercial name: "accofloc"; chemical formula: (na,ca) 0.33 (al 1.67 mg 0.33 )si 4 o 10 (oh) 2 n(h 2 o); american colloid company, arlington heights, il) was made homoionic with magnesium using the cation substitution technique described by moore and reynolds (1997) . the clay was initially washed in 5% sodium hypochlorite (bleach) to remove organic contaminants and then washed multiple times in distilled water to eliminate the bleach. large clay particles and nonclay minerals were removed by centrifugation at 3600 rpm (2700g) for 20 min. the supernatant comprising the fraction with an equivalent spherical stokes diameter less than 0.2 lm was collected for use in this experiment. the purified mmt was suspended in 0.1 mol/l mgcl 2 overnight and centrifuged at 1300 rpm (350g) for 30 min. the pellet was rinsed in distilled water 8-10 times and resuspended in distilled water. two drops of agno 3 were added to the suspension after the rinses to verify that all chloride was removed. the concentration (w/v) of the stock suspension was 2 mg ml à1 . the clay suspension was autoclaved just prior to addition of the viruses to ensure sterility. φ6 is the first isolated member of the cystoviridae family. the φ6 virion has a layered structure consisting of an inner nc surrounded by the lipid envelope (kenney et al. 1992 ). the φ6 bacteriophage host cell, p. syringae lm2333 growth medium is luria bertani, supplemented with ampicillin (100 lg/ml) to inhibit contamination. buffers a and acn were used for the suspensions of purified φ6. buffer a contains 10 mmol/l kh 2 po 4 (ph 7.5) and 1 mmol/l mgso 4 . buffer acn is buffer a with the addition of 200 mmol/l nacl and 0.5 mmol/l cacl 2 . plate lysates of φ6 were prepared by plating phage dilutions into soft agar with a culture of lm2333. the plates were incubated overnight at room temperature prior to collecting the phage-containing top agar. the cell debris and agar were removed by centrifugation in a sorvall gsa rotor (thermo scientific, asheville, nc) at 15,000 rpm (3.0 9 10 4 g) for 30 min at 4°c. virus was collected by centrifugation in a beckman t-1270 rotor at 33,000 rpm (7.5 9 10 4 g) for 2 h at 4°c. the pellet was suspended in 1 ml of buffer a. the bacteriophage samples were then layered on a 10-30% sucrose gradient in buffer a. sedimentation centrifugation was at 23,000 rpm (6.7 9 10 4 g) for 1 h at 15°c using a beckman sw 40 ti rotor, after which the virus particle band was visualized by light scattering and the band collected by needle puncture, pelleted by centrifugation at 33,000 rpm (7.5 9 10 4 g) for 2 h at 4°c, and resuspended in 1 ml of buffer a. final purification of the phage was by equilibrium centrifugation through 40-60% sucrose gradient in buffer a using a beckman sw 40ti rotor at 23,000 rpm (6.7 9 10 4 g) overnight at 4°c. the next day the phage band was again visualized by light scattering and collected by tube puncture. the phage sample was then centrifuged with a sorvall t-1270 rotor at 33,000 rpm (7.5 9 10 4 g) for 2 h at 4°c and the collected phage particles suspended in 100 ll buffer acn. turbidity measurements of φ6 following the procedure described by oster (1951) demonstrated that φ6 surface charge is negative at ph 7 (spectra not shown). the nc particles were isolated by removing the φ6 envelope with triton x-100 as described by steely and lang (1984) . the purified φ6 virus was mixed with 10% triton x-100 solution in buffer acn. acn buffer is recommended because of the presence of ca 2+ ions which stabilize the nc particles, preventing the loss of p8 proteins. the particles were centrifuged at 33,000 rpm (7.5 9 10 4 g) for 1.5 h at 4°c. pellets were washed and resuspended in acn buffer. although nc concentrations cannot be verified by plaque formation, triton x-100 envelope removal is essential complete and thus the nc concentrations are approximately equal to the initial whole-virion concentrations. sterilized mg-mmt (500 ll) suspensions were mixed in eppendorf tubes with 100 ll φ6 bacteriophage in acn buffer. tris buffer (10 ll) was added to bring the resultant mixture to 0.5 mmol/l cacl 2 and 1 mmol/l mgso 4 . nc-mmt mixtures were prepared in the same manner (500 ll of mmt, 100 ll of ncs in acn buffer and tris). the concentration of cations in the buffer was sufficient to initiate fast aggregation of mmt (katz et al. 2013) . visual inspection confirmed that the mixture aggregated in less than a few minutes. φ6 in acn buffer with tris, but without mmt was used as a control. the eppendorf tubes were incubated at room temperature (25°c) for an hour. the mixtures were then centrifuged for 10 min at 3600 rpm (1200g). the supernatant was collected and the clay pellet resuspended in 100 ll acn buffer. the pelleted aggregates were vortexed prior to plaquing to disaggregate the mixture and separate the viruses from the mmt. the supernatants and final, disaggregated, clay suspensions were spot checked on plates with growing p. syringae to determine fractionation of virus in the system. virus concentrations were calculated using the plaque assay technique. the nc populations used in the transmission electron microscopy (tem) analysis were made from comparable φ6 concentrations. phage infectivity was measured over a range of 6 orders of magnitude of mmt-to-phage ratios (mg of mmt/number of virions). samples were negatively stained with uranyl acetate. a minimal amount of stain was employed to reduce the effect of the stain on abundant viral proteins, which would impede identification of viral particles. tem grids were prepared using φ6 concentrations of 10 11 virions ml 1 and mmt concentration of 1.7 mg ml à1 . electron micrographs were acquired at the new york structural biology center with either a jeol 2100 (jeol inc., peabody, ma) or a tecnai fei 20 (fei, hillsborough, or) electron microscope, both operating at 200 kv. for the jeol 2100, micrographs were acquired at magnifications of 30,0009 or 50,0009 giving pixel sizes of 0.511 or 0.306 nm/pixel, respectively, on the 2k 9 2k ccd. for the tecnai f20, micrographs were acquired at magnifications of 29,0009 or 50,0009 giving pixel sizes of 0.592 or 0.340 nm/pixel, respectively, after 2 9 2 binning of the 4k 9 4k ccd. isolated φ6 virions and ncs are nearly spherical in shape and when negatively stained will appear in electron micrographs as~70 nm and~57 nm circles, respectively. φ6 virions have a clearly defined envelope that appears as a lower density region under negative staining. the nc outer icosahedral shell also appears as a brighter region in the micrographs when negatively stained. classification of intact φ6 virions and mmt-induced, enveloped-stripped ncs (mmt-nc) was determined by size and shape analysis. particles exhibiting nearly circular shape in the micrographs with a diameter between 68 nm and 75 nm were classified as nondistorted φ6. nearly circular particles with diameters between 55 and 63 nm diameters were classified as nondistorted mmt-ncs. virions deviating from circularity were classified as complete but distorted φ6 if the larger diameter was greater than 68 nm, or distorted mmt-ncs if the larger diameter was less than 63 nm. size distributions of the isolated φ6 and ncs were calculated using the particle analysis function of imagej software (u. s. national institutes of health, bethesda, md; schneider et al. 2012) . poor contrast between embedded virions and mmt hinders the ability to use the automatic particle size and counting functionality in standard image processing software. therefore, size analysis was performed by counting pixels and converting to distance using the calibrated pixel size of the microscopes. corel-draw was used to resample the insets shown in figure 3 at twice the pixel density, and autoadjust the contrast. tem of phage φ6, nc, and montmorillonite tem micrographs of φ6 and nc in figure 1a and b, respectively, reveal a consistency of particle size in the absence of clay that is not affected by uranyl acetate staining. the micrograph in figure 1a shows~10 2 φ6 virions with diameter 69 ae 2 nm for over 95% of the particles. the vast majority of the virions in figure 1a exhibit aspect ratios near unity (1.08 ae 0.07), confirming the spherical morphology of the particles. only two nc particles with diameters of 57 nm (labeled nc in fig. 1a ) are present in the field of view, confirming that the φ6 envelope rarely spontaneously disassembles. very few partially disassembled φ6 are visible in the micrograph. although nc particles are icosahedral, the nc particles appear spherical at the resolution of the figure 1b micrograph. the vast majority of the ncs in figure 1b have a diameter of 57 ae 2 nm and aspect ratios near unity (1.05 ae 0.02). therefore, size and aspect ratio clearly distinguish intact φ6 and ncs and can be used to gauge morphological changes in the virions as a result of interactions with suspended mmt. figure 2 shows a representative tem micrograph of mmt platelets. the clay platelets exhibit a sheet-like structure (platelet face) which in the tem aggregate results in a mosaic of sharp angular forms with discrete edges. the arrow in figure 2 indicates parallel edges which is indicative of platelet stacking. the platelet faces exhibit a mottled texture and lack geometric characteristics similar to those of viral particles. an important characteristic of the micrographs is the complete absence of either free virions or virion-only aggregates at sufficient mmt concentrations to produce visible aggregates (~1 mg ml à1 ). in all the tem grids, the entire φ6 phage population is sequestered within aggregates. a majority of the virions are sandwiched between platelets and partially obscured due to masking by thin mmt sheets. however, a noticeable fraction of the φ6 are attached to the edges of the platelets and are clearly discernible in the micrographs. a preponderance of edge-attached and face-attached virions exhibits extensive distortion in shape, in contrast to the uniformity of particles shown in figure 1a and b. the presence of smaller (~60-nm-diameter) particles in the micrographs is interpreted as clear evidence of disassembled envelopes leaving only ncs. approximately third of the φ6 in the micrographs are completely stripped of their envelopes and are morphologically similar in size to distorted nc particles. the microscopy evidence therefore reveals that the interaction with mmt results in the disassembly of the φ6 envelope. cation bridging between the negatively charged platelet faces and negatively surface-charged viruses facilitates a van der waals attraction causing virions to attach to platelet faces. this attraction is consistent with dlvo theory of colloidal aggregation. the attachment of virions to the positively charged edges likely includes both electrostatic and van der waals interactions. a tem micrograph (fig. 3) shows a population of φ6 in varying states of disassembly, and embedded in two well-defined clay particles. top plateleta distorted φ6 virion (dashed arrow) and several mmt-ncs (solid arrows) appear embedded within layers of clay at position labeled a. the particles near the platelet edge (far upper right) are distinctly smaller than triton-isolated ncs indicating partial disassembly. bottom plateleta distorted 57 nm mmt-nc attached to the clay edge is evident (particle labeled b). the mmt-nc distortion increases the contact area between the particle and the positively charged mmt edge, suggesting that the outer surface of the p8 protein shell acquired a negative charge during disassembly. a number of distorted φ6 (four are noted by dashed arrows) and mmt-ncs (five are noted by solid arrows) are located between platelets. a highly distorted virion (particle labeled c) is shown in inset c; particle size is 93 9 56 nm, corresponding to an aspect ratio of 1.7. the deformed virions indicate that either the envelope disassembly increases particle size as water infiltrates the lipid-protein membrane or compression of virions between platelets has caused distortion. the particle labeled d and shown in inset is nearly circular with a diameter~57 ae 1 nm indicating that the envelope has been completely removed. figure 4 shows an aggregate of clay platelets and partially disassembled virions (dashed arrows) and mmt-ncs (solid arrows). a pair of fused virions is observed in the micrograph (the elongated shape labeled a, at the bottom center). distorted φ6 and mmt-ncs are congregated near platelet edges (upper left of micrograph, labeled b, c, and d), further evidence that φ6 particles 100 nm are sequestered in the mmt aggregates. other virions with disrupted envelopes (e, f, g, h, and k), as well as deformed mmt-ncs (i, j, and l), are readily observable. the magnified inset depicts the repeating tetrahedraloctahedral mmt layering, visible when platelets are viewed edge-on. in this view, virions are shown to preferentially aggregate along the positive edges and wedged between vertically oriented platelets. φ6 adhered to mmt edges exhibits a distinct oval shape in which the major axis is parallel to the edges, increasing contact area between the virion and the platelet edges. the increased contact suggests that the positively charged edge of the mmt is electrostatically interacting with the negatively charged virus. two such virions are seen in the tem micrograph shown in figure 5a . a tracing of the outer boundary and the internal mmt-nc of virions labeled #1 and #2 are presented in the respective insets, b and c, respectively. virion #1 has a shorter axis normal to the platelet edge (71 nm), which corresponds to the diameter of an undistorted virion. however, the elongated axis stretches to 92 nm resulting in an aspect ratio of 1.3. in contrast to virions interlayered with platelets (fig. 4) , where the distortion may be due to either expansion in two or three dimensions, the departure from sphericity noted here includes a volume increase. the boundary layer that includes the envelope (bright border) between mmt-nc and the outer surface of the virion is of variable thickness, as much as 17 nm, compared to the 6 nm envelope thickness reported by kenney et al. (1992) , suggesting that water is infiltrating the virion. virion #2, while not appreciably different in size than an undistorted virion, deviates significantly from a spherical form. the envelope of virion #2 at the platelet edge also appeared to have undergone disassembly. the thicker region between the envelope and mmt-nc at the upper part of #2 may have resulted primarily from distortion or water absorption between the envelope and nc. the mmt-nc in virion #2 departs dramatically from a spheroidal geometry. mixing detergent-isolated nc particles with mmt creates aggregates that encompass virtually all the nc particles. the morphology of the nc particles in nc-mmt aggregates (tem micrograph in fig. 6 .) appears similar to that of detergent-isolated nc particles. this is in distinct contrast to the mmt-nc particles produced by mmtinduced envelope disassembly. in the present case, the nc particles are roughly circular with a 57 nm diameter, that is, nearly identical in appearance to detergent-isolated nc particles. as the detergent-isolated nc particles in the aggregates retain their original morphology, the interaction with the clay is likely weaker than that between the mmt and the partially disassembled virions. this is consistent with the hydrophobic interactions reported for nonenveloped viruses puls 1999, 2000; chrysikopoulos and syngouna 2012) . it also suggests that triton x100, which is nonionic, is less likely to produce negatively charged ncs in contrast to envelope-stripped mmt-nc particles produced by interaction with mmt. sds-page analysis was employed to confirm the tem results that the φ6 and ncs were nearly completely sequestered in the mmt aggregates. an sds-page gel showing the larger molecular-weight φ6 proteins (p1, p2, p3, and p4) (gottlieb et al. 1988 ) of φ6 and ncs is presented in figure 7 . lane 1 is from purified φ6. ponents remain sequestered in the mmt; and (2) the viral proteins are not being hydrolyzed by the mmt but remain intact. lane 4 is from purified nc isolated by detergent. effectiveness of nc isolation is confirmed by the absence of the p3 envelope protein band. lane 5 is from the extracted pellet and lane 6 is from the supernatant of a mixture of mmt and detergent-isolated nc, also at an nc concentration equal to that used in lane 4. the comparable band intensities in lanes 5 and 6, in conjunction with the absence of nc protein bands in lane 6, demonstrate that the nc is also almost totally sequestered in the mmt aggregate without noticeable hydrolysis of viral proteins. these results are similar to those found for the whole-virus-mmt mixture. the absence of p3 in the nc lanes verifies that the nc preparation did not contain whole virus. plaque assays of the supernatant (assuming 100% infectivity) reveal that over the entire experimental series the fraction of φ6 that remained in the supernatant after centrifugation ranged from 10 à4 to 10 à6 . in other words, more than 99.99% of the virions are in the pellet. this is in agreement with the tem and sds-page analysis. figure 8 plots the reduction in φ6 infectivity relative to a virus control (no mmt) for a range of ratios of mmt to φ6. for the 0.2 lm mmt fraction used in these experiments, a mmt platelet has a mass of~10 fg, yielding about~10 11 platelets mg à1 . at virus concentrations exceeding the number of platelets, moderate loss of infectivity (20-95%) was observed. at virus concentrations lower than the number of platelets, infectivity is reduced by a factor of 10 à1.5 -10 à5 , where the least amount of loss of infectivity corresponded to smaller amounts of clay and the larger degree of loss corresponded to larger amounts of clay. the variation in infectivity loss at comparable platelet and virion populations (10 à9 -10 à11 mg of mmt per virion) may be due to aggregation dynamics affecting the probability of virion-clay contact. this loss of infectivity resulted from either the partial disassembly or complete removal of the envelope. since essentially all the φ6 aggregates with the mmt independently of virion concentration, the differences in infectivity under different relative concentration can be directly attributed to the probability of direct contact between virions and platelets. at relatively lower φ6 concentration, the probability that virions are in direct contact with platelets increases, resulting in greater envelope disassembly. however, at higher φ6 concentrations, many of the virions are more likely to only be in direct contact with other virions rather than platelets and thus less likely to experience envelope disassembly. interactions between colloidal clay and suspended viral particles result in the stripping of the viral envelope and a consequent loss of infectivity of the phage φ6. as viral particles become embedded in growing clay aggregates, virions continue to be scavenged from the aqueous medium. tem captures images of the φ6 virions in varying states of envelope disassembly. the extensive shape distortion exhibited by the edge-bound φ6 virions indicates that the forces are stronger than those binding the face-attached virions. this observation suggests that an electrostatic interaction is involved. the sandwiched virions are likely held preferentially by van der waals forces to the negatively charged platelet faces as prescribed by dlvo theory. both binding mechanisms contribute to the disassembly and swelling of virions and indicate that the envelope is pliable. the irreversible loss of infectivity through the process of envelope disassembly by mmt is a novel and previously unreported mechanism and is relevant to predicting the response of pathogenic enveloped viruses to clays in the environment. here a virus, there a virus, everywhere the same virus? adsorption of bacteriophages on clay minerals forces dictating colloidal interactions between viruses and soil differential adsorption of occluded and nonoccluded insect-pathogenic viruses to soil-forming minerals attachment of bacteriophages ms2 and fx174 onto kaolinite and montmorillonite: extended-dlvo interactions theory of the stability of strongly charged lyophobic sols and of the adhesion of strongly charged particles in solution of electrolytes production of a polyhedral particle in escherichia coli from a cdna copy of the large genomic segment of bacteriophage phi 6 deposition and aggregation kinetics of rotavirus in divalent cation solutions fate and transport of viruses in porous media influence of cations on aggregation rates in mg-montmorillonite bacteriophage phi 6 envelope elucidated by chemical cross-linking, immunodetection, and cryoelectron microscopy ecology of viruses in soils: past, present and future perspectives enhancement of bacteriophage φx-174 plaques by homoionic clay minerals adsorption of reovirus to clay minerals: effects of cation-exchange capacity, cation saturation, and surface area effect of proteins on reovirus adsorption to clay minerals specificity of virus adsorption to clay minerals effect of kaolinite on the specific infectivity of reovirus bacteriophage phi 6: a unique virus having a lipid-containing membrane and a genome composed of three dsrna segments packaging, replication and recombination of the segmented genome of bacteriophage f6 and its relatives x-ray diffraction and the identification and analysis of clay minerals the isoelectric points of some strains of tobacco mosaic virus elution and inactivation of bacteriophages on soil and cation-exchange resin modification of the interaction between escherichia coli and bacteriophage in saline sediment effect of clay particle size on clay-escherichia coli-bacteriophage interactions analysis of bacteriophage inactivation and its attenuation by adsorption onto colloidal particles by batch agitation techniques adsorption of coliphages t1 and t7 to clay minerals nih image to imagej: 25 years of image analysis electron microscopy of bacteriophage phi 6 nucleocapsid: two-dimensional image analysis interaction between viruses and clays in static and dynamic batch systems particle-associated viruses in water: impacts on disinfection processes deposition kinetics of ms2 bacteriophages on clay mineral surfaces theory of the stability of lyophobic colloids interaction between bacteriophage pbs1 and clay minerals and transduction of bacillus subtilis by clay-phage complexes clay minerals protect bacteriophage pbs1 of bacillus subtilis against inactivation and loss of transducing ability by uv radiation interactions of poliovirus with montmorillonite clay in phosphate-buffered saline poliovirus adsorption to narrow particle size fractions of sand and montmorillonite clay effects of ph, ionic strength, dissolved organic matter, and flow rate on the co-transport of ms2 bacteriophages with kaolinite in gravel aquifer media ecology of prokaryotic viruses are viruses driving microbial diversification and diversity? transduction of escherichia coli by bacteriophage p1 in soil interactions between viruses and goethite during saturated flow: effects of solution ph, carbonate, and phosphate the authors this work is supported in part by a city seed grant #93370-09 from the city college of new york; an nih research centers in minority institutions (nih/ncrr/ rcmi) ccny/grant g12-rr03060; psc-cuny award #64577-00 42; and a national institute of general medical science grant sc1-gm092781. the electron microscopy facilities at the new york structural biology center are supported by grant c000087 from the new york state foundation for science, technology and innovation (nystar) and nih grant s10 rr017291. none declared. key: cord-318495-1w74wf02 authors: vignuzzi, marco; lópez, carolina b. title: defective viral genomes are key drivers of the virus–host interaction date: 2019-06-03 journal: nat microbiol doi: 10.1038/s41564-019-0465-y sha: doc_id: 318495 cord_uid: 1w74wf02 viruses survive often harsh host environments, yet we know little about the strategies they utilize to adapt and subsist given their limited genomic resources. we are beginning to appreciate the surprising versatility of viral genomes and how replication-competent and -defective virus variants can provide means for adaptation, immune escape and virus perpetuation. this review summarizes current knowledge of the types of defective viral genomes generated during the replication of rna viruses and the functions that they carry out. we highlight the universality and diversity of defective viral genomes during infections and discuss their predicted role in maintaining a fit virus population, their impact on human and animal health, and their potential to be harnessed as antiviral tools. v iruses are remarkably resilient microorganisms able to adapt and survive in the complex host physiological and immune environment. accumulating evidence demonstrates that viruses can generate modified forms of their genome during replication as tools to adapt to environmental challenges. while the standard genome encodes all the viral proteins required for sustaining viral replication, viral variants contain random mutations that can serve to enhance the ability of the virus to adapt to new conditions 1 . in addition, defective viral genomes (dvgs) and an expanding family of sub-viral particles (box 1) that result from either small mutations or drastic truncations and modifications of the viral genome render the virus unable to complete a full replication cycle in the absence of a helper full-length virus to complement the functions lost. dvgs were identified by preben von magnus in the late 1940s as incomplete influenza viruses able to interfere with the replication of the wild-type virus 2 . more than two decades after their discovery, alice huang and david baltimore coined the term defective interfering (di) particles, or dips, to define viral particles that contain normal structural proteins but only a part of the viral genome. in addition, they stipulated that dips can only replicate in the presence of helper virus and that they interfere with the intracellular replication of non-defective homologous virus 3 . huang and baltimore theorized that dips play a critical role in determining viral pathogenesis. follow-up studies using viruses grown to contain either a large content of dips, or depleted of them, revealed that dips reduced virulence in vivo 4, 5 , induced high levels of interferon (ifn) during infections in vitro [6] [7] [8] , and promoted viral persistence in vitro [9] [10] [11] [12] [13] [14] [15] [16] and in vivo 17, 18 . however, despite their ubiquitous presence and important functions, the lack of appropriate technology to identify dips in infections in vivo led to a widespread belief that dips and their dvgs were largely a product of in vitro virus replication and were not relevant in natural viral infections 19, 20 . by the late 1990s, the study of dvgs had slowed down drastically and was limited to their use as tools for studying viral replication or as potential antivirals. today, dvgs have been described in most rna viruses (table 1 ) and technological advances have contributed to establishing their role as de facto danger signals for triggering of antiviral immunity in many infections. in addition, we are beginning to appreciate their impact on the clinical outcome of natural infections and on the evolution of viruses. moreover, we are witnessing rapid advancements in the understanding of the molecular mechanisms that regulate the generation of dvgs and those explaining their paradoxical roles in promoting antiviral immunity and viral persistence. here, we review evidence accumulated over more than half a century of observations on dvg generation and activity during rna virus infections. we highlight recent advances that illustrate their critical impact on viral dynamics and evolution during both acute and long-term virus-host interactions. see box 2 for a glossary of relevant terms. different types of dvgs are defined by the type of genomic alterations present. next generation sequencing (ngs) has revealed a large variety of dvg species present in some infections, and recent studies have begun to elucidate the distinct functions of these different dvgs. point mutations, hypermutations and frame shifts. while the first dvgs to be identified and distinguished from the wild-type fulllength viral genome lacked large parts of the genome [21] [22] [23] [24] [25] , there are a number of dvg types that do not involve drastic genomic alterations. point mutations in rna viruses can result in detrimental alterations because of the highly constrained nature of their genome organization. indeed, a majority of randomly introduced mutations are either lethal or confer a significant fitness cost, as observed for vesicular stomatitis virus (vsv) 26 , poliovirus 27 and influenza virus 28 . while it is inherently understood that detrimental mutations give rise to defective genomes, such genome types have historically not been considered dvgs. a genome harbouring a detrimental mutation in a structural protein could replicate, but not assemble properly; while a detrimental mutation in the replicase would yield a genome that can produce proper structural and assembly proteins, but could not replicate. either of these genomes could potentially hijack the lacking functions from (and thus, interfere with) a fulllength co-infecting virus. indeed, studies where mutation rates are increased to perturb a viral population from viability to non-viability, revealed that the defective rnas that appear prior to population marco vignuzzi 1 and carolina b. lópez 2 * viruses survive often harsh host environments, yet we know little about the strategies they utilize to adapt and subsist given their limited genomic resources. we are beginning to appreciate the surprising versatility of viral genomes and how replicationcompetent and -defective virus variants can provide means for adaptation, immune escape and virus perpetuation. this review summarizes current knowledge of the types of defective viral genomes generated during the replication of rna viruses and the functions that they carry out. we highlight the universality and diversity of defective viral genomes during infections and discuss their predicted role in maintaining a fit virus population, their impact on human and animal health, and their potential to be harnessed as antiviral tools. dvg diversity during infection. historically, it was thought that only certain viruses generated dvgs and that only one or a few distinct dvgs existed for any given virus. most studies originally relied on high multiplicity of infection (moi) passaging conditions that favoured the emergence of larger deletions (and thus, smaller dvgs) that could rapidly outcompete the longer full-length genomes owing to reduced replication times. furthermore, these works relied on methods with low sensitivity of detection (such as visualization and purification on agarose gels) that would only isolate the most abundant dvgs. however, evidence for populations of distinct species of dvgs in a single infection has been reported since the 1980s 45, 47 . we now know the diversity of dvgs to be much larger than initially appreciated and that certain dvgs are better than others at reaching high abundance, likely by retaining certain properties that confer replication advantage such as packaging signals and replication elements, or by acquiring the ability to interfere with the replication of other variants. ngs has revealed that dvgs are present in virtually any and every virus population, and that the diversity of dvgs is immense; however, the relative abundance of any given deletion or rearrangement varies, likely indicating that a variety of factors drive dvg generation and accumulation. the use of single-cell sequencing technology will allow precise quantification of the frequency and diversity of dvgs in an infected cell and will provide data on the distribution of dvg variants among a cell population. since most ngs alignment tools filter out reads with more than two mismatches, reads corresponding to deletion breakpoints or rearrangement junctions are rejected from a typical virus genome alignment. a re-analysis of rejected reads would identify these dvg hallmarks. with growing interest in this neglected part of the viral population, informatics tools such as virema 48 , di-tector 49 and vodka 50 are emerging. these tools re-examine reads that potentially harbour jumbled or rearranged viral sequences and have enabled a broader appreciation of dvg diversity. a current challenge with ngs data is to determine how best to differentiate between true dvgs and background error of ngs, how to quantify the relative abundance of any given dvg with respect to full-length in addition to dvgs, a growing number of viral particle variants and sub-viral agents have been discovered in viruses of plants, arthropods and mammals. these include viroids, satellite viruses, virophages and viral-like extracellular vesicles (vlvs). the definition of these entities seems, at times, blurry, due to their largely intersecting properties. in general, sub-viral agents, similar to dvgs, depend on complementation with the standard virus to replicate and spread. however, differences in their requirements for complementation, target virus and sequence identity with their helper virus are used to sub-classify them. here, we provide current definitions of these sub-viral entities and highlight those aspects that differentiate them from dips and their dvgs. satellite viruses were originally described in plant viruses as linear or circular rnas (200-1,800 nt long) that require a helper virus to propagate but are unrelated in sequence to the helper virus. satellite viruses are generally dispensable for the replication of the helper virus, with some exceptions 192, 193 . satellite viruses differ from satellite rnas in that they encode a protein that packages the satellite rna into virions. satellite rnas can interfere with the replication of its helper virus and either attenuate or exacerbate disease. an example of a satellite virus is the satellite tobacco necrosis virus 194 . this positive-sense, singlestranded rna satellite virus suppresses the replication of its helper virus and ameliorates tobacco necrosis virus symptoms 195 . viroids differ from satellite viruses in that they do not encode any protein, do not require helper virus for replication, and are not encapsidated. viroids have a circular rna genome (200-400 nt long) that is highly complementary and structured, and are adapted to carry out their complete life cycle as a result from interactions with the host cell machinery 196, 197 . virophages are 15-30 kbp long dsdna viruses that normally produce icosahedral particles and parasitize from giant dsdna viruses (mimiviruses and others) for propagation. virophages infect the giant virus factory, thereby harming the giant virus 198 . the first virophage discovered, sputnik, was found together with acanthamoeba castellanii mamavirus and interferes with its propagation 199 . several other virophages, as well as a large number of virophage candidates, have been identified since then 195, 198, 200 . recent studies show that virophages could resemble bona fide dna viruses 201 and their reclassification as their own viral family has been proposed 195 . vlvs are produced during viral infection and, in contrast to other extracellular vesicles, contain viral proteins and nucleic acids but lack capsid protein or viral genomes and, therefore, are not infectious. vlvs have been described in both rna and dna viruses, including herpes simplex virus-1, hepatitis c virus and kaposi's sarcoma-associated herpes virus. vlvs play functional roles during viral infections by facilitating communication among cells and enhancing viral infection [202] [203] [204] . continued virus, and how best to normalize between samples and between sequencing runs-problems that are similar to transcriptome analysis of genetic isoforms. furthermore, with increasing data sets and samples, it is becoming evident that dvg species are not necessarily the same if generated in different host and cell types, and the factors dictating these differences remain to be uncovered. dvgs have been long considered the result of stochastic mistakes introduced by the viral rna polymerase that lacks proofreading activity. however, new evidence suggests that additional factors control dvg generation, opening the possibility of manipulating their generation for therapeutic purposes. random products or encoded in the viral genome? while dvgs are generated by many viruses, the molecular mechanisms that govern their generation are poorly understood. a predominant theory is that dvgs arise from random errors that occur during viral replication at high viral titers due to the combination of a lack of proofreading activity of the viral polymerase and the presence of lower fidelity variants that favour the generation of deletions. in support, analyses using deep sequencing approaches revealed that multiple species of dvgs are generated during infection. for example, in infections with flock house virus, a positive-sense (ps)rna virus, clickseq and nanopore sequencing identified a large and seemingly random population of deletion dvgs early after infection 48 . in addition, sequencing analysis of nasopharyngeal samples from influenza virus-infected humans or infections in vitro with human metapneumovirus or measles virus (mev) revealed multiple dvg species in these infections [51] [52] [53] . however, different from deletion and point mutation dvgs, copy-back dvgs are frequently found in discrete dominant populations in an infected cell or tissue, and the same copy-back dvg seems to arise in independent infections with the same parental virus 54, 55 or during infections with different virus strains 56 . the demonstration of hotspots for the generation of copyback dvgs from respiratory syncytial virus (rsv) and the identification of specific nucleotides that determine where copy-back dvgs rejoin further demonstrate that the generation of copy-back dvgs is not completely random, but instead that specific sequences encoded in the viral genome direct or facilitate their formation 50 in some infections, dvg generation is not a completely stochastic process and, instead, virus-encoded sequences favour the production and/or amplification of predominant dvgs. it remains to be determined whether conservation is a property of certain dvg types and which specific sequences and/or rna structures lead to dvg generation in these conditions. a number of viral proteins are implicated in the generation of dvgs, the best studied being the viral rdrp. engineered viral polymerases with a decreased fidelity and an increased mutation rate produce highly attenuated viruses 60, 61 which, in many instances, correlate with enhanced production of dvgs 62-65 (fig. 2a) . although the mechanism mediating dvg generation by mutant rdrp remains speculative, a few models are emerging. for example, in viruses with low-fidelity rdrp, such as sindbis virus or tombusvirus, an increased production of dvgs correlates with an enhanced rate of viral rna recombination 63, 65 . in addition, during influenza virus infection, variations in the elongation capacity of the polymerase associate with differential generation of dvgs 64 . a role for the polymerase multimerization activity was also recently proposed as a driver of dvg generation during influenza virus infection 62 . viral proteins implicated in the regulation of viral transcription and replication are also associated with the generation of dvgs. mutations in the influenza virus nuclear export protein (nep, also known as ns2), which regulates the synthesis of complementary rna, result in enhanced dvg production 66 . similarly, deletion or mutations of the paramyxovirus c proteins that regulate template switching from antigenomic to genomic replication result in enhanced copy-back dvg production 53, 56 (fig. 2a) . curiously, in infections with influenza virus lacking nep or paramyxovirus lacking c, dvg production is observed in conditions that normally restrict the generation of dvgs, such as infections at low moi. it is possible that the enhanced production of copy-back dvgs in these situations is related to higher template availability or to an indirect effect of viral polymerase activity, but the precise mechanism remains to be established. in addition, a single amino acid mutation in the sendai virus (sev) nucleoprotein that reduced the density of the ribonucleprotein associates with dvg generation 67 (fig. 2b) . lastly, in lymphocytic choriomeningitis mammarenavirus (lcmv) infection, the ppxy domain encoded within the matrix protein drives the production of viral particles containing defective genomes, but not those containing standard genomes 68 (fig. 2c) . replication-driven template-switching. rna recombination is a major driver of deletion dvg formation. a predominant model proposes that sequences at the break point or structural signals in the template rna promote the replicase to switch to the acceptor rna and resume synthesis (fig. 2d) . replicase-driven recombination was proven in biochemical assays using rdrps of a large number of rna viruses [69] [70] [71] . variations of this model include the forced template switch mechanism in which the replicase switches template after encountering the 5′ end of the template generating headto-tail rna dimers. if the templates are dvgs, these would lead to head-to-tail dvg dimers. the 5′ end could also be modified by endo-or exo-nucleases, leading to new versions of these genomes 72 . rna editing as a driver of dvg diversity. editing of viral rna leads to hypermutations and can result in the generation of dvgs, as reported in persistent measles infections 31 . in addition, a high rate of viral adenine-to-guanine (or uracil-to-cytosine) rna editing by adenosine deaminase acting on rna occurs in dvgs from vsv, human metapneumovirus and mev 38, 52, 53 . in some cases, rna editing regulates the immunostimulatory potential of dvgs 53 . however, the impact of dvg editing on viral replication seems to be virus-specific 73 . whether dvgs have a higher rate of editing than the standard viral genome, as well as the impact of editing generated diversity in dvg evolution and selection, remains to be determined. dvgs have three well-described functions that relate to their role in pathogenesis: interference with standard viral replication, immunostimulation and establishment of viral persistence (fig. 3a) . interference with viral replication and viral production. dvgs were discovered during the search for factors responsible for reduced infectivity of influenza virus after passages at high titers 2 . dvgs with the ability to interfere with the replication of their parental virus both in vitro and in vivo are found in most positive and negative-sense (ns)rna viruses 54, [74] [75] [76] [77] [78] [79] [80] [81] [82] . dvgs accumulate at higher rates than full-length viral genomes in co-infected cells due to their shortened length and, in the case of copy-back species, their highly efficient flanking trailer promoters 83, 84 . a predominant theory for how dvgs interfere with the replication of standard virus is based on the observed competition between defective and full-length viral genomes for viral components needed for replication (fig. 3b) . as dvgs accumulate to high levels, it is predicted that they can directly interfere with helper virus replication by monopolizing the viral polymerase and/or competing for structural proteins 84, 85 . it should be noted that while most evidence supports the notion that the shortest dvgs with largest deletions are best able to outcompete full-length virus because their much smaller size can be more rapidly replicated, most of these studies consist of high moi cell culture conditions which favour competition dynamics based on replication kinetics. the question arises as to whether a longer deletion dvg that retains more coding sequence, yet contains mutated proteins (presumably replicating faster than full-length genomes, yet slower than the shortest dvgs), could be a better competitor of wild-type virus in certain conditions because it additionally expresses defective proteins that, in turn, interfere with wild-type proteins (for example, in multi-component structures such as capsids or replicases). furthermore, recent studies have demonstrated that dvgs and full-length viral genomes dominate in different cells during infection, dips. defective interfering particles. viral particles containing a fraction of the viral genome able to replicate only in the presence of helper virus and interfere with the intracellular replication of non-defective homologous virus. dvgs. defective viral genomes. viral genomes with defective ability to replicate in the absence of a co-infecting standard virus. viral genomes can become defective due to mutations, deletions or a variety of gene rearrangements. rdrp. rna-dependent rna polymerase. viral enzyme that copies the viral rna during viral replication. tips. therapeutic interfering particles. synthetic dvgs with strong interfering activity proposed as therapeutics to outcompete standard viruses. conferring distinct functions to different infected cells 86, 87 . while cells dominated with full-length viral genomes are the predominant producers of viral particles containing either full-length or defective genomes, cells enriched in dvgs do not produce many particles of any species 86 . these data highlight the need to consider the single cell versus population level impacts of dvgs during interference. triggers of antiviral immunity. dvgs, especially those of the copy-back type, strongly induce the expression of type i and iii ifns, tumour necrosis factor (tnf), interleukin (il)-6, il-1β and other pro-inflammatory cytokines, and are the primary stimuli of antiviral immunity in many infections [6] [7] [8] 52, 54, 55, [88] [89] [90] (fig. 3c) . in addition, dvg stimulation optimizes the antigen presentation capacity of antigen-presenting cells 91, 92 . accumulating evidence indicates that the immunostimulatory activity of dvgs is maintained in vivo and during natural infections in humans. increased survival of infected mice in infections containing dvgs have been reported for multiple viruses 5, 19, 93 . in mice infected with the respiratory viruses sev, influenza or rsv, ifns and pro-inflammatory cytokines are strongly induced only after dvgs have accumulated to detectable levels 54, 55 . detection of dvgs in respiratory secretions of children infected with rsv correlates with expression of antiviral genes 55 , and highly pathogenic influenza virus isolates that fail to induce potent antiviral responses in humans have an impaired ability to generate dvgs 62 . immunostimulatory dvgs can be recognized by pattern recognition receptors (prrs), including toll-like receptors (tlrs) and rig-i-like receptors (rlrs). while tlr signalling is not essential for production of type i ifns in vitro in response to dvgs, rlr signalling is required 55, 88, [94] [95] [96] [97] [98] . sev copy-back dvgs are stronger immunostimulators than deletion dvgs 89 and are among the strongest known inducers of the antiviral response. therefore, much of what we know of dvgs immunostimulatory activity is based on the study of sev copy-back dvgs. copy-back dvg rna binds rig-i 94,97,99 and dvgs from sev, mev and rsv viruses strongly stimulate rig-i-dependent signalling 55, 92, 95 . rig-i is triggered by binding of 5′ di-or triphosphates present on uncapped rna or short regions of double-stranded (ds)rna. phosphatase treatment suppresses the ability of in vitro-transcribed copy-back dvgs to trigger ifn production following transfection supporting the role of rig-i in dvg sensing 91, 94, 96 . sev dvgs can also associate with melanoma differentiation-associated protein 5 (mda5) 88, 94 , but the role for mda5 in sensing other dvgs is less clear 99, 100 . other cellular proteins can also associate with dvgs. for example, mev dvgs associate with the dsrna binding protein named protein activator of the interferon-induced protein kinase to optimally activate rig-i 96 . dvg-induced rlr signalling is not simply a result of higher viral rna content in the infected cells, as increasing the amount of dvg-deficient virus does not increase the ifn response 54, 88, 92 . importantly, efficient sensing of copy-back dvgs occurs even in the presence of virus-encoded antagonists of the cellular sensing pathways 88, 92 . these observations suggest that unique features of dvgs favour their detection during infection. the predicted long dsrna stretch formed by the reverse complementary ends of copyback dvgs was thought to be a critical factor in their immunostimulatory activity 8, 99, 101 . however, recent evidence demonstrates the existence of additional features in dvgs that have a larger impact on their immunostimulatory potential. structural modelling identified a 44 nucleotide (nt)-long stem-loop motif (dvg ) in a sev dvg-546, a well-characterized and potent immunostimulatory copy-back dvg, which was absent in the sev genome and spans the unique junction formed between the genomic break and rejoin points that form this copy-back dvg. deletion of dvg reduces the dvg immunostimulatory activity, while introduction of the motif into an immunologically inert rna improves its ability to induce the expression of type i ifns and ifn-stimulated genes 94 . dvg acts in concert with the 5′-triphosphate motif to activate rig-i and allows for enhanced rlr polymerization, a marker of activation 94 . dvg lose immunostimulatory potential 94 . failure to detect dsrna through immunostaining during sev infection 102 and normal immunostimulatory activity following disruption of the complementarity between the 5′ and 3′ ends of copy-back sev dvgs 94 further support the notion that the predicted long complementary ends of copy-back dvgs have a minor role in the onset of anti-viral immunity. it remains to be determined when and how dvg 70-114 is exposed during infection, and whether similar motifs are present in other highly immunostimulatory dvgs. dvg immunostimulation is also an important factor in the modulation of infections in insects. similar to the rig-i and mda5 prrs in mammals, insects sense viral rna through the protein dicer-2. this protein processes the viral rna into small interfering rnas that protect insects from re-infection with the same virus 103 . dvgs are the primary targets for dicer-2 (ref. 103 ). these observations indicate that the immunostimulatory activity of dvgs is widespread and may have a significant impact on the spread of pathogens within and across species. 18, 108 , as well as one study reporting dvgs in the brain of human patients who had died due to post-mev subacute sclerosing panencephalitis 17 . dips and full-length viruses cycle asynchronously in many persistent infections in vitro 109, 110 and in vivo 19, 109 . cycling occurs in a predictable pattern and has been mathematically modelled using variations of the predator-prey model 111 . a theory to explain the asynchronous cycling of full-length viral genomes and dvgs during persistency was put forward in 1970 (ref. 3 ). this theory, which is based on the interference effect of dvgs on the replication of the full-length genome, proposed the following: dips accumulate during virus replication until they reach high concentrations and become predominant. in this condition, dvgs interfere with the replication of the full-length viral genome by competing for essential replication machinery, driving a reduction in the full-length virus. during this process, some previously uninfected cells are infected by standard virus and re-initiate the cycle. interestingly, in some persistent infections the amount of dips appears constant 112 . what drives these cyclic patterns in some viruses but not others, and whether host factors such as the infected cell type influences the cycling pattern, remain unknown. recent evidence indicates that the mechanisms involved in the establishment of persistence are more complex than simple intracellular competition for the replication machinery among different types of viral genomes 87 . using rna in situ fluorescent hybridization, xu et al. showed that during infection with sev or rsv containing dips, there is heterogeneity in the content of viral genomes in the infected population 87 . while some cells are enriched in dvgs, others are enriched in standard full-length viral genomes. the mechanisms for this heterogeneity are currently unknown, however, striking functional differences among these cell populations are beginning to emerge 86, 87 . cells enriched in dvgs engage the rlr sensing pathway and produce ifns and other pro-inflammatory molecules, including tnf. in addition, these cells induce a pro-survival program, also dependent on signalling through the rlr pathway. these programs protect dvg-high cells from tnfmediated death, while cells lacking dvgs die during infection. surviving dvg-high cells can be propagated for months as a persistent infection. this mechanism provides an explanation for the paradoxical stimulation of both antiviral immunity and establishment of persistence by dvgs. it remains unclear how the enhanced survival of dvg-high cells leads to persistence, and how this survival fits into the cycling of dvg and standard virus observed in many infections. cycling may occur at the intracellular level (where each infected cell goes through cycles of standard viral genome or dvg enrichment driven by competition and interference with the viral replication machinery) and/or at the population level, where the strong interfering and immunostimulatory activities of dvgs make them attractive candidates for vaccine adjuvants and antivirals 113, 114 . the ability of dips to interfere with the replication of standard viruses and diminish virus-associated disease has been extensively demonstrated in mice during infection with virus stocks containing dips 4, 54, 75, 77, 115 , even when the vaccine contained standard virus inactivated by ultraviolet treatment 116 . a similar protection has been reported in ferrets vaccinated with an influenza vaccine containing only dips 117 . synthetically engineered dips with strong interfering potential, or 'therapeutic interfering particles (tips)' , have been more recently proposed as a possible strategy to control viral infections. tips would theoretically replicate faster than the wild-type virus and therefore outcompete the virus hindering spread and transmission. tips would have the advantage of being active only in organisms already infected with the wild-type virus due to their dependence on a helper virus to replicate 118, 119 . although still in the exploratory phase, it remains to be determined how tips would impact virus persistence, the generation of adaptive mutations and the generation of new infectious viruses by complementation. whether protection elicited by natural or synthetic dips is due to direct interference with the standard virus replication or through strong immunostimulatory ability of dvgs is unclear. sev copyback dvgs augment the antigen presentation capacity of mouse and human dendritic cells, resulting in enhanced activation of t cells 91 . in addition, experimental vaccines against influenza virus and rsv adjuvanted with in vitro-transcribed sev dvgs delivered subcutaneously, intramuscularly or intranasally show improved antibody production and increased protection from virus challenge 91, 120 . sev dvg-derived oligonucleotides (ddos) containing the immunostimulatory motif dvg are effective adjuvants able to bias the humoral and cellular responses against inactivated virus and protein vaccines towards type i immune responses including antibodies of the igg2a/c isotypes, th1 cd4 + t cells and cytotoxic cd8 + t cells in mice 91, 121 . ddos also synergize with the emulsion antibody addavax and enhance its type i immunity-driving potential by mechanisms that depends on type i ifns 121 . notably, a dip influenza vaccine conferred protection to an unrelated virus through stimulation of type i ifn production 122 , suggesting that they can be used as prophylactic or therapeutic antivirals. dvgs are present in live attenuated vaccines against polio, measles and influenza viruses 76, [123] [124] [125] [126] . however, their impact in the development of protective immunity and vaccine efficacy has not been formally assessed. based on their interfering and immunostimulatory ability, it is speculated that dvgs may enhance the efficiency of the vaccine while enhancing safety of the virus by reducing its replication and spread. if correct, it would be important to carefully regulate the amount of dvgs in vaccine preparations to avoid complete interference and drastic reduction of the virus to a point of ineffectiveness. while recent ngs data reveal that hundreds of dvgs can arise within a single viral infection, the fact that a smaller subset of dominant dvgs are repeatedly detected in different samples 50 indicates that complex dynamics are at play within the viral population. these complexities include competition (and possibly compensation or cooperation) between different dvgs and positive selection of the best competitors that implicates their relative fitness in relation to the wild-type parental virus and other dvgs. parameters such as replication fitness, packaging, immunoregulation and other traits determine the virus dynamics. it is important to stress that most events leading to dvg formation, including mutations, deletions, recombination and translocations, are either non-viable or deleterious to the virus. in addition, although hundreds or even thousands of different dvgs are generated during a virus infection, the vast majority of these will be lost during the population bottlenecks that occur in vivo, for example when crossing anatomical barriers or during transmission from host to host 127 . however, there are instances where these genomes could make it through bottlenecks, such as during infections of hosts that are immunosuppressed or have comorbidities where founding populations are increased. in addition, infections may occur with virions that have co-packaged wild-type genomes and dvgs 128 or virions may aggregate during infection enhancing co-transmission, as seen for vsv 129 mathematical models have generally only considered one dominant dvg 131, 132 ; further development is required to incorporate the potential cooperation and competition among others. while historically dvgs have been considered replication waste, an artifact of cell culture passaging conditions or simply a nuisance to laboratory experimentation, a renewed interest in this part of the viral population may reveal a more functional or biological relevance to their existence. do dvgs exist for a reason? why keep waste lying around? given the notoriously high recombination and reassortment rates of some viruses, it is possible that for viruses that undergo recombination, dvgs can provide a repertoire of mutations that could feed back into the viable virus population to promote adaptation. it remains to be seen whether dvgs can provide a selective advantage to the virus, and whether the high moi or localized co-infection conditions are biologically relevant outside of cell culture. recent work in insects suggests that dvgs, which are a preferred template for the generation of the viral dna form of rna viruses, provide additional substrate to help boost the rna interference response that is responsible for viral persistence in insects 103 . indeed, it was shown that a change in the amount of viral dna generated during rna virus infection in drosophila, altered the persistence and kinetics of a wild-type rna virus infection. the authors suggested that evolution has perhaps fine-tuned the production of dvgs to balance wild-type infection and promote persistence (and ultimately, transmission of viruses, including arboviruses in mosquitoes). additionally, a closer examination of dvg dynamics within viral populations can help better understand the biology of standard viruses. indeed, in addition to distinguishing between the dispensable and indispensable nucleotide sequences, proteins and rna structures, we can identify what viral components can operate in cis or in trans. a recent study of hcv dvgs, for example, identified novel cis-acting rna structures that were required for replication and packaging 133 . emerging technologies that allow the identification of defective and standard viruses in natural infections, as well as those allowing the establishment of specific associations of dvgs with functional outcomes, have been crucial in reviving the interest in studying dvgs. recent work has provided new appreciation for dvg diversity and the potentially critical role of dvgs in defining the clinical outcome of infections; however, a number of questions remain unanswered: what are the molecular mechanisms driving the generation of dvgs? can dvgs be harnessed for the control of virus pathogenesis and spread? how do alterations in the dvg population impact virus evolution and adaptation to new hosts? how do host factors impact dvg accumulation and activity? further technological developments and interdisciplinary research will be required to obtain these answers. virus population dynamics during infection studies on interference in experimental influenza defective viral particles and viral disease processes modulation of semliki forest virusinduced infection 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viruses virus-associated small satellite rnas and viroids display similarities in their replication strategies what has been happening with viroids? the expanding family of virophages the virophage as a unique parasite of the giant mimivirus ecogenomics of virophages and their giant virus hosts assessed through time series metagenomics capsid protein structure, self-assembly, and processing reveal morphogenesis of the marine virophage mavirus virus-like vesicles of kaposi's sarcoma-associated herpesvirus activate lytic replication by triggering differentiation signaling ultrastructural and biophysical characterization of hepatitis c virus particles produced in cell culture extracellular vesicles released by herpes simplex virus 1 infected cells block virus replication in recipient cells in a sting-dependent manner this work was supported by the us national institutes of health national institute of allergy and infectious diseases (grants nos. nih ai083284, ai137062 and ai134862 to c.b.l.) and the darpa intercept program (to m.v.) managed by j. gimlett and administered though darpa cooperative agreement (grant no. hr0011-17-2-0023). in addition, this work was supported by a fulbright us scholar award to c.b.l. the views expressed in this article do not necessarily represent the position or the policy of the us government the authors declare no competing interests. reprints and permissions information is available at www.nature.com/reprints.correspondence should be addressed to c.b.l.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-312223-qgwzgazd authors: shafagati, nazly; narayanan, aarthi; baer, alan; fite, katherine; pinkham, chelsea; bailey, charles; kashanchi, fatah; lepene, benjamin; kehn-hall, kylene title: the use of nanotrap particles as a sample enrichment method to enhance the detection of rift valley fever virus date: 2013-07-04 journal: plos negl trop dis doi: 10.1371/journal.pntd.0002296 sha: doc_id: 312223 cord_uid: qgwzgazd background: rift valley fever virus (rvfv) is a zoonotic virus that is not only an emerging pathogen but is also considered a biodefense pathogen due to the threat it may cause to public health and national security. the current state of diagnosis has led to misdiagnosis early on in infection. here we describe the use of a novel sample preparation technology, nanotrap particles, to enhance the detection of rvfv. previous studies demonstrated that nanotrap particles lead to both 100 percent capture of protein analytes as well as an improvement of more than 100-fold in sensitivity compared to existing methods. here we extend these findings by demonstrating the capture and enrichment of viruses. results: screening of nanotrap particles indicated that one particle, nt53, was the most efficient at rvfv capture as demonstrated by both qrt-pcr and plaque assays. importantly, nt53 capture of rvfv resulted in greater than 100-fold enrichment from low viral titers when other diagnostics assays may produce false negatives. nt53 was also capable of capturing and enhancing rvfv detection from serum samples. rvfv that was inactivated through either detergent or heat treatment was still found bound to nt53, indicating the ability to use nanotrap particles for viral capture prior to transport to a bsl-2 environment. furthermore, both np-40-lysed virus and purified rvfv rna were bound by nt53. importantly, nt53 protected viral rna from rnase a degradation, which was not observed with other commercially available beads. incubation of rvfv samples with nt53 also resulted in increased viral stability as demonstrated through preservation of infectivity at elevated temperatures. finally, nanotrap particles were capable of capturing veev and hiv, demonstrating the broad applicability of nanotrap particles for viral diagnostics. conclusion: this study demonstrates nanotrap particles are capable of capturing, enriching, and protecting rvfv virions. furthermore, the use of nanotrap particles can be extended to a variety of viruses, including veev and hiv. rift valley fever virus (rvfv) belongs to the genus phlebovirus and family bunyaviridae. rvfv is composed of a tripartite singlestranded rna genome with large (l), medium (m), and small (s) segments [1, 2, 3, 4] . rvfv particles have icosahedral symmetry and are 90-110 nm in diameter [4] . the envelope is made up of a lipid bilayer that is embedded with the gn and gc glycoproteins. these glycoproteins, which are the most exposed components of the virus during infection, play a crucial role in the entry of the virus into the host cell. rvfv is a highly pathogenic arthropod-borne virus that is primarily transmitted by mosquitoes, particularly after heavy rainfall. although it can infect a wide range of vertebrate hosts, rvfv primarily affects livestock and humans [2] . animals are infected through mosquito bites and other arthropod vectors. humans are typically affected when they come in close contact with infected bodily fluids or tissues, but transmission via mosquito bites, as well as aerosolization may also occur. however, humans are dead-end hosts [1, 5] . since being first identified in 1930 in the rift valley of kenya, outbreaks have led to high mortality rates as well as significant economic loss [3] . rvfv has remained endemic in sub-saharan africa, causing major outbreaks throughout the continent over the last century [6] . in 1976, 200,000 individuals were infected and 600 fatal cases were reported in egypt [5] . most likely due to international livestock trade, it has since crossed the arabian peninsula into saudi arabia and yemen. over 30 mosquito species, mostly aedes and culex are vectors for rvfv [5] . of particular concern is that the aedes species is widely distributed in the eu countries and many of those countries (turkey, greece, italy, spain, portugal, and france) have high-risk vector habitat areas that may serve as emergent sites. moreover, in the unites states, this species has been found in 23 states [7] . since rvfv is capable of utilizing a wide range of mosquito vectors, the virus has the potential to spread further into non-endemic areas [5, 8] . mortality rates are dependent on species and age. in livestock, mortality rates are as high as 30%. mortality rates can reach as high as 95% in newborns and the young, while abortion rates are as high as 100% [5] . symptoms in humans are usually mild and include febrile illness resembling the flu, with a small percentage developing serious clinical manifestations such as retinal lesions, meningoencephalitis, hepatitis, severe hemorrhagic fever, coma and death. in recent years an increase in mortality amongst humans from 2% to 45% has been reported, suggesting evolving mechanisms of virulence and mutations [3] . due to its transmission via aerosolization, high pathogenicity, and classification as a group iii (bioterrorism potential) category a emerging infectious disease by the niaid, work with rvfv requires bsl-3 containment. it is highly suggested that laboratory staff working with rvfv be vaccinated. therefore, diagnosis of rvfv is restricted to a small number of laboratories. this limitation has led to some delay in diagnostics associated with virus isolation and identification techniques that may pose a problem for healthcare authorities in the event of an rvfv epidemic. there is a crucial need for rapid detection and identification of the virus [3, 5] . nanotrap particles are a novel technology that can address all the critical analytical challenges for pathogen identification and measurement. they are homogenous hydrogel particles of about 800 nanometers in size that have a shell made of polymers of nisopropylacrylamide (nipam) and co-monomers such as acrylic acid (aac) and allylamine (aa) with cross links of n,n9methylenebisacrylamide (bis). this shell can be modified to alter permeability or porosity by increasing or decreasing the percentage of bis [9, 10] . charge-based affinity baits are incorporated into the nanotrap particles by copolymerization and covalent binding to the shell [10] . the nanotrap particles are temperature-and ph-sensitive, decreasing in size with increased temperature and low ph. the molecular sieving properties of the particles depend on several aspects. the degree of cross-linking within the particles provides inclusion and exclusion of high abundance large molecules (e.g. albumin). affinity baits further facilitate the capture and concentration of the target protein, and prevent it from exiting the particle. they may be of negative or positive charge, therefore attracting analytes of opposite charge. this was seen in an early experiment performed by luchini et al. where the incubation of particles containing anionic affinity baits captured myoglobin, a protein with a positive charge [9] . some nanotrap particles are composed of nipam shells, and a few of these shelled nanotrap particles are also coated with vinyl sulfonic acid (vsa) [11] . nanotrap particles are able to perform three functions in one step: molecular size sieving, target analyte affinity sequestration, and complete protection of captured analytes from degradation. furthermore, nanotrap particles help to bridge the gap between detection and the limits of sensitivity. mass spectrometry (specifically liquid chromatography coupled with tandem mass spectrometry) is a favored technique for the discovery of candidate biomarkers in biological fluids. however, this technique only accepts a small input volume and complex solutions often lead to decreased sensitivity. the nanotrap particles concentrate protein analytes in small volumes to effectively amplify the sensitivity of mass spectrometry. in addition, their promiscuity allows for multiple analytes to be harvested from a single sample [9] . experiments conducted by luchini et al. demonstrated the capture and enrichment of small molecules spiked in complex solutions such as whole blood and serum [9, 10] . a 2011 study by douglas et al. on the detection of lyme disease demonstrated that nanotrap particles can improve sensitivity more than 100-fold (over existing methods) as well as lead to 100 percent capture and 100 percent elution yield of low abundance antigens in biofluids. lyme disease antigens at low abundance were detected in both urine samples as well as from a single infected tick [12] . the current library of commercially available nanotrap particles has been designed to specifically harvest proteins, peptides, metabolites and small molecules. we hypothesized that nanotrap particles would be able to capture whole virus through the interaction of the nanotrap particle with the positively charged residues on the surface of rvfv. our study demonstrates that nanotrap particles are capable of capturing whole virus, and can be assayed with both qrt-pcr and plaque assays. importantly, serial dilution studies and studies in serum indicate that nanotrap particles increase detection sensitivity at lower viral titers. furthermore, the virus can be inactivated with either heat or detergent, while the virus captured can still be detected with qrt-pcr. importantly, the nanotrap particles protect purified viral rna as well as stabilize the infectivity of rvfv. the studies described here expand upon the nanotrap particles repertoire to characterize the capture of viruses. the nipam/aa nanotrap particles were provided by ceres nanoscience, manassas, va. the vero cell line (kidney epithelial cells) was grown in dulbecco's modified eagle medium (dmem) supplemented with 10% fbs, 1% penicillin/streptomycin, and 1% glutamax there is a dire need for fast and efficient diagnosis of many viral diseases. our research specifically looked at rvfv, a virus that can only be worked with in biosafety level 3 (bsl-3) laboratories, and its capture with nanotrap particles. nanotrap particles are hydrogel particles that contain internal affinity baits. they have previously been used in the capture of several analytes, but never in the capture of whole virus particles. we were not only able to capture and detect rvfv at very low titers from both media and serum, but we were also able to inactivate the virus, which allows for its safe transport to bsl-2 laboratories. while there are other commercially available beads that can also capture virus, nanotrap particles are the only beads that can protect the viral rna from enzymatic degradation. furthermore, we demonstrated that whole virus detection with nanotrap particles is not limited to only rvfv, but that nanotrap particles can be used to detect other viruses such as human immunodeficiency virus (hiv) and venezuelan equine encephalitis virus (veev). (dmem+++). the j1.1 cell line, which are jurkat e6.1 suspension cells chronically infected with the la1 strain of hiv-1, were grown in medium containing advanced rpmi-1640, 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% l-glutamine. all cell lines were cultured in a humidified environment containing 5% co 2 at 37uc. the experiments used a live attenuated vaccine derived from the rvfv zh548 strain, known as mp-12, which had been isolated in 1977 from a patient with uncomplicated rvfv. the virus was generated by 12 serial passages in mrc5 cells, inducing 25 nucleotide changes across the viral genome [13] . both rvfv zh548 and mp12 strains were anonymized. mp12 was propagated by infecting vero cells at 80-90% confluency at an moi of 0.1 in dmem+++. cell culture medium was collected from the cells when ,75% cytopathic effect was observed (typically 72 hours post-infection (hpi)). cell culture medium was centrifuged at 10,000 rpm for 10 minutes to pellet the cellular debris. cell free-viral supernatants were then filtered using a 0.22 mm filter and viral titer determined by plaque assays. screening experiments for venezuelan equine encephalitis virus (veev) used the live attenuated vaccine tc-83, which had been derived from the trinidad donkey (trd) strain by 83 serial passages in fetal guinea pig hearts. this induced changes at 12 nucleotide positions across the viral genome [14] . the viral supernatant of chronically infected j1.1 cells was used in the hiv-1 screening experiments. the lav strain of hiv-1 had previously been used to infected jurkat e6 cells at a multiplicity of infection of 0.1 to 0.01 for 2 hours at 27uc and cultured for two weeks. the cells that survived the cytopathic effects of virus infection were cloned and the supernatant from growth-positive wells were screened for reverse transcriptase (rt) activity [15] . the j1.1 cells express viral rna and proteins at low levels. six commercially available beads -deae-sephadex (sigma-aldrich), dynabeads m-280 streptavidin (invitrogen), sephacryl s-200 beads (ge healthcare), biorex 70 resin (bio-rad laboratories), sp sephadex c-25 (ge healthcare), and bio-gel htp hydroxyapatite (bio-rad laboratories) were used to compare their capture to nt53. each bead was washed four times with water and a 33% percent slurry with water was prepared. according to a protocol standardized by ceres nanoscience, 100 microliters (ml) of sample was incubated with 75 ml of nanotrap particles for 30 minutes at room temperature. the sample was centrifuged at 10,000 rpm for 5 minutes and the supernatant was discarded. the pellet was washed with 100 ml of rnase-and dnase-free water four times. the pellet was then resuspended in the appropriate buffer. for lysis of mp12 with np-40, 1% np-40 was added to 100 ml of mp12 and incubated at room temperature for 30 minutes. a standard nanotrap particle incubation was performed followed by a qrt-pcr assay. for the rnase treatment, purified mp12 rna was treated with rnase a and incubated for one hour at 37uc. vero cells were plated in 6 well plates at 1.0e+06 cells/ml in order to achieve 100% confluency. after nanotrap particle incubation and subsequent washes, the pellet was resuspended in 100 ml of supplemented dmem and serial dilutions performed. four hundred ml of the serial dilution was added to each well in duplicate and incubated for 1 hour. three hundred milliliters (ml) of a primary overlay known as the cv mixture containing equal parts 0.6% agarose in distilled water and media containing 2x emem, 5% fbs, 1% minimum essential amino acids, 1% sodium pyruvate, and 1% glutamax was added directly to each well. the cells were fixed with 10% formaldehyde in water after 72 hpi. the cells were stained with 1% crystal violet in 20% ethanol and water. after two hours, the crystal violet stain was washed off and the plaques formed were counted to determine the plaque forming units per milliliter (pfu/ml). after nanotrap particle incubation and subsequent washes, the pellet was resuspended in 180 ml of lysis/binding solution (life technologies) containing guanidinium thiocyanate and incubated on ice for thirty minutes. the samples were spun at 13,000 rpm for 5 minutes at room temperature. the supernatant was transferred to a 96-well plate and rna extraction was performed with ambion's magmax 96-well viral rna extraction kit according to manufacturer's instructions. in order to determine the number of viral genomic copies produced, qrt-pcr with viral specific primers was performed using rna ultrasense onestep quantitative rt-pcr system (life technologies). the experiment was performed according to a standardized protocol using fifteen ml of master mix containing enzyme mix, 5x reaction mix, 50 mm magnesium sulfate (excluded for veev qrt pcr), rox reference dye, 10 mm taqman fluorogenic probe, 10 mm forward primer (aaaggaacaatg-gactctggtca), and 10 um reverse primer (cacttct-tactaccatgtcctccaat) added to five ml of extracted rna. the samples were heated at 50uc for 15 minutes, 95uc for 2 minutes, and at 95uc and 60uc for 40 cycles. rvfv was spiked into 100% bovine, sheep, and donkey serum (purchased from innovative research) at 1.0e+05 pfu/ml. one ml of spiked serum was used in a standard nanotrap particle incubation with nt53. after a standard nanotrap incubation was performed with nt53 and rvfv, the samples were incubated at room temperature with 0.1, 0.5, or 1% np-40 for 1 hour or heated at 57uc for 0.5, 1, or 2 hours. the samples were then analyzed by plaque assays and qrt pcr. a standard nanotrap particle incubation with one ml of hiv-1 supernatant from infected j1.1 cells and nanotrap particles was performed. an rna extraction was performed (as described above). a master mix was then prepared with the following components for a 25 ml reaction: superscript iii rt/platinum taq mix -0.5 ml, 2x reaction mix with rox -12.5 ml, and 0.5 ml of forward and reverse primer mix (ltr forward primer cgagcttgctacaagggact and ltr reverse primer gagattttccacactgactaaaag) at 10 mm. five ml of sample, 6.5 ml of water, and 13.5 ml of master mix were aliquoted out into pcr tubes. the pcr was conducted with the following cycles: 15 min at 50uc (cdna synthesis), 2 min at 95uc (prime reaction), and 35 cycles at 30 seconds at 95uc (denature), 30 seconds at 51uc (annealing), 30 seconds at 72uc (extend), 72uc for 10 min, and a hold at 4uc. a dna gel was prepared using 1% agarose powder in 1x tae buffer with the addition of ethidium bromide for a final concentration of 0.5 mg/ml. the gel was visualized on an ultraviolet transilluminator and the volume of each band was quantified. nanotrap particles can capture whole virus nanotrap particles have previously been shown to capture proteins. we hypothesized that the rvfv glycoproteins would be figure 1 . rvfv capture by nanotrap particles. a) seven different types of nanotrap particles were incubated with viral supernatants containing rvfv (1e+7 pfu/ml) for 30 minutes at room temperature and washed 4 times with water. viral rna was extracted from the particles with ambion's magmax viral rna extraction kit and quantitated by qrt-pcr assays. b) percent detected virus was calculated compared to a sample processed without nanotrap particle incubation. c) viral supernatants were incubated with nt46, nt53, and nt69 for 30 minutes at room temperature and washed 4 times with water. serial dilutions followed by plaque assays were performed to determine if full virus was bound by the particles. doi:10.1371/journal.pntd.0002296.g001 capable of interacting with the nanotrap particles, facilitating capture in a fashion similar to the way in which protein biomarkers interacted with the nanotrap particles. to test this hypothesis, seven different nanotrap particles were tested with rvfv. four nanotrap particles possessed shells (nt53, nt55, nt69, and nt71) whereas three did not (nt45, nt46, and nt75) ( table 1) . we incubated culture supernatants from rvfvinfected veros with each nanotrap particle. all seven nanotrap particles successfully captured virus, averaging 6.8e+07 genomic copies per reaction. specifically, nt46, nt53, and nt69 captured higher genomic copies than the other nanotrap particles, with each capturing approximately 1.0e+08 genomic copies per reaction ( figure 1a ). this corresponds to 78-83% capture ( figure 1b) of a sample containing a high titer of virus. in order to determine if the amplification observed in the qrt-pcr assay was due to the nanotrap particle capturing intact viral particles or association of viral rna (presumably due to lysed virus) with the particles, plaque assays were performed. if the particles captured viral rna or lysed virus, no plaques should be observed. plaque assays were performed on the three best candidates from the qrt-pcr screening. captured viruses were not eluted off of the nanotrap particles, but rather the samples were diluted and added directly to the vero cells during the plaque assay procedure. we hypothesized that the viral glycoproteins would have a greater affinity for the cellular receptor than the nanotrap particles and thus would enter the cells. nt53, which contains a cibacron blue bait with a shell, captured infectious rvfv virion six-and four-fold more than nt46 or nt69, respectively ( figure 1c ). these plaques were not due to cell death induced by the nanotrap particles themselves, as nanotrap particles alone did not produce plaques. therefore nt53 was chosen for all future experiments with rvfv. experiments were performed to determine potential elution methods that would release the virus without affecting the viral particle integrity. it had previously been found that sodium chloride (nacl) concentrations between 0.5m and 2m could effectively elute various analytes from cibacron blue dyes by disrupting electrostatic interactions between cibacron blue dyes (the bait molecule found within nt53; [16] ). we hypothesized that incubating the rvfv-bound nanotrap particles on ice would allow the particles to swell and, with the aid of vortexing, the virus would disassociate from the nanotrap particles. therefore, we tested a nacl based elution method coupled with an ice-swelling method. plaque assays were performed to determine the amount of virus eluted from the nanotrap particles and the amount that remained bound to the particles after a high salt elution. after nt53 incubation with rvfv, the pellets were resuspended in 2.0 m nacl in dmem and placed on ice for 30 minutes with vortexing every ten minutes. both the eluates and pellets were analyzed by plaque assay ( figure s1 ). the results showed that 5.5% of rvfv was detected after elution with 2.0m nacl. the addition of nacl coupled with incubation on ice only slightly released rvfv virions, demonstrating the virus' strong affinity for the nanotrap particles. however, as seen in figure 1b , rvfv-bound nanotrap pellets directly added to vero cells during the plaque assays procedure were capable of producing plaques. based on these results, we opted not to elute rvfv from the nanotrap particles, but rather to add the rvfv bound to the nanotrap particles directly during the plaque assay procedure. we next wanted to determine the limit of detection of rvfv in plaque assays with nt53. nt53 was incubated with rvfv at decreasing titers, from 2.5e+6 to 2.5e+1 pfu/ml, and plaque assays performed (figure 2a) . captured virus was detected down to 2.5e+1 pfu/ml for rvfv. these results show that nanotrap particles are capable of capturing whole virus even at low viral titers. we next determined the percentage of rvfv captured by nt53 in comparison to the total input amount. rvfv at 1.0e+6 and 1.0e+3 pfu/ml were added to nt53. at 1.0e+6 pfu/ml, 99.35% of the virus was bound to the nanotrap particles whereas at 1.0e+03 pfu/ml, ,100% of the virus appeared bound to the nanotrap particles ( figure 2b ). the results confirm that the nanotrap particles are efficient at capturing rvfv, especially at a lower titer. interestingly, the results also suggest that a small volume of rvfv can be captured with nanotrap particles and then recultured to grow more virus. rvfv detection is more sensitive with nanotrap particle incubation in clinical instances of infection, the viral titers in circulation during very early stages after exposure are expected to be low and therefore, hard to detect [17] . we wanted to determine if viral enrichment by the nanotrap particles (nt53) would enhance detection of rvfv when compared to detection in the absence of enrichment afforded by the nanotrap. we specifically wanted to see the enrichment potential at lower viral titers when detection would be most difficult. for these assays, we chose to utilize qrt-pcr based detection due to its increased sensitivity over plaque assays. to this end, we spiked rvfv into cell culture media that contained 10% fbs at various concentrations from 1.0e+5 to figure 2 . characterization of rvfv nanotrap particle capture. a) viral supernatants were serially diluted (2.5e+6 to 2.5e+1 pfu/ml) and incubated with nt53 for 30 minutes at room temperature. the pellets were washed 4 times with water and then particles were tested in plaque assays to determine if full virus was bound by the particles. b) viral supernatants at 1.0e+6 and 1.0e+3 pfu/ml were incubated with nt53 for 30 minutes at room temperature. the sample was spun at 10,000 rpm for 5 minutes and the unbound viral supernatant was saved separately. nt53 was washed 4 times with water and then particles were tested in plaque assays to determine how much virus were bound verses unbound by the particles. the percentage of bound virus at 1.0e+6 and 1.0e+3 pfu/ml was graphed. doi:10.1371/journal.pntd.0002296.g002 1.0e+1 pfu/ml. nt53 was then added to 1 ml of the spiked media. viral capture with and without nanotrap particles gave similar yields at higher viral titers ( figure 3a) . however, at lower viral titers, there was a significant increase in viral capture with the use of the nanotrap particles compared to samples without nanotrap particle capture. there was greater than a 100-fold increase of viral detection with the use of nt53 at 1.0e+1 pfu/ml. we next wanted to determine if we could capture and enrich virus from a clinically relevant matrix. rvfv was spiked into 100% bovine, donkey, and sheep sera at 1.0e+05 pfu/ml. incubation of rvfv spiked sera with nt53 resulted in enrichment by 13-, 3-, and 52-fold for bovine, sheep, and donkey sera, respectively ( figure 3b ). these results demonstrate that nt53 not only captures but also enriches virus found in complicated matrices such as animal sera. the complex analytes (e.g. albumin) found in the sera are likely excluded by the nanotrap particles and do not interfere with whole virus capture. however, we speculate that since the serum from each of the three animals contains different analytes, there may be interfering proteins that would lead to the observed enrichment differences. nanotrap particles are capable of capturing inactivated rvfv viral inactivation is crucial for its transport from the field or a bsl-3 facility to a bsl-2 environment for downstream analysis. however, after inactivation the virus may be susceptible to degradation. therefore, we wanted to determine if rvfv would remain bound to the nanotrap particles in an inactivation scenario. after nanotrap particle incubation with rvfv and subsequent washes, np-40 detergent was used to inactivate the virus (figure 4 ). plaque assays were performed to confirm viral inactivation. plaque assays demonstrated that 0.1% np-40 did not fully inactivate the virus incubated with or without nt53 ( figure 4a ). higher concentrations of np-40 (0.5% and 1%) fully inactivated rvfv in the presence or absence of nt53. while the plaque assays confirmed inactivity of rvfv, qrt-pcr data demonstrated that rvfv was still captured following np-40 addition ( figures 4b). in the presence of np-40 the levels of capture with nt53 were decreased as compared to the controls. this is likely due to the interference of the nanotrap particle binding to rvfv due to the presence of detergent. we next tested the ability of nt53 to function in another commonly employed viral inactivation procedure. the samples were heat inactivated at 57uc for three different time pointsthirty minutes, one hour, and two hours -and plaque assays were performed to confirm viral inactivation. at thirty minutes approximately 1.0e+4 and 1.0e+2 pfu/ml of rvfv with and without nt53, respectively, were still detectable. interestingly, rvfv was more resistant to heat inactivation in the presence of nt53, suggesting the nanotrap particles may have a slight protective effect on the virus. however, complete inactivation was achieved at one hour ( figure 4c ). while the plaque assays confirmed inactivation of rvfv, qrt-pcr data demonstrated the ability of nt53 to detect rvfv nucleic acids after heat inactivation ( figure 4d ). these data demonstrate that it is possible to fully inactivate the virus before applying downstream assays such as qrt-pcr. furthermore, these two experiments demonstrate the ability to inactivate a sample and transport it as a noninfectious sample, while still retaining capture. nanotrap particles are capable of capturing and protecting viral rna as we observed viral capture in the presence of np-40, we hypothesized that the virus was being lysed and the released viral rna recaptured with the nanotrap particles. if the nanotrap particles were not providing protection of the viral rna, there will be no possibility for any downstream assays using inactivated material, which is a critical step in diagnostics. to test this hypothesis, we first lysed the virus with 1% np-40 and followed by adding nt53 to the lysed material. results in figure 5a indicate that nt53 was able to capture the lysed virus. however, there was a 100-fold decrease in lysed virus with the addition of nt53 compared to the control (no nt53) with and without np-40. these results mirrored what was observed in figure 4b , where nt53 was capable of capturing virus to a lesser extent in the presence of np-40. these results demonstrated that rvfv could be initially inactivated by traditional inactivation methods and then captured with nanotrap particles. by incubating with the nanotrap particles, the viral rna will be protected and hence, can be used for downstream rna detection. in order to directly show that nt53 was able to capture and protect viral rna, we performed a nanotrap experiment with purified viral rna. we first incubated nanotrap particles with purified rna, and performed qrt-pcr assays. results in figure 3 . rvfv enrichment with nt53 incubation. a) rvfv was spiked into cell culture media (dmem+++) at various concentrations from 1.0e+5 to 1.0e+1 pfu/ml. nt53 was added to 1 ml of media and captured according to standardize protocols. the pellet was washed 4 times with water, followed by processing with ambion's magmax 96well viral rna extraction kit. rvfv-spiked media without nt53 were processed in parallel. viral rna was quantitated by qrt-pcr with viral specific primers. b) rvfv was spiked into 100% bovine, sheep, and donkey serum at 1.0e+5 pfu/ml. nt53 were added to 1 ml of serum and captured according to a standardize protocol. the pellet was washed 4 times, followed by processing with ambion's magmax 96-well viral rna extraction kit. serum without nt53 was processed in parallel. viral rna was quantitated by qrt-pcr. doi:10.1371/journal.pntd.0002296.g003 figure 5b demonstrate that nt53 is capable of capturing purified viral rna, albeit with less affinity than whole virus capture. nt53 was able to capture 0.01% of the input viral rna. while we screened the nanotrap particles for whole virus capture, we did not screen the nanotrap particles for rvfv viral rna capture. there is likely a nanotrap particle that captures viral rna with greater efficiency than nt53. as previous studies have shown that proteins captured by nanotrap particles were protected from trypsin degradation, we next aimed to determine if nanotrap particle capture could protect viral rna from rnase degradation [9, 10] . samples with and without nt53 were treated with rnase a at 140 or 1400 units/ml and incubated for one hour at 37uc. interestingly, the rna incubated with nt53 was protected from rnase a degradation, whereas the rna controls were subject to complete rnase a degradation ( figure 5b ). at 140 units/ml of rnase a, the captured rna was detected at the same level as the rnaseuntreated sample. even at a substantially higher rnase concentration (1400 units/ml), 1% of the viral rna input was still detected. our results demonstrate that the nanotrap particles are capable of capturing and protecting viral rna from enzymatic degradation. in some situations, it may be important to retain the infectivity of the captured virus to enable the virus to be propagated for further characterization. therefore, we evaluated the ability of the nanotrap particles to capture and preserve the infectivity of rvfv following capture. rvfv was spiked into bovine serum and incubated with or without nt53 at 25uc for 48 or 72 h. in the absence of nt53, the infectivity of rvfv was decreased by ,3 logs ( figure 5c ). in contrast, samples incubated with nt53 displayed only ,1.5 log decrease by 48 h. although ,3.5 log decrease was observed with nt53 at 72 h, this still resulted in increased virus detected as compared to the control samples due to the enrichment afforded by nt53. the infectivity of rvfv was also assessed for samples that were incubated at 37uc, which would likely result in a more rapid decline in viral infectivity and thus 24, 48, and 72 h time points were examined. as suspected, a ,4 log decrease was observed in samples incubated at 37uc for 24 h without nt53. in contrast, samples captured by nt53 only displayed ,2 log decrease as compared to the control nt53 sample. at extended time points a further decrease in infectivity was observed with and without nt53, but in all cases a higher amount of infectious virus could be rescued from samples . viral inactivation following nt53 capture. panels a and b: nt53 was incubated with viral supernatants containing rvfv at 2.7e+8 pfu/ml for 30 minutes at room temperature. samples were then incubated at room temperature with 0.1, 0.5 or 1% np-40 for 1 hour (np-40). control samples were not treated with np-40 nor captured with nt53. samples treated with np-40 without nt53 were processed in parallel (black bars). viral inactivation was assayed by plaque assays (a) and viral rna was extracted from the particles with ambion's magmax 96-well viral rna extraction kit and quantitated by qrt-pcr (b). panels c and d: nt53 was incubated with viral supernatants containing rvfv at 2.7e+8 pfu/ml for 30 minutes at room temperature. samples were then incubated at 57uc for 30 minutes, one hour, or two hours. control samples were not heat treated nor captured with nt53. samples heat-treated without nt53 were processed in parallel (black bars). viral inactivation was assayed by plaque assays (c) and viral rna was extracted from the particles with ambion's magmax 96-well viral rna extraction kit and quantitated by qrt-pcr (d comparison of capture efficacy of nanotrap particles and commercially available beads for rvfv capture nanotrap particles have unique properties not demonstrated in other beads that are used for protein purification and albumin exclusion such as dye baits that make them an ideal candidate in virus capture. therefore, we wanted to directly compare the ability of other beads to capture rvfv with nt53's rvfv capture capability. the capture of rvfv was tested with nt53 and six commercially available beads used in various assays. deae-sephadex beads are used in ion exchange chromatography for purifying and isolating proteins; dynabeads m-280 streptavidin are used for isolating nucleic acids and antibodies; sephacryl s-200 beads are used to purify protein and macromolecules; biorex 70 resin beads are used for purification and fractionation of peptides, proteins, and other cationic molecules; sp sephadex c-25 beads are used in chromatography to separate and purify protein, polypeptides, and other charged molecules; and bio-gel htp hydroxyapatite beads are used in chromatography to separate and purify proteins, nucleic acids, viruses, and other macromolecules. these calcium phosphate beads work by the cationic interaction of the ca 2+ functional groups with the carboxylate residues located on the protein surface and the anionic interaction of the po 4 22 functional groups with the basic protein residues. rvfv was incubated with each of these beads and plaque assays were performed to determine whole virus capture. bio-gel htp hydroxyapatite (htp) captured rvfv the most efficiently, averaging 1.0e+7 pfu/ml, while nt53 performed the second best averaging 2.5e+6 pfu/ml ( figure 6a ). the other four beads captured rvfv around or below 1.0e+5 pfu/ml. as we have demonstrated that nt53 not only captures intact rvfv, but can also capture and protect viral rna from rnase a degradation, we tested the ability of htp to act in a similar capacity. nt53 was able to fully protect the viral rna against rnase a degradation and genomic copies for nt53 with and without nt53 treatment were similar. however, htp beads were figure 5 . nt53 protects viral rna from degradation and preserves viral infectivity. a) rvfv was lysed with 1% np-40 for 30 minutes at room temperature and then incubated with nt53 for 30 minutes at room temperature. the viral rna was extracted from the particles with ambion's magmax 96-well viral rna extraction kit and quantitated by qrt-pcr. samples without nt53 and samples without np-40 were processed in parallel. b) nt53 was incubated with purified rna at 2.0e+8 genomic copies for 30 minutes at room temperature. following water washes, the samples were resuspended in water and treated with 140 or 1400 units/ml of rnase a. samples with no rnase a treatment were processed in parallel. the viral rna was extracted from the particles with ambion's magmax 96-well viral rna extraction kit and quantitated by qrt-pcr. samples without nt53 were processed in parallel (black bars). c) rvfv (1.4+e7 pfu/ml) was spiked into 1 ml of bovine serum, nt53 added, and samples incubated at 25uc (for 48 or 72 hr) or 37uc (for 24, 48, or 72 h). samples without nt53 were processed in parallel. samples were assayed for viral infectivity by plaque assay. control samples are samples that were processed immediately for plaque assays with no incubation period. doi:10.1371/journal.pntd.0002296.g005 unable to provide protection against rnase degradation, and no viral rna was detected ( figure 6b) . a control experiment with rna alone demonstrated that our rnase treatment was effective. we next compared the ability of nt53 and htp to capture rvfv during an inactivation scenario. for these experiments nt53 or htp were added to the samples followed by viral inactivation through treatment with 1% np-40 or heating at 57uc. the amount of virus captured was quantitated by qrt-pcr ( figure 6c ). as was observed in previous experiments, viral inactivation with either np-40 or heat treatment resulted in some loss of rvfv binding to the nanotrap (1.2 and 0.8 log, respectively). however, htp rvfv capture was more dramatically affected, resulting in a 2.5 log decrease with the np-40 treated samples and a 2.7 log decrease in the heat inactivated samples. collectively, our experiments demonstrate that while htp is capable of capturing whole virus, it cannot protect viral rna against rnase degradation and it displays a reduced ability to capture rvfv during an inactivation scenario. in contrast, nt53 is capable of capturing and protecting rvfv as well as capturing rvfv in samples that have been inactivated by heat or detergent treatment. we next asked the question if nanotrap particles were capable of capturing other viruses. for this, we selected veev and hiv-1. veev, which at approximately 70 nm in diameter is a smaller virus than rvfv, which is approximately 100 nm in diameter. veev viral supernatants were incubated with various nanotrap particles shown in table 1 and capture was measured by qrt-pcr. our data indicated that all six nanotrap particles successfully captured veev, averaging 9.9e+06 genomic copies per reaction ( figure 7a) , with a slight preference observed with nt45, nt46, and nt55 capturing 1.3e+07, 1.1e+07, and 1.1e+07 genomic copies per reaction, respectively. nanotrap particles capture was also tested using hiv-1. hiv-1 supernatants from infected j1.1 cells were incubated with nanotrap particles. rna extraction was performed, cdna was synthesized, and rt-pcr was performed. a semi-quantitative analysis shown in figure 7b , demonstrated that all seven nanotrap particles were able to capture hiv-1 with nt46 and nt53 demonstrating the best capture ( figure 7b ). these results indicate that nanotrap particles are capable of capturing multiple viruses. (7) for 30 minutes at room temperature. the sample was washed 4 times with water and then particles were tested in plaque assays to determine how much virus was bound by the particles. b) nt53 or htp was incubated with purified rna at 1.0e+7 genomic copies for 30 minutes at room temperature. following water washes, the samples were resuspended in water and treated with 380 units/ml of rnase a. samples with no rnase a treatment were processed in parallel. the viral rna was extracted from isolated particles and quantitated by qrt-pcr (black bars). samples without nt53 were processed in parallel (gray bars). c) nt53 and htp beads were incubated with viral supernatants containing rvfv at 1.7e+8 pfu/ml for 30 minutes at room temperature. samples were then inactivated by incubation at 57uc for one hour or incubating in the presence of 1% np-40 at room temperature for 1 hour. a ''no bead'' control processed in parallel was included for each condition. viral rna was extracted from the particles and quantitated by qrt-pcr. doi:10.1371/journal.pntd.0002296.g006 viral infections in nature do not occur in isolation and are often accompanied by other co-infections (bacterial and/or viral); therefore we sought to determine if the nanotrap particles could capture rvfv in a ''mixed'' infection setting. to this end, bovine serum was spiked with rvfv only or with both rvfv and hiv, followed by nt53 viral capture and quantification as measured by qrt-pcr. results indicated that nt53 was capable of capturing and enriching rvfv from samples that contained only rvfv or both rvfv and hiv ( figure 7c ). seven-fold enrichment was observed in samples containing rvfv only and 5-fold enrichment from samples containing both rvfv and hiv. these data provide evidence that the nanotrap particles could be used with clinical samples. therefore, in conclusion, the results demonstrate that nano-trap particles can capture and enrich rvfv from both cell culture media and clinically relevant matrices. the captured virus can then be inactivated and viral rna protected from enzymatic degradation. the bound rvfv can be eluted off the nanotrap particles, and used in downstream assays such as plaque assays and qrt-pcr. furthermore, nanotrap capture can be extended to other viruses as well, including veev and hiv. rift valley fever virus is a zoonotic virus that primarily affects livestock but has the potential to cause severe disease in humans. rvfv has led to outbreaks in egypt and the arabian peninsula with the potential to spread to the united states and europe. changes in climate, travel, and trade have made rvfv an emerging disease that can have deadly economic and social consequences. furthermore, rvfv is of biodefense interest due to its potential spread via aerosolization. there are currently no fda-approved vaccines, so there is a reliance on sensitive and specific diagnostics early on in infection. the current state of rvfv diagnostics includes virus isolation, nucleic acid techniques, and antibody detection. current rt-pcr-based assays require a critical amount of the virus circulating in the system. this can lead to misdiagnosis, especially falsenegative results, of the disease early on in infection. in contrast, our results demonstrate the ability of nanotrap particles to enrich for rvfv from both cell culture supernatants as well as more complex matrices such as animal serum. the capability of nanotrap particles to enrich virus is crucial early on in infection during which the virus can go undetected using other diagnostic methods. in our serum sample studies we noted different levels of enrichment depending on the source of the serum. for example, nanotrap particles incubated in donkey serum resulted in a 52fold increase in rvfv detection sensitivity, whereas incubation in sheep serum only displayed a 3-fold increase. the presence of other analytes found in serum may be competing for capture with nanotrap particles, which likely will differ between species as well figure 7 . capture of other viruses with nanotrap particles. a) six different types of nanotrap particles were incubated with viral supernatants containing veev for 30 minutes at room temperature and washed 4 times with water. viral rna was extracted and quantitated by qrt-pcr assays. b) seven different types of nanotrap particles were incubated with 1 ml j1.1 supernatant for 30 minutes at room temperature and washed 4 times with water. the pellets were diluted in 100 ul water and rna extracted. a cdna synthesis using 150 ng of each sample was performed and followed by pcr using 10 ul cdna. a dna gel was run to determine viral capture. a sample with no reverse transcriptase added and a sample with just water were used as negative controls. the volume of each band was quantified and graphed. c) bovine serum was spiked with rvfv (1.0e+6 pfu/ml) only or both rvfv (1.0e+6 pfu/ml) and hiv (100 ml of j.1. supernatants). samples were incubated with nt53 for 30 minutes, viral rna extracted and quantitated by qrt-pcr. samples without nt53 were processed in parallel (black bars). doi:10.1371/journal.pntd.0002296.g007 as between individual animals. importantly we have also demonstrated the ability to capture rvfv in samples that also contained hiv. this is an important area of investigation, as clinical samples will likely contain multiple pathogens, providing further competition for nanotrap binding. we hope to extend our spiked serum sample studies to experiments with serum samples taken from animals exposed to rvfv and human clinical samples. these studies will allow further optimization of the nanotrap particle collection. in our current study we used very stringent wash conditions to ensure that the virus captured was tightly bound to the nanotrap particles. in clinical samples, it may be necessary to decrease the number of wash steps to ensure that the maximum amount of virus is being captured from more complex samples. alternatively, different wash buffers (altering salt and detergent concentrations) could be utilized to allow more selective binding of analytes. due to the complex nature of clinical samples, it may also be necessary to increase the amount of nanotrap particles added to prevent saturation. nonetheless, our studies provide an important first step in the application of nanotrap particles as a sample preparation and enrichment process to improve diagnostics from serum samples. one important advantage of utilizing nanotrap particles is their ability to protect analytes from degradation. previous studies have indicated that protein captured by nanotrap particles are protected from trypsin degradation [9, 10] . in these experiments pdgf was incubated with an excess of trypsin. following incubation the majority of the trypsin was found outside of the nanotrap particles. however, even though some of the trypsin entered the particles, pdgf was completely protected from degradation. in the current study we extend these findings to demonstrate that viral rna was protected from degradation in the presence of rnase a. sample preservation is critical for stabilization of sample integrity both during field collection and during transported to diagnostic facilities. bio-gel htp hydroxyapitite, while able to capture rvfv was unable to protect viral rna from degradation, further demonstrating the advantage of using nanotrap particles over other commercially available chromatography beads. another critical aspect of the nanotrap particles is the ability to collect viral samples and inactivate them to render them noninfectious, while still retaining the ability to detect the analyte of interest. this was demonstrated by captured of rvfv by nt53 followed by inactivation of rvfv with np-40 (determined by plaque assays). following inactivation, viral genetic material was still detected with qrt pcr and to a higher level than that observed with htp beads. this is of particular importance in the transport of rvfv from a bsl-3 environment or field sample collection setting to a bsl-2 laboratory for diagnostic testing. given that bsl-3 laboratories are both difficult to access and work in a bsl-3 environment is time-consuming and expensive, the inactivation method will allow for fewer lapses in time between obtaining the samples and the determining the results. in addition, as the nanotrap particles allow the capture of the whole virus, the samples can then be analyzed with a variety of downstream analysis methods such as elisa for the nucleoprotein of rvfv, western blotting, plaque assays, and qrt-pcr. the exact mechanism of nanotrap binding to rvfv is unclear at this point. we hypothesize the nanotrap particle capture is occurring through interactions with rvfv's glycoproteins, gn and gc. gn and gc are the only viral proteins available for capture by virtue of being exposed on the outside of the virion. the fact that the binding observed with htp beads slightly exceeded nt53 in binding rvfv may provide insight into the mechanism of binding. htp is known to bind primarily through electrostatic interactions and similarly, cibacron blue, the affinity bait component of nt53 binds through electrostatic, hydrophobic or a combination of surface and electrostatic interactions. based on these results, we expect that electrostatic interactions may provide the dominant mode of nt53 binding to rvfv. due to the size of the virus (90-100 nm) as compared to the size of the nanotrap particles (800 nm), it is unclear if the viruses are entering inside the core of the nanotrap particle or binding to the outside of the nanotrap particles. we have observed preferentially binding of rvfv with nanotrap particles containing cibracon blue baits, suggesting that the bait plays at least a partial role in the binding. even if rvfv binding is partially or primarily found on the surface of the nanotrap particles, the particles provide a unique advantage over other commercially available beads, which is sequestration of analytes within the nanotrap particles. this is important as many enzymes (proteases, rnase,etc.) found in serum can rapidly digest protein and rna. however, the nanotrap particles can bind to small molecular weight proteins (such as trypsin) rendering them inactive [9] , thereby providing protection for other proteins and/or viruses captured by the nanotrap particles. nanotrap particles can be engineered with increased pore sizes to facilitate capture of rvfv inside the nanotrap particles. this approach has the added advantage of ensuring capture within the particles themselves, which would be predicted to further increase the stability of the virus as well as increase the viral binding capacity of the nanotrap particles. one potential disadvantage of larger pores sizes would be the loss of some of the sieve sieving capabilities of the particles. the size sieving is important for more complex samples (whole blood, sera, etc.), which would benefit from the ability of the nanotrap particles to enrich for certain analytes (i.e. viruses) while excluding high abundant proteins such as bsa, which may interfere with downstream assays or mask lower abundant molecules such as low levels of viruses. however, it may be possible to obtain a balance of larger pores with efficient size sieving if the appropriate level of cross-linking could be achieved. this is an active area of research and warrants further investigation. our results demonstrated that nanotrap particle capture was not limited to rvfv, but could be extended to other viruses including veev and hiv. all three of these viruses are enveloped rna viruses. future studies will focus on the capture and enrichment of different viral classes, including dna viruses and non-enveloped viruses. that capture of a wide range of viruses is especially important when multiple viruses cause the same type of disease and/or the symptoms of infection are very general. for example, respiratory infections can be attributed to multiple pathogens, including influenza a and b viruses, coronaviruses, and adenoviruses. for this type of application, the promiscuity of the nanotrap particles is particularly important, as it will allow the capture and enrichment of multiple viruses from the same sample. therefore, we believe that increasing sensitivity for respiratory viral infections is an important diagnostic issue that the nanotrap particles could address. in conclusion, our results have demonstrated that nanotrap particles are able to capture and enrich whole virus. while other commercially available beads can also capture virus, only nano-trap particles are capable of protecting the integrity of the virus after inactivation with detergent or exposure to rnase a. however, further research is needed to determine the exact mechanism by which the nanotrap particles capture and protect the virus. figure s1 elution with nacl as demonstrated by plaque assays. rvfv supernatants were incubated with nt53 for 30 minutes at room temperature. the sample was spun at 10,000 rpm for 5 minutes. nt53 was washed 4 times with water and then particles were eluted with dmem+2m nacl and incubated on ice for 30 minutes with vortexing every 10 minutes. the particles were tested in plaque assays to determine how much virus was eluted off of the particles. (tif) rift valley fever in rural northern senegal: human risk factors and potential vectors nonstructural nss protein of rift valley fever virus interacts with pericentromeric dna sequences of the host cell, inducing chromosome cohesion and segregation defects rift valley fever virus(bunyaviridae: phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention molecular biology of rift valley fever virus an inhibition enzyme-linked immunosorbent assay for the detection of antibody to rift valley fever virus in humans, domestic and wild ruminants rift valley fever virus infection in african buffalo (syncerus caffer) herds in rural south africa: evidence of interepidemic transmission identification and geographical distribution of the mosquitos of north america, north of mexico the pathogenesis of rift valley fever smart hydrogel particles: biomarker harvesting: one-step affinity purification, size exclusion, and protection against degradation nanoparticle technology: addressing the fundamental roadblocks to protein biomarker discovery multifunctional core-shell nanoparticles: discovery of previously invisible biomarkers the use of hydrogel microparticles to sequester and concentrate bacterial antigens in a urine test for lyme disease mapping of the mutations present in the genome of the rift valley fever virus attenuated mp12 strain and their putative role in attenuation attenuation of venezuelan equine encephalitis virus strain tc-83 is encoded by the 59-noncoding region and the e2 envelope glycoprotein an hiv-1-infected t cell clone defective in il-2 production and ca2+ mobilization after cd3 stimulation human serum albumin chromatography by cibacron blue f3ga-derived microporous polyamide hollow-fiber affinity membranes core-shell hydrogel particles harvest, concentrate and preserve labile low abundance biomarkers the authors thank dr. sina bavari (usamriid) for providing rvfv mp-12. the authors thank dr. cynthia de la fuente for critical reading of the manuscript. the following reagent was obtained through the nih biodefense and emerging infections research resources repository, niaid, nih: venezuelan equine encephalitis virus tc-83 (subtype ia/b), nr-63. conceived and designed the experiments: an fk cb bl kkh. performed the experiments: ns ab cp kf. analyzed the data: ns an kkh. contributed reagents/materials/analysis tools: bl. wrote the paper: ns an kkh. key: cord-336948-8yqdhcnz authors: löhner, rainald; antil, harbir; idelsohn, sergio; oñate, eugenio title: detailed simulation of viral propagation in the built environment date: 2020-08-05 journal: comput mech doi: 10.1007/s00466-020-01881-7 sha: doc_id: 336948 cord_uid: 8yqdhcnz a summary is given of the mechanical characteristics of virus contaminants and the transmission via droplets and aerosols. the ordinary and partial differential equations describing the physics of these processes with high fidelity are presented, as well as appropriate numerical schemes to solve them. several examples taken from recent evaluations of the built environment are shown, as well as the optimal placement of sensors. starting in wuhan, china, in the fall of 2019, the covid-19 pandemic has claimed and will continue to claim millions of infected patients and hundreds of thousands of deaths. the lockdowns that followed its outbreak have led to mass unemployment, stalled economic development and loss of productivity that will take years to recover. some changes in habits and lifestyles may be permanent: in the future, working from home or in a 'socially distanced manner' may be the prevalent modus operandi for large segments of society. the present paper gives a short description of computational techniques that can elucidate the flow and propagation of viruses or other contaminants in built environments in order to mitigate [31] or avoid [47] before addressing the requirements for the numerical simulation of virus propagation a brief description of virus propagation and lifetime are given. covid-19 is one of many corona-viruses. the virus is usually present in the air or some surface, and makes its way into the body either via inhalation (nose, mouth), ingestion (mouth) or attachment (eyes, hands, clothes). in many cases the victim inadvertedly touches an infected surface or viruses are deposited on its hands, and then the hands touch either the nose, the eyes or the mouth, thus allowing the virus to enter the body. an open question of great importance for all that will follow is how many viruses it takes to overwhelm the body's natural defense mechanism and trigger an infection. this number, which is sometimes called the viral load or the infectious dose will depend on numerous factors, among them the state of immune defenses of the individual, the timing of viral entry (all at once, piece by piece), and the amount of hair and mucous in the nasal vessels. in principle, a single organism in a favourable environment may replicate sufficiently to cause disease [86] . data from research performed on biological warfare agents [33] suggests that both bacteria and viruses can produce disease with as few as 1-100 organisms (e.g. brucellosis 10-100, q fever 1-10, tularaemia 10-50, smallpox 10-100, viral haemorrhagic fevers 1-10 organisms, tuberculosis 1). compare these numbers and consider that as many as 3,000 organisms can be produced by talking for 5 min or a single cough, with sneezing producing many more [48, 72, 78, 90, 95] . figure 1 , reproduced from [90] , shows a typical number and size distribution. (90) coughs [72] 3 virus lifetime outside the body current evidence points to lifetimes outside the body that can range from 1-2 h in air to several days on particular surfaces [45, 94] . there has also been some documentation of lifetime variation depending on humidity. in the sequel, we consider human sneezing and coughing as the main conduits of virus transmission. clearly, breathing and talking will lead to the exhalation of air, and, consequently the exhalation of viruses for infected victims [2] [3] [4] . however, it stands to reason that the size and amount of particles released-and hence the amount of viruses in them-is much higher and much more concentrated when sneezing or coughing [3, 4, 32, 44, 49, 90] . the velocity of air at a person's mouth during sneezing and coughing has been a source of heated debate, particularly in the media. the experimental evidence points to exit velocities of the order of 2-14 m/s [25, [36] [37] [38] [39] [87] [88] [89] . a typical amount and size of particles can be seen in fig. 1. if, for the sake of argument, we consider stoke's law for the drag of spherical particles, valid below reynolds numbers of re = 1, the terminal sink velocity (also known as the settling velocity) of particles will be given by [26] : where ρ p , ρ g , g, μ, d denote the density of the particles (essentially water in the present case), density of the gas (air), gravity, dynamic viscosity of the gas and diameter of the particle respectively. the equivalent reynolds' number is: with ρ p = 1 gr/cc, ρ g = 0.0012 gr/cc, g = 981 cm/s 2 , μ = 1.81 · 10 −4 gr/(cm s) this yields a limiting diameter for i.e. approximately 1/10th of a millimeter in diameter-a particle size that would still be perceived by the human eye. the corresponding sink velocity is given by: with d in cm, i.e. for re = 1 v s (re = 1) = 18 cm/s. note the quadratic dependency of the sink velocity with diameter. table 1 lists the sink velocities for water droplets of different diameters in air. one can see that below diameters of o(0.1 mm) the sink velocity is very low, implying that these particles remain in and move with the air for considerable time (and possibly distances). depending on the relative humidity and the temperature of the ambient air, the smaller particles can evaporate in milliseconds. however, as the mucous and saliva evaporate, they build a gel-like structure that surrounds the virus, allowing it to survive. this implies that extremely small particles with possible viruses will remain infectious for extended periods of times-up to an hour according to some studies. an important question is then whether a particle/droplet will first reach the ground or evaporate. figure 2 , taken from [96] [96], shows that below 120 µm the particles evaporate before falling 2 m (i.e. reaching the ground). a central question that requires an answer is then: how many viruses are in these small particles? an approximate answer may be obtained from the experiments that are being carried out on animals to trace and monitor infections. for ferrets [46] o(10 5 − 10 6 ) have been used to infect via intranasal swabs, while for mice [92] o(10 4 ) seem to suffice. viral titers can vary a lot, but one may assume on the order of o(10 6 ) viruses/ml for a nasopharyngeal swab [46, 92] . table 2 lists the number of viruses per droplet and the number of droplets needed to contain just 1 virus. note that while for a droplet with a diameter of 1 mm one can expect o(500) viruses, only every 2000th particle of diameter 10 μm does contain a single virus. similar numbers are seen in field studies as well. the size of viruses varies from 0.02-0.3 µm, while the size of bacteria varies from 0.5-10 µm. the influenza virus rna detected by quantitative polymerase chain reaction in human exhaled breath suggests that it may be contained in fine particles generated during tidal breathing and not only coughs [32, 48, 49, 78] . influenza rna and mycobacterium tuberculosis have been reported in particles that range in size from 0.5-4.0 µm ( [32, 48, 49, 78] and references cited therein). when solving the two-phase equations, the air, as a continuum, is best represented by a set of partial differential equations (the navier-stokes equations) that are numerically solved on a mesh. thus, the gas characteristics are calculated at the mesh points within the flowfield. however, as the droplets/particles may be relatively sparse in the flowfield, they can be modeled by either: (a) a continuum description, i.e. in the same manner as the fluid flow, or (b) a particle (or lagrangian) description, where individual particles (or groups of particles) are monitored and tracked in the flow. although the continuum (so-called multi-fluid) method has been used for some applications, the inherent assumptions of the continuum approach lead to several disadvantages which may be countered with a lagrangian treatment for dilute flows. the continuum assumption cannot robustly account for local differences in particle characteristics, particularly if the particles are polydispersed. in addition, the only boundary conditions that can be considered in a straightforward manner are slipping and sticking, whereas reflection boundary conditions, such as specular and diffuse reflection, may be additionally considered with a lagrangian approach. numerical diffusion of the particle density is eliminated by employing lagrangian particles due to their pointwise spatial accuracy. while a lagrangian approach offers many potential advantages, this method also creates problems that need to be addressed. for instance, large numbers of particles may cause a lagrangian analysis to be memory intensive. this problem is circumvented by treating parcels of particles, i.e. doing the detailed analysis for one particle and then applying the effect to many. in addition, continuous mapping and remapping of particles to their respective elements may increase computational requirements, particularly for unstructured grids. as seen from the experimental evidence, the velocities of air encountered during coughing and sneezing never exceed a mach-number of ma = 0.1. therefore, the air may be assumed as a newtonian, incompressible liquid, where buoyancy effects are modeled via the boussinesq approximation. the equations describing the conservation of momentum, mass and energy for incompressible, newtonian flows may be written as 3) here ρ, v, p, μ, g, β, t , t 0 , c p , k denote the density, velocity vector, pressure, viscosity, gravity vector, coefficient of thermal expansion, temperature, reference temperature, specific heat coefficient and conductivity respectively, and s v , s e momentum and energy source terms (e.g. due to particles or external forces/heat sources). for turbulent flows both the viscosity and the conductivity are obtained either from additional equations or directly via a large eddy simulation (les) assumption through monotonicity induced les (miles) [18, 34, 35, 40, 42, 98] . in order to describe the interaction of particles/droplets with the flow, the mass, forces and energy/work exchanged between the flowfield and the particles must be defined. as before, we denote for fluid (air) by ρ, p, t , k, v i , μ and c p the density, pressure, temperature, conductivity, velocity in direction x i , viscosity, and the specific heat at constant pressure. for the particles, we denote by ρ p , t p , v pi , d, c pp and q the density, temperature, velocity in direction x i , equivalent diameter, and heat transferred per unit volume. in what follows, we will refer to droplet and particles, collectively as particles. making the classical assumptions that the particles may be represented by an equivalent sphere of diameter d, the drag forces d acting on the particles will be due to the difference of fluid and particle velocity: the drag coefficient c d is obtained empirically from the reynolds-number re: as (see, e.g. [83] ): the lower bound of c d = 0.1 is required to obtain the proper limit for the euler equations, when re → ∞. the heat transferred between the particles and the fluid is given by where h f is the film coefficient and σ * the radiation coefficient. for the class of problems considered here, the particle temperature and kinetic energy are such that the radiation coefficient σ * may be ignored. the film coefficient h f is obtained from the nusselt-number n u: where pr is the prandtl-number of the gas as having established the forces and heat flux, the particle motion and temperature are obtained from newton's law and the first law of thermodynamics. for the particle velocities, we have: this implies that: where α v = 3ρc d /(4ρ p d). the particle positions are obtained from: the temperature change in a particle is given by: which may be expressed as: . equations (15, 16, 18) may be formulated as a system of ordinary differential equations (odes) of the form: where u p , x, u f denote the particle unknowns, the position of the particle and the fluid unknowns at the position of the particle. the last six decades have seen a large number of schemes that may be used to solve numerically the incompressible navier-stokes equations given by eqs. (6.1-6.3). in the present case, the following design criteria were implemented: -spatial discretization using unstructured grids (in order to allow for arbitrary geometries and adaptive refinement); -spatial approximation of unknowns with simple linear finite elements (in order to have a simple input/output and code structure); -edge-based data structures (for reduced access to memory and indirect addressing); -temporal approximation using implicit integration of viscous terms and pressure (the interesting scales are the ones associated with advection); -temporal approximation using explicit, high-order integration of advective terms; -low-storage, iterative solvers for the resulting systems of equations (in order to solve large 3-d problems); and -steady results that are independent from the timestep chosen (in order to have confidence in convergence studies). the resulting discretization in time is given by the following projection scheme [60, 63] : which results in θ denotes the implicitness-factor for the viscous terms (θ = 1: 1st order, fully implicit, θ = 0.5: 2nd order, crank-nicholson). α i are the standard low-storage runge-kutta the k − 1 stages of eq. (21a) may be seen as a predictor (or replacement) of v n by v k−1 . the original right-hand side has not been modified, so that at steady-state v n = v k−1 , preserving the requirement that the steady-state be independent of the timestep t. the factor γ denotes the local ratio of the stability limit for explicit timestepping for the viscous terms versus the timestep chosen. given that the advective and viscous timestep limits are proportional to: we immediately obtain or, in its final form: in regions away from boundary layers, this factor is o(1), implying that a high-order runge-kutta scheme is recovered. conversely, for regions where re h = o(0), the scheme reverts back to the usual 1-stage crank-nicholson scheme. besides higher accuracy, an important benefit of explicit multistage advection schemes is the larger timestep one can employ. the increase in allowable timestep is roughly proportional to the number of stages used (and has been exploited extensively for compressible flow simulations [43] ). given that for an incompressible solver of the projection type given by eqs. (20) (21) (22) (23) (24) (25) most of the cpu time is spent solving the pressure-poisson system eq. (24) , the speedup achieved is also roughly proportional to the number of stages used. at steady state, v * = v n = v n+1 and the residuals of the pressure correction vanish, implying that the result does not depend on the timestep t. the spatial discretization of these equations is carried out via linear finite elements. the resulting matrix system is rewritten as an edge-based solver, allowing the use of consistent numerical fluxes to stabilize the advection and divergence operators [63] . the energy (temperature) equation [eq. (6. 3)] is integrated in a manner similar to the advective-diffusive prediction [eq. (21)], i.e. with an explicit, high order runge-kutta scheme for the advective parts and an implicit, 2nd order crank-nicholson scheme for the conductivity. the equations describing the position, velocity and temperature of a particle [eqs. (15)(19) ] may be formulated as a system of nonlinear ordinary differential equations of the form: they can be integrated numerically in a variety of ways. due to its speed, low memory requirements and simplicity, we have chosen the following k-step low-storage runge-kutta procedure to integrate them: for linear odes the choice leads to a scheme that is k-th order accurate in time. note that in each step the location of the particle with respect to the fluid mesh needs to be updated in order to obtain the proper values for the fluid unknowns. the default number of stages used is k = 4. this would seem unnecessarily high, given that the flow solver is of second-order accuracy, and that the particles are integrated separately from the flow solver before the next (flow) timestep, i.e. in a staggered manner. however, it was found that the 4-stage particle integration preserves very well the motion in vortical structures and leads to less 'wall sliding' close to the boundaries of the domain [70] . the stability/accuracy of the particle integrator should not be a problem as the particle motion will always be slower than the maximum wave speed of the fluid (fluid velocity). the transfer of forces and heat flux between the fluid and the particles must be accomplished in a conservative way, i.e. whatever is added to the fluid must be subtracted from the particles and vice-versa. the finite element discretization of the fluid equations will lead to a system of ode's of the form: where m, u and r denote, respectively, the consistent mass matrix, increment of the unknowns vector and right-hand side vector. given the 'host element' of each particle, i.e. the fluid mesh element that contains the particle, the forces and heat transferred to r are added as follows: here n i (x p ) denotes the shape-function values of the host element for the point coordinates x p , and the sum extends over all elements that surround node i. as the sum of all shape-function values is unity at every point: this procedure is strictly conservative. from eqs. (15) (16) (17) (18) and their equivalent numerical integration via eq. (30), the change in momentum and energy for one particle is given by: for a large number of very small particles, it becomes impossible to carry every individual particle in a simulation. the solution is to: (a) agglomerate the particles into so-called packets of n p particles; (b) integrate the governing equations for one individual particle; and (c) transfer back to the fluid n p times the effect of one particle. beyond a reasonable number of particles per element (typically > 8), this procedure produces accurate results without any deterioration in physical fidelity. in order to achieve a robust particle integrator, a number of additional precautions and algorithms need to be implemented. the most important of these are: -agglomeration/subdivision of particle parcels: as the fluid mesh may be adaptively refined and coarsened in time, or the particle traverses elements of different sizes, it may be important to adapt the parcel concentrations as well. this is necessary to ensure that there is sufficient parcel representation in each element and yet, that there are not too many parcels as to constitute an inefficient use of cpu and memory. -limiting during particle updates: as the particles are integrated independently from the flow solver, it is not difficult to envision situations where for the extreme cases of very light or very heavy particles physically meaningless or unstable results may be obtained. in order to prevent this, the changes in particle velocities and temperatures are limited in order not to exceed the differences in velocities and temperature between the particles and the fluid [70] . -particle contact/merging: in some situations, particles may collide or merge in a certain region of space. -particle tracking: a common feature of all particle-grid applications is that the particles do not move far between timesteps. this makes physical sense: if a particle jumped ten gridpoints during one timestep, it would have no chance to exchange information with the points along the way, leading to serious errors. therefore, the assumption that the new host elements of the particles are in the vicinity of the current ones is a valid one. for this reason, the most efficient way to search for the new host elements is via the vectorized neighbour-to-neighbour algorithm described in [51, 63] . the techniques described above were implemented in feflo, a general-purpose computational fluid dynamics (cfd) code based on the following general principles: -use of unstructured grids (automatic grid generation and mesh refinement); -finite element discretization of space; -separate flow modules for compressible and incompressible flows; -edge-based data structures for speed; -optimal data structures for different architectures; -bottom-up coding from the subroutine level to assure an open-ended, expandable architecture. the code has had a long history of relevant applications involving compressible flow simulations in the areas of transonic flow [55, [73] [74] [75] [76] [77] , store separation [7, 10, 12, 14, 15] , blast-structure interaction [6, 8, 9, 11, 13, 16, 17, 58, 64, 82, 85, 93] , incompressible flows [5, 57, 60, 68, 79, 81, 91] , freesurface hydrodynamics [54, 61, 62] , dispersion [20] [21] [22] 59] , patient-based haemodynamics [1, 23, 24, 55, 65] and aeroacoustics [50] . the code has been ported to vector [56] , shared memory [53, 84] , distributed memory [52, 66, 79, 80] and gpu-based [27] [28] [29] [30] 67, 69] machines. the cases shown all simulate sneezing/coughing in different environments. the ambient temperature was assumed to be 20 • c. in order to simulate a sneeze/cough, the velocity and temperature in a spherical region of radius (r = 5 cm) near the patient's mouth was reset at the beginning of each timestep according to the following triangular function: the droplets were initialized with 4 different sizes and different velocities, and released every 0.005 s during 0.1 s. this resulted in a final number of particle packets of n p = 25, 662. the temperature was set to t p = 37 o c and the velocity to v = 5 m/s. table 3 summarizes the diameters and resulting mass distribution. in the cases shown different temporal scales appear: we have attempted to show these phases in the results, and for this reason the results are not displayed at equal time intervals. unless otherwise noted, the particles have been colored according to the logarithm of the diameter, with red colors representing the largest and blue the smallest particles. the examples given show clearly the dangers of dropletand aerosol-based infections in the built environment. one of the obvious vectors for viral contamination and spread are security and passport examination queues in airports. air flow is moderate, passengers are in very close proximity, and in some airports queues wind back and forth in narrow lanes. figure 3a, this case considers a typical hospital room. of interest here was the dispersion of particles in the first minute after cough-ing, in particular the reach into neighbouring halls and the amount of 'negative pressure' needed to keep all contaminants in the room. figure 4a shows the arrangement of the room, with patient and caregiver clearly visible. this particular mesh had 2.25mels. the distribution of particles over time can be discerned from figures 4c-l. as before, one can see that the large (red) particles follow a ballistic path. this 'ballistic phase' ends at about t = 1 s. the (green) particles of size d = 0.1 mm are quickly stopped by the air, and then sink slowly towards the patient. the even smaller (cyan, blue) particles rise with the cloud of warmer air exhaled by the sneezing individual, and disperse much further at later times, covering almost the entire room. the velocity distribution in the room may be inferred from figure 4m . a lingering question facing all levels of society is how and when to reopen facilities where people congregate in close proximity. one key technology that would allow opening is testing and sensing. we consider sensing in the sequel. several vendors have announced measuring devices for covid-19 in the next half year. given that these sensors are expensive, and that a hospital or university many need hundreds of these, the question becomes how best to deploy them. in other words: given an arbitrary number of contamination or infection scenarios, which is the minimum number of sensors needed to detect them, and where should they be placed? a partial answer to this non-trivial question was given in [59, 97] . if we assume a given number of sensors, every contaminant/infection scenario (location and amount of release, flow conditions, etc.) will lead to a sensor input. the data recorded from all the possible release scenarios at all possible sensor locations allows the identification of the best or optimal sensor locations. clearly, if only one sensor is to be placed, it should be at the location that recorded the highest number of releases. this argument can be used recursively by removing from further consideration all releases already recorded by sensors previously placed. the procedure is repeated recursively until no undetected release cases are left, or the available sensors have been exhausted. see [19, 41] for an in-depth analysis of robust sensor placement under uncertainty. fig. 3 tsa queue: a arrangement of pedestrians and surface mesh, b surface mesh and cut plane, c particle distribution at t = 0.02 s, d particle distribution at t = 0.10 s, e particle distribution at t = 0.20 s, f particle distribution at t = 0.40 s, g particle distribution at t = 0.80 s, h particle distribution at t = 1.60 s fig. 4 hospital room: a surface mesh, b particle distribution at t = 0.0 s, c particle distribution at t = 0.2 s, d particle distribution at t = 0.4 s, e particle distribution at t = 0.6 s, f particle distribution at t = 1.0 s, g particle distribution at t = 2.0 s, h particle distribution at this case considers the same hospital room as shown before. the boundary conditions determining the flow are assumed as steady, with air entering the room through vents 1-3 and exiting the room through the bathroom exhaust or the door. figures 5a-c show the outlay of the room, average velocities and the 'age of air' after 5 min. note the high values for the age of air in the corners and the back of the room. this particular mesh had 2.2mels. four contaminant release scenarios were considered: cases 1-3 assumed contaminant coming in through each of the vents (separately) during the first minute, while case 4 assumed virus production from the patient for a period of 10 s. the case was run for 5 min of real time, and the contaminant concentration was measured on all walls/ceilings. the maximum concentrations measured have been summarized in fig. 5d . note the different areas covered depending on the release scenario. it was assumed that sensors should only be allowed above a certain height, and should be located on a wall or the ceiling. table 4 summarizes the points that measured data above a set threshold. as one can see, none of the possible sensor locations is able to measure/detect all 4 cases, and many possible sensor locations do not detect even a single case. there are many possible pairs of sensors that can detect all 4 cases. the pair selected is the one that achieves the highest relative measurement values, and is shown in fig. 5e . note that this makes good sense: one sensor close the hvac exits, and one close to the patient. the present paper has summarized some of the mechanical characteristics of virus contaminants and the transmission via droplets and aerosols. the ordinary and partial differential equations describing the physics of these processes with high fidelity were given, as well as appropriate numerical schemes to solve them. several examples taken from recent evaluations of the built environment were given, as well as the optimal placement of 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combination of body-fitted and embedded grids for external vehicle aerodynamics temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study numerical modeling of long-duration blast wave evolution in confined facilities aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 airborne spread of infectious agents in the indoor environment how far droplets can move in indoor environments-revisiting the wells evaporation-falling curve optimal sensor placement for airborne contaminant detection in an aircraft cabin numerical simulation of coughed droplets in conference room publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-352743-goier9an authors: habchi, carine; ghali, kamel; ghaddar, nesreen title: transient transport model of particles resulting from high momentum respiratory activities: inter-personal exposure date: 2015-07-31 journal: build environ doi: 10.1016/j.buildenv.2015.07.028 sha: doc_id: 352743 cord_uid: goier9an in this work, a transient mathematical multi-region zonal transport model of particle behavior resulting from high momentum respiratory activities (hmra) is developed focusing on the transient inter-personal exposure (ipe) in indoor spaces ventilated by displacement ventilation (dv) systems. the developed model was validated by experimentation and by published empirical data. three stages are identified with respect to time for the variation of the ipe: a first stage dominated by the propagation and decay of the exhaled jet, a particles' redistribution stage, and a particles' removal stage. the inhaled dose is affected by the dv flow rate, cough velocity, particle diameter and distance between the occupants. the dv system with a flow rate of 100 l/s reduced significantly the inhaled dose during particle redistribution and removal stages decreasing the total inhaled dose by 83% compared to a flow rate of 50 l/s. ipe is higher when particle diameter is increased from 1 to 20 μm due to the opposition of particle removal by the upward dv. a comparison between steady and transient modeling of the ipe showed that steady modeling captures the physics affecting particle spread due to hmra but it over-predicts the inhaled dose. it is found that for a dv flow rate of 100 l/s and a cough velocity of 22 m/s during 1 s, and 1 μm particles, the minimum required distance between the occupants for a threshold inhaled dose of 10(−5) kg is nearly 0.5 m by transient modeling while it is 2.15 m by steady state modeling. with the increased time spent by people in indoor environments and the outbreaks of the severe acute respiratory syndrome (sars) [1] a lot of concerns have been raised on people health in indoor environments, particularly on the control and possible prevention of airborne disease transmission [2] . humans generate particles by the different respiratory activities (talking, breathing, sneezing, and coughing) [3] and through emissions from the skin, hair, and clothes etc. [4] . it is well established that one of the main contributors to cross-contamination between occupants in indoor environments are high momentum respiratory activities as sneezing or coughing [5e7] which will be studied in the current work. in fact, the intense upper respiratory activities generate a higher number of particles than the normal breathing [8] . furthermore, pathogens delivered by strong expiratory jets could be transmitted for large distances up to 3 m in the horizontal direction for high outlet velocities [9, 10] increasing the inter-personal exposure (ipe) between occupants. the transient behavior of sneezing and coughing complicates modeling the spread of particles resulting from these activities. for simplification, some researches modeled coughing as steady state using computational fluids dynamics (cfd) [10] or simplified modeling [11] to understand the physics affecting disease transmission resulting from coughing. however, steady-state simulations might not reflect the complete physics affecting particle distribution resulting from transient respiratory activities (tra). for instance, rim and novoselac [12] studied particle distribution caused by short term and continuous particle generation for different ventilation configurations. they found that for ventilation governed by buoyant flows, disease transmission resulting from a short-term source release differs from continuous source emission. furthermore, the exposure is higher under continuous generation than under particle release for a limited period of time. hence, contaminant exposure due to short-term indoor emissions differs from exposure estimated using steady-state source. from their observations, rim and novoselac [12] concluded that caution should be taken when making conclusions and recommendations about the risk of infection from short term sources emissions using steady-state simulations. since high momentum respiratory activities (hmra) as coughing and sneezing are transient, assessing the actual ipe from these activities requires transient modeling. steady state simulations over-predict the ipe which might lead to overdesigned ventilation flow rates to maintain acceptable indoor air quality (iaq) [12] . transient modeling not only allows assessing accurately the risk of infection but also can reveal the effect on ipe of transient parameters (e.g. period of injection, time) that cannot be captured by steady state modeling. for instance, xiaoping et al. [13] observed that particle concentration variation with time can be divided into two main stages: a first stage dominated by the exhaled jet propagation, and a second stage affected by the ventilation configuration. the observation of transient particle concentration variation cannot be deducted from steady-state simulation. furthermore, many efforts have been made to reveal the effect of ventilation types, relative orientation between the occupants, sneezing and coughing velocities, in addition to droplet size distribution on the transient spread of exhaled droplets in variable types of indoor environments using cfd and experimental simulations. mui et al. [14] investigated by numerical simulations variable velocities and orientations of sneezing under mixed ventilation (mv) and displacement ventilation (dv) schemes. they concluded that the spread of droplets in the room was faster under mv than under dv. gao and niu [15] conducted a numerical study in a room occupied by two persons sitting opposite to each other. they concluded that the propagation of the high momentum sneezed jet in the horizontal direction is one of the main causes of crosscontamination between the occupants [15] . licina et al. [16] investigated experimentally the effect of the distance of cough source from an exposed occupant, airflow velocity and direction on the ipe. they observed that increasing the separating distance between the exposed person and the location of the cough source decreases the peak exposure with an increased exposure delay time after the cough. they also reported that assisting flow from below (which is the case of dv) decreased the personal exposure with the increase of supplied air velocity. chen and zhao [8] studied particle distribution resulting from the different respiratory activities (breathing, coughing, and sneezing) for a wide particle sizes range extending from 0.1 to 200 mm. they showed that the exhalation velocity and the droplet size with initial diameter from 10 mm to 100 mm largely affected the particle distribution [8] . displacement ventilation is known to provide good iaq in the occupied zone [17e19]. it was reported to have a high efficiency in the reduction of disease transmission when particles were generated by low momentum respiratory activities (normal breathing) as the upward convective plumes of the infected person carried nearly 100% of the exhaled particles to be exhausted at the ceiling level [18, 20] . on the other hand, it was shown that higher momentum respiratory activities degraded the effectiveness of dv system in providing good iaq in the occupied zone because they lead to a horizontal spread and accumulation of infected particles at the breathing level [11, 21, 22] . the performance of dv system in terms of removal of particles resulting from tra was investigated by experimentation and cfd simulations. gao [15] . seepana and lai [6] observed experimentally and by cfd that concentration peaks at the breathing height in dv were relatively high compared to mv due mainly to high sneezing velocity and thermal stratification. however, experimental and cfd techniques have high cost and computational time. therefore, simplified modeling is an essential design tool for capturing the principal physics involved at low computational time [24] . habchi et al. [18] developed a simplified zonal model to assess the risk of cross-contamination between occupants resulting from normal breathing activities in office buildings ventilated by dv. they extended their model to account for hmra by coupling between two particles' transport models [11] . nevertheless, their developed models were steady hence they were not able to assess the variation with time of the ipe resulting from tra as sneezing and coughing. in this work, a transient zonal model is developed to compute particle distribution resulting from tra in indoor environment conditioned by dv systems. the model is validated with experimentation and literature data. this is followed by a parametric study conducted to assess the effect of variable factors on the crossinfection between occupants (dv flow rate, coughing velocity, particle diameter, and distance between the occupants). recommendations for reduced ipe in spaces ventilated by dv system are established based on understanding transient particle behavior resulting from short-term release respiratory activities as coughing and sneezing. the simulation of transient horizontal particle spread resulting from hmra requires coupling two transient sub-models extending the work done by habchi et al. [11] . a transient transport model of exhaled particles computes the percentage of generated particles penetrating the infected thermal plume and tracks the exhaled jet propagation with time and distance. this model is coupled with another transient transport model for particle exchange between the different affected layers and regions. the developed transient multi-region zonal model will be validated by experimental and literature data. occupants are represented by heated cylinders [11, 18, 25, 26] as shown in fig. 1 . the domain is discretized into horizontal layers with several lumped regions within each layer to capture the vertical motion of the air surrounding the thermal plumes and the effect of buoyant flows, [11, 18, 25] . vertically three main zones are identified [11, 18] : -zone i characterized by upward flow in the air surrounding the thermal plumes extends from the floor to the stratification height where the surrounding air and dv flow rates are equal [26, 27] ; -zone ii is bounded by the stratification and critical height where rising plumes expand [18, 28] ; and -zone iii is a recirculation zone where surrounding air and thermal plumes merge together [18, 28] . establishing the flow field in the ventilated space is essential for computing the particle distribution within the room. in zones i and ii below the recirculation zone, lateral flow is negligible compared to vertical air motion [11, 19, 25] . the stratification and critical heights are affected by the dv flow rate and the strength of convective buoyant plumes. the thermal plume upward mass flow rate, mp l, induced by the heat source for known room vertical temperature gradient dt ∞ /dz is determined using mundt correlation [27] as follows: m n (1a) m n ¼ 0:004 þ 0:039z n þ 0:38z 2 n à 0:062z 3 n (1b) where r is the air density, q h is the heat source strength, t ∞ is the room temperature, z is the plum height from the floor, z v is the virtual point source height [18] , and z n and m n are non-dimensional parameters defined by mundt [27] . a mass balance applied for each layer i gives the expression of the mass flow rate of the surrounding air m a,i at the interface between layers i and i þ 1: where ms is the mass supply flow rate, mw the wall plumes given by jaluria formulation [11, 29] , n is the number of vertical walls within a layer i, and m is the number of convective thermal plumes. the stratification height is the level at which m a,i ¼ 0. the upward velocity strength v up,i in zone i at layer i is given by: where a a,i is the cross-sectional area of the air surrounding the plumes. the critical height position from the virtual point source is given by: particle distribution within zones i and ii is computed in the horizontal level by dividing each layer into five principal regions [11] : infected plume region, exposed plume region, microclimate 1, microclimate 2, and macroclimate. a detailed description about the space discretization can be found in our previous work [11] . microclimate 2 is of particular interest in predicting crosscontamination between occupants since it constitutes a cylindrical region tangent to the occupants and limited by their convective upward plumes until their expansion [11] . the exhaled jet is highly unidirectional propagating and decaying with time from the infected to the exposed occupant. in order to capture the transient propagation of the exhaled jet and to assess accurately the ipe, microclimate 2 was discretized into a number n of tangent cylindrical sub-microclimates zones as shown in fig. 1 . the characterization of exhaled jets is very important for accurate prediction of the resulting particle distribution. there have been extensive reviews [7, 30] on the size distribution of human exhaled droplets. nicas et al. [30] found a correlation between the equilibrium diameter of the completely evaporated particle (deq) and the initial diameter (di) where deq ¼ 0.44 di and this correlation was adopted in several studies [31e33]. chen and zhao [8] found that when the normalized evaporation time was lower than 0.051, the evaporation time can be neglected in the modeling. the coughing process has been simulated in several works with variable cough velocities. gupta et al. [34] and kwon et al. [35] conducted experiments on a number of human subjects to determine initial velocities of a cough and characterize the coughing process. zhu et al. [10] showed that the peak cough velocity varied from 6 to 22 m/s. chao et al. [36] reported that the average expiration air velocity was 11.7 m/s for coughing which is close to the average value of 11.2 m/s found by gupta et al. [34] . accordingly, the cough velocity was varied in the range of [6 m/s; 22 m/s] in this study. furthermore, gupta et al. [34] found that the average mouth opening for males during a cough is equal to 4 cm 2 which was used in several cough studies [37, 38] . therefore, this value was adopted in the current work corresponding to an equivalent mouth diameter of nearly 2.25 cm. in order to model particle spread resulting from coughing or sneezing, the transient propagation of the exhaled jet should be accurately modeled. a top-hat profile was used for the generation velocity [10, 15] : where v gen (t) is the generation velocity function of time, v g is the generation velocity during the cough and t rl is the cough release time. chen et al. [39] modeled transient propagation of high momentum jets and validated their model for different ventilations configurations. chen formulation [39] is used in the current study and is summarized below. the horizontal jet peak v max decreases with the horizontal coordinate y from the source and is a function of the injection velocity v inj and the hydraulic diameter of the mouth d inj. v max is given by the following expression [39] : the propagation jet is stopped by an obstruction (e.g. wall, exposed occupant) or damped when the jet peak becomes lower than a reference velocity of 0.25 m/s [39] . thus, the maximal propagation distance y max is given by: where v r is the reference velocity and y ob is the obstruction distance. the time needed by the jet to reach a position y is given by ref. [39] : the decay of the velocity with time after the jet had reached a certain distance y along the unidirectional propagation line [39] (for y between 0 and y max ) is given by: where t is the time constant given by the following expression [39] : the unidirectional horizontal jet decay c(y,t) along the propagation line (illustrated in fig. 2 ) can be computed from: where v inj is the penetrating velocity computed from the exhaled particles model [11] . in fact, the upward plume of the infected person obstructs the propagation of the generated jet, thus the exhaled flow does not penetrate completely to the surrounding [11] . the penetrating portion l can be determined by resolving for the navierestokes equations within the thermal boundary to compute the penetrating velocity v p [11] . the propagation of the exhaled jet with time and distance affects the upward dv flow within each zone such that mass balances are conserved. the variation with time of the horizontal flow entering a sub-microclimate 2 number j (lc(y jà1 ,t)m g ) is different from the horizontal exiting flow (lc(y j ,t)m g ) due to the time delay and flow decay resulting from the jet propagation. the difference between the horizontal entering and exiting flows is compensated by the vertical air motion within the surrounding air. convective flows through a sub-microclimate 2 number j within the generation layer are illustrated in fig. 2 . the transient formulation of the jet propagation is used as boundary conditions for the transient multi-zonal model for spaces ventilated by dv system to predict particle spread resulting from hmra. the model computes the temperature, velocity and concentration fields targeting mainly the transient ipe. the complete formulation for predicting the vertical temperature and velocity in different zones and coupling between the transport model of exhaled particles and multi-zonal model can be found in our previous work [11, 18] . to predict particle distribution within the variable zones, the different physics affecting particle spread were included in the model [18, 25, 28, 40] . for instance, convective dv flows and diffusion between and within the different regions were considered [11, 18] . furthermore, interactions between upward convective plumes and the surrounding air by lateral diffusion and air entrainment were incorporated in the proposed physical model [25, 28] . since the developed model involves particles of variable diameters, deposition on walls of variable orientation and gravitational effect were included. the detailed steady state mass balances within the variable zones can be found in the work of habchi et al. [11] . in the current model, the same physics are involved but transient formulation is incorporated. the contribution of this work is that it simulates the propagation of the exhaled jet between the occupants at the breathing level and assesses the transient ipe. in order to capture the transient particle spread microclimate 2 was sub-divided into 20 sub-microclimates zones to verify grid independent results. a sub-microclimate 2 region number j interacts with the adjacent regions and the infected or/and exposed thermal plumes and the concentration in this zone is governed by the following equation the maximal function is introduced to account for the variation in the surrounding airflow direction allowing the equation to be applicable within the different zones defined in the vertical direction [11, 18] . for instance, the flow is upward below the stratification height while it is downward above the stratification height. the particle concentration is c, v s is the settling velocity, d p is the molecular brownian diffusion coefficient, d t is the turbulent diffusion coefficient, m is the mass flow rate, t is the time, r mic2.j is the radius of the sub-microclimate 2 number j. the subscripts: i refers to the layer number, a refers to the air outside the thermal plumes (surrounding air), pl refers to the air inside the thermal plume, ent refers to entrainment, int to interaction and g refers to generation. a a,mic2.j,i represents the cross sectional area of the sub-microclimate 2 number j at the interface between layers i and i þ 1, and a int,mic2.j/ pl(,i,k) its interaction area with plume k within the layer i. these interaction areas are found mathematically as the intersection between two surfaces of defined geometry (the conical shape for the plumes and the circular shape for the sub-microclimates 2). the terms of the right hand side of equation (3) represent respectively: i) the vertical convection flux of particles in the submicroclimate 2 number j resulting from the dv flow rate within the surrounding air affected by the propagation of the exhaled jet; ii) the gravitational flux; iii) the vertical diffusion flux; iv) the lateral particle diffusion fluxes presenting the interactions between the sub-microclimate 2 air region j and the plumes due to concentration gradients; v) the entrainment fluxes of the sub-microclimate 2 region j by the rising plumes; vi) the diffusive interactions of submicroclimate 2 number j with the adjacent regions; vii) the horizontal convective flux which is only present within the generation layer due to the exhaled jet uni-directional propagation. the left hand side of equation (3) is the transient storage term. in order to understand the effect of the different variables involved on the ipe, two parameters are defined and their variation in time is to be observed. the first parameter is the normalized concentration given by equation (4a): where the breathing level of the exposed person is simulated by the last sub-microclimate 2 which is adjacent to the exposed person at the nose level (centered at 1.05 m in height). the second parameter introduced is the infection index i in kg defined by the following equation [15] : where c in (kg/m 3 ) is the inhaled concentration by the exposed person equal to the concentration within the last sub-microclimate 2 zone, m in (kg/s) is the inhalation rate of the exposed person, c g rv a;mic2$j;i vc a;mic2$j;i ðtþ vt ¼ where t b,t and t e,t are respectively the beginning and end time of the considered period. in order to validate the ability of the current model in capturing the transient jet propagation and decay with distance, an experimental set-up was constructed in a controlled climatic chamber conditioned by a dv system (fig. 3a) . the experimental room is of inner dimensions 2.5 m â 2.75 m â 2.8 m with supply and return grills cross-sectional area of respectively 0.582 m (width) â 0.24 m (height) and 0.44 m (width) â 0.19 m (height). two occupants were represented by thermal cylinders of 0.3 m in diameter and 1.2 m in height generating each 100 w. due to the experimental constraint of highly insulated walls, the load was distributed between the lighting (100 w) and the heated cylinders such that the load per unit floor area is approximately 40 w/m 2 . the dv supply conditions were a flow rate of 100 l/s and a temperature of 22 c. a thermo anemometer (abk precision 731a) with an accuracy of ±0.6 c for temperature measurements and ± 2% of full scale for velocity measurements was used. in order to remove particles from the supplied air before introducing it to the chamber, high efficiency particulate filters (ks-ng-k1/2 hepa filters) were adopted. prior to each set of measurements, the light and the heated cylinders were turned on and the dv system was run for 3e4 h to reach steady-state conditions. then mono-disperse heavy 5 mm particles of density of 912 kg/m 3 were generated from a condensation aerosol generator (tsi model 3475) (fig. 3b) with a flow rate of 4 l/min at a velocity of 10 m/s during 4 s. the tsi generator used can generate monodisperse particles with an aerosol geometric standard deviation smaller than 1.1. a chronometer watch of 0.01 s accuracy was used to control the generation time. the validation consisted of comparing measured and predicted particles concentrations at different distances (0.5, 1, and 1.5 m) from the generation (fig. 3a) . particle concentrations were measured using an optical particle sizer (tsi model 3330) (fig. 3b) giving measurements with a relative error lower than 5%. a sampling time of 1 s was selected to track particle concentration variation with time. results were recorded automatically by a computer for post-processing. the measured concentrations were normalized by the generation concentration for comparison with the predictions of the simplified model. the developed mathematical model will be used to perform a parametric study to investigate cross-contamination between two occupants (75 w each) within a space of dimensions 3.4 m â 3.4 m â 2.6 m ventilated by a dv system. the frequently encountered heat sources (occupants, lighting, external heat from walls) are considered. the lighting load was set to 100 w and wall heat fluxes to 200 w. the case study is selected to represent typical office load at 40 w/m 2 of floor area based on upper limit of load removal by dv system [41, 42] . the generality of the obtained results is due to the fact that office spaces are usually configured in modular workstation units where each unit would have identical number of occupants as well as electrical load. one occupant generated particles transiently during 1 s simulating a cough released by an infected person while the second occupant was healthy simulating the exposed person. in order to assess the risk of hmra, the effect of different variables such dv flow rate, coughing velocity, particle diameter, and distance between the occupants on cross-infection is studied. the dv supply flow rate is varied in the range of [50 l/s e 200 l/ s] and the supply temperature is fixed to 22 c [11] . in fact, habchi et al. [18] have shown that a flow rate of 100 l/s is required to insure both thermal comfort and iaq for typical office spaces loaded by 40 w/m 2 ventilated by dv system considering normal respiratory activities. then they extended their model to consider hmra [11] but they considered continuous generation which might over-predict the ipe. in this study, a typical cough velocity in the interval of 6 m/s to 22 m/s and an exhalation area of 4 cm 2 [34, 43] are used. particle diameter is varied in the range of 1 mme50 mm and the distance between the occupants is varied between 0.5 m and 3 m [11] . interpersonal exposure was studied for different dv supply flow rates (50, 75, 100 l/s), cough velocities (6, 11, and 22 m/s), particles diameters (1, 20, 30 , and 50 mm), and distances between the occupants (0.5, 1, 2, and 3 m). each of these factors was varied while the other variables were fixed to capture the studied parameter effect on ipe. fig. 4 represents the comparison between the model and experiments of the variation with time and distance of the particle concentration normalized by the generation concentration. three cases were investigated at distances from generation of 0.5, 1 and 1.5 m while keeping occupants' separating distance fixed at 1.5 m. after generation, the normalized concentration increases with time reaching a peak and then decays. as the distance from generation increases, the concentration peak decreases due to the exhalation jet decay and the time needed to reach this peak increases because of the time delay resulting from the jet propagation. good agreement of the order of 10% with a maximum error of 16% was obtained between the model and experimental results validating the ability of the model in capturing the exhaled jet propagation and decay with time and distance. the research study of gao and niu [15] was used to validate the ability of the model in predicting the variation of the ipe with time. in their work, two persons (contaminated and exposed occupants) in the seated position facing each other and separated by a distance of 1.2 m occupy a chamber of dimensions (2.7 m â 2.6 m â 2.2 m). the chamber is ventilated by the dv system with a supply flow rate of 0.024 m 3 /s and a temperature of 22 c. one sneeze exhaled from the mouth of the infected occupant lasting for 1 s with a volume flow rate of 250 l/min is simulated [15] . the variation of the inhaled fraction by the exposed occupant is simulated during and after the sneeze. the case study of gao and niu [15] was simulated by our developed model. fig. 5 represents the comparison between gao and niu [15] and model results of the variation of the ipe with time. the results are in agreement with a relative error of the order of 10% with a maximum error of 15%. the obtained agreement validated the ability of the model in predicting the variation of the ipe with time by simulating it as the concentration within the last submicroclimate 2 which is at the breathing level of the exposed person. 3.3.1. dv flow rate effect table 1 summarizes the effect of the dv flow rate on different parameters. as the dv flow rate increases, the stratification and critical heights are shifted upward resulting in an increase in zones i and ii heights and a decrease in zone iii height. it is clear that the increment of the dv supply rate strengthened the occupants' thermal plume and the upward velocity in zone i. this velocity decreases from a maximum value at the floor level to zero at the stratification height due to air entrainment by the convective rising plumes. the vertical air motion by buoyancy effects leads to temperature stratification within the space resulting in a temperature increase from the floor to the ceiling levels (see table 1 ). as dv flow rate increases, room air temperature gradient decreases. ipe was studied for different dv supply flow rates (50, 75, 100 l/ s) for a cough velocity of 22 m/s, 1 mm particles in diameter, and 1 m separating distance between occupants. fig. 6a illustrates the effect of the dv supply flow rate on the variation with time of the normalized concentration at the breathing level of the exposed person. logarithmic scale was used for the figure's axis for clarity of illustration. three stages can be defined with respect to time for the different dv supply flow rates (fig 6) . the first stage is dominated by the propagation and decay of the exhaled jet (fig. 6a) and is very fast lasting for few seconds (table 2a ). in the second stage, particles are redistributed by the ventilation system for several minutes. a final stage is the removal stage by deposition and upward transport by the dv system and has the largest period (fig. 6a) . the observation of different exposure stages is in agreement with the findings of xiaoping et al. [8] . table 2a summarizes the periods of the three stages for each dv flow rate. the period of these three stages varied with the dv flow rate. the larger the flow rate is, the lower are the durations of the three stages ( fig. 6a and table 2a) . furthermore, the effect of the dv flow rate on the duration of a stage is highest for the third stage and lowest for the first stage (table 2a) . for the different flow rates, a concentration peak (penetration peak) is observed during the first stage due to the jet propagation. the higher the dv flow rate is, the lower is the penetration peak because a larger number of particles is convected upward by the dv system (fig. 6a) . this observation is in agreement with the findings of licina et al. [16] . a second inhaled concentration peak (redistribution peak) at dv supply flow rate below 75 l/s appears during the second stage since the stratification height is close to the breathing level which results in accumulating particles in the breathing zone (fig. 6a) . as the dv flow rate increases from 50 to 75 l/s, the second peak is reduced and completely disappears for a flow rate of 100 l/s while the influence of the dv flow rate is less significant on the first peak which is dominated by the horizontal propagation of the exhaled jet (fig. 6a) . the penetration peak (first stage peak) is two to three orders of magnitude larger than the redistribution peak (second stage peak). however, the duration of the first stage is much smaller than the second stage (table 2a) . fig. 6b represents the effect of the dv supply flow rate on the variation with time of the infection index. the rate of increase of the infection index is relatively high during the first stage and is reduced progressively with time to reach nearly zero at the end of the third stage. the profiles of variation with time of the normalized concentration and infection index are consistent with the work of xiaoping et al. [8] . within the set of studied conditions, the higher the dv flow rate is, the lower is the rate of increase of the infection index during the different stages. the inhaled dose during each stage for the different flow rates was calculated and results were summarized in table 2b . for the different flow rates, the first stage represented the highest inhaled dose. as the dv flow rate increases, the infection index is reduced for the three stages but the rate of reduction is highest for stage 3 and lowest for stage 1 due to the decay of the exhalation jet with time (table 2b) . therefore, stage 1 presents the highest probability of cross-contamination between occupants. for a dv flow rate of 50 l/s, stages 2 and 3 represented a significant percentage of the total exposure. the dv system with a flow rate of 100 l/s reduced significantly the inhaled dose during stages 2 and 3 decreasing the total inhaled dose by 83% compared to a flow rate of 50 l/s. the dv flow rate of 100 l/s was adopted for the rest of the study. ipe was studied for different cough velocities (6, 11 , and 22 m/s) for a dv flow rate of 100 l/s, 1 mm particles in diameter, and 1 m separating distance between occupants. fig. 7a and b illustrate respectively the effect of the cough velocity on the variation with time of the normalized concentration at the breathing level of the exposed person and the infection index. as the cough velocity increases, the exhaled mass flow rate is incremented proportionally (for the same mouth opening) and the proportion of particles penetrating the thermal plumes of the infected occupant increases, therefore the ipe is increased (fig. 7) . hence, the higher the cough velocity is, the faster and stronger is the attained concentration peak (fig. 7a) , and the higher is the infection index (fig. 7b) . the coughing velocity largely affected the exposure during the first fig. 8a and b illustrate respectively the effect of the particle diameter on the variation with time of the normalized concentration at the breathing level of the exposed person and the infection index. with the increase of particle diameter, the gravitational settling effect opposing the upward transport of particles within the microclimate zones is strengthened leading to particle accumulation within the occupied zone. on the fig. 7 . effect of the cough velocity on the transient of: a) the normalized concentration at the breathing level of the exposed person; b) the infection index. fig. 8 . effect of the particle diameter on the variation with time of: a) the normalized concentration at the breathing level of the exposed person; b) the infection index. other hand, particle deposition by gravitation increases acting as a sink of particles. these two counter effects of increased particle diameter results in non-monotone variation of the concentration with particle diameter. this explains the fact that the ipe is higher when particle diameter is increased from 1 to 20 mm due to the opposition of particle removal by the upward dv. for larger particle diameters, the deposition by gravitation becomes dominant playing a major role in decreasing ipe (fig. 8 ). ipe was studied for different distances between the occupants at 0.5, 1, 2, and 3 m for a dv flow rate of 100 l/s, 1 mm particles in diameter, and cough velocity of 22 m/s. fig. 9a and b illustrate respectively the effect of the distance between occupants on the variation with time of the normalized concentration at the breathing level of the exposed person and the infection index. as the distance between occupants increases, the microclimate zone 2 fig. 9 . effect of the distance between occupants on the variation with time of: a) the normalized concentration at the breathing level of the exposed person; b) the infection index. relating the occupants is larger. therefore, the ipe is reduced as the exhaled jet decay is higher reducing contaminant transmission to the exposed person with a larger time delay. these findings are consistent with the ones of licina et al. [16] . the effect of the distance on the ipe is reduced in stages 2 and 3 compared with stage 1 due to the decay of the exhaled jet. fig. 10 represents the variation of the total inhaled dose with distance between the occupants showing that as the separating distance is reduced the rate of increase of the total inhaled dose is incremented and becomes significantly high below 1 m. the ipe is largely reduced for a separating distance of 3 m which justify the common recommendation of keeping a large distance between occupants for reduced exposure. the physical effect of different parameters (dv flow rate, coughing velocity, particle diameter, and distance between the occupants) on particle behavior computed from the transient model is consistent with the conclusions made from the steadymodel [11] . therefore, steady modeling is helpful in understanding the physics affecting particle spread resulting from hmra. in order to compare the ability of steady and transient modeling in predicting the ipe, the inhaled dose during the first stage (which is dominated by the jet propagation) was calculated for both models. the inhaled dose computed by transient modeling was calculated using the concentration profile obtained from the transient simulation. on the other hand, the inhaled dose obtained by steady modeling was computed using the steady-state concentration [11] . fig. 11 illustrates the comparison of the inhaled dose predicted by transient and steady modeling for variable dv flow rate, cough velocity, and distance between the occupants. steady modeling significantly over-predicts the ipe (fig. 11) . for instance, if the threshold inhaled dose is set to 10 à5 kg, for a cough velocity of 22 m/s during 1 s, 1 mm particles in diameter, and a separating distance of 1 m transient modeling predicts a minimum required dv flow rate of 75 l/s while steady modeling predicts than even an oversized flow rate of 200 l/s is not enough (fig. 11a) . furthermore, for a dv flow rate of 100 l/s, 1 mm particles in diameter, and a separating distance of 1 m transient modeling predicts that the whole coughing velocity range is acceptable while steady modeling lead to the conclusion that the maximum possible coughing velocity is 10.25 m/s (fig. 11b) . finally, for a dv flow rate of 100 l/s and a cough velocity of 22 m/s during 1 s, and 1 mm particles in diameter, the minimum required distance between the occupants is nearly 0.5 m by transient modeling while it is 2.15 m by steady modeling (fig. 11c) . therefore, steady modeling should not be used to predict the inhaled dose since it leads to largely over-predicted values. a transient mathematical multi-zone transport model predicting particle behavior in spaces ventilated by dv system was developed in order to assess transient ipe. experimental validation was performed showing that the developed model is capable of predicting the transient jet propagation and decay with distance and time, and can be used to assess the transient ipe. the variation of the ipe with time can be categorized into three stages: a first stage dominated by the propagation and decay of the exhaled jet, a particles' redistribution stage, and a particles' removal stage. within the set of studied conditions, the larger is the flow rate, the lower are the durations of the three stages and the effect of the dv flow rate on particle behavior is the highest for the third stage and the lowest for the first stage due to the decay of the exhaled jet with time. the inhaled dose during the first stage is the highest and is largely affected by the cough velocity, the particle diameter and the distance between the occupants. as the cough velocity increases, the ipe is increased since the quantity of the generated droplets and their momentum increases. as the separating distance is reduced, the rate of increase of the total inhaled dose is incremented and becomes significantly high below 1 m. for particle diameters above 30 mm, the deposition by gravitation becomes dominant playing a major role in decreasing the ipe. steady modeling of the ipe due to hmra is able of capturing the physics of particle distribution but should not be used to predict the inhaled dose since it leads to largely over-predicted values compared to transient simulations. for example, for a cough velocity of 22 m/s during 1 s, 1 mm particles in diameter, and a separating distance of 1 m transient modeling predicts a minimum required dv flow rate of 75 l/s while steady modeling predicts than even an oversized flow rate of 200 l/s is not enough for a threshold inhaled dose of 10 à5 kg. this study investigated the effect of several parameters (dv flow rate, coughing velocity, particle diameter, and distance between the occupants) on exhaled particle distribution and cross contamination between occupants. however, many other factors affect respiratory droplets spread and are worthy of future studies as the evaporation of exhaled droplets, 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hybrid model for investigating transient particle transport in enclosed environments modeling particle distribution and deposition in indoor environments with a new drifteflux model indoor air quality in rooms with cooled ceilings: mixing ventilation or rather displacement ventilation? energy build a critical review of displacement ventilation transport of droplets expelled by coughing in ventilated rooms the authors would like to thank the lebanese national council for scientific research (cnrs) for their financial support. the support of the university research board (urb) at the american university of beirut is also acknowledged as well as the shammas phd fellowship. key: cord-329135-g8fuax6p authors: haig, c.w.; mackay, w.g.; walker, j.t.; williams, c. title: bioaerosol sampling: sampling mechanisms, bioefficiency and field studies date: 2016-04-01 journal: j hosp infect doi: 10.1016/j.jhin.2016.03.017 sha: doc_id: 329135 cord_uid: g8fuax6p investigations into the suspected airborne transmission of pathogens in healthcare environments have posed a challenge to researchers for more than a century. with each pathogen demonstrating a unique response to environmental conditions and the mechanical stresses it experiences, the choice of sampling device is not obvious. our aim was to review bioaerosol sampling, sampling equipment, and methodology. a comprehensive literature search was performed, using electronic databases to retrieve english language papers on bioaerosol sampling. the review describes the mechanisms of popular bioaerosol sampling devices such as impingers, cyclones, impactors, and filters, explaining both their strengths and weaknesses, and the consequences for microbial bioefficiency. numerous successful studies are described that point to best practice in bioaerosol sampling, from the use of small personal samplers to monitor workers' pathogen exposure through to large static samplers collecting airborne microbes in various healthcare settings. of primary importance is the requirement that studies should commence by determining the bioefficiency of the chosen sampler and the pathogen under investigation within laboratory conditions. from such foundations, sampling for bioaerosol material in the complexity of the field holds greater certainty of successful capture of low-concentration airborne pathogens. from the laboratory to use in the field, this review enables the investigator to make informed decisions about the choice of bioaerosol sampler and its application. recent outbreaks such as severe acute respiratory syndrome (sars), h1n1 influenza, and the h5n1 avian influenza pandemic have raised concerns among infection control teams about the importance of the aerosol transmission of pathogens and have been an impetus to investigate the transmission dynamics of bioaerosols including influenza. 1e3 studies of suspected airborne transmission routes of various pathogens have been undertaken with differing degrees of success. 4 bioaerosol material is derived from biological origins, including aerial suspensions of bacteria, viruses, fungi, enzymes, and pollen. the size range varies from submicron-sized viral particles to fungal spores and pollen grains up to 1 mm in diameter. if carried by a favourable air flow, bioaerosol material may be distributed over large distances with potentially fatal results. for example, a community-wide outbreak of legionnaires' disease, which resulted in 18 fatalities, had an outbreak source in industrial cooling towers 6 km from the affected community. 5 however, bioaerosols may be relatively delicate structures susceptible to damage due to environmental conditions, such as desiccation. 6 there are several different types of bioaerosol sampler available to investigators, which broadly fall into four categories including impingers, cyclones, impactors, and filters ( figure 1 ). impingers and cyclones collect airborne particles into a liquid collection medium, whereas impactors collect particles on to solid/semi-solid mediums and filters trap bioaerosol material on fine fibres or porous membrane surfaces. the mechanisms of each collection method and their associated benefits/drawbacks are discussed in detail in the subsequent section. this paper constitutes a review of bioaerosol sampling mechanisms and seeks to address practical issues such as choosing a bioaerosol sampling device, bioefficiency, and operational considerations. a comprehensive literature search was performed, using electronic databases to retrieve english language papers on bioaerosol sampling within hospitals and the wider environment. the databases searched were science direct, medline (web of science), proquest, and taylor & francis online. the primary search criterion was bioaerosol with secondary search criteria being sampling, hospital, pathogen, infection, environment, cyclone, impactor, impinger, and aerosol. the final search was on january 16th, 2016. initially papers were included from 1994 to present day; however, having received expert advice, the search was revised to included papers from 1940 onwards. patents and foreign language papers were excluded from the literature review. the search yielded 314 publications, of which 131 were included in the review. the software programme endnote was used for reference management. bioaerosols may be collected using passive or active sampling systems as described in the following sections, with active sampling devices involving a mechanical component. 7, 8 passive sampling passive sampling is arguably the most readily available, economic, and unobtrusive method of bioaerosol sampling and relies on particles settling by means of gravity, on a collection substrate housed in a settle plate. the collected particles are usually quantified in terms of the number of colony-forming units (cfu) within the area of the settling plates for the duration of a specified time-period (for example in units of cfu/m 2 /h). as no mechanical aids, such as a pump, are required, passive sampling has the benefit of not disturbing the surrounding air. the settling velocity of a particle describes the speed of the particle as it descends in still air and is dependent on particle size and density. 9 smaller, lighter particles will remain airborne for longer than larger, denser particles; and if the air speed exceeds the settling velocity the particle will remain suspended indefinitely. in addition, as airflow, even within an enclosed room, will be driven by subtle variations in temperature, the source volume of air for the passively collected sample will be unknown. the combination of these factors has allowed passive sampling to be regarded as both quantitatively and qualitatively inaccurate, and as a subordinate collection method to active sampling. however, this is an oversimplification to the invariably complex nature of bioaerosol sampling. if the area of interest is the dust contamination of surfaces, for example wounds or surgical instruments, then the assessment of the microbial fallout, as opposed to particles remaining suspended in the air, is the imperative. 10 substantial effort has been made to standardize the use of settle plates in the investigation of microbial surface contamination, with consideration given to plate size, position, and length of exposure. 11 the 1/1/1 scheme refers to the positioning of 90 mm diameter petri dishes at a height of 1 m above floor level, 1 m from a wall, and with an exposure time of 1 h. this standardized method also allows for description of the microbial contamination of the surrounding atmosphere through the use of an index of microbial air contamination (ima). more recently an investigation into contamination in modern operating theatres (ots) with turbulent airflows suggested that the ima value could lead to an underestimation of the risk. 12 in a study which compared air and surface sampling for aspergillus sp. using contact plates within a hospital ward, a significant difference between the collection of airborne and surface spores was noted, with aspergillus accounting for >25% of the fungi isolated in the air but <2% of fungi isolated from surfaces. 13 this study underlined the need to be aware of the fact that pathogens which have settled on to a surface (subsequently collected on a contact plate) or settle plate may not give an accurate reflection of the suspended airborne concentrations of that pathogen. another study concluded that although settle plates could demonstrate a close correlation with bioaerosol collection undertaken by active samplers, there were exceptions, with settle plates shown to be less sensitive to the collection of fungal spores. 14 to countermand this deficiency an increase of the exposure time of settle plates from 1 to 4 h was proposed. by contrast, using a 30 min settle plate exposure time, a study in post-flood central thailand demonstrated that settle plates can be used as an alternative to active sampling systems. 15 the study did, however, acknowledge that the higher than average fungal bioaerosol presence may have limited the generalizability of their findings. surface sampling in intensive care units has been undertaken using tryptic soy agar contact plates accompanied by air sampling using a sampl'air lite (aes chemunex, bruz, france). 16 the total viable count of collected microbes varied between the surface and air sampling methods, which suggested that the source of the contamination may be different. surface contamination is especially influenced by human activity such as touch. surface and air sampling both concluded that the bed areas were consistently highly contaminated. the effectiveness of nitrocellulose membranes as an alternative to replication detection and organism counting (rodac) plates for surface sampling has been demonstrated. 17 the membranes have the advantage of being more effective at removing microbes, while also enabling samples to be taken from curved surfaces. passive sampling can also be used to describe more unusual methods of collection of bioaerosol material such as solid phase microextraction (spme). 18 microbial volatile organic compounds (mvoc) have been successfully collected using an 85 mm stableflex carboxen/polydimethylsiloxane fibre contained in a commercial housing. active sampling cannot be discussed without reference to a particle's mass and inertia. the mass of a particle is equal to its volume multiplied by its density, so it is possible for two particles to have the same mass but differing volumes and densities. if, however, two particles have the same density but differ in volume, then the larger particle will have a greater mass than the smaller particle. the inertia of a particle can be described as follows: consider two particles with differing masses being carried by an air flow inside a pipe. on reaching a bend in the pipe, the particle with the smaller mass will be able to travel along the airflow streamline and continue beyond the bend, whereas the particle with the larger mass will be unable to turn as quickly and will hit the wall and potentially become attached (figure 2 ). when a particle collides with a wall because its mass is too great to allow it to travel with the airflow, the collision is known as inertial impaction. if particles of varying size all have the same density then the larger particles will succumb more readily than the smaller particles to inertial impaction, hitting the wall at a bend in the pipe. hence the smaller particles will be collected in the sampling devices but the larger ones will not. there are several active sampling devices including impingers, cyclones, and impactors, which will be discussed in detail later. however, all active bioaerosol sampling systems consist of five fundamental elements which are necessary to undertake accurate sampling: 19 1. inlet to the sampling device 2. transport of the air sample through the device 3. particle size selection (not always present) 4. collecting medium 5. pump and calibrated flow monitoring. first, the design of the inlet combined with the air flow rate is essential in the collection of a representative sample that correctly reflects the concentration and size distribution of the airborne particles. 9 in a moving air stream, such as a ventilation duct, this is achieved by isokinetic sampling. this method considers the ratio of duct and sampling probe diameters, air flows, and inlet orientation to the air stream to ensure successful capture of particles regardless of size or inertia, thus providing a representative sample. sampling in still air is also affected by particle inertia, with larger particles being susceptible to evading probe collection; as the particle nears the inlet its velocity increases, thus increasing its stopping distance which may permit the particle to bypass the probe, distorting the concentration in the sample collected. the situation in still air is more straightforward but the probe inlet must be positioned horizontally to prevent an over/underestimation sampling bias. air streamline figure 2 . inertial impaction in a pipe. once the air sample is within the device, the sample path should be as straight and direct as possible in order to minimize losses in the conducting tubing. 9 where this is not possible, for example in a cascade impactor, particle loss will occur through inertial impaction on the bends between collection stages. in devices with sampling probes, particle loss may also occur in the probe head or flexible connective tubing, as particles will be lodged on to the side wall and will not reach the sampling medium. examples of this include liquid-based bioaerosol samplers, which also experience collection losses through evaporation of the collecting liquid and adhesion of the particles to the device walls. when this occurs the particles do not enter the collection liquid and the sampling process may therefore yield lower or false-negative results. 20 regardless of the mechanism of particle loss, predicting the quantity of losses is problematic, as much depends on the interaction of the air/liquid flows, particle inertia and the design of the sampler. an estimation of losses can be achieved through appropriate laboratory validation, which is discussed in more detail below. identification of particle size selection is not always available in a sampling device; however, there are several ways of selecting by particle size, either by using a pre-classifying cyclone or a series of impaction plates. collection of the bioaerosol samples is normally on to agar or a filter or into liquid, with liquid collection placing less stress on the bioaerosol particles as they are not dried out and are more likely to maintain their viability than the other two methods. 6 calibrated flow monitoring and the pump are crucial to the collection procedure as they ensure that the sampling device operates with an accurate air flow rate. each device will have an optimum speed at which the air flow should pass through the inlet and subsequent tubing to ensure that the bioaerosol particles will be collected with maximum efficiency. bioaerosol samplers vary considerably in size from large static samplers to smaller, portable personal samplers. large static samplers operate with higher flow rates of about 12.5e800 l/min, allowing them to collect larger volumes of air more rapidly than personal samplers operating at about 2e4 l/min. 21e24 impingers impingers operate by channelling particle-laden air flow through nozzles that exit into a chamber containing liquid (example shown in figure 1a ). 6 as the particles exit the nozzles in a jet of air they enter the collection chamber ( figure 3 ). the distance from the nozzle outlet to the surface of the liquid along with the air flow rate influences the diameter of the particles that will be collected. collection on to liquid prevents desiccation of the collected particles; however, shear forces in the jet in conjunction with the turbulence caused by the air being forced into the chamber may result in loss of viability. this bioefficiency (the ability of the sampling device to maintain the viability of the bioaerosol during and after sampling) may also be reduced through evaporation, re-aerosolization (loss of previously collected particles) and adherence of particles to the internal walls of the collection chamber. 20,25e27 bioaerosol impingers that are widely used include the all-glass impinger (ace glass inc., vineland, nj, usa), the bio-sampler (skc inc., covington, ga, usa) and the multistage liquid impinger (burkard manufacturing co. ltd, rickmansworth, uk) among others. 24,28e30 cyclones in a cyclone sampler (figure 1b) the particle-laden air is forced by the shape of the collection chamber into a spiral, swirling flow ( figure 4 ). within this airflow, particles experience a centrifugal force proportional to their diameter, density, and speed. this centrifugal force carries particles with sufficient inertia towards the cyclone wall where they are separated from the air flow into a liquid. 31 this generally means that larger particles are more likely to be collected than smaller particles, and, as with all samplers, a calibrated airflow is essential to maintain the correct collection efficiency. on reaching the bottom of the cyclone, the air flow reverses its direction and carries the smaller, uncollected particles out of the cyclone through a vortex finder positioned in the cyclone roof. to increase the bioefficiency, a film of liquid is injected near to the cyclone's inlet, resulting in wetting of the cyclone walls; the liquid is then collected at the base of the cyclone for analysis. 32, 33 collecting the particles on a liquid film maintains their viability but shear forces may still reduce bioefficiency. collection losses may again arise from evaporation of the collection liquid, resulting in the re-aerosolization of liquid air streamline previously collected material or through liquid carryover where the liquid injected into the cyclone travels over the cyclone roof and vortex finder wall before escaping from the system, carrying with it collected particles. 34, 35 cyclones vary considerably in size and airflow rate, with both the cyclone geometry and the airflow rate affecting the collecting efficiency. depending on the scale of the cyclone they can be used for collecting large volumes of air while operating at high flow rates or as miniature cyclones that can be worn on a person's clothing in potentially hazardous environments, with the collected material being analysed at the end of each day to assess exposure. 36 cyclones are also frequently used as pre-classifiers, removing larger particles from an airflow before further size classification by other types of sampler. 37 cyclones that are widely used include the coriolis ã� m (bertin technologies, saint quentin en yvelines, france), sass 2300 (research international, inc., monroe, wa, usa), burkard cyclone sampler (burkard manufacturing co. ltd, uk) along with several other cyclones specifically designed for bioaerosol sampling. 24,29,38e43 impactors in common with cyclones, impactors use the inertia of a particle to facilitate collection. the air sample is passed through an array of nozzles that channel a jet of particle-laden air across a gap towards an agar culture plate, which lies perpendicular to the nozzle outlet. the air flow of the jet will follow a set of curves known as streamlines through the sampler; these curves lie tangentially to the velocity vectors of the flow. the plate deflects the streamlines by 90 , with the air flowing past the plate and through the passageway between the plate and the device walls ( figure 5 ). the particles with sufficiently low inertia will be carried by the streamlines and escape capture. however, particles with higher inertia will be unable to follow the 90 curve of the streamlines, and, under the influence of the centrifugal force, will impact on the agar plate. the collection efficiency of an impactor is therefore primarily dependent on the diameter and density of the particle and the diameter of the nozzle, along with the air velocity of the jet (hence the need to calibrate the air flow through the device). the efficiency of an impactor should have a sharp cutoff curve, with the ideal impactor acting like a sieve, with all particles above a certain size, known as the cut-off size, being captured by the agar plate. this feature makes impactors highly suitable as particle size classifiers, with particles greater than a given size being separated from the air flow, while smaller particles remain airborne. a single-stage impactor has one cut-off size, so only requires one set of nozzles and an agar plate (see example in figure 1c ). cascade impactors can be used to gain information on the particle size distribution of an aerosol, with the particle-laden air flow being passed through successive tiers of nozzles and impaction plates. each tier, known as a stage, will collect particles of a specific size with the smaller particles remaining airborne and passing on to the next stage. at each subsequent stage, the nozzle diameter will become progressively smaller, hence the jet velocity increases, and the particle cut-off size is reduced. finally the air flow will pass through a filter to allow remaining small particles to be captured. by weighing the impaction plates from each stage and the filter, before and after sampling, the fraction of the total mass in each particle size range can be established. after cultivation the number of cfu should be enumerated and the counts corrected by positive-hole correction method, which accounts for deposition of multiple bioaerosol particles at the same deposition area. 44e47 in practice, each stage in a cascade impactor will not behave entirely like a sieve, and some particles will be deposited in the passageways between the stages or may bounce off the impaction plates and avoid capture. the airflow over the impaction plates may also be disturbed by the build-up of deposited particles, leading to altered collection efficiencies; however, this can be overcome by the use of multi-jet impactors with >100 nozzles. the bioefficiency of impactors is air streamlines collection substrate reduced due to the shear forces on the bioaerosol particles within the jet and on impaction with the agar plates. the microbial species under investigation along with jet velocity and jet-to-plate distance have been found to play an important role in the enumeration of bioaerosols. 48 desiccation of the pathogen will also reduce bioefficiency, which can be overcome by mineral-oil-spread agar plates. 49 despite the drawback of reduced viability, impactors are frequently used in sampling for many airborne pathogens. 18, 50, 51 virtual impactors also use the centrifugal force and inertia to separate particles depending on their diameter. however, virtual impactors do not collect on to an agar plate but instead have a collection probe operating with a minor flow. this works by particles entering the impactor and being carried by the major flow around a bend ( figure 6 ). the smaller particles are able to follow the streamlines around the curve while the particles larger than the cut-off diameter of the apparatus have sufficient inertia to carry them into the collection probe. the minor flow in the collection probe carries these larger particles on to a collection filter. likewise the smaller particles are collected on a filter in a separate part of the device. virtual impactors usually have only one or two stages, as each separation stage requires control of both the major and minor flow rates. the use of a collection probe rather than an agar plate avoids issues of particle bounce and deposition build-up; however, virtual impactors suffer collection losses near to the size of the cut-off diameter at the inlet of the probe. a useful feature of these devices is that the airflow in effect concentrates the particles larger than the cut-off size into a smaller volume of air, making virtual impactors useful as particle concentrators. 52 slit impactors operate using the same principles of centrifugal force and particle inertia as described with regard to other impactors. rotating agar plates are especially useful as they provide a record of bioaerosol concentration over a specified time-period to enable certain activities to be monitored. bioaerosol particles enter the apparatus through a slit, causing the particles to impact on the slowly rotating agar plate below. the smallest particles will escape capture by following the streamlines of the air flows over the plates through the passageways to the outlet. impactors may also take the form of sticky plastic rods, such as rotorods (ted brown associates, los altos hills, ca, usa), or sticky glass plates where the airflow rate through the device can be adjusted in order to vary the collected particle size diameter, for example versatrap spore trap cassette (skc, inc.). 53 examples of impactors include various single-stage and multi-stage anderson impactors, aerotech n-6 impactor (aerotech laboratories, coventry, uk), air test omega (lcb, la salle, france), air samplair mas-100 (merck, lyon, france), and bioimpactor 100-08 (aes), biostage impactor (skc, inc.) among many others. 50,54e59 filters personal samplers are small, portable devices that are attached to workers' clothing to provide a representative sample of the exposure of the individual to hazardous aerosol. as with larger devices, personal samplers require a pump to draw air through the device, with a sample head, foam, or cyclone being used as pre-selectors for particle size. 6 the bioaerosol particles are collected on to filters from where they can be transferred on to plates or dissolved into a liquid solution for culturing, or examined by microscopy (e.g. immunofluorescence). sampling by filtration is commonplace in aerosol collection but less popular for the collection of bioaerosol particles due to the loss of bioefficiency through desiccation of the pathogen; however, there have been notable successes with filter collection of bioaerosols. 60, 61 fibrous filters consist of layers of fine fibres with relatively substantial gaps between the fibres that allow the filter to be between 70% and 99% air. as particles pass through the filter they are captured by the fibres. membrane filters have a complex pore-like structure and a porosity of about 50e90% less than fibrous filters. as particle-laden air enters the membrane filter, the particles are deposited on the pore structures, with the benefit that particles much smaller than the pore diameters may be successfully captured. personal samplers with various filters include the inhalable gsp samplers (cis; bgi, inc., waltham, ma, usa) used with teflon and polycarbonate filters, pas-6 sampling heads containing polytetrafluoroethylene (ptfe) filters (millipore, merck, france) and the button aerosol sampler containing gelatin filters (skc, inc.). 23, 60, 61 other bioaerosol sampling techniques less widely used bioaerosol sampling techniques include electrostatic precipitation and condensation techniques. on entering the inlet of an electrostatic precipitator, the bioaerosol particles are electrically charged at the inlet before progressing through an electric field, where they are separated from the air flow and deposited on to charged plates. although there is active research into the natural charge on bioaerosol particles and the efficiency and design of electrostatic precipitators, there is concern that the electric field undermines air streamlines collection probe figure 6 . particle-laden airflow in a virtual impactor. the viability of microbes and that more extensive investigations are required into this sampling technique. 53,62e65 sampling of bioaerosol through condensation techniques involves the air sample being processed through a humidifier. subsequently the warm, humid air is rapidly cooled with the bioaerosol particles acting as condensation nuclei. although this method can be used effectively to amplify small microbes, hence improving their chances of detection, the system is complex to use and heat transfer to the microbes may result in a loss of viability. 53, 55, 66 choosing the bioaerosol sampler considerations when choosing a bioaerosol sampler include the type and size of micro-organisms under investigation, the environment where the sampling is to be undertaken, and cost. other factors, more specific to active samplers, should include ease of cleaning/disinfection and precautions that need to be implemented to prevent exhaust air from contaminating the sampling environment. 8 manufacturers' websites usually provide information regarding the suitability and cost of their devices; however, more revealing is the practical use made of bioaerosol samplers by investigators in the field. sampling for airborne pathogens that may pose a health risk is not limited to healthcare environments. wastewater treatment plants (wwtps), farms and slaughterhouses, public and residential buildings, compost facilities and the general outdoor environment have all been the focus for bioaerosol studies and much can be learned from such research. 23,38,50,51,60,67e101 if the research is focused on an individual's risk of exposure to harmful airborne microbes, then the obvious choice of device is the personal sampler. these samplers can be worn on the person's clothing and have proven successful in capturing fungi, bacteria, and even viruses. 60, 98, 102, 103 one notable study investigated the potential for workers at a danish wwtp to be exposed to aerosolized noroviruses (novs), adenoviruses (advs), endotoxins, moulds, and bacteria. 60 this is consistent with previous studies reporting increased occurrence of gastrointestinal illness among wwtp workers compared with control groups. 104, 105 the study used inhalable gsp samplers (cis; bgi, inc.) to monitor the exposure of sixteen workers. teflon filters were fitted to the gsp samplers to allow endotoxin capture, whereas polycarbonate filters were successfully used for bacteria, mould, and virus collection. this study was the first to detect viruses, specifically norovirus gi, using gsp samplers. 47 the exposure risk experienced by workers in a slaughter house was also undertaken with the wearing of personal samplers. 23 the samplers consisted of pas-6 sampling heads containing 1 mm pore size polytetrafluoroethylene (ptfe) filters (merck millipore sas, molsheim, france) attached in the breathing zone and were connected to portable sampling pumps (gilian 3500, sensidyne, inc., st petersburg, fl, usa). the ptfe filters successfully captured wu polyomavirus and human papillomavirus 120 along with other pathogens. one drawback of this study, which was focused on analysing the inhaled breath of the workers, was that it became apparent that the filters were also sampling exhaled breath. this would have been overcome by the use of larger static samplers placed away from workers' immediate environment that were able to sample a bulk background air volume. on occasion, using more than one type of sampler may be necessary to overcome specific sampler limitations. 53 personal samplers have also been used as static samplers in a variety of situations. gelatin filters (3 mm pore size) fitted to a button inhalable aerosol sampler (skc, inc.) have successfully captured influenza a virus (h5n1) nucleotides, dermatophagoides allergens (der f 1 and der p 1), and bacillus subtilis in a laboratory setting; 61 whereas other studies have used gelatin filters with iom personal samplers (skc, inc.) to successfully capture airborne legionellae from a wwtp and shower rooms in nursing homes and methanobrevibacter species and saccharopolyspora rectivirgula (causative agents of farmer's lung) in a dairy barn. 21, 106 however, gelatin filters were noted to perform poorly in high-humidity environments, as they dissolved when sampling in a shower room for >30 min. 21 gelatin filters fitted to iom samplers have been shown to have good efficiency in capturing total and viable legionellae but perform very poorly in capturing culturable samples. 21 midget impingers (skc, inc.) have been successfully used to assess the effectiveness of a selection of surgical face masks against aerosolized influenza. 107 having undertaken field work, it may be possible to correlate field data to laboratory results to assess the potential risk to workers' health of exposure to other degrees of contamination. a study investigating organic dust toxic syndrome (odts) in a seed handling factory used gsp inhalable samplers attached to workers' clothing to collect bioaerosol samples during 8 h shifts. 108 in the laboratory it was determined that a rotating drum (hse rotating drum dustiness tester, j.s. holdings, hertfordshire, stevenage, uk) containing contaminated dust could successfully aerosolize bacteria and fungi. by comparing results with the samples collected by the personal samplers, it was possible to calculate the concentration of airborne microbes to which the workers would be exposed from the tested dust. one major limitation with personal samplers is their relatively low flow rate, which can be as low as 2 l/min. 21, 23, 93, 106 therefore longer sampling times are more appropriate to sample a significant volume of air and this may lead to loss of bioefficiency through desiccation of the pathogen, however the loss of bioefficiency is dependent not only on sampling time, but also the microbial species and relative humidity. 6, 109 in comparison with personal samplers larger static samplers, with their associated higher flow rates, enable the capture of larger, more representative air samples over the same time interval. their associated flows rates vary considerably from about 12.5 l/min for the biosampler (skc, inc.) and the agi-30 (ace glass, inc.) through 100 l/min for the coriolis cyclone (bertin technologies) and the mas-100/a (merck), to 800 l/min for a described impactor. 21, 22, 106 larger samplers have been used extensively in healthcare environments and in other indoor and outdoor environments to successfully capture viruses, bacteria, and spores. 50,110e113 investigating the aerial transmission dynamics of influenza gained impetus during the recent h5n1 avian influenza pandemic; however, prior to this and during the h1n1 pandemic, work was undertaken to assess the risk to healthcare workers carrying out aerosol-generating procedures (agps) on h1n1positive patients. 1e4 using glass may three-stage impingers (produced at health protection agency, porton down, uk), air was sampled 1 m from the head of the h1n1-positive patient while agps were being undertaken. the impinger operated at 55 l/min for 40 min intervals and classified the particles into three aerodynamic size ranges (>7.3 mm, 4e7.3 mm and 0.86e4 mm) to assess the respiratory fraction. the air was collected into 15 ml of phosphate-buffered saline and samples analysed using quantitative reverse transcriptionepolymerase chain reaction. the study showed that the may three-stage impinger proved successful in capturing h1n1 rna. other studies have used larger static samplers to investigate airborne microbial concentration in operating theatres and recovery rooms, the aerial spread of mrsa in hospitals and residential environments, along with investigations undertaken in non-healthcare environments. 50, 71, 82, 106, 110, 114, 115 determining the bioefficiency the most important aspect of bioaerosol sampling for the user to understand is bioefficiency. 7 the bioefficiency of the sampling device is affected by the mechanical stress and desiccation experienced by the pathogen and will vary with the type of sampling device chosen, the sampling time, the type of pathogen under investigation and environmental conditions. 48, 109 many studies have compared the effectiveness of various samplers but unless previous studies have examined the pathogen that you wish to investigate then such studies are of limited use in providing information on bioefficiency. 20e22, 24, 29, 30, 59, 116 it is therefore necessary to test the sampler/pathogen combination in a laboratory, preferably at a similar humidity to that which is expected in the field. this can be undertaken by spiking the sampler with a known concentration of the pathogen and then assessing the concentration collected. 60 however, earlier studies used various other methods, such as using two samplers in tandem or parallel, to assess sampling efficiency. 117 surrogate viruses may be used to limit the hazard when investigating high-risk pathogens, but it should be borne in mind that each pathogen responds uniquely to the conditions experienced. 118 the time-interval during which the sampler will operate should also be replicated during laboratory testing in order to identify any operational issues or time-related loss of bioefficiency. during such bioefficiency tests, inherent variations in performance of the sampler may also become evident over different particle size ranges. 119 with the limitations of different sampling devices being widely acknowledged and variation in collection efficiency between such devices being noted, establishing the bioefficiency of your chosen sampler against the target microbe in itself provides a valuable contribution to the field of bioaerosol sampling. 11, 53 if the target microbe is unknown and a general assessment of bioaerosol particles present in an environment is sought, then the use of different types of sampling devices will mitigate the limitations of individual samplers, making a comprehensive study more likely. finally this is also a good opportunity to test the storage, enumeration and identification procedure, be that through cultivation and visual enumeration of the cfu, various pcr techniques, metagenomics, mass spectrometry, epifluorescence microscopy, matrix-assisted laser desorption/ionization mass spectrometry or other means. 3, 21, 23, 50, 54, 60, 92, 93 these enumeration and identification methods, along with their advantages and limitations, have recently been discussed and are not repeated here; however, it should be noted that quantification of the pathogens captured by active samplers is normally expressed per cubic metre of air, which provides another reason to determine accurately the air flow rate of the device and the sampling time-period. 11, 50, 53, 92 bioaerosol sampling out in the field the statistical analysis relating to bioaerosol sampling varies considerably depending on the nature of the study, and an investigator would do well to consult a statistician when designing any study. 3, 106, 110 errors arising from bioaerosol sampling are typically threefold: random error of samples containing a finite number of discrete particles; errors due to non-uniformity of the bioaerosol distribution in the atmosphere; and errors due to sampling techniques. 117 the sampling period will be influenced by factors that include the operational limitations of the sampling devices, such as the rate of evaporation of the collecting liquid, or the amount of time one has access to a site. however, even with such matters taken into consideration, the sampling timeperiods used by investigators varied widely, from as low as 3 min to several hours. 60, 110 the longer the sampling period the greater the volume of air being collected, thus the higher probability of capturing airborne pathogens, as long as the bioefficiency of the sampler does not deteriorate with time. when using samplers with differing flow rates concurrently, it may be preferable to calculate the sampling time of each device so that the volume of air captured is the same. if short sampling periods are most suited to the device being used, then repeating the sampling in triplicate should be considered. 93 the overall length of the study may span from one day to a couple of years. 120 new techniques such as lightinduced fluorescence (lif) methodologies are being implemented in real-time online biological particle sensors, enabling continuous on-site detection of bioaerosol counts. 121 the height of the sampling device above floor level within an indoor environment is also important if the investigation is collecting samples from the breathing zone of patients. 3, 110 if a more general bioaerosol sampling regime is undertaken, then sampling at different heights within a room and at several spatial locations will provide good sampling coverage. 122 once the samples have been taken, they should be transported and stored in conditions that preserve their efficacy until cultivation and/or identification can be undertaken. having previously undertaken a bioefficiency study, the investigator is in a strong position to estimate with reasonable accuracy the concentration of the target bioaerosol in the sampled environment. combining this information with the genus of the captured microbe, the particle size range (informing on the penetration of the respiratory system), and the health effects on the human or animal population, conclusions can be made regarding bioaerosol concentration and health risk. presently there is no international consensus on the acceptable exposure limits of bioaerosol concentration, with a recent review drawing attention to this research deficit. 53 a lack of bioaerosol studies targeting viruses and archea has also been identified, further limiting our understanding of the impact of airborne microbes on human health. 123 several of the studies discussed in this review were based in bioaerosol-emitting facilities, such as wwtp and compost facilities, where the exposure to harmful microbes is a cause for concern for occupational safety reasons and for risk to health of the population in the surrounding area. in such cases the task for current research is to establish suitable doseeresponse relationships to enable health-based exposure limits for bioaerosols to be derived. 124 such exposure limits would be designed to protect the general population from the ill effects of long-term exposure to bioaerosols. the situation for healthcare studies is quite different with a wide array of 'at risk' groups needing to be considered, making the derivation of health-based exposure limits challenging. the staff, patients or their visitors may be the source of the bioaerosol health risk, such as with sars virus, respiratory syncytial virus, influenza, measles, mumps, or rubella viruses. 53 their stay in hospital may be brief and may not be contained to one ward, making it difficult to trace the source of an outbreak. the wider environment may also be a source of harmful bioaerosols, such as an increased risk of airborne aspergillus during construction activities or the risk of legionella bacteria in hvac (heating, ventilation, and air conditioning) or water systems. to gain a greater understanding of the transmission dynamics of certain airborne diseases and to increase hygiene standards through improved infection control, bioaerosol sampling studies have frequently focused on healthcare environments. bioaerosol sampling in operating theatres (ots) is motivated by the need to reduce the incidence of surgical-site infections. with the inclusion of high-efficiency particulate air (hepa) filters within ots, cleanroom technology standards have frequently been applied to these healthcare settings with the airborne particulate count being monitored. with the observations that bioaerosol sampling is time consuming, requires trained personnel and that results are not instantaneous, interest has grown in the correlation between microbiological and dust contamination, to the extent that it has been suggested that microbial sampling should be limited to epidemics, validation of protocols, or changes to the ot environment that may affect microbial content. 125 further investigations have been unable to establish a relationship between dust particles and microbes in ots, although a relationship between the number of airborne microbes and human activity was confirmed. 126 this relationship between increased airborne bacterial concentration and human activity is widely accepted. 127, 128 approximately 5e10% of human skin debris carries bacteria and skin shedding increases with physical activity, with millions of particles being shed per person each day. in addition to measuring the dust count using a light-scattering particle analyser, bioaerosol sampling was undertaken using both passive and active sampling. settle plates with a 90 mm diameter were placed at strategic locations, 1 m above floor level, throughout the ot and left exposed during 23 surgical operations. the active sampling was undertaken using a single stage slip-type impactor operating at 25 l/min for duration of 20 min, with samples taken during 13 operations. the study did observe an increased concentration of dust particles >5 mm during conventional surgery as opposed to scope procedures. an inverse relationship between dust and bacterial concentration was reported. as the ot door opened into the anaesthetic room, the turbulent airflow resulting from the pressure differential between the two rooms in effect removed dust from the ot. however, the bacterial concentration increased and it was proposed that this may be due to increased movement of the staff. this highlights the need for the investigator to be aware of airflow in and between areas under investigation, in addition to patterns of human activity. over a three-year sampling period, a study of surface and airborne microbial contamination was conducted in 29 ots. 14 both passive and active sampling was conducted during the commissioning of ots, during major renovations and surgical activities, as well as in adjacent corridors. passive sampling was undertaken using 90 mm diameter settle plates using the 1/1/1 scheme (explained in the section on 'passive sampling') with tryptic soy agar used for the total aerobic bacterial count, whereas sabouraud dextrose agar with chloramphenicol was used for fungal isolation. active sampling of airborne contamination was carried out using a duosas 360 sampler (pbi international, milan, italy) operating at 180 l/min. the study found a moderately strong correlation between the active and passive sampling methods, with the discrepancy between the two techniques being attributed to the relatively short sampling period and limited spatial collection zone of the active sampler compared with the longer exposure time of the settle plates. the investigation also concluded that bioaerosol sampling could be used for the evaluation of the ventilation and air conditioning system within the ot. comparing the results from sampling during different surgical procedures also had the potential to inform improved surgical hygiene practice. correlation between active and passive sampling was also described during a study comparing different ventilation regimes in ots. 12 using a surface air system sampler (sas, international pbi, milan, italy) operating at 180 l/min and settle plates, both with tryptic soy agar, the study showed that unidirectional airflows within ots did not guarantee low counts of airborne bacteria. the study also confirmed that an increased number of people and door openings in an ot influenced an increase in bacterial count. a year-long monitoring of airborne microbial contamination in ots and surrounding areas has also been studied using mixed effect models to assess the influence of air temperature, relative humidity, number of people in a space and different sampling locations on levels of co 2 , suspended particulate matter, and airborne bacteria. 110 bioaerosol sampling was undertaken using an andersen one-stage viable impactor (n6; andersen samplers, atlanta, ga, usa), with tryptic soy agar. the sampling period was 3 min, with duplicate samples taken at a height of 1.2e1.5 m from floor level to represent the breathing zone of healthcare workers. in concurrence with a previous study, bacillus spp., micrococcus spp., and staphylococcus spp. bacteria were frequently found in the operating theatre area. 116 the study found a positive correlation between airborne bacterial concentration and suspended particulate matter (pm 10 and pm 2.5 ). a positive correlation was also found between the number of people in a room and co 2 concentrations, but, when temperature, relative humidity and sampling location were accounted for, no significant correlation was found between the number of people and bacterial concentrations. one exception was the postoperative recovery room where there were a greater number of people, higher co 2 levels, and higher concentrations of bacteria. caution should be exercised when investigating the relationship between the number of human occupants and the concentration of airborne microbes. although a correlation has been noted within one room of this investigation and in previous studies mentioned in this section, a study carried out in an environmental chamber suggested that outdoor air had a greater influence on the bioaerosol composition. 129 airborne viral and bacterial concentrations were monitored in the outpatient area of a paediatric unit and in the paediatric emergency room twice a week for one year. 111 the sampled air was filtered through a closed face, threepiece disposable, plastic cassette containing a 0.2 mm polytetrafluoroethylene filter and operating at 12 l/min. the air within the outpatient area was sampled for 8 h a day whereas the air in the emergency room was monitored during 24 h periods. in both cases, the bioaerosol sampler was positioned in the breathing zone between 1.2 and 1.5 m above floor height. during the course of the study, 186 filter samples were taken and airborne adenovirus and mycoplasma pneumoniae were detected in both monitored areas, with greatest prevalence found in the outpatient area. the negative control was the use of filters with no air flow passing though the sampling device. no adenovirus and m. pneumoniae was found on the negative controls. the study did, however, passive sampling e settle plates e consider using the 1:1:1 scheme with 90 mm plates 11 e surface sampling e consider using membranes (e.g. nitrocellulose) as an alternative to contact plates on curved surfaces e surface and aerial contamination may have different sources e results from passive and active samplers should not be assumed comparable active sampling e impactors e collection on to agar plates e collection efficiency highly dependent on particle size (should be sieve-like in performance) e ideal as a particle size classifier e loss of bioefficiency: shear forces, desiccation, particle bounce, and deposition build-up e virtual impactors e collection into liquid, thus minimizing risk of desiccation e collection efficiency dependent on particle size e useful as particle concentrators e slit impactors e collection on to agar plates e loss of bioefficiency: shear forces, desiccation, particle bounce, and deposition build-up e records variation in bioaerosol concentration over a specified time-period e impingers e collection into liquid, thus minimizing risk of desiccation e loss of bioefficiency: shear forces, re-aerosolization, evaporation, adherence to device walls e collection efficiency dependent on particle size e cyclones (wetted) e collection into liquid, thus minimizing risk of desiccation e loss of bioefficiency: shear forces, liquid carryover, evaporation, adherence to device walls e may be used as pre-classifiers for particle size e collection efficiency dependent on particle size e vary considerably in size and airflow rate e filters e small, portable personal samplers e loss of bioefficiency: desiccation e collection efficiency dependent on particle size (sample head, foam, or cyclone being used as pre-selectors) in the laboratory e calibrate the flow rate of the active sampler e ensures the maximum collection efficiency e influences the size of particles collected e determine the bioefficiency of the sampler against the target pathogen e test in air conditions expected in the field (relative humidity and temperature) e spike sampler with known concentration of the target pathogen e each type of pathogen has a unique response to conditions experienced e surrogate viruses may be used in place of hazardous pathogens; however, response may differ from target pathogen e check that bioefficiency is maintained throughout planned sampling time e determine errors in numeration when sampling from a known, repeatable concentration of the target pathogen e ensure that the sampler exhaust is not a source of pathogen contamination to the environment e test the storage, enumeration, and identification procedure in the field e position of the inlet sampler e avoid strong airflows around the inlet of the sampler e if using an inlet nozzle, position horizontally e ensure that the sample position is beyond the range of droplet fallout from a source (e.g. coughing/vomiting patient) e aerial microbial concentration e expect non-uniformed concentration in the area studied (expect associated sampling errors) e consider taking samples at various locations in the area studied e note human/animal activity and number of humans/ animals present, as this may influence concentration of certain microbes e be aware of airflow patterns due to hvac (heating ventilation and air conditioning) and natural ventilation e note air quality: relative humidity, temperature (also consider co 2 and particle dust count) e there may be seasonal variation in concentration of the target pathogen e active samplers: quantification of pathogens e expressed as enumeration per cubic meters of air e need to know the collection time and flow rate of the sampler notice evidence of seasonal variation in this taiwanese hospital, with airborne adenovirus peaking in the summer months, whereas m. pneumoniae detection rates increased in the autumn and winter. identifying peaks in bioaerosol contamination during certain months allows for ventilation rates in affected areas to be increased to reduce the risk to patient health. effective ventilation and controlled airflow patterns within wards alongside improvements in hygiene and operational procedures are arguably the strongest defence against high concentrations of airborne microbial contamination. 4, 112, 130, 131 these hospital-based bioaerosol investigations highlight many of the issues facing the bioaerosol researcher. the methodologies applied differ between research teams. sampling devices vary with regards to their collection efficiency. concentrations of airborne pathogens are influenced by airflows within the hospital building and seasonal variation. the influence and correlation between human activity, air quality (humidity, temperature, co 2 concentration) and dust particle count on bioaerosol concentration is uncertain, with contradictory results being presented. the transmission dynamics of some pathogens are not fully understood and an airborne component to transmission should not be overlooked. even with a good understanding of the concentrations of bioaerosols in an environment, the health-based exposure limits for a diverse group of patients and staff may not be known. yet such research can inform on appropriate ventilation rates to maintain good air quality, assess the bioaerosol risk to patients and staff, gain a greater understanding of the transmission dynamics of pathogens, and suggest improvements to hygiene procedures. amid all the uncertainties and difficulties of bioaerosol research, the goal remains to gain a greater understanding of airborne pathogens and to provide safe healthcare environments for our patients and staff. a summary of the key points in bioaerosol sampling is presented in box 1. a wide variety of bioaerosol samplers have been used to investigate airborne pathogens in healthcare facilities and other environments. we have described the underlying principles behind bioaerosol sampling devices along with benefits and disadvantages of various designs. examples of bioaerosol sampling have been given to point to best practice and to highlight the wide array of devices used and pathogens captured. due to the unique response of each variety of pathogen to environmental conditions and the stresses experienced in differing sampling devices, the investigator should commence studies by determining the bioefficiency of the chosen sampler and the pathogen under investigation within laboratory conditions. from such foundations, sampling for bioaerosol material in the complexity of the field holds greater certainty of successful capture of low-concentration airborne pathogens. avian influenza h5n1 transmission in households, indonesia airborne transmission of influenza a/h5n1 virus between ferrets influenza aerosols in uk hospitals during the h1n1 (2009) pandemic e the risk of aerosol generation during medical procedures roles of sunlight and natural ventilation for controlling infection: historical and current perspectives a community-wide outbreak of legionnaires' disease linked to industrial cooling towers e how far can contaminated aerosols spread? the development of a bioaerosol sampler for the detection of enzymes in industry bioaerosol evaluation in indoor environments iso 14698-1:2003. cleanrooms and associated controlled environments e biocontamination control e part 1: general principles and methods aerosol technology environmental control of microbial contamination in the operating room the index of microbial air contamination operating theatre ventilation systems and microbial air contamination in total joint replacement surgery: results of the gisio-ischia study prospective survey of indoor fungal contamination in hospital during a period of building construction microbial air monitoring in operating theatres: experience at the university hospital of parma post-flood measurement of fungal bio-aerosol in a resourcelimited hospital: can the settle plate method be used? healthcare environments and spatial variability of healthcare associated infection risk: cross-sectional surveys comparative efficiency of nitrocellulose membranes versus rodac plates in microbial sampling on surfaces prediction of mold contamination from microbial volatile organic compound profiles using solid phase microextraction and gas chromatography/mass spectrometry physical and chemical properties of aerosols. london: blackie academic & professional investigation of inherent and latent internal losses in liquid-based bioaerosol samplers evaluation of sampling techniques for detection and quantification of airborne legionellae at biological aeration basins and shower rooms bioaerosol sampling with a wetted wall cyclone: cell culturability and dna integrity of escherichia coli bacteria metagenomic detection of viruses in aerosol samples from workers in animal slaughterhouses the impact of sampler selection on characterizing the indoor microbiome effect of sampling time on the collection efficiency of all-glass impingers long-term sampling of airborne bacteria and fungi into a nonevaporating liquid collection of airborne microorganism into liquid by bubbling through porous medium influence of storage on the fungal concentration determination of impinger and filter samples rapid quantification of bioaerosols containing l. pneumophila by coriolis ã� m air sampler and chemiluminescence antibody microarrays sampling port for real-time analysis of bioaerosol in whole body exposure system for animal aerosol model development reverse-flow centrifugal separators in parallel: performance and flow pattern the cyclone scrubber e a high efficiency wet separator the development of sampling methods for the assessment of indoor bioaerosols development of a biosensor for airborne proteases an improved wetted wall bioaerosol sampling cyclone a direct approach to the design of cyclones for aerosol-monitoring applications computational fluid dynamics (cfd) and empirical modelling of the performance of a number of cyclone samplers use of a culture-independent approach to characterize aerosolized bacteria near an open-freestall dairy operation correlation between olea europaea and parietaria judaica pollen counts and quantification of their major allergens ole e 1 and par j 1-par j 2 field evaluation of a personal, bioaerosol cyclone sampler wetted wall cyclones for bioaerosol sampling chamber evaluation of a personal, bioaerosol cyclone sampler development of a personal bioaerosol sampler based on a conical cyclone with recirculating liquid film new sampler for the collection, sizing, and enumeration of viable airborne particles positive-hole correction of multiple-jet impactors for collecting viable microorganisms inertial samplers: biological perspectives an alternative approach for the correction of bioaerosol data collected with multiple jet impactors effect of physical and biological parameters on enumeration of bioaerosols by portable microbial impactors enhancing bioaerosol sampling by andersen impactors using mineral-oil-spread agar plate reduction and characterization of bioaerosols in a wastewater treatment station via ventilation critical working tasks and determinants of exposure to bioaerosols and mvoc at composting facilities aerosol measurement: principle, techniques and applications review of bioaerosols in indoor environment with special reference to sampling, analysis and control mechanisms prediction of mold contamination from microbial volatile organic compound profiles using head space gas chromatography/mass spectrometry effects of condensational growth on culturability of airborne bacteria: implications for sampling and control of bioaerosols comparison of two biological aerosol sampling methods differences in detection frequency as a bioaerosol data criterion for evaluating suspect fungal contamination comparative performance of impactor air samplers for quantification of fungal contamination a comparison of the efficiencies of a portable biostage impactor and a reuter centrifugal sampler (rcs) high flow for measuring airborne bacteria and fungi concentrations exposure to airborne noroviruses and other bioaerosol components at a wastewater treatment plant in denmark use of gelatin filter and biosampler in detecting airborne h5n1 nucleotides, bacteria and allergens charge levels and gram (ae) fractions of environmental bacterial aerosols collection of airborne microorganisms by a new electrostatic precipitator design and development of an electrostatic sampler for bioaerosols with high concentration rate influence of secondary flows on the collection efficiency of a cylindrical electrostatic precipitator development and evaluation of a novel bioaerosol amplification unit (bau) for improved viral aerosol collection the efficient method for simultaneous monitoring of the culturable as well as nonculturable airborne microorganisms airborne enteric coliphages and bacteria in sewage treatment plants microorganisms in bioaerosol emissions from wastewater treatment plants during summer at a mediterranean site reducing bioaerosol dispersion from wastewater treatment and its land application: a review and analysis relationship between airborne detection of influenza a virus and the number of infected pigs the potential for community exposures to pathogens from an urban dairy food animal transport: a potential source of community exposures to health hazards from industrial farming (cafos) bioaerosols from municipal and animal wastes: background and contemporary issues airborne bacteria and carcass contamination in slaughterhouses distribution and identification of culturable airborne microorganisms in a swiss milk processing facility human occupancy as a source of indoor airborne bacteria /3)-b-d-glucan, fungi, and dust mite allergens in flood-affected homes of new orleans evaluation of respiratory symptoms and their possible association with residential indoor bioaerosol concentrations and other environmental influences /3)-b-d glucan, and endotoxin from flood-affected materials collected in new orleans homes characterization of seasonal indoor and outdoor bioaerosols in the arid environment of el paso. texas isolation of staphylococcus aureus and antibiotic-resistant staphylococcus aureus from residential indoor bioaerosols indoor and outdoor bioaerosol levels at recreation facilities, elementary schools, and homes sampling for indoor fungi autocorrelation and variability of indoor air quality measurements environmental assessment of aerosols, bioaerosols, and airborne endotoxins in a machining plant bioaerosol concentrations in noncompliant, compliant, and intervention homes in the midwest exposure to culturable airborne bioaerosols during noodle manufacturing in central taiwan workplace exposure to bioaerosols in pet shops, pet clinics, and flower gardens characterizations and relationships between outdoor and indoor bioaerosols in an office building bioaerosol emissions from open window composting facilities: emission characterisation and dispersion modelling improvements presence of legionella and free-living amoebae in composts and bioaerosols from composting facilities spatial variations in airborne microorganism and endotoxin concentrations at green waste composting facilities evaluating the quality of bioaerosol risk assessments for composting facilities in england and wales particle size distribution of airborne aspergillus fumigatus spores emitted from compost using membrane filtration sorting and recycling of domestic waste. review of occupational health problems and their possible causes ambient influenza and avian influenza virus during dust storm days and background days. environ personal sampler for monitoring of viable viruses; modelling of outdoor sampling conditions culturable airborne fungi in outdoor environments in beijing evaluation of four bioaerosol samplers in the outdoor environment london plane tree bioaerosol exposure and allergic sensitization in the adaptation of existing personal inhalable aerosol samplers for bioaerosol sampling performance characteristics of the button personal inhalable aerosol sampler health effects among workers in sewage treatment plants work related symptoms among sewage workers: a nationwide survey in sweden characterization of bioaerosols from dairy barns: reconstructing the puzzle of occupational respiratory diseases by using molecular approaches effectiveness of surgical masks against influenza bioaerosols microbial diversity in bioaerosol samples causing odts compared to reference bioaerosol samples as measured using illumina sequencing and maldi-tof effect of sampling time and air humidity on the bioefficiency of filter samplers for bioaerosol collection air quality monitoring of the post-operative recovery room and locations surrounding operating theatres in a medical centre in taiwan surveillance of airborne adenovirus and mycoplasma pneumoniae in a hospital paediatric department indoor air quality varies with ventilation types and working areas in hospitals collection efficiencies of aerosol samplers for virus-containing aerosols air and surface contamination patterns of meticillin-resistant staphylococcus aureus on eight acute hospital wards change in environmental bacterial flora in a new hospital building comparison of the collecting efficiency of microbiological air samplers a slit sampler for collecting and counting air-borne bacteria methods for sampling of airborne viruses the performance of the biotest rcs centrifugal air sampler airborne viable, non-viable, and allergenic fungi in a rural agricultural area of india: a 2-year study at five outdoor sampling stations on-line monitoring of airborne bioaerosols released from a composting/green waste site ventilation and transport; bioaerosols in healthcare environments non-culturable bioaerosols in indoor settings: impact on health and molecular approaches for detection evaluation of exposureeresponse relationships for health effects of microbial bioaerosols e a systematic review environmental controls in operating theatres risk factors for particulate and microbial contamination of air in operating theatres reduction of skin bacteria in theatre air with comfortable, non-woven disposable clothing for operating-theatre staff behaviours and rituals in the operating theatre chamber bioaerosol study: outdoor air and human occupants as sources of indoor airborne microbes the control by ventilation of airborne bacterial transfer between hospital patients, and its assessment by means of a particle tracer: ii. ventilation in subdivided isolation units detection of viruses in used ventilation filters from two large public buildings none declared. none. key: cord-314009-7t1bzc7f authors: barclay, t.; petrovsky, n. title: vaccine adjuvant nanotechnologies date: 2016-10-07 journal: micro and nanotechnology in vaccine development doi: 10.1016/b978-0-323-39981-4.00007-5 sha: doc_id: 314009 cord_uid: 7t1bzc7f the increasing sophistication of vaccine adjuvant design has been driven by improved understanding of the importance of nanoscale features of adjuvants to their immunological function. newly available advanced nanomanufacturing techniques now allow very precise control of adjuvant particle size, shape, texture, and surface chemistry. novel adjuvant concepts include self-assembling particles and targeted immune delivery. these individual concepts can be combined to create a single integrated vaccine nanoparticle-combining antigen, adjuvants, and dc-targeting elements. in the process, the concept of an adjuvant has broadened to include not only immune-stimulatory substances but also any design features that enhance the immune response against the relevant vaccine antigen. the modern definition of an adjuvant includes not only classical immune stimulators but also any aspects of particle size, shape, and surface chemistry that enhance vaccine immunogenicity. it even includes purely physical processes such as texturing of particle surfaces to maximize immunogenicity. looking forward, adjuvants will increasingly be seen not as separate add-on items but as wholly integrated elements of a complete vaccine delivery package. hence, vaccine systems will increasingly approach the complexity and sophistication of pathogens themselves, incorporating highly specific particle properties, contents, and behaviors, all designed to maximize immune system recognition and drive the immune response in the specific direction that affords maximal protection. reduced immunogenicity and thereby efficacy is an unfortunate downside to vaccines based on highly purified or synthetic antigens. this requires the use of vaccine adjuvants to improve vaccine immunogenicity. the term adjuvant has traditionally referred to as any substance that when added to a vaccine antigen increases its immunogenicity. 1 as such, the study of adjuvant action is made complicated by the wide diversity of agents including small molecule immune modulators, mineral salts, oil emulsions, and even whole viruses or bacteria. these agents all exhibit adjuvant action in vivo with no one common feature explaining this activity. almost certainly, these compounds work through many and varied different mechanisms to enhance immune responses to coadministered antigens. increasing use of small protein or peptide antigens allows greater control over vaccine design and synthesis but has further exacerbated issues of poor immunogenicity. this necessitates closer examination of how best to design vaccine delivery and adjuvant systems to maximize antigen immunogenicity, assist targeted immune delivery, and provide potent adjuvant activity. most importantly, this must all be done in a manner that does not compromise vaccine tolerability or safety. 2, 3 the immune system has evolved to protect against invading pathogens. as pathogens present as nano-to microsized particles, it should not be surprising that the immune system has evolved strategies to specifically recognize and respond to particles of particular sizes and shapes. 4, 5 this helps explain the boost to immunogenicity when antigens are presented to the immune system as virus-like particles (vlps) rather than as soluble proteins. particle size was shown to be important for uptake by dendritic cells (dcs), with spherical particles less than 500 nm in diameter having better uptake than larger particles. 6 size is also relevant to t cell maturation whereby poly(lactide-co-glycolide) (plga) microparticles modified with recognition and costimulatory ligands with similar dimensions to dcs were better able to induce t cell activation when compared with nanosized particles 7 that were rapidly phagocytosed. 8 while the idea that size is important to vaccine action is now well accepted, it is also likely that shape, texture, and surface chemistry are equally highly relevant to adjuvant action, 8 albeit less well-explored. shape is known to be important to particle uptake in vivo, half-life, and biodistribution. 9, 10 for example, cylindrical silicon particles exhibited strong liver accumulation, whereas discoidal particles accumulated away from the liver and spherical particles were distributed evenly across tissues. 11 decreasing the diameter of spherical particles from 3 to 0.7 µm resulted in increased accumulation in reticuloendothelial tissues. 11 these tissues are rich in various immune cells, and consequently accumulation in the reticuloendothelial system is an advantage from a vaccine standpoint. notably, high-axial-ratio nanoparticles were shown to better target draining lymph nodes and thereby enhanced antigen presentation and memory b cell responses. 12, 13 surface chemistry has also been shown to be important for adjuvant particle properties, with uptake of larger particles by dcs being enhanced by a positive surface charge. 6 furthermore, while 25 nm diameter polyhydroxylated and polymethoxylated poly(propylene sulfide) nanoparticles and polystyrene nanoparticles were all equally efficient in inducing dc uptake, only the polyhydroxylated particles induced high levels of dc maturation, 14 an effect attributed to the polyhydroxylated surface activating complement thereby acting as an immune danger signal for dc activation. 14 use of hydrophilic polymeric coatings such as polyethylene glycol and polyzwitterions with an overall neutral surface charge that bind water to the surface has also been shown to change particle behavior, 8 with the hydrophobic regions of amphiphilic systems considered to act as immune danger signals that provide adjuvant effects. 15 mechanical properties are yet another factor that need to be taken into account when assessing the immune effect of particles. macrophages and dc have been shown to preferentially phagocytose rigid particles even if these have identical surface chemistry to softer particles. 16 hence making particles more flexible so that they mimic red blood cell properties was shown to increase their half-life in vivo, 11, 17, 18 whereas making them more rigid should improve their uptake by dcs and thereby their immune action. increasingly sophisticated particle manufacturing approaches have enabled ever-tighter control of the size, shape, external chemistry, and mechanical properties of adjuvant particles. this has provided unique opportunities to modify vaccine and adjuvant particles with the aim to maximize their biological action. the remainder of this chapter will describe a wide variety of nano-and microparticulate vaccine and adjuvant systems, their method of manufacture, and how size, shape, and surface chemistry are all important to adjuvant action ( fig. 7.1 ). emulsions are composed of a dispersion of droplets of one liquid in another immiscible liquid, commonly described as either water-in-oil or oil-in-water emulsions. 21 in use as adjuvants, the droplets in an emulsion act as particles and are perceived by the immune system as such. there are two different forms of nanoscale emulsions, both of which are defined as having droplet radii from 1 to 100 nm, which is sufficiently small to generate transparent, isotropic liquid media. the first form of nanoscale emulsion has historically been termed a microemulsion but is better described as a nanodimensional lyotropic liquid crystalline phase. 22 in a microemulsion the structural organization of the two liquids is supported by a relatively large concentration of surfactant. formation is by spontaneous selfassembly with relatively gentle mixing, resulting in stable organization at thermodynamic equilibrium. 22, 23 by contrast, what is termed a nanoemulsion generally uses significantly less surfactant and hence requires high shear forces to make a metastable structure. 22, 24 emulsion adjuvants are thought to work through a depot effect, creating inflammation around the site of antigen injection and thereby enhancing humoral immune responses. 25 early examples of emulsion adjuvants were water-in-mineral-oil emulsions. 26, 27 however, such adjuvants were problematic as mineral oil is not biodegradable, is proinflammatory, and has long-term persistence in the body, thereby leading to major inflammatory side effects including injection site granulomas, pyrexia, and long-term safety issues. 28 care must also be taken with any emulsion adjuvant to avoid any oxidation of the oil component as this could otherwise lead to increases in vaccine reactogenicity. generally, oil-in-water nanoemulsions using biodegradable oils have a better tolerability profile than water-in-oil formulations. a well-known example of an oil-inwater nanoemulsion adjuvant is mf59, which is composed of squalene oil in a citrate buffer using nonionic surfactants. 29, 30 the nanoemulsion is key to the adjuvant effect of mf59 as when tested individually the various components of mf59 failed to demonstrate adjuvant activity. 31 mf59-based vaccine formulations have been used extensively in research, 32 and recent work has included tests as a cationic nanoemulsion delivery system for delivery of a self-amplifying mrna vaccine. 33 nonetheless, the bulk of research has been for influenza vaccines, 34 and while mf59 is licensed for this purpose in elderly humans 31 and has extensive safety data in the elderly population, more limited safety data are available for use in children. of potential relevance, a 2009 pandemic influenza vaccine containing as03, a squalene-based nanoemulsion that is similar to mf59 except for the addition of tocopherol, was associated with a rise in childhood cases of narcolepsy, 35, 36 an autoimmune sleep disorder. this has put the question of pediatric safety of squalene-based emulsion adjuvants back under the spotlight. the immunogenicity of emulsion adjuvants can be increased further by coformulation with additional immune-stimulatory components. interestingly, the original mf59 squalene emulsion adjuvant was initially designed as a delivery system for muramyl tripeptide phosphatidylethanolamine (mtp-pe), a modified mycobacterial peptidoglycan with potent adjuvant activity. 37 mtp-pe activates innate immune receptors including nucleotide-binding oligomerization domain-containing protein 2 (nod2) 38 and various toll-like receptors (tlr), 39 leading to nuclear factor kappa-b (nfκb) activation and induction of inflammatory cytokines. 40 however, the mtp-pe component was ultimately removed from mf59 because of excessive reactogenicity. 41 a more recent version of a combined adjuvant emulsion and immunostimulator approach is the as02 adjuvant, which is an oil-in-water emulsion combined with qs21 saponin and the tlr4 agonist, monophosphoryl lipid a (mpl). 42 intranasal delivery of vaccines provides advantages in terms of convenience and induction of mucosal immunity, a major benefit when protecting against respiratory or mucosal pathogens. 43, 44 a nasal vaccine containing a soybean oil nanoemulsion adjuvant successfully enhanced humoral and cellular responses in mice, [43] [44] [45] [46] [47] consistent with the utility of this approach. a library of more than 100 intranasal adjuvant candidate formulations consisting of oil-in-water nanoemulsions containing various cationic and nonionic surfactants were tested in mice and demonstrated that varying the physicochemical properties of the surfactant components (charge, surfactant polar head size, and hydrophobicity) and the surfactant blend ratio of the intranasal adjuvant formulations could modulate the strength and type of the immune response. 48 this indicates that there may be considerable scope for further optimizing nanoemulsion adjuvants for intranasal use. however, the tolerability and safety in humans of this approach have yet to be confirmed. it was recently shown that nanoemulsion adjuvants induce epithelial cell death via activation of caspases 1, 3, and 6-9. 49 furthermore, the safety of nasal vaccine adjuvants has remained under a cloud since the experience of nasalflu, an intranasal influenza vaccine containing a detoxified enterotoxin adjuvant, that was associated with increased cases of facial nerve palsy in clinical trials. 44 multiple or double emulsions, most often water-in-oil-in-water formulations, have also been used in vaccine research. 50 however, water-in-oil-in-water multiple emulsion proved to be less stable and induced an excessive inflammatory response when compared with an oil-in-water microemulsion of isopropyl myristate, which successfully enhanced humoral responses to rabies vaccine in mice. 51 these stability issues arise as there are two interfaces with high free energy to stabilize, generally requiring two surfactants that may interact, thereby interfering with stabilization. also, for multiple emulsions there is the potential for an osmotic pressure to develop, placing further strain on the system. 52 even simple emulsions are not without issues. nanoemulsions not at thermodynamic equilibrium have poor stability, and microemulsions suffer from the need for high concentrations of surfactants, which may cause toxicity in vivo. furthermore, the stability of any microemulsion can be easily compromised by dilution, heating, or ph changes. various nanoparticles constructed from disordered, precipitated polymers, also described as nanobeads, have been used for vaccine delivery. poly-ε-caprolactone nanoparticles of two sizes were made, with mean diameters of 61 and 467 nm, by solvent evaporation. only the smaller diameter particles were found to have an adjuvant effect, eliciting both cellular and humoral responses in mice. 53 polystyrene nanobeads covalently conjugated to ovalbumin were found to best activate cd8 t cells when they were 40-49 nm diameter when beads ranging in size from 20 to 2000 nm diameter were compared. 54, 55 another study found that larger beads with diameters from 93 to 123 nm were best at activating cd4 t cells 55 thus, it may be possible to specifically tune nanoparticle size to induce either cellular or humoral immunity. in another system poly(γ-glutamic acid) grafted with l-phenylalanine ethyl ester on 53% of the carboxylic acid groups self-assembled upon the addition of water from organic solution into nanoparticles with diameters of 150-200 nm. 56 in the presence of ovalbumin the nanoparticles encapsulated the antigen and targeted it to dcs, resulting in enhanced dc maturation and immunity. 56,57 inorganic nanoparticles have been used as vaccine delivery vehicles; aluminum oxide nanoparticles having distinctly better performance compared with other metal oxide nanoparticles. 58 such aluminum oxide nanoparticles of 113-355 nm diameter elicited adjuvant effect when peptomers derived from an hiv antigenic sequence and retaining the native α-helical conformation were covalently attached to the external surface. 59, 60 aluminum oxide nanoparticles were also functionalized with ovalbumin and shown to enhance dc activation of t cells via an autophagy-dependent mechanism. 58 particle size was also shown to be important as while both 60 and 200 nm nanoparticles were effective in vitro, the 60 nm aluminum oxide particles were much more effective in being transported to the draining lymph nodes in vivo. vlps can be made using recombinant protein techniques whereby antigenic proteins such as hbsag are synthesized in yeast, following which the antigen self-assembles into 22 nm vlps. 61 vlps are safe and immunogenic, making them ideal vaccine antigens. 62, 63 however, cell-culture systems used for manufacture of vlps can be low yielding and expensive, and hence synthetic methods have been developed for vlp production. for example, short linear synthetic proteins with coiled-coil domains were used for self-assembly of α-helical motif-containing nanoparticles able to present repetitive epitopes on their surface. 64, 65 these synthetic particles had a 25 nm diameter and elicited strong immune responses to surfaceexpressed epitopes. [64] [65] [66] a related nanoparticulate vaccine delivery system was also assembled from peptide chains containing a coiled-coil sequence to drive helical assembly. in this instance the peptide was modified at the n-terminus with a dual chain lipid and with synthetic epitopes at the c-terminus. upon dispersion in aqueous solution, the construct aggregated into ordered synthetic particles having a diameter of 17-20 nm and presenting multiple copies of the antigen on the surface that induced a humoral immune response. 67 in another synthetic system polypeptides designed to self-assemble into β-sheet-rich nanofibers with epitopes attached to the c-terminus generated strong antibody responses without inflammation. 68 similarly to vlp's, the self-assembled structure was critical to the adjuvant affect, the unassembled sequence having no adjuvant properties. 69 it has been shown that the nanofibers are internalized by dcs and macrophages at the injection site, resulting in increased expression of the activation markers cd80 and cd86 and enhanced production of t follicular helper cells and germinal center b cells. 68 nanostructuring of materials within tissue depots may also generate adjuvant activity. peptides having alternating hydrophobic and hydrophilic amino acids can undergo a sol-gel transition upon exposure to physiological conditions whereby they form a hydrogel made up of interconnected nanofibers. 70 in this way a mixture of biphasic oligopeptide, antigen, and adjuvants can be injected with the peptides, subsequently undergoing postinjection self-assembly into hydrated nanofiber gel matrices, forming an antigen and adjuvant depot in the aqueous phase. 71 a self-assembling nanofibrous hydrogel induced an antibody response when tested as a vaccine delivery platform, either alone or formulated with cpg adjuvant (tlr9 agonist) as a delivery system for recombinant hepatitis b surface antigen (hbsag). 71 similarly, the self-assembling peptide q11 conjugated to a cd8 t cell epitope of ovalbumin elicited a strong antigen-specific cd8 t-cell response. 72 liposome-based vaccines were an early form of particulate adjuvant formulation based on the use of cell-like spherical lipid bilayer vesicles containing antigens within the internal aqueous compartment, within the lipid bilayer, or attached externally. 73, 74 for example, 165 nm-diameter liposomes assembled from cationic lipid, cationic polymer, and plasmid dna were shown to target antigen to draining lymph nodes, resulting in enhanced dc activation and immunity. 75 coating of these liposomes with a cholesterol-modified mannan further enhanced the therapeutic anticancer action of an oncoprotein vaccine. 76 as liposomes themselves are generally poorly immunogenic, they may require combination with other immunostimulatory adjuvants to enhance their immunogenicity. 4 for example, liposomal nanoparticles self-assembled from a mixture of phospholipids containing 5% pegylated lipid to prevent protein binding were used to target cyclic diguanylate as an agonist of stimulation of inf genes to draining lymph nodes, thereby enhancing vaccine potency. 77 single bilayer liposomes have also been constructed using viral lipid envelopes, retaining the viral cell binding and fusion glycoproteins and resulting in 150 nm-diameter virosomes that promote attachment to respiratory mucosal surface and subsequent fusion release of contents into the cytoplasm of endocytosing cells. 78 further, the viral components enhance the efficiency of the interaction with antigen-presenting cells (apcs), making them an ideal vaccine delivery system. 79, 80 more recent virosome research has included the incorporation of additional components to improve the particle formation and stability characteristics as well as enhance immunogenicity. 81 for example, an amphiphilic saponin derivative (gpi-0100) was incorporated into a virosome and provided potent immunogenicity to allow the use of very low antigen doses. 82 electrostatic interactions are an important part of nanoparticle vaccine formulations, both between components of the formulation 83, 84 and between the formulation and the anionic cell membranes of apcs. 85 these charged interactions have demonstrated importance in various nanovaccines, including liposomal systems, 86 but perhaps are best exploited in polymeric systems. a mixture of an anionic poly(γ-glutamic acid) acid grafted with phenylalanine ethylester and a cationic ε-polylysine was self-assembled from aqueous solution into nanoparticles with 200-300 nm diameter. 83 when formed in the presence of ovalbumin, the particles were loaded with antigen and induced effective cellular and humoral immune responses in immunized mice. 87 poly(γ-glutamic acid) has also been eletrostatically attached to complexes of dendrigraft poly-l-lysine with plasmid dna. in this instance the poly(γ-glutamic acid) moderates the cytotoxicity of the electrostatic complex to enable delivery and expression of genes in the spleen, suggesting application in vaccine formulations. 88 this is related to a similar gene delivery formulation in which electrostatic interactions between positively charged polyethyleneimine and negatively charged plasmid dna and poly(γ-glutamic acid) were used to construct a nanoparticle dna vaccine. this formulation conferred protection against malaria infection in mouse models. 84 standard plga particles have negative charge, and electrostatic interactions have been identified as a critical aspect of the binding to protein antigens. consequently, protein binding can be tuned through adjusting the ph of binding conditions to optimize the charge differential between components. 89 plga nanoparticles have also been constructed with positive charge using print technology through the incorporation of cationic additives. these particles were specifically designed to electrostatically interact with commercial hemagglutinin antigens to generate an influenza vaccine with enhanced immune responses compared with the hemagglutinin alone. 12,13 lipid-like amphiphiles have been used in several self-assembling nanoparticulate-based self-adjuvanting antigen delivery systems because their structure mimics bacterial lipoproteins and as such can activate the immune system. 90 this type of vaccine formulation includes a hydrophobic core covalently attached to a hydrophilic peptide antigen such that the antigen epitope is presented on the surface of the nanoparticle. 91 a nontoxic, dendritic poly tert-butyl acrylate polymer core has been used and when conjugated to relevant peptide epitopes induced cellular responses able to kill human papillomavirus-infected cancer cells. 92 epitope packing on the particle surface was dense enough to maintain a native conformation and enable proper recognition by the immune system. 91, 92 in other examples the particle core was made up of either lipid-like peptides 93 or lipidated peptides. 94, 95 such formulations include a peptide epitope from a hookworm protein flanked by coil-producing sequences attached to a lipid-like peptide core. the native alpha helix structure of the epitope presented on the surface of the nanoparticle was able to generate antibodies in mice. 96 as previously suggested by their use in emulsion and liposome adjuvants, saponins can provide potent immunogenicity to vaccine formulations. a commonly used adjuvant is the saponin qs21, which is an acylated saponin at the 4-hydroxyl position on fucose with two linked 3,5 dihydroxy-6-methyloctanoic acids. 97 qs21 induces inflammatory cytokines and imparts a th1 bias in vaccine responses, 98,99 but because of its ability to lyse cell membranes, hemolysis and injection site pain are major limiting factors in its use. 100 qs21's toxicity can be reduced by forming it into an immune-stimulating complex (iscom), which is a spherical particle of ∼40 nm diameter that self-assembles from specific mixtures of cholesterol, phospholipids, and qs21. [101] [102] [103] an advantage of iscoms is that the toxic hemolytic effect of the saponin is moderated by its incorporation into the liposomal structure while its immune-stimulatory properties are retained. the liposomal particle also helps target the saponin adjuvant and antigen formulation to the draining lymph nodes. 5 while initially thought important for immunogenicity that antigens were loaded inside the iscom structure, it is now recognized that adjuvant action can be achieved by simple admixture of the antigen with the iscoms, a major advantage as many antigens are difficult to load into the particles. 104 many plant-based polysaccharides have been shown to have adjuvant activity, with the additional benefit that as sugar-based compounds polysaccharides are generally extremely safe and well tolerated. 20 while some polysaccharides (eg, mannan) may have adjuvant activity when in soluble form, most are only adjuvant active when present as insoluble particles. this is clearly seen with the polysaccharide inulin (β-dpoly(fructo-furanosyl)-d-glucose), a natural plant-derived storage carbohydrate of plants of the compositae family that has no immunological activity when in the usual soluble form but has potent adjuvant activity when crystallized into the nanostructured delta inulin microparticles. [105] [106] [107] [108] delta inulin particles have been shown to enhance humoral and cellular immune responses to a wide variety of viral, bacterial, and protozoan antigens as well as toxins and allergens. for example, enhanced vaccine protection was seen in models of influenza, 109, 110 japanese encephalitis, 111, 112 west nile virus, 113 hepatitis b, 114 human immunodeficiency virus, 115 anthrax, 116 sars coronavirus, 117 listeriosis, 118, 119 and african horse sickness. 120 its adjuvant effects are seen across a broad range of animal species including mice, rats, guinea pigs, rabbits, chickens, dogs, sheep, monkeys, horses, and camels. it has proved effective in human studies when combined with a recombinant pandemic influenza vaccine, 121 a hepatitis b vaccine, 122 or a bee sting allergy vaccine. 123 an interesting feature of delta inulin is its ability to enhance adaptive immune responses even when injected a day prior to the antigen, a feature not shared by alum adjuvant. 114 delta inulin does not work like other polysaccharide adjuvants, as it has not been found to activate innate immune receptors such as tlrs, dectin-1, or the inflammasome. instead, delta inulin adjuvant appears to work by directly modulating dc function, thereby resulting in enhanced antigen presentation to memory t and b cells. this was recently shown in human subjects to translate into enhanced b cell receptor affinity maturation with upregulation of activation-induced cytidine deamidase in day 7 postimmunized plasmablasts. 124 notably, delta inulin has proved safe and effective when administered to pregnant dams 125 or their 7-day-old mouse pups 126 and, in a large animal model, to either pregnant mares or their foals, 127 a feature that may be attributable to its noninflammatory mode of action. interestingly, a powder particle formulation of delta inulin has recently been shown to be safe and effective when administered with influenza vaccine directly into the lungs of mice, 128 opening up a further novel vaccine application of this technology. other polysaccharide particles with adjuvant activity include dextran, β-glucan, lentinan, zymosan, mannan, and chitosan. 20 these all work through the action of specific innate immune receptors expressed on apcs known as lectins that specifically recognize and respond to sugars. these include the β-glucan receptor, the mannan receptor, dectin 1, and tlr. activation of these innate immune receptors results in nfκb activation and production of proinflammatory cytokines that enhance adaptive immune responses. hence delta inulin currently appears to be the only polysaccharide particulate adjuvant that works through an alternative noninflammatory pathway. given the high expression of lectins on apcs, decorating the surface of vaccine or adjuvant particles with sugar groups can assist vaccine particle targeting to apcs. for example, mannose has been used to target plasmid dna-containing liposomes to macrophages. 129 coating of cationic liposomes with mannan significantly enhanced the ability of a dna vaccine to induce hiv-specific cellular immunity and also enhanced the activity of a dna vaccine against melanoma. 130, 131 mannosylated niosomes composed of span 60, cholesterol, and stearylamine (all coated with the modified polysaccharide o-palmitoyl mannan) have been used as orally administered dna vaccine carriers with a demonstration of enhanced mucosal immunity in mice. 132 a similar approach using o-palmitoyl mannan coating was used to target niosomes to langerhan's cells in the skin after topical delivery. 133 chitin, a linear β-1-4-linked polymer of d-glucosamine and n-acetyl-d-glucosamine extracted from shrimp, and chitosan, obtained by partial deacetylation of chitin, act by binding innate receptors including dectin-1, macrophage mannose receptor, and tlr-2. 134 chitosan particles produced by cross-linking with a counter ion were used to entrap antigen and enhance its immunogenicity in mice. 135 by virtue of their mucoadhesive qualities, particles coated with chitin and its derivatives have been extensively used as nasal adjuvants for delivery of inactivated 136 or even live viral vectors. hence mucosal delivery of adenoviral vectors microencapsulated in a chitosan microparticle not only helped to protect and improve the viability of the viral vector but also made its release dependent on cell surface contact. 137 given the ability of specific nanoparticles to target apcs, as discussed previously, such particles can also be loaded with immune stimulators to further increase their adjuvant potency. mdp (n-acetyl muramyl-l-alanine-d-isoglutamine), a mycobacterial peptidoglycan with potent adjuvant activity, 37 binds and activates nod2 38 and tlr receptors, 39 leading to nfκb activation, inflammatory cytokine production, and dc maturation. a modified form, mtp-pe, was the key immune stimulator for which the original mf59 emulsion adjuvant was designed as a delivery system, given the highly hydrophobic nature of mtp-pe. 40 ironically, the combined mtp-pe in mf59 formulation was abandoned because of excess reactogenicity, 41 but the mf59 vehicle was found to have some adjuvant activity in its own right, and hence its use in influenza vaccines was continued. in a similar fashion, liposomes formulated with dtp-gdp (n-acetylglucosaminyl-n-acetylmuramyl-l-ala-d-isoglu-l-ala-glyceroldipalmitate) had potent adjuvant activity and induced remission in human metastatic colorectal cancer, although reactogenicity with fever, chills, and hypotension was a problem at higher doses. 138, 139 the combination of chitosan microparticles with the mucosal toxin-based adjuvant ltk63 significantly enhanced the immunogenicity of an intranasal group c meningococcal polysaccharide vaccine in mice. 136 similarly, intranasal administration of alginate-coated chitosan nanoparticles loaded with antigen and cpg adjuvant enhanced antibody and cellular responses in mice. 140 cpg has also been incorporated as the hydrophilic head group into a lipid-like adjuvant macromolecule, the hydrophobic tail composed of cholesterol, simple monoacyl, or diacyl lipidic groups. this formulation was used to successfully target cpg adjuvant and the antigen to lymph nodes, thereby helping decrease systemic cpg toxicity while enhancing vaccine immunogenicity. 141 emulsions are not only useful as adjuvants, as discussed earlier, but they can also be used as templates to produce controlled-size solid adjuvant particles (fig. 7.2 ). using this method, the polysaccharide inulin was cosolubilized with an ovalbumin antigen into a water-in-oil emulsion with the inulin then precipitated by the slow addition of acetone. this resulted in formation of spherical 1.5 µm-diameter inulin particles encapsulating the ovalbumin with high efficiency. 142 these antigen-loaded inulin microparticles improved apc uptake of ovalbumin and enhanced antiovalbumin antibody responses in mice. emulsions have similarly been used as templates to precipitate many other biodegradable polymeric nanoparticles for use in vaccine delivery, including the formation of plga nanoparticles from a nanoemulsion. the plga nanoparticles acted as an antigen delivery system although, unlike inulin particles, the plga particles themselves appeared to have no intrinsic adjuvant properties with an immunomodulator being required to create a strong immune response. 15, 143 by contrast, poly(γ-glutamic acid)-based nanoparticles generated an immune response without the requirement of other adjuvants. 57 emulsion polymerization is another method whereby polymeric particles of size defined by the emulsion can be generated. 144 in this method the soluble polymer droplets within the emulsion are chemically cross-linked such that the droplet-sized particles remain solid when the emulsion is removed. poly(ortho ester) microparticles of 5 µm diameter produced in this way were used to deliver plasmid dna antigen to apcs. the acidic conditions in the apc lysosome degraded the orthoester bonds holding the particles together, thereby releasing the dna. the dna was then transcribed into protein that was presented to t and b cells, resulting in a cellular and humoral memory response. 144 in another example, size-tuneable cross-linked poly(propylene sulfide) nanoparticles with sizes ranging from 25 to 250 nm were prepared from emulsions, with monomers in the water phase stabilized by pluronic f-127. 145 particle size was then controlled by varying the concentrations of the monomer and surfactants in the emulsion. these particles presented pluronic hydroxyl groups at the surface, resulting in complement activation, dc maturation, and a strong adjuvant effect. 14 such particles are unstable and release any attached drug under oxidative conditions, such as those found in the lysosome. 14, 145 the ability to produce particles of different sizes enabled the effect of particle size on antigen delivery to be directly studied, revealing that small 20-25 nm nanoparticles delivered attached antigen to draining lymph nodes with 10-fold higher efficiency than 100 nm particles. 14, 19 other studies using these particles have shown that particle surface chemistry is important to the action of nanoadjuvants with, for example, polyhydroxylated surfaces performing much better than polymethoxylated surfaces 14 despite both particles being targeted to the dcs. controlled production of defined-size adjuvant nanoparticles can also be achieved through particle replication in nonwetting template (print) technology. 9,13 print techniques use photolithography to create a template that is then transferred to a perfluoropolyether elastomer mold. the mold is filled with the desired material under pressure and then allowed to set. release of the material from the mold produces particles in the nano-or microscale that can have complex and highly conserved morphology. 9 this technology has been used for a range of medical applications, including production of stimuli-responsive targeted drug delivery vehicles 9,146 and vaccine adjuvants. 13 the size, shape, and chemistry of print nanoparticles can be optimized to maximize their transport to lymph nodes, enabling their use as an antigen delivery vehicle. 12 this work found that anionic 80 x 180 nm cylinders covalently bound to ovalbumin through a 500-dalton polyethylene glycol spacer optimized delivery of antigen to lymph nodes and enhanced vaccine responses. 12 this chapter highlighted the increasing sophistication of vaccine adjuvant design driven by the convergence of improved immunological understanding of the importance of nanofeatures to adjuvant function together with advanced nanomanufacturing techniques that allow very precise control of particle size, shape, texture, and surface chemistry. principles from antigen design (eg, particle self-assembly) are increasingly being incorporated into adjuvant nanoparticle design. these individual concepts are now being applied to the design of a single integrated vaccine particle, incorporating antigen, adjuvant, and apc-targeting strategies. in the process, the concept of what is an adjuvant needs to be broadened to not only include specific immune-stimulatory substances but also to include any vaccine design features that enhance immune responses against a relevant antigen. thus, the definition of an adjuvant could potentially now include aspects of antigen formulation such as nanoparticle incorporation; aspects of particle size, shape, and surface chemistry that enhance immunogenicity; or even purely physical processes such as texturing of the particle surface to maximize immunogenicity. looking forward, adjuvants will increasingly not be seen as separate add-on items but as wholly integrated elements of a complete vaccine delivery package. vaccine systems are steadily approaching the complexity and sophistication of the pathogens they mimic, with particle properties and behaviors designed to maximize long-term immune protection while maintaining a high safety profile. microparticulate polysaccharide adjuvants 135 nanoparticle manufacturing methods 138 7.6.1 emulsions for template-based particle assembly 138 7.6.2 print-based adjuvant production vaccine adjuvants: current state and future trends new-age vaccine adjuvants: friend or foe? freeing vaccine adjuvants from dangerous immunological dogma particulate systems as adjuvants and carriers for peptide and protein antigens advances in vaccine adjuvants particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model a comprehensive platform for ex vivo t-cell expansion based on biodegradable polymeric artificial antigen-presenting cells engineering nano-and microparticles to tune immunity print: a novel platform toward shape and size specific nanoparticle theranostics nanofabricated particles for engineered drug therapies: a preliminary biodistribution study of print (tm) nanoparticles size and shape effects in the biodistribution of intravascularly injected particles rapid and persistent delivery of antigen by lymph node targeting print nanoparticle vaccine carrier to promote humoral immunity development of a nanoparticle-based influenza vaccine using the print® technology exploiting lymphatic transport and complement activation in nanoparticle vaccines peptide-based subunit nanovaccines fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform red blood cell-mimicking synthetic biomaterial particles in vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles carbohydrate-based immune adjuvants synthesis of nanomaterials in microemulsions: formation mechanisms and growth control nanoemulsions: formation, structure, and physical properties preparation of metal nanoparticles in water-in-oil (w/o) microemulsions formation and stability of nano-emulsions adjuvants designed for veterinary and hum vaccin sensitization and antibody formation after injection of tubercle bacilli and paraffin oil the mode of action of mineral-oil emulsion adjuvants on antibody production in mice outlining novel cellular adjuvant products for therapeutic vaccines against cancer design and evaluation of a safe and potent adjuvant for hum vaccin clinical and immunologic responses to human immunodeficiency virus (hiv) type 1sf2 gpl20 subunit vaccine combined with mf59 adjuvant with or without muramyl tripeptide dipalmitoyl phosphatidylethanolamine in non-hiv-infected human volunteers the adjuvant effect of mf59 is due to the oil-in-water emulsion formulation, none of the individual components induce a comparable adjuvant effect mf59™ as a vaccine adjuvant: a review of safety and immunogenicity a cationic nanoemulsion for the delivery of next-generation rna vaccines mf59 adjuvant: the best insurance against influenza strain diversity as03 adjuvanted ah1n1 vaccine associated with an abrupt increase in the incidence of childhood narcolepsy in finland increased incidence and clinical picture of childhood narcolepsy following the 2009 h1n1 pandemic vaccination campaign in finland adjuvant activity of 6-0-mycoloyl-nacetylmuramuyl-l-alanyl-d-isoglutamine muramyldipeptide and diaminopimelic acid-containing desmuramylpeptides in combination with chemically synthesized toll-like receptor agonists synergistically induced production of interleukin-8 in a nod2-and nodl-dependent manner, respectively in human monocytic cells in culture enhancement of tlr-mediated innate immune responses by peptidoglycans through nod signaling systemic cytokine profiles in balb/c mice immunized with trivalent influenza vaccine containing mf59 oil emulsion and other advanced adjuvants enhancement of humoral response against human influenza vaccine with the simple submicron oil/water emulsion adjuvant mf59 evaluation of the immune response to rts,s/as01 and rts,s/as02 adjuvanted vaccines: randomized, double-blind study in malaria-naive adults development of immune response that protects mice from viral pneumonitis after a single intranasal immunization with influenza a virus and nanoemulsion efficacy, immunogenicity and stability of a novel intranasal nanoemulsion-adjuvanted influenza vaccine in a murine model nanoemulsion nasal adjuvant w805ec induces dendritic cell engulfment of antigen-primed epithelial cells formulation and characterization of nanoemulsion intranasal adjuvants: effects of surfactant composition on mucoadhesion and immunogenicity safely and immunogenicity of a novel nanoemulsion mucosal adjuvant w805ec combined with approved seasonal influenza antigens formulation, high throughput in vitro screening and in vivo functional characterization of nanoemulsion-based intranasal vaccine adjuvants nanoemulsionbased mucosal adjuvant induces apoptosis in human epithelial cells use of a sustained-release multiple emulsion to extend the period of radio protection conferred by cysteamine evaluation of water-in-oil-in-water multiple emulsion and microemulsion as potential adjuvants for immunization with rabies antigen multiple emulsions: technology and applications fabrication of nanoadjuvant with poly-ε -caprolactone (pcl) for developing a single-shot vaccine providing prolonged immunity size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors type 1 and 2 immunity following vaccination is influenced by nanoparticle size: formulation of a model vaccine for respiratory syncytial virus preparation and characterization of biodegradable nanoparticles based on poly(gamma-glutamic acid) with 1-phenylalanine as a protein carrier targeting of antigen to dendritic cells with poly( -glutamic acid) nanoparticles induces antigen-specific humoral and cellular immunity alpha-alumina nanoparticles induce efficient autophagy-dependent crosspresentation and potent antitumour response peptomer aluminum oxide nanoparticle conjugates as systemic and mucosal vaccine candidates: synthesis and characterization of a conjugate derived from the c4 domain of hiv-1 mngpl20 immunization of mice with peptomers covalently coupled to aluminum oxide nanoparticles synthesis and assembly of hepatitis b virus surface antigen particles in yeast priming of class i-restricted cytotoxic t lymphocytes by vaccination with recombinant protein antigens immunization of human hiv-seronegative volunteers with recombinant pl7/ p24:ty virus-like particles elicits hiv-1 p24-specific cellular and humoral immune responses nanoparticles as novel immunogens: design and analysis of a prototypic severe acute respiratory syndrome vaccine protective antibody and cd8+ t-cell responses to the plasmodium falciparum circumsporozoite protein induced by a nanoparticle vaccine a nonadjuvanted polypeptide nanoparticle vaccine confers long-lasting protection against rodent malaria synthetic virus-like particles from self-assembling coiled-coil lipopeptides and their use in antigen display to the immune system the use of self-adjuvanting nanofiber vaccines to elicit high-affinity b cell responses to peptide antigens without inflammation a self-assembling peptide acting as an immune adjuvant controlled release of insulin from self-assembling nanofiber hydrogel, puramatrix (tm): application for the subcutaneous injection in rats vaccine self-assembling immune matrix is a new delivery platform that enhances immune responses to recombinant hbsag in mice antigenic peptide nanofibers elicit adjuvant-free cd8(+) t cell responses liposomes as immunological adjuvants immunogenicity and protectivity of a new liposomal hepatitis a vaccine dileep padinjarae vangasseri a, leaf h. immunostimulation mechanism of lpd nanoparticle as a vaccine carrier coating of mannan on lpd particles containing hpv e7 peptide significantly enhances immunity against hpv-positive tumor nanoparticulate sting agonists are potent lymph node-targeted vaccine adjuvants adjuvant activity of immunopotentiating reconstituted influenza virosomes (irivs) virosome-mediated delivery of protein antigens to dendritic cells pharmaceutical aspects of intranasal delivery of vaccines using particulate systems influenza virosomes as vaccine adjuvant and carrier system influenza virosomes supplemented with gpi-0100 adjuvant: a potent vaccine formulation for antigen dose sparing stabilization of polyion complex nanoparticles composed of poly(amino acid) using hydrophobic interactions immunogenicity of novel nanoparticle-coated msp-1 c-terminus malaria dna vaccine using different routes of administration nanoparticle vaccines liposomal cationic charge and antigen adsorption are important properties for the efficient deposition of antigen at the injection site and ability of the vaccine to induce a cmi response induction of potent adaptive immunity by the novel polyion complex nanoparticles biodegradable nanoparticles composed of dendrigraft poly-l-lysine for gene delivery an investigation of the factors controlling the adsorption of protein antigens to anionic plg microparticles handbook of biologically active peptides polyacrylate dendrimer nanoparticles: a self-adjuvanting vaccine delivery system selfadjuvanting polymer-peptide conjugates as therapeutic vaccine candidates against cervical cancer lipid core peptide system for gene, drug, and vaccine delivery structure-activity relationship of a series of synthetic lipopeptide self-adjuvanting group a streptococcal vaccine candidates a novel synthetic adjuvant enhances dendritic cell function peptidebased subunit vaccine against hookworm infection structure/function studies on qs-21, a unique immunological adjuvant from quillaja saponaria structural and immunological characterization of the vaccine adjuvant qs-21 a strong cd8+ t cell response is elicited using the synthetic polypeptide from the c-terminus of the circumsporozoite protein of plasmodium berghei together with the adjuvant qs-21: quantitative and phenotypic comparison with the vaccine model of irradiated sporozoites advances in saponin-based adjuvants the iscom: an immunostimulating complex iscoms and other saponin based adjuvants functional aspects of iscoms iscom, a delivery system for parenteral and mucosal vaccination inulin isoforms differ by repeated additions of one crystal unit cell the polysaccharide inulin is characterized by an extensive series of periodic isoforms with varying biological actions delta inulin: a novel, immunologically active, stable packing structure comprising beta-d-[2->1] poly(fructo-furanosyl) alpha-d-glucose polymers inulin crystal initiation via a glucose-fructose cross-link of adjacent polymer chains: atomic force microscopy and static molecular modelling advax, a polysaccharide adjuvant derived from delta inulin, provides improved influenza vaccine protection through broad-based enhancement of adaptive immune responses delta inulin polysaccharide adjuvant enhances the ability of split-virion h5n1 vaccine to protect against lethal challenge in ferrets an inactivated vero cell-grown japanese encephalitis vaccine formulated with advax, a novel inulin-based adjuvant, induces protective neutralizing antibody against homologous and heterologous flaviviruses je-advax vaccine protection against japanese encephalitis virus mediated by memory b cells in the absence of cd8+ t cells and pre-exposure neutralizing antibody an inactivated cell culture japanese encephalitis vaccine (je-advax) formulated with delta inulin adjuvant provides robust heterologous protection against west nile encephalitis via cross-protective memory b cells and neutralizing antibody a novel hepatitis b vaccine containing advax, a polysaccharide adjuvant derived from delta inulin, induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing induction of mucosal and systemic antibody and t-cell responses following primeboost immunization with novel adjuvanted human immunodeficiency virus-1-vaccine formulations advax-adjuvanted recombinant protective antigen provides protection against inhalational anthrax that is further enhanced by addition of murabutide adjuvant severe acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathology novel nanoparticle vaccines for listeriosis a gold glyco-nanoparticle carrying a listeriolysin 0 peptide and formulated with advax delta inulin adjuvant induces robust t-cell protection against listeria infection improving the dromedary antibody response: the hunt for the ideal camel adjuvant randomized clinical trial of immunogenicity and safety of arecombinanthlnl/2009 pandemic influenza vaccine containing advax polysaccharide adjuvant immunogenicity and safety of advax, a novel polysaccharide adjuvant based on delta inulin, when formulated with hepatitis b surface antigen: a randomized controlled phase 1 study immunotherapy-2076. a controlled study of delta inulin-adjuvanted honey bee venom immunotherapy delta inulin adjuvant enhances plasmablast generation, expression of activation-induced cytidine deaminase and b-cell affinity maturation in human subjects receiving seasonal influenza vaccine a single immunization with inactivated h1n1 influenza vaccine formulated with delta inulin adjuvant (advax) overcomes pregnancy-associated immune suppression and enhances passive neonatal protection advax delta inulin adjuvant overcomes immune immaturity in neonatal mice thereby allowing single-dose influenza vaccine protection safety and immunogenicity of a delta inulin-adjuvanted inactivated japanese encephalitis virus vaccine in pregnant mares and foals enhanced pulmonary immunization with aerosolized inactivated influenza vaccine containing delta inulin adjuvant mannose receptor-mediated gene transfer into macrophages using novel mannosylated cationic liposomes hiv-1-specific cell-mediated immune responses induced by dna vaccination were enhanced by mannancoated liposomes and inhibited by anti-interferon-gamma antibody development of an antigen-presenting cell-targeted dna vaccine against melanoma by mannosylated liposomes mannosylated niosomes as adjuvant-carrier system for oral genetic immunization against hepatitis b mannosylated niosomes as carrier adjuvant system for topical immunization chitosan-based systems for the delivery of vaccine antigens chitosan-based nanoparticles for improving immunization against hepatitis b infection the concomitant use of the ltk63 mucosal adjuvant and of chitosan-based delivery system enhances the immunogenicity and efficacy of intranasally administered vaccines encapsulation of adenoviral vectors into chitosan-bile salt microparticles for mucosal vaccination phase i trial of immther, a new liposome-incorporated lipophilic disaccharide tripeptide immunologic and toxicologic study of disaccharide tripeptide glycerol dipalmitoyl: a new lipophilic immunomodulator immune response by nasal delivery of hepatitis b surface antigen and codelivery of a cpg odn in alginate coated chitosan nanoparticles structurebased programming of lymph-node targeting in molecular vaccines development of soluble inulin microparticles as a potent and safe vaccine adjuvant and delivery system biodegradable nanoparticle delivery of a th2-biased peptide for induction of thl immune responses molecularly engineered poly(ortho ester) microspheres for enhanced delivery of dna vaccines oxidation-sensitive polymeric nanoparticles incorporation controlled release of silyl ether prodrugs from print nanoparticles key: cord-313971-czcs1y9w authors: zhao, yang; aarnink, andré j. a.; de jong, mart c. m.; groot koerkamp, peter w. g. title: airborne microorganisms from livestock production systems and their relation to dust date: 2014-04-16 journal: crit rev environ sci technol doi: 10.1080/10643389.2012.746064 sha: doc_id: 313971 cord_uid: czcs1y9w large amounts of airborne microorganisms are emitted from livestock production. these emitted microorganisms may associate with dust, and are suspected to pose a risk of airborne infection to humans in vicinity and to animals on other farms. however, the extent to which airborne transmission may play a role in the epidemic, and how dust acts as a carrier of microorganisms in the transmission processes is unknown. the authors present the current knowledge of the entire process of airborne transmission of microorganisms—from suspension and transportation until deposition and infection—and their relation to dust. the sampling and the mitigation techniques of airborne microorganisms and dust in livestock production systems are introduced as well. pathogenic microorganisms may occur in high concentrations in the air inside the livestock houses. along with ventilation, they can be emitted to the ambient environment and pose airborne infection risk to healthy animals on other farms and to humans living in the vicinity . 1072 y. zhao et al. the extent to which pathogenic microorganisms are transmitted to nearby recipients and cause disease spreading through the airborne route remains largely unclear. actually, airborne transmission only has been considered as a possible route in historical disease outbreaks in livestock production when the outbreaks could not be attributed to other known routes such as direct contact transmission or fecal-oral transmission (elbers et al., 2001; gloster et al., 2003) . attempts to link pathogen transmission (between farms) to prevalent wind directions-an apparent epidemiological proof of airborne transmission-have not been always successful, and the transmission is assumed to only be favored at picky atmospheric conditions mikkelsen et al., 2003) . all these facts induce doubts on the importance of the role of airborne transmission in disease outbreaks; however, no one can conclusively exclude this transmission route due to its potentially extensive and intensive impacts if truly involved in epidemics. given the lack of knowledge on airborne transmission it is important to stimulate relevant research in order to better understand what role it can play. here we define airborne transmission in livestock production as "an entire transmission process that involves pathogenic microorganisms releasing from the infected animal's excrement or secretion to air, transporting air, inhaled by a healthy animal, and eventually infecting the recipient." the airborne transmission of certain pathogenic microorganisms from animal to animal has been demonstrated in lab-scale experiments in which healthy animals separated physically but not aerially from infected animals became infected (berthelot-herault et al., 2001; brockmeier and lager, 2002) . furthermore, some microorganisms collected kilometers away from the source farm were found to be capable of infecting healthy animals intramuscularly or intratracheally (otake et al., 2010) . however, there is still uncertainty, because of the incomplete knowledge about the entire process of airborne transmission of microorganisms, from generation and transportation through inhalation and finally to infection (stark, 1999) . dust probably plays a role as the carrier of the microorganisms in the air. in 1987, the importance of the relationship between airborne microorganisms and dust from livestock production systems was reviewed by muller and wieser (1987) . the authors separately described the indoor properties (source, concentration, and constitute) of airborne microorganisms and dust, and the dispersion in ambient air outdoors. since then, much research has been done on specific processes involved in the transmission of the airborne microorganisms and dust. however, we lack an integrated overview of and insight into all the processes involved in the airborne transmission of microorganisms in association with dust. the objective of this article is to review current knowledge on airborne microorganisms from production systems for typical livestock species (swine, poultry and cattle), and their relation to dust. specifically, in section 2, we identify the sources, species, size distributions, and concentrations of identifying the source of microorganisms and dust in livestock production systems helps to elucidate how airborne transmission is generated, and ultimately can help to develop and implement strategies that prevent such transmission from beginning (bull et al., 2006; cambra-lopez, 2010) . sources of dust in livestock production systems have been identified and assessed qualitatively and quantitatively (aarnink et al., 1999; donham and gustafson, 1982) . it is generally accepted that all dust sources are also sources of airborne microorganisms because these source materials somehow contain certain microbial species that may be generated together with dust. however, the source identification of airborne microorganisms has not yet been extensively investigated, and it is thought to be more complicated than the source identification of dust in at least two ways. the first way is associated with the complexity of microbial species in a source. a source material always contains a microbial flora composed of many different microbial species. the second way is associated with the dynamic viability of microorganisms in the generation process (milne et al., 1989) ; microorganisms may either decay or multiply in the source material. thus, the source identification for microorganisms should also be dynamic. animals shed microorganisms mainly by means of fecal excretion, which may contain large amount of microorganisms (letellier et al., 1999; pell, 1997) . consider two common zoonotic bacterial species, salmonella and escherichia, both have been found in feces: salmonella at a concentration of 2-7 log cfu g −1 feces (gray and fedorka-cray, 2001; himathongkham et al., 1999) and e. coli at 2-6 log cfu g −1 feces (mcgee et al., 2001; omisakin et al., 2003) . feces are also an important pathway for virus shedding from infected animals (fouchier et al., 2003) . a list of viral species that may be excreted by cattle was proposed by pell (1997) , including infectious bovine rhinotracheitis and foot-and-mouth disease (fmd) virus. many other viruses have been recovered from feces of other animal species, such as avian influenza a virus (webster et al., 1978) and newcastle disease virus (spradbrow et al., 1988) in poultry, and swine fever virus (van oirschot, 1979) , hepatitis e virus (de deus et al., 2007) , and porcine reproductive and respiratory syndrome virus (prrsv) (yoon et al., 1993) in pigs. the microorganisms in feces can become airborne when dried fecal particles are disturbed by air flow or animal activity. some studies have managed to identify feces as the source of airborne microorganisms. a study by duan et al. (2009) found the airborne e. coli strains inside and downwind from the pig houses were closely associated with those isolated from pig feces. water content binds particles in feces and prevents their suspension, so, the microorganisms in dry feces that have low water content become airborne more easily than microorganisms in fresh feces. the water content of fresh feces (or manure, when urine is not separated) is the range of 60-90% (derikx et al., 1994) , depending on the animal species. under typical livestock housing environmental conditions, it may take hours or days to dry the feces to a water content less than 20%-the water content of airborne dust in livestock production systems (aarnink et al., 1999; zhao et al., 2013) . this means the microorganisms must undergo a latent period between the moment they are excreted in the feces and the moment they become airborne. during inhalation and exhalation the surface of the mucus in the respiratory tract is destabilized through an interplay between surface tension and viscous forces (edwards et al., 2004) . this can result in mucus's microorganisms to become airborne and expelled from the body via breathing, coughing, and sneezing. this source of airborne microorganisms is widely accepted in the human model of disease transmission, and pathogenic microorganisms have been frequently recovered from the exhaled aerosols (fabian et al., 2008; weber and stilianakis, 2008) . only a few studies have been carried out to directly detect the microorganisms in exhaled air of animals. by sampling exhaled air in masks placed over the heads of infected pigs, cho et al. (2006) recovered prrsv and hermann et al. (2008) recovered mycoplasma hyopneumoniae and bordetella bronchiseptica. in contrast, hermann et al. (2008) failed in recovering prrsv, porcine circovirus 2, swine influenza virus, and porcine respiratory coronavirus in the exhaled air of infected pigs, although they were found in the oral and nasal swabs. similarly, zhao et al. (2013) could not recover infectious brusal disease virus in the exhaled air of infected broilers. these results indicate that some microorganisms in animal respiratory tracts might not readily become suspended in the air or be expelled out of the body, which implies that this is perhaps not an major source of airborne microorganisms as compared to animal feces (zhao et al., 2013) . however, the reason the microorganisms may not be detected could be because the quantities of exhaled microorganisms were below the detection limit of the sampling devices. therefore, animal respiratory tracts can only be excluded as a source of airborne microorganisms until it has been incontrovertibly established that every single microorganism is detectable. organic materials such as feed may serve as carriers for variety of microorganisms. the microorganisms may originate from the soil and are transferred to standing crops by wind, rain, mechanical agitation, or insects (maciorowski et al., 2007) . both nonpathogenic and pathogenic microorganisms have been recovered from feed; analysis has revealed concentrations of gram-negative bacteria in feed as high as 5 log cfu g −1 (hofacre et al., 2001) . these microorganisms can be disseminated together with feed particles during feeding (andersson et al., 1999; chang et al., 2001) ; the extent of dissemination depends greatly on how the feed is given (e.g., dry vs. wet feed delivery and powder vs. pellet feed delivery; pearson and sharples, 1995) . litter is a mixture of bedding materials (e.g., wood shavings, chopped straw, sawdust, and rice hulls) animal feces, dander, and feed (torok et al., 2009) . the provision of litter in livestock production systems may improve animal welfare by increasing the incidence of natural behaviors (appleby and hughes, 1991) , which, however, may result in more microorganisms being present in the air than in housing systems without litter (madelin and wathes, 1989; vucemilo et al., 2007) . the microorganisms arrive in litter during the harvesting and processing of the bedding material, and through animal excretion and secretion. the concentrations of aerobic bacteria in poultry litter range from 3 to 9 log cfu g −1 (lu et al., 2003; martin et al., 1998) . most of the bacteria in the poultry litter are gram-positive. gramnegative bacteria and mold account for a small fraction of the total microbial count, but due to the high concentration of the total microorganisms, their numbers can still be high in some cases (martin et al., 1998) . surprisingly, some pathogenic bacteria that are commonly recovered from animal feces (e.g., e. coli, salmonella, and campylobacter) are not always detectable in litter (lu et al., 2003) . the reason could be the less favorable microenvironment for microbial survival in the feces-bedding mixture than in feces alone. microorganisms, including globicatella sulfidofaciens, corynebacterium ammoniagenes, corynebacterium urealyticum, clostridium aminovalericum, arthrobacter sp., and denitrobacter permanens, which may be involved in degradation of wood and cycling of nitrogen and sulfur have been identified in poultry litter (lu et al., 2003) . other possible sources of airborne microorganisms in livestock houses include animal skin (baird-parker, 1962; gailiunas and cottral, 1966; kloos et al., 1976) and animal products (e.g., broken eggs, spoiled milk (de reu et al., 2008; donaldson et al., 1983; doyle, 1984; doyle and roman, 1982) , farm workers and visitors (newell and fearnley, 2003; nishiguchi et al., 2007) , and ambient air martin et al., 1996) . sources of airborne dust include feed, animal skin and feather debris, feces, litter, microorganisms, pollen, and insect parts (aarnink et al., 1999; donham et al., 1986) . the contribution of these sources to airborne dust varies, depending on the animal species and the housing system. heber et al. (1988a) reported that the main source of airborne dust in pig houses was feed, which is consistent with the findings of donham et al. (1986) and aarnink et al. (1999) . muller and wieser (1987) found that 55-68% of the airborne dust in floor layer systems with litter originated from the bedding materials in litter, while 80-90% of the airborne dust in layer systems with battery system originated from feedstuff (table 1 ). in floor systems with wood shavings as litter for three-week old broilers, aarnink et al. (1999) found that airborne dust mainly (>10%) originated from down feathers and urine components. the contribution of feed to the airborne dust largely depends on its composition and how it has been processed (pearson and sharples, 1995m; e.g., crumbles or pellets). the contribution of feces is probably related to the housing system (e.g., with or without litter [straw bedding vs. liquid manure]). table 1 lists the main sources of dust and gives an estimation of their contributions. a large fraction of the airborne microorganisms in livestock production systems are bacteria, of which the most dominant are gram-positive bacteria, accounting for approximately 90% of the bacterial flora (zucker et al., 2000) . the most common species of these gram-positive bacteria are staphylococcus, streptococcus, and enterococci (clark et al., 1983; hartung, 1992; matkovic et al., 2007) . the gram-negative bacteria account for only a small fraction of airborne bacteria (zucker et al., 2000) . bakutis et al. (2004) reported that in terms of the total bacterial count, the proportion of gramnegative bacteria was approximately 10% in cattle houses, 4.9% in pig houses, and 2.6% in poultry houses. zucker et al. (2000) found that the airborne gram-negative bacteria in pig and cattle houses were aerobic and include enterobacteriaceae, pseudomonadaceae, and neisseriaceae; no culturable obligate anaerobic gram-negative bacteria were isolated. possible reasons for the smaller proportion of airborne gram-negative bacteria in livestock production systems are that their excretion by animals is less than their counterparts and these bacteria are more vulnerable to environmental stress such as oxidation, radiation, and dehydration, probably because of their thinner cell walls (pal et al., 2007; theunissen et al., 1993) . the proportion of fungi, molds, and yeasts in the airborne microbial flora in animal houses is low (hartung, 1992; lee et al., 2006) . the most frequently reported fungi in poultry, pig, and dairy houses are aspergillus sp., alternaria sp., cladosporium sp., penicillium sp., fusarium sp., scopulariopsis sp., and yeast (chang et al., 2001; cormier et al., 1990; martin et al., 1996; matkovic et al., 2007; vittal and rasool, 1995; wilson et al., 2002) . the size of an airborne particle determines its transportation, sedimentation, and resuspension, as well as its deposition in the respiratory tracts of recipients. investigations of the size distribution of microorganisms and dust in livestock production systems may provide a useful overview of their quantitative importance, indicate the health risk for human and animals, and facilitate the establishment and evaluation of control techniques. according to interests by different scientific sectors, sizes of airborne particles are differently categorized. for concerns in occupational health, particle sizes are categorized into three categories: inhalable (<100 μm), thoracic (<10 μm), and respirable (<4 μm or 5 μm; cambra-lopez et al., 2010; curtis et al., 1975; madelin and wathes, 1989; zhang, 2004) . in environmental science, recent research is increasingly focusing on dust with aerodynamic diameter smaller than 10 μm (pm 10 ) and 2.5 μm (pm 2.5 ). the size distribution of airborne dust has been expressed either in mass or in counts. zhao et al. (2011a) found that in three pig houses about 73-95% of the airborne bacteria were in the nonrespirable range ( figure 1) . a similar result was reported by curtis et al. (1975) : nonrespirable bacteria accounted for approximately 78-89% of the airborne bacteria in pig houses. the size distribution of microorganisms in poultry houses depends on the type of housing system. in broiler rooms with wood-shaving litter, most of the bacteria were nonrespirable in the full life cycle of broilers (eight weeks); a similar distribution pattern was found in broiler rooms with raised netting floor only after the birds were older than six weeks. when the birds were two to five weeks old, the proportions of airborne respirable and nonrespirable bacteria were similar (madelin and wathes, 1989) . heber et al. (1988a) reported that nonrespirable particles (>4 μm) in pig houses accounted for more than 80% in mass, but less than 30% in terms of count. expressed as percentage of total dust, lai et al. (2010) found the mass of pm 10 was 30-54% in pig houses, 41-69% in poultry houses, and 36% in cattle houses. in all three types of house, pm 10 count was more than 99% of the total dust. the equivalent figures for pm 2.5 mass were 1-3% in pig houses, 2-8% in poultry houses (2009), and 5% in cattle houses. for pm 25 counts the figures were 90-99% in pig houses, 88-92% in poultry houses and 99% in cattle houses. the difference in the mass and numeric size distribution is caused by the fact that small dust particles contribute little to mass. that more microorganisms and less dust particles are found in the nonrespirable range indicates that a nonrespirable dust particle is more likely to be loaded with microorganisms than a respirable one. this is a reasonable hypothesis, because the larger a particle is, the greater the chance it may contain microorganisms. nowadays, the size distribution of airborne microorganisms is normally determined with the andersen stage impactor (andersen, 1958; zhao et al., 2011a) . this sampler actually gives the counts of particles (in seven different size ranges) that contain microorganisms. in some cases, for instance a biosecurity assessment for occupational health, it is more important to quantify the individual microorganism in the collected particles rather than the microbial-containing particles themselves, because the former will give a better idea of the risk of infection. for this purpose, predetachment of microorganisms from dust particles or visually microbiological counting techniques (yamaguchi et al., 2012 ) may be required. concentrations of airborne microorganisms and dust in livestock production systems have been investigated in previous studies (kim et al., 2008; radon et al., 2002; zhao et al., 2011a) . due to the huge spatial and temporal variations in microorganism and dust concentrations and the difference in sampling techniques used, it is difficult to compare the data between studies. so far, the studies by seedorf et al. (1998) and takai et al. (1998) still provide the most representative concentration data on microorganisms and dust in livestock production systems, and sampling methods were detailed in the publications by phillips et al. (1998) and wathes et al. (1998) . these data are summarized in table 2 , together with the pm 10 and pm 2.5 concentration measured by lai et al. (2010) . for microorganisms, the highest concentrations of airborne bacteria and fungi were found in broiler houses. the concentrations found in layer, pig and cattle houses were lower than those in broiler houses, but still much higher than those in ambient air (wang et al., 2010) . for dust, the highest dust concentrations were found in poultry houses, and the lowest dust concentrations were in cattle houses. the concentrations of airborne microorganisms and dust in animal houses are affected by animal, housing system and management. in this section, these factors are discussed separately, but one should realize that these factors always interactively affect the concentration because they are intercorrelated. for instance, animal activity is associated with animal age, weight, and light schedule, and ventilation rate is affected by outdoor and set-point temperature, humidity, and animal species and age. the animal factor can be further detailed into subfactors such as age, weight, activity, and stocking density. the concentrations of airborne microorganisms and dust generally increase concomitantly with animal age and weight predicala et al., 2001; yoder and van wicklen, 1988) . however, an inverse relationship has also been found. madelin and wathes (1989) found a decrease of microorganisms and dust concentrations in the late fattening period of broilers. a similar result was reported by saleh et al. (2005) . the decrease in concentration of microorganisms and dust is probably because the older broilers occupied all the floor space, which limited their activity. in general, higher concentrations of bacteria, fungi, and dust are measured when the animals are more active, as can be inferred from the finding that their concentrations were higher in day time than at night takai et al., 1998) . image and infrared technology allows animal activity to be automatically detected (gloster et al., 2007; pedersen and pedersen, 1995) . using an infrared detector, haeussermann et al. (2007) demonstrated that the indoor concentrations of pm 10 were associated with pig activity. a similar study by heber et al. (2006) showed that both total dust and pm 10 were correlated with the pig activity. however, gloster et al. (2007) failed to establish the correlation between the concentration of airborne fmd virus and pig activity quantified by taking sequential pictures. the reason is not clear, but the authors explained that the virus production appears to be more closely associated with other factors (e.g., physiological symptoms; gloster et al., 2007) . hardly any other information is available on the relation between quantified animal activity and concentrations of microorganisms. compared to a cage system, an aviary system contained higher concentrations of microorganisms (de reu et al., 2005) and dust (appleby and hughes, 1991) . this is because in an aviary system, laying hens have more scopes for moving horizontally and vertically and perform dust bathing behavior in the litter. housing systems with bedded floors caused more air quality problems, although such housing systems are generally thought to be more beneficial for animal welfare (kim et al., 2008; madelin and wathes, 1989; quarles et al., 1970) . the type of bedding material also affects the concentration of microorganisms in the air. for instance, in broiler houses, straw bedding released less bacteria in the air than wood shavings did (banhazi et al., 2008a) . the authors argued that this was probably because wood shavings provided a better microenvironment for bacteria viability and multiplication. comparisons of dust concentrations between natural and mechanical ventilation systems showed that with mechanical ventilation, less respirable (phillips, 1986) and total dust was found in pig houses (chiba et al., 1985) and there was less total dust in turkey houses (janni and redig, 1986) . by contrast, concentrations of total bacteria and fungi were lower in naturally ventilated pig houses without bedding materials (deep-pit manure system with slats, and manure removal system by scraper) than in mechanically ventilated houses. the contradictory results found for the effect of type of ventilation (natural or mechanical) on microorganisms and dust are not fully understood, but it seems likely that the situations (including the management) of the ventilation systems vary between the different studies, making the data less comparable. feed management may play an important role in dust concentration in livestock production systems. previous studies have shown that dust concentrations were reduced by giving pelleted feed rather than powdered feed, wet feed rather than dry feed, and coated feed rather than uncoated feed (clark and mcquitty, 1988; pearson and sharples, 1995; zeitler et al., 1987) . the effect of feeding management on airborne microorganism concentration has not been extensively studied. maintaining good hygiene in livestock production systems may help to improve the air quality with respect to microorganisms and dust. for instance, cleaning (e.g., removing litter, scrubbing surfaces, and disinfecting the house) between two production circles may reduce both airborne microorganisms and dust (banhazi et al., 2008a) . investigations of the relation between hygiene and air quality have found they are not always positively correlated. duchaine et al. (2000) reported that a housing system that appeared cleaner contained more airborne bacteria than one that appeared dirtier. the probable explanation is that houses with more settled dust on the surfaces are more readily ranked as dirtier, but dust accumulated on surfaces is not an appropriate indicator of the concentration of bacteria in the air. ventilation management (e.g., adjusting the rate at which indoor air is exchanged with outdoor air by mechanical ventilation systems) is to control the temperature and other aerial variables such as humidity and gas concentrations inside livestock houses. previous studies have shown that lower concentrations of microorganisms and dust can be achieved by increasing the ventilation rate in livestock production systems (duchaine et al., 2000; hinz and linke, 1998; kim et al., 2007b) . however, a nonsignificant correlation between these variables has also been reported in livestock production systems (banhazi et al., 2008b; seedorf et al., 1998) . the probable reason for these contradictory findings is that ventilation affects the concentration of microorganisms and dust in two ways: by exhausting airborne microorganisms and dust to outdoors through air exchange, thereby reducing their indoor concentrations, and by producing airflow turbulence above surfaces, agitating the particles and causing them to suspend in the air, thus compromising the removal effect. smaller airborne particles seem to be more effectively removed from livestock houses by ventilation than bigger particles (kuehn, 1988) because they are readily transported in the air streams. a high relative humidity in the air reduced the dust concentration in livestock production systems (guarino et al., 1999) . in humid environments, the dust particles bind to the surface and are not easily suspended, and those in the air aggregate and to settle faster (heber et al., 1988b; takai et al., 1998) . in contrast, humidity seems not to affect the concentrations of total and gram-negative bacteria (attwood et al., 1987; banhazi et al., 2008b; nicks et al., 1993) . a high humidity favors survival and/or multiplication of dehydration-sensitive microbial species in the sources (de rezende et al., 2001) , which might trade off the physical settlement of airborne microorganisms. experimental validation of this hypothesis is of importance, because it may strengthen the understanding of the interaction between airborne microorganism concentration and air humidity. this will help to formulate effective air humidification strategies for reducing both, dust and microorganisms, instead of achieving one by compromising the other. this information will also be valuable for understanding the bioenvironment in livestock houses where air humidification (e.g., water spray and evaporation cooling pad) is used to ameliorate animal heat stress in summer time (brown-brandl et al., 2010; tao and xin, 2003) . indoor temperature management is well regulated for the poultry and pig industries, to optimize productivity. al homidan et al. (1997) reported that the total dust in broiler rooms increased when the temperature was set 2 • c above the recommended level. the correlation between dust concentration and temperature reverses from positive to negative when the temperature is extremely high, apparently because animal activity decreases at high temperature and thus fewer particles from surfaces are disturbed and suspended (donkoh, 1989; guarino et al., 1999; wylie et al., 2001) . as well, a high temperature triggers a series of events that may affect the dust concentrations in livestock production systems, such as increasing the ventilation rate and activating wet cooling systems (simmons and lott, 1996) . although some researchers (banhazi et al., 2008a) have suggested there is a relation between the concentration of microorganisms and temperature, little information is available so far. the decay of microorganisms and dust is a parameter that cannot be ignored in empirical and theoretical models of airborne transmission (lighthart and frisch, 1976; yu et al., 2004) ; it may help when assessing health impacts from exposure. the term "decay" encompasses both physical and biological means. physical decay is the physical elimination of a particle from the air by means of a series of processes such as gravitational sedimentation, impaction and electrostatic precipitation; biological decay is the loss of biological activity of an airborne microorganism owing to loss of enzyme activities or denaturing of membrane phospholipids, proteins or nucleic acids (cox, 1989) . for biological decay, the situation may be more complicated in some cases when the microorganisms go into a viable but nonculturable (mckay, 1992; oliver, 2010; trevors, 2011) or dormancy state (locey, 2010) . it is clear that physical decay applies to the elimination of dust particles, whereas both physical and biological decay apply to airborne microorganisms. mechanisms affecting physical decay of particles are listed in table 3 . in reality, these mechanisms may have collective effect on a particle, but the dominant mechanism varies mainly with particle size (e.g., decay of larger particles is prone to drive by gravimetric sedimentation whereas decay of smaller particles is mainly determined by diffusion (abadie et al., 2001; he et al., 2005) . physical decay can be quantified by the rate of deposition (deposition rate loss coefficient or deposition velocity; deshpande et al., 2009) . lai (2002) reviewed the deposition rate for particles ranging from 0.01 to 10 μm and found it had a u-shaped pattern. the lowest deposition rate was found for particles ranging from 0.1 to 1 μm because particles in this range were less affected by either sedimentation or diffusion than the mean transport of a particle by the mean motion of the atmosphere, and occurs when the spatial gradient is nonzero and the particle is transported along the mean wind (baldocchi et al., 1988) brownian diffusion the process of mass transfer of particles brought about by a random molecular motion (brownian motion) and associated with a concentration gradient (vaithiyalingam et al., 2002) thermophoresis the motion of a particle under the influence of a temperature gradient (langer and holcombe, 1999) gravitational sedimentation the separation of dispersed particles from gaseous phase under action of gravity (wunsch, 1994) impaction the deposition of particles due to their momentum causing them to deviate from airflow streamlines and impacting at bifurcations (katz et al., 2001) electrostatic precipitation the use of an electrostatic field for precipitating or removing charged particles from a gas flow in which the particles are carried (shen and pereira, 1979) [a] some definitions in this table that were originally for gases or molecules in nonaerial environments have been modified to make them suitable for describing the associating mechanisms of microorganisms and dust in the air. their counterparts do. besides particle size, deposition rate is affected by other factors such as room furnishing and air speed. thatcher et al. (2002) found increasing surface area (provide more furniture) and air speed (by means of elevating ventilation rate) increased the deposition rate of particles (0.5-10 μm) in an experimental room. the biological decay of airborne microorganisms has been expressed in different ways (e.g., decay rate [or death rate], survival, or half-life). the decay rate is the decrease in concentration of viable microorganisms over time. a proportionality constant (k) indicates the extent of decay rate, and is shown in equation 1, where c o is the initial concentration of airborne microorganisms, c t is the concentration of microorganisms at time t after initial (phillips et al., 1964) . the survival represents the percentage of viable microorganisms left at a certain moment vis-à-vis the initial microbial count (wu, 2009 ). the half-life, t 1/2 , is the time taken for the concentration of viable microorganisms in the air to decrease by half (see equation 2). previous studies showed that the biological decay of airborne microorganisms was species-dependent and was determined by many external factors, such as humidity, oxygen concentration, temperature, ozone concentration, radiation (uv, γ -ray, x-ray), air ions, and air pollutants (co, so 2 , and no x ; benbough, 1971; lighthart, 1973) . for long distance airborne transmission between farms, microorganisms may be exposed to unfavorable environmental conditions that induce death or dormancy of microorganisms (lennon and jones, 2011; locey, 2010; wu et al., 2012) . in this section we present an introduction of several environmental factors and their working mechanisms on biological decay of microorganisms. the effect of humidity on the biological decay of airborne microorganisms has been investigated since the 1950s. in the early studies, the measure most used for humidity was relative humidity (rh; the ratio of the actual water vapor pressure of the air to the water vapor pressure of saturated air at a certain temperature). the results of these studies showed that different microorganisms were prone to decay either at low rh (lighthart, 1973) , at median rh (wright et al., 1968b) , or at higher rh (songer, 1967; theunissen et al., 1993) . more recently, a few studies have used absolute humidity (ah; the actual water content of the air) as another measure of humidity. for instance, shaman and kohn (2009) reported that the survival of airborne influenza virus was more significantly constrained by ah than by rh. the authors argued that rh is a meaningful physical quantity and for certain organisms may affect biological response; however, the ah can be of greater biological significance for many organisms. some studies reported significant effects of other measures of humidity, such as evaporation potential (ep; the difference between actual water vapor content in the air and the water vapor content in saturated air at the same temperature), on microbial survival (zhao et al., 2012) . although a bunch of studies have been carried out, it is not yet fully understood how humidity influences microbial decay. temperature profoundly affects the biological decay of airborne microorganisms. in general, the higher the ambient temperature is, the faster the microorganisms decay. for instance, the decay rate of flavobacterium sp. is 0.007 log min −1 at -2 to 24 • c, but increases to 0.017 log min −1 at 29 to 49 • c (ehrlich et al., 1970a) . a faster decay at higher temperature has also been reported for other microbial species, such as e. coli, s. marcescens (ehrlich et al., 1970b) , and newcastle disease virus (kournikakis et al., 1988) . prescott et al. (2005) have stated that high temperature may damage microorganisms either by denaturing the enzymes, transport carriers, and other proteins, or by melting and disintegrating the lipid bilayer, or both. the ambient environment is full of various types of radiation, including uv (10-400 nm) and visible light (400-750 nm), which may inactivate the microorganisms. the wavelength of uv between 10 nm and 120 nm has the highest energy, but this type of uv is blocked by normal dioxygen in air and cannot reach the ground. the rest of uv radiation, from low to high energy, is categorized as uv-a (320-400 nm), uv-b (280-320 nm), and uv-c (100-280 nm), of which uv-c is considered to be the most germicidal. the uv-c wavelength between 250-270 nm can be effectively absorbed by microbial genetic material, and is the uv wavelength most lethal to microorganisms (keyser et al., 2008) . the germicidal effect of uv-c light on bacteria and viruses is primarily due to the formation of pyrimidine (thymine and cytosine) dimers that inhibit the replication and function of genetic material (giese and darby, 2000; prescott et al., 2005) . bacteria decay more readily under uv radiation than rna viruses (harris et al., 1987; hijnen et al., 2006) . the probable reason for this is that the thymine of bacterial dna is more vulnerable to dimerization induced by uv than the uracil of viral rna. the mechanism whereby uv-a and uv-b (also called near-uv) inactivates microorganisms is suspected to be the breaks in strands of genetic materials that are induced by the uv itself and toxic tryptophan photoproducts (prescott et al., 2005) . visible light may also damage microorganisms. microbial pigments become excited when they absorb light energy, and are transferred to o 2 , generating singlet oxygen ( 1 o 2 ), which is a highly reactive oxidant (valduga et al., 1993) . as a self-protecting mechanism, some microorganisms may possess carotenoids that can absorb the excitation energy and reduce the formation of singlet oxygen, thus preventing cells from being damaged by light radiation (mccambridge and mcmeekin, 1981) . oxygen may accept electrons forming other toxic derivatives such as superoxide radical, hydrogen peroxide, and hydroxyl radical, which may easily destroy the cellular constituents. many aerobic microorganisms contain enzymes such as superoxide dismutase and catalase, which protect the cell against oxidation by the derivatives; all the strictly anaerobic microorganisms lack these enzymes (prescott et al., 2005) . the effect of oxygen level on decay of airborne serratia marcescens 8uk and e. coli b was studied by cox (1989) , who reported that oxygen was toxic for these microorganisms only when rh was lower than 70%. the toxicity increased with oxygen concentrations up to 30%; higher concentrations produced no additional toxicity. this finding was generally in agreement with other similar studies (benbough, 1967; hess, 1965) . viruses seem to be less sensitive to oxygen than bacteria. the decay of airborne viruses that included semliki forest virus, langat virus, t7 coliphage, poliovirus, and encephalomyocarditis virus was no different whether they were aerosolized in the air or in nitrogen (benbough, 1971; de jong et al., 1975 ). bacteria exposed to ozone (o 3 ) may be inactivated due to damage to the cell surface (giese and christensen, 1954; scott and lesher, 1963) and destruction of the intracellular enzymes, protein and genetic material (barron, 1954; ingram and haines, 1949; kim et al., 1999) . ozone may damage the viral nucleic acids of viruses and alter the polypeptide chains of the viral protein coat kim et al., 1999; roy et al., 1981) . investigations of the ozone effect on decay of airborne microorganisms showed that fungi seemed more resistant to ozone than bacteria; gram-positive bacteria were more resistant than gram-negative ones (heindel et al., 1993; kowalski et al., 1998) . ozone alone can be toxic to airborne microorganisms; however, its toxicity is enhanced when ozone reacts with compounds in the ambient air, known as open air factors (oaf). studies have shown that mixture of ozone with olefins druett and packman, 1972) and ozone with negative air ions (fan et al., 2002) are more toxic to airborne microorganisms than ozone alone. the dust particles to which microorganisms adhere may protect them from biological decay. when microorganisms are carried by the dust, they may suffer less radiation and exposure to toxic gas, and less fluctuation in micro-climate (milling et al., 2005) . it has been found that individual bacteria are effectively inactivated by ozone; however, when these bacteria were covered with a coating of organic matter, as a bio aerosols, ozone in permissible concentration had no effect (elford and van den ende, 1942) . as well as providing physical protection, the composition of dust particles might give bio-chemical support to microorganisms. in order to metabolize, microorganisms require carbon, oxygen, nitrogen, phosphorus, sulfur, and other elements (maus et al., 2001) . compounds containing these elements are abundant in airborne dust from livestock production systems (aarnink et al., 1999; muller and wieser, 1987) . an interesting hypothesis is that microbial decay in particles differs, depending on the composition of the particles. actually, in some laboratory experiments, it appears that decay of microorganisms differs as they are aerosolized from liquid suspensions with different chemical compounds. (benbough, 1971; hess, 1965) . there have been extensive studies on the biological decay of microorganisms at different temperature and rh levels, using aerosol experiments. in these experiments, microorganisms were aerosolized in airspace that was sampled at certain intervals. the biological decay was indicated by the amounts of collected microorganisms at different sampling moments. the results of selected studies are summarized in tables 4 and 5. the selection of references follows the procedures and screening criteria as below: first, all references related to biological decay of any species of airborne microorganisms were searched in literature databases (web of science, scopus, google scholar); second, only references in which biological decay was investigated by excluding confounding effect of physical decay (e.g., using inert tracers or labeled microorganisms) were selected; third, due to the fact that some microorganisms showed a biphase biological decay (with a fast initial decay rate in the first few seconds or minutes after aerosolization, followed by a slow secondary decay) and the airborne transmission concerned is long distance and time (brankston et al., 2007; cox, 1971; songer, 1967; webb, 1959) , only the secondary biological decay is presented in the tables. the tables show the highest biological decay at the least favorable temperature/rh, and the lowest biological decay at the most favorable temperature/rh. the values of biological decay have been presented in three ways: decay rate, survival and half-life. by reviewing the results in tables 4 and 5, it proofs that the biological decay largely varies between the microbial species and is profoundly affected by temperature and rh. most of the previous studies have used wet aerosolization in which microbial suspensions were aerosolized. wet aerosolization does mimic the fate of microorganisms expelled from animal respiratory tracts in wet aerosols. however, after they have been generated, the large wet aerosols containing microorganisms settle on surfaces and the small ones shrink into dry nuclei; both processes are very fast, taking only seconds to minutes (kincaid and longley, 1989; sun and ji, 2007; wells, 1934) . therefore, the microorganisms in wet aerosols can only be transported over short distances and in brackets is the time span (in minutes) between which survival rate corresponds to. h = highest biological decay of airborne microorganisms (i.e., worst survival and shortest half-life time); l = lowest biological decay of airborne microorganisms (i.e., optimal survival and longest half-life time). [b] calculated by dividing the amount of virus collected in the last air sample (360 min) by that in the first air sample (15 min). [c] in brackets is the time span (in minutes) between which survival rate corresponds to. induce infections in limited areas (brankston et al., 2007) . for microorganisms released from dry sources such as feces and litter, which may be more closely associated with long distance transmission, dry aerosolization is recommended. dry aerosolization may give a picture of the biological decay of microorganisms that differs from the decay in wet aerosolization because the microorganisms may suffer either dehydration stress at low ambient rh or rehydration at high ambient rh (cox, 1971 ). according to heyder et al. (1986) , the probability of deposition will be different for each particle even if all the particles in the air inhaled in one breath are identical, because the inhaled air with particles penetrates the respiratory tract to different depths where it remains for different periods of time, and because of the stochastic nature of particle transport. therefore, particle deposition (in the respiratory tract) refers to the "mean probability" of an inspired particle being collected on airway surfaces. particle deposition in the respiratory tract depends on particle characteristics (e.g., size, shape, density) and breathing pattern (e.g., nasal/oral breath, respiratory flow rate, cycle period), and is commonly expressed as a function of particle size. particle deposition in the human respiratory tract has been well documented (brown et al., 2002; james et al., 1991; lippmann et al., 1980) . in principle, the deposition of particles is governed by the mechanisms of diffusion for particles <0.1 μm, or by diffusion and sedimentation for 0.1-1 μm particles, or by sedimentation and impaction for particles >1 μm (heyder, 2004) . on the basis of previous experimental studies, heyder et al. (1986) developed a semiempirical deposition model for particles ranging from 0.005 μm to 15 μm. the model-simulated deposition pattern for slow inspiration over a long period for both oral and nasal breathing is shown in figure 2 . the deposition has been shown for three regions of the respiratory tract-extra-thoracic, bronchial, and alveolar-based on how far down the tract particles may be deposited. in addition, total deposition is given (the sum of the deposition in the three regions). it can be seen that the total deposition is least for particles of 0.1-1 μm, and that the deposition increases as the size of small particles (<0.1 μm) decreases, and the size of the larger particles (>1 μm) increases. particles larger than 10 μm are mainly deposited in the extrathoracic region, and cannot penetrate the alveolar region during slow and fast oral and nasal inspiration. the deposition in the human respiratory tract cannot be extrapolated to livestock because of the discrepancy in morphology of human and animal respiratory systems (corbanie et al., 2006) . particle deposition in guinea pig head, trachea, and lungs was investigated by harper and morton (1953) . they found an increasing regional deposition in guinea pig head for larger particles: 35.7% of the 1 μm particles were deposited in the guinea pig head region, compared with 98.1% of the 10 μm particles. the reverse was true for lung deposition: 55.2% of 1 μm particles and 0.6% of 10 μm were deposited in this region. the particle size most deposited in the trachea was 2.5 μm. a model of particle deposition in the guinea pig respiratory tract was established by schreider and hutchens (1979) , who found that 99% of unit density particles of 10 μm or more could be deposited in the nasopharyngeal-tracheobronchial region (figure 3 ). the lowest deposition was 10% for a particle size of 0.8 μm. about 17% of particles ranging from 0.08 to 4 μm could be deposited in the pulmonary region. however, this model was compromised by several assumptions that were made by the authors (e.g., laminar airflow in the respiratory tract, equal expansion of all lobes and alveoli, and complete mixing of the particles in the alveoli. hayter and besch (1974) investigated the regional deposition of five particle sizes (0.091, 0.176, 0.312, 1.1, and 3.7 to 7 μm) in chicken. the particles deposited in the head and anterior trachea were in the 3.7-7 μm size range, those deposited in the lung and posterior air sacs were 1.1 μm size, and those deposited in the caudal regions of the birds were in the size classes 0.091 and 0.176 μm. those of size 0.312 μm were deposited mainly figure 3. deposition of unit density spherical particles in the respiratory tract of guinea pig at a tidal volume of 4.44 cm 3 and a respiratory rate of 60 breaths min −1 . np-tb: nasopharyngeal-tracheobronchial region. p = pulmonary region. adapted from schreider and hutchens (1979). in upper airways. these early data of particle deposition in chicken airways are suspected to be compromised by the use of anesthetized chickens, because anesthesia alters the animals' breathing pattern. corbanie et al. (2006) investigated the deposition of particles in a wider range (1, 3, 5, 10, and 20 μm) in unanaesthetized chickens of three ages. unlike the definition of deposition that was proposed by heyder et al. (1986) , corbanie et al. (2006) defined deposition as the percentage of particles deposited in a particular region of the respiratory tract among those in the entire tract. they found that particles larger than 5 μm were too large to be deposited in the lungs and air sacs in 2-and 4-week-old chickens, as low percentages of particles were recovered in these regions (figure 4 ). for 1-day-old chicks, however, the particle deposition in lungs and air sacs was independent of particle size and even particles of 20 μm were deposited in the lower airway, possibly due to the chicks having a different breathing pattern than older chickens. the deposition pattern of monodispersed particles (3.3 μm) in calf airways was studied by jones et al. (1987) . they found those particles were preferentially deposited in the trachea and major bronchi. inhalation of pathogens may result in infection of recipients. infection is likely to fit a single-hit model, which means that one pathogenic microorganism may trigger an infection in a recipient. from this point of view, the infective dose (id; or occurrence of infection) is therefore related to the probability that a recipient becomes infected after taking in a certain dose of pathogenic microorganisms, following a poisson distribution. the id of pathogenic microorganisms to human and animals has generally been expressed in two ways. in one way, id is expressed as the number of infected recipients out of a population after a dose of microorganisms has been administered. the other way is to determine the microbial concentrations required to infect 50% of a population (id 50 ). table 6 lists the id of several pathogenic microorganisms. the id of the same microorganism varies, depending on the recipient animal species. for instance, a lower id of fmd virus is needed to infect sheep and cattle than to infect a pigs (alexandersen and donaldson, 2002; donaldson et al., 1987; gibson and donaldson, 1986) . it can be also seen that a certain dose of a microorganism is not always capable of infecting all recipients, probably because of a difference in the resistance of individual recipients (due to e.g., age, breed; roy, 1980) . furthermore, the route by which the microorganisms are administered may also be responsible for variation in id (cafruny and hovinen, 1988; zimmerman et al., 1993) . in previous studies, the administration routes were either nasally or orally, or via aerosols, and these reflect different deposition situations and regions for microorganisms in the respiratory tract. nasal administration simulates infection because larger microbial particles are deposited in the upper airways, the oral ministration may simulate oral breathing, and the aerosol administration simulates infection because smaller microbial particles are deposited in deeper airways. it was reported that the microorganisms had preferential sites for multiplication and infection because of the complex vulnerability of regions in respiratory tracts (cafruny and hovinen, 1988; druett et al., 1953; druett et al., 1956) . this being so, infection occurs more readily when the microorganisms are administered to the more vulnerable region. baskerville (1981) summarized the preferred infection regions of some microorganisms to animals by categorizing nose, pharynx, and tonsils as the upper respiratory tract, and the trachea, bronchi and bronchioles, and alveoli as the lower respiratory tract (table 7) . sampling protocols for dust in ambient air have been legislated by the u.s. environmental protection agency (2006) and the european committee for standardization (european commission, 1998 . nowadays, the official sampling protocols focus increasingly on small particles (e.g., pm 10 and pm 2.5 ) because these have the potential to be suspended for longer time, transported for longer distance, and deposited in the lower respiratory tract, thus are more hazardous to human and animal health. to ensure unbiased sampling, these protocols specify many details, such as sampling duration, type of sampler, and sample handling. the protocols for ambient air may not be directly applicable for dust sampling in livestock production systems where the dust concentrations are much higher than those in ambient air. in addition, to date there is no gold standard for sampling airborne microorganisms. given that the sampling of microorganisms and dust in livestock production systems is increasingly being performed for assessing the biosecurity of air environments and for evaluating mitigation techniques, the sampling protocol must be well designed in order to assure reliable data. subsequently, sampling strategies and samplers for airborne microorganisms and dust are highlighted, taking into consideration their sampling in livestock production systems. isokinetic sampling is the ideal sampling method, because it has been devised to sample the true numbers of particles in the air. such sampling can be achieved if the sampler inlet is in alignment with and facing the direction of air flow and if the air velocity within the sampler is the same as the ambient air velocity (zhang, 2004) . however, true isokinetic sampling is impossible in practice due to variations in the surrounding air flow pattern (air direction and velocity) and the limitations of some samplers (liu and pui, 1981) . as a concession, the current legislation aims to reduce the sampling bias in nonisokinetic samplings by stipulating the range of conditions under which the samplings may be performed. the sampling location should be chosen bearing in mind the research purpose. when human health is of concern, sampling should be carried out near the human breathing zone. one option is to fit a portable sampler on a worker's body at a height of 150-170 cm above floor level and within a radius of 30 cm around the mouth ouellette et al., 1999) . there are difficulties in doing the same with animals, therefore stationary samplers in their breathing zones are recommended. the recommended level of the breathing zone is 30-40 cm above floor level for pigs, 10-25 cm for poultry, and shoulder height for cattle (kim et al., 2007a; topisirovic, 2003; zhao et al., 2011d) . for growing animals these figures should be adjusted according to the animal height at a certain age. when emissions of microorganisms and dust are of interest, the best sampling location is in or near the air outlet. care should be taken not to place the samplers at a location where the air speed is too high, because the efficiency of the sampler (the ratio of the aerial pollutant concentration calculated from the air samples to the true concentration in the air) may drift far from 100% (grinshpun et al., 1994; hofschreuder et al., 2007) . for ambient air, the daily and annual thresholds for pm 10 have been set at 50 and 40 μg m −3 , respectively (european commission, 1999) ; the annual threshold for pm 2.5 has been set at 25 μg m −3 (european commission, 2008). to assess the dust concentration, the sampling period is generally 24 hr. the sampling duration for ambient air may also be applied when sampling dust in animal houses to obtain daily mean concentrations. for studies collecting information on dust fluctuations during the day, continuous samplings for short periods should be performed. this kind of sampling can be achieved by interval sampling or sampling with real-time optical samplers. due to the lack of a standard, the sampling duration for airborne microorganisms in different studies varies, but is normally less than 1 hr. the duration is determined by taking account of the estimated concentrations of microorganisms and the characteristics of the sampler in use. the sampling duration can be set at a short period (a few minutes) when the total microorganism is abundant in livestock houses. when the microorganism of interest is sporadically present, the period should be set long enough to collect enough microorganisms for further quantification analysis. some samplers have not been designed to be used for long sampling duration. for instance, severe evaporation of liquid medium in all glass impinger (agi-30) occurs when the sampling period is long and this may affect its sampling efficiency. the recommended maximum duration of the sampling period for the agi-30 is 30 min. impactors may easily become overloaded when samples are taken in livestock houses (thorne et al., 1992) , therefore, the sampling duration is limited to minutes or even to seconds. filtration method would not encounter the problems (evaporation of sampling liquid and overloading) of impingers and impactors; however, long sampling duration by filtration may not benefit in microorganism collection because of the biological decay of microorganisms owing to dehydration (griffin et al., 2011; wang et al., 2001) . in order to collect detectable amounts of microorganisms and to get representative samples over a longer period, some high-volume and durable samplers that can be continuously operated over hours have been developed and applied, including omni-3000 and high-volume impinger (griffin et al., 2011; kesavan and schepers, 2006) . other aspects in sampling and detection strategies include practical and economic considerations. the portability of the instrument and its ancillaries (e.g., weight and dimensions) are factors that should be considered. a highquality pump is required for dust filtration sampling, and it should be able to provide a constant airflow when ambient temperature changes or when pressure differences increases because of dust accumulation on the filter. the culture-dependent techniques for microorganism numeration are time consuming and particularly expensive; culture-independent techniques such as quantitative pcr provide possibilities for rapid and accurate analysis which is critical for biological emergency (postollec et al., 2011; ståhl et al., 2011) , but its incapability to distinguish between viable and dead microorganisms needs to be considered (yáñez et al., 2011). current samplers for airborne microorganisms are generally based on one of three main principles (i.e., impaction, impingement, or filtration; table 8 ). these different sampling principles have their advantages and disadvantages. samplers using the impaction principle (e.g., andersen six stage impactor) can be used to distinguish the microorganisms according to their sizes (andersen, 1958) . in addition, the bacteria impacted on agar plates may be directly incubated for viable counts. however, its susceptibility to overloading limits its sampling in livestock houses to short periods, which may result in nonrepresentative samples. the problem of overloading can be overcome by using samplers with the impingement principle, because the liquid samples may be decimally diluted and analyzed after sampling. a disadvantage of this sampler is that it may not be able to sample for a long time due to evaporation of the collection liquid (lin et al., 1997) . filtration is a user-friendly method in a practical situation, but not suitable for sampling microorganisms that are vulnerable to dehydration stress. a sampler with known efficiency is a prerequisite for a reliable evaluation of the microbial concentration. the efficiency of a bioaerosol sampler includes physical and biological efficiency. the physical efficiency describes how well nonviable airborne particles are aspirated by the device's inlet and transported to the collection medium, referred to as inlet sampling efficiency, and how well the bioaerosol sampler retains these particles in its medium, referred to as collection efficiency (griffiths and stewart, 1999; nevalainen et al., 1992) . for particles in the range from 1 to 10 μm and an airflow rate between 0 and 500 cm s −1 , the inlet sampling efficiency of the andersen six stage impactor is 90-150% when the opening faces in direction of the air flow and 8-100% when it is oriented perpendicularly to the horizontal aerosol flow (grinshpun et al., 1994) . the inlet efficiency of the agi-30 for particles of 1 μm is close to 100%, but it is reduced to 70-90% for 5 μm particles and to 20-30% for 10 μm particles (grinshpun et al., 1994) . when the 50% collection efficiency of the andersen six stage impactor and agi-30 was investigated it was found that the andersen six stage impactor has 50% collection efficiency for 6.6-7.0 μm particles at the first stage and 0.57-0.65 μm particles for the last stage (andersen, 1958; nevalainen et al., 1992) . the 50% collection efficiency of agi-30 was for particles of 0.31 μm (nevalainen et al., 1992) . filters vary in their physical efficiency. some are highly effective. by measuring the particle concentration upstream and downstream of filters with an optical particle counter, polytetrafluoroethylene (ptfe) filters and gelatin filters were confirmed to collect more than 93% of particles, even down to 80 nm (burton et al., 2007) . if the particles are living organisms, they may be inactivated during sampling due to impaction stress (stewart et al., 1995) , impingement stress , and/or dehydration stress (li et al., 1999) . therefore, in order to indicate how well a bioaerosol sampler maintains the microbial viability and prevents cell damage during sampling, the concept of biological efficiency has been introduced (griffiths and stewart, 1999) . in some studies, the efficiency has been evaluated by comparing samplers side by side in an environment with unknown microbial concentration (engelhart et al., 2007; henningson et al., 1982; thorne et al., 1992) . this method easily ranks the performance of different samplers; however, it neither reveals whether the amount of microorganisms collected in the samples accurately represents the microorganism content of the air nor distinguishes between the physical and biological efficiency. other studies separately investigated the physical and biological efficiency of bioaerosol samplers in aerosol experiments, in which a known amount of microorganisms together with an indicator (either labeled microorganisms, or inert tracer compound) were nebulized in an isolator. the physical efficiency can be determined by comparing the amount of tracer collected by a bioaerosol sampler with that collected by the reference sampler (a sampler that has a high physical efficiency); the biological efficiency is subsequently indicated by the change in the ratio of microorganisms/indicator. using this method, zhao et al. (2011b; 2011c) reported the efficiency of the andersen six stage impactor, agi-30, omni-3000, and gelatin filter in sampling enterococcus faecalis, e. coli, campylobacter jejuni, mycoplasma synoviae, and gumboro vaccine virus. european reference samplers use the filtration principle to collect dust from the air (european commission, 1998; european commission, 2005) ; while in us, a list of reference and equivalent samplers, including tapered element oscillating microbalance (teom), filter, and optical sampler, have been proposed by the u.s. environmental protction agency (1997). for sampling dust in certain size fractions (e.g., pm 10 and pm 2.5 ), a preseparator for separating the coarse dust from the target dust particle sizes has to be installed in front of the filter/sensor/dust collector. sampling systems with pre-separators using the impaction principle have been legislated as reference methods for measuring dust in ambient air in the united states (u.s. environmental protction agency, 1997) and european countries (european commission, 1998 . these systems show steep collection curves for sampling fine dust in ambient air where dust load is low (kenny et al., 2000) . however, the preseparator with impaction principle may become overloaded when sampling in dusty livestock production systems, thereby resulting in overestimated concentrations of fine dust (zhao et al., 2009) . in contrast, a preseparator with cyclone principle has been found to be less vulnerable to overloading in dusty environments than preseparators based on impaction (zhao et al., 2009) . optical dust samplers are now commercially available. these dust samplers can monitor the real-time dust concentrations, and no further process is needed after sampling (unlike the gravimetric method, in which filters must be weighed). moreover, some optical samplers may separately record the concentrations of dust in different size ranges. however, the optical samplers have limitations in humid environments, because it cannot distinguish between droplets and dry dust. another limitation of using optical samplers lies to the difficulty in converting count to mass concentrations without knowing the true density of the particles in concern. 6. mitigation techniques for airborne microorganisms and dust many techniques have been applied in practice to reduce the concentrations and emissions of airborne microorganisms and dust in the livestock industry. they vary in their utility, but can be grouped into two main principles. the first principle, to control particles at source, includes techniques such as feed coating and oil spraying, which has been stated to be "the most effective means" of controlling airborne particles in the space (pearson and sharples, 1995) . the second principle is air purification. ionization (electrostatic) and air scrubbers are examples. the fatty substances often added to feed to increase metabolizable energy content may reduce the airborne microorganisms and dust in animal houses (pearson and sharples, 1995) . gore et al. (1986) reported that concentrations of airborne bacteria were reduced by 27%, and settled dust by 45-47%, when 5% soybean oil was added to the pig diet. this result is consistent with the study by welford et al. (1992) , who found a 31% reduction of inhalable dust with 2% oil addition to the feed. other substances, such as tallow, lecithin and lignin have also been used as effective feed additives for the purpose of particle control in animal houses (dawson, 1990; pearson and sharples, 1995) . spraying techniques reduce the particle concentrations mainly by coating the surfaces, thereby, preventing particles from being suspended or resuspended from their sources (takai, 2007) . kim et al. (2006) reported that spraying 60 ml m −2 of tap water, salt water, treated manure, microbial additive, soybean oil, artificial spice, and essential oil may all reduce particles in pig houses. these authors found an average reduction of 53% for airborne bacteria, 51% for fungi, and 30% for total dust. the substance for spraying found to be the most effective additive for reducing dust was soybean oil. in broiler rooms, aarnink et al. (2009) reported that pm 10 reduction increased linearly from 55 to 85% when daily rapeseed oil application rates increased from 6 to 24 ml m −2 . the pm 2.5 reduction was not related to application rate and was about 80%. in aviary systems for layers, a 34% reduction for pm 10 and 50% reduction for pm 2.5 were achieved by daily spraying with 20 ml m −2 . although a high oil application rate achieves high pm 10 reduction in broiler rooms, it may adversely affect animal health . when spraying 24 ml m −2 daily, there was a tendency for statistically higher footpad lesion in broilers. aarnink et al. (2009) recommended limiting oil application to 16 ml m −2 . the ionization (also referred to as electrostatic) technique produces negative ions in the air, which causes airborne particles to become negatively charged. the negatively charged particles are attracted to earthed or positively charged surfaces. previous studies have shown promising reduction effects on dust in animal houses using ionization techniques, total dust was reduced by 13-61% in poultry houses (lyngtveit and eduard, 1997; mitchell et al., 2000; mitchell et al., 2004; richardson et al., 2003) , and by 45-58% in pig houses (rosentrater, 2003; tanaka and zhang, 1996) . cambra-lopez et al. (2009) reported that the technique produced average reductions of 36% for pm 10 and 10% for pm 2.5 . the disparity is probably caused by the different charging mechanisms to particles. the small particles (<0.1 μm) are charged by the thermal charging mechanism, which is proportional to the diameter; large particles (>0.5 μm) are charged by field charging mechanism, which is proportional to the square of the diameter (bundy, 1984) . ionization has the potential to prevent airborne transmission of microorganisms holt et al., 1999) , however, no reduction of airborne bacteria, fungi and mold was found in broiler houses by this technique ). knowledge of ionization on reduction of airborne microorganisms in animal houses is still limited and needs to be augmented. end-of-pipe techniques, such as dedusters, air filters, and scrubbers have been installed at air outlets of animal houses to minimize emissions of air pollutants. zhang et al. (2001) found a deduster removed 90% total dust from the air exhausted from a pig house. because dedusters are based on the centrifuge principle, they are more effective at removing larger particles than smaller ones. the deduster studied by zhang et al. (2001) had a removal efficiency of 90% for particles larger than 10 μm, 77% for 7 μm particles, and 50% for 4 μm particles. the low removal efficiency for small particles was assumed to be particle re-entrainment due to high air turbulence. acid and biological air scrubbers were originally developed to reduce ammonia and odor emissions, and they also appear to be effective in reducing particle emissions. an acid scrubber that uses sulfuric acid may achieve a 70% reduction of total bacteria . in a lab-scale experiment, aarnink et al. (2011a) found that e. faecalis and gumboro virus could be reduced by 100% when per-acetic acid was used as the circulation solution in a scrubber. the biological scrubbers are not consistent in reducing microorganisms (seedorf and hartung, 1999) , probably because the microorganisms for digesting odorous compounds can also be emitted to the ambient air. the removal of total dust by biological scrubbers was found to be 22-96% (seedorf and hartung, 1999) . combined techniques have been applied in practice: for instance, oil spraying combined with feed coating (takai and pedersen, 2000) and multistage air scrubbers . these techniques are more consistent and effective in reducing emissions of airborne microorganisms and dust, as well as other gaseous pollutants (ogink and bosma, 2007; zhao et al., 2008) . a disadvantage of combined scrubbing techniques is the relatively high energy use and the complexity for use on practical farms. therefore, further research is needed to develop energy-saving and simple-to-operate combined scrubbing techniques. • the most important source of airborne microorganisms is the animals themselves by means of excretion. the sources of dust include excrement, litter, feed, skin, and feathers. • airborne microorganisms in animal houses are mostly bacteria, with grampositive bacteria predominating; fungi account for only a small proportion of microorganisms. • concentrations of both microorganisms and dust are high in animal houses. they are affected by animal type, housing system, management, and environmental factors. because these factors play an interrelated role on the concentrations of microorganisms and dust, integrated research on the effects is required. • the microorganisms transmitted in the air suffer physical and biological decay. the physical decay largely depends on their size, and the biological decay is mainly determined by environmental factors, such as humidity, temperature, radiation, and toxic gases. in airborne transmission, microorganisms may be carried by dust particles that may protect microorganisms from biological decay. knowledge of the role of dust in the transportation of microorganisms is still lacking, and needs to be expanded. • microorganisms are deposited on different regions in the respiratory tract, mainly depending on their size. they have different preferred infection regions in the respiratory tract. the amount of microorganisms needed to induce an infection varies with microorganism species and animal species. • reference methods for microorganism and dust sampling in animal houses need to be legislated. these methods should be resistant to highly microbial and dusty environments, and the efficiency of the sampling devices needs to be investigated. • different techniques have been applied to reduce airborne microorganisms and dust in and from animal houses. combining several abatement techniques may achieve higher and more consistent reduction. energysaving and 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livestock houses influence of dose and route on transmission of encephalomyocarditis virus to swine airborne gram-negative bacterial flora in animal houses key: cord-258762-vabyyx01 authors: garbey, marc; joerger, guillaume; furr, shannon title: a systems approach to assess transport and diffusion of hazardous airborne particles in a large surgical suite: potential impacts on viral airborne transmission date: 2020-07-27 journal: int j environ res public health doi: 10.3390/ijerph17155404 sha: doc_id: 258762 cord_uid: vabyyx01 airborne transmission of viruses, such as the coronavirus 2 (sars-cov-2), in hospital systems are under debate: it has been shown that transmission of sars-cov-2 virus goes beyond droplet dynamics that is limited to 1 to 2 m, but it is unclear if the airborne viral load is significant enough to ensure transmission of the disease. surgical smoke can act as a carrier for tissue particles, viruses, and bacteria. to quantify airborne transmission from a physical point of view, we consider surgical smoke produced by thermal destruction of tissue during the use of electrosurgical instruments as a marker of airborne particle diffusion-transportation. surgical smoke plumes are also known to be dangerous for human health, especially to surgical staff who receive long-term exposure over the years. there are limited quantified metrics reported on long-term effects of surgical smoke on staff’s health. the purpose of this paper is to provide a mathematical framework and experimental protocol to assess the transport and diffusion of hazardous airborne particles in every large operating room suite. measurements from a network of air quality sensors gathered during a clinical study provide validation for the main part of the model. overall, the model estimates staff exposure to airborne contamination from surgical smoke and biological material. to address the clinical implication over a long period of time, the systems approach is built upon previous work on multi-scale modeling of surgical flow in a large operating room suite and takes into account human behavior factors. there is a large debate on the possible airborne transmission of coronavirus 2 (sars-cov-2) in closed buildings [1, 2] . we are still understanding the sars-cov-2 spreading but scientists support the hypothesis of airborne diffusion of infected droplets from person to person at a distance that can be greater than two meters [3] . in the unfortunate case an elective surgery is practiced on an asymptomatic covid-19 patient and who was not tested positive, one may ask if the virus can escape an operating room (or) kept under positive pressure and expose staff in peripheral area to the disease. this question is particularly important to healthcare staff who spend multiple long hour shifts in a hospital system that manages covid-19 patients. to quantify airborne transmission from a physical point of view, we consider surgical smoke as a marker of airborne particle diffusion-transportation emitted from the surgical table area. surgical smoke is 95% water or steam and 5% particle material and therefore surgical smoke can act as a carrier for tissue particles, viruses, and bacteria [4] . today, the risk of surgical smoke has clearly been established [5] [6] [7] [8] [9] [10] [11] . one of the main difficulties is that surgical smoke carries ultra fine particles (ufp) as small as 0.01 microns, which are able to bypass pulmonary filtration, and small particles up to several microns [10] . it was recently shown in a study that air quality, especially concentration of fine particles, is associated with an increase in covid-19 mortality [12] . respiratory protection devices are used to protect staff in healthcare facilities with various degrees of success [13, 14] . we propose to construct a rigorous multi-scale computational framework to address these questions and use measurements of diffusion-transportation of surgical smoke particles with off-the-shelf portable sensors to calibrate the model. this methodology addresses only the physical side of the problem and therefore does not answer the effectiveness of airborne particles to induce covid-19. some of the difficulties encountered in such studies are that air sampling and infection may or may not be strongly correlated [15, 16] . however, it is an important step to quantify the level of exposure in order to estimate the corresponding viral load in part. transport and diffusion mechanisms are very effective for ufp to travel a long distance from the source in a short period of time. a 2020 report from china demonstrated that sars-cov-2 virus particles could be found in the ventilation systems in restaurants [17] and in hospital rooms of patients with covid-19 underlining how viable virus particles can travel long distances from patients [18] . clinical environments are too complex to model with the traditional modeling method of airflow and particle transportation because both the source intensity of surgical smoke [19] as well as the mechanism of propagation via door openings [20] are largely dominated by human factors. the geometric complexity of the infrastructure and of the heating, ventilation, and air conditioning (hvac) system limit the capability of computational fluid dynamics (cfd) [20, [20] [21] [22] [23] [24] [25] [26] to predict indoor air quality and health [27] . last but not least, droplet behavior depends not only on their size, but also on the degree of turbulence and speed of the gas cloud, coupled with the properties of the ambient environment (temperature, humidity, and airflow) [2] . we present in this paper a mathematical framework and experimental protocol to assess the transport and diffusion of hazardous airborne particles in any large or suite. human behavior factors are taken into account by using a systems and cyber-infrastructure approach [28] [29] [30] coupled to a multi-scale modeling of surgical flow in a large or suite [31] . overall, the model estimates staff's exposure to airborne contamination, such as surgical smoke or biological hazard. validation is provided by a network of wireless air quality sensors placed at critical locations in an or suite during the initial phase of the surgical-suite-specific study. a step-by-step construction of the model scaling up from the or scale to the surgical suite scale will be presented; the model integrates the transport mechanism occurring at the minute scale with the surgical workflow efficiency simulation over a one year period. to assess potential contamination from one or to another, the extent of the propagation of surgical smoke in the area adjacent to the or will be checked-this might be more significant than the level of concentration itself. to simulate the airflow and dispersion of surgical smoke, an or that is representative of the surgical suite shown in figure 1 was used. measurements for calibrations and verifications were conducted in a real or of this dimension when there were no surgeries taking place. figure 2 provides the schematic of all boundary conditions and geometric parameters. this cfd approach is used to justify and build a simplified large-scale model of the airflow in the surgical suite. a 3d cartesian coordinate system was used with length along the x-direction, width along the y-direction, and height along the z-direction. the or is 7.5 m long, 6 m wide, and has a height of 2.7 m (see figure 2 ). the model takes into account the architecture of the room, the operating table location, and the hvac system design in the or as well as in the hallway. the corridor was modeled as a rectangle of 12 m long, 2.5 m wide and a height of 2.7 m. the operating table is displayed as a rectangle in the middle of the or, and the anesthesia equipment is also simulated by a rectangle that is close to the table. the computation of the flow was done by using a pressure based solver and an ansys fluent solver in steady states first and then transient mode after. the model's geometry was meshed using an unstructured tetrahedral grid with about 10 6 elements. the exact size of the mesh depends on the angle of the door with its initial closed position since the mesh gets refined at the interfaces. the airflow is assumed to be turbulent [25] and was modeled using the k − turbulence model, taking into account gravity to introduce the boussinesq approximation in the navier-stokes equation. it is the most common model for indoor airflow simulation in which the turbulent kinetic energy k and turbulent dissipation rate are modeled. the temperature and pressure boundary conditions in the model were measured and are reported in the result section of this paper. typically, the or is kept cooler than the hallway, and the inlet vent inside the or blows air at a temperature as low as 13 • c. to match the ventilation infrastructure, the model has three different rows of inlet vents in the ceiling. the first row of 4 inlets is in the middle of the room. it blows on top of the surgical table a laminar flow directly on the surgical table to remove as fast as possible any contaminant close to the patient. in our model, one inlet vent is represented by 2 rectangles of 0.5 by 0.1 m each. then, there are two rows of three inlet vents on each side of the one on top of the or table: one on the left (1.7 m from the center of the or table) and one on right (1.5 m from the middle of the or table, see figure 2 . those are present to avoid any flow returning towards the operating table. the slightly different velocity flows for each inlet were implemented (see result section) in order to replicate the anemometry measurement (via peak meter ms6252b with an accuracy of ±2.0%) obtained near these inlets-it was noticed that the large surgical lights over the surgery field have the tendency to obstruct part of the inlet flow. the or also contains two outlet vents represented by two rectangles of 0.9 by 0.6 m placed on the wall at 0.1 m from the ground with the coordinates of the middle of the bottom part: (−3.35 m, 3.35 m, 0.1 m) and (3.35 m, −2.68 m, 0.1 m) taking the center of the or table at ground level as reference (0, 0, 0). these outlet of that suck out the air in the or with such a velocity that the outlets pump out less volume than the volume injected by the ceiling inlets, which creates a positive pressure of about 8 pa. pressurization is a key factor in controlling room airflow patterns in a healthcare facility. positive pressurization is used to maintain airflow from clean to less-clean spaces. the appropriate airflow offset to reach the desired pressure differential depends mostly on the quality of the construction of the room. it is difficult, if not impossible, to know what the room's leakage area is before finishing the construction and doing measurements of airflow. the facility management service (fms) of the hospital was able to supply the values of volume per minute that the inlets are blowing (1.15 m 3 /s) and of the volume going through the outlets (0.55 m 3 /s). as the surface of the outlets is known, the velocity of the outlet vents were 0.5 m/s for the left one and 0.4 m/s for the right one, reported on figure 2 . the volume of extra air in the or is the difference: 1.15 − 0.55 = 0.6 m 3 /s. due to the positive pressure of 8 pa present in the or, this additional volume leaks out of the or through either the door being left open or the narrow gap around the door when it is closed. in that case, the free boundary surface was estimated to be 0.17 m 2 . for the hallway, a uniform inflow boundary condition of 0.1 m/s was imposed in order to take into account the anemometry measurement mentioned above. this upstream boundary condition is completed by a free outlet boundary condition at the other end of the hallway. to be as realistic as possible, the two existing inlet vents' boundary conditions were respectively added for the inlet vent located on the ceiling and the other inlet located close to the entry door of the next or, both with a velocity of 1. to validate the model, measurements were done of velocity flow and of the concentration of particles at various locations that were close to specific regions of interest-the door-frame location in particular, measurements are reported in the result section. it is unrealistic, in practice, to build a cfd model of the whole surgical suite and run this model for an extensive period of time. next, an upscale model will be presented that will use the present cfd simulation to verify some of the key parameter values, especially relating to transmission parameters between ors and the hallway. as an example, consider a system of 10 identical ors aligned on one side of a hallway. each or has one door access to the hallway. this system is part of a standard or suite and represents one half of the facility in figure 1 that has an architectural design almost symmetric with its two circulation side-halls. computing the concentration of a so-called "marker," which can be a specific gas or set of airborne particles in the air of this or suite, is particularly of interest. this marker is generated from the location of the or's surgical table, where a surgeon is using an electrosurgical instrument that produces smoke from the thermal destruction of tissues. the marker can also be the particles resulting from evaporation of any alcohol-based chemical used either to prep the patient or to clean the or. the model has two parts: first, a compartment-like model that can monitor the indoor pollution [32]; second, a multi-scale agent-based model (abm) that simulates the surgical flow activity and the impact on the indoor air quality either from the source of surgical smoke or from door openings affecting the dispersion of pollutants [31] . staff movement throughout the or suite via door openings and closings will manifestly be a key mechanism for propagation of markers. the indoor air quality is a linear set of differential equations that will be slightly more complex than a standard compartment model since the coefficient will be stochastic, the sources and output/leaks of the particles term will have a time delay built in, and the hallway will require a transport equation. the rationale for building this specific model will come out of the set of experiments described hereafter. next, the description of the acquisition process to identify the production of airborne contaminants will be explained. for this experiment in a surgical training facility, electrosurgical energy was delivered on the surface of two pieces of pork meat, each 2 cm thick, placed on an or table. three types of energy delivery systems were compared: electrosurgery (conduction) via the covidien forcetriad monopolar device(medtronic, minneapolis, mn, usa), ultrasonic (mechanic) with the ethicon harmonic scalpel p06674 device (ethicon inc., somerville, nj, usa), and laser tissue ablation with erbe apc (argon plasma coagulation) 2 device (erbe elektromedizin gmbh, tübingen, germany). to keep the tissue burn superficial, a pattern of parallel lines was followed with each device and always used unburned areas of the meat. the energy was delivered for a period of 30s up to 60s in order to produce a large quantity of smoke and thus particles. the measurement was done by several laser particle counters from dylos corp (riverside, ca, usa) placed at various distances from the source (http: //www.dylosproducts.com/dc1700.html). they give an average particle count every minute in a unit system with units u d that correspond to 0.01 particles per cubic foot or 0.00028 particles per cubic meter (1 cubic foot = 0.0283168 cubic meter). a traditional problem with the validation of particle count in laboratory conditions is that particles are not all the same uniform size. according to smartair (http://smartairfilters.com/cn/en/), the dylos system output is highly correlated (r = 0.8) to a "ground true" measurement provided by a high-end system such as the sibata ld 6s (sibata scientific technology ltd., tokyo, japan) that is claimed to be accurate within 10% in controlled laboratory conditions. according to smartair, the dylos system seems particularly accurate at the lower concentration ends, which is of interest for this study's purpose. semple et al. [33] also compared the dylos system with a more expensive system: the sidepak am510 personal aerosol monitors (tsi incorporated, shoreview, mn, usa). they concluded that the dylos' output agrees closely with the one produced by the sidepak instrument with a mean difference of 0.09 µg/m 3 . the dylos sensors were set up to track particles of small size in the range from 0.5 to 2.5 microns, which are the sizes of biological material. the results were checked systematically by comparing the measures of several sensors at the same location to show consistency, as well as checked that the particle count lowers back down to nearly zero in a clean-air room with ac equipped with high efficiency particulate air (hepa) filters. each experiment was started from an initial clean-air condition of a small particle count, fewer than 50 units, which is much less than the number of particles counted during energy delivery. it took about 6 min to reach the initial clear-air count after each experiment. for each experiment, the concentration increased to a maximum after a short time delay s from the time the energy was delivered; this delay depends on the distance to the source. the concentration then exponentially relaxes to zero in time. consequently, the model of source dispersion is an exponential function as follows: the least squares fitting technique was used to interpolate the data with this function. the amplitude of the source a (see figure 3 ), the delay s on particle diffusion and transport to reach the sensor and the rate of "diffusion decay" ρ > 0 were identified. the accuracy on s, which measures the time interval between the source production and the peak of the signal, cannot be faster than one minute since the sensor only works at a one-minute timescale. a delay of s ≤ 1 was found to be a good approximation for all three energy devices. each experiment was done 4 to 5 times depending on the variability of the results. therefore, about 24 to 30 data points were available to identify the parameters a, s, and ρ for each energy device that was tested. now, the protocol experiment to assess the transport and diffusion of particles in different areas of a large or suite will be described. this set of experiments, as opposed to the previous one, was done in a large or suite late at night and on weekends when the ors were empty and had clean air with high-efficiency hvac. a hairspray product (lamaur vitae, unscented) was used as the marker and sprayed for a duration of 1 to 2 s to track its small particles while keeping the same positions of the dylos systems. the experiment first tested the propagation in a closed-door or with the source above the or surgical table. the spray nozzle was held facing the near-vertical direction, pointing to the ceiling. a distribution of sensors as displayed in figure 4 was used. the initial observation was that all four sensors distributed along the central line of the whole or space were getting a particle count of the same order of magnitude after an average of 15 s. the mixing of particles was quite extensive within a minute by reason of the hvac input/output design in the or, and that the concentrations on each sensor quickly relaxed to zero. this observation is also coherent with the results of the cfd model of the flow circulation described above. a method identical to the previous one was used to identify the key parameters a, s, and ρ characteristic of the dispersion of hairspray in the or. the model for or diffusion of particles is then where q denotes the global concentration of particles in each or, s(t = 0) denotes the source production that is non zero at time zero and ρ or denotes the diffusion decay inside the or. this simple ordinary differential equation (ode) model provides an average of particle concentration in the or at the minute timescale. a first-order implicit euler scheme with a time step dt of one minute is used: an entirely similar technique is used to describe the dynamic of particle diffusion and transport in the hallway, except that the hallway is discretized as a one-dimensional structure of consecutive hall blocks located at the same level as the or block. in this part of the experiment, the source is set in the hallway-see figure 1 . as noticed earlier, there is a slow but significant air flow speed v 0 in the hall, pointing in the direction of the main entrance of the surgical suite, situated on the right of the map in figure 1 . naturally, the high pressure of the or is designed to drive the airflow out and the front corridor seems to be a significant outlet. on the opposite end of the hall, situated on the left of the map, figure 1 , this velocity is close to zero. it is assumed that v 0 (x) is an affine function, with a linear growth from 0 to 0.1 m/s at mid-hall, and a constant value beyond. the model of hallway diffusion of particles is then d dt where d dt denotes the total derivative ∂ ∂t − v 0 ∂ x using the x coordinate system in the one space dimension hall model. to assess the transmission of particles from an or to the adjacent hallway with closed or doors, the same experiments were run with some of the sensors placed in the hallway either facing the closed door or sitting at a location in the hallway (see figure 4 ). as a matter of fact, the door of the or is not perfectly sealed due to the difference between the pressure inside the or and the lower pressure in the hallway, a significant airflow with velocity around the order of 1 m/s exists at the gap located between the door's edge and the door frame. [sentence removed]. a similar technique is used to represent the diffusion coefficient as well as the delay s that is now interpreted as the time it takes for the particles to flow from the or to the hallway right outside the door. this transmission condition will be entered into the model to couple equations (2) and (3). finally, an entirely similar approach is used to get the transmission in the compartment model when the door of a specific or is wide open. in such cases, the gradient of pressure between the or and the hallway nearly vanishes. at the doorstep, we are observing buoyancy-driven effects due to the difference in temperature between the or (cold air) and the hall (warm air). there is a convective flow exchange with cold air at the bottom going out of the or and hot air at the top going into the or [34] . during our experiment with particle sensors, we were able to validate the propagation of aerosol traveling into the or from outside when the door is left open. with the cfd model and taking into account the gravity, we simulated the contamination by adding a source of co 2 from the inlet at the beginning of the hallway and keeping the door open. it took 18 s for the gas to reach the door and start contaminating the or. this proved the importance of keeping the door closed to maintain the positive pressure in order to control the contamination rate and nosocomial propagation in the or suite. now, the simple compartment-like model to monitor, in time and in space, the diffusion and transport of particles with intermittent source production in each or will be assembled. such a source of pollutants corresponds to either the use of some chemicals or the use of electrosurgical instruments during surgery. the goal is to get the average rate at which the staff working in the or suite is getting exposed to particle concentration emanating from surgical smoke throughout the day. potential propagation of particles that may carry biological material from one or to another is also of interest. as discussed earlier, the concentration is tracked in time and in space with a coarse time step of one minute. this time step scale is coherent with the measurement system used for particle counting. one minute is also roughly the time that the particles emitted from a point source next to the or table need to transport and diffuse throughout the or block once released. the compartment model computes the global concentration of the particles in each or as well as in each section of the hall adjacent to the or. these concentrations are denoted respectively q j (t) for or number j at time t and p j (t) for the corresponding section of the hall-see figure 1 . the source of particles is denoted as s j (t). in principle, s j (t) should be non-zero for a limited period of time and follow a statistical model based on the different phases of the surgery and the knowledge of electrosurgical instrument used during a surgical procedure [19] . the coefficients of decay are defined inside different parts of the model (ρ or and ρ hall ) as well as the coefficients of transmission between these spaces (α or from the or to the hall and γ hall for the opposite). β or represents the flow from the or to the hall when the door is open. the frequency of door openings is following a statistical model based on where the surgery is at; δ door j is a function of time and is 1 if the door is open, 0 otherwise. the simulation of the surgery schedule uses data from the smartor project [30] , which will be detailed later on. only the door openings of the order of a minute will be counted and γ hall = β or will be assumed because the gradient of pressure between the or and hallway vanishes. the system model of marker transport-diffusion in the or suite is: an additional unknown to track back-flow of marker in the or coming from the hallway can be introduced with: using this equation, the number of particles going from one or to another can be separately counted. this number is expected to be very low-see "results" section. the model (4) and (5) is not a standard box model. first, the source term has a delay built-in to simulate the transmission conditions observed. second, equation (5) is a pde, more precisely a linear transport equation. third, most of the coefficients are stochastic, especially those related to door openings and sources that are linked to human behavior. because the system of equations is linear, the superposition principle has been implicitly used to retrieve each unknown coefficient from the experimental protocol. let us describe our surgical flow model more precisely in order to provide an accurate description on how we manage to compute the source term s j (t). for each of the standard or stages of the surgery, an attributed state value is given as follows: • phase 1: anesthesia preparation label as state = 1. the type of airborne marker expected to release depends on those state values. for example: in state 0, cleaning crew uses a lot of chemical products that quickly evaporate in the or. similarly, a different type of sterilization product is used to prep the patient in state 1. in state 2, cauterization is often used for a short period of time. in state 3, various phases of the surgery will require energy delivery instruments to cut tissue and access specific anatomy or tumors. a stochastic model of energy delivery is used that consists of delivering short time fractions of energy in several consecutive minutes. the parameters of that model are: the frequency of energy delivery denoted f , the duration of the impulse denoted ξ, and the number of repetition r. a uniform probabilistic distribution of events is used within these intervals of variation for each parameter. figure 5 provides a typical example of the number of door openings observed in the or at a 15-min interval. both the detection of door openings and a patient bed coming in and out were provided by the sensors of the cyber-physical infrastructure [28] . a stochastic model of door openings will be used based on a uniform frequency of door opening during surgery, even though this distribution is non-uniform in practice and tends to concentrate at the beginning and the end of a case. • and x on the horizontal axis corresponds respectively to the entering and exiting time of the bed of the patient. this example has two procedures. the model of air pollution in the surgical suite will first be tested with a simplified model of surgical flow as follows: to provide the timeline of events, the model assumes there are three surgical procedures in each or. the timeline of each surgery will be such that: phase 1 and phase 5 last 12.5 min ± 5 min, phase 2 and phase 4 last 15 min ± 5 min, phase 3 is the surgery itself that lasts 65 min ± 25 min. phase 6 corresponds to a turnover time between surgeries that lasts 30 min ± 10 min. this simplified model of surgery scheduling has the correct order of time-length for each phase. its simplicity allows a sensitivity analysis to run with respect to the key parameters of the indoor air quality model that can be easily interpreted. next, the model will be coupled on an abm of an existing large general-surgery suite in the hospital that has been calibrated by tracking about 1000 procedures over one-year [31] . this model is complex and specific to a 20 ors surgical suite of a 1700 bed hospital, which has been monitored for over two years. to asses, the impact of human behavior on the transport and diffusion of surgical smoke in the surgical suite over a period of one year, a realistic abm of the surgical flow and of people behavior is now used. a byproduct of this study is the assessment of the air quality and the risk factors associated with surgical smoke by coupling it to the present model of transport and diffusion of airborne particles generated by surgical smoke. the method to construct this model is briefly explained in this paragraph. the exact description of the model goes beyond the scope of this paper's focus on air quality and has been detailed in garbey et al. [31] . the mathematical model of surgical flow is built upon observations and robust clinical data covering 1000 procedures with a noninvasive array of sensors that automatically monitor the surgical flow. to this end, several ors were equipped with sensors that capture timestamps [28, 29, 35] corresponding to the different states described in the previous section. overall, the model can simulate the or status of a large surgical suite during any clinical day and can be run over a long period of time. the model is able to reproduce the statistic distribution pattern over a year of performance indicators: turnover time, induction of anesthesia time, the time between extubation, and patient exit. the model classifies the human factors impact and limitation of shared resources on flow efficiency. in the end, communication delays and sub-optimal or awareness in large surgical suites have significant impacts on performance and should be addressed. this paper concentrates on the duration of surgery state 3 that corresponds to how long surgical smoke is generated, and how behavior inducing or door openings are responsible in part for the spread of surgical smoke and other agents. the output of the abm model of surgical flow coupled to the air quality model is the number of hours per year that staff gets exposed to surgical smoke in the or and hallway. various scenarios have been run related to the rate of adoption of vacuum systems for surgical smoke and or door openings to discuss the influence of human behavior on those results. following are the results on the circulation of surgical smoke in a surgical suite starting from a local source of emission in the or and ending on global dispersion in the suite. a detailed cfd model of the airflow in the or along with its immediate adjacent structure will be used to build an upper-scale, simplified model. a series of air quality measurements based on the density of particles at specific locations will be used for calibration of the model and for validation purposes. the measured rate of particles generated by various energy sources, such as monopolar cautery, argon plasma coagulation (apc), and harmonic sources, are found by testing them in an or space allocated to training, i.e. without patients. the unit used for the source of the emission is 0.01 particles per cubic foot; it gives the measurements an order of magnitude from ten to thousands by which they can be compared. small particles are found in the range of 0.5 to 2.5 microns, which are the sizes of biological-material particles like viruses. as opposed to the results reported in weld et al. [36] , our off-the-shelf particle sensor does not give us access to the ufp count. a conservative estimate from weld et al. [36] results would be that the concentration number of ufp is 2 to 3 orders of magnitude larger than what is measured for the small particles. in table 1 , each source's mean, standard deviation, and diffusion coefficient are reported -more precisely α is the rate at which the pollutant concentration decreases, which is obtained from fitting a simple exponential decay model source exp −αt to the experimental data. there was no significant statistical difference between the rates of diffusion of the particles emitted by the monopolar versus the apc instruments. the coefficient of diffusion corresponding to the harmonic instrument is lower but has strong variation. we interpret this result with the fact that the range of size of particles produced by the harmonic instrument is wider and the distribution of size is random. in some trials the particles emitted were then too small, they can go down to 0.06 microns [36] , to be detected by our sensors while in other only detectable particle were produced. as mentioned earlier, covering the sensor with a surgical facemask dropped the number of large particles to some extent, but there are always leaks on the sides. as noticed in the literature standard, surgical facemasks do not protect from ufp. a 3-dimensional (3d) cfd model is used to simulate the dispersion of a single source of pollutant in an or. the dimensions used in the model are the ones in the surgical suite where the clinical study and validations were made. every or is different, but the order of magnitude of the physical quantities is the same for each or in our clinical study. figure 2 provides the geometric details of the simulation setup that takes into account the geometry of the room, location of air conditioning ducts, location of the doors, and air leaks due to positive pressure despite closed doors. table 2 lists the boundary conditions on velocities and temperatures of the or and its adjacent hallway obtained from measurements. the surgical smoke plume in the cfd model was simulated using an injection of co 2 at the location of the or table for a duration of 10 s. the co 2 phase was tracked in the multi-phase cfd simulation as a marker of pollution. the smaller the particle, the best the dispersion model would be based on gas transportation. figure 6 shows the dispersion of the plume inside and outside the or, while the door is closed. dispersion into the hallway was due to the air leaks between the door of the or and its frame. verification of the simulation was obtained by refining the mesh and time step until it reached a numerical convergence on the quantities of interest -in particular, the density of co 2 and velocity of flow at specific locations. table 3 provides a comparison between the different velocity values found by the model and by the direct measurement obtained at those locations. the time intervals were also computed: between the emission of the pollutant and the time when an air sensor detected the pollutant inside the or, close to the door, and in the hall outside the door. the results of table 3 provides the first level of validation of the cfd simulation. in table 3 , r 3 1 is the ratio of pollutant phase concentration between the sensor location 1 and 3 in the or in figure 4 , and is interpreted as a small particle density ratio as well. it is particularly interesting to notice that the flow at the door has a 3-dimensional component that is driven by the pressure gradient as well as the temperature difference between the or and the hallway. while the or is kept under positive pressure, it loses this pressure as soon as the door is opened. because the temperature of the or is generally cooler than the temperature of the hall, we observe from the cfd that the buoyancy effect causes back-flow between the adjacent hallway when the door is opened. this might be part of the mechanism of contamination between ors. this result is consistent with air quality measurements done in controlled experimental conditions presented hereafter. it was found that the mixing of contaminants from a burst source to the rest of the or is reached within a minute; applying a simplified compartment model to describe the or's contribution to pollutants using a time step of one minute became apparent. this upper-scale model described in the methodology section will be calibrated next. the identification of the model parameters from the experimental data-set corresponding to the setup in figure 4 is explained below. the experiment was designed first, to assess the delay of pollutant transmission between the or and the hallway depending on if the door was opened or closed, see figures 7 and 8 . second, to compute the rate at which pollutant concentration decreases. an exponential decay was observed in the or, which is consistent with the fact that diffusion is the main mechanism due to the small velocities present inside the model. however, the hallway behaves more like a duct with a combination of convection and diffusion running down the hallway. these measurements are consistent with the cfd simulation results shown previously. fitting the simplified model to the controlled experiment with a single source of smoke, the coefficients of diffusion in the or and in the hallway can be retrieved, as well as the convection velocity in the hallway-see table 4 . figure 7 . source in closed-door or and its impact on hallway air concentration: a 2 min delay in transmission from or to hallway and an exponential decay for each signal was observed. the diffusion coefficient in the or and the hallway are dependent on the hvac system that is, by design, more effective in the or than in the hallway. therefore, the rate of decay in the or is twice as large as the rate of the decay in the hallway. as reported before, the diffusion coefficient for the particle tracking setting is about the same for the spray source as it is for the monopolar or apc sources. the transmission condition with a closed or door is not negligible: it is about 4 times less than with an open door. from figure 9 , the traveling wave velocity is reconstructed and travels about one or width in a minute, v 0 is about 0.1 m/s at mid-hall location. this small velocity in the hallway could not be directly measured, but it is in agreement with the cfd simulation reported earlier. table 4 . parameters of the model obtained by fitting the outcome on single-source controlled experiments with injection source locations; 9 measures were used to obtain this table. the coefficients of decay are (ρ or and ρ hall ) as well as the coefficients of transmission between these spaces (α or ) from the or to the hall. β or represents the flow from the or to the hall when the door is open. 9 . effect of door opening and closing on propagation of marker from one or to the next or down the hall: • is the sensor close to the source in the or, is 10 times the concentration down the hall, and is 100 times the measured concentration in the next or down the hall-for convenience, we have plotted in solid line the exponential model fitting for the experimental datasets in the main or and in the hall. the most important result is summarized in figure 9 : surgical smoke emitted in a single or can rapidly reach the hallway within a minute due to the or door opening and is diluted by a factor of roughly 10. in the unfortunate event that the door of the next or is opened, then some trace of the surgical smoke emitted by the or upstream, can flow inside the next or down the hall; while the level of exposure to surgical smoke would be insignificant in this second or, it is clear that the standard positive pressure established in these ors cannot guarantee those airborne particles do not propagate from one or to another. over a period of several months, this rare event might be capable of propagating an airborne disease. in fact, the frequency of door openings of each or can be very high, as shown in figure 5 , that the probability of propagating airborne diseases and contaminating other ors seems inevitable. next, to systematically assess long-term exposure, the result obtained by coupling the air quality model with an agent-based model (abm) of the surgical flow will be reported on. figure 10 shows a measurement done during a clinical study with 3 consecutive laparoscopic procedures during the day. the red curve accounts for the number of particles detected by the sensor inside the or, while the blue curve provides the corresponding measurement from the hallway. patients' registration starts at 7 a.m., before any surgery occurs, and lasts all day until all surgeries are complete. large peaks of particle concentrations were observed during the times the or was being cleaned. these peaks were removed from the or acquisition curve that corresponds to the use of detergent. similarly, during the preparation and closing of the patient, the sensor sometimes captured the use of chemicals when preparing the sterile field or the leak of anesthetic gas. the red peak during the third procedure in figure 10 most likely corresponds to an excess of surgical smoke. as expected, the concentration of particles in the hall is not strictly correlated to the emission of surgical smoke in the or. the hallway collects pollutants from a number of ors under positive pressure at the same time. because of this, it is difficult to separate out surgical smoke from other sources in the hallway measurement, such as chemicals used in the preparation of patients located in ors upstream. the model is built to qualitatively reproduce the concentration of surgical smoke inside an or and its adjacent section of the hall. in our simulation (see figure 11 ), the emission of surgical smoke is restricted during the time the patient is intubated. this simulation was done on the whole surgical suite with a stochastic production of smoke in each or similar to the one reported in meeusen et al. [19] . one observation is the same pattern of pollutant concentration in the hallway as seen in the clinical dataset. in particular, there is no obvious correlation between the source of smoke in the or of that simulation and the concentration in the section of the adjacent hallway. in fact, while exposure to surgical smoke in the or is relatively intense in a short period of time and then vanishes, the pollutant is stagnant in the hallway for a much longer period of time and therefore contributes to long-term exposure. overall, the delay ∆t 2 in the transmission conditions in (4) and (5) has very little influence on the result and can be neglected. to expand the study, this simplified air quality model was then coupled to the abm of surgical flow that reproduces the daily activity of a large surgical suite over a long period of time [31] . the model was calibrated using custom-made sensor systems placed at key locations of the surgical suite to capture the daily activity over a period of a year [29] . this or suite, dedicated to general surgery, has about 20 ors distributed in a layout as seen in figure 1 and is rather typical of the activity in a large urban hospital. a simple stochastic model is assumed for the source of surgical smoke in each or, similar to the previous one. the probability p door ∈ (0, 1) of the number of or door openings per minute is a parameter of the model. on average, one door opening every two minutes during a surgery is rather standard. this is mainly due to the fact that the staff may have to support logistics in various ors at the same time, and that coordination of team activity is still done by a direct conversation in the surgical workflow. in fact, it is common knowledge that a door opening every 8 min on average would correspond to a very strict policy controlling traffic in the surgical suite, but it would only reduce the exposure in the hallway by half. from the simulation, it is concluded that long-term exposure to surgical smoke in the hallway is about the same order of magnitude as the one in the or. figures 12 and 13 demonstrate the effect of the frequency of door openings on the average concentration of pollutants that a staff member is exposed to during the day. there was a noticeable low concentration at the upstream end of the halls, which was confirmed by direct measurement with the particle counter. enforcing strict control on door openings may reduce the level of transport and diffusion of hazardous airborne particles in the hallway by half. the model can be ran to test a fictitious situation as in figure 14 where every other or has an ideal practice and generates no surgical smoke at all. the usage of ideal exhaust ventilation devices during surgery in half of the ors has a direct linear correlation with the rate of exposure in the hallway, and it seems to be the most efficient technique to reduce long-term exposure; it cuts down the staff's exposure to surgical smoke in the hallway by half. about half a million healthcare professionals are exposed, daily, to surgical smoke in their clinical activities. transport and diffusion of hazardous airborne particles such as virus, generated by surgical smoke in particular, and its long-term effects on staff have not been studied systematically yet. the debate on the impact of surgical smoke on patients' and staff's health is reminiscent of the incident involving airborne hazards from anesthetic gas [23, [38] [39] [40] [41] . the national study led by the american society of anesthesiologists established that "female members in the operational room-exposed group were subject to increased risks of spontaneous absorption, congenital anomalies in their children, cancer and hepatic and renal disease." while the link with waste anesthetic gas (wag) was not clearly established at that time, except in animal studies, the stream of work initiated in the 70's [23, 41] eventually ended up in a better management of wag "by always using scavenging systems, by periodically testing anesthetic machines for gas leaks, and by not emptying or filling vaporizers" [42] . unfortunately, the efficiency of surgical masks to prevent virus transmission or surgical smoke breath intake is usually tested using non-biological markers while their use in hospitals is mostly against airborne biological particles. standard surgical masks and filtration techniques are not effective on ufp, which include viruses and bacteria. for example the sars-cov-2 as a size comprised between 0.06 to 0.14 microns [43] . long et al., showed in a new meta-analysis that there was no significant difference in effectiveness between surgical masks and n95 masks against laboratory-confirmed respiratory viral infections [13] especially at higher inhalation flow rate [14] . seongman et al., also showed that for viruses, effectiveness of surgical mask (and cotton based masks in the paper) are dependent to virus concentration and flow rate of inhalation but also showed a higher concentration of viral load on the outside of the mask compared to the inside [44] . yang et al., built a multi-criteria decision-making method based on the novel concept of the spherical normal fuzzy to assist healthcare staff in the decision of which mask to wear [45] . an italian research team is underlining the possible correlation between concentration of particulate matter (pm) with propagation of the virus like, for example, in the north part of italy where industrial pollution is high [46] . high concentration of pm could be a vector of propagation and needs to be carefully observed inside building, especially hospitals. being able to know where these particles are and in what concentration seem then the best protection and awareness for staff to avoid staying too long in contact. deposition rate of these particles is not addressed in this paper and researchers are still debating about lifetime on surface of the sars-cov-2 virus [47] . a method to construct a surgical-suite-specific model of the transport-diffusion of airborne particles can quickly be calibrated with cost-effective wireless particle counters. coupling this indoor quality model to the previous multi-scale model of surgical flow [28] [29] [30] allows quantification of surgical smoke exposure across long periods of time and provides a rationale for recommendations. as a matter of fact, the abm of surgical workflow allows insight into the human behavior factor, which can be included in the analysis. this work may expose rare events, such as contamination from one or to another, which when accumulated over the months becomes a tangible risk. this study has potential because it can run for long periods of time and can address the complexity of hundreds of staff's spatiotemporal behaviors in a large or suite. the cfd model requires detailed geometric and boundary conditions to be reliable; the k − model is an approximation that has its own limit as well. running a cfd model is a tedious process both in setting up the mesh and simulation parameters, as well as in terms of central processing unit (cpu) time required. cfd was used here only to test the components of a hybrid stochastic compartment model that incorporates the mechanism of diffusion-transport of airborne particles at the surgical suite scale over a one-year period. a coarse statistical model was used for the source of surgical smoke: the actual generation of surgical smoke depends on the surgery team, type of procedure, and many more parameters. however, the capability to non-invasively monitor such parameters using appropriate sensors via the cyber-physical system is available. the hybrid partial differential equation (pde) compartment model provides a first-order approximation of average exposure at the room scale. the delay in the transmission conditions between the or and the hall in equations (4) and (5) is not essential to reproduce the result on daily exposure to smoke. in the meantime, the uncertainty of the hvac input/output provides a much larger error. furthermore, deposition of particles on the or's surfaces was not taken into account. the deposition of ufp may be expected to be negligible [22] . a low accuracy model that carries an error of the order of 20% could, however, be conclusive for this study. as a conclusion, it is particularly important to recognize the impact of door design and human behavior when considering hazardous airborne particles spreading throughout a surgical suite. or doors constantly leak air depending on the difference of pressure with the outside hall, and they contribute to the transport of particles throughout the surgical suite. the door opening effect depends on the motion of the door and also the difference of temperature between the or and the hallway. some of these negative impacts can be controlled by a better design of the door and of the temperature control in order to work with a more cost-effective hvac design. the benefit of positive pressure in the or is still canceled by door openings, inducing possible back-flow and contamination from the hallway, especially when the door stays open for several minutes. therefore, efficient movements by personnel may improve indoor air quality and should be quantified. the architectural design of the or suite should optimize the circulation of staff and patient movement activities. the next important step in the modeling to address the complementary aspect of biological transmission versus physical transportation is to correlate the database of staff's pulmonary events with the study's findings to recognize these rare events. we can then translate the quantitative model of surgical smoke transport into a risk assessment for staff's health. the model should be surgery-specific: an efficient cyber-physical infrastructure should non-invasively monitor energy usage and smoke presence to instantly deliver awareness on practices that can improve air quality. a factor that has been neglected is the deposition of surgical smoke in the common storage area, see figure 1 , in which all ors have personal access to via their back-doors. this may offer a different mechanism of propagation of biological material. in the current study, quantification of surgical smoke concentration in the hallway, the duration of exposure along the year, and the mechanism of propagation of hazardous airborne particles from one or to another was feasible. on the practical side, an automatic sliding or door seems to be a better solution over a traditional door's rotation that acts as a pump. the analysis can also be extended to address the problem of the optimum placement of uv lights in the hallway to improve air quality in an efficient and controlled way. finally, the importance of aorn's guideline in the use of a vacuum system during surgery needs to be reinforced at a time when elective surgery may involve asymptomatic covid-19 patients. author contributions: this project is highly interdisciplinary and required all co-authors contributions to establish the concept and reach the goal of the paper. m.g. leads the project, designed the overall framework, including the hybrid model, agent-based clinical model, and matlab code implementation. g.j. did the cfd model at the or scale, participated in the design of the overall method and ran the experiments with air quality sensors required for validation. s.f. participated in the experiments with air quality sensors and with or door activity sensors. all authors contributed to the redaction of this publication. all authors have read and agreed to the published version of the manuscript. airborne transmission of sars-cov-2: the world should face the reality turbulent gas clouds and respiratory pathogen emissions: potential implications for reducing transmission of covid-19 airborne transmission route of covid-19: why 2 meters/6 feet of inter-personal distance could not be enough review awareness of surgical smoke hazards 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recent data summary and its importance for medical and dental settings funding: this research received no external funding. the authors declare no conflict of interest. key: cord-311311-rmv5rfst authors: harper, martin title: recent advances in occupational exposure assessment of aerosols date: 2020-09-18 journal: int j environ res public health doi: 10.3390/ijerph17186820 sha: doc_id: 311311 cord_uid: rmv5rfst exposure science is underpinned by characterization (measurement) of exposures. in this article, six recent advances in exposure characterization by sampling and analysis are reviewed as tools in the occupational exposure assessment of aerosols. three advances discussed in detail are (1) recognition and inclusion of sampler wall deposits; (2) development of a new sampling and analytical procedure for respirable crystalline silica that allows non-destructive field analysis at the end of the sampling period; and (3) development of a new sampler to collect the portion of sub-300 nm aerodynamic diameter particles that would deposit in human airways. three additional developments are described briefly: (4) a size-selective aerosol sampler that allows the collection of multiple physiologically-relevant size fractions; (5) a miniaturized pump and versatile sampling head to meet multiple size-selective sampling criteria; and (6) a novel method of sampling bioaerosols including viruses while maintaining viability. these recent developments are placed in the context of the historical evolution in sampling and analytical developments from 1900 to the present day. while these are not the only advances in exposure characterization, or exposure assessment techniques, they provide an illustration of how technological advances are adding more tools to our toolkit. the review concludes with a number of recommended areas for future research, including expansion of real-time and end-of-shift on-site measurement, development of samplers that operate at higher flow-rates to ensure measurement at lowered limit values, and development of procedures that accurately distinguish aerosol and vapor phases of semi-volatile substances. exposure science is underpinned by exposure assessment [1, 2] . exposure assessment includes modeling of exposures, but most importantly characterization (measurement) of exposures. in this article, six recent advances in sampling and analysis in the occupational exposure assessment of aerosols will be addressed. three advances to be discussed in detail are (1) recognition and inclusion of sampler wall deposits; (2) development of a new sampling and analytical procedure for respirable crystalline silica that allows non-destructive field analysis at the end of the sampling period; and (3) development of a new sampler to collect the portion of sub-300 nm aerodynamic diameter particles that would deposit in human airways. three additional developments will be described briefly: (4) a multi-fraction size-selective aerosol sampler; (5) a miniaturized pump with interchangeable sampling heads to meet different size-selective sampling criteria; and (6) a novel method of sampling bioaerosols including viruses while maintaining viability. in order to understand the rationale behind these recent advances, it is first necessary to recognize the historical context of prior advancements. aerosols have been measured in the occupational environment since before 1900. dust collection for investigation probably occurred even before the invention of the aëroconiscope in 1870 [3] . mining is one of the dustiest occupations and much research has been undertaken and many developments have arisen from consideration of aerosols in mining. the procedures in use at the beginning of the twentieth century are not the same as those used today. changes have been driven by advances in our understanding of aerosol behavior and toxicity in air and in the human airways, and by technological innovations. one of the first procedures to be used in mining was to pull a measured volume of air through a tube containing granulated sugar [4] . the passage of humidified air caused the surface of the sugar to become sticky, trapping particles carried in the airstream. after sampling, the sugar could be dissolved and filtered off and the particles ignited to remove organic material. then the particles could be segregated by size, if necessary, by sedimentation and then counted under a microscope in order to obtain a particle number concentration in particles per cubic centimeter or millions of particles per cubic foot. the sugar tube was used in mines prior to 1900 until its replacement by the greenburg-smith impinger in 1922 [5] . in this device, air is accelerated by passing through a nozzle immersed in water. the particles contained in the air have enough momentum to be carried to an impaction plate where their momentum is arrested and the particles are stopped, wetted and remain in the water for subsequent analysis. the original device operated at 1 cubic foot per minute (28.3 l min −1 ), but in 1934 it was miniaturized (the "midget impinger") to operate at flow rates in the range of ml min −1 [6] . impingers had the problem that they contained liquid, which was cumbersome in the field, but also it was known that very fine aerosol (original experiments used magnesium oxide fume and tobacco smoke) was not collected efficiently-such aerosol would attach to the surface of the air bubbles and be released through the bubbles bursting at the outlet. in 1944, the us bureau of mines investigated glass fiber filters [7] , and in 1957, the us public health service (the precursor of the national institute for occupational safety and health) published on the virtues of the polymeric membrane filters they had been using for several years [8] , contained in brass holders similar to those being used by the us atomic energy commission for airborne radionuclide sampling. also, in the 1950s it was realized that it was the finer or "respirable" particles that were most toxic [9] , so that size-selection curves were developed along with size-selective sampling devices (cyclones) for fine particle sampling. for several years, different respirable fractions were measured in different jurisdictions, which eventually were reconciled under iso [10] . for larger particles than those considered for pneumoconiosis in mining, it was felt that all particles should be collected, and since the critical health issue is absorption and systemic toxicity, particle mass is considered more important than particle number (with the exception of mineral fibers). in 1956, a plastic cassette to house filters was developed for "clean-room" particle sampling, and, in 1960, it was featured in the first edition of the air sampling instruments handbook from the american conference of governmental industrial hygienists [11] . it was made by the millipore corporation and known as the "millipore monitor", but is now widely manufactured and is known more generally as the "closed-face cassette" (cfc), although it can also be used with a ring piece in place of the cap, where it is known as an "open-face cassette" (ofc). both styles are manufactured to be used with filters of 25, 37, or 47 mm diameter. the cap on the cfc includes a small (4 mm) entry orifice, an advantage in preventing accidental or deliberate tampering with the filter, which was important since by that time pumps had been developed of size and weight suitable to be carried by a person for "personal sampling" [12] . it was believed that the cassette and filter, now known as a "sampler," collected all particles in the air that would be relevant to inhaled dose onto the filter. the filter was the only part analyzed and considered to be an analysis of total particulate mass (tpm). however, only particles up to a certain size were collected efficiently [13] . further research in the 1980s showed that larger particles up to 100 micrometers aerodynamic equivalent diameter (aed) could enter the nose and mouth while breathing [14] . since mass is related to the cube of the diameter, even small numbers of large particles could have a profound impact on the inhaled mass dose. note that most studies have considered solid particles; the behavior of liquid particles needs further study the "inhalable" sampling convention developed out of this research [15] and an "inhalable" sampler was developed at the institute of occupational medicine in scotland, which became known as the iom personal inhalable sampler [16] . during the development of this sampler it was clear that a substantial portion of the collected sample either did not reach the filter or bounced off the filter and was deposited on the internal surfaces [17] . the same issue affects other samplers, and its importance has now been studied over 30 years with clear conclusions bolstered by the research. that many occupational hygienists and the laboratories that serve them are either unaware of, or slow or even unwilling to accept the consequences is a puzzle. samplers for aerosols typically consist of a filter or other collection substrate, for example an impaction plate or foam, supported in a container or holder. the entire device typically is considered an aerosol sampler. part of the aerosol entering a sampler will deposit on the internal surfaces of the sampler prior to reaching the collection substrate. there are a number of mechanisms by which this can occur, including direct inertial impaction, gravitational settling, interception resulting from eddies during transport, electrostatic attraction and bounce from the filter [18] . all of these mechanisms can occur simultaneously, with the relative importance depending on factors such as particle size, shape, and density; inertial velocity; wind speed and orientation; etc. in addition, after sample collection, if the collection substrate is transported while mounted in the sampler, it is possible that particles originally deposited on the collection substrate may dislodge during transportation. such particles can thereby contribute to deposits on the walls, as well as on the base of any cover plate or plug [19] . all particles found elsewhere than on or in the collection substrate are often loosely termed "wall deposits". deposits on internal surfaces are often invisible to the naked eye, even with transparent cassettes; they often comprise a large fraction of the aerosol that enters the sampler [20] (updated with additional information from the authors, personal communication, october 2007), [21] [22] [23] [24] and can even exceed the quantity collected on the filter. example data for cfcs are presented in table 1 . if the sample of interest entails the entire aspirated air particulate into the container or holder (sampler), it is necessary to account for these wall deposits, especially if it cannot be shown that they should be disregarded. similar data for the iom sampler are given in table 2 . table 1 . summary of findings of internal wall deposits as a percentage of total catch (filter plus walls) in field samples using the 37-mm cfc sampler. copper smelter [25] 18 cu 21% 55% lead ore mill [26] 9 pb 19% 35% solder manufacture [27] 30 pb 29% 74% battery production [20] 16 pb 28% 66% battery recycling [26] 54 pb 29% 54% welding [28] 10 cr(vi) 5% 55% plating [28] 12 cr(vi) 12% 17% paint spray [28] 29 cr(vi) 7% 12% zn foundry [20] 9 zn 53% 62% zn plating [20] 18 zn 27% 91% cast iron foundry [20] 18 fe 22% 46% grey iron foundry [20] 18 fe 24% 77% bronze foundry [29] 6 cu, pb, zn 19%, 13%, 15% 45%, 17%, 21% cuproberyllium alloying [20] 4 cu, be 31%, 12% 40%, 39% solder manufacturer [30] 50 pb 45% 77% solder manufacturer [30] 47 sn 56% 93% table 2 . summary of findings of internal wall deposits as a percentage of total catch (filter plus walls) in field samples using the iom sampler. lead ore mill [26] 8 pb 19% 30% copper smelter [25] 17 cu 16% 38% copper refinery [19] 48 cu 18% 36% battery production [31] 11 pb 8% 33% welding [31] 18 al 3% 13% cast iron foundry [20] 18 fe 8% 69% grey iron foundry [20] 18 fe 5% 16% bronze foundry [29] 6 cu, pb, sn, zn 0%, 0%, 0%, 3% 10%, 3%, 23%, 6% most research exploring the extent of the wall loss phenomenon has considered inert particles [32, 33] , and metalliferous particulates [20, 21, [26] [27] [28] [29] [30] [31] . however, the issue has also been studied for airborne organic materials, including bacterial endotoxin [34] , wood [35] , and pharmaceutical dusts [22] ; another relevant study reported results from investigations in thermosetting plastics, wood, paper, and animal breeding [36] . except in the case of very large wood dust particles, there is no evidence to suggest that wall deposited particles are sufficiently different from those found on the collection substrate to warrant their exclusion [37, 38] . wall deposits are not limited to aerosol samplers for larger airborne particles but may also be found in samplers for finer particles [39, 40] . there is a justification for excluding wall deposits where the performance of an aerosol sampler tested to european standard en 13205 [41] shows appropriate compliance with the relevant iso 7708 size-selective convention without their inclusion, but it should not be assumed. the sampling and analytical methods published in the niosh manual of analytical methods (nmam) represent state-of-the-art methods for assessing worker exposures to toxic chemicals. niosh considers that all particles entering the cfcs and the iom sampler should be included as part of the sample whether they deposit on the filter or on the inside surfaces of the sampler. this is published policy [42] and is further stated in a section on the manual web page. the us occupational safety and health administration (osha) has the same policy [43] , which has specifically been addressed by their gravimetric method [44] since inception, and which is now explicitly written into new methods for metals sampling and analysis [45] . all aerosol particles entering an air sampler should be considered potential contributors to exposure, and this extends to gravimetric and chemical analyses. appropriate sample preparation procedures are necessary to account for material on the internal surfaces. a number of techniques have been proposed, depending on the analytical finish: (1) sample extraction within the cassette; (2) rinsing of internal surfaces and adding the rinse to the filter preparation and analysis; (3) wiping the interior of the capsule and adding the wipe to the filter preparation and analysis; and (4) analysis of internal sampling capsules or cartridges (sampler inserts). very few procedures have been developed and validated for carrying out sample extraction within the cassette. a procedure for in situ (that is, within-sampler) extraction in france [46] uses a 3-piece polystyrene cassette as the container for both sampling and extraction. originally the filter used consisted of quartz fiber media, but the high background of some metals in these filters, together with the need for large quantities of hydrofluoric acid for complete dissolution of the filter, has led to replacement with a cellulose support pad and mixed cellulose ester (mce) filter [47] . after sampling, the cassette is inverted and opened from the rear and the support pad removed, leaving the mce filter in place. acids (perchloric first, followed by nitric and hydrochloric, with hydrofluoric where necessary) are pipetted into the cassette, which is then sealed and placed in an ultrasonic bath for 10 min with the cassettes being upended after 5 min. rinsing has not been shown to be a procedure with efficient recovery in most cases [43, 47] . osha methods use wiping, for example with a clean "smear tab" (or 1 × 2 inch section of "ghost wipe") that has been moistened with deionized water and placed in the same digestion beaker with any rinse of the interior and the sample filter [48] . the use of a single wipe without rinse has been validated by a niosh study [47] . however, wiping is cumbersome and potentially subject to operator variability. that leaves sampler inserts as the most practical and valid method for either gravimetric analyses or digestion with analysis for metals. as noted above, osha uses a sampling method for gravimetric analysis, which is a cfc containing an internal capsule comprising a polyvinyl chloride (pvc) filter and aluminum foil cone. the cone has an inlet hole at its apex directly under the cassette inlet and the edge of the cone is tightly fixed to the filter. all particles are thus included on the filter or otherwise within the cone, which is pre-and post-weighed without removing the filter [44] . the same system is used with a cyclone pre-selector for respirable dust and respirable crystalline silica, in which case the filter is used to wipe the interior of the foil capsule before being digested for redeposition and analysis [49] . these capsules are expensive and a proposal for a cheaper all-pvc version (capsule and filter) was first suggested in 1992 for the collection of pharmaceutical dusts [50] . these sampler inserts are now available for both the 25 mm and 37 mm cfc and are known as gravi-serts™ (zefon international, inc., ocala, fl, usa), and they can be used in niosh method 0501 [51] . the disposable inhalable sampler (dis; zefon international, inc.) is a dimensional copy of the iom sampler (patent pending) and similar pvc capsules and filters are available for the dis. since the geometric design of the dis sampler is identical to that of the iom sampler it has identical size-selective sampling performance characteristics. this was tested in bakeries with identical gravimetric results for side-by-side dis and iom samplers [52] . cellulose acetate inserts with mce filters have been extensively tested [30, [53] [54] [55] and are intended for acid digestion and subsequent analysis for metals in niosh method 7306 [56] . commercially available products include solu-serts™ (zefon international, inc.) and solu-caps™ (skc inc., eighty four, pa, usa). again, a similar product is available for the dis (zefon international, inc.). the dis has other advantages over the iom sampler-for example, it is inexpensive and intended for single use, thus avoiding the need for clean-up and the possibility of cross-contamination, an important concern for trace metal analysis. background levels of the cellulosic dis after microwave digestion in a combination of acids (nitric-hydrochloric-hydrofluoric) with hydrogen peroxide, by magnetic sector inductively coupled plasma-mass spectrometry are presented in table 3 . . the method reports a bias of 0.058 and an overall precision of 0.059, for an accuracy of ± 15.5%. weight stability over 28 days was verified for both blanks and spiked capsules. independent laboratory testing on blanks and field samples verified long-term weight stability and uncertainty estimates. the working range is given as 0.25 to 5 mg per sample, with an estimated limit of detection of 0.075 mg per sample and a precision of 0.031 at approximately 2 mg per sample. an inter-laboratory study of niosh method 7306 [56] for 33 elements, gave recoveries better than 80% for all elements at both low and high spike levels, except for silver at the higher level. accuracy of analysis, calculated from a combination of precision and bias, was 25% or less for all elements except silver. niosh methods 0501 and 7306 should be used in place of older methods and the analyzing laboratory has a duty to inform their clients in this regard; iso 17205 section 7.1.2 states "the laboratory shall inform the customer when the method requested by the customer is inappropriate or out of date" [57] . consideration of internal sampler wall deposits is included in related international voluntary consensus standards, published by iso and astm international (formerly american society for testing and materials), which describe the sampling and analysis of airborne metals and metalloids in occupational atmospheres. other large particle samplers have been shown to have internal deposits other than on the filter [25] . samplers that collect both aerosols and vapor where, for example, the sampler consists of a filter cassette and sorbent tube in series may be similarly affected. where cyclone-cassette assemblies are used for fine-particle, "respirable", internal non-filter deposits are still found [39, 40] but it is more difficult to account for them. cassettes made of conductive materials dissipate the charges induced by charged fine particles, minimizing losses to the internal surfaces and should be used in place of non-conductive cassettes [42] . exposure to respirable fraction of crystalline silica (rcs) by inhalation can cause silicosis, lung cancer, other respiratory diseases, and kidney diseases [58] . silica, especially quartz, is a common constituent of mined and quarried rocks and mineral deposits and materials used in construction including concrete, cement, bricks, aggregates, granite, slate and limestone. exposure to rcs can occur during typical mining and construction. while the number of miners is relatively small, osha estimates about two million construction workers are exposed to rcs in over 600,000 us workplaces [59] . studies of construction exposures have reported excessive exposures associated with certain tasks. for example, exposures ranging as high as 100 times the niosh recommended limit of 0.05 mg/m 3 have been reported. the new osha comprehensive standard for silica in construction in effect from 23 september 2017, alongside a similar rule for general industry which took effect 23 june 2018, lowered the permissible exposure level (pel) for rcs to 0.05 mg/m 3 rcs and added a concentration level where exceedances would drive further enforcement actions (i.e., an "action level") of 0.025 mg/m 3 [59] . osha estimates that more than 840,000 construction workers are exposed to rcs levels that exceed the new pel. osha's preliminary economic analysis and initial regulatory flexibility analysis expects a net benefit between $2.8 and 4.7 billion annually over the next 60 years by preventing between 579 and 796 annual fatalities from rcs exposure in all industries [59] . demonstrating compliance with exposure limits for rcs requires the collection of respirable dust. respirable dust is sampled from air using a cyclone or impactor to separate the respirable fraction from aerosol. evaluation of rcs in the respirable dust has, until recently, required the use of off-site sophisticated laboratory analysis, but this can involve results being returned up to several weeks following the period sampled. long lag times can lead to unacceptable conditions persisting during the interim without being recognized or addressed. while x-ray diffraction (xrd) is used extensively for the determination of silica in air samples, results from a proficiency test scheme for laboratories did not show any strong bias between xrd analysis and analysis by fourier transform infra-red (ftir) spectroscopy [60] . ftir spectrometers today are manufactured sufficiently robust and small enough to be taken to the field allowing on-site analysis. a methodology to quickly determine monitoring exposure results, even if the accuracy is outside of that required for compliance purposes, can be very useful. niosh has developed a field-ready methodology capable of an end-of-shift (eos) measurement for rcs contained in airborne dusts in the mining sector [61] , and this has been found also to provide results comparable to xrd [62] . rcs is collected on a direct-on-filter (dof) sampler (eos™ silica cassette, zefon international, inc.) attached to any one of several different cyclones to select the respirable dust fraction. following sample collection, the cassette is removed from the cyclone and placed in a holder in any one of four different models of ftir spectrometer, which have been evaluated for the purpose [63, 64] . in a complex matrix of minerals, the estimation of quartz content may be subject to interference. work is continuing to minimize these interferences by means of mine-specific correction factors [65] . however, since ftir is a non-destructive analysis, samples can still be submitted to a laboratory for further analysis. this method promises to be applicable to monitoring rcs in the construction sector. however, the construction samples contain not only silica but other components, which are typically different from those found in mines and quarries. the presence of such components also may interfere with the ftir response from rcs in a manner which might require corrections to be applied to obtain a valid result. a recent pilot study evaluated possible interferences from different types of construction dusts-including drywall, plaster, cement, and brick-in laboratory-generated mixed dust samples [66] . results from a set of prepared samples analyzed by portable ftir showed that a) plaster and drywall dusts do not interfere substantially with the quartz measurement; b) cement does not interfere with the quartz measurement, but it does change the background absorbance of the filter; and c) in addition to having a substantial quartz content that has to be carefully evaluated in any study, brick dust may also contain an additional material, probably a silicate mineral, which interferes additionally with the quartz peak. in the range of interest from 20-110 µg per filter sample (bracketing the niosh rel/osha pel for a 1 m 3 air sample), 83% of the quartz contents predicted from the averaged calibration data agreed within 50% of the adjusted nominal loadings and 91% agreed within 100%. this result is encouraging given the high levels (500 or 1000 µg) of interfering dusts. an on-site reading of 100 µg is highly likely to be above the pel, and a reading of 50 µg is highly likely to be above the action level so that appropriate action could be taken prior to receiving a definitive analysis from the laboratory. samples loaded with smaller amounts of all four dusts in combination gave even better results, with all nine results within the range of interest falling within 25% of the adjusted nominal loadings. both cement and brick could be correctable interferences once the identity of the interference is revealed, in the same way that correction is made for interfering mineral dusts in mines, and this is one of the further investigations suggested by this pilot study. exposure through inhalation of both incidental and engineered nanoparticles is a primary concern for worker health and safety, since nanoparticles are considered to have greater reactivity and thus toxicity compared to larger particles of similar composition [67] . particles in this range can be engineered, for example, the metals and metal oxides and sulfides manufactured for commercial purposes, or incidental, including welding and other fumes. although it was once thought that many sub-micrometer particles quickly agglomerate and attain particle sizes that are larger than 0.5 µm, there is growing evidence that a significant fraction of particles remains in singular or small number agglomerates in the nanoparticle (<100 nm) range. around 300 nm there is a minimum in particle deposition in human airways, so that a particle of this size is far more likely to be breathed back out into the ambient atmosphere than to be deposited internally [68] . below 300 nm the deposition efficiency begins to increase with decreasing particle size as a result of the importance of particle movement by diffusion resulting from brownian motion, and interception. this deposition mechanism, unlike impaction or gravitational settling, occurs as much in the head airways as the lower respiratory tract. based initially on work on the collection efficiency of nylon screens [69] , a lightweight (60 g), personal nanoparticle respiratory deposition sampler (nrd sampler, zefon international, inc.) was developed to selectively collect particles smaller than 300 nm [70] . most samplers are designed to sampling conventions that are based on penetration, but in this case the sampler was designed to collect nanoparticles with efficiency matching their deposition in the respiratory tract, in order to provide a physiologic relevance to sampler's performance. a new sampling criterion was devised to provide the relevant deposition efficiency for the sampler [70] . the sampler operates at 2.5 lpm and consists of a respirable cyclone fitted with an impactor and a diffusion stage. the cut-point diameter of the impactor is 300 nm with a sharpness σ = 1.53. the diffusion stage collects particles smaller than 300 nm according to the proposed convention. impactor separation performance was not affected in experiments of loading at particle levels typically encountered in workplaces. the pressure drop of the nrd sampler is sufficiently low to permit its operation with conventional, belt-mounted sampling pumps. the initial design used nylon screens as the diffusion stage, and the limit of detection of common metals in welding fume using low-level spikes was determined to be 0.3 ug ni, 0.4 ug cr, and 0.9 ug fe. both laboratory [71] and field [72] trials in welding environments produced excellent results for short term samples, although it is possible that agglomerated particles, such as those which characterize welding fume, could with loading affect the porosity of the nylon screens, altering performance. studies found that interception did become important as a collection mechanism as the collection of agglomerated nanoparticles progressed to higher loadings [73] . performance of the nylon screens for agglomerated particles was found to be affected when the accumulated nanoparticle fraction loadings exceeded 1 mg. the change in performance also was accompanied by an increase in pressure drop across the screens to 14.3 kpa, and this could cause many commercial sampling pumps to fault. at the american conference of governmental hygienists (acgih ® ) threshold limit value (tlv ® ) for welding fume of 5 mg/m 3 , a one-hour sample at 2.5 l min −1 collects 0.75 mg. the nanoparticle fraction of welding fume is typically less than half the total mass [70] , so nylon screens are effective in sampling welding fume for one-hour or less. nylon screens were also used for area samples of metal gouging and lancing side-by-side with inhalable, thoracic and respirable samplers [74] . nanoparticles averaged around 170 nm in fume during gouging and around 30 nm during lancing. nrds iron mass concentration was about 21% of inhalable for gouging and 28% for lancing. unsurprisingly nanoparticles surface area dominated the total surface area. the sampler now comes with a choice of two different diffusion stages. an alternative diffusion collection substrate, polyurethane foam, has characteristics more closely resembling human airways and may be preferable for collecting agglomerated materials, such as welding fume, in higher loading scenarios [70] . polyurethane foam is easily digested in acids [75] and, unlike nylon, does not contain titanium allowing this sampler to be used to assess nanoparticle titanium dioxide [76] . values for the median background of elements on small pieces of commercially available foam (n = 10) cleaned by a proprietary procedure, have been determined [77] after microwave digestion in a combination of acids (nitric-hydrochloric-hydrofluoric) with hydrogen peroxide, by magnetic sector inductively coupled plasma-mass spectrometry and are presented in table 4 . results have been scaled to the size of the foam used in the nrd sampler. note that tin (approximately 10 micrograms per foam piece) is a cross-linking agent and is not removed by washing. the effective deposition to the foam was tested using sodium chloride aerosol, and up to 19 mg loading of metal fume was generated from welding rods using spark discharge. field studies included testing against a device providing aerosol size distributions in three workplace situations [78] . good correlations were found in the two workplaces (a heavy vehicle machining and assembly facility and a shooting range) where the aerosol was not dominated by large particles, but the correlation was not so good at an iron foundry where 95% of the particles were >1 µm aerodynamic equivalent diameter. use of nanoparticle respiratory deposition samplers is described in the astm international (formerly american society for testing and materials) standard practice [79] . as noted, particles that penetrate to the gas-exchange region of the lungs are known as respirable. the diseases caused by inert respirable particles, including rcs, asbestos, coal dust, talc, bauxite, etc. cause, respectively, silicosis, asbestosis, "black lung", talcosis and shavers' disease, known collectively as pneumoconiosis [80] . the exposures that cause these diseases traditionally have been assessed by measuring only those particles that penetrate to the gas-exchange region by means of a size-selector that only allows the fine particles to collect on the filter for analysis [81] . these size-selectors are designed to mimic size-selection in the lungs, and the most common technology is the miniature cyclone. on the other hand, particles of any size that can enter the mouth and nose that are soluble and can be absorbed, either in the lungs or in the gastrointestinal tract following expectoration and swallowing, can contribute to the dose of a systemic poison, for example, as with lead or cadmium. these particles are assessed by measurement of all particles that can enter the nose and mouth; a fraction termed "inhalable" [14] . some metals are also thought to affect the lower reaches of the lung and so occupational exposure limit values for both inhalable and respirable fractions (e.g., for nickel compounds) are beginning to appear. there are many instances where knowledge of both inhalable and respirable particulates can be important and, traditionally, it has been necessary for the worker to wear two sets of sampling equipment (samplers and pumps) for the purpose of making the two determinations. expensive multi-fraction samplers exist but are not widely used. a cheap alternative is the dis with a foam insert as size-selector. with the correct type of foam, it is possible to match the iso respirable convention. the filter collects the respirable faction, and anything between respirable and inhalable is caught in the foam, so that adding the analysis of the foam to that of the filter gives the inhalable fraction. the size-selective performance of foams has been intensively studied and the concept of using foam has been thoroughly researched [82] [83] [84] [85] [86] [87] . in a field study in several industries [86] the iom dual-fraction sampler yielded similar results as personal cyclones with an explained variance (r 2 ) of 0.8 and an association (β) of 0.93, not different from unity. the size-selective performance of the foam used in the dis has been tested at the health and safety executive in the uk. over 96% of the aerosol size-distributions tested under en13205 were separated by the dis foam with less than 10% bias from ideal separation as shown in figure 1 . a potential issue of using foam as a size-separator is the effect of progressive particle deposition within the foam on its separation characteristics. penetration tests were carried out using iom samplers with a respirable foam separator for 10 foams previously used to sample dust (aloxite f800) in the laboratory at very high concentrations for short periods of time to produce loadings up to 50 mg, 20 foams previously used in sampling from a range of industries, selected to cover different particle types and dust loadings (on the foam) of up to 20 mg, and 3 blank foams [86] . a slight trend for the 50% cut-point to decrease with increasing loading was evident, but the effect was generally small, even with the exceptionally high loadings. the rapidly loaded laboratory samples were not more affected than the gradually loaded workplace samples. loading effects were independent of particle type and small in comparison to the inherent variability of the foams. in a european study [87] , two different foam plugs were tested with loading. the 10 mm diameter plugs with a mass load of 10 mg showed a decrease in d 50 of some 7%, while 30 mm diameter plugs had a decrease of only 1%. since the plugs in the dis are 16 mm diameter, the expected reduction for a load of 10 mg is expected to be less than 5%. this study reported a limit of detection (lod) of 0.015 mg based on 3 times standard deviation and a limit of quantitation (loq) of 0.050 mg based on 10 times standard deviation of foams similar to the dis foam weighed in a glove box with controlled temperature (peltier element with small fan) and saturated salt bath to control humidity. a different set of experiments in the same study but in a laboratory room with less control on the temperature and humidity gave an lod of 0.069 mg and an loq of 0.230 mg. a potential issue of using foam as a size-separator is the effect of progressive particle deposition within the foam on its separation characteristics. penetration tests were carried out using iom samplers with a respirable foam separator for 10 foams previously used to sample dust (aloxite f800) in the laboratory at very high concentrations for short periods of time to produce loadings up to 50 mg, 20 foams previously used in sampling from a range of industries, selected to cover different particle types and dust loadings (on the foam) of up to 20 mg, and 3 blank foams [86] . a slight trend for the 50% cut-point to decrease with increasing loading was evident, but the effect was generally small, even with the exceptionally high loadings. the rapidly loaded laboratory samples were not more affected than the gradually loaded workplace samples. loading effects were independent of particle type and small in comparison to the inherent variability of the foams. in a european study [87] , two different foam plugs were tested with loading. the 10 mm diameter plugs with a mass load of 10 mg showed a decrease in d50 of some 7%, while 30 mm diameter plugs had a decrease of only 1%. since the plugs in the dis are 16 mm diameter, the expected reduction for a load of 10 mg is expected to be less than 5%. this study reported a limit of detection (lod) of 0.015 mg based on 3 times standard deviation and a limit of quantitation (loq) of 0.050 mg based on 10 times standard deviation of foams similar to the dis foam weighed in a glove box with controlled temperature (peltier element with small fan) and saturated salt bath to control humidity. a different set of experiments in the same study but in a laboratory room with less control on the temperature and humidity gave an lod of 0.069 mg and an loq of 0.230 mg. foam is easily soluble in an oxidizing acidic digestion [75] . the background and loq (based on 10 times the standard deviation of the blank) has been established through analysis by microwave digestion in a combination of acids (nitric-hydrochloric-hydrofluoric) with hydrogen peroxide, using magnetic sector inductively coupled plasma-mass spectrometry. sodium (na) and tin (sn) are normal components of the foam and loqs are 3.8 and 2.1 µg/foam, respectively. for other elements the results are presented in table 5 . table 5 . limits of quantitation (blank corrected) of elements in dis sampler foam (mean of ten foam pieces, data from wisconsin state hygiene laboratory, madison, wi, usa). foam is easily soluble in an oxidizing acidic digestion [75] . the background and loq (based on 10 times the standard deviation of the blank) has been established through analysis by microwave digestion in a combination of acids (nitric-hydrochloric-hydrofluoric) with hydrogen peroxide, using magnetic sector inductively coupled plasma-mass spectrometry. sodium (na) and tin (sn) are normal components of the foam and loqs are 3.8 and 2.1 µg/foam, respectively. for other elements the results are presented in table 5 . pvc filters are preferred for gravimetric analysis because they are more weight stable than other filters but do not digest entirely in acids, and this has led to the use of mixed cellulose-ester (mce) filters in most published methods for metals. however, complete dissolution of the filter is not required so long as the dissolution of the sampled particles is efficient and the presence of undissolved filter does not compromise the sample entry to the instrument or interfere with the analysis. niosh has evaluated method 7304 [88] for processing pvc filters using microwave digestion and osha has a method [48] for processing pvc filters using hot-block digestion. while niosh showed that some elements such as antimony, silver, and tin do not form stable solutions in nitric acid when chloride from the pvc filters is present (and tin is also present in the background of pvc as a polymer cross-linking additive), these elements are not the most commonly evaluated in industrial hygiene investigations. while the osha method does not report any validation of the procedure, there is a back-up data report for the niosh method documenting recovery [88] . in a test on pvc filters with pvc capsules, the wisconsin state hygiene laboratory (madison, wi, usa) was able to digest samples with a nitric-hydrochloric-hydrofluoric acid mixture plus hydrogen peroxide in a microwave oven. although the background quantities of tin, sulfur and sodium are relatively high, all other elements analyzed were present below 1 µg, with many <1 ng. however, recovery studies are still to be performed. using pvc filters allows for gravimetric analysis, and on-filter xrd or ftir analysis for silica, prior to digestion for metals analysis. this leads to the possibility of a single, disposable (single use) sampler with determination of inhalable and respirable particulate, inhalable and respirable metals, and respirable crystalline silica. assessing personal exposure to air pollution is limited to a degree by the technology of samplers themselves. wearable aerosol samplers are often noisy and burdensome, especially those designed to give real-time output of concentration, i.e., monitors. the ultrasonic personal aerosol (air) sampler (upas; access sensor technologies, fort collins, co, usa) has been developed to overcome many of these limitations [89] . the upas features a novel micropump with virtually silent operation. onboard environmental sensors measure and record mass airflow (0.5-2.0 l min −1 , accurate within 5%), temperature, pressure, relative humidity, light intensity, and acceleration. there is a mobile phone application available, which can be used to program and track the upas. pump flow and pressure measurements, temperature and relative humidity, global positioning system coordinates, and semi-quantitative continuous particle mass concentrations based on filter differential pressure can be uploaded to a central server automatically whenever the mobile phone is connected to the internet, with sampled data automatically screened for quality control parameters. filters and filter cartridges that are weighed pre-and post-sampling are barcoded to minimize sample collection errors and potentially track sources of contamination. interchangeable cyclone inlets provide a close match to the epa pm2.5 (particles below 2.5 µm in aerodynamic diameter) mass criterion (within 5%) for device flows at either 1.0 or 2.0 l min −1 . battery life varies from 23 to 45 h depending on sample flow rate and selected filter media. laboratory tests of the upas prototype demonstrated excellent agreement with equivalent us federal reference method samplers for gravimetric analysis of pm2.5 across a broad range of concentrations [89] . approximately 250,000 measurements of household and personal pm2.5 exposure using the upas were made in a multi-country cohort study [90] , specifically to measure pm2.5 exposures for participants in rural communities in ten countries with high levels of indoor solid fuel use. pilot study field evaluation of cooking area measurements indicated high correlation between the upas and reference harvard impactors (r = 0.91; 95% ci: 0.84, 0.95; slope = 0.95). additional inlets with collection efficiencies that match criteria for workplace respirable or thoracic mass sampling have also been developed and tested against polydisperse compressor oil aerosol [91] . the respirable mass inlet includes both an impaction stage and a cyclone, whereas the thoracic mass inlet utilizes a circular slot impactor. both the respirable mass inlet and the thoracic mass inlet have been designed to be interchangeable with the pm2.5 inlet. both inlets tested in the laboratory resulted in sample collection within ±5% of the respective criterion specifies for aerosols with reasonably broad size distributions. for dusts with mass median diameters smaller than about 6 µm the error in using the respirable inlet is under 5%. as is typical for respirable size-selectors, the error becomes larger with monodisperse dusts at larger particle sizes, but dusts with these characteristics are not commonly encountered. bias of the thoracic inlet is generally within ±5% but increases to between 10 and 20% for dusts with median diameters between about 13 and 20 µm in diameter when the geometric standard deviation is under 2.0. further validation work should include laboratory calibration with solid particles and characterization of field performance. although the presence of viable microorganisms in air has been recognized for centuries [92] , the development of quantitative sampling methods for bioaerosols only really took off in the 1990s when it was recognized that bacteria and fungi could play a role in building-related sickness [93] . the mix of species growing in damp buildings is typically different from the mix of outdoor species, so the development of sampling techniques focused primarily on the identification of bacterial and fungal species to detect problematic situations. in the absence of limit values, lesser attention was paid to the numbers, but viability was considered important in the assessment of pathogenic species, particularly spurred by the deliberate spread of anthrax bacteria in the usa. unfortunately, many species of bacteria are fragile under the stress of normal impaction or filtration sampling [94] and specific techniques have been developed for the maximal preservation of viability [95] . recent outbreaks of viral disease (sars, h1n1, h1n5, mers, sars-cov-2) have spurred investigations of airborne viruses. the dynamics and significance of aerosol transmission of respiratory viruses are still controversial. one possible reason for this may be that collected viruses are inactivated by the collection method leading to inaccurate infection risk analyses. the viable virus aerosol sampler (biospot-vivas™ sampler; aerosol devices, inc., fort collins, co, usa) is a novel device designed to sample bioaerosols while preserving their viability to the greatest extent possible [96] . the vivas operates via a water vapor condensation process to enlarge aerosolized virus particles to facilitate their capture. it consists of eight parallel, wet-walled tubes termed growth tubes. during operation, the first half region of each tube is cooled to 6 • c, and the second half region is warmed to 45 • c. in the heated region, water vapor diffuses from the wet walls into the air stream faster than the air heats creating a supersaturation condition. particles as small as 5 nm that can be wetted serve as condensation sites for the excess vapor from the supersaturated air and thus droplets are formed between approximately 10 µm and up to 2 mm in diameter. the flow exiting the tubes is directed by nozzles to a small volume of collection material in which the particles are collected, with a collection efficiency which was shown for inert particles from 8 nm to 10 µm to be greater than 95%. for viruses, the collection medium can be the same as that used typically for dilution in the generation of virus aerosols such as phosphate-buffered saline plus 0.5% (w/v) bovine serum albumin fraction v. in an evaluation of the apparatus, fine aerosols (<500 nm) containing ms2 coliphage were generated from a collison nebulizer, conditioned by a dilution dryer and collected by a vivas and a sampler commonly recommended for bioaerosol viability preservation, the biosampler (skc, inc. eighty four, pa, usa). the vivas collected >93% of the inlet virus particles compared to about 10% for the biosampler [97] . viable counts of the vivas-collected viruses were also one order of magnitude higher than those of the biosampler (p = 0.003), so that overall efficiency of the vivas exceeded that of the biosampler for the viable collection of ms2 viruses by a factor of 10-100. another set of experiments was performed using a more representative wild-type influenza, a h1n1 pandemic 2009 strain [98] . on average, the lower limit collection efficiency of the vivas was 74 ± 12% compared to 5.6 ± 3.0% for the biosampler. the higher recovery from the vivas is attributed to the higher physical collection efficiency, slower rehydration of the infectious virus, and the gentle (non-destructive) impaction onto the collection medium. a further pilot study was performed to determine whether infectious (viable) respiratory viruses in aerosols could be collected from air in a real-world environment and to determine the efficiency of viable collection compared to the biosampler. a variety of viable human respiratory viruses, including influenza a h1n1 and h3n2 viruses and influenza b viruses, were collected by the vivas located at least 2 m from seated patients, during a late-onset 2016 influenza virus outbreak [99] . the biosampler did collect virus aerosols, butt was considered less successful. these results using the vivas indicate that respiratory virus aerosols are more prevalent and potentially pose a greater inhalation biohazard than previously thought. the vivas thus appears to be a useful apparatus for microbiology air quality tests related to the detection of viable airborne viruses. in a very recent (not yet peer-reviewed) study with the vivas in a hospital, viable virus was isolated from air samples collected 2 to 4.8 m away from patients showing that patients with respiratory manifestations of covid-19 produce aerosols that contain viable sars-cov-2, and these aerosols may serve as a source of transmission of the virus [100] . anyone wishing to assess exposures to aerosols needs to understand that the science and technology exists in a continuum of research and response resulting in a ten-to twenty-year cycle of improving methodology; for example, from sugar tube to impinger to midget impinger to glass fiber filter to polymer membrane filter to closed-face cassette housing to "inhalable" samplers has taken about 100 years. even more rapid recent advances have been made possible by new technologies. a selection of novel technologies described in more detail here includes procedures to include wall deposits, on-site analysis of silica, nanoparticles deposited in the airways, multi-fraction samplers, miniaturization of samplers and preservation of viability of microorganisms, especially viruses. this is only a sub-set of current developments, being those with which the author has had recent personal experience. it is expected that many of these and other research themes will continue in the future. perhaps the most important theme is the development of samplers operating at higher flow rates [100] , necessitated by lowered limit values and made possible by advances in pump and battery technology. it is likely that investigations will continue into real-time and end-of-shift on-site analyses, for example for silica [101] and metals [25] . another situation for research is the characterization of semi-volatile aerosols. the equilibrium that exists between aerosol and vapor for semi-volatile substances has been known for a long time and there have been efforts to design samplers appropriate to the situation. however, recognition of the numbers of chemicals and situations where semi-volatile substances can occur has grown and resulted in further research into sampling methods [102] . the difficulty of obtaining an accurate separation of aerosol and vapor phases is profound, but the rewards would be a better understanding of physiological consequence of exposure and potential measures for controlling exposure, so, hopefully there will be continued research in this area. it is important to comprehend this continuum of research because it is not appropriate to ignore scientific advances and nor is it ethical to refuse to act upon them. those who are reluctant to accept change because they mistakenly believe "things have always been done this way" need to recognize that "this is not your parent's sampler" and likely, neither will it be your children's. establishing exposure science as a distinct scientific discipline commentary on a more comprehensive vision and strategy for the discipline of exposure science on the apparatus for collecting atmospheric particles the sugar tube method of determining rock dust in air a new instrument for sampling aerial dusts bureau of mines midget impinger filter-paper method for obtaining dust-concentration results comparable to impinger results use of membrane filters in air sampling dust sampling and lung disease air quality-particle size fraction definitions for health-related sampling american conference of governmental industrial hygienists (acgih). air sampling instruments 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different samplers: final results, summary and conclusions a comparison of xrf and icp-oes for lead on air filter samples from a lead ore concentrator mill and a lead-acid battery recycler a comparison of x-ray fluorescence and wet chemical analysis for lead on air filters from different personal samplers used in a secondary lead smelter/solder manufacturer occupational safety and health administration (osha) a comparison of x-ray fluorescence and wet chemical analysis for lead on air filters from different personal samplers used in a bronze foundry comparison of lead and tin concentrations in air at a solder manufacturer from the closed-face 37 mm cassette with and without a custom cellulose-acetate cassette insert sampling efficiencies of the american 25-mm personal sampler. presented at the airmon: modern principles of workplace air monitoring large particle and wall deposition effects in inhalable samplers; health and safety executive (hse) contract research report no performance of personal inhalable aerosol samplers in very slowly moving air when facing the aerosol source endotoxin deposits on the inner surfaces of closed-face cassettes during bioaerosol sampling: a field investigation at composting facilities evaluation of six inhalable aerosol samplers personal sampling in parallel with open-face filter cassettes and iom samplers for inhalable dust. implications for occupational exposure limits size distributions of 0.5 to 20 µm aerodynamic diameter lead-containing particles from aerosol sampler walls and filters comparison of filter and wall deposits from samplers used to collect airborne lead-containing dusts at field sites evaluation of quartz residue on cassette interiors of aiha proficiency samples air sampling filtration media: collection efficiency for respirable size-selective sampling en 13205, workplace exposure. in assessment of sampler performance for measurement of airborne particle concentrations closed face filter cassette (cfc) sampling-guidance on procedures for inclusion of material adhering to internal sampler surfaces on wiping the interior walls of 37-mm closed-face cassettes: an osha perspective occupational safety and health administration (osha) institut national de recherche et de sécurité pour la prévention des accidents du travail et des maladies professionnelles (inrs). métaux et métalloïdes, m-124 mise en solution à froid des membranes en ester de cellulose dans le cadre de l'analyse des aérosols comparison of a wipe method with and without a rinse to recover wall losses in closed face 37-mm cassettes used for sampling lead dust particulates method id 125g: metal and metalloid particulates in workplace atmospheres (icp analysis); updated 2002 method id 142: crystalline silica (quartz and cristobalite); version 4 gravimetric determination of airborne dust by using a filter cartridge inside a closed-face, 37-mm polystyrene cassette national institute for occupational safety and health (niosh) info-labo 2019-06: la cassette inhalable: nouveau média d'échantillonnage pour les poussières de fraction inhalable preliminary studies on the use of acid-soluble cellulose acetate internal capsules for workplace metals sampling and analysis acid-soluble internal capsules for closed-face cassette elemental sampling and analysis of workplace air interlaboratory evaluation of cellulosic acid-soluble internal air sampling capsules for multi-element analysis national institute for occupational safety and health (niosh) 17205-general requirements for the competence of testing and calibration laboratories one agent, many diseases: exposure-response data and comparative risks of different outcomes following silica exposure occupational safety and health administration (osha), department of labor. occupational exposure to respirable crystalline silica, final rule assessment of respirable crystalline silica analysis using proficiency analytical testing results from 2003-2013 promoting early exposure monitoring for respirable crystalline silica: taking the laboratory to the mine site evaluating portable infrared spectrometers for measuring the silica content of coal dust a comparison of respirable crystalline silica concentration measurements using a direct-on-filter fourier transform infrared (ft-ir) transmission method versus a traditional laboratory x-ray diffraction method performance comparison of four portable ftir instruments for direct-on-filter measurement of respirable crystalline silica evaluating the use of a field-based silica monitoring approach with dust from copper mines application of end-of-shift respirable crystalline silica monitoring to construction human health implications of nanomaterial exposure international commission on radiological protection (icrp) particle collection efficiency for nylon mesh screens a personal nanoparticle respiratory deposition (nrd) sampler a novel method for assessing respiratory deposition of welding fume nanoparticles a field study on the respiratory deposition of the nano-sized fraction of mild and stainless steel welding fume metals porous polyurethane foam for use as a particle collection substrate in a nanoparticle respiratory deposition sampler particle size and metal composition of gouging and lancing fumes a novel method using polyurethane foam (puf) substrates to determine trace element concentrations in size-segregated atmospheric particulate matter on short time scales accurate quantification of tio 2 nanoparticles collected on air filters using a microwave-assisted acid digestion method sampling and analysis of sub-micron metal particulate for respiratory dose estimation particle concentrations in occupational settings measured with a nanoparticle respiratory deposition (nrd) sampler d8208, standard practice for collection of non-fibrous nanoparticles using a nanoparticle respiratory deposition (nrd) sampler occupational lung diseases size selective samplers for estimating the "respirable" 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for enumeration of viable aerosols survival of microorganisms during swirling aerosol collection highly efficient collection of infectious pandemic influenza h1n1 virus (2009) through laminar-flow water based condensation efficient collection of viable virus aerosol through laminar-flow, water-based condensational particle growth collection of viable aerosolized influenza virus and other respiratory viruses in a student health care center through water-based condensation growth viable sars-cov-2 in the air of a hospital room with covid-19 patients quartz measurement in coal dust with high flow rate samplers: laboratory study analysis of crystalline silica aerosol using portable raman spectrometry: feasibility of near real-time measurement development and field testing of a miniaturized sampling system for simultaneous sampling of vapours and droplets this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license funding: this research received no external funding. the author is an employee of a company (zefon international, inc.) and previously an employee of another company (skc, inc.), whose equipment is cited in this work. this article was written in the course of the authors' employment with zefon international, inc. no other funding received. key: cord-022538-1g9kmpdi authors: makino, hisao; emi, hitoshi; yamaguchi, akimasa; iritani, eiji; namiki, norikazu; myojo, toshihiko; yamamoto, kenji title: environmental and safety issues with nanoparticles date: 2008-05-20 journal: nanoparticle technology handbook doi: 10.1016/b978-044453122-3.50010-6 sha: doc_id: 22538 cord_uid: 1g9kmpdi this chapter evaluates the relationship between nanoparticles and the environment, and describes the trouble caused by nanoparticles as well as the safety issues. the relationship between nanoparticles and the environment is clarified from the viewpoint of the kind of influence nanoparticles generated either artificially or naturally have on the environment, such as in atmosphere, groundwater, wastewaters, and exhaust gases. indoor nanoparticles originate from the several sources such as products of chemical reactions, nonvolatile residues (nvrs) of liquid droplets, printers/photocopiers, combustion, bioaerosols, and infiltration of outdoor air. the influence of nanoparticles on the indoor environment is discussed in the chapter. it describes the sources of nanoparticle generation in general industrial processes such as grinding processes, and in cleanroom or controlled environment industrial processes, such as exhaled air, ionizers, and haze by chemical reaction on solid surfaces. the chapter discusses safety issues related to nanoparticles such as possibility of dust explosion, health risks and biological effects of nanoparticle materials such as carbon nanotubes, fullerenes, nanosized metal oxides, and carbon black. the chapter also discusses methods for removing nanoparticles from gas and liquid as technology to control the influence of nanoparticles on the environment. since nanoparticles have superior surface activity and can be applied to the production of particles with various functions, they are extremely important for the future development of sophisticated material technologies. on the other hand, this superior activity of nanoparticles is a cause of trouble from the perspective of safety, and does not always have a positive influence on the environment. attention must also be paid to impact on health. nevertheless, all technologies have negative aspects, and overcoming these kinds of problems, we will be able to utilize the superior characteristics of nanoparticles for practical purposes. to achieve this goal, it is necessary to fully understand the influence of nanoparticles on the environment and the relevant safety issues. this chapter evaluates the relationship between nanoparticles and the environment, and also describes the trouble caused by nanoparticles as well as the safety issues. the relationship between nanoparticles and the environment will be clarified from the viewpoint of what kind of influence nanoparticles generated either artificially or naturally have on the environment. the influence on the indoor environment, where nanoparticles are produced, will also be clarified. the safety of nanoparticles will be clearly described from the perspective of the trouble caused by the superior surface activity of nanoparticles; the effect of the compositional characteristics of nanoparticles, and also the influence on health. a method for assessing the influence of nanoparticles using quantum dots is also explained. in the final section, methods for removing nanoparticles from gas and liquid are described as technology to control the influence of nanoparticles on the environment. in our atmospheric environment, particles ranging from several nanometers to several tenth micron orders are suspended. they are emitted into the atmosphere at the rate of 2.5 billion tons every year. emission sources are classified as either natural or artificial. natural particles occupy 60% of total particles, consisting mainly of salt particles (ϳ1 billion ton) from the sea and soil particles (ϳ0.5 billion ton) from the land. on the other hand, the latter particles are brought about by human activities. although occupying only 16% of the total emitted particles, their size is mostly of submicron order and because they contain hazardous chemical components such as nitrates, sulfates, hydrocarbons, heavy metals, etc. in high concentration, their effects on the ecosystem are serious. fig. 7 .2.1 shows an overview of the size and concentration ranges of various aerosol particles. as it can be seen, the number concentration of atmospheric aerosol which we inhale every day ranges from several thousand particles per cm 3 in clean area to several hundred thousands in dusty areas, and the size range lies between 10 nm and several tens of micrometer. fig. 7 .2.2 shows mass-based size distribution of atmospheric aerosol particles. since the size distribution in the nanosize range appears only when the sources of particle generation exist, the size distribution is usually bimodal with peaks in the size range of a few to 10 micron and submicron. the former peak consists of naturally generated coarse particles such as soil dust, sea salt spray, and so on. on the contrary, the latter contains plenty of artificially generated particles, some of which grow from molecules (in most cases vapor state) exhausted by human activities through chemical reaction, condensation, and coagulation. particle growth rarely leads to particles larger than 1 m unless high concentration of vapors or particulate matters which cause the above-mentioned growth mechanisms exist in the atmosphere. as it can be seen from the differences in the particle generation process, fine particles generated from molecules or nanoparticles are much more complicated in their chemical component than the coarse particles, and sometimes have serious adverse health effects. such fine particles are called pm2.5, which is defined for particles less than 2.5 m including nanosized particles. recent epidemiologic investigation reports that the concentration of pm2.5 showed a positive correlation to the mortality due to pulmonary diseases [1] . various research techniques are used in order to understand the process of particle growth and to trace back to the source of pollution. an example is shown in fig. 7.2.3 where a characteristic function of sulfur dioxide is shown taking into account all possible factors related to particle growth. where f is the characteristic function that expresses particle size, particle concentration, particle composition, and so on [2, 3] . particulate materials in water are present in the form of colloids. these colloid particles are classified into inorganic colloids. examples of the former are oxides of aluminum, silicon and other substances, and typical examples of the latter are substances such as humic acid and fulvic acid. while the structure and molecular weight of particles vary depending on the area of water, it is known that what are usually present in water are comparatively small colloids (particles smaller than 500 nm). the number concentration of colloid particles in ground water, or a typical water area environment, ranges from 10 11 to 10 20 (number / m 3 ) and varies significantly depending on the geochemical conditions of the aquifer. it is known that in moving water, colloid particles sometimes act as a medium in conjunction with water and in some cases move faster than water. homogeneous porous layer such as a sand layer, and most of the colloid particles are trapped. the mechanism of partical trap in this layer is explained by the sand filtration theory. c and d are a gravel layer and a rock bed, respectively, and both have high water permeability with large gaps and cracks. particles can also pass through easily. safety and movement characteristics of colloid particles have a significant influence on the movement of materials such as ionized molecules in aquatic environments. since fine particles such as nanoparticles in particular are highly stable as colloid particles, it will be very important in the future to understand their influence. at the same time, these characteristics are considered to have a high potential to be developed for further application of nanoparticles. in most cases, nanoparticles in exhaust gases are studied from the viewpoint of the influence of total particulate matters on the environment. the term "nanoparticles" is used only in a few cases, "fine particles" is usually used for investigation. since nanoparticles are part of fine particles, this section will be described from this perspective. major sources of combustion exhaust gases are stationary large-scale combustors and diesel engines for stationary and portable use. for stationary combustors, fuels such as coal, oil, and gas are used. lighter fuels have a lower rate of particulate emission, but have a higher fine particle content including nanoparticles. fig. 7 .2.5 [1] shows the frequency distribution in combustion of coal and heavy oil. fig. 7 .2.5a and b are the distributions on a number and mass basis, respectively. as these figures clearly show, the total weight of particles of a size of 1 m or smaller is extremely low, while their total number is, on the contrary, very large. it is clear that, while the total quantity of particulate material is far larger in coal combustion than in oil combustion, the difference is less when it comes to particles 1 m or less in diameter, including nanoparticles. most of the particles contained in pulverized coal combustion exhaust gases are considered to be formed as particulate materials directly from ash content, which is originally contained in coal and also includes some unburned carbon. particularly, almost all large-size particles are considered to be this type of particle. on the other hand, fine particles include two types. one type is formed in the process by which low boiling point metal contained in coal ash is evaporated and vaporized in a high-temperature combustion field and then becomes particles in the exhaust gas cooling process. the other includes carbon particles formed in the gas phase, or so-called soot, which is generated due to the delay in oxygen supply for combustion of evaporated volatile matter in the initial stage. fig. 7 .2.6 [2] shows the relationship between the trace metal content in coal ash and the particle diameter. aluminum with a high boiling point has a constant concentration regardless of the particle diameter. however it is obvious that in the case of metals with a lower boiling point, the smaller the particle diameter, the larger the content. with regard to particles with sizes 1 m or smaller in the nano domain, it has been clarified that the generated amount is increased rapidly by reducing combustion air supply or by weakening the oxidation atmosphere in the volatile matter combustion area, for example, when air supply from a burner is reduced in twostage combustion. this also demonstrates the significant contribution of carbon particles formed in the gas phase. also in the case of ash from heavy oil combustion, there are large particles of a carbon residue type generated from sprayed liquid particles and particles formed in the gas phase as well. as in the case of coal, trace metal contained in heavy oil with a low boiling point is concentrated into fine particles and discharged. also in the case of the combustion of liquefied natural gas, carbon particles formed in the gas phase are generated, albeit in trace amounts. in contrast, only in the case of diesel engines, fuel is injected into the high-temperature and highpressure atmosphere produced by compressing only air to induce spontaneous ignition, and combustion continues with a heterogeneous mixture of fuel and air in the combustion chamber. therefore, particulate materials mainly consisting of unburnt carbon are generated due to incomplete combustion. fig. 7 .2.7 [3] shows changes in the diameter of particles according to changes in the diesel engine load. it is obvious that the overall concentration of particles increases with the increase in the load rate of the engine. according to observations using sem, fine particles in diesel engine exhaust gases have also been found to comprise fine primary particles of a size several tens of nanometers, and coarse particles with carbon hydride condensed on the surface of secondary aggregates of primary particles. influence of particle diameter on trace element contents. the volume of industrial and domestic wastewater is increasing significantly year by year with the change in the lifestyle based on mass consumption and mass disposal brought about by the dramatic development of the economy and industry. effective advanced wastewater treatment is required because wastewater contains a variety of constituents such as particles, organic materials, and emulsion depending on the resource. inorganic nanoparticles are not generally stabilized in the liquid because they form aggregates of some sort more or less. for wastewater reclamation and reuse, these nanoparticles can be removed from the liquid by the advanced treatment processes such as membrane filtration following biological treatment processes. organic materials such as macromolecules are regarded as soft nanoparticles judging from their sizes, in contrast with hard inorganic particles. chemical mechanical polishing (cmp) is one of the fastest growing processes in semiconductor industry, and it has become an integral part of the state-of-the-art fabrication line for the multilayer wiring board of large-scale integrated circuit (lsi). besides the semiconductor devices, cmp is widely applied to the magnetic head, the magnetic memory, and the imaging devices. the process is primarily used for polishing the device side of a semiconductor wafer through the mechanical downforce of slurry abrasive in combination with chemical oxidation of wafer surface. in general, colloidal silica is used as abrasive slurry to planarize the oxide wafer surface. particles in slurry are highly charged to avoid aggregations between particles or between particles and wafer surfaces. during the process, large volumes of ultrapure water are consumed to clean the surface of the wafer, which generates large quantity of cmp wastewater typically having high solid content resulting from slurry abrasive particles of sio 2 , al 2 o 3 , or ceo 2 , depending on the nature of the cmp application. the quantity of cmp wastewater generated is expected to increase proportionally with the growing needs of the cmp processes. as a result, the treatment and reuse of cmp wastewater has become increasingly necessary. the cmp wastewater has been generally treated with the conventional chemical coagulation-sedimentation process, producing large quantity of sludge. currently, a membrane filtration process coupled with chemical pretreatment is used to separate the nanoscale particles from the cmp wastewater to reclaim the water [1, 2] . wastewater of nanosized metal colloid, which is hard to be removed by coagulation-sedimentation process, is discharged in such diverse fields as metalworking factory, electronic components factory, and pigment-manufacturing factory [3] . it is reported that various trace elements of heavy metal are contained in wastewater discharged from a pulp production plant [4] . a spent emulsion, which contains nanosized copper colloid, is discharged from plants manufacturing copper cables for electrical industry, and the treatment for purification of effluents is examined by the integrated membrane system based on ultrafiltration (uf) and nanofiltration (nf) [5] . a glass company generates the wastewater containing fine clay and glass particles from the grinding process of glass surfaces during production of crt glass used for tvs and monitors. separation of fine clay and glass particles by microfiltration (mf)/uf is examined in order to treat glass industry wastewater for reuse in the manufacturing process [6] . the colored substances are free from regulatory constraint of water quality so far because they are not considered hazardous substances. however, water color is being recently used as a standard for the judgment of the purity in water because the removal of color becomes important for wastewater reclamation and reuse. dye works are scattered across the country as the industry with local tradition. the dyehouse effluent is discharged in large quantity, and it has extremely complex composition because it contains not only dye but also dyeing aid and finishing agent. in general, dye cannot be removed by standard biological treatment because of its low environmental biodegradability. dye wastewater is treated by coagulation-sedimentation and activated sludge processes, and nanoparticles produced in the course of the treatment are released into the environment [7] . the color is often imparted by organic substances, predominantly humic substances. aquatic humic substances including humic and fulvic acids are a term referring to a broad class of naturally occurring mixture of organic compounds, ubiquitous in surface waters, ground waters, and soil pore waters. they are a complex mixture of heterogeneous organic materials in terms of elemental composition, chemical functionality, and molecular size distribution since humic substances can be derived from any organic materials, including plant and animal debris, microfauna, biowaste, pesticide, and others. the molecular weights of humic acids range from several thousands to several tens of thousands daltons, and those of fulvic acids range from several tens of thousands to several hundred thousands daltons. because of this versatility, humic substances are known to significantly affect the behavior of some pollutants in natural environments, such as trace metal speciation and toxicity, solubilization and adsorption of hydrophobic organic compounds, and disinfection by-product formation [8] . melanoidins are natural polymeric compounds of dark brown color, and they are closely related to humic substances. they are produced by a set of consecutive and parallel nonenzymatic reactions taking place between amino compounds and carbohydrates during a maillard reaction [9] . they are contained in the molasses wastewater from alcohol distillery, sugar processing and refinery industry, and glutamate processing industry. such wastewater containing melanoidins has frequently caused a coloration problem of water environment, and thus the suitable decolorization treatment is required in many fermentation and sugar industries using molasses. treatments by flocculation, ozonation, and electrolysis are promising in color removal [10] . food-processing wastewater usually contains a variety of organic materials in varying degree of concentration. in cheese-making in the dairy products industry, only ϳ10% of the initial milk volume becomes product, cheese, and the other 90% becomes by-product, liquid cheese whey. since cheese whey is a protein-and lactose-rich by-product of cheese production, its cost-effective utilization is becoming increasingly important. recent developments in membrane technology have provided exciting new opportunities for large-scale protein and lactose fractionation in whey treatment [11] . in textile industry, typically it takes over 100 l of water to process just 1 kg of textile material. not only the washing water must be treated to recover important by-products such as lanolin, but bleaching and dyeing chemicals must also be removed before discharge back to the rivers [12] . surfactants are a primary constituent of the detergent used in the household routinely, and also they are widely used in industry and agriculture because they have several functions such as washing, emulsification, and dispersion. the surfactants are usually present in the solution in the form of the micelle, and large amounts of surfactant wastewater are discharged in the rivers [13] . pesticides whose molecular weight ranged from 200 to 400 da (ϳ1 nm) have been used in great quantities not only for agricultural use but also in golf links and resort. therefore, the wastewater and effluent treatments have become an important issue, and pesticide separation by nf membranes is found to be very efficient [14] . the potential reclamation of high-quality water produced by the advanced treatment of the secondary effluent of the municipal sewage has come a long way in recent years. the sewage contains various components such as virus [15] , pharmaceutical substances [16] , and endocrine disrupting compounds derived from zoonotic excretory substances [17] . the advanced treatment of such chemical contaminants at low level becomes increasingly important. as mentioned above, the removal of nanoparticles contained in wastewater is stringently required to recycle the reclaimed wastewater in a wide variety of industries such as chemical industry, textile industry, pulp and papermaking industry, food-processing industry, dairy products industry, and pharmaceutical industry. also for domestic wastewater, the reuse of the reclaimed wastewater for nonpotable purposes is becoming more and more important, and this is expected to raise awareness of the behaviors of nanoparticles contained in wastewater in order to upgrade the water treatment processes. in recent urbanized lifestyles people tend to spend more time in enclosed buildings or residences than outdoors. therefore, it is of great importance to characterize indoor particles and correlate between indoor and outdoor ones from the viewpoint of evaluating the influence of indoor air quality (iaq) on human health. as shown in table 7 .2.1, indoor nanoparticles originate from the several sources such as products of chemical reactions, nonvolatile residues (nvrs) of liquid droplets, printers/photocopiers, combustion, bioaerosols, and infiltration of outdoor air. 7.2.5.1 secondary particle formation by gas phase ozonolysis for particle formation resulting from chemical reaction via ozone, the reaction of terpenes is very common in indoor environments as well as atmospheric ones. terpenes are emitted from fragrance-containing vegetable oils such as pine oil and citrus oil, and wooden materials including woody furniture [1] . meanwhile, sources of emission of ozone are air cleaners, air-conditioners, laser printers using corona discharge, and infiltration of outdoor air. terpenes are generic terms of unsaturated organic compounds that are composed of isoprene as unit (e.g. -pinene and limonene). these compounds used for household applications readily react with ozone because they have one or more double bonds. it has been proposed that, as shown in fig. 7 .2.8, the reaction mainly proceeds to form less volatile pinonic acid via pinonaldehyde of intermediate [2] . furthermore, the acid-catalyzed reaction allows the products to convert into higher molecular weight compounds by the polymerization via carbonyl groups in the aldehydes and the aldol condensation [3] . it has been reported that the resultant generated particles have a size distribution with a peak diameter of about 30 nm, and that the products by terpenes ozonolysis irritate human airways [4] . nanoparticles are also generated from air humidifiers or negative air-ion generators in which water is atomized. in general, humidifiers are mainly categorized into vaporization type and atomization type [5] . the former does not entrain impurities in water when it is fed into indoor spaces of interest. meanwhile, the latter has the drawback that nvrs are suspended in spaces to be humidified by feeding water via spraying and sonication. the nvrs in tap water include colloidal particles and soluble fraction such as silicates, sulfates, carbonates, and chlorides. the size of nvr particles, d p _ r can be estimated from the following equation: where dp_ m is the droplet size, c the mass fraction of nvr in the droplets, m the droplet density, and r the nvr particle density, respectively. assuming that 2 m-sized droplets ( m ϭ 1,000 kg/m 3 , r ϭ 2,500 kg/m 3 ) are formed by an ultrasonic nebulizer, and the mass fraction of nvr in city water is 100 ppm (10 ϫ4 ), the nvr particle size, d p_r is estimated to be 68 nm. recently, a wide variety of negative ion generators using the lenard effect, corona discharge, uv/photoelectron emission and electrospray have been commercialized and attention has been focused on features such as air purification and physiological activation [6] . among them there are the ion generators that atomize water based on the lenard effect and electrospray form the nvr particles as by-product in addition to ion products if the supplied water contains nonvolatile impurities. fig. 7 .2.9 shows an example of electrical mobility distribution for ions generated by the electrospray method (positive in this case) [7] . this method atomizes liquid fed to a tip of a capillary electrode to form fine droplets with large amounts of charge by applying high voltage between the tip and the downstream counter electrode. when water in the generated droplets evaporates and their surface charge density attains the charge limit called "rayleigh limit", this phenomenon induces their self-fragmentation followed by the formation of a high concentration of cluster ions. in this figure, the high-mobility peak on the right side corresponds to the cluster ions. these ions are 2 nm in size assuming that they are singly charged. meanwhile, another peak on the figure results from nvr in water and then its height increases with the increase in the fraction of tap water in the fed liquid. the electrical-mobility-equivalent size of nvr particles measured by differential mobility analyzer (dma) -condensation nucleus counter (cnc) method ranges from 10 to 20 nm and their concentration is on the order of 10 4 particles/cm 3 . comparing the forementioned electrical mobility distribution with the particle size one, the nvr nanoparticles are estimated to hold about 100 charges. accompanying the recent proliferation of computers, the use of inkjet printers and electrophotographic machines such as laser printer and photocopier is becoming common in homes as well as offices. it has been reported that these devices emit various sorts of pollutants. the eco-friendlinessoriented standards such as blue angle standard [8] regulate the maximum permissible limits of benzene, styrene, total volatile organic compounds (tvoc), ozone and particles. as the regulation of particles is based on the emitted mass per hour, mainly the relatively coarser particles such as toner and dust adhering to paper have been targeted. however, some reports have revealed that nanoparticles are emitted from inkjet printers or laser printers [9] . fig. 7 .2.10 depicts the size distribution of particles emitted from a laser printer measured by a scanning mobility particle sizer (smps). as seen in the figure, nanoparticles with a peak diameter of around 30 nm are generated in printing mode, whereas the emission in the case of feeding paper without printing is about one third of the normal printing mode. furthermore, these particles were dried by passing them through a diffusion dryer because they are thought to originate from the nucleation of water vapor emitted from papers in the fixation process. as a result, it was found that most particles formed in the paper feed mode evaporated and then vanished, while particles in the printing mode contained nonvolatile components as well as water. from these results it is anticipated that the particles are derived from styrene remaining in the toner even though their composition has still not been identified. meanwhile, it is thought that during ink discharge ink-jet printers emit not only the main ink droplets but also their satellites (about 1 m) to result in nanometer-sized nvrs during printing. one of the most significant source of indoor nanoparticles relevant to combustion is cooking such as frying and sautéing [10] . some reports said that over 90% of the particles by number were in the ultrafine fraction range during cooking with bimodal peaks at 60 and 10 nm, attaining the number concentration on the order of 10 5 particles / m 3 and the emission rate of 10 14 particles / h. owing to lifestyles in asian countries, cigarette smoke, incense, and mosquito coils also contribute to indoor nanoparticle levels [11] . it was reported that especially in the indian subcontinent the combustion of biofuels such as straw and dried cattle manure used for cooking could have a significant impact on climate change in the south asian region [12] . sidestream cigarette smoke also contains nanoparticles, having a concentration distribution with the main peak between 0.1 and 0.2 m [13] . in addition, it was found that nanoparticles a peak size of 30 nm formed by the nucleation of vapor fraction in filtered sidestream smoke immediately after burning when the dilution of smoke by air was insufficient [14] . attention should be paid to air cleaners when a high concentration of cigarette smoke has to be treated by the cleaners using a single unit air filter. airborne virus particles or virions are typically in the 20-400 nm size range, and are a good example of nanoparticle bioaerosols. smaller viruses typically contain one subunit, which consists of an outer protein capsid, internal nucleic acid (e.g. dna and rna), and other internal proteins. corona virus that causes sars and influenza are good examples of them. the viruses most often are transmitted through direct contact with an infected person, such as by shaking hands, hugging or kissing, while sometimes it is spread by nasal droplets. however, it is still unknown how these virus particles behave in the case of airborne infection. recently, the studies that attempt to elucidate the behavior have been progressing [15] . this section describes the sources of nanoparticle generation in industrial processes by categorizing them into specific processes where a cleanroom is used and other general ones. the sources of emission of unwanted nanoparticles in general workplaces are categorized as fumes from hot processes (e.g., smelting, refining, and welding) and from (incomplete) combustion processes. favorable conditions required for the generation of nanoparticles are found in workplaces where there is (1) presence of vaporizable material, (2) sufficiently high temperature to produce enough vapor, followed by condensation to form an independent aerosol, and (3) rapid cooling and a large temperature gradient. there have been so many studies on occupational exposure to fine particles in the field of public health. in general, high spikes of nanoparticle concentration are observed during active operations, followed by a gradual decay after the operation, primarily because of coagulation, evaporation, dilution, and/or deposition. the fraction of the total number of nanoparticles generally decreases, whereas that of the number of submicrometer particles increases with time and distance from the point of emission. in order to accurately estimate exposure, the effects of spatial and temporal changes will need to be evaluated. therefore, it is important to identify the time required for the concentration to decline to the normal or background levels. as an example of reports on grinding processes, fig. 7.2.11 shows the case where a steel substrate was ground upon using a high-speed grinder [1] . from the figure the distribution of concentration of generated particles has a distinct bimodality, one with the finer peak at around 10 nm and the coarser one at around 1 m. the former results from within the grinder motor and the volatilization or combustion of amenable ground substrate and/or grinding materials, the latter from the mechanical abrasion and attrition. however, the resultant total concentration on the order of 10 5 particles/cm 3 is not so high. cleanrooms and associated controlled environments (e.g., in the case of an iso class 3 cleanroom, the maximum permissible airborne particle concentration is less than 10 3 particles/m 3 for particles with the size of 0.1 m or larger, while the airborne particle concentration in ordinary indoor environments is on the order of 10 9 particles/m 3 or higher) are usually adopted to avoid particle contamination in industrial processes where precision products such as engineered nanoparticles, semiconductors, and other electronic or optical devices are fabricated because the deposition of particles onto product surfaces causes their yield reduction and quality deterioration. the emission sources in cleanroom environments are tabulated in table 7 .2.2. since some of the listed emission sources emit trace amounts of nanoparticles, these nanoparticles are not regarded as particulate contaminant but as chemical or molecular one. in this section, these nanometer-sized solid substances formed on solid surfaces by chemical reaction are also included. size distribution of nanoparticles generated when a steel plate was ground with a high-speed grinder. (1) air exhaled by humans emissions from human bodies are a minor contribution in ordinary indoor situations because airborne particle concentration in such places is quite high, whereas the emission cannot be seen as negligible in cleanroom environments. the major human emissions are thought to be atmospheric dust deposited on clothes and skin fragments, and most of these particles are submicrometer in size. meanwhile, particles in exhaled air are composed of fine liquid droplets from spittle (99.5% of water), and then evaporate to form nanoparticles of nvr. in fig. 7 .2.12 an example of size distributions of particles in exhaled air before and after smoking is shown [2] . when measuring particles in exhaled air, the air was introduced into a measuring device after drying them by passing them through a diffusion dryer. the size distribution of particles in exhaled air before smoking (n db ϭ 0) has a bimodality, one with the peak size of 0.2 m, and the other of 20 nm or smaller. the former peak comes from atmospheric aerosols as it decreases with the increase in number of deep breaths in a clean booth (n db ). the latter originates from nvr particles of spittle droplets. incidentally, since smoking induces the rapid increase in number concentration of particles 0.1 m or larger by 10 4 times or more and for nanoparticles by about double, special attention should be paid to the management of personnel's clothes such as face mask when they enter a cleanroom after smoking. (2) emission from ionizers ionizers are commonly used in cleanrooms to eliminate electrostatic charge on substrates for precision electronic devices. the most popular ionizer is a corona-discharge type. corona-discharge type ionizers are categorized into the following three groups; ac, dc and pulsed-dc types. the issues of emission of contaminants such as ozone, no x and particles have been pointed out [3] . these issues are also applicable to air cleaners using a corona discharger. among these problems is that the particle emission has a potential for particle contamination onto product surfaces and eventually decline in product yield. the particle emission, which has been studied since the 1990s, is caused by foreign particle deposition onto electrodes, electrode erosion, and gas-to-particle conversion. the issue of electrode erosion can be solved by the improvement of electrode materials, whereas for the issue of gas-to-particle conversion, the airborne molecular contamination (amc) control to be ionized has to be made. it was reported that exhaled air of human corona-discharge ionizer (e.g. gas-to-particle conversion of low-molecular-weight cyclosiloxane) boron-containing particles from borosilicate glass fibers of hepa filter haze by chemical reaction on solid surfaces (precipitation of ammonium salt and silica) watermark on wafer surfaces at drying leakage from thin film and nanoparticles processing equipment silicon-containing compounds that precipitated on the electrodes result from the gas-to-particle conversion of low-molecular-weight cyclosiloxane (lmcs) from silicone sealant via corona discharge [4] . (3) boron-containing particles from hepa filter with borosilicate glass fibers the use of hepa and ulpa filters made of borosilicate glass fibers prevails in the most cleanrooms. it has been known that owing to chemical reaction in equation (7.2.2) bf 3 vapor is formed from glass fiber filters by passing hf gas leaking from wet cleaning equipment through the filters. boron, which is a dopant element for semiconductors, has been thought to be a contaminant that might cause failure in semiconductor devices if it comes from the surroundings. in addition, it has been revealed that trace amounts of boron in the form of boric acid (h 3 bo 4 ) are also formed from the fibers via the reaction with moisture in the surrounding air (equation (7.2.3)). fig. 7 .2.13 depicts the change in volatilized boron mass from various filters in terms of airborne boron concentration. especially, at the initial stage just after the initiation of ventilation, the volatilized boron mass increases with increase in relative humidity [5] . boric acid, which is solid at room temperature, is surmised to form in the particulate form. however, its existence was identified only by chemical analysis because it is present only in trace amounts. haze might form on glass surfaces of lenses and mirrors for optical instruments if they are exposed for a long time to cleanroom environments where amcs are not controlled properly. the haze is more likely to bring about the insufficient light delivery onto a surface to be exposed in photolithographic processes. one of the reasons is that ammonium sulfate ((nh 4 ) 2 so 4 ) is formed, which then precipitates on the glass surfaces via chemical reaction of sulfur dioxide (so 2 ) with ammonia or amines. for another reason, hexamethyldisilazane (hmds) used as additive in resist coating or lmcs from silicone sealant is adsorbed, and then decomposed to form silica precipitates on glass surfaces by photochemical reaction during laser irradiation, followed by the unwanted decline in laser penetration [6] . as another example a report said that tiny projections, which are also known as "haze", with a size of 0.2 m or smaller were formed on silicon wafer surfaces owing to the adsorption of organosilicate compounds in thin film formation processes with cvd. it is similarly caused by the precipitation of sio 2 [7] . (5) watermarks on solid surfaces during drying when a silicon wafer surface is cleaned with deionized water and then dried in air, a watermark is formed on it via the mechanisms demonstrated in fig. 7 .2.14. oxygen in air is dissolved and diffused into water droplets or adsorbed water on a wafer surface, followed by the formation of silicate compounds via silanol reaction. the watermark on a wafer surface is detected in the form of nanometer-sized particles by an electron microscope [8] . (6) leakage from nanoparticle production processes in regard to the risks to processing equipments by nanoparticle leakage from production processes, the vdi report in germany [9] has been described in detail. the production of engineered nanoparticles can be generally categorized into two approaches. one is a "top-down" approach that is initiated with a bulk material and then breaks it into finer pieces using some form of energy such as etching, ball milling, sputtering, and laser ablation. the other approach is to synthesize materials from the atomic or molecular level by growth and assembly to form the desired nanoparticles. processes included in this "bottom-up" category are sol-gel, chemical vapor deposition, flame synthesis laser pyrolysis, and so on. most of these processes are performed in a closed reaction chamber installed in a cleanroom or associated controlled environment. human exposure to these engineered particles does not take place during synthesis unless there is an unexpected system failure (e.g. rupture of a seal). human exposure is more likely to occur after the manufacturing when opening the reaction chamber, drying the products, or in the post-process handling of the products. the release of nanoparticles during production chamber cleaning operations is another critical point. cleaning typically involves using water or some solvent. brushes, sponges, or tissues used in the cleaning will carry nanoparticles into the waste stream. disposal of the waste and wastewater may become a source of nanoparticle release into the environment. further, conditioning of nanoparticles such as compression, coating, and composition to form final products may also result in the release to the environment and resultant exposure although very few studies have been carried out on this subject. recent studies [10] to evaluate the aerosol discharge during the handling of carbon nanotubes showed that the generation of nanoparticles occurred under vacuum to remove spilled nanotube materials or vigorous mechanical agitation. however, they reported that the concentrations were very low. in addition, measurements of nanoparticle levels during final packaging of carbon black, which is a typical engineered nanoparticle material, showed that there was no increase in nearby air [11] . study on the safety of nanoparticles has started only recently, and no sufficiently systemized results have been obtained. what should be noted in particular is that the possibility of radial troubles caused by particulate matters are considered to increase by the decrease of particle diameter in nanoparticles. one typical example is the problem of dust explosion, caused by the high surface reactivity of fine particles. in other words, since nanoparticles are extremely fine particles, dust explosion is more likely to occur. explosion is more likely to occur because fine particles are different in their composition, in that low boiling point metal can be easily condensed, as described in section 7.2. however, of particular note here is that, since all particles do not necessarily exist independently in the form of a single particle, the possibility of dust explosion does not simply increase as particles become finer. fine particles with sizes of 1 m or smaller such as nanoparticles have an extremely high agglomeration propensity and secondary particles can be easily generated. therefore, in some cases they conversely behave like large particles. these are the points to be taken into consideration when studying the problems caused by nanoparticles. as shown in fig. 7 .3.1 [1] , the effect of the particle diameter on dust explosion tends to be that the smaller the particle diameter of the dust, the lower the minimum explosion concentration. in other words, explosion can be induced under conditions of lower concentration of particles in air as the particle diameter becomes smaller. due to the difficulty of conducting experiments to suspend particles with the same size in a uniform concentration, this result was obtained from particles far larger than nanoparticles; however, it has been clarified qualitatively that the smaller the particle diameter, the higher the possibility of dust explosion. from the perspective of composition, the possibility of explosion increase if materials that react easily with oxygen at low temperature are condensed into particles of small diameter. therefore, with regard to the effect of particle diameter on dust explosion, more careful attention needs to be paid in the case of combined materials than in the case of uniform materials, as assumed in fig. 7. 3.1. as described before, however, since nanoparticles are considered to exist often as agglomerates, it is necessary from the perspective of the particle diameter to take into consideration the diameter of not only primary particles but also of particles after agglomeration. to address the safety of nanoparticles, it will be important in the future to elucidate their behavior in detail including these factors. the terms 'nanoparticles' and 'nanomaterial' have been used for particles of which one representative dimension, for example, diameter of particles on cross-sectional diameter of fibers has at least 100 nm or less. some people hold that the majority of such fine particles are exhaled without depositing in the respirator tract, and that therefore the particles may not cause pulmonary diseases. however, the properties of nanoparticles are known to be different from the bulk material they are derived from. in cases where the biological effects of bulk materials have been reported, nanosized particles of these materials may be expected to have stronger dose response for the health effects. every effort must be made to clarify the uncertainty on the risks of these nanomaterials [1] . at the present time, there is no regulation or standard for assessing the biological effects of nanomaterials, and therefore there is a paucity of toxicological data concerning nanomaterials. much more systematic and strategic studies are needed to enable risk assessments for human health [2] [3] [4] [5] [6] . as regards risk assessment and risk management of nanomaterials, the characterization and identification of anticipated risks should be first determined for chemical substances or foods. conventional assessment methods are applicable for water-soluble particles. for insoluble nanoparticles, the assessment of potential health hazards should be made based on their properties or toxicity and dose-response relationship. the risk is a product of hazard and exposure; even if a nanoparticle has a hazard, the risk is lower when the possibility of exposure to the nanoparticle is small [2] . influence of particle diameter on lower limit of particle concentration for explosion. (1) exposure routes for nanoparticles nanoparticles can either be deliberately introduced into the body for medical purposes (drug delivery systems) or absorbed involuntarily from the environment (inhalation of nanoparticle-containing dust in the air). a distinction should also be drawn between nanoparticles manufactured for industrial application and those unintentionally generated and released in the environment, such as welding fumes or diesel exhaust particles (dep). in the fields of environmental science and toxicology, numerous studies on the potential health hazards caused by ultrafine particles have been conducted. practically, there are several definitions of nanoparticles or ultrafine particles, however, findings regarding biological effects of the ultrafine particles are useful as a starting point for estimating the effects of nanoparticles on human health. human and animals contact with nanoparticles through various routes: nanoparticles can be inhaled in the air, swallowed in the water, ingested in food, and absorbed via the skin in cosmetics. for successful risk assessment, it is important to determine how nanomaterials or nanoparticles are used, such as composites, surface coating, or powders. coatings or powders have the potential to release a part of their nanomaterials into the environment. workers who come into contact with nanomaterials have the possibility of exposure to nanoparticles at the workplace. consumers of products using nanotechnology can also be exposed to them. attention needs to be paid to the environments and ecosystems in which nanoparticles and nanomaterials are released. nanoparticles in the products may change their size, quantity, and composition during their life-cycle of manufacturing, use, transportation, and disposal. (2) respiratory uptake of nanoparticles inhalation is the main route of exposure to nanoparticles. particles inhaled with the air through the mouth and nose pass through the throat (nasopharynx and oropharynx) and tracheobronchial tree before reaching the alveolar region where oxygen moves from the alveoli to the blood and carbon dioxide moves from the blood to the alveoli. how deeply particles can penetrate and where they become deposited on each respiratory airway such as the nasal cavity, tracheobronchial tree, and the alveoli depend on their size under the various deposition mechanisms: inertial impaction, gravitational sedimentation and diffusion, etc. the respiratory airway includes the anterior nasal passage, posterior nasal passage, pharynx, larynx, trachea, main bronchi, bronchi, bronchioles, terminal bronchioles, alveolar duct, and alveoli, as shown in fig. 7. 3.2. in the human lungs, the trachea divides asymmetrically into the right main bronchus that enters the right lung where it divides into three lobes, that is, an upper, a middle, and a lower, and the left main bronchus that enters the left lung where it divides into two lobes, that is, an upper and a lower. the trachea divides into two branches, dividing progressively to the terminal alveolus. to quantitatively assess the pulmonary particle deposition needs human lung morphology models, respiratory physiology based models of the entire lung airway system and aerosol deposition models based on many experimental findings. in 1994, the icrp task group on lung dynamic (icrp: the international commission of radiological protection) published their revised lung model [7] . the deposition, clearance, and translocation of particles in each of the compartments were described. while the model has been widely used in the nuclear field, it is applicable to conventional aerosols as well as radioactive aerosol. in the nuclear field, aerosols including radon progeny that used to be nanoparticles have been studied. fig. 7.3.3 shows the deposition fractions of inhaled particles per adult nasal respiration of 1.2 m 3 /h in each region re-calculated for the nasopharynx, tracheobronchial, and the pulmonary (alveolar) region based on the model. inhaled aerosol particles deposit on different regions depending on their size; for example, nanoparticles larger than 10 nm deposit mostly in the alveoli and those less than 10 nm deposit in the nasal cavity. how deeply particles penetrate into the lung depends on their size. nanoparticles can reach pulmonary region in the lung and deposit more intensively and this, therefore, has become one of the reasons for concern about the effects of nanoparticles on human health. however, deposition of nanoparticle chain-like agglomeration and fibrous particles such as carbon nanotubes cannot be estimated by this model. most inhaled nanoparticles are deposited on the surface of the respiratory tract. generally, if insoluble particles are deposited in the ciliated airspaces which are lined with a mucous layer, they are transported to the digestive tract with the mucous flow by mucociliary movements. particles deposited on nonciliated bronchioles and alveoli are phagocytosed by macrophages, which is a kind of white blood cell. as a result, their residence time is longer, however, they are usually transported to the ciliated upper part of the respiratory tract. removal of deposited particles described above is called clearance. when the amount of deposited particles is below a certain value, no health effect is produced. in relation to a macrophage response to particles, crystalline silica is hazardous, whereas titanium dioxide is not. pneumoconiosis is a well-known lung disease that is caused by exposure to dust particles of several micrometers in diameter. silicosis is a typical form of pneumoconiosis resulting from exposure to crystalline silica dust and characterized by a progressive fibrosis of the lungs. the macrophage-mediated clearance (phagocytes) was effective for micron and submicron particles. it has been reported that only 20% of deposited nanoparticles were removed by the clearance mechanism [8] . it has been suggested that the remaining nanoparticles may pass through the alveolar walls, penetrate into the blood or lymphatic circulation, and be transported to other organs. many studies have shown that the smaller the particle size the greater the mobility or they pass easily through the alveolar wall and enter into the bloodstream. it is further presumed that the mechanism of health effects of nanoparticles on cardiovascular system other than the respiratory tract is similar to that in the airborne ultrafine particles from dep. in japan, two reference values, the 'administrative control levels' (acl) and the 'occupational exposure limits' (oels), are used for the regulation of hazardous chemicals as well as dust (particle matters). oels are recommended and revised every year by the japan society for occupational health. oel (oel-mean) for mean concentration of a chemical substance is defined as the reference value to the mean exposure concentration at or below which adverse health effects caused by the substance do not appear in most workers working for 8 h a day, 40 h a week under a moderate workload [9] . the 'threshold limit value' (tlv) has the same definition (but may not be the same values as oel of the same substance) provided by the american conference of governmental industrial hygienists (acgih). acl is an index to determine the control class to judge the propriety of the working environment control based on the results for working environment measurement, which have been implemented for the unit work area in accordance with the working environment measurement standard. the results of working environment measurement are evaluated by classifying the working environments concerned into three control classes (control class i, ii, and iii). these classes are used as the standards to classify the level of the working environment concerned. among those subject to working environment measurement, these standards apply to workplace where dust, lead, organic solvent, and specified chemical substances are used. article 65 of the industrial safety and health law stipulates that certain workplaces in which harmful substances are involved or harmful work operations are performed shall be the subject to working environment measurement. a 50% cut-off size of the dust particle is set at 4 m for both standards and is far larger than nanosized particles. oel or acl [10] for particulate matters are usually based on mass concentration, that is, milligram per cubic meter. therefore, if the particulate matters have broad size distribution, the contribution of nanoparticles is not large to in terms of mass of particles. evidence from a number of toxicological studies on insoluble particles indicates that the primary determinant of the health effect of particles depends on the surface area of particles deposited [11, 12] . on the basis of the results from a number of in vitro studies of insoluble nanoparticles, a hypothetical cellular interaction has been proposed [13] : inflammation and oxidative stress can be mediated by several primary pathways: (1) the particle surface causes oxidative stress resulting in increased intracellular calcium and gene activation; (2) transition metals released from particles result in oxidative stress, increased intracellular calcium, and gene activation; (3) cell surface receptors are activated by transition metals released from particles, resulting in subsequent gene activation; or (4) intracellular distribution of insoluble nanoparticles to mitochondria generates oxidative stress. in the workplace, the concentration of nanoparticles may be at a high level and most of the nanoparticles become agglomerates, while nanoparticles will form single nanoparticle at low levels in the general environment. it is a matter of debate whether agglomerates of nanoparticles react as a larger particle or a single nanoparticle in the lung or other organs. if insoluble particles are retained in the lung for a longer time without enough clearance mechanisms, they can cause pulmonary inflammation or pneumoconiosis. it is of interest that nanoparticles deposited in the lung can move into the blood vessel through alveolar epithelium and they can damage vessels or produce blood clots [14, 15] . in a recent study, nanoparticles deposited in the nose may move directly to the brain via the olfactory bulb [16] . (2) biological effects of fullerene the biological effects of fullerene have being investigated intensively. in rats dosed orally with radioisotope-labeled c 60 fullerenes, most were excreted in the feces and some were found in the urine. a small amount of them can be absorbed via the gastrointestinal tract. in contrast, in the same study, 90% of the same labeled fullerenes administered intravenously were retained after 1 week, with most found in the liver [17] . ld50s (acute toxicity) by intraperitoneal injection in mice and rats were 1.2 and 0.6 g/kg, respectively. the dose of 2.5 g/kg orally in rats did not result in death. the reproductive translocation of fullerenes was also observed in mice. fullerenes have shown mutagenic activity in ames tests. fullerenes have shown no skin irritation or allergic reactions [18] . on the other hand, fullerenes are being tested for possible medical use. fullerenes are basically hydrophobic but water-soluble derivatives have been synthesized to be used as drugs or its carrier. the derivative can be anticipated as drugs, for example, anti-aids drug. it has been stated that the toxicity of fullerenes changes due to slight structural changes including chemical modification [18] . (3) biological effects of carbon nanotubes carbon nanotubes are chemically stable and are similar in form and size to asbestos; these characteristics have given rise to concern that carbon nanotubes may have the potential to cause pulmonary diseases such as lung cancer and mesothelioma similar to asbestos. a few data are available concerning the biological effects of carbon nanotubes. the biological effects of carbon nanotubes are being researched. epithelioid granulomas and interstitial inflammation are induced in mice and rats following exposure to single-walled carbon nanotubes [18] [19] [20] . untreated carbon nanotubes contain the nanoparticles of transition metals such as iron and nickel, which are used as catalysts in forming carbon nanotubes. these nickel-containing carbon nanotubes have been reported to be toxic [19] . the concentration of airborne asbestos fibers is expressed as a number concentration, that is, fibres per cubic centimeter or fiber per liter. when fiber concentrations are determined by phase contrast light microscopy, the fibers with a diameter of less than 3 m, a length longer than 5 m, and a lengthto-diameter ratio (aspect ratio) greater than 3 are counted [21] . asbestos fibers having nanosized diameter were often observed in analyses of environmental samples using electron microscopy. international agency for research on cancer (iarc) rated asbestos as a known human carcinogen (group 1) [22] and the concentration of chrysotile asbestos is expressed as a risk level of 0.15 fiber/cm 3 [9] . health effects of vitreous fibers and other asbestos substitutes have been assessed to determine their oels or their carcinogenicity in humans. the health effects of carbon nanotubes are being intensively investigated now. the oel for carbon black respirable dust is 1 mg/m 3 and these for activated charcoal and graphite are 0.5 mg/m 3 in each [9] . in the ref. [23] , while rats and mice inhaled carbon black with a particle diameter of ϳ30 nm at a concentration of 5-13 mg/m 3 did not produce any specific changes, particles (agglomerate of small particles) of ϳ450 nm at a concentration of 2-6 mg/m 3 produced early pulmonary changes. micronsized titanium dioxide particles are thought to have almost no toxicity and often used as a negative control substance. the oel for titanium dioxide is 1 mg/m 3 for respirable fraction [9] . however, the results of a series of studies by oberdörster et al. [3, 11, 24, 25] on submicron-and nanosized titanium dioxide suggested that as size decreases, inflammatory effects are intensified, and normally nontoxic substances may assume hazardous characteristics. fig. 7.3.4 shows a part of the results by oberdörster et al. in which rats and mice were exposed to anatase titanium dioxide particles [25] . their results have been frequently cited in the discussion of whether the health effects of fine particles should be based on its mass or its surface area. in fig. 7.3.4 , percentages of neutrophils in lung lavage of rats are shown as indicators of inflammation after intratracheal instillation of different mass doses of 20 and 250 nm tio 2 particles. the steeper dose response of nanosized tio 2 particles is observed than for submicron tio 2 particles when the dose is expressed as mass (fig. 7.3.4a ). if the same dose-response relationship as in fig.7. 3.4a is indicated as particle surface area (fig. 7. 3.4b), the particle surface area seems to be a more appropriate dosimetric for comparing effects of different-sized particles of the same chemical structure. zinc oxide is a white powder and used in pigments. the oel is 1 mg/m 3 for its respirable fraction [9] , and the value for zinc oxide fume which causes metal fume fever is under consideration. nanoparticles of transition metals and rare earth elements and their oxides will be used widely. since many of these metals and their oxides have biological effects, particular attention should be given to them. nickel compounds are rated as a human carcinogen (group 1) by iarc. in particular, nickel oxide is particularly insoluble among the nickel compounds and remains longer in the lung. nanosized nickel oxide particles have greater toxicity to the lung than larger particles [26, 27] . pulmonary inflammatory responses induced by nanosized cobalt particles have been reported [28] [29] [30] . biological effects of nanosized particles of other transient metals such as iron and manganese have received attention [3] . rare earth elements are a general term of 17 chemical elements consisting of scandium (sc), yttrium (y), and a lanthanide series of 15 elements from lanthanum (la) to lutetium (lu). these elements have been used in magnetic alloy, fluorescent and hydrogen storage alloy. particularly cerium oxide nanoparticles are frequently used as a fuel additive and are incorporated in cosmetics formulation. the potential biological and environmental effects of these elements have not sufficiently been investigated. it has been demonstrated that ld50s for these elements in oral and intravenous administration are in a range from several dozens to several thousands milligrams per kilograms, indicating that none of these elements has high toxicity. in the results of studies in which the biopersistence and the distribution of rare earth compounds in the body were investigated, for example, the compounds deposited in bones and teeth, and organs including lung, liver, spleen and kidney following intratracheal, oral, intravenous, and intraperitoneal administration. although the compounds deposited predominantly in the liver other than bones, it has also been reported that the distribution of the compounds in the lung and spleen increased when the dose was increased [31] . the demand for indium compounds has been sharply increasing. the compounds have been used in the materials for transparent electrodes for flat panel displays. in japan, the cases of pulmonary interstitial pneumonia and pulmonary fibrosis have been reported in workers engaged in cutting and grinding of sintered indium-tin oxide (ito) and potentially having inhaled the dusts released from ito [32, 33] . the biological effects of indium arsenic compounds and indium phosphorus compounds also have been investigated. the acgih has proposed a value of 0.1 mg / m 3 for their tlv, and the value has been applied tentatively in japan. we have been experiencing amazing progress of the technology on the processing for the nanometer-sized materials. the applications cover even biomedical engineering in addition to information technology, material, environmental science, and energy production [1] [2] [3] [4] [5] . as the result, many kinds of new materials have been designed, fabricated, and discarded. from now on, this movement will be accelerated and even more new functional materials will be distributed in the world. here, we should not forget the safety of those materials in the process of the production, usage, and discard. without this safety assessment, we will go into the same problems as that of asbestos just we are facing now. first of all, we have to conduct experiments to reveal the minimal concentration for emergence of the toxicity. in other words, we have to fix the standard value for the threshold concentration for each material first of all. if we do not fix it, we should not use the material at any concentration, which means any engineering process could not be carried out. the applications in the various fields have started all over the world, and the safety assessment is urgently needed. in this article, we introduce the methods of the safety assessment of the semiconductor nanoparticles and describe the safety and the threshold depending on the surface treatment. the production process and the surface treatment play one of the most important roles for the safety of the nanoparticles. cd and se semiconductor nanoparticles coated with zns are one of the most widely used for the strong intensity of the fluorescent activity. cd oxide and se compounds once dispersed in the tri-octhil-phosphine oxide (topo) heated up to 300њc generate nanoparticles by self-assembly. then zns enhances the stability of the structure and raises up the fluorescent intensity than without the coated one. nanoparticles, thus manufactured, as materials for a novel memory in the field of intelligence technology (it), and as super-micro devices for laser in the field of optics, have been developed all over the world [1] [2] [3] [4] [5] . these nanoparticles cannot be dissolved into water, but dissolved into organic solvents like toluene. therefore, for biological and medical applications, various technologies for surface-conjugations to make them hydrophilic [6, 7] have been developed. for example, nanoparticles covered with topo are hydrophobic because an alkyl group on them is hydrophobic. therefore, a technology for replacing this alkyl group with hydrophilic carbonic acid (making the whole particles soluble in water) has been developed [6] . nanoparticles, thus surface-treated, can be dissolved into water, like sodium salt or potassium salt. with this method, various kinds of materials have been surface-conjugated. the mtt assay method is a way to evaluate the hazard assessment of nanoparticles, in which the activation metabolism in a mitochondrium in a cell is measured and the influence of nanoparticles on the prolification of the cell is qualitified. the mtt is a kind of tetrazolium, whose molecular formula is c 18 h 16 brn 5 s. taken into a cell, it is decomposed by a dehydrogenase enzyme in a mitochondrium into a pigment called 'hormazan'. the measurement of the fluorescence intensity of the pigment shows the prolification of the cell [8] [9] [10] . fig. 7 .3.5. shows the hazard assessment of vero cells and kidney cells of the african green monkey against cdse/zns nanoparticles. the horizontal axis shows the concentrations of the nanoparticles and the vertical axis the fluorescence intensity of the hormazan at 460 nm, that is, the metabolism of the cells. the figure indicates that cyto-toxicity is not observed for 10 5 cells when the concentration is less than 0.5 m. this result means that this concentration is the threshold of the cell toxicity. likewise, cyto-toxicity is not observed for the hela cells and for the human primary cells. further, in order to find out how the sizes of nanoparticles influence the cyto-toxicity, the cyto-toxicities were evaluated with three kinds of quantum dots; one whose fluorescence wavelength is 640 nm, red, one with 570 nm, yellow, and the other with 520 nm, green. the following results were obtained. the largest quantum dots whose fluorescence wavelength is 640 nm show a tendency to give cyto-toxicity. cyto-toxicity is observed at concentrations more than 1 m [11] . another method to evaluate cyto-toxicity is the flow cytometry [12] . the mtt assay alone cannot tell whether the toxicity observed is lethal to the cells or just restrains the prolification of them. in the flow cytometry, the nuclei of dead cells are dyed with propidium iodide (pi) after the nanoparticles are taken in and the ratios of the dead cells are measured. fig. 7.3.6 shows the lethal cyto-toxicity of mua conjugated nanoparticles (520 nm, green) against vero cells. the vertical axis indicates the numbers of the cells, and the horizontal axes show the fluorescence intensities and the cyto-toxicities. these experiments also show that dead cells cannot be observed at concentrations less than 0.5 m even though nanoparticles are taken in, as was shown in the mtt assay. however, at concentrations more than 2 m, the nanoparticles taken in cause damage to more than the half of the cells. that is, the cyto-toxicity of mua quantum dots against cells is lethal [11] . nanoparticles have been surface-conjugated for applications for various uses. some surface-conjugations cause more grave cyto-toxicity than others. therefore, relations between surface-conjugations and their safety for cells have to be considered. in order to find out the relations, the safety evaluations of nanoparticles surface-conjugated with two materials were made; one is with mua (quantum dots-cooh) and the other is with glycerol (quantum dots-oh), and their purified and unpurified particles. fig. 7.3.7 shows that the purification reduces the cyto-toxicity for the quantum dots-oh, and that the toxicity remains the same after the purification for the quantum dots-cooh. mua itself, a material with which particles are conjugated, has cyto-toxicity. this experiment shows that toxicity against cells is connected not only with particles themselves but also with kinds of surfaceconjugations and degrees of purification. it is shown in the above experiments that the toxicity of nanoparticles is lethal to cells. next, we found out by the comet assay method whether the toxicity is derived from damaged dna. the method is a way to evaluate quantitative damage of dna by electrophoresis. the fragmented dna seeps out of their cells by treating cells, whose dna has been fragmented, with agarose-gel to break their cell membrane, and then electrophoresing them. it looks like the "tales of comets". cells, whose dna has not been fragmented, have their nuclei keeping their spherical shape after electrophoresis, and the tales of comets cannot be observed [13] [14] [15] [16] . fig. 7 .3.8 shows the results of the experiments with quantum dots conjugated with cooh (both purified and unpurified) to wtk-1 cells, a human lymphoblast mutation strain [17] . the vertical axis shows the lengths of the tails, and the horizontal axis the concentrations of the quantum dots. the concentration of the nanoparticles is 2 m. the results are as follows: the unpurified quantum dots with cooh caused damage to dna. on the other hand, the electrophoresis with the purified quantum dots does not show dna damage. this is probably because the dna damage has been repaired during the longer cultivation time. conducted with quantum dots conjugated with mua, and quantum dots-nh 2 with an amino group. dna damage was not observed in either case. the results indicate that as far as particles themselves do not break down, the cyto-toxicity of quantum dots is derived from the chemical properties of the materials covering the quantum dots. the safety evaluation of nanoparticles has not been conducted sufficiently. as indicated above, the procedure for surface-conjugation could apply not only to cd/se nanoparticles but also to other nanoparticles. today, various kinds of techniques for surfaceconjugations have been available in academic papers, proceedings, and on the internet. some of them are widely known and others are patented. those techniques are all shared among the human race. even at this moment, the human beings are making breakthroughs in various fields and developing different kinds of technologies. sharing these technologies will lead to still more speedy developments of yet more advanced technologies. to achieve it, these technologies should be so structured that different fields, for example, bioimaging and biotechnology, structured on their own, can be linked to each other. such structured knowledge will play an essential part in merging different fields. in order to prevent nanoparticles release from a system so as to maintain environmental safety, the removal technique of nanoparticles must be established. in this section, separation techniques of particles from exhausted or suspended gas and liquid are described focusing on particles less than 100 nm. generally, as shown in fig. 7 .4.1, all particle separators for a dispersed system employ either one of three basic forms of particle separation. on the left hand side of the figure lie the separation methods in which particles are collected only by force field (electrostatic force, centrifugal force, gravity force, etc.), and the representative separator is electrostatic precipitator (esp). if some obstacles (collectors) are placed into the particle laden stream, particle separation is facilitated because particles are collected on obstacles with a smaller deviation from the fluid flow by the force exerting on the particles compared to the case without obstacles. typical collectors of this form are air filter, deep bed filter, etc. on the right hand side of the figure lie separators that collect particles utilizing only sieving effect of obstacles without any force field. in this case, geometrical size of channel between the obstacles must be smaller than that of particles. membrane filter, fabric filter, etc. belong to this group. when we apply the above collection forms to nanoparticles, the major collection mechanisms are brownian diffusion and electrostatic force for particles in gas, while sieving effect and interception/adhesion forces for those in liquid. as mentioned above, most airborne particles are collected by separators utilizing various kinds of forces such as gravity, centrifugal force, electrostatic forces, inertia, brownian diffusion force, and so on. therefore, the migration velocities or displacement of a particle per second due to the individual forces gives the basis for the comparison of removal efficiencies due to each force. in fig. 7 .4.2, migration velocities of particles due to various forces are depicted against particle diameter at normal temperature and pressure for particle density of 1 g/cm 3 [1] . as seen from the figure, the velocities due to gravity, centrifugal force, and inertia monotonically decrease with decreasing particle diameter, suggesting that the removal of nanoparticles with these forces is difficult. on the contrary, the velocities due to brownian diffusion and electrostatic forces increase with decreasing particle diameter for particles less than 100 nm. this suggests that brownian diffusion and electrostatic forces are most effective in collecting nanoparticles. fig. 7 .4.3 summarizes typical conventional dust collectors. among them, the effective collectors for nanoparticles are esp and fabric/air filter. however, for the case of esp, which relies on only electrostatic force, nanoparticles (ͻ 10 nm) fail to carry even one electron resulting in low collection efficiency. in this case electrically charged filters are effective because we can expect the combined effects of electrostatic forces and brownian diffusion. among charged filters, so-called electret filter, which consists of permanently polarized fibers, is the most favorable filter because of its charge stability. particle penetration data of electret filter are plotted against particle diameter in fig. 7 .4.4 and compared with that of uncharged filter with the same structure. for the three combinations of charged states of fiber and particle there exist the most penetrating particle diameters. for the uncharged fiber, collection efficiency of uncharged particle has a minimum at 100 nm and increases with decreasing particle diameter, showing that 100 nm is the most penetrating particle size. for the charged fiber, particle collection efficiency is very high even for uncharged particle, and the efficiency for charged nanoparticles is extremely high because of strong coulombic force between fiber and particle. the experimental data plotted in fig. 7 .4.4 are qualitatively in good agreement with the theoretical prediction following the particle size dependency on particle migration velocity (shown in fig. 7.4.2 ). however, as particle size becomes smaller and comparable with the size of a molecule, particles may rebound on a collector surface, and the adhesion probability of particles drops, resulting in a decrease in collection efficiency. fig. 7 .4.5 is an example of experimental data that confirm the particle rebound [2] . the figure shows the penetration of nanoparticles through a grounded circular tube. the solid curve is the theoretical line derived by assuming that particles are deposited from a laminar flow in a tube by brownian diffusion. it is evident that experimental penetration deviates from the theoretical line for particles less than 2 nm. this means that molecular behavior begins to appear when the particle size becomes as small as 2 nm, and as a result, the collection efficiency is reduced. it should be noted that considerable amounts of nanosized particles are contained in diesel exhaust particles (dep), possibly penetrating through the honeycomb type (tubular channel) diesel particulate filters (dpf). there are two types of methods that differ in the way the nanoparticles in liquid are collected. the first group, called membrane filtration, constrains the particles by a membrane, and the liquid is allowed to flow freely through the membrane. in the second group of ultracentrifugation, the liquid is constrained in a rotating vessel, and the particles move freely within the liquid by an external field of acceleration caused by ultracentrifugal field. these methods have been quite extensively used in separation of macromolecules and molecules from liquid, and they are recently becoming important also in separation of nanoparticles from liquid. in pressure-driven membrane filtration processes, the pressure gradient across the membrane would force solvent and smaller species through the pores of the membrane, while the larger molecules/particles would be retained. membrane filtration processes are usually classified into three general categories according to the size of separating components, as shown in fig. 7 .4.6. microfiltration (mf) is designed to retain suspended particles in the range of 50 nm-5 m. ultrafiltration (uf), on the other hand, retains macromolecules or nanoparticles in the range of 5-50 nm (nominal molecular weight cut-off (nmwco) ranging from 5,000 to 5,000,000 da). nanofiltration (nf) is a relatively new process that uses charged membranes, and it covers molecular sizes ranging from 0.1 to 5 nm (nmwco ranging from 200 to 1,000). it is useful in that it can separate dissociated forms of a compound from the undissociated form. one of the major factors limiting the use of membrane filtration is membrane fouling, resulting in a dramatic decline in flux with time of operation. to account for the membrane fouling, the resistancein-series model is frequently employed. the resistance model becomes (7.4.1) where u 1 is the permeate flux, v the filtrate volume per unit membrane area, the filtration time, p the applied transmembrane pressure, the viscosity of the permeate, r t the total resistance, r bm the resistance of the membrane per se plus the clogging of the membrane pores, r c the resistance of the filter cake, and r cp the resistance of the concentration polarization layer. significance of each resistance in membrane filtration is as follows. the membrane, even in the absence of any suspended particle, has a natural flow resistance. during membrane filtration, particles become attached to the pore channel of the membrane thereby reducing the flow channel dimension, or pores become blocked off completely. the last two effects lead to resistances that are due to adsorption and pore blocking. the blocking filtration model introduced by hermans and bredée [1] , and grace [2] is most commonly used as an interpretation of such phenomena. the clogging of the membrane pores is strongly influenced by such solution environment as ph and the ionic strength. the permeate flux of bovine serum albumin (bsa) (pi 5.1, molecular weight 67,000, stokes-einstein diameter 3.55 nm) solution by permeable mf membrane (nominal pore size 0.05 m) is lowest at around the isoelectric point [3] . as the bsa molecule carries no net charge at the isoelectric point, the molecule is in its most compact state at that point. the bsa molecules deposit themselves rather densely onto the pore walls of the membrane to form a compact configuration with a smaller lateral electrical interaction between the molecules. as a result of this, the flow resistance increases markedly at around the isoelectric point. in dead-end membrane filtration, which has a feed and permeate stream, each with the same mass flow rate, the resistance of the filter cake plays a major part in the filtration resistance. therefore, the cake filtration theory can be applied, and thus the permeate flux u 1 is described by (7.4.2) where m is the ratio of wet to dry cake masses, s the mass fraction of solids in the solution, av the average specific filtration resistance, the density of the permeate, and v m the fictitious filtrate volume per unit membrane area, equivalent to the flow resistance of the membrane [4] . for fine particle suspensions, colloidal forces which arise from interaction between the suspended particles control the nature of the filter cake. the average specific filtration resistance av and the average porosity av of the filter cake are strongly affected by the solution properties, including ph and electrolyte strength. for instance, in mf of suspensions of the titanium dioxide (pi 8.1, the original mean specific surface area size 470 nm), av goes through a minimum, and av is much larger near the isoelectric point [5] , as shown in fig. 7 .4.7. the titanium dioxide particles are destabilized around the isoelectric point where the van der waals attraction is more dominant. consequently, the particle tends to come together, that is, to flocculate, and the very porous flocs are then formed. thus, it is speculated that the filter cake formed from such porous flocs has often loose and wet structures. on the other hand, the filter cake becomes compact and dry when the particle carries the charge. since the most loose filter cake forms around the isoelectric ph, the filter cake is most permeable. it is interesting to note that the results in protein uf had a distinctly different behavior. in protein uf of bsa solution, the filter cake is in its most compact state around the isoelectric point [6] , as shown in fig. 7 .4.8. since the bsa molecules are hydrophilic colloids, their stability in the solution would appear to be influenced not only by the presence of a surface charge on the protein but also by hydration of the surface layers of the protein. the bsa molecules, because of hydrated layers surrounding them, are not destabilized by such consideration as depression of the electrical double layer. thus, the bsa molecules have water bound to them even around the isoelectric point. the hydrophilic bsa molecules maintain a dispersed state in the solution due to hydration of the surface layers of the protein even around the isoelectric point. when a bsa molecule acquires a charge, the filter cake becomes loose and wet due to electrostatic repulsion between the charged bsa molecules. this contrasts to the compact filter cake around the isoelectric point. the average specific filtration resistance av has a definite maximum around the isoelectric point since a compact filter cake provides a large hydraulic flow resistance. most membrane filtration processes are operated in the cross-flow mode, in which the feed is moved tangentially to the membrane surface so that the filter cake is continuously sheared off. during membrane filtration, particles in the feed are brought to the upstream surface of the membrane by convective transport, and this results in a higher local concentration of the rejected particles at the membrane surface as compared to the bulk solution which is referred to as concentration polarization [7] . the particle concentration in the solution adjacent to the membrane varies from the value at the membrane surface, c m , to that in the bulk feed solution, c b , over a distance equal to the concentration boundary layer thickness . the resulting concentration gradient causes the particles to be transported back into the bulk solution due to diffusional effects. at steady state, the rate of convective transport of particle toward the membrane is balanced by the rate of particle transport through the membrane plus the rate of the diffusive back transport of particle. thus, the permeate flux u 1 is given by (7.4.3) where cp is the particle concentration in the permeate, k (ϭd/ ) the mass transfer coefficient, and d the diffusion coefficient. the osmotic pressure model assumes that the deviation from pure water flux occurs solely due to the osmotic pressure difference across the membrane, and thus the permeate flux u 1 is given by (7.4.4) where is the osmotic pressure, which is a function of the concentration. equation (7.4.4) means that the effective driving force across the clean membrane is pϫ{ (c m )ϫ (c p )}. replacing pϫ{ (c m )ϫ (c p )} by the hydraulic pressure at the membrane surface, p m , equation (7.4.4) reduces to the cake filtration equation. to minimize the effects of cake buildup and concentration polarization, membrane filtration is usually conducted using the cross-flow geometry in which the feed flow is parallel to the membrane and perpendicular to the filtrate flow [8] . however, especially in mf the energy requirements associated with pumping the feed (plus any recirculation flow) along the membrane surface are typically very high. thus, there have been some innovations in recent years with cakeless filtration. the rotating disk module in which the membrane disk is stationary is suited for large-scale operation [9] . it is possible to enhance the permeate flux by using the vibrating modules [10, 11] . in the rotating cylinder device with the membrane on the inner rotating cylinder, counter-rotating taylor vortices within the annular gap are available [12, 13] . dean vortices that twist and spiral in the direction of flow inside a highly curved channel, similar to vortices in rotary modules can result in enhanced flux [14] . these vortices, or flow instabilities, induce turbulence into the system. periodic removal of the formed filter cake is also effective for enhancing the permeate flux. recently, several methods have been investigated: back washing using the filtrate or air pressurization [15] , periodic rotation of the cylindrical membrane [16] , pulsatile flow [17] , high-frequency transmembrane pressure pulsing with a frequency around 0.1-1 hz [18] . dead-end upward filtration, where the filtrate flow is in the opposite direction to gravity, and dead-end inclined filtration, where the membrane is inclined, can reduce the cake formation onto the membrane in uf of nanoparticulate suspension and protein solutions. in upward uf of silica sol (mean diameter 6.2 nm) and bsa solution, a sustained permeate flux is achieved, as shown in fig. 7 .4.9 [19, 20] , because the filter cake overlying the membrane is exfoliated continuously under the gravitational force acting on the particles comprising the filter cake. another approach for enhancing the permeate flux is to employ external force fields. electrofiltration, in which an applied electric field is used to drive charged particles away from the membrane surface, has been developed. in electrofiltration, the accumulation of the particles on the membrane surface is limited by the imposed electrophoretic force. in addition, the permeate flux through the filter cake is dramatically enhanced due to electroosmosis as a secondary electrokinetic phenomenon. this method can be applied to a broad combination ranging from mf of particulate suspension such as bentonite [21] to uf of protein solution. fig. 7 .4.10 shows the reciprocal permeate flux (d /dv) versus the permeate volume per unit membrane area, v, for various values of the strength of the dc electric field, e [22] . the steady permeate flux increases noticeably with the magnitude of the imposed field strength. also, a higher electric field strength causes the permeate flux to equilibrate more rapidly. a method has been developed for removing humic substances by hybrid uf combined with both flocculation and adsorption treatments, as shown in fig. 7 .4.11 [23] . flocculation by use of polyaluminum chloride (pacl) is particularly effective for the removal of humic acids, which constitute the relatively high molecular weight fractions of humic substances, whereas adsorption by use of powdered activated carbon (pac) is able to remove fulvic acids of relatively low molecular weight effectively, which cannot be fully flocculated by pacl. hybrid uf in combination with flocculation and adsorption treatments exhibits high permeate quality because the flocs and pac are easily retained by the uf membrane. in ultracentrifugal sedimentation, ultracentrifugal force field of several tens of thousands of revolutions per minute is applied to a rotor. in recent years, ultracentrifugal sedimentation is employed for concentrating dilute protein solutions and for separating proteins and other large biological molecules from low-molecular-weight solutes or from much larger particles. fig. 7.4.12 shows the results for ultracentrifugal sedimentation of an aqueous solution of the mixtures of bsa and egg white lysozyme (pi 11.0, mw 14,300) measured using schlieren optics in an analytical ultracentrifuge [24] . the angular acceleration of the rotor is 5,445 rad/s. the symbol r i and r i0 in the figure represent the distances from the center of rotation to the sedimentation boundary at time and 0 , respectively. the electrical nature of macromolecules plays a significant role in determining the sedimentation behavior in ultracentrifugation of binary protein mixtures. in the ph range where both protein molecules were electropositive, the molecules sediment independently due to the electrostatic repulsive force acting between bsa and lysozyme molecules. denpun kagaku filtr. sep institute for quality assurance and certification: basic criteria for the award of the environmental label (printer ral-uz 85) proc. air cleaning contam proc. air cleaning contam proc. air cleaning contam chemical contamination in semiconductor processing environments and its countermeasures the japan society of industrial machinery manufactures: report on behavior control of individual sort of contaminants -1993 report on introduction of advanced technologies to environmental equipment industry industrial application of nanomaterials -chance and risks. future technologies, division of vdi technologiezentrum nanoscience and nanotechnologies: isbn 0 85403 604 0 recommendation of occupational exposure limits who: environmental health series 4. world health organization summaries & evaluations -nickel and nickel compounds rapid colorimetric assay for cellular growth and survival ouyou earozorugaku crossflow filtration: theory and practice encyclopedia of fluid mechanics: slurry flow technology filtr. sep key: cord-292928-a4bn30ul authors: ghosh, bipasha; lal, himanshu; srivastava, arun title: review of bioaerosols in indoor environment with special reference to sampling, analysis and control mechanisms date: 2015-10-03 journal: environ int doi: 10.1016/j.envint.2015.09.018 sha: doc_id: 292928 cord_uid: a4bn30ul several tiny organisms of various size ranges present in air are called airborne particles or bioaerosol which mainly includes live or dead fungi and bacteria, their secondary metabolites, viruses, pollens, etc. which have been related to health issues of human beings and other life stocks. bio-terror attacks in 2001 as well as pandemic outbreak of flue due to influenza a h1n1 virus in 2009 have alarmed us about the importance of bioaerosol research. hence characterization i.e. identification and quantification of different airborne microorganisms in various indoor environments is necessary to identify the associated risks and to establish exposure threshold. along with the bioaerosol sampling and their analytical techniques, various literatures revealing the concentration levels of bioaerosol have been mentioned in this review thereby contributing to the knowledge of identification and quantification of bioaerosols and their different constituents in various indoor environments (both occupational and non-occupational sections). apart from recognition of bioaerosol, developments of their control mechanisms also play an important role. hence several control methods have also been briefly reviewed. however, several individual levels of efforts such as periodic cleaning operations, maintenance activities and proper ventilation system also serve in their best way to improve indoor air quality. as a class of airborne pollutants "bioaerosols" are particulate matter usually associated with compounds of biological origin. this definition includes all pathogenic or non-pathogenic, live or dead fungi and bacteria, bacterial endotoxins, mycotoxins, peptidoglycans, β (1, 3)-glucans, viruses, high molecular weight allergens, pollens, etc. (douwes et al., 2003) . they are ubiquities, highly variable and complex and are natural or manmade in origin. air contains a significant number of bioaerosol particles which vary in size and composition. the majority of bioaerosols are of respirable size, namely of the order of 0.003 μm for viruses (taylor, 1988) , from 0.25 to 20 μm for bacteria (thompson, 1981) , from 17 to 58 μm for plant pollens (stanley and linskins, 1974) , and from 1 to 30 μm for fungi (gregory, 1973) . the inhalable fractions are of primary concern as major portions of bioaerosol are susceptible to reach the deeper parts of the respiratory system. composition mainly depends on the source, aerosolization mechanisms and environmental conditions prevailing at the site (pillai and ricke, 2002) . two types of factors that influence the bioaerosols according to several studies are physical characteristics and environmental factors. physical characteristics includes size, density, and shape of droplets or particles while environmental factors include the moisture content of building material, density of the air, relative humidity and temperature (droffner et al., 1995; foarde et al., 1993; pasanen et al., 2000) , air exchange rates (kulmala et al., 1999) , and human activities (buttner and stetzenbach, 1993) . high average temperature and higher relative humidity favor microbiological growth on proper substratum thereby acting as a proper source of bioaerosol (jones and harrison, 2004) . light intensity, magnitude of air currents, wind direction and wind speed also play major roles in bioaerosol concentration and their transportation and displacement from one environment to another . bioaerosols ranging in size between 1.0 and 5.0 μm generally remain in the air, whereas larger particles are shortly deposited on surfaces (mohr, 2001) . as environmental factors play a major role in bioaerosol distribution, several studies related to spatial and temporal distribution of bioaerosol have also been conducted in different parts of the world. in beijing, china the highest airborne bacterial concentration was observed in summer and fall (fang et al., 2007) . almost similar results were observed in washington d.c., montreal of canada and moscow, wherein bacterial concentrations were at their peak in the summer and lowest in the winter season due to regional climatic conditions (jones and cookson, 1983; kelly and pady, 1954; vlodavets and mats, 1958) . in case of fungal bioaerosol, their distribution nature had also been studied wherein in suburban areas of washington airborne fungal concentration was similar to bacterial concentration, i.e., the lowest in winter and the highest in summer and fall (jones and cookson, 1983) . similar pattern of fungal distribution was also observed in beijing with the highest in summer and autumn and the lowest in spring and winter (fang et al., 2007) . seasonal variability of airborne mold was conducted in single-family residence in ny with no visible water damage or leaks and wherein 15-40% rh and 17-18°c temperature was maintained in winter while 40-80% rh and 22-28°c temperature in summer. the study revealed that fungal levels in summer were 26 times higher in comparison to winter (lebouf et al., 2012) . similar to the above studies, seasonal variability of airborne bacteria were conducted in the restrooms different indoor environments such as a shopping centre, hospital, subway system, public library and old and new university lecture building in republic of korea. among the six locations, all five sites revealed significantly smaller bioaerosol concentration in winter than in summer except the restroom in hospital lobby which may be due to the maintenance of almost similar indoor air conditions by air conditioning system artificially in both summer and winter (lee et al., 2012) . several studies have revealed various sources for bioaerosol in different indoor environments. indoor upon outdoor (i/o) ratio in several studies has shown that outdoor concentration had always acted as one of the sources of bioaerosol in different indoor environments . apart from outdoor concentration building condition, occupancy level and human activities also determine the varying concentrations of bioaerosols (nasir and colbeck, 2010) . in fact human beings have been attributed to be one of the most important sources of airborne bacteria (stetzenbach, 1997) . increased human shedding of skin cells and activities such as talking, coughing and sneezing force air under pressure through nose. this process ejects microbes from upper respiratory tract into the air (terkonda, 1987) . sneezing is the most vigorous of these mechanisms, by generating as many as one million droplets lass than 0.1 μm in diameter (campbell et al., 1991) , and presence of such organic particulates in air gives added protection to bacterial cells and result in enhanced survival of the air borne microbes. sweeping of floors and dusting of objects, movement of people and air currents also have been found leading to the suspension of dust particulates and generation of airborne microorganisms (kallioski et al., 1996) . food stuffs, house plants, flower pots, pets and their beddings, furniture stuffing also release various fungal spores into the air (cox and wathes, 1995; kalogerakis et al., 2005) . according to certain studies higher level of dust and macromolecular organic components (proteins) were found in carpet dust than floor dust, hence it can be said that human movement across such a carpeted floor covering ejects trapped debris and dust into the air (gravensen, 1987) . as indoor air is highly dynamic so several studies have been carried out to keep a check on the indoor air quality (iaq) especially for occupational and public health (bonetta et al., 2010) . during different iaq studies it has been found that bioaerosols contribute to about 5 to 34% of indoor air pollution (wanner et al., 1994; srikanth et al., 2008) . hence it is an important criterion to take into account the microbiological air quality so as to provide a safe environment while designing indoor workplaces. this review provides a brief description of various sampling and analytical techniques that are generally used to characterize bioaerosols. this review also provides the information on the concentration levels of various airborne microorganisms in different indoor environments, their associated health effects as well as various bioaerosol control mechanisms worked upon till now. the objective of this review is not only to acquire knowledge about bioaerosol and their associated health effect in order to apply measures to reduce them by various controlling mechanisms, but also to find out the advantages and limitations of each sampling and enumeration technique from all the previously assembled works so as to facilitate future researchers in deciding upon the correct bioaerosol assessment protocol accordingly. at present a wide variety of bioaerosol sampling methods are in use and numerous other methods are in the developmental stage (grinshpun and clark, 2005; muilenberg, 2003; reponen et al., 2009) . till now there is neither a single sampling method suitable for the collection of different types of airborne microorganisms nor a standard protocol available (grishpun et al., 2007) . in any bioaerosol monitoring design depending upon the objective of sampling the method used and incorporated are often aimed at documenting the specific sources of the bioaerosols collected. active sampling method is usually used for collecting bioaerosol from air. in general the sampling efficiency of any sampling devise is the product of the aspiration, transmission and collection efficiency; each of which depends upon particle aerodynamic diameter, wind velocity, direction as well as inlet characteristic, which itself is dependent upon bioaerosol particles sampled under various conditions (grishpun et al., 1994) . the three major collection methods used for the sampling of bioaerosols are impaction, impingement and filtration (grishpun et al., 2007) . apart from the principle techniques alternative methods such as gravity sampling, electrostatic precipitation and cyclone have also been employed in many cases with respective advantages and disadvantages as shown in table 1 . impactors are economically feasible samplers due to their low costs and easiness of handling (zollinger et al., 2006) . the basic principle behind impactors is impaction that collects microorganism and particles in the air. the impaction sampler draws in air and forces to change its direction which causes particles with high inertia to get impacted over collecting surfaces (henningson and ahlberg, 1994) . generally particles which are larger than a particular aerodynamic size get impacted onto a collection surface forcing the smaller particles to proceed through the sampler (hinds, 1982) . a number of samplers available commercially using impaction based methods are one of the common approaches to collect bioaerosols (gangneux, 2001; pillai and ricke, 2002) . impactors differ in the characteristics of inlet size and shape, number of collection 'chambers' within the sampler and whether the microorganisms are impacted onto a solid (glass slide) or semi-solid (agar plate) surface, or, a filter or gelatin (macher and hansson, 1987) . collection and recovery efficiencies of the samples are strongly influenced by the inlet characteristics of the samplers where inlet (and collection) efficiency highly depend on wind velocity at the time of sampling, and the orientation of sampler during sampling. with wind velocity above 5 ms -1 and the inlet aperture facing the wind almost 50% of overestimation of 10 μm sized particles are done while when the sampler is in an upright position (with wind blowing across the inlet aperture), less than 5% of 10 μm particles were collected (willeke et al., 1993) . it has been seen that the mass of particles smaller than cut-diameter (d 50 ) that are generally collected are equal to the mass of particles larger than the d 50 that pass through the impactor. in fact it has been found that the collection efficiency of the impaction samplers is 100% when the aerodynamic diameter is greater than d 50 (hinds, 1982) . it has been seen that for the same aerosol sample which shares the same geometric standard deviation, the mass and count particle distributions will show distinctive good collection efficiency because of reduced particle bounce and loss through re-entrainment sterilization process easy due to evaporation of liquid medium problem of loss may be encountered thermal precipitator good collection efficiency for smaller sized particles and helps in determining size distribution of the particles. air flows freely through the sampler, thereby pressure drop is small and vacuum source is not needed. collection rate very low. collection area small. high temperature affects the viability of the microorganisms collected. major processing time period is very less ultrafine bioaerosol particles can also be sampled and detected easily. viability of the microorganisms maintained throughout. complex system requiring expertise to handle. means and medians. the mass median aerodynamic diameter (mmad) describes the mass distribution which equals the diameter where particles larger than mmad contribute half the collected mass while those particles smaller than mmad contribute the other half. the median of the number of particles in the particle distribution is named as count median aerodynamic diameter (cmad). sampling time and impaction velocity also plays a very important role in the collection and viability of the airborne collected microorganisms. at a constant time if impact velocity is increased it may cause particle bounce, decreasing the actual collection efficiency (especially for spores) and also affects the viability of stress sensitive microorganisms (juozaitis et al., 1994) . although aerosolized fungal spores are often collected over glass slide and semi-solid agar surface is used to collect bacteria (hinds, 1982) however, generally agar plates are mostly used for the collection of both fungi and bacteria predicala et al., 2002; sanchez-monedero and stentiford, 2003) and are called culture based impactors. as seen the collection medium is that the collected microorganisms are cultured and then counted on the plates requiring no post-sampling processing to determine the numbers of microorganisms present (except for incubation at the required temperature) nesa et al., 2002) . disadvantage of using agar plates is of becoming over loaded by microorganisms making enumeration of the colonies difficult as they overlap and become indistinguishable from one another. a statistical "positive hole correction" is thus needed to evaluate highly loaded plates (feller, 1950; andersen, 1958) . desiccation related problems are also associated with impaction sampling as surface moisture is removed by air stream passing over the agar plate limiting the ability to impact more particles due to reduced surface stickiness (cox and wathes, 1995) . a number of impactor samplers are commercially available each of which show differences in number of nozzles, nozzle dimension and shape, jet-to-plate distance and number of stages. when a single nozzle is used to suck in air the shape is usually rectangular as in slit sampler (e.g., burkard manufacturing co. ltd., hertfordshire, united kingdom; new brunswick scientific co., edison, n.j.; casella london ltd). in this sampler for proper spreading out of the collected bioaerosol particles the collection surface may be moved under the slit. such sampler has been used for both indoor and outdoor sampling of airborne microorganisms . cascade impacter such as anderson sampler (graseby andersen, smyrna, ga, usa) have several stages with successively smaller nozzles such as the six stage anderson sampler used to quantify both fungal and bacterial bioaerosol in different indoor environments such as hospital, student dorm, laboratory, hotel room etc., in beijing (xu and yao, 2013) . this allows separation of bioaerosols according to their aerodynamic diameter. mas 100 (merck, germany) is one of the single stage impactors which consists of a perforated plate with 400 holes each of 0.7 mm diameter through which the collected air is aspirated either vertically or horizontally and with the speed of 10.8 ms -1 is propelled onto a solid agar plate. the sampling volume is adjusted to 100 l/m 3 after passing through an airflow meter (meir and zingre, 2000) . consistent performance was found when mas 100 was used for sampling aspergillus fumigatus and other thermotolerant fungal bioaerosol (engelhart et al., 2007) .when the number of nozzles increases usually circular in shape it takes the appearance of a sieve thus forming another sampler named sievetype sampler (spiral system instruments, bethesda, md.). rotorod sampler (ted brown associates, los altos hills, ca) and rotoslide sampler (oak ridge reproduction service, oak ridge, tn) are rotating impactors generally used for outdoor sampling collects particles larger than 15 μm (mandal and brandl, 2011) . sampling by such samplers are done by sweeping the collecting surface which may be a rod or a slide through the air. fraction of particles impacted on the collecting surface from the volume of air swept by the rotating surface defines the collection efficiency of the samplers (juozaitis et al., 1994) . among all the above-mentioned impactor samplers slit sampler was specifically described for collecting airborne microorganisms long back (bourdillon et al., 1941) . this type of sampler is best applicable to monitor the effect of "people activity" as well as operational variation and material movements over the production and distribution of bioaerosol and hence have been widely used in settings such as agricultural environments that are highly contaminated (blomquist et al., 1984) as well as in domestic environments (verhoeff et al., 1990) . for collecting large airborne particles (such as large fungal spores, clumped spores and pollen grain) slit sampler is very useful and hence had been mostly employed to determine the fungal bioaerosol in domestic settings (kozak et al., 1980) instead of bacterial cells and small spores. as slit sampler provides information about bioburden in respect to time and activity without regarding particle size, both cascade impactor and sieve type sampler determine the particle size distribution of the bioaerosols. cascade impactor and stacked sieve six stage anderson viable impactor can be used to collect both bacterial and fungal spores and fragments unlike slit sampler. in case of six stage anderson viable impactor, sampling of airborne bacterial and fungal with mass median aerodynamic diameter (mmad) less than 4 may result in an overloaded sample if the concentration is greater than 5000-7000 cfu/m 3 while for the same diameter range sieve personal sampler can be used satisfactorily for sampling (jensen and schafer, 1998) . hence sieve sampler seems to best fit for sampling in highly contaminated environments while anderson sampler can be used when positive hole correction factors are used for proper calculation of the number of particles collected when overloaded (blomquist et al., 1984; macher, 1989) . in comparison to the above-mentioned rotorod sampler mainly helps in qualitative assessment of bioaerosols rather than quantitative (cox and wathes, 1995) . rotorod has been found to collect greater number of large sized airborne particles such as pollen grains instead of bioaerosol spores per unit of air (cage et al., 1996) . impingement-based methods operate almost similar to impactionbased approaches, except that the micro-organisms are collected into a liquid medium (mandal and brandl, 2011) . generally air is sucked in through a narrow inlet tube onto the collecting medium where the flow rate of the sampled air depends on the diameter of the inlet nozzle. suspended particles get impinged on the collecting liquid as soon as the air strikes the liquid. on completion of sampling the aliquots are cultivated on proper growth media to enumerate viable microorganisms. impingers are generally needed to be sterilized before re-use. in fact replicate samples require a 'fresh' impinger, which increases costs of impingement based sampling than impaction-based sampling (cartwright et al., 2009) . collection and recovery efficiencies of impingers are found to be influenced by the inlet characteristics of the sampler. reduced recovery efficiency has been found to be correlated with increased flow rate (ogden and raynor, 1967) . similar to impaction-based samplers, the collection and recovery efficiency of impingers are highly affected by wind speed at the inlet. studies carried out by scientist in may (1966) revealed that at 4.5 m s −1 wind speed across the inlet, the collection efficiency turned out to be 9.6%, whereas after creating a still air condition by using a baffle, the collection efficiency jumped upto 99% (sanchez-monedero and stentiford, 2003) . although impingement-based samplers generally use a liquid collection medium, the type of liquid used sometimes vary with only one common element i.e., the liquids should be an isotonic or buffered solution so as to avoid osmotic stresses being imposed on micro-organisms following their collection. as for example betaine or peptone solutions are recommended so as to protect bacterial cells from osmotic shock (eduard and heederik, 1998) . use of an improper buffered water medium such as ringers solution affects the collection efficiency of the device as buffer gets lost from the sampler through evaporation when the sampler is used for long periods of time (willeke et al., 1998) . a number of commercial impinger samplers are currently available which include the all glass impinger 30 (agi-30), the skc biosampler, the burkard multistage sampler, the modified personal impinger (mpi), the multi-orifice impinger (moi), and the multi-stage liquid impinger (mli). as most of the impinger samplers are made up of glass they are cheaper than metal samplers such as the andersen sampler, but affecting their robustness in the field. agi-30 is a single stage impinger with a cylindrical reservoir under vacuum that contains a suitable collection liquid for concentrating bioaerosol from the air through a central jet raised 30 mm from the base of the cylinder. agi-30 consists of an electrical powered pump capable of drawing 12.5 l/min at a pressure drop across the impinge jet of 50 kpa with a typical sampling time of 10-30 min. the agi-30 sampler (ace glass inc., n.j., usa) is a cheap but less efficient sampler (ding and wang, 2001) . agi-30 comes with a disadvantage of foaming that can be induced in several collection solutions through the impingement process (dillon et al., 2005) . the most popularly used sampler is "biosampler" liquid impinger (skc, eight four, pa, usa). this sampler is a good example of an all-glass, swirling aerosol collector. it mainly consists of an air inlet, three tangentially arranged nozzles and a collection vessel . the biosampler also consists of a pump capable of drawing in air at the same rate as that of agi-30 but the sampling duration is very high 0.5 to 4 h. the greatest advantage of the sampler is its ability to be used in highly contaminated environment with sampling duration up to 30 min with aqueous based sampling media and up to several hours by using a viscous sampling medium (dillon et al., 2005) . filtration based samplers are relatively simple and less expensive. they are highly effective means of collecting bioaerosols. in filtration method airborne microorganisms are collected by passing air through porous membrane filters made of glass fiber, polyvinylchloride (pvc), polycarbonate or cellulose acetate (incubated by transferring onto the surfaces of growth agar media) or gelatin. out of these gelatin filters offers a much better environment (mandal and brandl, 2011) . the forces that are responsible for collection of particles are inertial forces, diffusion and electrostatic attraction (gilbert and duchaine, 2009 ). the filter method is generally used in personal samplers (such as worn by workers at relevant facilities) rather than in general sampling due to their small size. in fact filter-based samplers when used as nonpersonal samplers were more suitable for qualitative assessments of airborne micro-organisms only (predicala et al., 2002) . according to size fraction bioaerosol collection can be done by using polyurethane foam inserts (kenny et al., 1998) . when filtration based samplers are used in highly contaminated environment enumeration of bioaerosol becomes impossible just similar to impaction samplers due to overloading of filters with microorganisms (eduard and heederik, 1998) . desiccation/drying off of microorganisms on the filters post collection is another problem related to filtration based samplers (hinds, 1982) . though some microorganisms such as fungi and spore forming bacteria are sometimes found alive on the filters, vegetative bacterial cells such as gram negative bacteria cannot tolerate the desiccation stress at all . in fact sampling time and relative humidity plays a major role in determining the loss incurred due to desiccation. it has been reported that when temperature exceeds 30°celsius and relative humidity increases from 30 to 85% fungal propagules are still viable while many vegetative bacterial cells become nonviable . the filters when vortexed help in the recovery of bacterial cells (douglas, 2012) . gravity sampling which is a non qualitative method is done by exposing an agar medium to the environment over which airborne microorganisms are collected by gravity (grishpun et al., 2007) . due to this large particles mostly settle down on the collection surface rather than smaller particles resulting in the misrepresentation of the airborne microorganisms due to exclusion of smaller particles (burge and solomon, 1987; solomon, 1975) . gravitational settling method gives information about the total number (or mass) of the collected bioaerosol only and does not quantify their concentration as the volume of air from which the bioaerosols originated is unknown. however some scientists also believe that this method is reproducible and reliable along with the fact that many places in an environment can be checked at the same time helping the operators to compare and understand without disturbing the air (pasquarella et al., 2000) . electrostatic precipitation sampler follows the basic principle of particle precipitation in which airborne particles are precipitated from an airstream by the application of an external force such as electrical force on charged particle (knutson and whitby, 1975) . in an electrostatic precipitation sampler the biological particles are charged at the inlet. the charged airborne biological particles are then exposed to an electrical field inside the sampler resulting in their cross sectional migration eventually depositing over charged plates. finally from the charged plates the microorganisms are extracted and analyzed. this technique provides a much better means of collection especially for stresssensitive microorganisms as the particle velocity component perpendicular to the collection medium is almost two to four order lower than those found in bioaerosol impactors and impingers at comparable sampling flow rate (mainelis et al., 1999) . the collection efficiency is found to be dependent on applied voltage, flow rate, dimension of the precipitators and initial particle charging level (mainelis et al., 2001; mainelis et al., 2002a mainelis et al., , 2002b . by limiting the initial charge on airborne microorganism at the inlet the loss due to viability can be controlled (mainelis et al., 2002c) . generally low power is required for maintaining the sampling flow through an electrostatic precipitator as it is an open channel. moreover since very little power is also needed to create precipitation voltage, this method turns out to be highly feasible for low power monitoring of bioaerosol in a counter bioterrorism network. several studies have shown that electrostatic precipitation can also be implemented without the use of additional charging at the inlet of the sampler (mainelis et al., 2002d) . a recently developed electrostatic precipitator had no charging unit in the inlet while the physical collection efficiency strongly depended on the precipitation voltage which eventually depended on the charge present on the airborne microbes naturally due to aerosolization (kunkel, 1950; flagan, 2001 ) thereby making collection possible by differentiating between the positively and negatively charged microorganisms by adding a signature to the bioaerosol particle sampled (lee et al., 2004a; ; lee et al., 2004b) . during the effort made for electrostatic sampler's development and evaluation, important information was found regarding the electro biological properties of microorganisms that is related to electric potential of their membrane which is further determined by the transmembrane potential between the extracellular fluid and cytoplasm, and 2 surface potentials at the external and internal interfaces of the membrane. according to the information gained bacteria that are dispersed from a liquid through pneumatic nebulization generally possess a wide and bipolar electric charge distribution. the viability of the bacteria was also found to be affected by the electric charge imposed on it during aerosolization due to removal of some fragments of bacterial surface and counter ions (mainelis et al., 2001) . since the basic metabolic activity of the bacterial cells depend on the membrane potential (cevc, 1990) , ion transporters/channels and metabolically essential proteins, atpase are significantly affected by this change in membrane potential (bond and russel, 2000) eventually making the microbes nonviable. due to the limitations related to the viability of the collected bioaerosol through impactors and impingers new bioaerosol samplers such as niosh one-stage bioaerosol cyclone, cip 10-m, niosh twostage cyclone, coriolis®, wwc, and pas-5 by rct & hrb were developed. in cyclone samplers microorganisms are captured into a liquid (aerosol to hydrosol) using swirling air and centrifugal force. such samplers are advantageous as they are less susceptible to particle 'bounce' and re-entrainment (willeke et al., 1998) . these samplers are relatively easy to sterilize and play an important role when multiple samples are needed to be taken (cartwright et al., 2009) . since water is used as the sampling medium in both the samplers cyclones and impingers, studies related to their efficiency was done revealing the recovery efficiency of cyclone samplers to be 100 ± 10% relative to agi-30 impinger for gram negative bacteria (henningson et al., 1988) . guidelines that are generally looked upon for matching the appropriate technique (depending upon their advantages and disadvantages) with the bioaerosol of interest are given in table 1 . the bioaerosol of interest is mostly categorized as culturable bioaerosol sampling and non viable bioaerosol sampling with subcategories of free bacteria and fungi. free bacteria (i.e., mostly single cells), free fungi (i.e., mostly single spores) as well as clumped bacteria and fungi with mmad 4 μm are in general bioaerosols of interest in environmental investigations hence it is noted that the samplers must collect these aerosol (wright et al., 1969; lee et al., 1973) . this is one of the oldest sampling techniques based on thermophoresis principle in which air laden with aerosol and bioaerosol is passed through a narrow channel containing a temperature gradient perpendicular to the air flow. on entering the temperature gradient the airborne particles tend to move away from the hot surface towards the cooler surface and depositing over it, a phenomenon known as thermophoretic motion (waldmann and schmitt, 1966) .the hot surface is generally heated up to 125°c while the other collecting surface is cooled by a circulating water heat exchanger. immediate microscopic examination can be done is glass microscopic cover slips that are used as the cooler collection surface or if collected over filter paper from which the deposits could be transferred to agar plates to allow colonies to grow and examined thereafter (kethley et al., 1952 and orr et al., 1956) . the collection efficiency of such sampling technique is very high for small particles ranging from b0.01 μm to 5 μm, when the temperature gradient is sufficiently maintained all throughout the sampling time, and thus this method is used to determine the particle size distribution (watson, 1958) . although its collection efficiency for smaller particles is high yet its collection rate is very low in comparison to other samplers ranging from 7 cm 3 min − 1 to 1 l min − 1 (cox and wathes, 1995) . thus because of such low rate sampling and requirements of very precise adjustments, such samples are not commonly used in industries (kang and frank, 1989 ). bioaerosol sampler using condensation technique consists of a number of parts attached together such as vacuum pump, humidifier, a heating source, a liquid source, amplifier, a cooling source and a biomass spectroscopy system for proper sampling and analysis of bioaerosol collected. in such sampler, air is drawn in through a vacuum pump into humidifier first. the humidifier consists of a heating source that evaporates liquid source such as water (usually a biocompatible source) to create a humid environment (with relative humidity 90% or higher) to temperature higher than the room temperature i.e., 35°c but lower than 45°c so as to prevent the deactivation of microbes collected. the air sample is then drawn into the amplifier via vacuum action. the amplifier consists of a cooling source that eventually reduces the temperature to as low as 10°c. due to this low temperature the air volume in the amplifier is subjected to condensation with supersaturated vapors. here the sampled bioaerosol acts as the condensation nuclei from which the particle grows or amplifies, however maintaining the viability of the microorganism. particle size amplification of bioaerosols due to condensation of super saturated vapors can occur at the order of nanometer and/or submicron to a greater size such as 10 μm and above. the time period required for condensation amplification is as less as 1 s or even lesser. thus amplification increases the bioaerosols dimensions indirectly increasing the detection efficiency. the amplified bioaerosol then can directly sent into a biomass spectroscopy system (such as maldi-tof) for in-line and continuous identification. due to such technique very small bio-species/ bioaerosols such virus that cannot be sampled or detected by conventional systems can be amplified and studied upon (wu et al., 2013) . enumeration of microorganisms forms the second major step in the monitoring strategy. the technique is divided into two broad groups namely culturable and non culturable approaches. culture based approach is a simple and low cost method that involves collection of airborne microorganisms and culturing of the sampled microorganisms on some semisolid growth medium with results expressed as colony forming units after a proper incubation (conditions including time, temperature and available oxygen). as it is assumed that single colony is formed from a single microorganism so the cfus give the information of the number of microorganisms present in the sample. the major limitation of this approach is that a very small proportion (almost 10%) of the microorganisms present in the environment can be cultured and identified (heidelberg et al., 1997; torsvik et al., 1994) . several studies have shown that culture conditions also limit the growth of the viable and culturable microorganisms with examples such as mesophilic bacteria namely escherichia coli and bacillus subtilis exhibit proper colony formation at temperatures between 5 to 55°c (droffner et al., 1995; pillai and ricke, 2002) while thermophilic microorganisms namely thermoactinomyces sp. prefer culture temperature above 50°c (neidhart et al., 1990) . several studies have been conducted across the world till date in order to evaluate microbial load (isolating, quantifying and identifying) in various indoor environments such as occupational, residential and educational using culture based techniques and their important findings have been shown in table 5 . airborne biological particles sampled from air onto glass slides, semisolid media and filters fitted to samplers can be enumerated and identified using microscopic examination. after processing the sample through a proper technique designed identification of taxa or species can be done. identification is mainly based on the morphological characteristic of the microorganisms and their spores (especially for fungi). in classical microscopy various types of stains available are used for identifying and describing fungal spores so as to differentiate between different fungal spores and organic debris (burge, 1995) . as shown in table 2 although classical microscopy id one of the easy performing technique that has the advantage of identifying specific taxa of both fungi and bacteria, however does not act as a representative of all the microbes in bioaerosol. such methods are mostly used only for quantification of microorganisms present in liquid samples where actual counting is not done and mostly depends on statistical calculations (makkar and casida, relatively swift and easy to perform being a statistical test it does not measure actual numbers of micro-organisms. as the micro-organisms are grown in liquid media such technique is less susceptible to the culturability issues that affect selective isolation plate methods . aggregates of cells may affect the result, thereby limiting the suitability of this method to analysis of bioaerosols. sometimes difficult to quantify due to collisional quenching of the excited state and potential photochemical effects. measurements are spatially resolved and can be further extended to laser imaging. not all excited species fluoresces causing improper measurements. cheap technique and easy to operate the compound (such as proteins) to be analyzed should be in the databases. highly sensitive this technique is generally not suitable for compounds less than 600 da in size due to intense matrix signal. very mild ionization technique used, thereby making analysis of mixture possible there is limitation in the resolution of this technique which can only be increased significantly by a reflector and or a delayed extraction. very little or no sample preparation is required that results in increased throughput, greater convenience and fewer opportunities for contamination to occur. limited usage due to increased cost and system complexity very sensitive and requires very small amount of sample (thus sometimes referred to as "nondestructive" method) sometimes regarded as semi quantitative technique as obtaining suitable standards is difficult. possibility of multi-elemental analysis simultaneously there are possibilities of large interference effects that include matrix interference as well as potential interference of particle size in case of aerosol. has the potential for direct detection in aerosols less precision ranging from 5 to 10% depending upon excitation properties of laser, sample homogeneity and sample matrix simple process with rapid analytical capability as in a single step ablation and excitation process is carried out. both culturable and nonculturable cells can be counted making the results more representative of total numbers of micro-organisms in the bioaerosol. restricted ability to identify specific taxa of micro-organisms relatively cheap operating costs fluorochromes if binds to abiotic particles may result into false positive results. high throughput of samples possible if image analysis system used image analysis system may count abiotic particles within the same size parameters as microbial cells. not suitable for counting aggregates of cells overestimation due to binding to abiotic material may take place pcr technique remarkably sensitive technique the efficiency and size ranges of bioaerosol high volume samplers should be completely characterized which can otherwise affect the quantification by quantitative pcr. applicable to any biological matter containing nucleic acid possibility of inaccurate bioaerosol quantification due to improper sample preparation steps like filter elution/concentration and nucleic acid extraction detection and identification can be made independent of culturing thereby removing the need of specialized labs to perform cell cultures which require extensive biosafety infrastructure. results may get affected by the presence of inhibitory pcr compounds in the samples. results are provided rapidly on the order of hours as compared to days or weeks. certain taxa of micro-organisms can be identified. no standard approach available for monitoring of biomarkers so as to provide certain information. as whole cells are not measured, this technique is not prone to many of the limitations of culturable or nonculturable methods lal assay types biomarker tests which are significantly used in bacterial bioaerosol analysis are affected by dust or other microbial cell components. this is likely to be a significant problem. 1987). in this method serial dilution of the sample is done in order to quantify the density of microorganisms present on the probability the basis of the probability that positive results will emerge after incubation from each microorganisms. possibility of detection of microorganisms by this method is more as the liquid medium used for growth imposes much less stress over the microbes rather than the semisolid medium. as mostly there is good probability that aggregates of cells will be found in the inoculation medium, this applicability of this method for enumeration ends as it is only used when single cells are found. laser induced fluorescence (lif) is a spectroscopic method mostly used for studying the structure of molecules and detection of selective species (especially bacteria). after the air samples are collected through impaction, the cultivated bacterial species is excited by a laser light whose wavelength that is selected is often the one at which the species has its largest cross section. within a few nanoseconds or microseconds the species de-excite and emit light (with wavelength longer than the excitation wavelength) (zare, 2012) . this emitted fluorescent light is then recorded by a photomultiplier tube (pmt) or filtered photo diode. lif is highly advantageous over absorption spectroscopy because the detection sensitivity is very high as the signals are observed against dark background and two to three-dimensional images can be obtained as emitted fluorescent radiation can be obtained in all possible angle (zare, 2012) . the classified fluorescent signals can be confirmed by correlating with morphology, gram staining or family (rösch et al., 2005) . as seen in table 2 in lif as analysis and detection is done on the basis of emitted fluorescence light from the species, probability of improper measurement exists as all the species do not fluoresce. moreover lif technique is mostly used for bacterial identification. matrix-assisted laser desorption/ionization (maldi) is a soft ionization technique that is mostly used in mass spectrometry for analyzing biomolecules (microorganism and biopolymers such as dna, proteins, peptides etc.) as well as large organic molecules which generally becomes fragile and fragment when conventional ionization methods are applied. maldi is a two step process where uv laser beams are used to trigger desorption first. the uv laser light is absorbed by the matrix material leading to ablation of the upper layer (~1 μm) of the material. during ablation the hot plume that is produced contains several species such as neutral and ionized matrix molecules, protonated and deprotonated matrix molecules, nanodroplets and matrix clusters. in the second step the matrix molecules get ionized in the hot plume. the matrix mainly consists of crystallized molecules with 3, 5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), 2,5dihydroxybenzoic acid (dhb) and α-cyano-4-hydroxycinnamic acid (alpha matrix) most commonly used (strupat et al., 1991; beavis et al., 1992) . with the use of highly purified water and an organic solvent which is usually acetonitrile or ethanol a solution of any of these molecules are made and used as a matrix. tof (time of flight mass spectrometer) has large mass range, it is mostly used with maldi. in the maldi-tof instrument an 'ion mirror' is present that reflects ions using an electric field, thereby doubling the ion path and increasing the resolution. for the identification of bacteria and fungi maldi-tof spectra can be used. after having been collected of airborne microorganisms on growth media via impaction and after their proper growth, a colony in question is smeared on the sample target directly. it is then overlaid by the matrix and mass spectra generated. the spectra are then analyzed with software present with the instrument. for proper identification comparison with the stored profiles are done. however even in the absence of prior cultivation it is possible to obtain a mass spectrum of a single airborne particle by on-line measurements using instrumental improvements (kleefsman et al., 2008) . in comparison to other immunological and biochemical tests identification of species by this process is highly economical and accurate (seng et al., 2009 ) however as shown in table 2 it can only analyze compounds greater than 600 da in size such as proteins, and peptides, available in database forms due to intense matrix signal thereby restricting its identification range. recently libs is being considered as a process for rapid real time detection of microbes responsible for biological warfare attacks and illness both in the fields and in laboratory settings. it is a time resolved atomic emission spectroscopic analytical technique based on optical emission following pulsed laser ablation of a sample (radziemski and cremers, 2006; schechter et al., 2006) . in this process when the laser is focused onto a small area at the surface of the specimen, it ablates a very small amount of material (in nanograms to picograms) which generates a plasma plume with temperature in excess of 100,000 k. at such high temperature the ablated materials breakdown into excited ionic and atomic species. during this time, the plasma emits a continuum of radiation which does not contain much information about the species present which is useful. during this emission of radiation within a very small time frame the plasma expands at supersonic velocities and cools. at this point the characteristic atomic emission lines of the elements can be observed which gives the information about the specimen. libs is mostly used for bacterial detection. it is important to note that libs analysis does not depend upon identifying the genetic differences between the species (the libs analysis does not rely on the elements that comprise dna or proteins such as carbon, nitrogen, hydrogen and oxygen), it is rather the difference in the inorganic chemical composition of the outer membrane that is detected. in fact the chemical composition varies between bacterial species as a function of the genetic variation between those species. in recent studies by using libs with both nanosecond and femtosecond laser pulses bacterium e. coli was identified (baudelet et al., 2006; diedrich et al., 2007) . e. coli a nonsporulating gram negative bacterium has specific outer membrane that contains mg 2+ and ca 2+ (singleton, 1997) . the ionized and neutral mg and ca emission lines act as the dominant spectral features in the libs spectrum of e. coli. thus for a particular bacterium for creating a "spectral fingerprint", measurement of emission lines from these and other trace inorganic elements such as iron, potassium, sodium, phosphorus and manganese are done. however, there are chances of less precision of result obtained ranging from 5 to 10% depending upon excitation properties of laser, sample homogeneity and sample matrix as mentioned in table 2 . as already mentioned above all of the culturable techniques can analyze microbes by isolating and characterizing them over commercially available growth media such as nutrient agar, and luri-bertani agar (kirk et al., 2004) . less than 1% of total microbial species in any environmental sample can be analyzed by this technique (hugenholtz, 2002) while the rest though viable in nature are nonculturable in laboratory conditions remaining in the "viable but nonculturable" stage (oliver, 2005) . with the evolution of fluorochromes staining the microorganisms collected in liquid medium quantification of all viable microbes (both culturable and nonculturable) were possible. a major shift in the paradigm of microbial analysis was seen with the advancement in the fields of genomics and sequencing technologies as well as analysis of microbial community using nonculturable molecular techniques such as genetic fingerprintings, metagenomics and next generation sequencing helping in not only identifying and quantifying the microbial load but also helps in understanding the probable changes taking place in the community as well. a wide array of scope of microbial identification also evolved with the analyses of metabolites and constituents of microorganisms without directly counting them (such as mycotoxins, and endotoxins) by advanced techniques like chromatographic, immunoassay and pcr based methods. the following are the few nonculturable techniques applied. by using fluorescent stain (a fluorochrome), microorganisms collected in liquid buffer solution or filters are stained and counted by epifluorescent microscopy (eduard and heederik, 1998) . different types of fluorochromes are available that indicate cell viability by showing differences in electron transport chain activity, cytoplasmic redox potential, enzymatic activity, cell membrane potential and membrane integrity (kepner and pratt, 1994) . dapi (4,6-diamidino-2-phenylindole) and acridine orange (3,6bis[dimethylamino]acridinium chloride) two fluorochromes that are applied for bioaerosol monitoring are nucleic acid stains and allow microorganisms to be distinguished on the basis of color. in general acridine orange binds to dna and rna and depending upon single stranded and double stranded nucleic acid different colors fluoresces such as orange and green respectively. in case of dapi when it binds to dna it fluoresces blue or bluish-white and yellow when bound to non dna material (kepner and pratt, 1994) . the basic advantage of this technique is that it facilitates all viable cells of both bacterial and fungal bioaerosol (culturable and nonculturable cells) to be counted as it is a nonculturable approach as mentioned in table 2 . moreover if it is attached with a computer based image analysis system, the counting gets automated and a high throughput of samples are achieved (kildeso and nielsen, 1997) . however the disadvantage of this technique (as shown in table 2 ) is binding of the fluorochrome to abiotic material, error in counting the microorganisms by human as well as differentiating between microbial cells and abiotic material such as dust (pillai and ricke, 2002) . although as mentioned before by using an image analysis automated system one can improve enumeration yet limitation still persists as it will only count particles which will fall within the size parameters of the programmer (crook and sherwood-higham, 1997). thus the overlapped cells are most likely not to be counted. in order to minimize the "false positive" results generated from binding of fluorochromes to abiotic particles baclight fluorescent stain can be used as this stain is less susceptible to binding to such materials in respect to acridine orange that is found to bind with humic material (kildeso and nielsen, 1997) . thus for enumeration of airborne microorganisms both baclight and acridine orange are used maximally (terzieva et al., 1996) . in the pcr technique a specific region of a genome is copied and amplified to a millionfold making them available for further analysis (georgakopoulos et al., 2009) . conventional polymerase chain reaction (pcr) assay has been used as an alternative method for analyzing total bacterial load in bioaerosol samples which also provide qualitative assessment when gel electrophoresis is used to visualize the resulting pcr amplicon (saiki et al., 1985) . in order to analyze air samples for the presence of endemic microorganisms (alvarez et al., 1994) , biowarfare agents (higgins et al., 2003) , airborne mycobacteria (schafer et al., 2003) and fungi generally associated with health effects (cruz-perez et al., 2001; williams et al., 2001) conventional pcr assay has been applied. identification of a particular microbe can also be done by using specific primer set and rapidly produce results on the order of hours in comparison to days and weeks. however as mentioned in table 2 possibility of inaccurate bioaerosol quantification is one of the major disadvantages of pcr technique mainly due to improper sample preparation such as filter elution and nucleic acid extraction. in recent phase real-time pcr (rt-pcr) is evolving as a technique capable of giving accurate measurements of total microorganism concentrations in environmental samples. unlike the conventional pcr, rt-pcr analysis is not done via gel electrophoresis. it is in fact attached to a thermal cycler coupled to an optical module which measures the fluorescence intensity of reactions generated by hybridization probes (such as taqman, melocular beacon, fluorescence resonance energy transfer) as well as by double stranded dna dyes such as sybrgreen green (sybr) (stetzenbach et al., 2004) . depending upon the background fluorescence the data analysis software provided then calculates the cycle number ct (at which the fluorescence in the sample crosses the threshold) which is inversely correlated with the microorganisms concentration in the sample. studies have proven that standard curves can be produced by using known microorganism concentration as templates. these standard curves can then help to quantify the total microorganism concentration in the unknown sample (ana et al., 2006) . in recent past two real time pcr systems were developed in order to quantify levels of cladosporium, one of the most common molds found both in indoors and outdoors environments (zeng et al., 2006) . flow cytometry is a technique in which both fungi and bacteria can be quantified on the basis of component or structural features of cells via optical means (muirhead et al., 1985; porter et al., 1997) . in flow cytometry the cells are required to be in aqueous solution, hence the types of sampling technique used to collect the bioaeorosols are impingement, filtration or cyclone. once the cells are in suspension, a continuous flow of a fine stream of the suspension in the form of a single file moves through a laser beam. as the stream passes through the laser beam the amount of light scattered by each cell is measured which is dependent on the size of the particles and the presence or absence of specific cell surface features. in different environmental samples such as bioaerosols light scatter characteristics are not sufficient to differentiate cells so different types of fluorochromes are required. hence combination of flow cytometry with fluorescent in situ hybridization (fish), where fish labels specific nucleic acid sequences inside intact cells using so-called phylogenetic stains for quantifying and differentiating cells ( porter et al., 1997) . according to certain studies the major advantage of flow cytometry is that it can count thousands of cells within seconds (davey and kell, 1996) similar to epifluorescence microscopy (table 2) . again just like epifluorescence microscopy, along with the microbial cells, counting of abiotic particles having the same size as of cells is one of the limitations of this technique. the other disadvantage is that it can only count microorganisms that are found as single cells. in case of bioaerosols along with single cells aggregates of cells can also be sampled, which will then be required to be vortexed or agitated in order to break the cell clusters before analyzing using flow cytometer. this process may again lead to some other disadvantages such as affecting the viability of some of the cells present as well as increasing the cell numbers (terzieva et al., 1996; jensen et al., 1992) . although flow cytometry has been used for quantification as well as identification of airborne bacteria (day et al., 2002) , when applied along with different dye stains it actually provided more rapid and accurate viability assessment (chen and li, 2005) . quantitative analytical comparison was carried out between flow cytometry (fcm) and culture method by analyzing bacterial bioaerosols (especially pseudomonas aeruginosa) collected from swine barn wherein it was found that in comparison to fcm, microorganism concentration was underestimated by 2 orders of magnitude by the culture method (lange et al., 1997) . metagenomics also known as community genomics or environmental genomics is a powerful centerpiece that helps ion genomic analysis of a population of uncultured microorganisms directly from environmental samples. metagenomic analysis involves various steps such as isolation of dna from the required environmental sample followed by cloning of the extracted dna into a suitable vector. soon after that the clones are transformed into a host bacterium. the transformants are then screened such as 16s rrna and rec a for phylogenetic markers, or for multiplex pcr (stein et al., 1996) , or for specific trait expression as for example enzyme activity (lorenze et al., 2002) , or can be randomly sequenced (tyson et al., 2004) . in recent studies "shotgun" is being largely used to get unbiased samples of genes from the sample community members (eisen, 2007) . advancement in the refinement of dna amplification as well as proliferation of computational power have helped the analysis of dna sequences extracted from environmental samples thereby allowing the adaptation of shotgun sequencing to metagenomic samples. although previously clone libraries were used yet at present cloning step is omitted due to the advancement of next generation sequencing techniques that yields greater sequencing data without labor intensive step. thus with the help of high throughput sequencing technologies shotgun metagenomics not only provide information about the type organisms present in the environmental sample but also informs about the possible metabolic processes in the community (segata et al., 2013) . whole genome sequencing has also been applied to study the airborne microbial community in various indoor and outdoor environments of nyc after collecting air samples using a wet cyclone portable air sampler at the flow rate of 450 l/min (yooseph et al., 2013) . next generation sequencing (ngs), a catch-all term describing all the modern sequencing technologies that help in quicker and cheaper sequencing of dna and rna in comparison to traditional sanger sequencing (table 2) is also known as high-throughput sequencing and is applicable for both bacteria and fungi. the throughput requirement also seems to be quite less, only one or two instruments for the completion of the experiment. the work process of next generation sequencing-ready libraries consists of a few steps. firstly, ligation of specific adaptor oligos at both ends of the dna fragment and hence prepared for sequencing. in ngs it is important to note that relatively very little dna input (only a few grams at the most) is required and by using slightly modified library processes the platforms can also sequence the paired ends of the of a given fragment. moreover in comparison to the capillary sequencers (where long read lengths of 650-800 bp are produced), depending upon the platforms shorter read lengths (35-250 bp) are produced by the next generation sequencers. apart from theses generally shared features, the commercially available sequencers significantly differ from each other such as illumina sequencing is based on "sequencing by synthesis" i.e., sbs (ansorge, 2009) , rocha (454) sequencing operates on the principle of "pyrosequencing" (margulies et al., 2005) , solid sequencing (mardis, 2008) , etc. thus as seen in table 2 the obvious disadvantage of ngs apart from high startup cost and requirement of multiple days of run the major disadvantage is limited phylogenetic characterization capability as it works on only short read lengths (b600 bp). the comparative metrics and performance of the above-mentioned next generation sequencers are given in table 3 . in the past few years ngs has been used to characterize microbial community in various environmental samples by using illumina sequencing (bartram et al., 2011) and pyrosequencing (monard et al., 2013) . illumina sequencing has also been used in studying the microbial diversity in airborne microbiological samples causing organic dust toxic syndrome (odts), where sequencing data revealed the presence of more than 150 bacterial and 25 fungal genera in the air sample (madsen et al., 2015) . the understanding of the pattern of genetic diversity in a microbial community has been provided by genetic fingerprinting techniques such electrophoretic separation of low molecular weight rrna molecules such as 5s rrna and trna that were extracted from natural samples in high resolution polyacrylamide gels for more than a decade (hofle, 1990) . recently, dgge of ribosomal dna fragment amplified by pcr has been introduced as another genetic fingerprinting technique in microbial ecology (muyzer et al., 1993) . once the dna fragments of multiple organisms are extracted and amplified using pcr, they are subjected to dgge where dna fragments of different sequences but same length are separated. in dgge a constant heat of 60°c and an increasing concentration of dna denaturants i.e., mixture of urea and formamide are used to unwind the dna molecules. since in electrophoresis molecular weight, shape and electrical charge of dna, rna and proteins play the major role in the separation process (creighton, 1999) , similarly in dgge, positive electrodes attract the negatively charged dna fragments and are forced to migrate through the polyacrylamide gel. while moving through the gel they encounter the denaturing reagent mixture which in the presence of constant temperature breaks the hydrogen bonds between the base pairs unwining them or as termed partially melting them (muyzer et al., 1993) . the melting domains are thus determined which are eventually defined as stretch of base pairs having specific identical melting temperatures (muyzer et al., 1993; muyzer and kornelia, 1998) . difference in melting temperatures due to variation of sequences within theses melting domains causes the differential migration of the sequences to different positions in the gel (muyzer et al., 1993) . almost 50% of sequence variant up to 500 bp can be detected in dna fragments using dgge (myers et al., 1985) , which can be increased to almost 100% by attaching on one side of fragment a gc clamp i.e., a gc rich sequence which prevents the complete dissociation of the double stranded dna into single stands due to high melting domain (sheffield et al., 1989) . pcr dgge as a culture independent approach was used in assessing the seasonal effect of winter and summer on bacterial bioaerosol community in few swine confinement buildings (scb) in canada, wherein the dgge profile showed similar patterned dna bands for each scb even though the indoor temperature and ventilation rate differed from each other in both the seasons suggesting that the major microbial community such as lactobacillales and clostrida did not change with season as their origin remains the same (nehme et al., 2008) . in another study carried out in a dairy barn in eastern quebec, dna from the airborne dust was extracted using a qiagen qiaamp dna extraction kit and was analyzed using pcr dgge along with gc clamps to reveal several archeal (such as 95-98% representation of methanobacteriaceae group while 100% representation of methanobrevibacter of all the dgge bands sequenced) and bacterial (such as 94-99% homology with staphylococcus gallinarum, agrobacterium tumefaciens, crocebacterium ilecola, oxalobacter sp., cornynebacterium variabile, agrobacterium sp., clostridium quinii, staphylococcus sp. and cornynebacterium xerosis) species from both the domains (lecour et al., 2012) . as one of the alternatives to direct counting or culture-based techniques, constituents or metabolites of the microorganism can be measured as an estimate to microbial exposure (pillai and ricke, 2002; crook and sherwood-higham, 1997) . biomarkers generally measured include fatty acids, ergosterol, muramic acid (marker of peptidoglycan, therefore bacterial biomass) (pillai and ricke, 2002) and microbial volatile compounds (dillon et al., 2005) . other agents that are also measured due to their toxic potency are β (1 ➙ 3) glucans and bacterial endotoxin (aketagawa et al., 1993; douwes et al., 1996) . thus not only toxic (such as mycotoxins) or pro-inflammatory (such as endotoxins) components are measured as biomarkers but nontoxic components also serve as markers of either large groups of microorganisms or specific microbial genera or species. the use of various advanced methods such as polymerase chain reaction (pcr) based reaction, immunoassays, chromatographic techniques etc. for measuring biomarkers, thereby helping in detection and speciation regardless of whether the organisms are culturable or not. table 4 gives an overview of different assessment methods for microbial constituents and markers. limitations of the 'whole cell' techniques such as culturability and non specific binding of fluorochromes can be easily avoided my measuring biomarkers and other microbial constituents. moreover as most of the health effects are caused due to exposure to the microbial products such as endotoxins, mycotoxins, etc. instead of the viable microorganism themselves, so in order to assess the exposure monitoring the levels of such compounds are more relevant than measuring the microorganisms themselves. bioaerosol poses serious health hazards for people and animals living in their vicinity. microbiological pollution is spread in the form of bioaerosol containing viruses, bacteria, actinomycetes and fungi (fernando and fedorak, 2005) . it is important to note that, the small size of these particles means that they can enter the lungs easily if inhaled. hence, it may become a potential cause of respiratory and various other infections in people. in addition, these small particles can be very easily carried away by the wind to long distances ranging from a few hundred meters to several kilometers (recer et al., 2001) , again posing a potential biological hazard not only to the nearby areas but also to residents of distant areas. bioaerosols, especially with pathogenic or allergic micro-organisms, may cause respiratory and other health disorders. the potential health hazard caused by bioaerosols depends on the pathogenicity of specific micro-organisms as well as other factors such as the environmental conditions which determine the survival of the microorganisms in the air (mohr, 2001) , the meteorological conditions (especially wind speed and wind direction) which controls the airborne dispersion from the emissions points (katzenelson et al., 1976) , provides the pathway to bioaerosol to enter the body and also the immunologic response of the body. the main pathways for transmission of micro-organism to humans are: by direct contact with contaminated sources such as through mucous membranes or skin, by ingestion through hands or accidentally and by inhalation process. and, there are evidences which indicate that enteric diseases prevailed in communities that may be associated with aerosols generated from waste water containing enteric pathogens (katzenelson et al., 1976) . as it is known that, enteric bacteria are the good indicator of water pollution. bioaerosol transmission is a key mode of transport for some of the world's most contagious, lethal and infectious diseases, such as tuberculosis (al-jahdali et al., 2003) , severe acute respiratory syndrome and influenza (klontz et al., 1989) . the major groups of diseases associated with bioaerosol exposure are infectious diseases, respiratory diseases and cancer (moreno-lopez, 1990; . bioaerosols are also associated with non-infectious diseases such as hypersensitivity, allergies, and asthma (olenchock, 1994) . a number of studies have already indicated the important role of bacterial and fungal airborne micro-organisms as potential opportunistic human pathogens. for instance, continued exposure to large concentrations may lead to a sensitization and to the development of occupational diseases, such as allergical veolitis, asthma and organic dust toxic syndrome in humans (lacey, 1991; lacey and dutkiewicz, 1994) . infectious diseases may be categorized into bacterial, fungal and viral diseases. such infectious diseases arise from viruses, bacteria, fungi, protozoa and helminthes and involve the transmission of an infectious agent from a reservoir to a susceptible host through airborne transmission. legionellosis, tuberculosis and anthrax are bacterial diseases that cause significant public health concern even due to bacterial bioaerosol low infectious dose (hussong et al., 1987) . legionella pneumophila causes human legionellosis, an airborne disease often caused as a result of active aerosolizing processes such as aeration of contaminated water. the transmission of tubercle bacilli occurs through the inhalation of aerosolized bacilli in droplet nuclei of expectorated sputum-positive tuberculosis patients during coughing, sneezing and talking. the transmission of anthrax occurs due to inhalation of the spores of bacillus anthracis and its outbreaks are often linked to bioterrorism. viruses readily transmitted by airborne route include, severe acute respiratory syndrome (sars) virus , enteric viruses of intestinal origin produced at sewage treatment facilities, respiratory syncytial virus source: mardis (2008) . (rsv), hantavirus from rodent feces (mojica, 1998) , varicella -zoster virus, measles, mumps and rubella viruses. sars, caused by novel corona virus, is a highly contagious and responsible for respiratory infection of significant morbidity and mortality, and may also cause very severe atypical pneumonia. besides the above-mentioned diseases, airborne fungi are also often reported to be an important cause of respiratory complaints in atopic individuals (howard, 1984) . atopy is the genetic predisposition of an individual to produce high quantities of ige in response to allergens in the environment (pollens, house dust mites, molds, cat dander, foods, etc). a great threat is also connected to the presence of microbial allergens and endotoxins, lipopolysaccharide which are produced by gramnegative bacteria that is considered as the most important health hazard. studies have demonstrated that endotoxins could be the cause of airway and intestinal inflammation and work-related symptoms (for example: diarrhea, fatigue and nose irritation) in various occupational sectors. in fact non-allergic work related asthma symptoms known as "irritant induced asthma" (bernstein et al., 1999) was found in farmrelated occupations and were assumed to be caused by bioaerosol exposure (anonymous, 1998) . airborne fungi causing respiratory infections and allergic reactions include penicillium, aspergillus, acremonium, paecilomyces, mucor and cladosporium (kanaani et al., 2008) . most infections, specifically aspergillosis can occur in immune compromised hosts or as a secondary infection, which is caused due to inhalation of fungal spores or the toxins produced by aspergillus fungus (swan et al., 2002) . fungal metabolism produces many volatile compounds that are capable of inducing sensory irritation to eyes and upper respiratory tract. aspergillus species that can grow indoors include aspergillus fumigatus and aspergillus flavus and can cause nosocomial infections, allergic broncho-pulmonary aspergillosis (abpa) and sinusitis. cladosporium, alternaria, penicillium, and aspergillus are the genera of fungi which cause many diseases in human beings and are mostly found in various environments as shown in table 5 . alternaria sp., cladosporium sp., and penicillium sp., are three fungi which have been associated with causing asthma and rhinitis. penicillium species with spores of 2 to 3 μm (mm) have apparently been responsible for several hypersensitivity pneumonitis epidemics (kreiss and hodgson, 1984) . the "moldy" or "mildew" odors in some indoor environments are associated with low levels of volatile organic compounds (vocs) in the air produced by fungi (kaminske et al., 1974) . health effects have not been directly attributable to these vocs to date, but the vocs and/or the organisms which produce them may be contributory factors to complaints of headache, eye and throat irritation, nausea, dizziness, and fatigue in subjects occupying contaminated interiors (burge, 1990a) . bacterial bioaerosols are responsible for diseases such as tuberculosis (mycobacterium), legionnaires' disease (legionella pneumophila), and hypersensitivity pneumonitis (thermoactinomyces). airborne transmission occurs when an infected person is coughing, sneezing, actively shedding fresh organisms into air close to susceptible individuals, or even talking or singing (burge, 1990b) . thermophilic bacteria such as saccharopolyspora rectivirgula or thermoactinomycetes vulgaris have been found to contaminate hay and act as a source of allergen to farmer's lungs (reboux et al., 2001) as well as to mushroom growers (van den bogart et al., 1993) . some examples of viral bioaerosols which infect humans and are spread by aerosols, rather than by direct contact only, are influenza (influenza a and b), measles (rubella), mumps, and chicken pox (kundsin, 1980) . till date although no uniform international standard have been established in relation to levels and acceptable limits of bioaerosol loads (wong et al., 2007) yet certain terminologies are used that are different in different countries such as "maximum acceptable values" (de aquino neto and de góes siqueira, 2004) , "orientation values" (suva, 2007) , "acceptable maximum value, amv" (becher et al., 2000; jo and seo, 2005) , "threshold limit value, tlv" (american conference of governmental industrial hygienists (acgih), 2009). in fact due to lack of data in accordance to exposure-response relationships acgih has totally nullified the general tlv for culturable bioaerosol concentration (american conference of governmental industrial hygienists (acgih), 2009). since no-observed-adverse-effect-level (noael) or lowest-observed-adverse-effect level (loael) depending upon dose-response approach has not been established for bioaerocol concentration, health effects in relation to exposure limits on the basis of data from epidemiological and toxicological studies could not be developed till date (swan et al., 2003) . however, several published values in relation to acceptable concentrations of fungal and bacterial bioaerosol have been found that differ from country to country such as for total bioaerosol concentration in korea and netherlands are 800 cfu/m 3 and 10,000 cfu/m 3 respectively (jo and seo, 2005; eduard, 2009) ; fungal concentration in brazil, germany, portugal and switzerland are 750 cfu/m 3 , 10,000 cfu/m 3 , 500 cfu/m 3 and 1000 cfu/m 3 respectively (de aquino neto and de góes siqueira, 2004; institut für arbeitsschutz der deutschen gesetzlichen unfallversicherung (ifa), 2004; pegas et al., 2010; suva, 2007) and bacterial concentration in finland, germany and netherlands are 4500 cfu/m 3 , 10,000 cfu/m 3 and 10,000 cfu/m 3 respectively (nevälainen, 1989 ; institut für arbeitsschutz der deutschen gesetzlichen unfallversicherung (ifa), 2004; eduard, 2009 ). most of the guidelines are found to be in relation to specific microorganism such as penicillium (eduard, 2009) or specific group microorganism such as gram negative bacteria (suva, 2007) . thus it is very evitable that till date no work has been done that can describe health effects solely on the basis of overall fungal or bacterial concentration present, rather many research have worked in the direction revealing that health effects are dependent on the combination of three factors namely, the genera of the airborne microbe, their size range (depicting till what level they may penetrate in the respiratory system) and their concentration in the concerned environment. this statement can be supported by the study done on 11 sawmill workers exposed to 0.2-1.5 × 10 6 fungal spores/m 3 mainly of rhizopus and penicillium by roponen et al., wherein a noaec in relation to nasal inflammation was suggested i.e., mere exposure to high microbial concentration does not evoke inflammation, rather the type of microbe or microbial product in the environment determines the potential of proinflammation of microbial exposure (roponen et al., 2002) . although all the three factors work in a combined way, yet most of the studies have been carried out emphasizing a single effect or combination of two. certain health effect studies in relation to concentration reveal that threshold concentration 100 spore/m 3 of alternaria were found to evoke allergic symptoms (gravensen et al., 1986) while in some other sick building syndrome were found to be potentially associated more 50 spores/m 3 of aspergillus sp. (holmberg, 1987) . in case off size dependent and genera specific health effects bacterial bioaerosol such a. lwoffi and a. johnsonii mostly found in 0.55-1 μm size range have been associated with bacteremia and meningitis (ku et al., 2000) while streptococcus mitis and streptococcus pneumoniae generally found in the particle size ranging from 0.55 to 7.9 μm, apart from meningitis have been found to cause acute otitis, pneumonia as well as bacterial sinusitis (balsalobre et al., 2006) . among other airborne bacteria haemophilus parainfluenzae of particle size range of 1-1.6 μm have been linked to acute bacterial meningitis to young children and infants as well as effects adults by causing chronic pulmonary disease (foweraker et al., 1993) . sphingomonas species falls in higher size range of n7.9 μm which is an opportunistic human and plant pathogens and induces various nosocomial infections (ammendolia et al., 2004) . various microbial compounds such as endotoxins, mycotoxins, microbial volatile compounds (mvcos), have also been found to induce several diseases as well. among all the three, standard for the exposure concentration of 50 eu/m 3 for endotoxin was published by dutch expert committee on occupational standards (decos) in 1998 (decos, 1998) which was re-evaluated to 90 eu/m 3 in 2010 on the basis of respiratory effects such as inflammation of airway (samadi et al., 2013) . apart from decos, in netherlands for general population an exposure limit of 30 eu/m 3 was also suggested by health council of the netherlands (health-council-of-the-netherlands, 2012). study carried out in several buildings of mid-western usa correlated pulmonary and respiratory problems with endotoxin levels in the indoor, some reports being as low as 1 eu/m 3 (reynolds et al., 2001) . mycotoxins, as the name suggests are secondary metabolites of fungi (fungal specific) that are highly toxic to both animal and human health. among different mycotoxins aflatoxin b1 released from aspergillus sp., (bennet and klich, 2003) have been found to cause liver cancer, hepatitis (ross et al., 1992) ; deoxynivalenol released from fusarium graminearum (bennet and klich, 2003) have been found to cause vomiting and nausea (rotter et al., 1996) while fumonisin b1 released from fusarium nygamai (bennet and klich, 2003) has the ability of probable esophageal cancer in humans (bucci et al., 1996) . in comparison to mycotoxins, mvocs have always received less attention although some studies revealed mvocs associated with "sick building syndrome" (molhave, 2009 ) as well as headache, lethargy, sore throat, nasal congestion, cough and wheezing (araki et al., 2010) . among different mvocs, cytotoxicity study of 1-octen-3-ol when exposed to human volunteers for 2 h have reported minor irritation of nose, eye and throat (walinder et al., 2008) . interestingly, bioaerosols is also used as biological weapons. the deliberate release of pathogenic bioaerosols has become an act of terrorism or warfare that has become a troubling possibility and a frightening reality. in 2001, bacillus anthracis spores were mailed in envelopes in u.s. around the country and its outbreak resulted in killing 5 people, sickening 17 others, and contaminating several senates, post, and media offices (klietmann and ruoff, 2001) . smallpox (variola virus) is considered to have the greatest bioweapon potential (henderson, 1999) . other potential bioweapons capable of getting released into the air include francisella tularensis, yersinia pestis, brucella spp., variola virus, and coxiella burnetii (atlas, 2002) . bioweapons are predicted to be the weapons of mass destruction (wmd) of the future due to many reasons for example: they are inexpensive to use, provide high probability of delivering considerable devastation and large scale panic (henderson, 1999) . in order to prevent or reduce adverse health effects of bioaerosols along with detection immediate controlling mechanism is also essential which includes inactivation, removal or collection at specific locations. in recent past many methods have been developed in order to control bioaerosol each of which has advantages as well as weakness regarding their economic requirements and environmental impacts. thermal energy has been used to control bioaerosol for a larger period of time in two forms such as moist heat (using steam under pressure) and dry heat (high temperature without moisture). the potential applicability of thermal energy has been studied by several researchers (jung et al., 2009; grinshpun et al., 2010) . bioaerosol treatment by thermal energy released from electrical heating coils is highly advantageous due to easy installation in buildings as well as low production of byproducts. research has shown that exposure to temperature of 100-140°c for sub seconds can inactivate airborne bacteria (lee and lee, 2006) decrease the size of fungal bioaerosol their concentration as well as reduce the amount of (1 → 3)-β-d-glucan (a key agent in bioaerosol-induced inflammatory responses) (jung et al., 2009) . denaturation of proteins followed by damage of microorganism is also seen when exposed to very high temperature (madigan and martinko, 2006) . although thermal energy was used to control bioaerosol by louis pasteur around 150 years ago and its use continued thereafter for a longer period of time yet currently due to the need of energy conservation its use has been restricted (lee, 2011) . as bioaerosols exhibit similar physical behavior as that of nonbiological aerosols, air ion emission technique can be definitely used to transfer bioaerosols from air to walls, ceiling and floor as proven by research revealing that when air ions of density 10 5 -10 6 e ± cm -3 are emitted for 30 min removes 97% and 95% of 0.1 μm and 1 μm particles respectively in indoor air along with the natural effect of decreasing aerosol concentration due to gravity and diffusion (lee et al., 2004a; lee et al., 2004b) . the biocidal effect of air ions on bacterial and fungal species has been proved by several scientists kerr et al., 2006) with few suggesting that in addition to ozone exposure electro poration mechanism played a primary role (fletcher et al., 2007; kim et al., 2011) . however most the studies revealed effect of air ions on static microorganisms rather than airborne microorganisms. hence more experimental work is needed to find out the effect of air ions on bioaerosol as well as to treat the side effect of bioaerosols being deposited on ceiling and walls where they grow and re-emit additional bioaerosols in the air. apart from thermal energy the other most commonly used method for controlling bioaerosols in indoor environment is ultraviolet (uv) irradiation. the germicidal effect of uv was found to be dependent on irradiation dosage, moisture content of the air and the movement pattern of the air along with the size of the room (kujundzic et al., 2006; beggs et al., 2006) . ultraviolet germicidal irradiation (uvgi) disinfects air by using ultraviolet light at sufficiently short wavelength between 220 and 300 nm destroying the nucleic acid of the organism leaving them unable to perform any vital cellular functions, eventually killing the microorganism (madigan and martinko, 2006) . several studies related to the dosage response of uvgi have been done revealing that high doses are required to inactivate fungal bioaerosol than vegetative bacterial bioaerosols (lee, 2011) . as for instance uvgi dosage of 1.6 × 10 4 μw s/cm 2 was found to be required for 2 log decrease in the concentration of fungal bioaerosol (kujundzic et al., 2007) while 12 fold reduction in bacterial bioaerosol were seen at 290 μw sec/cm 2 uvgi exposure (lidwell, 1994) . in comparison to thermal treatment uv irradiation technique utilizes very less energy along with simplified installation technique of uv lamps. hence due its user friendliness uv lamps are usually installed and used to inactivate bioaerosols in indoor environments. generally though common filters are useful in removing aerosols from indoor environments yet in case of bioaerosols they act as breeding ground where once trapped they grow by absorbing air moisture and nutrients in the dust and on instances of reverse airflow they get introduced back into the air. hence several researchers have developed filters with anti-microbial components such as iodine and other membrane breaking enzymes (lee et al., 2008a; eninger et al., 2008) . such anti-microbial filters are however useful for only short period of time because of its ineffectiveness caused by the accumulated dust particles over them. hence by combining different bioaerosol control methods, hybrid methods are developed by scientists, as for example deposition of silver nanoparticles over filters rendered 99% inactivation of bacterial bioaerosol (lee et al., 2008b) while under low relative humidity condition the death rate tolled up when exposed to high number of silver nanoparticles . apart from use of silver nanoparticles over filters, integration of thermal energy and uv irradiation in a single method had also proven to enhance the inactivation and control of bioaerosols compensating the respective weakness of the constituting methods (hwang et al., 2010) . apart from the already mentioned controlling mechanisms renovation and periodic mechanical cleaning operations have also shown to reduce both bacterial and fungal aerosols by approximately 80% and 50% respectively (berent et al., 2011) . along with these maintenance activities increasing the ventilation rate (dilution ventilation) by various mechanical or natural systems are few individual levels of efforts that can play an important role in improving the indoor air quality (cox and wathes, 1995) . bioaerososl is present in most of the enclosed environments due to its ubiquitous nature (jones and harrison, 2004) . in general when we breathe we inhale 0.5 l of air, thereby taking in almost 10 6 microbial cells per day (mandal and brandl, 2011) . moreover as potential health effects of bioaerosol are highly diverse including acute toxic effects, allergies, infections and cancer, assessment of bioaerosol is highly essential. without detailed information about sampling and enumeration technique interpretation of exposure level is very difficult. hence both advantages and disadvantages of all the methods should be known before deciding upon the suitable one for use. high bacterial count as well as presence of several allergenic fungal genuses in indoor environment represents a highly allergic environment. hence apart from assessment, suitable steps are also necessary for controlling the airborne microbes. although several control techniques have evolved as has been already mentioned controlling mechanisms such as periodic cleaning operations, maintenance activities as well as increasing the ventilation rate by various mechanical or natural systems are few individual efforts that can eventually improve the indoor air quality. although several studies in relation to health effects of bioaerosol have been conducted and have also been reported in this review yet none of the studies have been found providing suitable dose-response relationship that could eventually describe the exposure limits of bioaerosol that could be internationally accepted and followed mainly due to lack of valid dose-response data set, employment of diverse measuring methodologies for bioaerosol, insufficient real time quantification and identification of airborne microbes as well as due to the heterogeneous range of the health effects. thus studies are needed to be carried out to provide proper exposure limits in relation to health in various indoor environments for which experimental studies upon animals could be conducted as has been used in several toxicological studies for other hazardous substances. moreover, as seen in this review different detection methods 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