Our study was conducted using a 2005 Wheeled Coach Type III ambulance which met Federal Specification KKK-A-1822D when constructed.⁽²³⁾ For the purposes of evaluating surface disinfection, this specification is very similar to the construction standards for ambulances that are maintained by the National Fire Protection Association⁽²⁴⁾ and ASTM International.⁽²⁵⁾ A schematic of the ambulance patient compartment is shown in Figure 1. The patient cot was removed from the ambulance for all experiments.

The ultraviolet germicidal light fixture used in these experiments was custom-built. It consisted of ten UV-C lamps with a primary wavelength of 254 nm (TUV PL-L 60 W/4P HO 1CT/25, Philips Lighting). Each lamp had a nominal wattage of 60 watts and a UV wattage of 12.4 watts. The lamps were mounted vertically in two circles (one upper, one lower) around an 11.1 cm (4 3/8”) diameter aluminum-covered post. The lamps were powered by five electronic ballasts (PureVOLT IUV-2S60-M4-LD, Philips Lighting). Photographs and a diagram of the fixture are included in the on-line supplemental material.

The light fixture was placed in three positions in the patient compartment for testing: Front, with the light 81 cm (32“) from the left interior wall and 217 cm (85 1/2”) forward of the rear doors of ambulance; Middle, with the light centered laterally between the interior walls and 132 cm (52”) from the rear doors; and Back, with the light position centered laterally between the interior walls and 48 cm (19") from the rear doors. The light fixture positions are shown in the ambulance patient compartment schematic in the on-line supplemental information.

Irradiance measurements were made at 49 locations throughout the patient compartment as shown in Figure 1. A descriptive list of the locations is given in Table 1; photographs and an interactive schematic showing the sensor locations are included in the on-line supplemental material. The sensor locations were chosen to emphasize two areas: 1) surfaces that are frequently touched such as controls, handles, latches and hand rails, and 2) surfaces that were not directly exposed to light from the UVGI fixture, such as between the primary care seat and the wall, and in the passageway leading to the front cab.

The irradiance measurements were made using 17 UV-C sensors (SED033–25, International Light Technologies) routed through three automated switch boxes (A803, International Light Technologies) connected to three radiometers (ILT-1700, International Light Technologies). A scan delay of 3 seconds was used for the switch boxes to prevent measurements from one sensor from being carried over to the next sensor reading.

To perform irradiance measurements, the sensors were placed at 17 locations. The UVGI light fixture was turned on and allowed to stabilize for 30 minutes. Separate irradiance measurements were collected with the fixture in the back, middle and front positions. The fixture was then turned off and 16 of the sensors were moved to different locations; one sensor was left in the same location (B2) for all tests to allow comparison of irradiance levels from different tests. The measurements were collected using the three light fixture positions. The fixture was turned off, 16 sensors were moved to the final set of locations and the measurements were repeated. The entire set of measurements was repeated three times with the sensors rotated among the locations, so that measurements were collected three times with different sensors at each location for each UVGI fixture position (153 data points).

To prevent exposure of personnel to UV-C, the exterior of the patient compartment windows were covered with aluminum foil, warning signage was placed on the ambulance doors and access to the ambulance was restricted during experiments. One person was allowed to enter the ambulance to move the UVGI fixture while it was on; this individual was completely covered in protective clothing and wore a UV-C absorbing face shield.

To examine the effects of surface reflectivity, three different ambulance interior surface conditions were tested. The first condition (called “Original”) consisted of the ambulance interior surfaces as originally supplied by the ambulance manufacturer. The side panels and ceiling were primarily white melamine with aluminum trim and vinyl-covered padding on the edges. The cabinet sliding doors were clear plastic, the access doors were largely covered in diamond-plate aluminum, and the floor was a dark grey non-skid plastic material. Seat cushions were covered with blue vinyl.

For the second interior surface condition (called “Reflective”), many of the vertical white melamine surfaces were covered in diamond-plate aluminum sheets to increase the surface reflectivity for UV light within the patient compartment and thereby increase the irradiation of shadowed areas. Diamond-plate aluminum sheets also were added to the floor beneath the rearfacing primary seat and the patient cot release handle. The positions of the added reflective material are shown in the on-line supplemental material.

For the interior surface condition (called “UV paint”), the added aluminum sheets on the walls were removed, while the original aluminum surfaces and the aluminum added beneath the rearfacing primary seat and the patient cot release handle were left in place. All of the white melamine interior surfaces, including the walls, ceiling and counters, were then painted with UV-C reflecting paint (UVC-MAX, product # W15984, Lumacept, Inc.). The floor, the original aluminum sheets and trim, and the seats and padding were not painted and remained in their original state.

The antimicrobial capabilities of the UVGI system were tested using Bacillus subtilis spores as a surrogate for pathogenic microorganisms. B. subtilis spores are recommended for use in the testing of sterilizing agents by the US Environmental Protection Agency (EPA) and the European Committee for Standardization (CEN).^{(26, 27)} B. subtilis spores also tolerate drying, washing and other experimental stresses well, which helps provide consistent experimental results. Since it is not a human pathogen, B. subtilis can be handled using Biosafety Level 1 precautions.

The EPA defines a disinfectant as “a substance, or mixture of substances that destroys or irreversibly inactivates bacteria, fungi and viruses, but not necessarily bacterial spores, in the inanimate environment.⁽²⁸⁾ Published results for a variety of pathogenic vegetative bacteria and viruses indicate that a UVGI dose sufficient to inactivate 99.9% of B. subtilis spores on a solid surface would also inactivate at least 99.999% of many pathogens, including Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enteritidis, Serratia marcescens, Mycobacterium tuberculosis and human cytomegalovirus (although it should be noted that fungi are reported to be more resistant to UVGI).⁽¹⁵⁾ Thus, for our experiments, we defined the surface disinfection dose as the UV-C dose sufficient to inactivate 99.9% of B. subtilis spores.

Bacillus subtilis subsp. spizizenii were purchased (Catalog #6633, American Type Culture Collection) and spores were produced following the procedure described in European Standard EN 14347:2005.⁽²⁷⁾ A detailed protocol for spore production is provided in the on-line supplemental material.

To determine the dose-response relationship between UV-C and B. subtilis spore inactivation, spores were exposed to UV-C using a system described previously.⁽²⁹⁾ The system was modified by adding a shutter that covered the UV-C lamp until its output stabilized. For exposure tests, 100 μl droplets of water containing 3--4 × 10⁷ spores were placed on 2.5 cm x 1.9 cm coupons made of type 316 stainless steel and allowed to dry at room temperature. The coupons were then exposed in sets of 3 to UV-C doses of 8, 16, 32, 64 or 128 mJ/cm² The experiment was repeated 3 times for a total of 9 coupons exposed to each UVGI dose.

After exposure, each coupon was placed in a 6-well culture plate. Five ml of sterile 0.1% Tween 20 solution (Fisher Scientific) was placed in each well and the plates were shaken for 15 minutes using a titer-plate shaker (Model 4625, Thermo Scientific) at intensity level 5. Twenty μl of each control sample and 100 μl of each UV-exposed sample were then plated onto tryptic soy agar plates (236950, Becton Dickinson) in an exponential spiral using an automated plater (Autoplate, Spiral Biotech). The plates were incubated overnight at 30 °C and colony-forming units were enumerated using an automated plate counter (Qcount Model 510, Spiral Biotech).

The UVGI irradiance (intensity) and spore inactivation data were fitted to an exponential decay model of the form ^(15,30) S=e−kIt where

S = fraction of spores surviving after UV-C exposure

k = rate constant (cm²/mJ)

I = UV-C irradiance (mW/cm²)

t = exposure time (seconds)

The UVGI dose in mJ/cm² was determined by integrating the irradiance over the exposure time. The UVGI dose required to disinfect a surface was calculated based upon the k-value found in the laboratory exposure experiments. This surface disinfection dose was divided by the irradiance measurements in the ambulance to calculate the exposure time required to disinfect each location in the compartment (the disinfection time) with the different fixture positions and surface reflectivities. The disinfection dose was used to convert irradiance levels to disinfection times when presenting the results because the disinfection time is easier for the reader to interpret. Irradiance levels for all experiments are provided in the on-line supplemental materials.

To test the ability of the UVGI system to inactivate microorganisms in the ambulance, spore-loaded coupons were fastened next to the inlet domes of the ultraviolet light sensors. Ten coupons were placed in the locations in the ambulance with the lowest irradiance levels for each interior surface type. Two control coupons were not exposed to UVGI but were otherwise treated in the same manner. The test was performed 6 times with the original interior surfaces, 3 times with the reflective interior surfaces, and 3 times with the UV-reflective paint on the interior surfaces. For all of these experiments, the UVGI fixture was placed in the back position for first half of the exposure time and in the front position for the second half.

The dose-response curve for Bacillus subtilis spores dried onto stainless steel coupons resulted in a rate constant (k) of 0.131 cm²/mJ for the best-fit exponential decay curve with a goodness-of-fit of R² = 0.930. The average temperature in the exposure box during these experiments was 22.2 °C (SD 0.3) and the average relative humidity was 57.0% (SD 2.5%). Based on these results, a UV-C dose of 52.6 mJ/cm² was required to inactivate 99.9% of the spores on a coupon (0.1% survival). This disinfection dose was used with the UV-C irradiance levels measured with the radiometers to calculate the disinfection times in the results presented below.

The shortest, median and longest exposure times required for disinfection among the different locations in the ambulance are shown in Table 2 for each UVGI fixture position and interior surface type. Locations that were further away from the UVGI fixture or in shadowed areas received much lower UV-C doses than did locations closer to the UV fixture and directly in the line-of-sight. For this reason, the longest disinfection times were 220 to 3400 times larger than the shortest times (Table 2). As a practical matter, the longest disinfection time determines the estimated overall time required to disinfect all locations in the interior of the ambulance. The overall disinfection time ranged from 16.5 hours for the poorest performing configuration to 59 minutes for the best.

Figure 2 shows the disinfection times at each of the 49 test locations in the ambulance with the UVGI light in the middle position and with the original interior surfaces. In this experiment, the disinfection times ranged from 20 seconds at location G6 (on the left wall directly facing the UVGI fixture) to 216 minutes at location B6 (between the rear-facing primary patient care seat and the left wall, facing the wall), indicating that location G6 receives 652 times as much UV-C light as does location B6 with this configuration.

The disinfection times when the UVGI fixture was in different positions in the ambulance and with the original interior surfaces are shown in Figure 3. The locations presented in the figure include the 5 worst locations (that is, the locations with the longest disinfection times) for each light position. Note that, as the light is moved to different positions, the disinfection times for some locations improve while others become worse. For example, when the light was moved from the front to the back, the disinfection time for location B12 (vertical hand rail on left rear door) decreased from 72 minutes to 7 minutes, while for location W1 (bottom step at side door) the disinfection time increased from 32 minutes to 398 minutes. For the original interior surface condition, the worst overall disinfection time of 990 minutes occurred when the UVGI fixture was in the front position in the ambulance, and the best overall disinfection time of 216 minutes occurred when the fixture was in the middle position. The results for all interior surface conditions are summarized in Table 2.

The effects of adding aluminum sheets or UV-reflective paint to the interior surfaces are shown in Figure 4. The results presented in this figure were calculated assuming that the UVGI fixture was placed in the front, middle and back positions for one-third of the exposure time each. With the original interior surfaces, the time needed to disinfect all locations was 234 minutes. The overall disinfection time was reduced to 79 minutes by adding the reflective aluminum surfaces, and to 59 minutes by painting the interior with the UV-reflective paint.

The survival fraction vs. UVGI dose for Bacillus subtilis spores on coupons placed in the ambulance is shown in Figure 5, along with the predicted survival curve from the laboratory experiments. The correlation coefficient between the laboratory model and the ambulance results was 0.804. After 20 minutes, the survival fraction of the spores in the ambulance ranged from 0.1% to 17.9% with the original interior surfaces and from below detection to 1.9% with the UV-reflective paint. The average temperature during these experiments was 21.1 °C (SD 4.6) and the average relative humidity was 43.9% (SD 8.4%).

UVGI irradiation levels varied greatly from place to place within the ambulance compartment. As seen in Figure 2, this caused the disinfection times to vary considerably with surface location as well. Since the purpose of the UVGI system is to disinfect as much of the patient compartment as is practically possible, the time required for overall disinfection is driven by the locations with the lowest irradiance levels, not by the average irradiance. For this reason, interventions that increase the irradiance of the worst locations will reduce the overall disinfection times for the ambulance compartment and allow for a more rapid turnaround of the ambulance.

For each combination of UVGI fixture position and surface reflectivity, the median disinfection time among all locations was much less than the longest time (Table 2)., indicating that the distribution of the disinfection times among the various locations was skewed. This can be seen in Figure 2; half of the locations had disinfection times of 7 minutes or less, and 90% had disinfection times of 69 minutes or less, but the exposure time needed to disinfect the worst location was 216 minutes. These results show that, when testing a UVGI system, UVGI measurements must be made at multiple locations. In addition, a particular effort should be made to find the locations with the lowest irradiance levels, since these locations will determine the exposure time needed to disinfect the entire compartment. On the other hand, this also suggests that the overall disinfection time can be lowered substantially by increasing the irradiance of a small number of areas that are most shadowed from the UVGI fixture, or by determining that some areas either do not require disinfection or that those areas will need to be disinfected by other means.

The position of the UVGI fixture had a substantial effect on the overall disinfection time for the compartment. With the original interior surfaces, moving the fixture from the front to the middle position reduced the disinfection time from 16.5 hours to 3.6 hours. The ratio of the longest time to the median time was also reduced from 148 to 31, suggesting that the light was more evenly distributed. Similar results were seen with the reflective material and the UV-reflective paint. Thus, determining the optimal location for the UVGI fixture is important when designing a UVGI system for an ambulance.

An additional step that can be taken to better distribute the UV light within the compartment is to move the UVGI fixture during disinfection. We examined two possibilities: placing the fixture in the front position for half of the disinfection time and the back position for the other half; and placing the UVGI fixture in the front, middle and back position for one-third of the disinfection time each. With original interior surfaces, moving the fixture did not improve the disinfection time compared to leaving the fixture in the middle position. However, with the reflective aluminum, the disinfection time was reduced from 156 minutes with the light in the front position to 81 minutes by splitting the exposure time between the front and back positions. With the UV-reflective paint, splitting the exposure time between the front and the back reduced the disinfection time from 79 minutes to 67 minutes compared to leaving the fixture in the middle, and splitting the time between the front, middle and back reduced the time to 59 minutes. Further reductions in the disinfection time could be achieved by using a more powerful UVGI fixture or using multiple fixtures for disinfection; using two fixtures, for example, would potentially cut the overall disinfection time in half.

One way to enhance the efficacy of UVGI fixtures is to increase the UV reflectivity of surfaces in the compartment so that more UV irradiation reaches locations that are not directly exposed. Most common materials reflect only a small amount of the UV-C. For example, ordinary white water-based paint is reported to reflect about 10--23% of UV-C light, while stainless steel reflects about 28% and glass only reflects about 4%.⁽¹⁵⁾ By comparison, bare aluminum has a surface reflectivity of about 74--84% depending upon the finish, and the UV-C reflective paint that we used has a reflectivity of about 65%. Increasing the UV surface reflectivity with reflective aluminum sheeting or UV-C reflective paint both substantially reduced the disinfection times. As seen in Table 2, the best overall disinfection time with the original surfaces was 216 minutes. This was reduced to 79 minutes with the reflective aluminum and 59 minutes with the UV-reflective paint.

The UV-reflective paint reduced the disinfection times for all individual locations with all fixture positions, and gave the lowest overall disinfection times. The use of reflective surfaces was not quite as effective, probably because more surfaces were covered with the UV-reflective paint than with the aluminum sheeting, most notably the ceiling. The UV-reflective paint also provides a diffuse reflection, which may help distribute the UV light better, while the reflection from the aluminum sheets is specular (mirror-like). More surfaces could have been covered with the aluminum sheets, but the aluminum sheets have the disadvantage of creating visible light reflections and glare in the compartment. This might be reduced by using aluminum with a brushed or matte finish, but probably can’t be eliminated entirely. On the other hand, the UV-reflective paint that we used is intended for hospital rooms and will not tolerate the repeated scrubbings that need to be performed on some surfaces in the ambulance compartment. Thus, a combination of aluminum sheeting and UV-reflective paint might provide the best method to optimize the performance of a UVGI system.

Our study has several limitations. First, and most important, it is not feasible to perform UV irradiance measurements at every possible location in the ambulance. We attempted to find and measure the locations that received the lowest amounts of UV irradiation, but it is entirely possible that some unmeasured places received less. It is also important to note that covered locations, such as underneath seat cushions or behind cabinet doors, receive no UV exposure at all.

Second, the disinfection time in our paper is based on the use of Bacillus subtilis spores as a surrogate for pathogenic microorganisms. Although bacterial spores are generally considered to be hardier when exposed to UV light than vegetative bacteria or viruses,⁽¹⁵⁾ data on the susceptibility of surface pathogens to UVGI are limited, and some pathogens may survive higher doses of UV-C light than Bacillus subtilis spores. Fungi also are reported to have a much higher tolerance for UV exposure than bacteria.⁽¹⁵⁾

Third, the aluminum sheets and UV-reflective paint were freshly applied before our experiments. In a real-world application, these materials would likely lose some reflectivity over time due to dirt, corrosion, abrasion, and other factors.

Fourth, we did not examine the effects of soiling such as by blood droplets or fecal matter on the efficacy of the UVGI system. Such opaque materials are known to block UV-C, and thus it is important that the ambulance be cleaned of any visible contamination before using UVGI as a final step in the disinfection process. Similarly, materials such as patient cots, sheets, papers, clipboards, clothing and other items should be removed from the patient compartment before beginning a UV disinfection cycle, since they will also shield surfaces from UV light.

Finally, although our general conclusions should be widely applicable, the quantitative results in our study are specific to the ambulance compartment that we tested. The irradiance levels, disinfection times, worst locations and other results that we found could be substantially different in ambulances with different UVGI fixtures, cabinet and seat arrangements, surface materials, etc. Thus, any ambulance UVGI system needs to be validated with the actual ambulance configuration for which it is to be used.