This is a cross-sectional descriptive study, in which a convenience sampling was carried out including emergency ambulances from the city of Pereira (Risaralda Department), belonging to three different EPSPH. Two ambulances were randomly selected by each EPSPH, for a total of six ambulances analyzed in the study. A qualitative component was implemented by carrying out interviews with the personnel in charge of CPDs of each emergency ambulance, including questions being assessed, such as: types of products implemented, frequency of implementation of CDP, among others.

Three sampling sites were selected in each ambulance, recognized as noncritical points according to the risk stratification (2), that is: rear door handle, stretcher and wall adjacent to the patient. At each point, samples were collected at two moments: i) during the initial contact with each ambulance, after their transportation shift, and ii) after the usual cleaning and disinfection procedure ‘CDP’ carried out by the personnel in charge of each ambulance. Sampling moments were named as pre-CPD and post-CDP, respectively. (Figure 1).

Figure 1

Samples for each sampling point and at each assessment time were collected with a sterile swab and transported through Stuart media (Oxoid, CM 0111). Subsequently, each person in charge applied the CDP usually implemented in each ambulance. After a waiting period (15-20 min after completing the CDP), the second sample was collected in each of the three sampling points previously described. Samples were processed in the Microbiology Laboratory from Universidad Libre (Pereira branch).

The three samples pre-CDP and three post-CDP taken in the transportation services were carried to the laboratory and cultured through a streaking technique using a bacteriological loop in Blood Agar (BA; used as enrichment medium), Red Phenol Mannitol Saline Agar (RPMSA; for Gram-positive bacteria) and MacConkey Agar (MCK; for Gram-negative bacteria). In addition, the samples taken with saline solution were cultured in Tryptic Soy Agar (TSA) and cultured afterwards with Drigralskly loop massively on surface. All the cultured media were incubated for 48 hours at 37º C.

After the count of colonies forming units (CFU) was performed, a macroscopic description of the colony’s morphology was carried out in all the media implemented. Representative colonies from different morphologies were recovered, in a saline solution drop on slides. The microscopic morphology was described by routine inspection, after the implementation of Gram stain.

Taking the data obtained in the bacterial growth results before and after the disinfection process, presence or absence (percentages)/reduction, increase or evenness (percentages) of microbial load and site of higher microbial load, with their respective confidence intervals at a 95% reliability (95% CI).

This study was approved by the research center of the Faculty of Health Science of the Universidad Libre. This research did not use animal nor human material or data.

Interviews with the personnel responsible for carrying out CDPs allowed identifying that EPSPH in the city of Pereira use between 2 and 5 different chemical products to carry out their routine CDP. The frequency of CDP implementation was very similar among EPSPH, being predominant the completion of a thorough ambulance cleaning once a week, a general cleaning a day at the shift end and a cleanup in case of fluid spills showing biological risk, the latter limited to the affected area. Although the objective in which CDPs are carried out are common to all EPSPH, the CDP implementation strategy varied according to the number of chemical products used. All the companies claimed to have disposable materials, certified suppliers, guaranteed by the national vigilance entity (Instituto de Vigilancia de medicamentos y alimentos - INVIMA-), as well as biosecurity elements for the operating team. CDP information for each EPSPH is described in Table 1.

Table 1

Question	Providing company of the emergency ambulance service
	1	2	3
Which type of chemical products use for carrying out CDP procedures?	-Bactidin: Surfaces	-Benzaldine: Surfaces and biological fluids	-Deterganium: Surfaces
	-Benzidine: Equipment	-Deteridin: General cleaning	-Sulfanium: Surfaces and fluids inactivation
		-Enzidin: Metallic utensils	-Endozime: Instruments
		-Bactidin: Equipment	-Hydrogen peroxide: Fluids inactivation
			- Ambu: Plastic utensils
How frequently do you use CDP?	General cleaning per week Simple daily cleaning, at the end of the shift	General cleaning per week Cleaning at the night when the shift change.	A thorough cleaning at the end of each shift during the night
	When fluids are spilled the affected area is cleaned	A quick cleaning is done when fluids are spilled	A quick cleaning in the morning
			Between transfers, only if there is a spill of some type of fluid
What is the type of CDP that you carry out?	- Apply bactidin on all surfaces, and benzidine to clean equipment	Initially apply deterdine on all surfaces, followed by	-Apply deterganium on all surfaces, leaving it to act for 5 minutes, then apply sulfanium and let dry. Sulfanium and hydrogen peroxide are used in case of a spill, such as vomiting or any biological fluid. Endozime is used to clean the equipment, at a concentration of 15 mL in 4 liters of water. For plastic material, ambu is used. If mud has entered the ambulance, it is washed with plenty of water. Operators use topical disinfectant gel
		benzaldine, which is left on for about 5 minutes and removed with sterile towels
How do you carry out the CDP?	All companies carry out the process of disinfection from the inside out and top from down in a zigzag direction

A total of 36 samples were analyzed in this study between pre- CDP and post-CDP from six ambulances (Figure 1). Results showed that 77.8% of the total samples collected presented microorganisms (n= 28; 95% CI: 60.8-89.8).

The macroscopic characteristics assessment on blood agar revealed two types of colonies; the first group corresponded to small, punctiform colonies with a beige-yellow coloration. The second group of colonies identified showed greater size, dry appearance and irregular edges. Colonies showed beta-hemolysis in some cases.

The growth of colonies on MCK agar showed whitish colonies with large size and variable texture (some slimy and others dry), some of them lactose positive colonies, which could initially indicate the presence of enterobacteria species. When samples were grown on RPMSA agar colonies with punctate and mucous morphologies appeared, some of them mannitol positive which turns out to be presumptive for Staphylococcus aureus.

At the microscopic level, three groups of microorganisms were isolated corresponding to: Gram-positive G(+) cocci (n: 13, 36.1%), G (+) bacilli (n: 11, 30.6%) and Gram negative G(-) bacilli (n: 8, 22.2%). Additionally, a group of samples from which microorganisms with ‘mixed’ morphology were recovered (n: 4, 11.1%) were identified.

The percentage of positive samples was assessed according to the collection moment of the samples, finding that 94.4% (n: 17) of the samples collected pre-CDP were positive, while 61.1% (n: 11) were positive post-CDP. Pre-CDP the group of microorganisms most frequently found were G(+) bacilli (44.4%, n: 8), however, this group was the one, which showed the greatest reduction after CDP implementation (27.8%). G(+) cocci showed a smaller reduction after CDP implementation (post-CDP). The positivity frequency per group of microorganisms according to the collection time of samples is described in Figure 2a.

Figure 2

A total of 12 samples were processed for each risk zone in the analyzed ambulances (pre-CDP and post-CDP, six ambulances). The area with the highest detection frequency was in the ambulance stretcher with 91.7% (n= 11, 95% CI: 61.5-99.7), followed by the ambulance door with 75.0% (n= 9; 95% CI: 42.8-94.5) and finally, the ambulance wall with 66.7% (n= 8; 95% CI: 34.8-90.0) of isolated microorganisms. The frequency of microorganisms’ isolation in each risk zone is shown in Figure 2b.

The analysis according to the risk zones assessed showed that all the ambulance areas presented bacterial growth pre-CDP, being G(+) cocci frequently detected in the door (83.3%), followed by G(+) bacilli in the wall (66.7%). The detection frequency of pre-CDP microorganisms in the different risk areas sampled is described in Figure 2c. Although the frequency of all microorganisms was reduced post-CDP, an elimination of G(-) bacilli and mixed detections in the wall was observed; for the other groups of microorganisms and sampling areas, growth was detected after the usual CDP application (Figure 2d).

Two categories were defined to identify microbial diversity. Being ‘single’ the microorganisms detection with unique microscopic morphology, while ‘mixed’ was defined for microorganisms recovery that show different morphologies from a sample, according to the previously established criterion.

Although most of the samples revealed growth with single morphology, the risk zone with the highest number of isolated microorganisms with mixed morphology was the wall (16.7%; 95% CI: 9.9-29.1) (Figure 3a). A decrease in the number of isolates was found with CDP implementation (94.4% vs. 61.1%), with the door being the ambulance area with the greatest decrease (100% vs. 50.0%) (Table 2). Although these findings reveal some efficiency of the CDPs usually implemented, it is evident that the reduction of the detection frequencies of post-CDP microorganisms was limited. Total elimination was presented in the case of mixed morphologies in the stretcher and door (Figure 3b).

Table 2

Ambulance risk zone				Stretcher (n=6)		Wall (n=6)		Door (n=6)
		n	%	n	%	n	%	n	%
Pre-CPD (n=18)	Bacterial detection	17	94.4	6	100.0	5	83.3	6	100.0
	Sample with unique morphology	14	77.8	5	83.3	4	66.7	5	83.3
	Sample with mixed morphology	3	16.7	1	16.7	1	16.7	1	16.7
Post-CPD (n=18)	Bacterial detection	11	61.1	5	83.3	3	50.0	3	50.0
	Sample with unique morphology	10	55.6	5	83.3	2	33.3	3	50.0
	Sample with mixed morphology	1	5.6	0	0.0	1	16.7	0	0.0

Figure 3

For surface cleaning, each EPSPH used a specific disinfectant substance, Disinfectant 1 (Bactidin), Disinfectant 2 (Benzaldin) and Disinfectant 3 (Deterganium). In this context, the decrease in microorganisms observed post-CDP was assessed. On a general level, the results showed a relative decrease in bacteria percentage between 50.0% and 66.7% (Figure 4a). For disinfectant 1, a decrease was observed for all microscopic morphologies observed (Figure 4b), for disinfectant 2 and 3 the decrease was observed for Gram-positive bacilli (Figure 4c and 4d, respectively).

Figure 4