Gradual decrease, by 3.4-fold, of dynamic compliance was detected after 48 hours for the comparison group, while in the main study group there were no such changes in the dynamic compliance (Table 3).

Table 3

Parameter	At the beginning of MV	After 24 hours of MV	After 36 hours of MV	After 48 hours of MV	After 72 hours of MV
Group of patients without using decamethoxin (comparison group), n = 15
C dyn (mL/cm H2O)	3.81 ± 0.2	1.12 ± 0.1*	1.38 ± 0.3*	2.23 ± 0.1*	4.01 ± 0.2
C dyn/kg (mL/cm H2O/kg)	1.06 ± 0.1	0.87 ± 0.2	0.96 ± 0.1	0.98 ± 0.1	1.04 ± 0.1
C20/C	3.26 ± 0.1	2.24 ± 0.8*	2.44 ± 0.7*	2.87 ± 1.3*	3.42 ± 0.9
Rpk (cm H2O/L/kg)	189.0 ± 9.1	401.0 ± 9.0*	280.0 ± 9.1*	202.0 ± 7.4	198.0 ± 8.0
PIP (cm H2O)	12.4 ± 1.8	14.6 ± 2.4*	21.2 ± 2.2*	18.6 ± 1.9	14.0 ± 2.0
PEEP(cm H2O)	3.0 ± 0.06	5.2 ± 0.04*	4.9 ± 0.08	5.0 ± 0.08*	2.4 ± 0.08
Group of patients using decamethoxin (main study group), n = 15
C dyn (mL/cm H2O)	4.02 ± 0.1	4.03 ± 0.2	4.06 ± 0.2	4.02 ± 0.3	4.02 ± 0.2
C dyn/kg (mL/cm H2O/kg)	1.14 ± 0.3	1.08 ± 0.4	1.09 ± 0.2	1.11 ± 0.4	1.12 ± 0.6
C20/C	3.68 ± 0.6	3.49 ± 0.8	3.62 ± 0.4	3.70 ± 0.8	3.68 ± 0.6
Rpk (cm H2O/L/kg)	208.0 ± 9.6	209.0 ± 10.0	208.7 ± 9.6	211.2 ± 8.6	206.8 ± 8.2
PIP (cm H2O)	11.9 ± 2.0	12.2 ± 1.8	12.0 ± 2.4	13.4 ± 2.3	12.0 ± 2.2
PEEP (cm H2O)	3.1 ± 0.04	2.2 ± 0.04	2.4 ± 0.04	2.6 ± 0.04	2.2 ± 0.08

Note:* p<0.05 when comparing patients in two groups

An increase of resistance (by 2.4-fold) and significant increase in PIP values (20-22 cm water column) were determined in patients of the comparison group. Maximum changes of the parameters on the graphic screen were also established in this group. These changes were accompanied by prolonged purulent-inflammatory process in the lungs, which was possibly caused by the lack of efficacy of antimicrobial therapy or as a result of the persistence of resistant microorganisms, in comparison with patients who received additional nebulization with DCM in the early VAP period (Table 3).

From the results of the microbiological evaluation of the 30 samples of biological material, which were received from VAP patients (n=30) at the early period of the disease, 49 strains of opportunistic microorganisms were isolated and identified by their morphological, tinctorial and biochemical properties (Figure 1).

Figure 1

In patients with VAP at the onset of the disease, the microbial spectrum of pathogens was predominantly represented by gram-positive (S. aureus — 28.6%, S. pyogenes — 10.2%), and gram-negative (P. aeruginosa — 16.3%; A. baumannii — 12.24%; K. pneumoniae – 10.2%) bacteria. Yeast-like fungi of the genus Candida were found in less quantity (10.2%). Opportunistic pathogenic microorganisms were isolated from the respiratory tract through the endotracheal tube in monoculture (20.4%) and in microbial associations (79.6%) before the initiation of antimicrobial therapy (Figure 2).

Figure 2

The clinical strains of opportunistic gram-positive microorganisms S. pyogenes, S. epidermidis, Kocuria spp., and gram-negative representatives of genus Enterobacter were isolated in association with other bacteria and yeast-like fungi of the genus Candida. In the vast majority of cases, Klebsiella and non-fermenting gram-negative bacteria (A. baumannii, P. aeruginosa) were isolated from patients as part of microbial associations. According to the results of the observation of patients with VAP, qualitative changes in the conditional pathogenic microflora of the respiratory tract during treatment were seen.

On the 3^rd day after treatment initiation in both groups, the predominance of microbial associations in the sputum of patients was noted (study group — 85.2%; comparison group — 87.1%), in comparison with isolated pathogens in monoculture (study group — 14.8%; comparison group — 12.9%). However, significant differences were found between the composition of stains in the two groups of patients. In patients who received additional inhalation therapy with Decasan for 3 days, S. aureus (29.63%) and P. Aeruginosa (22.22%) were found as leading isolates from the tracheobronchial tract. S. pyogenes and C. albicans were also isolated (11.11% in each case). The presence of A. Baumannii and K. pneumoniae (7.41% each) were rarely observed. In the comparison group, Pseudomonas aeruginosa prevailed (22.58%). Furthermore, S. aureus was determined only in 19.35% of cases. Also, Acinetobacteria and Klebsiella were isolated from patients in the comparison group (12.9%), which were somewhat more frequent than in the main group. Candida spp. were registered within the 3 days as part of the microbial associations in 9.68% of cases. The incidence of other types of gram-positive and gram-negative bacteria was negligible in both groups (3.23-3.7%) (Figure 3).

Figure 3

After the 7^th day of observation of the group of patients who received additional inhalation administration of DCM, it was noted that within the tracheobronchial tract were S. aureus (27.78%), A. baumannii (11.11%) and E. cloacae (5.56%), which were predominantly in monoculture (61.11%). These pathogens were isolated from 11 patients. Associations of gram-negative bacteria were determined (P. aeruginosa — 33.33%; K. pneumoniae — 5.56%; E. aerogenes — 5.56%), as well as C. albicans (11.11%) in the tracheobronchial aspirate of the 4 cases of the same group (Figure 3).

P. aeruginosa (a pathogen of airway infections) was found in monoculture only in 1 patient in the comparison group. The 7^th day microflora of the respiratory tract was represented mainly by associations of microorganism in 14 (93.33%) patients with VAP; this consisted of 89.29% of all isolated microorganisms in the comparison group.

On the 14^th day, in the sputum of 5 seriously ill patients, the main pathogens of infectious complications that were identified were the following: in 1 case, S. Aureus was found (its quantity did not exceed log 2 CFU/ml); A. baumannii was also found in 1 of the patients (who had a significant regression of the clinical disease); and in 3 patients, a small number of P. aeruginosa was found (log 3-6 CFU/mL).

Microbiological examinations of the biological materials received from the airway of patients demonstrated that at the beginning of treatment, the average values of the number of microorganisms were conditionally expressed in log; the results for the main group (6.73±0.32 CFU/mL) did not differ significantly from the level of microbial colonization in the respiratory tract of patients in the comparison group (5.86±0.28 CFU/mL; p>0.05). During treatment, there was a microbiologically determined gradual decrease in the quantitative content of microorganisms in the samples obtained from the patients in both groups. In the group of patients who received inhalation administration of DCM, a significant decrease in microbial colonization was observed, starting from the 3^rd day of treatment (log (5.49±0.27 CFU/mL) (Figure 4).

Figure 4

Microbial colonization of the respiratory tract of patients in the comparison group on the 3^rd day of treatment was 1.1 log higher, indicating 14-fold increase in the absolute amount of microorganisms in the sputum from the respiratory tract of the patients in the comparison group (p<0.001). A significant reduction of the microbial count in the tracheobronchial secretions, by 3 orders of magnitude (log (2.64±0.43) CFU/mL), were found in patients after decamethoxine use, as compared to the baseline microbial colonization (p<0.001), which in absolute amount corresponded to their decrease in CFU per 1 mL of 10⁴ times. The values of microorganisms in the investigated material did not exceed the critical-permissible level of microbial loading, which is in decimal logarithm consisting of 4 CFU/mL. The main group of VAP-causing pathogens were not determined in the sputum from most of the patients after 14 days of therapy (Figure 5).

In patients of the comparison group, i.e. who received only standard systemic antibiotic therapy, higher rates of microbial airway colonization were observed during the 1st week of treatment. On the 7^th day, the number of opportunistic pathogens that were determined in these patients with VAP reached log (6.49±0.35) CFU/mL, exceeding by 4 orders of magnitude the amount of microorganisms in the inflammation locus in patients of the observation group (p<0.001).

Figure 5

A significant decrease in the microbial count in the tracheobronchial secretion of patients in the comparison group was observed only after 10 days of systemic antibiotic therapy (p<0.001). The total microbial count was expressed in log in 1 ml of tracheobronchial secretion. After 14 days of treatment, there was about a 2-fold decrease in total microbial count, in comparison with the 7^th day data, and amounted to 3.44±0.23 CFU/mL, which exceeded this indicator in the study group by 13-fold.

A smaller increase in serum TLR-4 levels in the early period of treatment was observed in patients who received inhalations of decamethoxine. Indeed, TLR-4 level was noted to be 1.42 times lower in patients of the study group on the 3rd day after treatment, when compared to the comparison group (p<0.001; (Figure 6).

Figure 6

A significant increase (by 1.7 fold) of serum TLR-4 level (p<0.001) was detected together with an increase in colonization rates by gram-negative bacteria in the tracheobronchial aspirate on the 3^rd day (r-Pearson=0.880) in the comparison group.

The positive dynamics of the microbial count in the aspirates obtained from the patients in the main group were accompanied by the decrease in TLR-4 level after the 7^th day of treatment. A strong correlation between the reduction of gram-negative pathogens of respiratory infections and the decrease of TLR-4 levels, compared to baseline indices (r-Pearson=0.893), has been established as proof of the efficacy of the additional application of DCM in the complex treatment of patients with VAP. In comparison, TLR-4 serum levels in VAP patients receiving only systemic therapy remained two times higher (p<0.001) than that of the primary group, and correlated with an increased number of gram-negative microorganisms in the tracheobronchial aspirates of patients (r-Pearson=0.801).

From a microbiological point of view, it is well-known that VAP, which occurs after 96 hours of mechanical ventilation (late-onset VAP), is usually caused by antibiotic-resistant bacteria and requires rapid initiation of broad-spectrum antibacterial therapy. However, recent data suggests that in the vast majority of cases, the infection in patients with early VAP onset is caused by a nosocomial microflora. In this case, infectious complications are usually caused by bacteria, resistant to antimicrobial agents 10111226.

According to the results of our study, it was found that the spectrum of pathogens in patients with VAP, at the onset of the disease, is predominantly represented by gram-positive and gram-negative bacteria. As well, there is representation of a smaller number of yeast-like fungi of the genus Candida, S. aureus, S. pyogenes, A. baumannii, P. aeruginosa and K. pneumoniae which were isolated from patients even at the early manifestation of VAP. Candida colonized the respiratory tract of patients with VAP somewhat less frequently (10.2%). Only in 1 of the patients was S. maltophilia identified in the monoculture and considered as the etiological agent of VAP. Indeed, the microbial spectrum of pathogens isolated from VAP patients was mostly well-known prominent microorganisms associated with this complication in critically ill children.

According to some experimental and clinical trials, one of the most important points to emphasize is that among many kinds of antimicrobial therapy, administration of antimicrobial agents in inhalations by means of a nebulizer in patients undergoing MV is highly effective. The use of antimicrobial therapy through a nebulizer during MV ensures rapid direct delivery of a medicinal antimicrobial drug directly into the lung parenchyma, creating its high concentration directly at the site of the largest microbial colonization, which in turn leads to an effective rapid destruction of the VAP pathogens. Moreover, the inhalation administration of drugs reduces the risk of the potential systemic toxic effects of the drugs 141516.

According to the results of our study’s clinical observation, the inhalation use of an antiseptic based on DCM showed significant benefits in the treatment of severe patients with VAP. Clinical data of respiratory functions showed that in the group of patients who received only standard systemic antibiotic therapy (without DCM use) during 48-72 hours of MV, a gradual decrease of dynamic complication (by 3.4-fold) and resistance (by 2.4-fold) were determined, when compared to the group of patients who used DCM (p<0.05). This is in line with the findings of the high efficiency of inhalation use of DCM in the complex treatment of respiratory infections as demonstrated by other researchers 21.

Our study show that patients with VAP who received inhalation of quaternary ammonium antiseptic decamethoxine in the complex management of respiratory infectious complications, had a significant decrease in microbial colonization. Meanwhile, for patients receiving only standard systemic antibiotic therapy, higher rates of airway colonization by opportunistic microorganisms were observed during the 1^st week of treatment. On the 7^th day, the quantitative content of pathogens in the respiratory tract of patients in the comparison group exceeded (by four orders of magnitude) the number of microorganisms in situ in inflammation of patients who additionally received nebulizer therapy with DCM (p<0.001).

According to our findings, the advantages of additional inhalation management with DCM in the enhancement of microbial eradication and normalization of some factors of innate immune response in patients with VAP have been demonstrated. Our results revealed that respondents who received inhalations of DCM antiseptic drug for 7 days had a decrease of the microbial count in the tracheobronchial secretion, which was accompanied by a decrease of nonspecific immune response factor levels (TLR-4). The occurrence was mediated and maintained by gram-negative bacteria persistence. Consistent with the analysis of current studies, which suggest a strong correlation between the decrease of TLR-4 serum concentrations and colonization degree by gram-negative microorganisms in the respiratory tract of patients in the observation group (r-Pearson=0.801), the DCM administration led to an early recovery period of patients with VAP.

The conducted clinical and microbiological study herein, of the effectiveness of inhalation use of an DCM-based antiseptic drug in the complex treatment of VAP, showed an early decrease in the absolute number of opportunistic pathogens in the respiratory tract. Moreover, their rapid eradication, minimal changes and rapid recovery of respiratory parameters after mechanical ventilation corroborate the positive effect of DCM on the clinical course of VAP.

The most obvious finding to emerge from this study was that early inhalation of quaternary ammonium decamethoxine-based antiseptic drug Decasan®, through a nebulizer during the first 7 days in complex antimicrobial therapy, provides gradual improvement (by 3.4-fold) of the dynamic compliece and a 2.4-fold reduction of lung resistance during 48-72 hours of mechanical ventilation. This was accompanied by effective early (on the 3^rd day) reduction of the absolute amount of microorganisms (by 10⁴-fold) in 1 mL of tracheobronchial sputum, compared to that of the initial level of microbial colonization. Our findings demonstrate significant qualitative changes in the microbial spectrum, as characterized by prevalence of monoculture (61.11%) on the 7^th day, but not in microbial associations (93.33%). There was an observed complete eradication of pathogens of infectious complications from the respiratory tract of patients after 14 days of management, in comparison with traditional treatment (p<0.001).

In summary, the decrease of gram-negative microorganism counts in the respiratory aspirates of patients with VAP (who received DCM inhalations) was accompanied by a slight increase of TLR-4 serum level in the early period (3^rd day), and was closely correlated with a faster (1.42-fold) reduction of rate TLR-4 levels to baseline on the 7^th day of treatment (r-Pearson=0.893). This confirm the positive influence of inhalation use of quaternary ammonium antiseptic on respiratory functions of critically ill children, as well as on the clinical course of VA in those patients.

The authors declare that they have no competing interests.

NOA: made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved involved in drafting the manuscript or revising it critically for important intellectual content.

DDV: made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved involved in drafting the manuscript or revising it critically for important intellectual content.

DKD: made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved involved in drafting the manuscript or revising it critically for important intellectual content.

C_dyn — Dynamic compliance CDC — Centers for Disease Control and Prevention CFU — Colony-forming unit DCM — Decamethoxine ICU — Intensive care unit LLC — Limited Liability Company MV — Mechanical ventilation NNIS — National Nosocomial Infection Surveillance PEEP — Positive end-expiratory pressure PIP — Peak inspiratory pressure QACs — Quaternary ammonium compounds TLR — Toll-like receptors US — Ultra sound VAP — Ventilator-associated pneumonia