Identifying Agents of Early-onset and Late-onset Nosocomial Pneumonia in Patients in Intensive Care Through Genetic Analysis of Bacterial DNA and Determination of Their Distribution.

Introduction. Hospital-acquired pneumonia is one of the most frequent and most severe medical complications in patients hospitalized in intensive care units. It develops mainly in association with invasive airway management and mechanical ventilation. It is an important factor affecting health care economy and often related to bacterial resistance. Understanding the etiology of pathogens and their potential resistance to antimicrobial agents has a major impact on both patients' morbidity and mortality and the economic aspect of their treatment. Pneumonia is diagnosed by assessing clinical signs and detecting etiologic agents. Clinical signs are assessed especially by finding fresh or progressive infiltrates on chest radiographs, together with at least two other signs of respiratory tract infection. From the epidemiological point of view, pneumonia may be either community-acquired (CAP) or hospital-acquired (HAP). CAP is defined by developing in the community outside health care facilities and, if the patient is hospitalized, clinical manifestations within 48 h of hospital admission. HAP may be characterized as pneumonia developing 48 h or more after admission to a health care facility or within 14 days after discharge. More precise classification according to the time of the onset of clinical signs distinguishes early-onset (from 2 to 4 days after admission) from late-onset (from day 5 after admission) HAP. The most common pathogens causing CAP are Streptococcus pneumoniae, Chlamydophila pneumoniae and Mycoplasma pneumoniae. HAP is mostly caused by endogenous bacterial strains, originating from primary microflora or secondary colonizing bacteria with a higher level of resistance in the early- and late-onset forms, respectively. This knowledge is used to select the proper therapy since the proportion of individual pathogens determines the strategy of initial antibiotic treatment. Adequate antibiotic therapy is an important factor influencing the effectiveness of therapy and, of course, the patient's survival. The prospective study aimed at identifying etiologic agents of pulmonary inflammation in patients with the most severe forms of respiratory insufficiency requiring mechanical ventilation. The objectives were to describe the prevalence of respiratory tract pathogens, to determine their susceptibility to a range of tested antibiotic agents, and to acquire information on the proportion of resistant strains in the population of these pathogens. The impact of adequate antibiotic therapy was assessed, with respect to patient mortality and morbidity rates.

Hypothesis. Most sever types of nosocomial bacterial pneumonias, required artificial lung ventilation are often exogenous. The infection source is transferred to patient´s airways from outsider during hospitalisation. The object of the research is bacterial agent direct recognition, its originality identification based on genome restrict fragment analysis.

Material and Methods. The group comprised patients hospitalized in the Intensive Care Departments of cooperating hospitals between 1 May 2013 and 30 April 2015 who developed signs of HAP. Also included are patients with positive findings in their tracheal secretion on admission to the department if transferred from another ward of the hospital. In intubated patients, secretion samples for microbiology culture tests are obtained by aspiration from the lower airway. Mortality is statistically analyzed by Fisher's exact test. The clinical signs of pneumonia are defined as the presence of newly developed or progressive infiltrates on chest radiographs plus at least two other signs of respiratory tract infection: temperature >38 °C, purulent sputum, leukocytosis >10x103/mm3 or leukopenia <4x103/mm3, signs of inflammation on auscultation, cough and/or respiratory insufficiency with oxygenation index≤300 mm Hg. Each sample is processed by semiquantitative method based on the four-quadrant streak technique using a calibrated loop. The etiologic agent is considered relevant if cultured in the tracheal secretion at a enough quantity. Tracheal secretion samples are processed by traditional microbiological methods. The microorganisms are identified by biochemical dentification tests, and automated systems. The yeasts are identified using commercial kit. If an identical culture, determined as the etiologic agent, is repeatedly isolated from multiple samples obtained from the same patient, only the first isolated culture is included in the study. Susceptibility to antibiotics is determined by the standard microdilution method. Adequate initial therapy is defined as administrative of at least one antibiotic (suitable for treating pneumonia) shown in vitro to be effective against the isolated causative agent.

Progress chart. The collection and processing of the patient´s microbiological samples at cooperatived clinic departments between 1 May 2013 and 30 April 2015 will be performed. The co-operative centers sent the samples to microbiological departments of their own hospitals and there the samples will processed and safed. Collected data will continuously to the central database be stored. The microbiological samples collection will finish 30 April 2015 and between 1 May 2015 and 31 december 2015 the data will be analysed and assessed. The processing of the data allows express conclusions and prepare proposals to event. hygienical arrangement changes.

Way of data acquiring. In intubated patients, secretion samples for microbiology culture tests were obtained by aspiration from the lower airway. The group comprise patients hospitalized in the Intensive Care Department of Hospitals, cooperated in study, between 1 May 2013 and 30April 2015 who developed signs of HAP. Also included are patients with positive findings in their tracheal secretion on admission to the department if transferred from another ward of the hospital. Predicted number of patients is 300 (400 - 500 positive isolates). Considerate number is estimated according the pilot study. Predicted number of patients in all cooperated centers is about 15 per month. There the data analysis will be made with statistic normality test Shapiro-Wilk and Mann-Whitney U test. Computer program SPSS version 15 to statistic assessment will be used.