Isolation Rate and Patterns of Pathogens Isolated from Hospitalized Patients with Community-Acquired Pneumonia (CAP) in Clinical Trials in Central O. Stetsiouk* V. Khokhlova A. Karelin M. Belotserkovsky (Smolensk, RU; Zug, CH) and Eastern Europe (CEE)
P2014
Introduction and Purpose New antibiotic research and development (R&D) is of extreme importance due to emergence of antimicrobial resistance, and there are very few agents in the pipeline that can address this problem. Therefore every new antimicrobial should be investigated properly in clinical trials to assess its potential for treating different types of infections in various geographical locations. Countries of Central and Eastern Europe (CEE) are becoming increasingly involved in clinical trials of new antimicrobials, and there can be unique scientific, regulatory and logistic challenges associated with antibiotic R&D process in these countries. Community-acquired pneumonia (CAP) in hospitalized adults is one of the primary cornerstones in evaluation of new antibiotics. Landscape of pathogens in adult hospitalized CAP patients is of essential interest for selection of countries for these studies, as there is a need to demonstrate efficacy of novel antibiotics against the local CAP pathogens. The purpose of our study was to investigate aetiology of CAP in hospitalized patients participating in clinical trials in different CEE countries.
Methods Analyzed clinical studies We performed an analysis of the joint database that included the results of microbiology investigation of clinical material obtained from patients participating in two identical phase 3 clinical trials of novel cephalosporin in patients hospitalized for CAP during 2007-2009 to determine «typical» bacteria patterns in CEE countries. Thirty-two (32) clinical sites located in six (6) CEE countries – Bulgaria (6 sites), Latvia (2 sites), Lithuania (4 sites), Romania (7 sites), the Russian Federation (5 sites) and Ukraine (8 sites) – were involved in these studies (Figure 1). Yekaterinburg St. Petersburg 687 km
1785 km
Smolensk
1073 km
Lviv
Kharkiv Poltava Dnipropetrovsk
Odesa
Rostov-on-Don
agent; respiratory tract infections attributable to sources other than community-acquired bacterial pathogens (e.g., ventilator-associated pneumonia, nosocomial pneumonia, aspiration pneumonia, suspected viral or fungal infections); non-infectious causes of pulmonary infiltrates (e.g., pulmonary embolism, chemical pneumonitis from aspiration, hypersensitivity pneumonia, congestive heart failure); active tuberculosis of the lung; pleural empyema (not including sterile parapneumonic effusions). Patients with coexisting diseases requiring the administration of systemic antibiotics or antifungals were not eligible for participation in the study, as well as patients with different immunodeficient conditions (HIV infection, neutropenia, etc.), complicated coexisting kidney, liver, cardiovascular, hematogenic, nervous, endocrine, electrolyte balance pathologies, cancer, and known hypersensitivity to study medication. Pregnant or lactating women, as well as patients not using effective contraception, did not participate in the study. Patients receiving concomitant therapy with high-dose glucocorticosteroids or drugs able to interact with the investigated antimicrobials were not enrolled in the study. It is important to note that patients who had received therapy with more than one dose of systemic antibiotic potentially effective for CAP within 96 hours prior to enrolment in the study were not allowed to participate, with the exception of cases of unequivocal clinical evidence of failure and isolation of an organism resistant to the prior non-study drug therapy, but not to the study drug. Procedure for investigation of microbiology samples Blood and respiratory cultures were obtained before initiation of the study therapy from all hospitalized CAP patients. Local laboratories at the study sites prepared Gram-stained smears of sputum, assessed the quality of sputum samples (sputum sample was considered acceptable if there was ≤10 squamous epithelial cells per low-power field at microscopy) and processed sputum samples for shipment to regional microbiology laboratories. As there were some challenges with microbiology investigations of respiratory samples in local laboratories (significant differences in capabilities, equipment, culture media supplies, quality assurance processes) it was decided to select one regional laboratory for each participating CEE country, with the exception of Baltic countries, where one regional laboratory located in Riga (Latvia) was used for both Latvia and Lithuania. This logistic approach was used to improve the quality and uniformity of microbiology investigations of respiratory samples in hospitalized CAP patients participating in clinical trials in different CEE countries. Logistics scheme of interactions between sites, local, regional and central microbiology laboratories is presented on Figure 2.
Luhansk
Donetsk
Local Laboratories at Study Sites Microbiology sample preparation and shipment to regional labs
Gram Stain Regional laboratory
Regional Laboratories: Moscow (Russia) Kiev (Ukraine) Bucharest (Romania) Sofia (Bulgaria) Riga (Baltic Countries)
Figure 1. Participating CEE countries with Regional Microbiology Laboratories
The main inclusion criteria for patients were the following: 1) male and female patients aged ≥ 18 years old; 2) signed informed consent form to participate in the study; 3) radiographically and clinically confirmed diagnosis of CAP (i.e. pulmonary infiltrate(s) on chest X-ray or CT and acute (≤ 7 days’ duration) illness with clinical signs or symptoms consistent with a lower respiratory tract infection; 4) hospitalized CAP patients with PORT risk class II-IV. Patients with the following conditions were not enrolled into the studies: severe CAP with PORT score of V or requiring admission to an intensive care unit; CAP suitable for outpatient therapy with an oral antimicrobial
Kit building and laboratory documents development
Shipment organization
Samples receipt and testing
Reporting to sites
Isolate shipment to Covance
Central Laboratory Supply of regional labs with laboratory materials
Isolate receipt
Isolates re-identification, susceptibility testing
Reporting to sites and regional labs
Figure 2. Interactions between sites, local, regional and central microbiology laboratories.
Contact information: Dr. Olga Stetsiouk olga.stetsiouk@antibiotic.ru Dr. Maxim.Belotserkovsky maxim.belotserkovsky@psi-cro.com
more microorganisms were identified in 19% of samples. The results of microbiology investigation of sputum samples in the Russian Federation and Romania were very similar (Table 1), with identical numbers of positive, negative or invalid cultures, monomicrobial and polymicrobial infections, and ratio of Gram-positive and Gram-negative pathogens. Streptococcus pneumoniae was isolated on average from 6% of samples, with maximum rate in Romania (17%) and only 3% in Russia. No cases of pneumococcal CAP were detected in Baltic countries. Haemophilus influenzae was isolated more frequently than S. pneumoniae – on average from 11% of samples, with the highest frequency in Romania, Bulgaria and Ukraine (14-15%). Staphylococcus aureus was frequently isolated from sputum samples of hospitalized CAP patients in the Russian Federation and Ukraine (11-12%), but in significantly fewer numbers of samples in other CEE countries. Patients in the Russian Federation had the highest rate of CAP caused by Klebsiella pneumoniae and Escherichia coli (10% and 6%, respectively); these bacteria were also isolated from 9% of sputum samples in Romania and Ukraine. Moraxella catarrhalis and Pseudomonas aeruginosa were revealed in CAP samples in isolated instances only. Regional microbiology laboratories in all CEE countries frequently reported also non-pathogenic bacteria Results for CAP, such as coagulase-negative staphylococci (CNS), StreptococRespiratory samples were obtained in 339 CAP patients. Using re- cus spp., Haemophilus parainfluenzae and Candida spp. gional microbiology labs pathogens were cultured on average from Regional differences in microbiology landscapes of prevalent CAP 75% of samples. The rate of positive cultures varied from 94% in pathogens in CEE countries are represented on Figure 3. Ukraine to 45% in Baltic countries (Table 1). Collected sputum samples were placed in the Port-A-Cul (BBL, USA) transport system and subsequently shipped in thermo-stabilized conditions along with the sputum Gram-stains to a regional microbiology laboratory for further examination. In regional microbiology laboratories a standard microbiology investigation of collected samples of clinical material was performed following a uniform protocol targeted at isolation of aerobic microorganisms. The identification of isolated microorganisms was carried out to the species level in accordance with the identical standard operating procedures accepted in the regional laboratories. Eventually, all microorganisms deemed clinically significant for CAP by investigators were shipped to the central laboratory for re-identification and antimicrobial susceptibility testing. The collected data were entered into the joint database which contains information regarding unique isolates from sputum samples obtained at screening from hospitalized patients with CAP. Thus, the database contains the information on the original microbiology landscape in the Intent-to-Treat (ITT) population.
18
Table 1. Results of microbiology investigation of sputum samples in regional laboratories All CEE Ukraine countries Pts with CAP samples obtained (N)
339
140
Russia 117
Romania Bulgaria 35
36
Samples with negative or invalid cultures, N (%) 86 (25%) 8 (6%) 49 (42%) 14 (40%) 9 (25%)
16
Baltic countries (Latvia, Lithuania)
14
11
12
6 (55%)
10 8
Monomicrobial infections (%)
56
74
36
37
72
45
Samples with ≥ 2 microbes isolated (%)
19
21
22
23
3
0
22
Gram-positive pathogens only (%)
32
14
14
9
8
6 4 2 0
38
Gram-negative pathogens only (%)
9
Mixed flora (%)
40 11
32 8
34 11
53
36 0
3
All CEE countries
Ukraine
Russia
Romania S. pneumoniae H. influenzae
Bulgaria S. aureus Enterobacteriaceae
Figure 3. Microbiology landscapes of prevalent CAP pathogens in CEE countries. Table 2. Landscape of pathogens in adult hospitalized CAP patients in CEE countries All CEE Ukraine countries
Russia
Romania
Bulgaria
Baltic countries (Latvia, Lithuania)
Streptococcus pneumoniae, %
6
7
3
17
6
0
Streptococcus spp., %
14
24
10
0
3
9
Staphylococcus aureus, %
9
12
11
3
3
0
Coagulase-negative staphylococci, %
4
6
2
6
0
0
Haemophilus influenzae, %
11
15
6
14
14
0
Haemophilus parainfluenzae, %
8
10
0
6
28
0
As it is clear from Figure 3, patterns of «typical» bacteria isolated from hospitalized patients with CAP in different CEE countries are notably distinct. Romania represents «classical» microbiological landscape with prevalence of S. pneumoniae, followed by H. influenzae and Enterobacteriaceae. In other CEE countries rates of H. influenzae overcome frequency of isolation of pneumococci. High rates of isolation of S. aureus from hospitalized patients with CAP were noted in Russia and Ukraine, and very high prevalence of Enterobacteriaceae was observed in CAP patients from sites in Russian Federation, followed by Ukraine and Romania.
Conclusions
There are specific challenges facing sponsors, contract research organizations (CROs) and investigators while conducting clinical trials 0 3 9 16 9 11 Enterobacteriaceae (K.pneumoniae, E.coli), % of new antibiotics developed for the treatment of community-acquired 0 3 0 1 1 1 Pseudomonas aeruginosa, % pneumonia in emerging regions (e.g. in CEE). Establishing regional referent microbiology laboratories ensures a reliably high level of Gram-negative microorganisms predominated among CAP pathogens pathogen identification in CAP (75% on average). Patterns of «typiin hospitalized patients in all CEE countries. On average, Gram- cal» bacteria isolated from hospitalized patients with CAP in different negative bacteria were isolated from 38% of samples, Gram-positive CEE countries are notably distinct. These data should be taken into bacteria – from 22% of samples and mixed flora – from 9% of samples. consideration while planning and conducting clinical trials in patients One pathogen only was isolated from 56% of sputum samples, two or with CAP in CEE. Moraxella catarrhalis, %
1
0
1
3
0
0