Microbiological Diagnostics of Community-Acquired Pneumonia

Pneumonija je najvažnija upalna bolest u dišnom sustavu iako sudjeluje samo jednim postotkom u svim akutnim infekcijama dišnog sustava. Kao i u većini drugih organa i sustava organa, imunosni obrambeni mehanizmi u plućima baziraju se na mehanizmima specifične i nespecifične imunosti i njihovim komponentama koje uključuju anatomske barijere te staničnu i humoralnu imunost. Prvu liniju obrane od infekcija donjega respiratornog sustava čine elementi nespecifične imunosti, među kojima najvažniju ulogu imaju epitelne stanice i makrofagi. Mehanizmi specifične imunosti imaju snažnu ulogu u obrani od različitih patogena, visoko su sofisticirani i ovise o prepoznavanju specifičnih antigena na površini mikroorganizama te tvore snažnu obranu protiv različitih patogena koji uzrokuju pneumonije. Međutim, za razliku od nespecifične imunosti, indukcija i razvoj imunoreakcija koje sudjeluju u specifičnoj imunosti mogu trajati danima i tjednima nakon ekspozicije patogenu, a važna komponenta tog procesa jest i stvaranje memorijskih stanica koje sudjeluju u snažnoj obrani pri ponovnim susretima s istim patogenima. Mehanizmi nespecifične i specifične imunosti u pneumonijama jesu kompleksni i bit će potrebna još brojna istraživanja da bi se bolje shvatili i rabili u kreiranju novih lijekova i cjepiva te bolje primijenili u uporabi postojećih lijekova. Osim važnog antimikrobnog djelovanja antibiotika ne smijemo nikako zanemariti ni njihovo protuupalno i imunomodulatorno djelovanje koje može pomoći u racionalnijem pristupu liječenju pneumonija.Community-acquired pneumonia is an important cause of morbidity and mortality. Even with microbiological investigation done, the etiology frequently remains unknown. In the last 20 years the interest in and the need for microbiological tests in patients with community-acquired pneumonia have decreased, so today the majority of patients do not have the causative agent detected. Limitations regarding the use of microbiological tests are also found in the guidelines for diagnostics and treatment of community-acquired pneumonia. Microbiological investigation is indicated in hospitalized patients in order to administer pathogen-directed treatment, ensure rational administration of antibiotics and achieve savings in the cost of treatment. Urinary antigen tests for pneumococcus and legionella have a specific place among microbiological tests. Their greatest advantages are tests results obtained in only 15 minutes and their simplicity. Molecular methods have not been standardized, but the detection of pneumococcus in respiratory samples in patients previously treated with antibiotics has been improved. As a result of the imperfections of microbiological tests, biomarkers are investigated as indicators of community-acquired pneumonia etiology. The values of such biomarkers have been used for assessing the length of therapy and differentiating between bacterial and viral infections. Research has shown that procalcitonin levels can assist in making a decision about the duration of antimicrobial treatment. The future of microbiological diagnostics will most probably be point-of-care tests which can easily be performed by medical staff outside the laboratory, as well as further development and standardization of molecular tests to enable their routine use

Microbiological Diagnostics of Community-Acquired Pneumonia

Pneumonija je najvažnija upalna bolest u dišnom sustavu iako sudjeluje samo jednim postotkom u svim akutnim infekcijama dišnog sustava. Kao i u većini drugih organa i sustava organa, imunosni obrambeni mehanizmi u plućima baziraju se na mehanizmima specifične i nespecifične imunosti i njihovim komponentama koje uključuju anatomske barijere te staničnu i humoralnu imunost. Prvu liniju obrane od infekcija donjega respiratornog sustava čine elementi nespecifične imunosti, među kojima najvažniju ulogu imaju epitelne stanice i makrofagi. Mehanizmi specifične imunosti imaju snažnu ulogu u obrani od različitih patogena, visoko su sofisticirani i ovise o prepoznavanju specifičnih antigena na površini mikroorganizama te tvore snažnu obranu protiv različitih patogena koji uzrokuju pneumonije. Međutim, za razliku od nespecifične imunosti, indukcija i razvoj imunoreakcija koje sudjeluju u specifičnoj imunosti mogu trajati danima i tjednima nakon ekspozicije patogenu, a važna komponenta tog procesa jest i stvaranje memorijskih stanica koje sudjeluju u snažnoj obrani pri ponovnim susretima s istim patogenima. Mehanizmi nespecifične i specifične imunosti u pneumonijama jesu kompleksni i bit će potrebna još brojna istraživanja da bi se bolje shvatili i rabili u kreiranju novih lijekova i cjepiva te bolje primijenili u uporabi postojećih lijekova. Osim važnog antimikrobnog djelovanja antibiotika ne smijemo nikako zanemariti ni njihovo protuupalno i imunomodulatorno djelovanje koje može pomoći u racionalnijem pristupu liječenju pneumonija.Community-acquired pneumonia is an important cause of morbidity and mortality. Even with microbiological investigation done, the etiology frequently remains unknown. In the last 20 years the interest in and the need for microbiological tests in patients with community-acquired pneumonia have decreased, so today the majority of patients do not have the causative agent detected. Limitations regarding the use of microbiological tests are also found in the guidelines for diagnostics and treatment of community-acquired pneumonia. Microbiological investigation is indicated in hospitalized patients in order to administer pathogen-directed treatment, ensure rational administration of antibiotics and achieve savings in the cost of treatment. Urinary antigen tests for pneumococcus and legionella have a specific place among microbiological tests. Their greatest advantages are tests results obtained in only 15 minutes and their simplicity. Molecular methods have not been standardized, but the detection of pneumococcus in respiratory samples in patients previously treated with antibiotics has been improved. As a result of the imperfections of microbiological tests, biomarkers are investigated as indicators of community-acquired pneumonia etiology. The values of such biomarkers have been used for assessing the length of therapy and differentiating between bacterial and viral infections. Research has shown that procalcitonin levels can assist in making a decision about the duration of antimicrobial treatment. The future of microbiological diagnostics will most probably be point-of-care tests which can easily be performed by medical staff outside the laboratory, as well as further development and standardization of molecular tests to enable their routine use

Molecular characteristics of MRSA strains and patient risk factors in vascular surgery

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major pathogens in hospitals, and since the 1990s it has been recognized as an important pathogen in community infections. (1) The aim of this study was to analyze MRSA strains from a vascular surgery ward over a five-year period, since the vascular ward is considered to be a high-risk site for different multi-resistant pathogens, among which MRSA is very important. The method used for the microbiological identification and susceptibility testing of strains was the Vitek2 system. For the detailed characterization of the MRSA strains, we used the following molecular methods: SCCmec typing, pulse-field gel electrophoresis (PFGE), spa typing and Panton-Valentine leukocidin (PVL) detection. During the 5-year period, 77 MRSA strains were isolated. Antimicrobial susceptibility: 100% of MRSA isolates were susceptible to oxazolidinones and glycopeptides, 55% were susceptible to gentamycin, and 98% were susceptible to tetracyclines. SCCmec typing: 43 of 77 (55.8%) strains were typed as SCCmec I. The number of isolates with SCCmec II was 28 (36.4%). Three isolates carried SCCmec III. After the PFGE analysis, the isolates were grouped into six similarity groups: A-F. The largest number of isolates (80.6%) belonged to one of two groups: A: 35 (46.8%) and D: 25 (33.8%). Conclusion: The analysis of MRSA strains in the vascular surgery ward revealed high homogeneity among the strains, the majority of which belonged to SCCmec type I. This type, together with the susceptibility profile and PFGE grouping, is considered to be typical of Hospital-Acquired (HA) MRSA

Nationwide Survey of Klebsiella Pneumoniae Strains Producing CTX-M Extended-spectrum b-lactamases in Croatia

Extended-spectrum β-lactamases (ESBL) producing bacteria have been increasingly reported in both hospital and community patients. Production of ESBLs is the major mechanism of resistance to oxymino-cephalosporins and aztreonam in Gram-negative bacteria 1,2. Recently a new family of ESBLs with predominant activity against cefotaxime (CTX-M β-lactamases) has been reported. Over 80 CTX-M enzymes have been described so far, which can be grouped into five main subgroups according to amino acid sequence identity (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25) 3 . In some countries, CTX-M β-lactamases are the most prevalent types of ESBLs, for instance in Russia 4, Greece 5 , Spain 6 , Switzerland 7, Japan 8, Taiwan 9, China 10 and Argentina 11 . These enzymes have been identified in countries near Croatia such is Italy 12, Hungary13 and Austria14 The aim of this study was to determine the prevalence and the types of CTX-M β lactamases produced by Klebsiella pneumoniae clinical isolates collected from October 2006 to January 2007 from both community- and hospital –based isolates were included (Figure 1.). 128 ESBL isolates were subjected to further analysis: screening with double disc diffusion test and confirmed by ESBL E test 15

Molecular Variants of Human Papilloma Viruses Type 16 and 6 in Women with Different Cytological Results Detected by RFLP Analysis

HPV infections are common and the presence of the same high-risk type in cervical specimens can be due to reinfection or persistence. Persistent infection is the most important predictor for development of cervical carcinoma. The aim of this study was to validate PCR-RFLP with two sets of primers: MY09/MY11 that amplify a fragment of L1 and P1/P2 that amplify a fragment of E1 ORF. PCR product of MY09/MY11 was digested with a set of 6 restriction enzimes (RE) and PCR product of P1/P2 with a set of 12 RE. Cervical samples from 110 women patients of the University Gynecologic Clinic CHC Zagreb were analyzed. There were 98 (89.1%) PCR positive samples detected with P1/P2 primers, and 94 (85.5%) PCR positive samples detected with MY09/MY11 primers. Seven HPV types were detected with P1/P2-RFLP technique and 17 with MY09/MY11-RFLP. PCR positive samples amplified with both primer pairs agreed with each other in 82 samples; 16 samples were only positive with P1/P2 and 12 samples were only positive by MY09/MY11. HPV 16 was detected in 39 samples with MY09/11-RFLP, out of these two variants (two different patterns) were found with P1/P2 using Dde I, Hae III and Eco I. HPV 6 was detected in 9 samples with MY09/11-RFLP, out of these two variants were found with P1/P2 using HinfI. Combining these two PCR-RFLP methods subtypes of HPV 16 and HPV 6 were detected

Evaluacija imunokromatografskog testa za otkrivanje galaktomanana Aspergillus spp. u uzorcima seruma i bronhoalveolarnih lavata – preliminarni rezultati

Background: Detection of biomarkers, such as galactomannan (GM), has proven to be of great significance in early recognition of invasive aspergillosis (IA). The aim of our study was to evaluate the lateral flow assay (LFA) for the detection of GM on serum and bronchoalveolar lavage (BAL) samples previously proven positive by enzyme-linked immunosorbent assay (ELISA). Methods: The study was performed on serum and BAL samples obtained from patients with suspected IA in the period from February 2019 to January 2020, which were previously GM positive by ELISA (Platelia Aspergillus Ag, Biorad, Hercules, USA). Samples were then tested by LFA (Aspergillus Galactomannan LFA, IMMY, Oklahoma, USA) with test line intensity visually read as 1+, 2+, 3+, or 4+. Results: A total of 45 GM ELISA positive serum and/or BAL samples were obtained from 41 patients; 25 (55,6 %) were BAL and 20 (44,4 %) serum samples. LFA showed a positive result in 39 out of 45 (86,7%) GM ELISA positive samples; 22/25 (88.0 %) BAL samples and 17/20 (85.0 %) serum samples tested positive. In BAL samples, low intensity test line of 1+ was significantly more frequent in GM ELISA positive samples with optical density index (ODI) 4.0) had low intensity line of 1+ when tested with LFA. Conclusions: Results obtained by LFA are comparable to GM ELISA. Since low intensity lines were found in serum samples with high ODI, this potentially makes BAL a superior sample for LFA, at least when visual and not automated reading is done.Uvod: Određivanje galaktomanana (GM) igra značajnu ulogu u ranom otkrivanju invazivne aspergiloze (IA). Cilj je ovog istraživanja bila evaluacija imunokromatografskog testa (LFA) za određivanje GM u uzorcima seruma i bronhoalveolarnih lavata (BAL) s pozitivnim rezultatom koji su utvrđeni prethodno napravljenom ELISA metodom. Metode: Istraživanje je napravljeno na uzorcima seruma i BAL-a pacijenata sa sumnjom na IA prikupljenim u razdoblju od veljače 2019. do siječnja 2020. godine. Uzorci s pozitivnim rezultatom utvrđenim ELISA metodom (Platelia Aspergillus Ag, Biorad, Hercules, USA) testirani su s LFA (Aspergillus Galactomannan LFA, IMMY, Norman, Oklahoma, USA) s vizualnim očitavanjem testne linije u rasponu 1+, 2+, 3+, ili 4+. Rezultati: Od 41 bolesnika dobiveno je 45 pozitivnih uzoraka seruma i/ili BAL-a testiranih GM ELISA metodom; 25 (55,6 %) uzoraka BAL-a i 20 (44,4 %) uzoraka seruma. LFA je pokazao pozitivni rezultat kod 39 od 45 (86,7%) uzoraka pozitivnih GM ELISA metodom; 22/25 (88.0 %) uzoraka BAL-a i 17/20 (85.0 %) uzoraka seruma. Kod uzoraka BAL-a, testna linija slabog intenziteta 1+ bila je značajno češća kod pozitivnih uzoraka testiranih GM ELISA metodom s ODI 4.0) imala su pri testiranju s LFA testnu liniju slabog intenziteta 1+. Zaključak: Rezultati dobiveni s LFA usporedivi su s GM ELISA metodom. S obzirom da su testne linije niskog intenziteta utvrđene u uzorcima seruma s visokim ODI, uzorci BAL-a su potencijalno pogodniji uzorci za LFA, barem kad se radi o vizualnom, a ne automatiziranom očitavanju testa

Value of Rapid Aetiological Diagnosis in Optimization of Antimicrobial Treatment in Bacterial Community Acquired Pneumonia

In 80 adult patients with community acquired pneumonia (CAP) conventional microbiological methods, polymerase chain reaction (PCR) and serum C-reactive protein (CRP) levels were performed and the appropriateness of the empirical antimicrobial treatment was evaluated according to bacterial pathogen detected. The aetiology was determined in 42 (52.5%) patients, with Streptococcus pneumoniae as the most common pathogen. PCR applied to bronchoalveolar lavage (BAL) provided 2 and PCR on sputum samples 1 additional aetiological diagnosis of CAP. The mean CRP values in the S. pneumoniae group were not significantly higher than in the group with other aetiological diagnoses (166.89 mg/L vs.160.11 mg/L, p=0.457). In 23.8% (10/42) of patients with determined aetiology, the empirical antimicrobial treatment was inappropriate. PCR tests need further investigation, particularly those for the atypical pathogens, as they are predominant in inappropriately treated patients. Our results do not support the use of CRP as a rapid test to guide the antimicrobial treatment in patients with CAP

Nationwide survey of Klebsiella pneumoniae strains producing CTX-M extended-spectrum b-lactamases in Croatia [Nacionalno istraživanje sojeva bakterije klebsiella pneumoniae koji proizvode CTX-M prošireni-spektar b-laktamaza u Hrvatskoj]

Extended-spectrum β-lactamases (ESBL) producing bacteria have been increasingly reported in both hospital and community patients. Production of ESBLs is the major mechanism of resistance to oxymino-cephalosporins and aztreonam in Gram-negative bacteria. Recently a new family of ESBLs with predominant activity against cefotaxime (CTX-M β-lactamases) has been reported. Over 80 CTX-M enzymes have been described so far, which can be grouped into five main subgroups according to amino acid sequence identity (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25). In some countries, CTX-M β-lactamases are the most prevalent types of ESBLs, for instance in Russia, Greece, Spain, Switzerland, Japan, Taiwan, China and Argentina. These enzymes have been identified in countries near Croatia such is Italy, Hungary and Austria. The aim of this study was to determine the prevalence and the types of CTX-M β lactamases produced by Klebsiella pneumoniae clinical isolates collected from October 2006 to January 2007 from both community- and hospital-based isolates were included (Figure 1.). 128 ESBL isolates were subjected to further analysis: screening with double disc diffusion test and confirmed by ESBL E test

1,3-Β-D-glucan in invasive fungal infection diagnostics – first Croatian experience

Invazivne gljivne infekcije (IGI) važan su problem suvremene medicine. Razlog tomu jesu rastući broj imunokompromitiranih bolesnika te visoke stope morbiditeta i mortaliteta zbog ovih infekcija. Pravodobno postavljena dijagnoza IGI-ja od presudne je važnosti jer odgađanje primjene antifungalne terapije utječe na ishod liječenja bolesnika. Kultivacija kao konvencionalna dijagnostička metoda ima nisku osjetljivost, dugo traje i nalaže uzimanje invazivnog uzorka. Zbog toga se posljednjih dvadesetak godina radi ranije i osjetljivije dijagnostike istražuju i primjenjuju fungalni biomarkeri. 1,3-β-D-glukan (BDG) fungalni je biomarker odrediv u serumu bolesnika kojim se može dokazati prisutnost ovih gljivnih patogena: Candida spp., Aspergillus spp., Acremonium, Coccidioides immitis, Fusarium spp., Histoplasma capsulatum, Trichosporon spp., Sporothrix schenckii, Saccharomyces cerevisiae i Pneumocystis jirovecii. Zbog niske razine odnosno nepostojanja BDG-a u staničnoj stijenci ovim se testom ne mogu dokazati vrste roda Cryptococcus spp. i reda Mucorales. Visoka negativna prediktivna vrijednost BDG-a u slučaju negativne vrijednosti može se iskoristiti za donošenje odluke o prekidu antifungalne terapije i biti dio strategije upravljanja primjenom antifungalnih lijekova u jedinicama intenzivnog liječenja. Kod hematoloških bolesnika BDG se može primjenjivati radi probira i u sklopu dijagnostičke obrade pri sumnji na IGI. Pouzdanost testa kod pojedinog bolesnika veća je u slučaju dvaju ili više uzastopno pozitivnih rezultata. Utjecaj antifungalne profilakse na rezultate testa BDG-a još je nejasan. Kinetiku BDG-a za sada je teško korelirati s kliničkim ishodom. Pedijatrijskim bolesnicima još nisu definirane granične vrijednosti za interpretaciju vrijednosti BDG-a iako su o tome objavljena brojna istraživanja. Trenutačno vrijedeće smjernice i dalje ne preporučuju primjenu ovoga fungalnog biomarkera kao rutinskoga dijagnostičkog testa u djece, premda može poslužiti u određenim situacijama uzimajući u obzir njegova ograničenja. BDG kao fungalni biomarker važan je napredak u dijagnostici IGI-ja te uz istodobnu primjenu ostalih dijagnostičkih metoda, ispravnu interpretaciju i racionalnu primjenu može pomoći ranijem i uspješnijem postavljanju dijagnoze i liječenju bolesnika s IGI jem.Invasive fungal infections (IFI) are an important problem of modern medicine. The reason is growing population of immunocompromised patients and high morbidity and mortality of these infections. Timely diagnosed IFI is of utmost importance because the delay of antifungal treatment has impact on treatment outcome. Cultivation as a conventional diagnostic method has low sensitivity, long duration and demands obtaining invasive samples. Therefore, in the last two decades fungal biomarkers are investigated for earlier and more sensitive diagnostics. 1,3-β-D-glucan (BDG) is a fungal biomarker in patients’ sera that enables detection of the following fungal pathogens: Candida spp., Aspergillus spp., Acremonium, Coccidioides immitis, Fusarium spp., Histoplasma capsulatum, Trichosporon spp., Sporotrix schenckii, Saccharomyces cerevisiae and Pneumocystis jirovecii. Low level and absence of BDG in the cell wall unables the detection of Cryptococcus spp. and order Mucorales with this test. High negative predictive value of BDG can be used when deciding to stop antifungal treatment and be a part of strategy for antifungal stewardship in intensive care units. In hematological patients BDG can be used as a screening method or as a part of diagnostic work-up when IFI is suspected. Reliability of test result is higher when two or more consecutive samples are positive. Influence of antifungal prophylaxis on BDG test results is still unclear. BDG kinetics and its relation to clinical outcome are still investigated. For pediatric population cut-off values for interpretation are still not defined, although many studies have been published investigating this issue. Although still not recommended by pediatric guidelines, this test can help in certain situations having in mind its limitations. BDG as a fungal marker represents the significant progress in IFI diagnostics. With simultaneous application of other diagnostic methods, exact interpretation and rational use, it can help earlier and more successful diagnostics and treatment of IFI