Antimicrobial Sensitivity Profile of Chlamydia trachomatis isolates from Croatia in McCoy Cell Culture System and Comparison with the Literature.

BACKGROUND: Chlamydia trachomatis (C. trachomatis) is the most common bacterial agent of sexually transmitted infections around the world, but susceptibility testing of this pathogen is rarely pursued due to its intracellular niche. The principal aims of this research were to determine in vitro sensitivity profile of urogenital chlamydial strains isolated from Croatian patients and to compare obtained concentration values of different antimicrobial drugs mutually and with the literature. METHODS: Forty strains of C. trachomatis isolated during 2010-2012 at the National Reference Laboratory for Chlamydia and two reference strains were subjected to susceptibility testing in 96-well microtiter plates containing McCoy cell monolayers. Minimal inhibitory concentration (MIC) and minimal chlamydicidal concentration (MCC) were determined for azithromycin, doxycycline, and levofloxacin. Briefly, strains were inoculated on McCoy cells, followed by addition of serially diluted antimicrobial drugs. Upon incubation, growth of C. trachomatis was detected using fluorescein-conjugated antibody to the lipopolysaccharide genus antigen under the inverted fluorescent microscope. RESULTS: All chlamydial strains were susceptible to the antibiotics tested (MIC < 4 pg/mL), thus the pattern of homotypic or heterotypic resistance has not been found. MCC values were equal or 1-5 dilutions higher than MIC values. Statistically significant differences in the effectiveness of antimicrobial agents in vitro have been proven. Significant correlation has been found for MCCs in the case of two antimicrobial pairs: azithromycin and levofloxacin, and doxycycline and levofloxacin. Comparison of medians for different clinical samples did not reveal any significant difference. CONCLUSIONS: Although resistant strains have not been found in this study, several literature reports of unsuccessfully treated genitourinary infections caused by C. trachomatis require our alertness for possible discovery of resistant strains. Considering the overall antibiotic burden worldwide, pursuing this kind of research is crucial in order to detect possible decreased susceptibility (or even resistance) of chlamydial strains, despite the laborious and time-consuming methodology.

Serum levels of metalloproteinases and their inhibitors during infection with pathogens having integrin receptor-mediated cellular entry.

BACKGROUND: Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes with numerous roles in the normal immune response to infection. However, excess MMP activity following infection may lead to immunopathological processes that cause tissue damage. Their activity in normal tissues is subject to tight control, which is regulated by its specific endogenous tissue inhibitors (TIMPs). It is known that MMPs bind to cell surface proteins (e.g. integrins) and that such interactions can have modulatory effects on MMP functionality. The objective of this study was to determine whether there are differences in MMP and TIMP production during the acute phase of infection with different pathogens that use ß-integrins as their receptors for cell entry. METHODS: We measured the total amounts of soluble MMP-2, MMP-9, TIMP-1, and TIMP-2 in the sera from patients infected with Dobrava virus (DOBV), Coxiella burnetii, or uropathogenic Escherichia coli. Statistical analyses were used to correlate MMP/TIMP serum levels with different clinical laboratory parameters. RESULTS: The results showed that both of the bacterial infections generally manifested the stronger effect on MMP production, while in contrast, viral infection introduced stronger changes to metalloproteinase inhibitors. MMPs and TIMPs were significantly correlated with some of the clinical laboratory parameters in both bacterial infections, but no correlations were found for DOBV infection. CONCLUSIONS: These findings suggest diverse mechanisms by which MMP activity could be implicated in the pathology of these 2 bacterial infections versus the viral DOBV infection, despite the type of their cellular entry receptors.

Critical issues in chronic prostatitis.

In the last decade, an impressive amount of clinical research data has shed new light on pathogenesis and management of the chronic prostatitis syndrome. A new classification and a validated symptom score have enabled urologists worldwide to speak a "common language", thus greatly improving the amount and quality of focused research in this field. In Europe, a large number of groups and experts have been actively involved in this research, and have developed in many cases a genuine view on prostatitis and chronic pelvic pain etiology, diagnosis and treatment. The present paper, written by a panel of researchers from Europe and Far East Russia, reviews the most recent findings, discusses the most controversial contemporary topics on prostatitis syndromes, and highlights a number of unresolved issues requiring further research and study.

Research of urinary tract infections in family medicine physicians' offices--empiric antimicrobial therapy of urinary tract infections--Croatian experience.

In the period between October 1st and November 30th, 2006, we investigated a total of 3188 episodes of UTI (802 among males; 2386 among females) recorded in 108 family medicine offices in 20 cities in Croatia. The most common UTIs in women were acute uncomplicated cystitis (62%), complicated UTIs - cystitis and pyelonephritis (14%), urethritis (9%), acute uncomplicated pyelonephritis (6%), recurrent cystitis (5%), asymptomatic bacteriuria (3%) and recurrent pyelonephritis. The most common UTIs in men were complicated UTIs - cystitis and pyelonephritis (48%), urethritis (25%), prostatitis (24%) and asymptomatic bacteriuria (3%). Etiological diagnosis was made in 999 (31%) UTI episodes before antimicrobial therapy was given. The most frequently isolated causative pathogens were Escherichia coli (77%), Enterococcus faecalis (9%), Proteus mirabilis (5%), Klebsiella spp (3%), Streptococcus agalactiae (3%) and Enterobacter (1%). Antimicrobial drug was administered in 2939 (92.19%) UTI episodes, in 1940 (66.01%) as empirical therapy, and in 999 (34%) as targeted antimicrobial therapy. The most commonly administered drug in empirical therapy for acute uncomplicated cystitis, recurrent cystitis and urethritis in women was cephalexin, for acute uncomplicated pyelonephritis and complicated UTIs in women co-amoxiclav, and for UTIs in males ciprofloxacin. The results of this research of 3188 UTI episodes in family medicine physicians' offices provide a confirmatory answer to question whether empirical antimicrobial therapy of UTI prescribed by Croatian family practitioners is in accordance with the national guidelines.

[ISKRA guidelines on antimicrobial treatment and prophylaxis of urinary tract infections--Croatian national guidelines]. / ISKRA smjernice antimikrobnog lijecenja i profilakse infekcija mokracnog sustava--hrvatske nacionalne smjernice.

These guidelines refer to diagnosis, antimicrobial treatment and prophylaxis of urinary tract infections in adults and children older than 12 years of age and cover lower urinary tract in females, uncomplicated pyelonephritis, complicated UTI with or without pyelonephritis, asymptomatic bacteriuria and recurrent UTI. These guidelines do not cover sexually transmitted diseases. The guidelines are primarily intended for use by general practitioners and specialists working in primary health care and hospitals. The members of the Working Group for the development of guidelines on antimicrobial treatment and prophylaxis of urinary tract infections were appointed by the Croatian Ministry of Health and Social Welfare. The project was financially supported by the Dutch government and professional assistance was provided by international consultants. The evidence for this guidelines is based on a systematic review of the literature, local antibiotic resistance data, the existing clinical protocols on the treatment and prophylaxis of UTIs, as well as suggestions and comments made by colleagues physicians during more than 50 continuous medical education courses held in the last three years on antimicrobial treatment and prophylaxis of UTIs. Draft version of the guidelines was available for comments on the web site http://iskra.bfm.hr and during the two-month piloting period the guidelines were widely presented to general practitioners, specialists working in primary care and hospitals--urologists, gynecologists, infectious disease specialists, nephrologists. The final version of the guidelines was approved by the Intersectoral Coordination Mechanism for the Control of Antimicrobial Resistance (ISKRA) Board.

Effectiveness and Pharmacokinetic evaluation of Geonistin® (Oxytetracyline and Nystatin) Vaginal Tablets for Unspecific and Mixed Vulvovaginal Infections (GENIE Study).

The GENIE study was performed to evaluate the effectiveness and systemic exposure to oxytetracycline in local treatment of unspecific and mixed vulvovaginal infections characterized by vaginal discharge with Geonistin® vaginal tablets (100 mg oxytetracycline and 100 000 IU nystatin). The total number of subjects enrolled was 189. The treatment had beneficial effects in 100% of the study population. According to the Nugent score, the treatment had a positive effect in 89.2% of participants. The microbiological cure rate was 78.8%. Oxytetracycline concentration levels were from 13.3 to 32.2 ng/mL in 11 out of 15 subjects, and in four subjects the levels were below 10 ng/mL. Geonistin® had a beneficial effect on the unspecific and mixed vulvovaginal infections characterized by vaginal discharge in all efficacy and safety outcomes. Microbiological and the Nugent score efficacy measures confirmed clinical effectiveness. Beneficial efficacy results were achieved with only a few non-serious adverse events.

The role of Chlamydia trachomatis in prostatitis syndrome--our experience in diagnosis and treatment.

Since the beginning of 1999, over 1500 patients with symptoms of chronic prostatitis were examined at Dr. Fran Mihaljevic University Hospital for Infectious Diseases in Zagreb. In almost all of these patients urethral swabs and quantitative segmented bacteriologic cultures and microscopy of expressed prostatic secretion (EPS) or voided bladder urine3 (VB3) were performed as described by Meares and Stamey. Urethral swabs, EPS or VB3 were examined for the presence of Chlamydia (C.) trachomatis by McCoy culture and Lugol stain or by immunofluorescent typing with monoclonal antibodies. In the majority of patients C. trachomatis was demonstrated in parallel in EPS or VB3 by DNA/RNA hybridization method. Normal white blood cell count viewed per high power field<10 was found in 362 (68%) of 536 patients with symptoms of chronic prostatitis and C. trachomatis detected in EPS or VB3. These findings additionally suggest that C. trachomatis can be suspected as a causative pathogen in all categories of chronic prostatitis syndrome. Furthermore, this paper summarizes the results of five previously published clinical studies on the efficacy and tolerability of various treatment schemes for chronic chlamydial prostatitis, conducted from the beginning of 1999 until the end of 2003.

Detection of sexually transmitted pathogens in patients with chronic prostatitis/chronic pelvic pain: a prospective clinical study.

In <10% of patients with prostatitis syndrome, a causative uropathogenic organism can be detected. It has been shown that certain organisms that cause sexually transmitted infections can also cause chronic bacterial prostatitis, which can be hard to diagnose and treat appropriately because prostatic samples obtained by prostatic massage are not routinely tested to detect them. We conducted a clinical study to determine the prevalence of Chlamydia, mycoplasma, and trichomonas infection in 254 patients that were previously diagnosed and treated for chronic prostatitis/chronic pelvic pain syndrome due to negative urethral swab, urine, and prostate samples. Urethral swabs and standard Meares-Stamey four-glass tests were done. Detailed microbiological analysis was conducted to detect the above organisms. Thirty-five (13.8%) patients had positive expressed prostatic secretions/VB3 samples, of which 22 (10.1%) were sexually transmitted organisms that were not detected on previous tests.

Septic abortion caused by Campylobacter jejuni bacteraemia.

A 20-year-old female patient, 14 weeks pregnant, was admitted to hospital with anamnestic and clinical features of acute pyelonephritis. Clinical signs of septic abortion developed and after obstetric examination the therapy was changed to ampicillin, gentamicin and clindamycin. Campylobacter jejuni was isolated from blood cultures. Pathohistological findings confirmed diagnosis of purulent chorioamnionitis. After 2 weeks of ciprofloxacin administration the patient fully recovered. Campylobacter jejuni was not isolated from stool culture and no signs of acute enteritis were registered during the illness. Invasive forms of Campylobacter disease without enteritis are not unusual in immunocompromised hosts but they are restricted to C. fetus rather than C. jejuni isolates.

Platelet satellitism in infectious disease?

BACKGROUND: Platelet satellitism is a phenomenon of unknown etiology of aggregating platelets around polymorphonuclear neutrophils and other blood cells which causes pseudothrombocytopenia, visible by microscopic examination of blood smears. It has been observed so far in about a hundred cases in the world. CASE SUBJECT AND METHODS: Our case involves a 73-year-old female patient with a urinary infection. Biochemical serum analysis (CRP, glucose, AST, ALT, ALP, GGT, bilirubin, sodium, potassium, chloride, urea, creatinine) and blood cell count were performed with standard methods on autoanalyzers. Serum protein fractions were examined by electrophoresis and urinalysis with standard methods on autoanalyzer together with microscopic examination of urine sediment. Erythrocyte sedimentation rate, blood culture and urine culture tests were performed with standard methods. RESULTS: Due to typical pathological values for bacterial urinary infection, the patient was admitted to the hospital. Blood smear examination revealed phenomenon, which has persisted for three weeks after the disease has been cured. Blood smears with EDTA as an anticoagulant had platelet satellitism whereas the phenomenon was not observed in tubes with different anticoagulants (Na, Li-heparin) and capillary blood. DISCUSSION: We hypothesize that satellitism was induced by some immunological mechanism through formation of antibodies which have mediated platelets binding to neutrophil membranes and vice versa. Unfortunately we were unable to determine the putative trigger for this phenomenon. To our knowledge this is the second case of platelet satellitism ever described in Croatia.

The role of unusual pathogens in prostatitis syndrome.

A total of 1442 patients with symptoms of chronic prostatitis were examined over a 4-year period at the Outpatient Department for Urogenital Infections, University Hospital for Infectious Diseases "Dr. Fran Mihaljevic", Zagreb, Croatia. An infectious aetiology was determined in 1070 (74.2%) patients. In 561 of 1070 (52.4%) patients the inflammatory finding (>10 WBC/hpf) was found in expressed prostatic secretions (EPS) or voided bladder urine (VB(3)). Normal, <10 WBCs/hpf was found in 362 of 536 (67.5%) patients with symptoms of chronic prostatitis in whom Chlamydia trachomatis was detected in EPS or VB(3), in 51 of 151 (33.8%) patients with isolated Trichomonas vaginalis and in 40 of 72 (55.6%) patients with isolated Ureaplasma urealyticum. Escherichia coli was the causative pathogen in 95, Enterococcus in 68, Proteus mirabilis in 37, Klebsiella pneumoniae in 16, Streptococcus agalactiae in 19, and Pseudomonas aeruginosa in 3 patients with chronic prostatitis. Other patients had a mixed infection. In patients with chronic bacterial prostatitis (CBP) caused by E. coli, P. mirabilis, K. pneumoniae, E. or S. agalactiae, an inflammatory finding was regularly found in EPS or VB(3).

Comparative randomized pilot study of azithromycin and doxycycline efficacy in the treatment of prostate infection caused by Chlamydia trachomatis.

The study included 125 adult patients (> 18years of age) who had symptoms of chronic prostatitis and proven presence of Chlamydia trachomatis. The presence of C. trachomatis was confirmed in expressed prostatic secretion or in voided bladder urine collected immediately after prostatic massage by a DNA/RNA hybridization method and/or by isolation on McCoy culture and then by immunofluorescent typing with monoclonal antibodies. The patients were randomized in the ratio 2/1; azithromycin/doxycycline, to receive a total of 4.0 g azithromycin over 4 weeks, given as a single dose of 1 x 1000 mg weekly for 4 weeks or doxycycline 100 mg b.i.d. for 28 days. Patients' sexual partners were treated at the same time. Clinical and bacteriological efficacy was evaluated 4-6 weeks after the end of therapy. In the group of patients with chlamydial infection of the prostate, there was no significant difference between the eradication rates (azithromycin 65/82, doxycycline 33/43; P = 0.82) and the clinical cure rates (azithromycin 56/82, doxycycline 30/43; P = 0.94) of the two antimicrobials.

Comparative analysis of azithromycin and ciprofloxacin in the treatment of chronic prostatitis caused by Chlamydia trachomatis.

A total of 89 patients, (>18 years), with symptoms of chronic prostatitis and inflammatory findings as well as the presence of Chlamydia trachomatis confirmed by DNA/RNA DIGENE hybridization method and/or by isolation, McCoy culture and Lugol stain in expressed prostatic secretion or in voided bladder urine collected immediately after prostatic massage, were examined. The patients were randomized to receive a total of 4.5 g of azithromycin for 3 weeks, given as a 3-day therapy of 1 x 500 mg weekly or ciprofloxacin 500 mg b.i.d. for 20 days. Patients' sexual partners were treated at the same time. Clinical and bacteriological efficacy were evaluated 4-6 weeks after the end of therapy. Significantly higher eradication (36/45: 17/44; P=0.0002) and a significantly higher clinical cure (31/45: 15/44; P=0.0021) were achieved in the group of patients treated with azithromycin than in the ciprofloxacin group.

Aetiology of chronic prostatitis.

A total of 388 patients with symptoms of chronic prostatitis and inflammatory findings in expressed prostatic secretion (EPS) or in a urine sample collected immediately after prostate massage, were examined over a 2 year period at the Outpatient Department for Urogenital Infections, University Hospital for Infectious Diseases 'Dr Fran Mihaljevic', Zagreb, Croatia. The infective aetiology was determined in 276 (71.13%) patients. Chlamydia trachomatis was the causative pathogen in 109 patients, Trichomonas vaginalis in 52, Escherichia coli in 26, enterococci in 25, Proteus mirabilis in 14, Klebsiella pneumoniae in six, Streptococcus agalactiae in eight, Ureaplasma urealyticum in seven patients with chronic prostatitis. Other patients had a mixed infection.

[Classification, diagnosis and treatment of prostatitis syndrome]. / Klasifikacija, dijagnostika i lijecenje sindroma prostatitisa.

The term prostatitis syndrome refers to a number of conditions affecting the prostate. Prostatitis syndrome is clinically manifested through symptoms of the lower urogenital tract and perineum. Basic factors in the classification of prostatitis syndrome are clinical symptoms and signs, and the presence of leukocytes and bacteria in selectivelly collected urine samples and in expressed prostatic secretion obtained by the Meares and Stamey localization technique. Antimicrobial therapy is indicated in patients with acute bacterial prostatitis, chronic bacterial prostatitis and chronic inflammatory nonbacterial prostatitis, which also includes bacterial prostatitis unproved by classical methods. Empirical antimicrobial treatment should be initiated immediately in patients with acute bacterial prostatitis and in patients with acute exacerbation of chronic bacterial prostatitis. Targeted antimicrobial therapy is administered in patients with chronic bacterial prostatitis after obtained microbiological results, and empirical antimicrobial therapy lasting for 2-6 weeks in patients with chronic inflammatory nonbacterial prostatitis. Because of their broad spectrum of activity and pharmacodynamic and pharmacokinetic characteristics, fluoroquinolones, ciprofloxacin and ofloxacin are first choice antimicrobial drugs for the treatment of prostatic inflammatory diseases. The efficacy of administered antimicrobial treatment should be followed up 4-6 weeks (early follow-up) and 6 months (late follow up) after the end of antimicrobial therapy. The treatment of a noninflammatory chronic pelvic pain syndrome without proved infection includes phytotherapy, hygienic-dietetic measures, microwave thermotherapy, alpha-adrenergic blocking agents, muscle relaxants, analgesics, non-steroidal antiflogistics, 5-alpha-reductase inhibitors, modified living habits, psychotherapy and antispasmodic analgesics. All patients with chronic types of prostatitis syndrome should avoid drinking alcohol, carbonated beverages, spices, cycling, colds, especially sitting on cold surfaces.

[Guidelines for antimicrobial treatment and prophylaxis of urinary tract infections]. / Smjernice antimikrobnog lijecenja i profilakse infekcija mokracnog sustava.

Recommendations for antimicrobial treatment and prophylaxis of urinary tract infections (UTI) have been made according to the results of investigation of resistance of the most frequent causative agents of UTI to antimicrobial drugs. This investigation has been conducted for the past seven years by the Committee for monitoring bacterial resistance to antibiotics in the Republic of Croatia, with consensus of eight professional societies of the Croatian Medical Association. Uncomplicated cystitis is treated 1, 3, or 7 days, complicated 7 days, pyelonephritis 10-14 days, and complicated UTI 7 to 14 days, rarely longer. For the treatment of cystitis fluorokinolons, nitrofurantoin, betalactam antibiotics, and in the fields of lower resistance trimethoprim/sulfamethoxazol are being used. Single treatment with fluorokinolons is administered to otherwise healthy young women with normal urinary tract in whom cystitis symptoms have been present for less than 7 days. Empiric antimicrobial treatment of pyelonephritis, recurrent and all complicated UTI must be reviewed after urine culture finding is obtained. In the treatment of bacterial prostatitis and febrile UTI in males, the drug of first choice is ciprofloxacin. Asymptomatic bacteriuria (AB) is treated in pregnant women, newborns, preschool children with urinary tract abnormalities, before invasive urologic and gynecologic procedures, in kidney transplant recipients, and in the first days of short term urinary bladder catheterization. Recommendations for the treatment of AB in patients with diabetes mellitus have been controversial in the past two years. Antimicrobial prophylaxis is administered mostly one hour prior to the diagnostic or therapeutic invasive urological procedure, using selected antimicrobial agents.

The significance of Chlamydia trachomatis in prostatitis syndrome.

Research and publication expenses were supported in part by the Croatian Science Foundation and PLIVA Croatia Ltd. (project no. 04/30 'Research on the aetiology, epidemiology, diagnostics, and treatment of patients with prostatitis syndrome').

In vitro susceptibility of urogenital Chlamydia trachomatis strains in a country with high azithromycin consumption rate.

Although Chlamydia trachomatis resistance is not of great concern due to its excellent sensitivity to the currently recommended first-line antibiotics (azithromycin and doxycycline), clinical treatment failures have been reported and some of them were linked to laboratory proved resistance. The aim of this study was to determine in vitro susceptibility to azithromycin and doxycycline for 24 urogenital chlamydial strains isolated in Croatia-a country with the highest consumption of azithromycin in Europe and with very high antibiotic prescription rates. Fourteen isolates from cervical swabs, nine from male urethral swabs, and one isolate from expressed prostatic secretion were tested in McCoy cell culture system. All strains were susceptible to azithromycin and doxycycline with minimal inhibitory concentration for azithromycin and doxycycline ranging from 0.064 to 0.125 µg/mL and 0.016 to 0.064 µg/mL, and minimal chlamydicidal concentration ranging from 0.064 to 2.0 µg/mL and 0.032 to 1.0 µg/mL, respectively. Since we still lack information on whether C. trachomatis is evolving in vivo in response to antibiotic selection pressure, this kind of surveillance for resistance is essential in detecting shifts in antimicrobial susceptibilities.