Prevalence of occult hepatitis B virus infection and characterisation of hepatitis B surface antigen mutants among adults in western Croatia.

INTRODUCTION AND OBJECTIVES: Occult hepatitis B virus (HBV) infection (OBI) is characterised by low levels of hepatitis B virus (HBV) DNA in the blood/liver of patients with negative hepatitis B surface antigen (HBsAg). This study aimed to determine the OBI prevalence and virological characteristics (viral genotypes and HBsAg mutants) in patients with an "anti-HBc only" serological profile. MATERIALS AND METHODS: A total of 24 900 serum samples were routinely screened for hepatitis B markers over a five-year period. All anti-HBc-positive/HBsAg-negative/anti-HBs-negative sera were selected and analysed for the presence of HBV DNA. Mutational analyses of the HBs gene and polymerase gene sequences were performed. RESULTS: 1749 (7.02%) sera were anti-HBc positive, and 113 (0.45%) sera had an "anti-HBc only" serological profile (HBsAg/anti-HBs negative). HBV DNA was detected in 12/113 (10.61%) "anti-HBc only" positive sera, representing 0.048% of all routinely tested samples. Due to extremely low viremia, HBV genome was successfully sequenced in only two sera where subgenotype D3 was confirmed. Mutational analyses of the S gene revealed multiple missense mutations. In addition to the M133I, Y134F, and G145R mutations, already associated with diagnostic escape, we also found nine novel OBI-related S-gene mutations - S136Y, F158L, K160N, E164G, S167L, A168V, L175S, S210I and F212C. CONCLUSIONS: We detected multiple known and novel S gene mutations in 2/12 (16.6%) OBI cases, nevertheless, further studies are required to determine their role in the pathogenesis of OBI. Understanding the frequencies of clinically relevant HBV mutations may contribute to improvement of diagnostic protocols.

Small intestinal bacterial overgrowth and non-alcoholic fatty liver disease diagnosed by transient elastography and liver biopsy.

BACKGROUND: We aimed to determine if there was a higher incidence of small intestinal bacterial overgrowth (SIBO) in non-alcoholic fatty liver disease (NAFLD) than in patients without NAFLD. Moreover, we assessed whether patients with significant fibrosis (SF) had a higher incidence of SIBO compared with patients with non-significant or no liver fibrosis. METHODS: NAFLD was diagnosed in 117 patients by using Fibroscan with a controlled attenuation parameter (CAP) as well as liver biopsy (LB). SIBO was defined by esophagogastroduodenoscopy with an aspiration of the descending duodenum. RESULTS: Patients with non-alcoholic steatohepatitis (NASH) and those with SF on LB had a significantly higher incidence of SIBO than patients without NASH and those without SF, respectively (P < .05). According to histological characteristics, there was a higher proportion of patients in the SIBO group with higher steatosis and fibrosis grade, lobular and portal inflammation, and ballooning grade (P < .001). In multivariate analysis, significant predictors associated with SF and NASH were type 2 diabetes mellitus (T2DM) and SIBO. Moreover, in multivariate analysis, significant predictors that were independently associated with SIBO were T2DM, fibrosis stage and ballooning grade (OR 8.80 (2.07-37.37), 2.50 (1.16-5.37) and 27.6 (6.41-119), respectively). The most commonly isolated were gram-negative bacteria, predominantly Escherichia coli and Klebsiella pneumoniae. CONCLUSION: In this relatively large population of patients, we used a gold standard for both SIBO (quantitative culture of duodenum's descending part aspirate) and NAFLD (LB), and we demonstrated that NASH patients and those with SF had a higher incidence of SIBO. Moreover, significant predictors independently associated with SIBO were T2DM, fibrosis stage and ballooning grade. Although TE is a well-investigated method for steatosis and fibrosis detection, in our study, independent predictors of SIBO were histological characteristics of NAFLD, while elastographic parameters did not reach statistical significance.

THE PREVALENCE OF ONYCHOMYCOSIS IN THE ZAGREB AREA - THE ROLE OF SABOURAUD AGAR WITHOUT CYCLOHEXIMIDE IN DIAGNOSIS AND THERAPY.

- Mycological analysis is considered the gold standard in the diagnosis of very common fungal diseases of nails. The purpose of the article is to present the prevalence of onychomycosis obtained by using direct microscopic examination (DME) and cultivation on Sabouraud media without cycloheximide. The quantitative retrospective research was conducted on 2706 patients of both sexes (mostly middle-aged to elderly) with various toenail lesions, who had undergone mycological analyses in the Mycological Laboratory of our Department of Dermatovenereology in 2013 and 2014. Analysis of the results obtained revealed that there were 35.4% of positive DME results; of these, there were 39.2% of dermatophytes, 59.1% of yeasts and 1.7% of molds. The percentage of dermatophytes (Trichophyton spp.) was twice as high in men as in women. A considerable percentage (12%) of mixed isolates was also observed. Particularly worrisome was the high percentage (one-third) of suppressed and insufficiently developed dermatophytes (a possible indicator of inappropriate preparation of patients for mycological analysis). Cultivation on culture media without cycloheximide requires frequent inspections because of the fast-growing molds while not allowing sporulation for a great number of dermatophytes. However, it has certain advantages, i.e. it offers an insight into the wide range of the present fungi. As part of the biofilm, they can be potentially pathogenic in dystrophic nails under certain circumstances because, according to our results, the percentage of dermatophytes does not change substantially with aging; however, what does change is the percentage of molds, yeasts and mixed isolates.

Three New Lactobacillus plantarum Strains in the Probiotic Toolbox against Gut Pathogen Salmonella enterica Serotype Typhimurium.

The benefits of probiotic bacteria have been widely explored. However, fermented foods and digestive system of humans and animals are an inexhaustible source of new potentially probiotic microorganisms. In this study we present three new Lactobacillus plantarum strains isolated from different dairy products: cow's cheese, sheep's cheese and whey. In order to determine the antibacterial activity of yet unexplored L. plantarum strains against Salmonella enterica serotype Typhimurium, in vitro competition and co-culture tests were done. Furthermore, adhesion of these strains to Caco-2 cells and their influence on the adhesion of Salmonella were tested. Results showed the potential probiotic activity of isolated strains. L. plantarum strains survived in the presence of 1% bile salts, they possessed acidification ability, antibacterial activity and significantly attenuated the growth of S. Typhimurium in brain heart infusion broth. All tested L. plantarum strains were able to adhere to Caco-2 cells and significantly impair the adhesion of S. Typhimurium. All three L. plantarum strains exhibited significant probiotic potential and anti-Salmonella activity; therefore, further testing on in vivo models should follow.

Superior induction and maintenance of protective CD8 T cells in mice infected with mouse cytomegalovirus vector expressing RAE-1γ.

Due to a unique pattern of CD8 T-cell response induced by cytomegaloviruses (CMVs), live attenuated CMVs are attractive candidates for vaccine vectors for a number of clinically relevant infections and tumors. NKG2D is one of the most important activating NK cell receptors that plays a role in costimulation of CD8 T cells. Here we demonstrate that the expression of CD8 T-cell epitope of Listeria monocytogenes by a recombinant mouse CMV (MCMV) expressing the NKG2D ligand retinoic acid early-inducible protein 1-gamma (RAE-1γ) dramatically enhanced the effectiveness and longevity of epitope-specific CD8 T-cell response and conferred protection against a subsequent challenge infection with Listeria monocytogenes. Unexpectedly, the attenuated growth in vivo of the CMV vector expressing RAE-1γ and its capacity to enhance specific CD8 T-cell response were preserved even in mice lacking NKG2D, implying additional immune function for RAE-1γ beyond engagement of NKG2D. Thus, vectors expressing RAE-1γ represent a promising approach in the development of CD8 T-cell-based vaccines.

Sequence types 235, 111, and 132 predominate among multidrug-resistant pseudomonas aeruginosa clinical isolates in Croatia.

A population analysis of 103 multidrug-resistant Pseudomonas aeruginosa isolates from Croatian hospitals was performed. Twelve sequence types (STs) were identified, with a predominance of international clones ST235 (serotype O11 [41%]), ST111 (serotype O12 [15%]), and ST132 (serotype O6 [11%]). Overexpression of the natural AmpC cephalosporinase was common (42%), but only a few ST235 or ST111 isolates produced VIM-1 or VIM-2 metallo-ß-lactamases or PER-1 or GES-7 extended-spectrum ß-lactamases.

In vivo modulation of Campylobacter jejuni virulence in response to environmental stress.

Campylobacters have developed a number of mechanisms for responding to environmental conditions, although the different virulence properties of these cells following exposure to stress are still poorly understood. We analyzed in vitro stress responses and the consequent in vivo modulation of Campylobacter jejuni pathogenicity in BALB/c mice, as a result of the exposure of the C. jejuni to environmental stress (starvation, oxidative stress, heat shock). In vitro, the influence of starvation and oxidative stress was milder than that of heat shock, although the majority of the stress conditions influenced the survival of C. jejuni. During starvation, C. jejuni viability was maintained longer than its culturability. Additionally, starvation elicited transformation of stressed bacteria to coccoid forms. In contrast, bacteria exposed to oxygen remained culturable, but their viability decreased. Pre-starvation did not contribute to improved survival of C. jejuni cells during oxygen exposure. Changes in bacteria numbers and the levels of several cytokines (interleukins 6 and 10, tumor necrosis factor-α, interferon-γ) were followed in vivo, in liver homogenates from the mice intravenously infected with either control (untreated) or stressed C. jejuni. The systemic infection with the control or stressed C. jejuni occurred with different production dynamics of the cytokines investigated. Starvation was the most powerful stress factor, which significantly decreased infectious potential of C. jejuni during the first 3 days postinfection. The most pronounced differences in cytokine production were found in interferon-γ and interleukin-10 production, which indicates that these have roles in the immune response to C. jejuni infection. These in vivo studies of environmental impact on bacterial virulence reveal that microbial adaptation during stress challenge is crucial not just for pathogen survival out of the host, but also during host-pathogen interactions, and thus for the bacterial pathogenicity.

New Insights into Foodborne Bacteria-Host Interactions: Evolving Research and Discoveries.

Given the growing concern about foodborne diseases, intensive research and the development of new approaches are crucial [...].

Borderline SARS-CoV-2 patients: the trace behind.

BACKGROUND: Effective and precise SARS-CoV-2 detection assays are crucial for maintaining regular hospital routines and identifying infected hospital employees and infected patients before hospital admission. Inconclusive PCR test results of potentially infectious borderline SARS-CoV-2 patients can confuse clinicians and delay appropriate infection control. OBJECTIVES AND STUDY DESIGN: In this retrospective study, we followed up borderline SARS-CoV-2 patients who were tested (from the second sample with the same method) at the Clinical Department of Clinical Microbiology. We aimed to determine the positivity conversion ratio within 7 days after inconclusive PCR test results. RESULTS: Out of 247 borderline patients, who were resampled and retested in the same laboratory, 60 patients (29.4%) showed conversion of the borderline viral load (inconclusive RT-PCR test) to a positive RT-PCR test result. CONCLUSIONS: Our results highlight the need for retesting of borderline patients with inconclusive SARS-CoV-2 results. Follow-up testing of inconclusive PCR results within 7 days can identify additional positive results and reduce the potential risk of intrahospital transmission.

Coxiella burnetii (Q-fever) outbreak associated with non-occupational exposure in a semi-urban area of western Croatia in 2022.

In March 2022, an outbreak of Q fever (Coxiella burnetii) with non-occupational exposure was confirmed in a semi-urban area in Cavle, Croatia. Veterinary and human epidemiological investigations were conducted to identify the source of the outbreak and to implement appropriate control measures. Three farms were settled next to each other near the homes of the first human cases at the end of the street. The closest farm was less than 500 meters away. These farms contained 161 adult sheep and goats. Among the animal samples analysed, all 16 goats (100%) and 24/50 sheep (48%) tested positive for C. burnetii IgM/IgG antibodies, phase I and II. One out of five sheeps' vaginal swabs were C. burnetti DNA positive. Human testing revealed 20 confirmed and three probable cases (9/23 pneumonia, 2/23 hepatitis, 21/23 fever), with three hospitalizations, and one death. Twenty-seven cases were discarded following negative laboratory results. The epidemiological investigation revealed airborne transmission as the most likely route of transmission. Multiple logistic regression analyses were used to evaluate risk factors for Q fever infection. Persons who were near the farms (≤750 m) (OR 4.5; 95% CI = 1.1-18.3) and lived in the nearest street to the farms had the highest risk of contracting Q fever (OR 3.7; 95% CI = 1.1-13.6). Decreased rainfall compared to monthly averages was recorded in the months prior to the outbreak with several days of strong wind in January preceding the outbreak. This was the largest Q fever outbreak in the county in the last 16 years, which was unexpected due to its location and non-occupational exposure. To stop the outbreak, numerous intensive biosecurity measures were implemented. The outbreak highlights the importance of urban development strategies to limit the number of animal housing near residential areas while providing regular biosecurity measures to prevent infections in livestock.

Breast abscess in a man due to Salmonella enterica serotype Enteritidis.

Nontyphoidal salmonellae can cause breast infection only exceptionally. A case of breast abscess in a 70-year-old man due to Salmonella enterica serotype Enteritidis (Salmonella Enteritidis) is reported. The infection was successfully treated with a combination of surgical and antibiotic treatment.

Alloiococcus otitidis-Cause of Nonspecific Acute Sinusitis: First Case Report and Review of Literature.

Although most sinus infections are viral, potential bacterial pathogens such as Streptococcus pneumoniae, Haemophilus influenza and Moraxella catarrhalis can migrate during a viral respiratory infection from the nasopharynx into the sinus cavity causing sinusitis. Alloiococcus otitidis is a commensal of the external auditory canal and is considered one of the potential middle ear pathogens. Unlike most otopathogens, A. otitidis is rarely found in the nasopharynx of healthy individuals. This difficult-to-culture organism has not previously been described as a causative agent of sinusitis. Here we describe one case of acute sinusitis due to A. otitidis and review previous knowledge of this controversial organism based on recent literature.

The effect of nickel ions on the susceptibility of bacteria to ciprofloxacin and ampicillin.

To explore the interaction effects of ciprofloxacin and ampicillin with nickel on the growth of bacteria, Staphylococcus aureus strain ATCC 29213, Enterococcus faecalis ATCC 29212 and Escherichia coli ATCC 25922 were used. Minimum inhibitory concentrations (MICs) were determined for nickel, ciprofloxacin and ampicillin, and the checkerboard method was used to assess their cumulative effects on bacterial growth. The interactions between the metal and antibiotics were assessed by the fractional inhibitory concentration (FIC). The MICs for ciprofloxacin and ampicillin were 0.31 and 1 mg/L for E. faecalis, 0.62 and 1 mg/L for S. aureus and 0.005 and 2.5 for E. coli, respectively. The MIC for nickel was 1000 mg/L for all bacteria. The FIC results for ciprofloxacin and nickel demonstrated an antagonistic effect of the two agents on the growth of E. coli and E. faecalis and an additive effect on S. aureus. The FICs for ampicillin and nickel demonstrated a synergistic effect on the growth of E. faecalis and E. coli. Different interactions of metals and antibiotics were observed depending on the bacteria and the type of antibiotic.

Epidemiologic characteristics of human campylobacteriosis in the County Primorsko-goranska (Croatia), 2003-2007.

The aim of the study was to investigate campylobacteriosis incidence in the County Primorsko-goranska (Croatia) between 2003 and 2007 and to find out possible connection with environmental factors (the average monthly temperature and total monthly precipitation). The data (number of stool samples examined, age and sex distribution of patients, monthly distribution of isolates and distribution of isolates according to the species) from the Laboratory for Diagnostics of Enteric Infections of the Teaching Institute of Public Health of the County Primorsko-goranska (Croatia) were analyzed retrospectively. During the observed period 30,164 stool samples were examined for Campylobacter spp. Campylobacters were identified in 1,242 (4.12%) samples. The overall annual campylobacter incidence rate was 81.3 +/- 21.9/100,000 population. Campylobacter jejuni was found in 1,093 (88%) and C. coli in 149 (12%) patients. Our findings showed age distribution of patients typical for developed countries. The patients were mostly children under 5 years (484.4 +/- 129.1/ 100,000, p < 0.001) and between 5 and 9 years of age (226.5 +/- 60.5/100,000, p < 0.05). Male consistently experienced higher rates, but the difference between genders was significant in the age groups from birth till late twentieth (p < 0.001). Campylobacter rates were significantly associated with monthly average temperatures (p < 0.05), but not with precipitation. Further investigations into the incidence of campylobacteriosis on the national level are necessary. The causes of the noticed monthly distribution, sources of infection and connection with the routes of transmission in humans need to be elucidated as well.

Diagnostic accuracy of three SARS-CoV2 antibody detection assays, neutralizing effect and longevity of serum antibodies.

Evidence is currently insufficient to know whether SARS-CoV-2 antibodies (Abs) protect from future infection and how long immunity will last. The kinetics of the immune response to SARS-CoV-2 infection and role of serology in estimating individual protective immunity is yet to be established. We evaluated diagnostic performances of three serological assays - Abbott Architect CMIA IgG, bioMerieux VIDAS ELFA IgG/IgM, and Diesse Chorus ELISA IgG/IgM, and analyzed longevity and potential neutralizing effect of SARS-CoV-2 Abs in COVID-19 patients. Clinical sensitivities of assessed IgG tests two to three weeks post symptom onset (PSO) were very high: 96.77 % for Architect, 96.77 % for Chorus, and 100.00 % for VIDAS. Sensitivities of two assessed IgM assays were moderate: 74.07 % for Chorus, and 76.92 % for VIDAS. Specificities were excellent for all assessed IgG assays: 99.01 % for Architect and 100 % for Chorus and VIDAS. Chorus and VIDAS IgM assays also achieved excellent specificity of 99.01 % and 100 %, respectively. In most cases IgG Abs were still present eight months PSO. Neutralizing antibodies were detected in majority of serum samples from convalescent patients. Serum samples from severe COVID-19 patients had higher antibody titers and higher neutralizing activity. We observed a strong positive correlation among SARS-CoV-2 IgG antibody titer and neutralizing activity. The strongest positive correlation to neutralizing activity was found for VIDAS IgG assay.

An outbreak of ertapenem-resistant, carbapenemase-negative and porin-deficient ESBL-producing Klebsiella pneumoniae complex.

INTRODUCTION: Carbapenem-resistant Klebsiella pneumoniae is an emerging healthcare-associated pathogen with dynamic molecular epidemiology. This study presents a retrospective analysis of the distribution and antibiotic resistance patterns of ertapenem-resistant ESBL-producing K. pneumoniae strains recovered during an outbreak from 2012 to 2014 in a Croatian University hospital. METHODS: We aimed to estimate genetic relatedness of clinical isolates and underlying mechanisms that conferred the ertapenem-resistant phenotype. RESULTS: Expression analysis of genes involved in the antibiotic resistance showed reduced expression of major non-selective porin channel OmpK35. Reduced expression of OmpK36 porin channel in isolates resistant to at least one more carbapenem, apart from the ertapenem, was found to a lesser degree. Pulsed-field gel electrophoresis analysis of genomic DNA revealed that almost all isolates belonged to the same genetic clone. CONCLUSIONS: Caution regarding ertapenem-resistant, carbapenemase-negative porin-deficient mutants of K. pneumoniae is required as they are widespread, and under selective pressure this could result in a local clonal outbreak.

Bacterial Exposure to Nickel: Influence on Adhesion and Biofilm Formation on Orthodontic Archwires and Sensitivity to Antimicrobial Agents.

The presence of nickel could modify bacterial behavior and susceptibility to antimicrobial agents. Adhesion and biofilm formation on orthodontic archwires can be a source of bacterial colonization and possible health hazards. Staphylococcus aureus was subjected to exposure and adaptation to various sub-inhibitory concentrations of nickel. Five strains of bacteria adapted to nickel in concentrations of 62.5-1000 µg/mL were tested for adhesion and biofilm formation on nickel-titanium archwires. Archwires were previously incubated in artificial saliva. Bacteria were incubated with orthodontic wire with stirring for 4 h (adhesion) and 24 h (biofilm formation). The number of adherent bacteria was determined after sonication and cultivation on the Muller-Hinton agar. Disk diffusion method was performed on all bacteria to assess the differences in antimicrobial susceptibility. Bacteria adapted to lower concentrations of nickel adhered better to nickel-titanium than strains adapted to higher concentrations of nickel (p < 0.05). Biofilm formation was highest in strains adapted to 250 and 500 µg/mL of nickel (p < 0.05). The highest biofilm biomass was measured for strains adapted to 250 µg/mL, followed by those adapted to 1000 µg/mL. Bacteria adapted to lower concentrations of nickel demonstrated lower inhibition zone diameters in the disk diffusion method (p < 0.05), indicating increased antimicrobial resistance. In conclusion, bacteria adapted to 250 µg/mL of nickel ions adhered better, demonstrated higher biofilm formation and often had higher antimicrobial resistance than other adapted and non-adapted strains.

Does Fluoroquinolones and Third-Generation Cephalosporins Restriction Reverse Extended-Spectrum ß-Lactamases Klebsiella pneumoniae Resistance Rates?

Aim: To decrease the incidence and resistance rates of extended-spectrum ß-lactamases (ESBL) Klebsiella pneumoniae (KP) by restriction of the use of third-generation cephalosporins (3GCs) and fluoroquinolones. Methods: Consumption of 3GCs, fluoroquinolones, and carbapenems in association with ertapenem and fluoroquinolone-resistant KP isolates, were analyzed in 21 months by autoregressive integrated moving average models. A follow-up analysis was performed 5 years later. Results: Consumption of 3GCs decreased significantly during the postintervention period. Their restriction was associated with a decrease in ertapenem-resistant KP isolates by 17.5%. Fluoroquinolone, 3GCs, and carbapenem use did not significantly predict the percentage of ertapenem-resistant KP isolates. Fluoroquinolone, but not cephalosporin use, significantly predicted the percentage of fluoroquinolone-resistant isolates, with an increase of 1 defined daily dose (DDD) of fluoroquinolone/100 occupied bed-days (OBDs) corresponding to a 0.32% increase of fluoroquinolone-resistant isolates (p = 0.008). A decrease of 1 DDD of carbapenem/100 OBD was associated with a 16.94% increase of fluoroquinolone-resistant isolates (p = 0.007). Five years later, the consumption of all three antimicrobial classes increased significantly compared with the 2011-2013 period, whereas ertapenem-resistant KP rates significantly decreased. Conclusion: This study may bring a valuable contribution to the understanding of the intricate association between antibiotic consumption and bacterial resistance. Reporting a spectrum of different results could present a useful basis for more profound research of various interventions' effects.

Listeria monocytogenes (delta-actA mutant) infection in tumor necrosis factor receptor p55-deficient neonatal mice.

Using TNF receptor 1 knock out (TNFR1KO) mice, we investigated the role played by TNFR1 in immune regulation during neonatal listeriosis. Induction of protective immune response in wild type pups resulted in the prompt control of infection with an attenuated DeltaactA mutant Listeria monocytogenes, accompanied by enhanced hepatic expression of mRNA for IFN-gamma, TNF-alpha, and IL-10. Conversely, the lack of TNFR1 signalling in TNFR1KO neonatal mice resulted in substantial changes in the profile of inflammatory mediators and ultimately fatal outcome of the infected pups. Despite remarkable increase in indoleamine 2, 3-dioxygenase (IDO) and inducible nitric oxide synthase (iNOS) mRNA detected in the liver of TNFR1KO mice, bacterial proliferation was unrestrained. Increased mRNA expression of IDO, iNOS, TNF-alpha, IFN-gamma, MCP-1, and MIP-1alpha was found in the spleens of infected KO mice, and in the brains mRNA encoding iNOS, IDO, IFN-gamma, IL-12p40, IL-10, and RANTES was also upregulated. Large necrotic lesions consisting of granulocytes and macrophages were scattered throughout the liver of these mice. TNFR1KO neonates were unable to clear neutrophils and switch from the innate immune response to a specific reaction mediated by T cells. These results prove that TNF-alpha signalling is crucial and irreplaceable in antilisterial protection during the neonatal period.