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.

Impact of Appropriate Empirical Antibiotic Treatment on the Clinical Response of Septic Patients in Intensive Care Unit: A Single-Center Observational Study.

The appropriate antibiotic treatment of patients with bacterial sepsis in the intensive care unit (ICU) remains a challenge. Considering that current international guidelines recommend 7 days of antibiotic therapy as sufficient for most severe infections, our primary outcome was a comparison of clinical response to initial empirical therapy on day 7 and mortality between two groups of septic patients-with appropriate (AEAT) and inappropriate (IEAT) empirical antibiotic therapy according to the in vitro sensitivity of bacteria detected in a blood culture (BC). Adult patients admitted to the ICU between 2020 and 2023, who were diagnosed with sepsis according to the Sequential Organ Failure Assessment (SOFA) score ≥ 2 in association with a suspected or documented infection, were selected for the study. Of the 418 patients, 149 (35.6%) died within 7 days. Although the AEAT group had a lower mortality rate (30.3% vs. 34.2%) and better clinical improvement (52.8% vs. 47.4%) on day 7 after starting empirical antibiotic therapy, there was no significant difference. A causative organism was isolated from BCs in 30% of septic patients, with gram-negative bacteria (GNB) predominating in 60% of cases, and multidrug-resistant (MDR) or extensively drug-resistant (XDR) bacteria predominantly detected in the BCs of the IEAT group. Although the AEAT group had slightly worse clinical characteristics at the onset of sepsis than the IEAT group, the AEAT group showed faster improvement on days 7 and 14 of sepsis. In this retrospective cross-sectional study, the AEAT group was associated with better clinical response at day 7 after sepsis onset and lower mortality, but without a significant difference. Comorbidities and the type of bacterial pathogen should also be taken into account as they can also contribute to the prediction of the final outcome. These results demonstrate the importance of daily assessment of clinical factors to more accurately predict the clinical outcome of a septic patient.

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.

[Comparative urinary bactericidal activity of oral antibiotics against gram-positive pathogens]. / Usporedna urinarna baktericidna aktivnost oralnih antibiotika prema gram-pozitivnim urinarnim patogenima.

In routine bacteriological laboratories the antibacterial activity of antibiotics is determined by in vitro testing, usually by disk-diffusion test. However, in vitro testing does not always reflect antibacterial efficiency of antibiotics in vivo. In this investigation, the urine samples obtained in a single oral dose pharmacokinetic study were examined for their bactericidal activity against a range of relevant Gram-positive urinary tract pathogens. Urinary bactericidal activity of linezolid had been previously compared with ciprofloxacin but not with other oral antibiotics such as beta-lactams. Linezolid showed satisfactory urinary bactericidal titres throughout the whole testing period against all Gram-positive cocci. Fluoroquinolones displayed high and persisting levels of urinary bactericidal activity against staphylococci, but their activity against enterococci was weaker. According to the results of ex-vivo testing amoxycillin could be recommended only for infections caused by E. faecalis. Amoxycillin combined with clavulanic acid can be considered as a therapeutic option for infections caused by S. saprophyticus and E. faecalis. Older cephalosporins had high titres only against S. saprophyticus. Their drawback is a short elimination half-time in urine resulting in rapid decrease of urinary bactericidal titers during dosing interval. Furthermore, they do not show activity against enterococci due to their intrinsic resistance to cephalosporins.

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.

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.

Polyclonal spread of colistin resistant Klebsiella pneumoniae in Croatian hospitals and outpatient setting.

INTRODUCTION: Recently, a marked increase in the rate of colistin resistant Klebsiella pneumoniae was observed in Croatian hospitals and the outpatient setting. This prompted us to analyze the molecular epidemiology of these isolates and the mechanisms of spread. METHODS: In total 46 colistin-resistant K. pneumoniae isolates from five hospitals and the community were analyzed. The presence of genes encoding broad and extended-spectrum ß-lactamases, plasmid-mediated AmpC ß-lactamases and carbapenemases was determined by PCR. Plasmids were characterized by PCR based replicon typing. Isolates were genotyped by pulsed-field gel electrophoresis. Virulence traits such as hemolysins, hyperviscosity and resistance to serum bactericidal activity were determined by phenotypic methods. RESULTS: High resistance rates were observed for cefuroxime, ceftazidime, cefotaxime, ceftriaxone and ertapenem, ciprofloxacin and gentamicin. The majority of OXA-48 producing isolates were resistant to ertapenem but susceptible to imipenem and meropenem. Nine strains transferred ertapenem resistance to E. coli recipient strain. Thirty-nine strains were phenotypically positive for ESBLs and harbored group 1 of CTX-M ß-lactamases. OXA-48 was detected in 39 isolates, KPC-2 in four and NDM-1 in one isolate. The isolates belonged to six PFGE clusters. All isolates were found to be resistant to serum bactericidal activity and all except four strains positive for KPC, produced ß-hemolysins. String test indicating hypermucosity was positive in only one KPC producing organism. CONCLUSIONS: The study demonstrated the ability of K. pneumoniae to accumulate different resistance and virulence determinants. We reported dissemination of colistin resistant K. pneumoniae in five hospitals, located in different geographic regions of Croatia and in the outpatients setting. mcr genes responsible for transferable colistin resistance were not found, indicating that resistance was probably due to chromosomal mutations.

Challenges to antimicrobial susceptibility testing of plant-derived polyphenolic compounds.

As multidrug resistance gains momentum, the last two decades have seen an ever-growing interest in the antimicrobial properties of plant extracts and plant-derived compounds. Most of the focus is on polyphenols - a large and diverse group of phytochemicals with strong antibacterial activity. Testing methods provide reliable results as long as they follow standard procedures. However, methods and procedures used in antimicrobial susceptibility testing (AST) are often too diverse to allow comparison of results. The lack of uniformity and comparability is much owed to the absence of guidelines. The focus of this review is to give a critical overview of different methods used in the assessment of polyphenols antimicrobial efficacy and to highlight the importance of their standardisation.

Tick-borne encephalitis outbreak following raw goat milk consumption in a new micro-location, Croatia, June 2019.

In June 2019, the Croatian Institute of Public Health was informed of a cluster of patients with laboratory confirmed tick-borne encephalitis (TBE) from the Gorski Kotar region. Five of the six patients with TBE reported consuming raw (unpasteurized) goat milk in the two week period before symptom onset, and one reported a recent tick bite. To assess risk factors for infection, we selected six control individuals from among healthy family and community members, and conducted a case-control analysis. None of the cases or controls were vaccinated against TBE. Individuals with TBE (cases) had 25 (95 % CI 0.8-1410.2, p = 0.021) times higher odds of raw goat milk consumption compared to healthy controls. Milk samples from 12 goats from the implicated farm were tested for the TBE virus (TBEV) using RT-PCR. TBEV RNA was not detected in the milk, but serological testing of goats and other farm animals yielded evidence of exposure to the virus: Six goats from the flock had TBEV neutralizing antibodies. Our findings suggest that the vehicle for the outbreak was raw goat milk from a single farm. Following public health advice to cease consumption of raw dairy products, no further cases have been reported.

Epidemic spread of OXA-48 beta-lactamase in Croatia.

PURPOSE: A dramatic increase in OXA-48 ß-lactamase was observed recently not only in large hospital centres, but also in smaller suburban hospital centres in geographic areas bordering Croatia. The aim of the study was to analyse the epidemiology, the mechanisms of antibiotic resistance and the routes of spread of OXA-48 carbapenemase in Croatia. METHODS: Carbapenemase and other ß-lactamase and fluoroquinolone resistance genes were detected by PCR and sequencing. Whole-genome sequencing (WGS) was performed on five representative isolates. The isolates were genotyped by PFGE. RESULTS: Forty-eight isolates positive for OXA-48, collected from seven hospital centres in Croatia from May 2016 to May 2017, were analysed (40 Klebsiella pneumoniae, 5 Enterobacter cloacae, 2 Escherichia coli and one Citrobacter freundii). Thirty-three isolates were ESBL positive and harboured group 1 CTX-M 1 ß-lactamases. In addition to the ß-lactam resistance genes detected by PCR (blaSHV-1, blaOXA-48 and blaOXA-1), WGS of five representative isolates revealed the presence of genes encoding aminoglycoside resistance, aadA2 and aph3-Ia, fluoroquinolone resistance determinants aac(6)Ib-c, oqxA and oqxB, the sulfonamide resistance gene sul1, and fosA (fosfomycin resistance). IncL plasmid was found in all isolates. Two K. pneumoniae isolates belonged to ST16, two E. cloacae to ST66 and E. coli to ST354. K. pneumoniae isolates were allocated to five clusters by PFGE which occured in different hospitals, indicating epidemic spread. CONCLUSIONS: The OXA-48-positive organisms found in this study showed wide variability in antibiotic susceptibility, ß-lactamase content and PFGE banding patterns. This study revealed a switch from the predominance of VIM-1 in 2012-2013 to that of OXA-48 in the 2015 to 2017.

Mechanisms of Carbapenem Resistance in Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa from a Croatian Hospital.

Pseudomonas aeruginosa is an opportunistic pathogen, one of the leading causes of nosocomial infections such as pneumonia, urinary tract infections, and bacteraemia. The bacterial resistance to structurally unrelated antibiotics and its spread within hospitals limits the efficient antimicrobial options and patients' outcome. Carbapenems are important agents for the therapy of infections due to multidrug-resistant (MDR) P. aeruginosa; hence, the development of carbapenem resistance severely hampers effective therapeutic options. The aim of this investigation was to examine mechanisms of carbapenem resistance and genomic diversity in carbapenem-resistant MDR strains of P. aeruginosa, which caused an outbreak among patients in Clinical Hospital Rijeka. Most of the isolates showed decreased expression of porin that is important for the entry of carbapenems (oprD). Overexpression of MexAB-OprM, MexCD-OprJ, and MexEF-OprN efflux systems was observed in many of the isolates. Production of metallo-ß-lactamases was not detected. Typing by pulsed-field gel electrophoresis discriminated the isolates into five clusters. The clonal distribution of the strains was related to the location of hospital departments where the isolates were collected, which implies that most of the infections were caused by spread of the epidemic strains within the hospital.

Listeriosis in pregnancy: case report and retrospective study.

BACKGROUND: Listeria monocytogenes usually causes mild maternal illness, but can be devastating to the fetus. CASE: Listeriosis in a pregnant patient successfully treated with empiric antibiotic therapy is described. After induced delivery, despite severe placental infection, a healthy infant was born. Additionally, archived placental specimens with similar pathological manifestation were evaluated for evidence of L. monocytogenes. CONCLUSION: It is important to emphasize the value of associating maternal data and clinical and laboratory findings of the newborn with the pathologic placental findings to get better results in the etiologic diagnosis of the listeriosis.