A Snapshot of Drug-Resistant M. tuberculosis Strains in Croatia.

Tuberculosis caused by resistant M. tuberculosis strains poses a serious threat as it requires prolonged and costly treatment and has high mortality rate. In order to investigate resistance to antituberculous drugs in Croatia, we analysed all resistant M. tuberculosis strains isolated from patients' samples in period 2010-2014 (1 strain per patient). Out of 2384 M. tuberculosis strains, we identified 88 (3.69 %) resistant strains. The analysis included resistance patterns, resistance conferring mutations and, according to MIRU-VNTR analysis, clustering and global lineages distribution. Relatively high number of strains was monoresistant, especially to isoniazid, while there were only six multiresistant strains. Among 59 strains with any pattern that includes resistance to isoniazid, a total of 22 (37.29 %) had resistance conferring mutation in katG gene (S315T), 23 (38.98 %) in inhA promoter region (C-15T) and 14 (23.73 %) had none of these mutations. The observed clustering rate of resistant strains was 28.41 %, and the most common global lineage was Euro-American (75 %).

Nosocomial pseudo-outbreak of Mycobacterium gordonae associated with a hospital's water supply contamination: a case series of 135 patients.

Nontuberculous mycobacteria (NTM) are opportunistic pathogens found in natural and human-engineered waters. In 2009, a relative increase in the isolation of Mycobacterium gordonae from pulmonary samples originating from General Hospital Zabok was noted by the National Mycobacteria Reference Laboratory. An epidemiological survey revealed a contamination of the cold tap water with M. gordonae and guidelines regarding sputum sample taking were issued. In addition, all incident cases of respiratory infection due to NTM reported from 2007 to 2012 at General Hospital Zabok were included in a retrospective review. Out of 150 individual NTM isolates, M. gordonae was the most frequently isolated species (n = 135; 90%) and none of the cases met the criteria of the American Thoracic Society for pulmonary NTM disease. While concomitant Mycobacterium tuberculosis infection was confirmed in only 6 (4%) patients, anti-tuberculosis treatment was initiated for a significant portion of patients (n = 64; 42.6%) and unnecessary contact tracing was performed. This study points out the need to enhance the knowledge about NTM in our country and indicates the importance of faster NTM identification, as well as the importance of good communication between laboratory personnel and physicians when evaluating the significance of the isolated NTM.

[Which tuberculin skin test hyperreactive child should be treated with -our experience]. / KOJIM PPD-HIPERREAKTORIMA TREBA DATI PROFILAKSU - NASA ISKUSTVA.

Since persons with latent tuberculosis infection (LTBI) represent a huge reservoir of potential tuberculosis (TB) disease, accurate diagnosis and treatment of LTBI is essential for TB control and eradication. The aim was to assess the diagnostic value of determination of interferon-gamma release assay in school children with hyperreactive tuberculin skin test (TST) reaction. A total of 120 BCG-vaccinated children were investigated due to a hyperreactive TST results. The QuantiFERON-TB Gold In-Tube test (QFT-GIT) was performed. Fifteen children (12.5%) had positive QFT-GIT and 105 (87.5%) children had negative QFT-GIT. There was no statistically significant difference in TST reaction (21.5 mm u QFT+ vs. 20.9 mm u QFT-group, p=0.458). The children with positive QFT-GIT had a statistically higher level of interferon-gamma (IFN-γ) than children with negative QFT-GIT. There were no statistically significant differences in concentrations of IFN-y either basic or upon stimulation with mitogen phytohemagglutinin. After isoniazid prophylaxis QFT-GIT remained positive in two children (p=0.019). In a difficult procedure for diagnosing LTBI in BCG-vaccinated children determination of IFN-γ could be the key factor in making decision whether to use preventive therapy or not.

First insights into the molecular epidemiology of tuberculosis in Croatia during a three-year period, 2009 to 2011.

BACKGROUND: Mycobacterium tuberculosis still represents a serious cause of morbidity and mortality worldwide. The aim of this study was to determine the transmission rate and genetic lineages of M. tuberculosis circulating in Croatia during a 3-y period, between 2009 and 2011. METHODS: A total of 1587 M. tuberculosis strains (1 strain per tuberculosis patient) isolated in Croatia from 2009 to 2011 were genotyped using 15-locus mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) analysis. RESULTS: The majority of tested isolates (66.73%) belonged to the Euro-American global lineage. The most prevalent sub-lineages were Haarlem (48.64%), followed by S (6.05%), Cameroon (3.72%), and Latin American-Mediterranean (3.4%). Among the total 1587 tested isolates, 996 (63%) were included in 1 of 236 clusters. The cluster size ranged from 2 (114 clusters) to 45 (1 cluster) patients, the mean cluster size being 4.2. These results indicate that 47.83% of tuberculosis cases during the period analyzed were the result of recent transmission. CONCLUSIONS: The most prevalent global lineage in Croatia is Euro-American (sub-lineages Haarlem, S, Cameroon, and Latin American-Mediterranean). The high clustering rate and high medium clustering size of 4.2 tuberculosis cases could indicate a possible failure in interrupting the transmission of infection and points to the need for improvements in national and local tuberculosis control activities. This is the first study describing the molecular epidemiology of tuberculosis in Croatia.

Large Anthrax Outbreak in a New Locality in Croatia, 2022.

Bacillus anthracis is a well-known zoonotic pathogen that can cause disease in both animals and humans. Moreover, it has a high bioterrorism potential as its lethal spores are resistant to inactivation, are easy to produce in large quantities, and are easily spread over large areas. Anthrax cases occur in different parts of the world, including most European countries. Specific areas of Croatia are long known as anthrax districts, but with sporadically reported cases over the years. Here, we present a major outbreak of animal and human anthrax in the region of Lonjsko Polje in Croatia, a region not known to have anthrax cases in the past. The outbreak started in July 2022 and lasted several months, but most human and animal cases were reported in the first month. During the outbreak, there were 17 reported human cases of cutaneous anthrax and 29 laboratory-confirmed animal cases. However, due to issues in reporting in animals and the late finding of the carcasses, which made laboratory diagnostics challenging, the actual number of animal cases was probably significantly higher.

Proinflammatory Chemokine Levels in Cerebrospinal Fluid of Patients with Neuroinvasive Flavivirus Infections.

Tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) are the most important neuroinvasive arboviruses detected in Europe. In this study, we analyzed cerebrospinal fluid (CSF) concentrations of 12 proinflammatory chemokines (CCL2, CCL3, CCL4, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, and CXCL11) in 77 patients with neuroinvasive diseases (NIDs). Flavivirus infection was confirmed in 62 patients (TBEV and WNV in 31 patients each), while in 15 patients the etiology of NID was not determined (NDE). Similar patterns of high-level expression of chemokines regulating monocyte/macrophage responses (CCL2), neutrophil recruitment (CXCL1 and CXCL8), and interferon-inducible chemoattractants for leukocytes (CXCL10 and CXCL11) have been observed in WNV and TBEV groups. None of the tested chemokines significantly differed between patients with TBEV or WNV. Concentrations of CCL17, CCL20, CXCL5, CXCL10, and CXCL11 were significantly lower in both WNV and TBEV groups compared to NID NDE patients. The logistic regression model showed that CSF concentrations of CXCL11, CXCL5, and CXCL10 could potentially be used for the classification of patients into the WNV or TBEV group versus groups with other NIDs. This study identified, for the first time, similar patterns of CSF chemokine expression in WNV and TBEV infections, suggesting common immunopathogenic mechanisms in neuroinvasive flavivirus infections that should be further evaluated.

SARS-CoV-2 Omicron Variant in Croatia-Rapid Detection of the First Case and Cross-Border Spread.

BACKGROUND: Due to rapid spread, the Omicron variant has become the dominant SARS-CoV-2 variant responsible for infections worldwide. We present the first detection of the Omicron variant in Croatia which resulted in rapid cross-border spreading. METHODS: Whole-genome sequencing was performed using the Illumina MiniSeq sequencing system. SARS-CoV-2 lineages were identified using the PANGOLIN and GISAID databases. RESULTS: The first case of the Omicron variant (BA.1.17) emerged in Croatia after a workshop held in Zagreb in November 2021. The patient reported a history of previous COVID-19 and received two doses of an mRNA vaccine. Three additional cases were detected among Croatian participants of the workshop. At the beginning of December, SARS-CoV-2 infection was confirmed in one participant from Montenegro and her husband. Phylogenetic analysis showed that the detected Omicron variants were closely related to the first Croatian case, confirming the connection with the workshop outbreak and rapid cross-border spreading. Subsequent analyses of SARS-CoV-2 positive samples in Croatia showed the rapid introduction of the Omicron variant and depletion of the Delta variant resulting in the fifth pandemic wave. CONCLUSIONS: Genomic monitoring and early detection of novel SARS-CoV-2 variants are essential to implement timely epidemiological interventions and reduce further transmission in the population.

A fatal Mycobacterium chelonae infection in an immunosuppressed patient with systemic lupus erythematosus and concomitant Fahr's syndrome.

We present a case of systemic Mycobacterium chelonae infection in an immunosuppressed patient with systemic lupus erythematosus (SLE), idiopathic hypoparathyroidism, and hypothyroidism. The patient was treated for 3 months for skin infection with clarithromycin monotherapy. Since her condition deteriorated, the antibiotic therapy was switched to intravenously administered clindamycin, cloxacillin, and meropenem. Due to further deterioration and isolation of M. chelonae from the blood culture, antimicrobial therapy was changed to azithromycin and amikacin. Drug-test sensitivity was performed, and the isolate was susceptible to clarithromycin only. The patient's deteriorating status prevented orally administered medication with clarithromycin (parenteral formulation is not registered in Croatia). The same antibiotic regime was continued until the isolation of Pseudomonas aeruginosa and Candida albicans. In addition, extensive calcifications in her brain were found on a computed tomography (CT) scan, which suggested Fahr's syndrome. Despite all measures and supportive care, the patient developed multiorgan failure and eventually died. There has been an increase in the number of infections by rapidly growing mycobacteria, but only a few cases of severe systemic infection with M. chelonae have been described. If the infection is diagnosed early and a patient is treated with appropriate drugs, dissemination can be avoided despite immunosuppression. For serious skin, bone, and soft-tissue disease, a minimum of 4 months of a combined drug therapy is necessary. This is the first report of M. chelonae infection in Croatia and the first-described M. chelonae infection in a patient with concomitant Fahr's syndrome.

Neglected tuberculous trochanteric bursitis in an adolescent girl: A case report and literature review.

Tuberculous trochanteric bursitis (TTB) is an extremely rare form of extrapulmonary tuberculosis. Due to a low clinical suspicion and poor collaboration among medical professionals, the diagnosis of TTB can be often delayed. In this report, we describe a case of neglected TTB in an adolescent girl that initially presented with right thigh swelling and fluctuance. The patient underwent repeated unsuccessful surgical treatment; however, dull pain and periodic wound drainage remained for eight years. Complete excision of fistula and trochanteric bursa and one year of oral antituberculous drug therapy led to complete recovery. This case report highlights tuberculosis as a diagnostic challenge, when rare localizations are affected. In addition, this report addresses several diagnostic pitfalls and reviews the literature regarding TTB in adolescent patients. Orthopedic surgeons need to consider TTB, when swelling, fluctuance or repeated wound drainage are present on the thigh.

Risk factors for nontuberculous mycobacterial pulmonary disease (NTM-PD) in Croatia.

BACKGROUND: The incidence, geographical distribution and clinical relevance of different nontuberculous mycobacteria (NTM) in Croatia are well described. There are few data on the risk factors for developing NTM pulmonary disease (NTM-PD) in this setting. METHODS: We conducted a retrospective cohort study on all Croatian residents with NTM isolated from respiratory samples in the period from 2006 to 2015 with follow-up to 2018. The American Thoracic Society/Infectious Diseases Society of America (ATS/IDSA) guidelines were used to establish NTM-PD diagnosis. Clinical, radiological and treatment data were collected from hospital records. RESULTS: Risk analysis calculations were made on the 439 isolation episodes that were classified as definitive NTM-PD (n = 137) or no disease (n = 302). Female gender, presence of bronchiectasis, low BMI and long-term systemic corticosteroid treatment were independent risk factors associated with NTM-PD. Hemoptysis and malaise were presenting symptoms independently associated with NTM-PD. Chronic obstructive pulmonary disease (COPD) and low/moderate dose inhaled corticosteroid (ICS) treatment were not associated with NTM-PD. High dose ICS treatment was a significant risk factor for developing NTM-PD (aOR = 4.73, CI 1.69-13.23 p = 0.003). CONCLUSION: The NTM-PD patients in Croatia are similar to those in other published cohorts in terms of their characteristics and risk factors. The significant dose-dependent association between ICS use and NTM-PD adds to the body of evidence suggesting that high dose ICS use is associated with NTM-PD.

Emerging Trends in the Epidemiology of COVID-19: The Croatian 'One Health' Perspective.

During the four pandemic waves, a total of 560,504 cases and 10,178 deaths due to COVID-19 were reported in Croatia. The Alpha variant, dominant from March 2021 (>50% of positive samples), was rapidly replaced by Delta variants (>90%) by August 2021. Several seroprevalence studies were conducted in different populations (general population, children/adolescents, professional athletes, healthcare workers, veterinarians) and in immunocompromised patients (hemodialysis patients, liver/kidney transplant recipients). After the first pandemic wave, seroprevalence rates of neutralizing (NT) antibodies were reported to be 0.2-5.5%. Significantly higher seropositivity was detected during/after the second wave, 2.6-18.7%. Two studies conducted in pet animals (February-June 2020/July-December 2020) reported SARS-CoV-2 NT antibodies in 0.76% of cats and 0.31-14.69% of dogs, respectively. SARS-CoV-2 NT antibodies were not detected in wildlife. Environmental samples taken in the households of COVID-19 patients showed high-touch personal objects as most frequently contaminated (17.3%), followed by surfaces in patients' rooms (14.6%), kitchens (13.3%) and bathrooms (8.3%). SARS-CoV-2 RNA was also detected in 96.8% affluent water samples, while all effluent water samples tested negative. Detection of SARS-CoV-2 in humans, animals and the environment suggests that the 'One Health' approach is critical to controlling COVID-19 and future pandemics.

Neglected disease in mentally ill patients: Major tuberculosis outbreak in a psychiatric hospital.

As tuberculosis incidence decreases, the possibility of overlooking the disease increases, especially in vulnerable populations. We describe here a major tuberculosis outbreak among mentally ill patients in Croatia, focusing on 1 regional hospital where most patients were hospitalized. The outbreak emphasizes the vulnerability of mentally ill patients to tuberculosis infection and the complexity of infection control measures in psychiatric institutions. The awareness of tuberculosis in these settings should be maintained to interrupt prolonged exposure and avoid unnecessary infection.

Evaluation of Xpert MTB/RIF assay for rapid molecular diagnosis of tuberculosis in a two-year period in Croatia.

Mycobacterium tuberculosis remains a major global health problem and is currently killing 1.5 million people every year. One of the most important steps in tuberculosis control is the rapid and accurate laboratory diagnosis. The Xpert MTB/RIF assay is a novel molecular, easy-to-use assay, which can lead to tuberculosis identification in less than 2h. In this study, the Xpert MTB/RIF assay performance for rapid diagnosis of tuberculosis was evaluated in comparison with conventional culture methods; 361 pulmonary and extrapulmonary patient samples were collected between October 2010 and October 2012 and were analyzed at the National Reference laboratory for Mycobacteria, Zagreb, Croatia. For pulmonary samples the sensitivity and specificity were 86% and 100%, while for extrapulmonary samples the sensitivity and specificity were 75% and 99%, respectively. It was concluded that Xpert MTB/RIF assay has high sensitivity and specificity for both pulmonary and extrapulmonary specimens.

Zoonoses and vector-borne diseases in Croatia - a multidisciplinary approach.

Emerging and re-emerging infectious diseases create constant and serious concerns for public health. The majority of emerging infectious diseases (EID) are wildlife zoonotic diseases and vector-borne diseases. Croatia has a long tradition in the control, management and research of EID zoonotic diseases and vector-borne diseases. There has also been a long and advantageous tradition in the collaboration of different experts and professionals in EID research in Croatia involving physician clinicians in infectious diseases, microbiologists, pathologists, veterinarians and animal scientists, ecologists, forestry experts, wildlife scientists, public health specialists and epidemiologists and laboratory scientists. The University Hospital for Infectious Diseases in Zagreb established the Centre for Emerging and Re-emerging Infectious Diseases in liaison with national and international partners from Europe and the United States. This Centre is working in line with the 'One Health initiative' which recognises the inter-relationships between human, animal and environmental health.