The most common type of periodontal disease is chronic periodontitis, an inflammatory condition caused by pathogenic bacteria in subgingival plaque. The aim of our study was the development of a ...real-time PCR test as a diagnostic tool for the detection and differentiation of five periodontopathogenic bacteria,
,
,
,
, and
, in patients with periodontitis. We compared the results of our in-house method with the micro-IDent
semiquantitative commercially available test based on the PCR hybridization method. DNA was isolated from subgingival plaque samples taken from 50 patients and then analyzed by both methods. Comparing the results of the two methods, they show a specificity of 100% for all bacteria. The sensitivity for
was 97.5%, for
96.88%, and for
95.24%. The sensitivity for
and
was 100%. The Spearman correlation factor of two different measurements was 0.976 for
, 0.967 for
, 0.949 for
, 0.966 for
, and 0.917 for
. In conclusion, the in-house real-time PCR method developed in our laboratory can provide information about relative amount of five bacterial species present in subgingival plaque in patients with periodontitis. It is likely that such a test could be used in dental diagnostics in assessing the efficacy of any treatment to reduce the bacterial burden.
Recently, emergence of carbapenem-resistance, in particular due to
Klebsiella pneumoniae
carbapenemase (KPC), was observed among
K
.
pneumoniae
causing urinary tract infections in Croatia. The aim of ...the study was to characterize, antimicrobial susceptibility, carbapenem resistance, virulence traits and plasmid types of the urinary KPC positive isolates of
K. pneumoniae
. The antimicrobial susceptibility to a wide range of antibiotics was determined by broth microdilution method. The transferability of meropenem resistance was determined by conjugation (broth mating method) employing
Escherichia coli
J63 strain resistant to sodium azide. Genes encoding broad and extended-spectrum β-lactamases, plasmid-mediated AmpC β-lactamases, group A and B carbapenemases, and carbapenem hydrolyzing oxacillinases (bla
OXA-48like
), respectively, were determined by Polymerase chain reaction (PCR). In total 30 KPC-positive
K
.
pneumoniae
urinary isolates collected from different regions of Croatia were analysed. The isolates were uniformly resistant to all tested antibiotics except for variable susceptibility to gentamicin, sulphamethoxazole/trimethoprim, and colistin, respectively. Four isolates were resistant to colistin with MICs values ranging from 4 to 16 mg/L. All tested isolates were susceptible to ceftazidime/avibactam. Sixteen isolates transferred meropenem resistance to
E. coli
recipient strain by conjugation. Other resistance markers were not co-transferred. PCR was positive for bla
KPC
and bla
SHV
genes in all isolates whereas 13 isolates tested positive also for bla
TEM
genes. PCR based replicon typing (PBRT) revealed the presence of FIIs in 13 and FIA plasmid in two strains. The study showed dissemination of KPC-producing
K. pneumoniae
in urinary isolates, posing a new epidemiological and treatment challenge. Sulphamethoxazole/trimethoprim, colistin, and ceftazidime/avibactam remain so far, as the therapeutic options.
Resistance to carbapenems in
has become a matter of the highest concern in the last decade. Recently,
harboring multiple carbapenemases were detected in three hospital centers in Croatia and in the ...outpatient setting, posing a serious therapeutic challenge for clinicians. In this study, we analyzed eight
and two
complex isolates with multiple carbapenemases, with regard to antibiotic susceptibility, β-lactamase production and plasmid content. The isolates demonstrated uniform resistance to amoxicillin/clavulanate, piperacillin/tazobactam, cefuroxime, ceftazidime, cefotaxime, ceftriaxone and ertapenem. Among novel β-lactam/inhibitor combinations, ceftazidime/avibactam exhibited moderate activity, with 50% of isolates susceptible. All isolates demonstrated resistance to imipenem/cilastatin/relebactam, and all but one to ceftolozane/tazobactam. Four isolates exhibited a multidrug-resistant phenotype (MDR), whereas six were allocated to an extensively drug-resistant phenotype (XDR). OKNV detected three combinations of carbapenemases: OXA-48+NDM (five isolates), OXA-48+VIM (three isolates) and OXA-48+KPC (two isolates). Inter-array testing identified a wide variety of resistance genes for β-lactam antibiotics:
,
,
,
,
,
, aminoglycosides:
6,
,
,
and
, fluoroquinolones:
A,
B and
S, sulphonamides:
1 and
2 and trimethoprim:
.
genes were reported for the first time in Croatia. This study demonstrated the ability of
and
to acquire various resistance determinants under the selection pressure of antibiotics widely used during the COVID-19 pandemic. The novel inter-array method showed good correlation with OKNV and PCR, although some discrepancies were found.
This paper presents the chronology, experiences, and challenges in introducing COVID-19 RT-PCR testing in Split, Croatia. We describe the processes from March 12, 2020 to May 26, 2020, starting from ...the initial knowledge transfer, expert team formation and management, testing implementation, and concluding with the standalone testing facilities, which used automated processes sufficient to meet testing requirements at that time. In the case presented, the COVID-19 unit was organized by joining human and laboratory resources from five clinical departments at the Split University Hospital Centre. Sample preparation procedures and analyses were launched within the restricted time frame while simultaneously training and organizing new laboratory staff and completing equipment requirements. As a result, the process that started with 30 tests per day was constantly improved over time and reached up to 160 tests per day when MagNA Pure was added to automatize RNA extraction at the end of April. At that pace, the cumulative number of samples soon exceeded the first thousand, and by the end of May it exceeded 4000. The case presented provides an example of good practice for crisis response and organization that successfully enabled sufficient COVID-19 testing capacities within the restricted time frame, human and technical resources. Despite limited understanding of COVID-19 at that time, appropriate management, transfer of knowledge, previous experiences in related laboratory and diagnostic work, as well as interdisciplinary and interdepartmental cooperation proved appropriate to overcome the above limitations and ensure adequate healthcare response. Keywords: COVID-19, RT-PCR, Split, Croatia
Carbapenemases involved in acquired carbapenem resistance in Enterobacteriaceae belong to Ambler class A serin β-lactamases, class B metallo-β-lactamases (MBL) or class D OXA-48-like β-lactamases. ...The aim of the present study was to analyse the molecular epidemiology and the mechanisms and routes of spread of class B and class D carbapenemases in Croatia.
In total 68 isolates were analyzed. Antibiotic susceptibility was determined by broth microdilution method. PCR was used to detect antibiotic-resistance genes. Genotyping was performed by rep-PCR and MLST.
Sixty-five isolates were found to harbour VIM-1 carbapenemase, seven of which were positive also for NDM-1, while two strains harboured only NDM-1. OXA-48 was detected in three isolates, two of which coproduced VIM-1. Thirty-six strains possessed additional CTX-M-15 β-lactamase whereas 64 were positive for TEM-1. CMY was found in 18 Citrobacter freundii isolates and DHA-1 in one Enterobacter cloacae isolate. Four different plasmid-incompatibility groups were found: A/C, L/M, N and FIIAs. Unlike C. freundii and E. cloacae, Klebsiella pneumoniae showed high diversity of rep-PCR patterns. E. cloacae and C. freundii predominantly belonged to one large clone which was allocated to ST105 and ST24, respectively.
Three different types of carbapenemases were identified showing the complexity of CRE in Croatia.
•In total 68 carbapenem-resistant isolates were analyzed: 34 Enterobacter cloacae, 18 Citrobacter freundii, 15 Klebsiella pneumoniae and 1 Klebsiella oxytoca•Sixty-five isolates were found to harbour VIM-1 carbapenemase, seven of which were positive also for NDM-1, while two strains harboured only NDM-1.•Three different types of carbapenemases were identified showing the complexity of carbapenemase producing Enterobacteriaceae in Croatia.•Four different plasmid-incompatibility groups were found: A/C, L/M, N and FIIAs.•K. pneumoniae isolates showed high diversity of rep-PCR patterns in contrast to E. cloacae and C. freundii.
...simplex or multiplex PCR, real-time PCR, or hybridization tests could significantly improve detection of carbapenemase genes in clinical laboratory bypassing the sensitivity and specificity ...problems with phenotypic tests. ...facing the global crisis in antibiotic resistance, presented by rapid dissemination of carbapenemase producing Gram-negative bacteria, many issues remain controversial, especially detection methods and treatment options.
Hepatitis C virus (HCV) genotyping is an important part of pre-treatment diagnostic algorithms as it guides the choice of therapeutic regimens. The aim of this study was to analyse the distribution ...of HCV genotypes in patients with chronic hepatitis C from Croatia in the period 2008-2015.
The study enrolled 3,655 anti-HCV positive patients with available results of HCV genotyping from the three largest national HCV genotyping laboratories.
The majority of HCV-infected individuals enrolled in the study were male (70.7%). Analysis of age distribution in a subset of 2,164 individuals showed a mean age of 40.9 years (SD 11.77 years). Croatian patients were mostly infected with HCV genotype 1 (56.6%), followed by genotype 3 (37.3%), genotype 4 (4.2%) and genotype 2 (1.8%). Genotype 1 subtyping in a subset of 1,488 patients showed 54% (803/1,488) of 1b infections and 46% (685/1,488) of 1a infections. Percentages of genotype 1 were the highest in Central/Northwestern and Eastern Croatia and the lowest in the Central/Southern Adriatic Region. Genotype 3 was most frequently found in the Central/Southern Adriatic Region (49.1%) but represented only 17.5% of infections in Eastern Croatia (p < 0.001).
The results of this nine-year retrospective analysis on the distribution of HCV genotypes and subtypes in 3,655 HCV-infected individuals from Croatia showed that the majority of infections can be attributed to genotypes 1 and 3 with absence of major changes in the molecular epidemiology of the two most frequent HCV genotypes infection in Croatia in the past 20 years.
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