Emese Juhász

HVS-I polymorphism screening of ancient human mitochondrial DNA provides evidence for N9a discontinuity and East Asian haplogroups in the Neolithic Hungary

Analysis of mitochondrial mutations in the HVS-I region is an effective method for ancient human populational studies. Discontinuous haplotype data between the first farmers and contemporary Europeans has been described before. Our contribution is based on a survey initiated on the Neolithic skeletons from Hungarian archaeological sites in the Alföld. This Lowland, the Hungarian Plain, is well excavated as an important region for spread of Neolithic culture from Near East and Balkans toward Central and Western Europe, started circa 8000 years ago. HVS-I sequences from nt15977 to nt16430 of 11 such specimens with sufficient mitochondrial DNA preservation among an extended Neolithic collection were analysed for polymorphisms, identifying 23 different ones. After assigning all single-nucleotide polymorphisms, a novel, N9a, N1a, C5, D1/G1a, M/R24 haplogroups were determined. On mitochondrial control mutations at nt16257 and nt16261, polymorphic PCRs were carried out to assess their distribution in remains. Neolithic data set was compared with contemporary Vác samples and references, resulting in higher frequency of N9a in Alföld as a remarkable genetic discontinuity. Our investigation is the first to study mutations form Neolithic of Hungary, resulting in an outcome of Far Eastern haplogroups in the Carpathian Basin. It is worth further investigation as a non-descendant theory, instead of a continuous population history, supporting genetic gaps between ancient and recent human populations.

In vitro biofilm production of Candida bloodstream isolates: any association with clinical characteristics?

Candida spp. are a leading cause of bloodstream infection (BSI) and are associated with high mortality rates. Biofilm production is a virulence factor of Candida spp., and has been linked with poor clinical outcome. The aim of our study was to assess biofilm production of Candida bloodstream isolates at our institute, and to determine whether in vitro biofilm production is associated with any clinical characteristics of infection. During the four-year study period, 93 cases of Candida BSI were analysed. The most frequently isolated species was C. albicans (66.7 %), followed by C. glabrata (9.7 %), C. parapsilosis (9.7 %), C. tropicalis (9.7 %) and C. krusei (4.3 %). Biofilm production was more prevalent among non-albicans Candida spp. (77.4 %) than C. albicans (30.6 %) (P = 0.02). Abdominal surgery was identified as a risk factor of BSI caused by biofilm producing non-albicans Candida isolates. No risk factors predisposing to bloodstream infection caused by a biofilm producing C. albicans isolate were identified. Biofilm production was not verified as a risk factor of mortality.

Emergence of VIM-4- and SHV-12-producing Enterobacter cloacae in a neonatal intensive care unit

In order to reveal colonization with multidrug-resistant bacteria early, routine screening is done on samples of all patients of the neonatal intensive care units at Semmelweis University, Hungary. Due to the extended-spectrum β-lactamase (ESBL) screening examinations, emergence of multidrug-resistant Enterobacter cloacae isolates was found with suspicion of clonal transmission, therefore active microbiological surveillance was initiated. The aim of our study was to characterize 60 E. cloacae isolates recovered in a 7-month period in 2010. MIC values of antibiotics were determined and ESBL and carbapenemase production was tested. Metallo-β-lactamase (MBL) genes, ESBL genes, and class-1 integrons were characterized, and the possible clonal relationship between isolates was investigated. The isolates showed increased MIC values for carbapenems and cephalosporins. All 60 E. cloacae strains recovered from 16 neonates proved to be VIM-4 MBL producers. Fifty-three strains were SHV-12 ESBL producers also. In all cases, the bla(VIM-4) gene was a part of class-1 integron, In238a. XbaI-macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) revealed identical patterns for the isolates. Our study supports the importance of active microbiological surveillance as well as molecular epidemiology at the NICUs as a part of infection control.

Taguchi optimisation of a multiplex pneumococcal serotyping PCR and description of 11 novel serotyping primers

Recently, a PCR-derived method for serotyping Streptococcus pneumoniae has been devised to substitute the conventional antiserum phenotypic method. The method initially used a multiplex PCR reaction, dividing the isolates into 6 different groups based on the detected PCR gel pattern. In order to optimise and refine this crucial step, the Taguchi technique was employed, which can evaluate the individual effect of six parameters (in this case: primers, MgCl2, nucleotide mix, polymerase and buffer), with only 18 experiments; varying the parameter levels in an orthogonal matrix which suppresses the interactions between them. With this method, clear and sharp bands were observed in 5 experiments out of the 18, while the PCR did not work reliably in the remaining cases. In addition, the PCR-based technique could be rendered more economic by the 10-fold lowering of the quantities of two primers. The modified reaction yielded identical results to those obtained with the original method. Furthermore, we have designed serotype-specific primers for 11 new serotypes. The most important ones are those that can distinguish the very closely related, but equally important serotypes 6A and 6B.

EMERGENCE OF ANTIRETROVIRAL DRUG RESISTANCE IN THERAPY-NAIVE HIV INFECTED PATIENTS IN HUNGARY*

Mutations in the HIV-1 genes associated with resistance to antiretroviral drugs were detected also in primary HIV infected individuals who did not receive antiretroviral treatment. Drug resistance genotyping of HIV pol gene was done by in situ DNA hybridization using a Line Probe Assay and by direct sequencing. Viral variants harbouring resistance mutations such as: M41, T69R, K70R, M184V, T215Y in the pol gene were detected in 14% of the subjects. HIV mutants resistant to NRT inhibitors were found in 10 and 20% of patients infected before and after the year 2000, respectively. Multiple drug resistant viruses (2-3 drug classes) were present in 3.5% of the mainly recently infected patients. In protease gene only minor resistant mutations were found such as L101 and A71V. These findings indicate the evolution of drug resistance showing a correlation with the time of introduction of combination therapy in our country, where more than 70% of HIV infections were by homo/bisexual transmission. This confirms the transmission of drug-resistant HIV shown by genotype testing during primary infection in therapy-naive patients and initiates serious clinical and public health consequences.

Significance of yeasts in bloodstream infection: Epidemiology and predisposing factors of Candidaemia in adult patients at a university hospital (2010–2014)

The incidence of Candida bloodstream infection (BSI) has increased during the past decades. Species distribution is changing worldwide, and non-albicans Candida spp. are becoming more prevalent. Acquired resistance to antifungal agents has been documented in several reports. The aim of our study was to assess the epidemiology and antifungal susceptibility of Candida isolates from BSI at our institute. The incidence of Candida BSI increased during the first four years of our investigation, from 1.7 to 3.5 episodes / 10 000 admissions, then dropped to 2.66 episodes / 10 000 admissions in the last year. The most frequently isolated species was C. albicans (63%), followed by C. glabrata (13%), C. parapsilosis (10.2%), C. tropicalis (9.3%), and C. krusei (3.7%). One isolate each of C. kefyr, C. fabianii and C. inconspicua were detected. The percentage of C. albicans remained stable throughout the study period. The most frequent risk factors of Candida BSI in our patient population were intensive care treatment (60.4%), abdominal surgery (52.5%), and solid malignancy (30.7%). All isolates were wild-type organisms, no acquired antifungal resistance was detected.

The Consequence of a Founder Effect: CCR5-∆32, CCR2-64I and SDF1-3'A Polymorphism in Vlach Gypsy Population in Hungary.

Frequencies of genetic polymorphisms of the three most frequent HIV-1 resistance-conferring alleles playing an important role in HIV-1 pathogenesis were analysed in Vlach Gypsy populations living in Hungary, as the largest minority. Mutations in the encoding genes, such as CCR5-∆32, CCR2-64I and SDF1-3’A are shown to result in protective effects against HIV-1 infection and disease progression. 560 samples collected from Vlach Gypsy individuals living in 6 North-East Hungarian settlements were genotyped by PCR-RFLP method. Overall allele frequencies of CCR5-∆32, CCR2-64I and SDF1-3’A were found as 0.122, 0.186 and 0.115 respectively. All the observed genotype frequencies were in accordance with Hardy-Weinberg equilibrium . In regions, however, Vlach Gypsies live in majority and in ethnically homogenous communities, a higher CCR5-∆32 mutations were found, with allele frequencies of 0.148 and 0.140 respectively, which are remarkably higher than those in general Hungarian people, and ten times higher than in regions of North-Western India from where present day Hungarian Gypsies originated in the Middle Ages. In the background of this higher CCR5-∆32 allele frequency in the population analysed in our study a genetic founder effect could be assumed. Allele frequency of CCR2-64I was found to be among the highest in Europe. SDF1-3’A allele frequency in Vlach Gypsies was significantly lower than in ethnic Hungarians. 63% of the total 560 individuals tested carried at least one of the mutations studied. These results could partially explain the low incidence of HIV/AIDS among Vlach Gypsies in Hungary.

Colistin resistance among blood culture isolates at a tertiary care centre in Hungary

OBJECTIVES The emergence of colistin resistance has been detected worldwide in recent years. Whilst colistin susceptibility has been tested in carbapenem resistant Enterobacteriaceae as well as multidrug-resistant Pseudomonas spp. and Acinetobacter spp. during routine laboratory practice, the overall rate of colistin resistance was unknown in our centre. The aim of this retrospective study was to reveal the prevalence of colistin resistance among clinically significant blood culture isolates in two different periods (2010-2011 and 2016) in our laboratory. METHODS Consecutive non-duplicate strains (n=776) were screened for colistin resistance using agar plates containing 4mg/L colistin. Strains cultured on colistin-containing plates were further examined. Minimum inhibitory concentrations (MICs) of colistin-tolerant subcultures and original cultures were determined in parallel by the broth microdilution method. Screening for mcr-1-mediated colistin resistance was performed by PCR. RESULTS The rate of colistin resistance was 0.6%, 1.3% and 2.6% in Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp., respectively; colistin-resistant subpopulations were found in 17%, 27% and 20% of isolates, respectively, with low frequency. Seven colistin-resistant strains were found, among which was an mcr-1-positive Escherichia coli isolated from a blood sample of a haemato-oncology patient in 2011. All Stenotrophomonas maltophilia isolates were resistant to colistin. CONCLUSIONS The low prevalence of colistin resistance was in accordance with European data. The prevalence of heteroresistance was significantly higher, but the clinical significance of the phenomenon is unclear. We have identified the first mcr-1-positive E. coli strain in Hungary. mcr-1 has been in Hungary since 2011 but has not yet expanded.

[Special application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry in clinical microbiological diagnostics].

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry as a new possibility for rapid identification of bacteria and fungi revolutionized the clinical microbiological diagnostics. It has an extreme importance in the routine microbiological laboratories, as identification of the pathogenic species rapidly will influence antibiotic selection before the final determination of antibiotic resistance of the isolate. The classical methods for identification of bacteria or fungi, based on biochemical tests, are influenced by many environmental factors. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry is a rapid method which is able to identify a great variety of the isolated bacteria and fungi based on the composition of conserved ribosomal proteins. Recently several other applications of the method have also been investigated such as direct identification of pathogens from the positive blood cultures. There are possibilities to identify bacteria from the urine samples in urinary tract infection or from other sterile body fluids. Using selective enrichment broth Salmonella sp from the stool samples can be identified more rapidly, too. The extended spectrum beta-lactamase or carbapenemase production of the isolated bacteria can be also detected by this method helping the antibiotic selection in some cases. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry based methods are suitable to investigate changes in deoxyribonucleic acid or ribonucleic acid, to carry out rapid antibiotic resistance determination or other proteomic analysis. The aim of this paper is to give an overview about present possibilities of using this technique in the clinical microbiological routine procedures.A mikrobiológiai diagnosztika területén kevés technikai fejlesztés volt az elmúlt évtizedekben, amely olyan rohamos fejlődést hozott volna a baktériumok és gombák fajszintű (speciesszintű) identifi kálásában, mint a „matrix-assisted laser desorption ionization time-of-fl ight” tömegspektrometria. A klinikai mikrobiológiai gyakorlatban ennek jelentősége felbecsülhetetlen, hiszen a kórokozó ismerete jelentősen befolyásolja a terápiás választást még az antimikrobás szerrel szembeni rezisztencia meghatározása előtt. A hagyományos speciesmeghatározás számos, a környezeti hatások által befolyásolt biokémiai reakción alapszik és sok esetben igen időigényes folyamat. A speciális tömegspektrometriás módszer néhány perc alatt elvégzi a kitenyésztett baktérium vagy gomba pontos identifi kálását a konzervált riboszomális fehérjék tömegspektrometriás mérése alapján. Emellett a módszer alkalmazásának lehetőségét számos más új területen is kutatják. Így például a pozitív hemokultúrákból történő direkt kórokozó-meghatározás segítségével hamarabb megkezdhető a szeptikus beteg célzott antibiotikumkezelése. Lehetőség van a kórokozó direkt azonosítására pozitív vizeletmintából, esetleg egyébként steril testnedvekből, vagy megkísérelhető szelektív dúsítást követően Salmonella kimutatása székletből. Az izolált baktériumok „extended spectrum beta-lactamase” és karbapenemáztermelésének gyors kimutatása segítheti a terápiás választást. Ez a tömegspektrometriás módszer a közeljövőben a klinikai mikrobiológiai diagnosztika más területein is teret nyerhet, így például használható lehet a dezoxiribonukleinsav és a ribonukleinsav analízisére, gyors komplett rezisztencia meghatározására és más proteomikai alkalmazásokra is. A közlemény rövid áttekintést kíván adni ennek az új technikának a klinikai mikrobiológiai diagnosztikában való jelenlegi alkalmazhatóságáról. Orv. Hetil., 2014, 155(38), 1495–1503.

Ureaplasmas: From commensal flora to serious infections

The two existing human pathogen Ureaplasma species containing 14 serovars are associated with a variety of maladies. Colonizing the human urogenital tract, ureaplasmas cause infections mainly in these organs. However, in the special population of immunosuppressed patients or preterm and very low birth weight newborns such serious Ureaplasma infections such as meningitis or pericarditis have been described. Because they lack a cell wall, ureaplasmas are very susceptible to drying and other adverse environmental conditions; therefore, careful attention has to be given to specimen collection and transportation. Commercially available culture based tests made the laboratory diagnosis of Ureaplasma species much easier. PCR methods provide further facilities to get faster diagnosis, simultaneously with serovar identification, quantitation of Ureaplasma or detection of other sexually transmitted infectious pathogens. Although fluoroquinolone, macrolide, and tetracycline resistant strains are known, antibiotic treatment of Ureaplasma infections is not problematic for now. The mechanism of antibiotic resistance has not been entirely understood till now. Biofilm-forming ability of Ureaplasmas can be important in chronic infections and antibiotic resistance. The basic microbiology of Ureaplasma species, such as clinical manifestations, the diagnostic opportunities, and the therapeutic options are reviewed in the current article.