Lack of guidelines and translational knowledge is hindering the implementation of psychiatric genetic counseling and testing within Europe - A multi-professional survey study.

Genetic research has identified a large number of genetic variants, both rare and common, underlying neurodevelopmental disorders (NDD) and major psychiatric disorders. Currently, these findings are being translated into clinical practice. However, there is a lack of knowledge and guidelines for psychiatric genetic testing (PsychGT) and genetic counseling (PsychGC). The European Union-funded COST action EnGagE (CA17130) network was started to investigate the current implementation status of PsychGT and PsychGC across 35 participating European countries. Here, we present the results of a pan-European online survey in which we gathered the opinions, knowledge, and practices of a self-selected sample of professionals involved/interested in the field. We received answers from 181 respondents. The three main occupational categories were genetic counselor (21.0%), clinical geneticist (24.9%), and researcher (25.4%). Of all 181 respondents, 106 provide GC for any psychiatric disorder or NDD, corresponding to 58.6% of the whole group ranging from 43.2% in Central Eastern Europe to 66.1% in Western Europe. Overall, 65.2% of the respondents reported that genetic testing is offered to individuals with NDD, and 26.5% indicated the same for individuals with major psychiatric disorders. Only 22.1% of the respondents indicated that they have guidelines for PsychGT. Pharmacogenetic testing actionable for psychiatric disorders was offered by 15%. Interestingly, when genetic tests are fully covered by national health insurance, more genetic testing is provided for individuals with NDD but not those with major psychiatric disorders. Our qualitative analyses of responses highlight the lack of guidelines and knowledge on utilizing and using genetic tests and education and training as the major obstacles to implementation. Indeed, the existence of psychiatric genetic training courses was confirmed by only 11.6% of respondents. The question on the relevance of up-to-date education and training in psychiatric genetics on everyday related practice was highly relevant. We provide evidence that PsychGC and PsychGT are already in use across European countries, but there is a lack of guidelines and education. Harmonization of practice and development of guidelines for genetic counseling, testing, and training professionals would improve equality and access to quality care for individuals with psychiatric disorders within Europe.

New in vitro findings about halogenated boroxine cytotoxicity and deregulation of cell death-related genes in GR-M melanoma cells.

Anti-proliferative effects of halogenated boroxine - K2(B3O3F4OH) (HB) - have been confirmed in multiple cancer cell lines, including melanoma, but the exact mechanism of action is still unknown. This study aimed to determine its cytotoxic effects on human Caucasian melanoma (GR-M) cell growth in vitro as well as on the expression of cell death-related genes BCL-2, BECN1, DRAM1, and SQSTM1. GR-M and peripheral blood mononuclear (PBM) cells were treated with different HB concentrations and their growth inhibition and relative gene expression profiles were determined using the Alamar blue assay and real-time PCR. HB significantly inhibited cell growth of both GR-M and PBM cells but was even more effective in GR-M melanoma cells, as significant inhibition occurred at a lower HB concentration of 0.2 mg/mL. GR-M BCL-2 expression was significantly downregulated (P=0.001) at HB concentration of 0.4 mg/mL, which suggests that HB is a potent tumour growth inhibitor. At the same time, it upregulated BCL-2 expression in normal (PBM) cells, probably by activating protective mechanisms against induced cytotoxicity. In addition, all but the lowest HB concentrations significantly upregulated SQSTM1 (P=0.001) in GR-M cells. Upregulated BECN1 expression suggests early activation of autophagy at the lowest HB concentration in SQSTM1 cells and at all HB concentrations in PBM cells. Our findings clearly show HB-associated cell death and, along with previous cytotoxicity studies, reveal its promising anti-tumour potential.

Halogenated boroxine increases propensity to apoptosis in leukemia (UT-7) but not non-tumor cells in vitro.

A hallmark of the development of solid and hematological malignancies is the dysregulation of apoptosis, which leads to an imbalance between cell proliferation, cell survival and death. Halogenated boroxine [K2 (B3 O3 F4 OH)] (HB) is a derivative of cyclic anhydride of boronic acid, with reproducible anti-tumor and anti-proliferative effects in different cell models. Notably, these changes are observed to be more profound in tumor cells than in normal cells. Here, we investigated the underlying mechanisms through an extensive evaluation of (a) deregulated target genes and (b) their interactions and links with main apoptotic pathway genes upon treatment with an optimized concentration of HB. To provide deeper insights into the mechanism of action of HB, we performed identification, visualization, and pathway association of differentially expressed genes (DEGs) involved in regulation of apoptosis among tumor and non-tumor cells upon HB treatment. We report that HB at a concentration of 0.2 mg·mL-1 drives tumor cells to apoptosis, whereas non-tumor cells are not affected. Comparison of DEG profiles, gene interactions and pathway associations suggests that the HB effect and tumor-'selectivity' can be explained by Bax/Bak-independent mitochondrial depolarization by ROS generation and TRAIL-like activation, followed by permanent inhibition of NFκB signaling pathway specifically in tumor cells.

Novel boron-containing compound, halogenated boroxine, induces selective cytotoxicity through apoptosis triggering in UT-7 leukemia.

Apoptosis induction is a promising approach in targeting tumor cells. As halogenated boroxine (HB) shows antitumor activity, but its mechanism of action in hematological tumors remains unclear, in this study, we aimed to analyze apoptosis triggering in normal and UT-7 leukemia cells by HB. Methods for assessing cell viability and cytotoxicity, apoptosis detection, relative expression of 84 apoptosis-associated genes and BCL-2, and functional analysis were applied. Pronounced HB activities in inhibition of cell viability, cytotoxicity, and apoptosis induction with measurable differences between tumor and normal cells were found. HB modulated the expression of 21 genes, predominantly downregulated the antiapoptotic genes in leukemia. The functional association revealed HB's impact on inhibition of NF-κB signaling pathway. BCL-2 expression decreasing was found only in UT-7 leukemia. This study identified HB as an apoptosis inducer affecting leukemia but not normal cells considering mechanisms of selective activity that may be a great advantage of HB applications.

The alternative serotonin transporter promoter P2 impacts gene function in females with irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a gut-brain disorder in which symptoms are shaped by serotonin acting centrally and peripherally. The serotonin transporter gene SLC6A4 has been implicated in IBS pathophysiology, but the underlying genetic mechanisms remain unclear. We sequenced the alternative P2 promoter driving intestinal SLC6A4 expression and identified single nucleotide polymorphisms (SNPs) that were associated with IBS in a discovery sample. Identified SNPs built different haplotypes, and the tagging SNP rs2020938 seems to associate with constipation-predominant IBS (IBS-C) in females. rs2020938 validation was performed in 1978 additional IBS patients and 6,038 controls from eight countries. Meta-analysis on data from 2,175 IBS patients and 6,128 controls confirmed the association with female IBS-C. Expression analyses revealed that the P2 promoter drives SLC6A4 expression primarily in the small intestine. Gene reporter assays showed a functional impact of SNPs in the P2 region. In silico analysis of the polymorphic promoter indicated differential expression regulation. Further follow-up revealed that the major allele of the tagging SNP rs2020938 correlates with differential SLC6A4 expression in the jejunum and with stool consistency, indicating functional relevance. Our data consolidate rs2020938 as a functional SNP associated with IBS-C risk in females, underlining the relevance of SLC6A4 in IBS pathogenesis.

Molecular and histopathological profiling of imiquimod induced dermatosis in Swiss Wistar rats: contribution to the rat model for novel anti-psoriasis treatments.

Imiquimod (IMQ) induced human-like psoriasis in mice has been shown to be effective in testing and development of novel treatments. The IMQ psoriasis model has become widely used animal model, however, it is not completely characterized in different rat strains. We aimed to evaluate IMQ and betamethasone treatment for induction and reversal of psoriatic lesions on macroscopic, histological, genetic as well as cytokines and chemokines activation levels. Wistar rats were treated topically with IMQ. Adopted Psoriasis Area Severity Index (PASI) was calculated at the baseline, after the IMQ-symptoms induction and after betamethasone-symptoms reversal. Systematic effects were studied on cytokines and chemokines levels in plasma. Skin biopsy was taken to assess histological symptoms and selected inflammatory cytokines and receptors genes expression levels. Reversal of skin lesions, after betamethasone treatment, was significant (p = 0.03). Histological differences between untreated and IMQ-treated skin were significant for some markers (p < 0.05) though not significantly decreased by betamethasone treatment. Fourteen genes were significantly up-regulated after the IMQ and four genes were down-regulated after skin lesions reversal by betamethasone. This work provides new insights on biological effects of imiquimod induced psoriasis and its reversal by betamethasone treatment in Wistar rats. It also contributes to general knowledge of the rat model usage for testing of novel anti-psoriasis drugs.

Molecular Variation of Rh, MN, Duffy, Kidd, Kell, and Lutheran Blood Groups in the Human Population of Bosnia and Herzegovina.

Six blood groups (Rh, MN, Duffy, Kidd, Kell, and Lutheran) were investigated among three major ethnic groups (Bosniaks, Bosnian Croats, and Bosnian Serbs), as well as 10 regional subpopulations across Bosnia and Herzegovina (B&H): Krajina; Posavina; northeastern, eastern, middle, and central Bosnia; Sarajevo region; eastern, central, and western Herzegovina. This is the first study that introduces the molecular genetic typing of five blood groups within the B&H population, with the exception of the RhD blood group. The sample consisted of 450 buccal swabs from unrelated individuals. Five blood group systems (RhD, RhC, RhE, Kidd, MN) were genotyped by PCR with sequence specific primers, while three blood group systems (Kell, Duffy, Lutheran) were genotyped by the PCR-restriction-fragment-length polymorphism method. Minor variation of genetic diversity was observed within the three major B&H ethnic groups, as well as within the 10 subpopulations stratified according to geographical criteria. No genetic differentiation among ethnic groups was noticed. These results are in agreement with the results of previous studies based on different molecular genetics markers, which indicate that the three B&H ethnic groups belong to the same gene pool. A similar level of genetic variance was observed within regional subpopulations, with no significant genetic differentiation among them. Comparison of intrapopulation genetic diversity of the B&H population with other European and non-European populations, based on three loci (RHD, MN, and KEL), clearly show that the level of genetic diversity of the B&H population is within the European range.

A novel flavivirus strain detected in phlebotomine sandflies in Bosnia and Herzegovina.

Aim Phlebotominae sandflies are primary vectors of phleboviruses, causing the sandfly fever disease. The aim of this study was to detect and report the presence of flaviviruses in Phlebotominae sandflies captured in Bosnia and Herzegovina. Methods After a microscopic and morphometric analysis, the final identification of collected Phlebotomus specimens was confirmed by PCR, using a hemi-nested polymerase chain reaction on extracted and reversely transcribed RNA. Results We obtained a 155 nt long fragment of the viral non-structural protein 5 (NS5) gene (GenBank accession no. MN090154). The acquired nucleotide sequence, provisionally named as Dreznica, showed a maximum of 70-80% identity in 70-88% (110-137 nucleotides) of the query coverage with several Anopheles, Sabethes, Calbertado and Culex flaviviruses. Maximum likelihood phylogenetic analysis showed that the new flavivirus Dreznica clusters together with the flavivirus isolated from Culiseta annulata mosquitos. Conclusion We report the presence of flavivirus in Phlebotominae sandflies, captured in Dreznica, Herzegovina for the first time. The next phase of research will be directed towards virus cultivation, obtaining a longer or complete virus sequence and clarifying the medical and epidemiological importance of the Dreznica virus.

Current state and prospects of biotechnology in Central and Eastern European countries. Part II: new and preaccession EU countries(CRO, RO, B&H, SRB).

Innovation holds the potential for economic prosperity. Biotechnology (BT) has proved to be a viable vehicle for the development and utilization of technologies, which has brought not only advances to society, but also career opportunities to nation-states that have enabling conditions. In this review, we assess the current state of BT-related activities within selected new and preaccession EU countries (NPA) of CEE region namely Croatia, Romania, Bosnia and Herzegovina and Serbia, examining educational programs, research activity, enterprises, and the financing systems. The field of BT covers a broad area of activities, including medical, food and agriculture, aquaculture or marine, environmental, biofuels, bioinformatics, and many others. Under the European Commission (EC), member-states are to set their Research and Innovation Strategies for Smart Specialization (RIS3), to identify priorities or strengths in order to develop knowledge intensive economies. As the four countries highlighted in this review are in the early stages of implementing RIS3 or have not yet fully formulated, it presents an opportunity to learn from the successes and failures of those that have already received major structural funds from the EC. A critical point will be the ability of the public and private sectors' actors to align, in the implementation of RIS3 as new investment instruments emerge, and to concentrate efforts on a few select target goals, rather than distribute funding widely without respect to a long-term vision.

Influence of Plasminogen Activator Inhibitor -1 Gene Polymorphism on Renal Scarring After First Febrile Urinary Tract Infection in Infants.

BACKGROUND: The pathogenesis of renal scarring (RS) after first febrile urinary tract infection (UTI) in children is multifactorial. In addition to well-known risk factors, a role for genetic predisposition has been suggested. AIMS: To determine whether deoxyribonucleic acid (DNA) polymorphisms at the plasminogen activator inhibitor -1 (PAI-1) gene were associated with evolution to RS following a febrile UTI in infants. MATERIALS AND METHODS: Our research included 100 infants, 84 girls and 16 boys, ages up to 1 year with a first febrile UTI, increased inflammatory parameters and positive urine culture treated at the Pediatric Clinic II of the University Clinical Center Sarajevo (UCCS). The diagnostic was based on the imaging studies: ultrasonography, voiding cystourethrography (VCUG) and initial and control static renal scintigraphy (DMSA renal scan), to assess the renal parenchymal damage (RPD). The polymorphisms of the PAI-1 were determined based on polymerase chain reaction technique. The distribution of PAI-1 genotypes and the allele frequencies were compared between different groups of patients with febrile UTI. RESULTS: Results presented that 66 infants had acute pyelonephritis (APN) and 22 had vesicoureteral reflux (VUR). On initial DMSA renal scan examination, we detected no RPD in any patient. After 6 months, the repeat DMSA renal scan revealed the presence of RPD in 18 (27%) out of 66 infants with APN. Distribution of PAI-1 genotypes was not different between various groups of patients with febrile UTI. CONCLUSIONS: The results of our study have not shown that individual genetic variation in PAI-1 is an independent variable that predispose same of children for RS after first febrile UTI. Maybe that yet unknown gene polymorphisms together with geographical and /or socio-economic differences can influence on the development of RS.

Gene set enrichment analysis and expression pattern exploration implicate an involvement of neurodevelopmental processes in bipolar disorder.

BACKGROUND: Bipolar disorder (BD) is a common and highly heritable disorder of mood. Genome-wide association studies (GWAS) have identified several independent susceptibility loci. In order to extract more biological information from GWAS data, multi-locus approaches represent powerful tools since they utilize knowledge about biological processes to integrate functional sets of genes at strongly to moderately associated loci. METHODS: We conducted gene set enrichment analyses (GSEA) using 2.3 million single-nucleotide polymorphisms, 397 Reactome pathways and 24,025 patients with BD and controls. RNA expression of implicated individual genes and gene sets were examined in post-mortem brains across lifespan. RESULTS: Two pathways showed a significant enrichment after correction for multiple comparisons in the GSEA: GRB2 events in ERBB2 signaling, for which 6 of 21 genes were BD associated (PFDR = 0.0377), and NCAM signaling for neurite out-growth, for which 11 out of 62 genes were BD associated (PFDR = 0.0451). Most pathway genes showed peaks of RNA co-expression during fetal development and infancy and mapped to neocortical areas and parts of the limbic system. LIMITATIONS: Pathway associations were technically reproduced by two methods, although they were not formally replicated in independent samples. Gene expression was explored in controls but not in patients. CONCLUSIONS: Pathway analysis in large GWAS data of BD and follow-up of gene expression patterns in healthy brains provide support for an involvement of neurodevelopmental processes in the etiology of this neuropsychiatric disease. Future studies are required to further evaluate the relevance of the implicated genes on pathway functioning and clinical aspects of BD.

Lessons learned--resolving the enigma of genetic factors in IBS.

IBS is the most prevalent functional gastrointestinal disorder and phenotypically characterized by chronic abdominal discomfort, pain and altered defecation patterns. The pathophysiology of IBS is multifactorial, albeit with a substantial genetic component. To date, studies using various methodologies, ranging from family and twin studies to candidate gene approaches and genome-wide association studies, have identified several genetic variants in the context of IBS. Yet, despite enlarged sample sizes, increased statistical power and meta-analyses in the past 7 years, positive associations are still scarce and/or have not been reproduced. In addition, epigenetic and pharmacogenetic approaches remain in their infancy. A major hurdle is the lack of large homogenized case-control cohorts recruited according to standardized and harmonized criteria. The COST Action BM1106 GENIEUR (GENes in Irritable Bowel Syndrome Research Network EURope) has been established to address these obstacles. In this Review, the (epi)genetic working group of GENIEUR reports on the current state-of-the-art in the field, highlights fundamental flaws and pitfalls in current IBS (epi)genetic research and provides a vision on how to address and improve (epi)genetic approaches in this complex disorder in the future.

Effects of dipotassium-trioxohydroxytetrafluorotriborate, K2[B3O3F4OH], on cell viability and gene expression of common human cancer drug targets in a melanoma cell line.

Recently it was found that dipotassium-trioxohydroxytetrafluorotriborate, K2(B3O3F4OH), is a potent and highly specific inhibitor of precancerous cell processes. We conducted gene expression profiling of human melanoma cells before and after treatment with two concentrations (0.1 and 1 mM) of this boron inorganic derivative in order to assess its effects on deregulation of genes associated with tumor pathways. Parallel trypan blue exclusion assay was performed to assess the cytotoxicity effects of this chemical. Treatment with K2(B3O3F4OH) induced a significant decrease of cell viability in melanoma cellline at both tested concentrations. Furthermore, these treatments caused deregulation of more than 30 genes known as common anti-tumor drug targets. IGF-1 and hTERT were found to be significantly downregulated and this result may imply potential use of K2(B3O3F4OH) as an inhibitor or human telomerase and insulin-like growth factor 1, both of which are associated with various tumor pathways.

Frequency of main western-Euroasian mtDNA haplogroups and paleolithic and neolithic lineages in the genetic structure of population of northeastern Bosnia.

Mitochondrial DNA (mtDNA) variations were analyzed in a sample of 245 individuals of Bosnian-Herzegovinian population from the area of Northeastern Bosnia (also known as Tuzla region). Haplogroup affiliation was determined using RFLP method (Restriction Fragment Length Polymorphism) analyzing haplogroup-specific markers of mtDNA coding region, characteristic for the main Western-Eurasian haplogroups. Additional analyses of two sequenced hypervariable segments (HVSI and HVSII) of mtDNA control region were performed in order to identify U subhaplogroups. The study revealed that 95.51% of the analyzed individuals belonged to the typical Western-Eurasian haplogroups: H, I, J, K, T U, V, W or X. The most frequent haplogroup in the analyzed population was the haplogroup H (52.65%) which, due to its increased frequency, represents a marking haplogroup of the population of Northeastern Bosnia. The results of intergroup genetic analysis showed that Bosnian-Herzegovinian population is genetically closer to previously studied populations of Herzegovinians (part of Bosnia and Herzegovina), Slovenians and Croats in relation to other neighboring populations located in Southeastern Europe. Our study also suggests that population genetic structure of Tuzla region is dominated by mutations that are classified as "Paleolithic". These mutations were probably brought to the area of northeastern Bosnia through waves of prehistoric and historic migrations, but the impact of any pre-Neolithic, Neolithic or some "later" migrations, with a slightly lower contribution to the genetic structure of this population, also cannot be neglected.

Association of dopamine receptor gene polymorphism and psychological personality traits in liability for opioid addiction.

There is a clear evidence that same psychoactive substance may cause various individual physiological reactions in same environmental conditions. Although there is a general attitude on equal liability to opioid addiction, latest genetic analysis findings imply there are certain quantifiable factors that could lead to elevated individual liability towards development of opioid addiction. The goal of this study was to investigate association of certain personality traits and genetic factors (separately and in combination) with heroin addiction. Total of 200 individuals participated in the study: 100 patients on Metadone Maintenance Treatment (MMT) and 100 age and sex matched healthy volunteers. All were medically examined, interviewed and psychologically evaluated using Eysenck personality questionnaire (EPQ) and genotyped for DRD2 (rs1800497) using PCR-RFLP method. Overrepresentation of certain personality traits (neuroticism, psychoticism and extraversion/ intraversion), together with environemental risk factors such as: upbringing within incomplete families and familial history of psychotropic substances abuse, are associated with high-risk development of opioid addiction.

Molecular-genetic variance of RH blood group system within human population of Bosnia and Herzegovina.

There are two major theories for inheritance of Rh blood group system: Fisher - Race theory and Wiener theory. Aim of this study was identifying frequency of RHDCE alleles in Bosnian - Herzegovinian population and introduction of this method in screening for Rh phenotype in B&H since this type of analysis was not used for blood typing in B&H before. Rh blood group was typed by Polymerase Chain Reaction, using the protocols and primers previously established by other authors, then carrying out electrophoresis in 2-3% agarose gel. Percentage of Rh positive individuals in our sample is 84.48%, while the percentage of Rh negative individuals is 15.52%. Inter-rater agreement statistic showed perfect agreement (K=1) between the results of Rh blood system detection based on serological and molecular-genetics methods. In conclusion, molecular - genetic methods are suitable for prenatal genotyping and specific cases while standard serological method is suitable for high-throughput of samples.

Polymorphic Alu insertions in human populations of Bosnia and Herzegovina.

BACKGROUND: From a demographic and genetic perspective, Bosnia and Herzegovina is interwoven with a number of differentially isolated local populations of indigenous people with different population and religious backgrounds. AIM: In order to estimate their genetic structure, this study investigated the frequencies of 10 Alu polymorphic loci in 10 regional populations distributed across Bosnia and Herzegovina. Genetic differentiation among the three major population groups in Bosnia and Herzegovina was estimated. SUBJECTS AND METHODS: DNA from 506 unrelated individuals was extracted from buccal swabs using the salting-out extraction method. Each DNA sample was PCR-amplified using locus-specific primers. RESULTS: Gene diversity values showed similarity in all analysed populations and ranged from 0.305-0.328. FST values for all loci showed that most variability is found within populations. Overall FST for all loci and AMOVA indicated that most variability was detected within populations. CONCLUSION: Results of this study are in agreement with the previous studies, indicating that the three populations in Bosnia and Herzegovina have the same genetic background. There is no significant differentiation among regional populations, pointing to absence of geographic influence. The Bosnian population is clearly located within the European gene pool.

Assessment of relatedness between neurocan gene as bipolar disorder susceptibility locus and schizophrenia.

Large scale genetic association meta-analyses showed that neurocan (NCAN) gene polymorphism rs1064395 is susceptibility locus for bipolar disorder. These studies also included patients with bipolar disorder originated from Bosnia and Herzegovina. Followed by theory of shared genetic elements between bipolar disorder and schizophrenia susceptibility, other studies explored several genetic factors with schizophrenia vulnerability as well. In this work, authors investigated the association between previously confirmed bipolar disorder genetic risk factor- neurocan with schizophrenia in a population sample of Bosnia and Herzegovina. Ethical aspects of this research were assessed by Ethics Committee of Clinical Center University of Sarajevo. Blood samples for DNA extraction were taken from the total of 86 patients and healthy individuals who previously signed informed consent. Genotyping for rs 1064395 was done using direct sequencing method. A case-control analysis of common genetic polymorphism within neurocan gene and schizophrenia status in a consecutively sampled patient cohort have been done using Fisher-exact test with odds-ratio calculation. No statistically significant allele and genotype association with disease status was found (p>0.05). Our finding supports the fact that large-scale genetic association studies approach need to be employed when detecting the variants with small additive effect in phenotypes with complex ethiology.

Investigation of IVS14 + 1G > A polymorphism of DPYD gene in a group of Bosnian patients treated with 5-Fluorouracil and capecitabine.

Adverse drug reactions still pose an important clinical problem. Dihydropyrimidine dehydrogenase (DPD) is an enzyme that regulates 5-FU quantities available for anabolic processes and hence affects its pharmacokinetics, toxicity and efficacy. There are several studies describing a hereditary (pharmacogenetic) disorder in which individuals with absent or significantly reduced DPD activity may even develop a life-threatening toxicity following exposure to 5-FU. The most common mutation is known as the DPYD*2A or as the splice-site mutation (IVS14 + 1G A) leading to creation of a dysfunctional protein. An objective behind the study was to ascertain existence of the IVS14 + 1G A mutation among the population of Bosnia and Herzegovina. Our research has undeniably attested to existence of one heterozygote for the DPYD gene mutation, i.e. one heterozygote for IVS14 + 1 G > A, DPYD*2A mutation.

Tannerella forsythia detected in infected root canals using nested PCR.

PURPOSE: This study assessed the occurrence of Tannerella forsythia in patients with acute and chronic primary endodontic infections. METHODS: Clinical samples were collected from 40 patients with acute and chronic periradicular disease. Nested polymerase chain reaction (PCR) assay technique was used to detect the presence of T. forsythia in primary endodontic infections. The first round of PCR amplification used universal primers to detect the 16S rDNA sequence. Product from the first round was then used to amplify T. forsythia specific fragment with species-specific pairs of primers. RESULTS: T. forsythia was found in 12 of 27 chronic and 5 of 13 acute infected patients for an overall occurrence frequency of 42.5%. No significant correlation was found between patients with the T. forsythia positive genotype and the occurrence of clinical symptoms in the primary endodontic infections (P < 0.05) (P = 0.496). Also, no significant relationship was found between the occurrence of T. forsythia and the patient's age (P = 0.61) or gender (P = 0.239).