Restriction Fragment Length Polymorphism Genotyping Research Articles

Genetic Variability in Leishmaniasis-Causing Leishmania infantum in Humans and Dogs from North-East Spain.

Leishmania infantum is the primary cause of visceral and cutaneous leishmaniasis in the European Mediterranean region. Subspecies-level characterization of L. infantum aids epidemiological studies by offering insights into the evolution and geographical distribution of the parasite and reservoir identity. In this study, conducted in north-east Spain, 26 DNA samples of L. infantum were analyzed, comprising 21 from 10 humans and 5 from 5 dogs. Minicircle kinetoplast DNA (kDNA) polymerase chain reaction assays using primers MC1 and MC2, followed by sequencing, were employed to assess intraspecific genetic variability. Single-nucleotide polymorphism (SNP) analysis detected seven genotypes (G1, G2, G12*-G15*, and G17*), with five being reported for the first time (*). The most prevalent was the newly described G13 (54%), while the other currently identified genotypes were predominantly found in single samples. The in silico restriction fragment length polymorphism (RFLP) method revealed five genotypes (B, F, N, P, and W), one of them previously unreported (W). Genotype B was the most prevalent (85%), comprising three SNP genotypes (G1, G2, and G13), whereas the other RFLP genotypes were associated with single SNP genotypes. These kDNA genotyping methods revealed significant intraspecific genetic diversity in L. infantum, demonstrating their suitability for fingerprinting and strain monitoring.

Role of Inflammatory/Immune Response and Cytokine Polymorphisms in the Severity of Chronic Hepatitis C (CHC) before and after Direct Acting Antiviral (DAAs) Treatment.

Host regulatory immune response is involved in the hepatic inflammatory process caused by the hepatitis C virus (HCV). We aimed to determine if HCV clearance with direct-acting antivirals (DAAs) changes the hepatic fibrosis stage, biochemical parameters of liver injury, and inflammatory/immune responses. Sample: 329 chronic hepatitis C (CHC) patients, 134 of them treated with DAAs. Liver fibrosis was evaluated by transient elastography (FibroScan), biochemical and cellular parameters were determined by standard methods, cytokine concentration by enzyme-linked immunoabsorbent assay (ELISA), and genetic polymorphisms by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or endpoint genotyping. Before DAA treatment, severe fibrosis or cirrhosis (F3/4) was associated with higher values of tumor necrosis factor-alpha (TNF-α) and genotypes transforming growth factor-beta-509 C/T_CC (TGF-β-509 C/T_CC), interleukine-10-1082 T/C_CC (IL-10-1082 T/C_CC), and IL-10-592 G/T_GT. After DAA treatment, fewer F3/4 patients and lower values of TNF-α were found. Patients with TNF-α-308 G/A_GG and IL-10-592 G/T_GT were at risk for F3/4. Lack of improvement of liver fibrosis was associated with lower baseline values of platelet count for genotypes TNF-α-308 G/A_GG and haplotype TT/GG of IL-10-1082 T/C and IL-10-592 G/T. Our study showed decreased liver fibrosis/inflammation and normalization of liver injury biomarkers after DAA treatment. It also points to the importance of suppressing the pro-inflammatory response by DAAs in the resolution of hepatitis C, contributing to the improvement of liver damage evaluated by transient elastography.

Abortion outbreak in a sheep flock caused by Toxoplasma gondii clonal type III.

Toxoplasma gondii is a major cause of reproductive losses in small ruminants in several countries. We describe here an outbreak of T. gondii-associated abortion in sheep in Southern Brazil. The flock was comprised of 55 adult sheep, and late-term abortions and stillbirths were detected in 15/36 (41.66%) gestating ewes. Serum samples collected from 45 sheep were tested for T. gondii through indirect immunofluorescence assay; IgM and IgG positive results were detected in 44.44% (20/45) and 86.67% (39/45) of the cases, respectively. Four fetuses and two placentas were pathologically evaluated. Gross changes were restricted to fetal membranes and were characterized by multifocal white areas in the cotyledons. Microscopically, these areas corresponded to necrotic foci affecting the chorionic epithelium accompanied by rare cysts of T. gondii. The main histological change in fetal tissues consisted of well-demarked and sparsely distributed necrotic foci in the central nervous system. Tissue samples from all four fetuses and one placenta had positive PCR results for T. gondii. Restriction fragment length polymorphism (RFLP) genotyping using ten markers (SAG1, 5'-3'SAG2, alt.SAG2, SGA3, BTUB, GRA6, c22-8, c29-2, L358, and PK1) was carried out on one sample, and results were consistent with T. gondii clonal type III (ToxoDB-PCR-RFLP genotype #2, TgCpBr4).

A Molecular Analysis Of Prion Protein Expression In Alzheimer's Disease

In Prion Diseases, misfolding of neuronal prion protein (PrPC) to a pathogenic isomer (PrPSC) is associated with neuronal death. Previous pathological studies have demonstrated increased immunoreactivity of PrPC at Aβ plaques in Alzheimer's Disease, and it has been suggested that this either reflects a role for PrPC in the neuronal response to stress or is a feature of the neuropathogenesis of atypical subtypes of Alzheimer's disease. In this paper we utilised western blotting to examine the molecular characteristics of PrP in frozen Hippocampal tissue from 7 cases of Alzheimer's Disease in which prion protein expression was demonstrated by immunohistochemistry, before using Restriction Fragment Length Polymorphism (RFLP) methodology to define the genotype of the codon 129 polymorphism of PRNP in each case. We observed PrP accumulating as globular structures at A plaques, and within ependymal cells lining the lateral ventricle. Immunohistochemistry also showed that PrPC and Superoxide dismutase-1 where deposited in a similar pattern at Aβ plaques. Western blotting revealed that PrP in Alzheimer's disease is composed of the same 208-residue peptide expressed in non-diseased brain. Quantitative western blot analysis demonstrated increased levels of PrPC in a short duration case of Alzheimer's Disease, while, in the remaining cases, levels of PrPC decreased in parallel with increasing disease duration and decreasing brain mass. RFLP genotyping revealed that all codon 129 genotypes (M/M, M/V, V/V) were represented in our study cohort. Our data suggest that increased levels of PrPC may account for PrP immunoreactivity at plaques in Alzheimer's disease, and that PrP deposition is not restricted to certain atypical subtypes of Alzheimer's disease.

173 The role of an intronic single nucleotide polymorphism within the dopamine receptor type-2 gene on pituitary prolactin protein expression in bulls

Abstract Detrimental effects to male reproductive physiology have been observed due to changes in prolactin (PRL) serum concentration. Regulation of PRL by dopamine binding to the dopamine type-2 receptor (DRD2) is well defined and associations between male physiology and single nucleotide polymorphisms (SNPs) within the DRD2 gene have been observed. The objective of the study was to evaluate association of a DRD2 SNP to PRL protein expression in bulls. Testis and epididymis were collected from bulls grazing a forage containing or lacking a dopamine agonist at the end of a 126 d study (n = 14). Bovine pituitaries (n = 587) were collected randomly over 3 mo from a local abattoir which processes cull cows and bulls. Sex of pituitaries was verified (n = 259 males) by duplex PCR for amplification of SRY and b-actin followed by Southern blotting of PCR products for selection of male. Prolactin protein expression was assessed in testis, epididymis, and pituitary by western blotting. Expression of PRL protein was below detection range in reproductive tissues but was present in pituitary, therefore experiments continued in pituitary. Restriction fragment length polymorphism genotyping was performed on pituitaries by amplification of the DRD2 SNP region followed by digestion with a Tfil enzyme. Digested of products produced 3,2, or 1 band (AG, AA, GG, respectively). A subset of male pituitaries was blotted by slot blot manifold and PRL protein expression assessed by immunodetection and densitometry analysis normalized to GAPDH expression. Pituitary genotype distribution was 17.4% AA (n = 16), 63% AG (n = 58), and 19.6% GG (n = 18). Prolactin protein expression in the pituitary was similar across genotype (P = 0.23). These findings indicate that the DRD2 SNP has no genotypic effect on PRL protein expression in bovine pituitary.

Isolation and genotyping of Toxoplasma gondii in the Midwestern Brazil revealed high genetic diversity and new genotypes

We aimed to describe the genetic diversity of Toxoplasma gondii strains isolated from domestic animals, wildlife and humans in the Midwestern Brazil. For this purpose, fragments of tissue samples (heart, brain and lung) from 35 dogs, four cats, 105 wildlife, and amniotic fluids from eight pregnant women were collected and submitted to mouse bioassay test. In a total, 22 isolates from nine dogs, one cat, ten wild animals and two women were obtained. The DNA was extracted from T. gondii isolates (lungs and brains of infected mice) and from “primary samples” (aliquots of tissue homogenate from wild animals and amniotic fluids from pregnant women) in order to screen using a Polymerase Chain Reaction (PCR) targeting a repeated 529-base pairs fragment of the T. gondii genome. All positive PCR samples were genotyped using restriction fragment length polymorphism (RFLP) analysis. To the best of our knowledge, this was the first study to report isolates of T. gondii from Leopardus pardalis, Crax fasciolata, and Dasyprocta azarae. Moreover, multilocus PCR-RFLP revealed 11 T. gondii RFLP genotypes, comprising nine previously described, including the archetypal lineage #2 type III (n = 1); two clonal Brazilian lineages, #6 type BrI (n = 1) and #8 type BrIII (n = 5); #14 (n = 2), #41 (n = 1), #108 (n = 1), #140 (n = 2), #166 (n = 4), #190 (n = 1), one potentially mixed, and two new described genotypes in two isolates. Our results confirmed the high diversity of T. gondii strains in Brazil, including identical genotypes circulating among humans, domestic dogs and wildlife.

A multi-restriction fragment length polymorphism genotyping approach including the beta-tubulin gene as a new differential nuclear marker for the recognition of the cryptic species Anisakis simplex s.s. and Anisakis pegreffii and their hybridization events

The detection of Anisakis simplex s.s./A. pegreffii putative hybrids has been a controversial issue in spite of the fact that natural hybridization is an extended process across free living and parasitic organisms. Differential traits of biomedical and ecological importance, such as invasive and allergenic potential have been demonstrated in both cryptic species. Therefore, in this work, we discuss about the potential for hybridization between these anisakid species in sympatric zones, implementing a multi-marker Restriction fragment length polymorphism (RFLP) genotyping approach based on the ribosomal DNA internal transcribed spacer 1 (ITS1), the mitochondrial cytochrome C oxidase 2 (Cox-2) and a new nuclear marker, the highly conserved β-tubulin gene (β-TUB). The two cryptic species differed at least in 7 bp in the β-TUB gene and some larvae with heterozygous genotypes at the 7 diagnostic nucleotide positions were found. Taxonomic, population and genealogical analyses served to support the occurrence of hybridization between both species. Predicted restriction endonucleases enzymes were assayed for Cox-2 and β-TUB markers. The implemented multi-marker PCR-RFLP allowed us to detect the two pure parental species, F1 hybrids, hybrid backcrossed progeny and individuals with nuclear-mitochondrial discordance, being a useful, simple and reproducible procedure in any laboratory for epidemiological studies.

Toxoplasmosis Outbreak Associated With Toxoplasma gondii-Contaminated Venison-High Attack Rate, Unusual Clinical Presentation, and Atypical Genotype.

During 2017, in response to a physician's report, the Wisconsin Department of Health Services, Division of Public Health, began investigating an outbreak of febrile illness among attendees of a retreat where never frozen, intentionally undercooked, locally harvested venison was served. Preliminary testing tentatively identified the illness as toxoplasmosis. Confirmatory human serology panels and testing of the venison to confirm and categorize the presence and type of Toxoplasma gondii were completed by French and American national reference laboratories. All 12 retreat attendees were interviewed; medical records were reviewed. All attendees were male; median age was 51 years (range: 22-75). After a median incubation period of 7 days, 9 (82%) of 11 exposed persons experienced illness lasting a median of 12 days. All 9 sought outpatient healthcare for symptoms including fever, chills, sweats, and headache (100%) and ocular disturbances (33%). Testing confirmed the illness as toxoplasmosis and venison as the infection source. Multiple laboratory results were atypical for toxoplasmosis, including transaminitis (86%), lymphocytopenia (88%), thrombocytopenia (38%), and leukopenia (63%). One exposed but asymptomatic person was seronegative; the other had immunity from prior infection. The T. gondii strain was identified as closely related to an atypical genotype (haplogroup 12, polymerase chain reaction restriction fragment length polymorphism genotype 5) common in North American wildlife but with previously uncharacterized human clinical manifestations. The T. gondii strain contaminating the venison might explain the unusual clinical presentations. In North America, clinicians and venison consumers should be aware of risk for severe or unusual presentations of acute toxoplasmosis after consuming undercooked game meat.

Impact of Genetic Variation in TLR4 3'UTR on NSCLC Genetic Susceptibility.

Toll-like receptors (TLRs) are expressed not only in immune cells but also in a variety of tumor cells. Single-nucleotide polymorphisms (SNPs) located in the TLRs' promoter or the 3′ untranslated region may affect gene expression by affecting the activity of the promoter or regulating the binding of mRNA to miRNA. This study aimed to investigate the association of the SNPs in TLR genes with the susceptibility to NSCLC. This case-control study involved 700 lung cancer patients and 700 healthy controls. All individuals were genotyped for all selected SNPs in TLR genes using polymerase chain reaction (PCR) test-based restriction fragment length polymorphism (PCR-RFLP) and TaqMan SNP genotyping assay. The association of genetic variations in TLRs with the susceptibility to NSCLC was evaluated by unconditional logistic regression with OR (95% CI). After evaluating transcriptional factor or miRNA binding capability by bioinformatics methods, six TLRs were identified for further analysis. We did not find that TLR3 rs5743303, TLR4 rs1927914, TLR4 rs11536891, TLR5 rs1640816, and TLR7 rs3853839 were associated with NSCLC risk (P > 0.05). Our data showed that TLR4 rs7869402 C > T polymorphism reduced the risk of NSCLC with OR (95% CI) of 0.63 (0.45–0.89). When stratified by gender and age, the individuals carrying at least one rs7869402T allele significantly decreased the NSCLC risk among males (OR = 0.58, 95% CI = 0.38–0.87) and among youngsters (OR = 0.43, 95% CI = 0.27–0.69). Smoking stratification analysis showed that the rs7869402T allele-containing genotype reduced the risk of NSCLC with OR (95% CI) of 0.50 (0.29–0.87) among smokers but not among nonsmokers (P > 0.05). When the individuals were classed by the pathological type, we found that the rs7869402T-containing genotype was associated with the risk of adenocarcinoma (OR = 0.62, 95% CI = 0.41–0.92) but not with that of squamous cell carcinoma (OR = 0.71, 95% CI = 0.44–1.13) and other types (OR = 0.23, 95% CI = 0.03–1.70). Compared with the TLR4 Ars1927914-Crs7869402-Trs11536891 haplotype, the Grs1927914-Trs7869402-Trs11536891 haplotype was associated with a decreased risk for developing NSCLC with OR (95% CI) of 0.57 (0.41–0.80). These results indicated that the TLR4 rs7869402 variation affects the genetic susceptibility to NSCLC.

Prevalence of Toxoplasma gondii Infections in Chicken Hearts from Farmers' Markets and Supermarkets in the Tai'an Region of China

The detection of Toxoplasma gondii in quick-frozen chickens is a good indicator of the possible transmission risk that this parasite poses to human consumers. The aim of this study was to investigate the occurrence of T. gondii in quick-frozen chicken hearts using a nested PCR assay. Heart samples (n = 720) from farmers' markets and supermarkets in the Tai'an region, People's Republic of China, were collected, and the DNA extracted was analyzed for the presence of T. gondii. The overall prevalence of T. gondii in all samples was 10.7%, but the rates for samples from farmers' markets (19.2%) and supermarkets (2.2%) were significantly different (P < 0.01). Nested PCR restriction fragment length polymorphism genotyping was performed on the 77 positive samples based on the T. gondii SAG3 and GRA6 gene loci. SAG3 genotyping revealed a mixed infection rate of 89.6% for type I and type I/II strains, and GRA6 genotyping revealed an infection rate of 98.7% for type I strains. Our results revealed a high prevalence of T. gondii in chicken hearts from farmers' markets, and most strains were mainly type I strains. Further studies are needed to determine whether quick-frozen chicken hearts are involved in disease transmission to human consumers in this area.

Stray dogs in the tropical state of Chiapas, Mexico, harbour atypical and novel genotypes of Toxoplasma gondii

Genotyping of Toxoplasma gondii remains a relevant topic of study, since genotypes can be related to the presentation and severity of toxoplasmosis. To date, 292 restriction fragment length polymorphism genotypes have been described around the world. Serosurveys in southeastern Mexico have documented exposure in over 70% of people and certain animals. Recently, we have described new genotypes and mixed infections in feral cats from Quintana Roo. Thus, the aim of this study was to genotype T. gondii and to describe its genetic variability, from naturally infected stray dogs of Chiapas, which has different geographical and climatic conditions from those found at the Yucatan Peninsula and the other parts of the country. Eleven stray dogs were captured and bled to obtain DNA, and then they were euthanized to perform necropsies and to collect target tissues. Diagnosis of T. gondii was done by quantitative real-time PCR (qPCR) and endpoint PCR. Genotyping was carried out, amplifying 12 polymorphic markers and 15 microsatellites. Atypical SAG3 gene products were cloned and sequenced. All blood samples of dogs were positive to T. gondii DNA by PCR. Two isolates were obtained from pooled heart and diaphragm tissue of two dogs. Two complete PCR-RFLP genotypes were identified (type BrIII and #28). Four animals had mixed infections. A new RFLP atypical allele for the SAG3 marker was observed; cloning and sequencing analysis of this locus revealed mixed infection by a strain identical to GT1, and one type I × II intragenic recombinant. The microsatellite analysis revealed that both isolates are atypical. Thus, atypical new genotypes of T. gondii and mixed infections were found in dogs of Chiapas. The results found here and in genotyping studies in México suggest that the southeastern region favours wide genetic diversity of T. gondii and the possible presence of virulent genotypes such as those found in central and South America.

Vascular Endothelial Growth Factor Receptor Polymorphisms and im Treatment Response in Chronic Myeloid Leukemia Patients

Abstract Background Imatinib mesylate (IM) is the gold standard drug for chronic myeloid leukemia (CML) treatment. However, intrinsic and acquired resistance against IM has become a problem in CML treatment. Vascular endothelial growth factor receptor 2 (VEGFR2) plays a pivotal role in leukemia associated angiogenesis. Its expression is a good predictor of poor survival in CML. Genetic polymorphisms in VEGFR2 result in varied VEGFR2 expression and may lead to inter individual variation in disease progression and outcome to IM therapy. Aim of this study was to investigate the frequencies of VEGFR2 polymorphisms rs1531289 and rs2305948 in Malaysian CML patients and to determine the impact of these SNPs on IM treatment response in these CML patients. Methods DNA extracted from 230 CML patients (121 IM resistant and 109 IM good response) were genotyped using restriction fragment length polymorphism analysis and genotypes were categorized into homozygous wildtype, heterozygous and homozygous variants. The difference in genotype frequencies of the two SNPs, between the two groups of CML patients were compared using x2 test and the association between genotypes and treatment response was assessed using logistic regression analysis and deriving ORs with 95% CI. Results The SNP rs1531289 did not show any significant association with IM resistance. For rs2305948, the heterozygous variant genotype (CT) and variant allele (T) showed significant association with IM resistance (OR, 2.38; CI,1.33-4.25, p = 0.003 and OR,1.48; CI, 1.02-2.14,p=0.039 respectively). Haplotype analysis of the two SNPs did not show any association with treatment response. Conclusion VEGFR2 polymorphism rs2305948 which is associated with cytogenetic response and development of resistance to IM therapy might be a potential prognostic predictor of IM treatment response in CML patients. This study was financially supported by Research University Grant of Universiti Sains Malaysia, 2017.

Genetic diversity and phylogenetic relationships between Leishmania infantum from dogs, humans and wildlife in south-east Spain.

Leishmania infantum causes human and canine leishmaniosis. The parasite, transmitted by phlebotomine sand flies, infects species other than dogs and people, including wildlife, although their role as reservoirs of infection remains unknown for most species. Molecular typing of parasites to investigate genetic variability and evolutionary proximity can help understand transmission cycles and designing control strategies. We investigated Leishmania DNA variability in kinetoplast (kDNA) and internal transcribed spacer 2 (ITS2) sequences in asymptomatically infected wildlife (n=58) and symptomatically and asymptomatically infected humans (n=38) and dogs (n=15) from south-east Spain, using single nucleotide polymorphisms (SNPs) and in silico restriction fragment length polymorphism (RFLP) analyses. All ITS2 sequences (n=76) displayed a 99%-100% nucleotide identity with a L.infantum reference sequence, except one with a 98% identity to a reference Leishmania panamensis sequence, from an Ecuadorian patient. No heterogeneity was recorded in the 73 L.infantum ITS2 sequences except for one SNP in a human parasite sequence. In contrast, kDNA analysis of 44 L.infantum sequences revealed 11 SNP genotypes (nucleotide variability up to 4.3%) and four RFLP genotypes including B, F and newly described S and T genotypes. Genotype frequency was significantly greater in symptomatic compared to asymptomatic individuals. Both methods similarly grouped parasites as predominantly or exclusively found in humans, in dogs, in wildlife or in all three of them. Accordingly, the phylogenetic analysis of kDNA sequences revealed three main clusters, two as a paraphyletic human parasites clade and a third including dogs, people and wildlife parasites. Results suggest that Leishmania infantum genetics is complex even in small geographical areas and that, probably, several independent transmission cycles take place simultaneously including some connecting animals and humans. Investigating these transmission networks may be useful in understanding the transmission dynamics, infection risk and therefore in planning L.infantum control strategies.

Identification of novel polymorphism in buffalo stanniocalcin-1 gene and its expression analysis in mammary gland under different stages of lactation

Stanniocalcin-1 (STC1) is secreted by the variety of tissues having a major role in the regulation of calcium ions in the involuting mammary gland. The present work aims to sequence and structural characterization as well as expression profiling of STC1 gene in buffalo. Polymorphism identified in the 3-untranslated region (UTR) was analysed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) genotyping in riverine and swamp buffaloes. Expression profiling of STC1 was performed in different lactation stages of mammary gland and peripheral blood mononuclear cells to study the impact of 3'-UTR polymorphism on its expression. Different polymorphic sites were detected in the entire coding and noncoding regions of riverine and swamp buffaloes, including two INDELs. An identified polymorphic nucleotide locus A324G, having target sites for two miRNAs, namely bta-miR-2382 and bta-miR-1343, reported in cattle, was genotyped by PCR-RFLP to reveal variable allelic distribution among swamp and riverine buffaloes. Gene expression profiling across buffalo mammary tissues representing different lactation stages showed maximum expression of the STC1 gene in the involuting mammary gland. Ruminants' specific genetic variation has been observed in STC1 and its implication in buffalo mammary gland involution as well as coregulation of gene expression through miRNA binding in the 3'-UTR is suggested.

Association between ABCB1 polymorphisms and response to first-generation antiepileptic drugs in a Tunisian epileptic population

ABSTRACTPurpose: We aimed in this study to investigate the association between the ATP-Binding Cassette sub-family B, member1 (ABCB1) polymorphisms: C1236T (rs1128503), G2677T (rs2032582) and C3435T (rs1045642), and the resistance to antiepileptic drugs (AEDs).Materials and methods: The Polymerase Chain Reaction–Restriction Fragment Length Polymorphism genotyping of ABCB1 polymorphisms was conducted on 153 Tunisian epileptic patients treated with AEDs.Results: Two genetic polymorphisms of the ABCB1 gene seemed to influence the response to AEDs. In fact, the G2677T T and the C3435T T alleles appeared to increase the risk of developing AEDs resistance (ORs* = 3.13; 95%CI = [1.16–8.98]; p = 0.024 and ORs* = 3.10; 95%CI = [1.15–8.37]; p = 0.025), respectively. However, the C1236T T allele did not seemed to influence the response to AEDs (ORs* = 1.14; 95%CI = [0.53–3.88]; p = 0.471). Haplotypic analysis indicated high-degree linkage disequilibrium of ABCB1 polymorphisms. Our results showed a synergic effect, in fact patients with the CTT and TTT haplotypes were more likely to be drug resistant than patients with the CGC haplotype, these associations remained significant even after adjustment for confounding parameters (ORs* = 2.68; 95%CI = [1.11–8.25]; p = 0.033 and ORs* = 3.76; 95%CI = [1.69–21.05]; p = 0.006, respectively).Conclusion: The G2677T T and C3435T T alleles as well as the TT, CTT and TTT haplotypes seemed to be significantly associated with drug-resistance epilepsy in our population. Genetic predisposition, involved in this resistance, may contribute to the establishment of a personal optimized therapy for newly diagnosed epileptic patients.

Molecular typing of Trichomonas vaginalis isolates by actin gene sequence analysis and carriage of T. vaginalis viruses

BackgroundThe protozoan parasite Trichomonas vaginalis is the most common non-viral, sexually transmitted pathogen. Although T. vaginalis is highly prevalent among women in Kenya, there is lack of data regarding genetic diversity of isolates currently in circulation in Kenya.MethodsTyping was performed on 22 clinical isolates of T. vaginalis collected from women attending the antenatal care clinic at Kilifi County Hospital, Kenya, in 2015. Genotyping followed a previously proposed restriction fragment length polymorphism (RFLP) scheme, which involved in silico cleavage of the amplified actin gene by HindII, MseI and RsaI restriction enzymes. Phylogenetic analysis of all the sequences was performed to confirm the results obtained by RFLP-analysis and to assess the diversity within the RFLP genotypes. Additionally, we determined carriage of the four different types of Trichomonas vaginalis viruses (TVVs) by polymerase chain reaction.ResultsIn silico RFLP-analysis revealed five actin genotypes; 50.0% of the isolates were of actin genotype E, 27.3% of actin genotype N, 13.6% of actin genotype G and 4.5% of actin genotypes I and P. Phylogenetic analysis was in agreement with the RFLP-analysis, with the different actin genotypes clustering together. Prevalence of TVVs was 43.5% (95% confidence interval, CI: 23.2–65.5). TVV1 was the most prevalent, present in 39.1% of the strains and 90% of the T. vaginalis isolates which harbored TVVs had more than one type of TVV. None of the isolates of actin genotype E harbored any TVV.ConclusionThe presence of five actin genotypes in our study suggests notable diversity among T. vaginalis isolates occurring among pregnant women in Kilifi, Kenya. Isolates of the most prevalent actin genotype E lacked TVVs. We found no association between T. vaginalis genotype, carriage of TVVs and symptoms. Further studies with higher number of strains should be conducted in order to corroborate these results.

Detection of newly defined superantigenic toxin genes and coagulase gene polymorphism in Staphylococcus aureus isolates.

The current study aims to use coagulase (coa) polymorphism gene to identify Staphylococcus aureus isolated from stool samples, evaluate the efficiency of these methods in discriminating variable strains, and compare these subtypes with antibiotypes. A total of 100 specimens were collected from patients in Babylon province, Iraq, between July 2016 and September 2016. Twenty S. aureus strains were isolated and identified using standard laboratory microbiological tests. The bacterial isolates were then examined by coa gene restriction fragment length polymorphism genotyping. Out of 20 isolates, coa gene types were classified, and the amplification products showed multiple size bands (500, 600, 700, 800, and 900-bp bands). Coa gene PCR restriction fragment length polymorphisms exhibited seven patterns that ranged from one to four fragments with AluI digestion. The results have demonstrated that many variants of the coa gene are present. At least one type of S. aureus newly described enterotoxin gene (staphylococcal enterotoxins) was harboring in all 20 (100%) of the isolates. The most frequently encountered gene were sei (100.%), seh (5%), seg (65%). Many S. aureus isolates carry at least one of the enterotoxin genes, and (95%) strains harbored more than one toxin gene coding.

NEWLY DESCRIBED TOXOPLASMA GONDII STRAIN CAUSES HIGH MORTALITY IN RED NECKED WALLABIES (MACROPUS RUFOGRISEUS) IN A ZOO.

This manuscript describes an outbreak of fatal toxoplasmosis in wallabies. Ten adult red necked wallabies (Macropus rufogriseus) were imported from New Zealand to the Virginia Zoo. Agglutination testing upon admission into quarantine showed all animals to be negative for antibodies to Toxoplasma gondii. Nine of these wallabies died from acute toxoplasmosis within 59-565 (average 224) days after being moved onto exhibit. Clinical signs included lethargy, diarrhea, tachypnea, and ataxia that progressed rapidly; death without premonitory signs occurred in one case. Histopathologic examination revealed interstitial pneumonia, encephalomyelitis, myositis, enteritis, and myocarditis. The diagnosis was confirmed through serologic, histopathologic, and polymerase chain reaction (PCR) testing. Multilocus PCR-RFLP (restriction fragment length polymorphism) genotyping revealed that the first six animals were infected by a previously undiscovered Toxoplasma gondii genotype, designated as ToxoDB PCR-RFLP genotype No. 263. These six cases survived for an average of 118 days on exhibit before succumbing to toxoplasmosis. The other three wallabies were infected with a Toxoplasma gondii strain of ToxoDB PCR-RFLP genotype No. 4, which is a common strain type circulating in wild animals in North America. These three cases survived for an average of 435 days on exhibit before succumbing to toxoplasmosis. The outbreaks of toxoplasmosis in these wallabies are likely from two different sources. Furthermore, the results highlight Toxoplasma gondii PCR-RFLP genotyping in parasite diagnosis and understanding parasite transmission and potential mitigation procedures.

Association of β-Secretase Functional Polymorphism with Risk of Schizophrenia.

The NRG1-ERBB4 neurotransmitter signaling pathway plays a key role in the pathogenesis of schizophrenia (SZ). The intronic single-nucleotide polymorphism rs707284 in ERBB4 links to PI3K-AKT suppression in SZ. Another protein indirectly affecting NRG1-ERBB4 signaling is β-secretase, which is encoded by the BACE1 gene, and activates NRG1 by proteolytic cleavage. In this study, we aimed to investigate the association of ERBB4 rs707284 and BACE1 rs490460 with the risk of SZ in an Iranian population. A total of 973 subjects, including 480 SZ patients and 493 healthy controls, matched by ethnicity, age, and gender, were recruited in a case-control study. Genomic DNA was extracted from peripheral blood of all subjects and genotyping of rs707284 and rs490460 was performed using polymerase chain reaction (PCR)-restriction fragment length polymorphism and tetra-primer amplification refractory mutation system (tetra-ARMS) PCR genotyping assays, respectively. A significant association was observed between the rs490460 T allele and SZ (p = 0.0002, odds ratio 0.69, 95% confidence interval 0.57-0.84). There was no association between the risk of SZ and rs707284. Our data indicate that rs490460 is associated with the risk of SZ. In silico analysis indicates that rs490460 may be a potential splicing site, which affects protein structure. Replication studies are needed to confirm our data.

Genetic variations of DICKKOPF family genes might not be associated with gastric cancer susceptibility.

BackgroundRecent studies have implicated that members of the DICKKOPF (DKK) were causally involved in large number of human cancers. This study was designed to investigate the relationship between the genetic variations of DKK family genes and the risk of gastric cancer (GC).MethodsSix SNPs (single nucleotide polymorphisms) of DKK family genes, including rs2241529 in DKK1, rs3733635, rs17037102 and rs419764 in DKK2, rs3206824 in DKK3 and rs2073664 in DKK4, were selected and genotyped by restriction fragment length polymorphism (RFLP) and TaqMan SNP genotyping methods in 409 GC cases and 554 cancer-free controls in the Han population in eastern China.ResultsNone of the six SNPs achieved significant association with the overall GC risk and stratified analysis by age, gender, smoking status, drinking status, tumor location and pathological classification confirmed these non-significant associations.ConclusionsOur study indicated that the studied six SNPs of DKKs would not be the risk factors for GC in this Han Chinese population. Studies of larger population for different ethnicities will be needed to warrant our findings.