PCR Restriction Analysis Research Articles

Isolation of potential zoonotic Mycobacterium spp. from diseased freshwater angelfish (Pterophyllum scalare) from an aquarium.

Nontuberculous mycobacteria infection is one of the most common chronic bacterial diseases in ornamental aquarium fish and appears to be directly related to stressful husbandry practices. Furthermore, it also represents zoonotic potential. Here we present the isolation and characterization of non-tuberculous mycobacteria from diseased freshwater angelfish (Pterophyllum scalare) in São Paulo, Brazil. Nine discarded breeding females with signs of disease were evaluated. The fish exhibited lethargy, loss of appetite, cachexia, skin ulcers, and exophthalmia. At necropsy, four fishes presented macroscopic granulomas in the spleen. Mycobacterium chelonae, M. fortuitum, M. gordonae, M. intracellulare and M. peregrinum were isolated and identified by hsp65 PCR restriction analysis. Histopathological analysis revealed microscopic lesions compatible with mycobacteriosis, and Mycobacterium bacillus were observed by Ziehl-Neelsen stain. Notably, all Mycobacterium species identified in this study have already been reported in human patients; therefore, diseased animals may be a source of infection for people who handle fish and aquariums.

The application of the self-probing primer PCR for quantitative expression analysis of R607Q (un)edited GluA2 AMPA receptor mRNA

Adenosine deaminase-dependent RNA editing is a widespread universal mechanism of posttranscriptional gene function modulation. Changes in RNA editing level may contribute to various physiological and pathological processes. In the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor GluA2 subunit, A-I editing in the Q607R site leads to dramatic changes in function, making the receptor channel calcium-impermeable. A standard approach for quantifying (un)edited RNAs is based on endpoint PCR (Sanger sequencing or restriction analysis), a time-consuming and semiquantitative method. We aimed to develop RT-qPCR assays to quantify rat Q607R (A-I) edited/unedited mRNA in samples in the present work. Based on self-probing PCR detection chemistry, described initially for detecting short DNA fragments, we designed and optimised RT-qPCR assays to quantify Q607R (un)edited mRNA. We used self-probing primer PCR technology for mRNA quantification for the first time. Using a novel assay, we confirmed that Q607R GluA2 mRNA editing was increased in 14-day- (P14) or 21-day-old (P21) postnatal brain tissue (hippocampus) compared to the embryonic brain (whole brains at E20) in Wistar rats. Q607R unedited GluA2 mRNA was detectable by our assay in the cDNA of mature brain tissue compared to that derived through classical methods. Thus, self-probing primer PCR detection chemistry is an easy-to-use approach for RT-qPCR analysis of RNA editing.

Association of SOD2 p.V16A polymorphism with Parkinson’s disease: A meta-analysis in Han Chinese

Oxidative stress could participate in the pathogenesis of Parkinson's disease (PD). However, the role of genetic variation of superoxide dismutase 2 (SOD2), an important regulator against oxidative stress, in PD remains to be elucidated. We screened SOD2 gene variation by sequencing cDNA from 72 patients with early onset PD. A cohort of PD (n=609) and ethnically matched controls (n=681) were further examined for the identified sequence variant by PCR and NaeI restriction analysis. Only a reported c.47T>C polymorphism (rs4880, SOD2 p.V16A) was found by cDNA sequencing. Case-control study of c.47T>C revealed that genotype and allele frequencies were in Hardy-Weinberg equilibrium in both patients and healthy controls. In a recessive model, those with CC genotype had a 2.61-fold increased risk of PD (95% CI: 1.08-6.30, P=0.03) compared to subjects with TT and TC genotypes. Significant association between CC genotype and PD in non-smokers was also observed after stratification according to the history of smoking (3.54-fold increased risk of PD, 95% CI: 1.17-10.72, P=0.02). Meta-analysis by combining studies of Chinese in China, Singapore, and Taiwan (total 2302 cases and 2029 controls) consistently showed CC genotype with increased risk of PD (OR=1.77, 95% CI: 1.15-2.71, P=0.01). Our findings demonstrate that SOD2 p.V16A may play a role in the susceptibility of PD in Han Chinese.

MiR-9-1 gene methylation and DNMT3B (rs2424913) polymorphism may contribute to periodontitis.

Genetic and epigenetic changes have been associated with periodontitis in various genes; however, little is known about genes involved in epigenetic mechanisms and in oxidative stress.Objective:This study aims to investigate the association of polymorphisms C677T in MTHFR (rs1801133) and −149C→T in DNMT3B (rs2424913), as well as the methylation profiles of MTHFR, miR-9-1, miR-9-3, SOD1, and CAT with periodontitis. The association between polymorphisms and DNA methylation profiles was also analyzed.Methodology:The population studied was composed of 100 nonsmokers of both sexes, divided into healthy and periodontitis groups. Genomic DNA was extracted from the epithelial buccal cells, which were collected through a mouthwash. Polymorphism analysis was performed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while methylation-specific PCR (MSP) or combined bisulfite restriction analysis techniques were applied for methylation analysis.Results:For DNMT3B, the T allele and the TT genotype were detected more frequently in the periodontitis group, as well as the methylated profile on the miR-9-1 promoter region. There was also a tendency towards promoter region methylation on the CAT sequence of individuals with periodontal disease.Conclusion:The polymorphism −149C→T in DNMT3B (rs2424913) and the methylated profile of the miR-9-1 promoter region are associated with periodontitis.

THE ROLE OF METHYLATION OF A GENE GROUP’S ОF THE SHORT ARM OF CHROMOSOME 3 IN DIAGNOSTICS AND PROGNOSIS OF BIOLOGICAL SUBTYPES OF BREAST CANCER

Breast cancer (BC) is the multifactorial disease where different genes play the major pathogenic role with tumor-suppressor genes in particular. These genes methylation is supposed to be the common regulation mechanism. The aim of the present paper is to study methylation in group of tumor-suppressor genes: RASSF1A, SEMA3B, RARP2, RHOA, GPX1 and NKIRAS1 in luminal and mioepithelial breast cancer, and to evaluate their correlation with clinical subtype's course of this disease. Methods. Analysis of methylation was done in group of 174 malignant/normal breast tissue pairs and of 10 health breast tissue samples. Two independent methods a methyl-specific PCR (RASSF1A, RARP2, SEMA3B) and methylation-sensitive restriction analysis (RHOA, GPX1, NKIRAS were used. Results. A statistically significant high methylation frequency for genes RASSF1A, SEMA3B and RAR-p2 were shown (the positive ratio ranged from 13.2% to 37.9%) compared with normal breast tissue (the positive ratio ranged 0-6.9%, respectively), (p<0.00001). We revealed high frequent methylation level of RAR-P2 gene that was significantly higher in the luminal subtype B with the over expression of Her2 and Ki-67 compared with Her2-negative type and low level of Ki-67 (44% vs. 23.1%, p = 0.0458 and 38.5% vs. 16.2%, p = 0.0059, respectively). Interestingly we report that 5 and 10 years survivability without methylation was 93.5%, 85.5%, and 91.3%, 83.7%, respectively, compared ones with methylation where was lower 80.3 %, 65.3% (p = 0.007) and 84.3%, 67.2% (р=0,038) respectively. Conclusion. Methylation of RASSF1A, SEMA3B, RAR-P2, GPX1, RHOA и NKIRAS1 genes plays important role in tumorigenesis of luminal breast cancer by modulating their functions. Methylation of the RASSF1A and RAR-P2 genes is associated with an unfavorable the disease outcome. Identified epigenetic alterations in luminal subtypes BC could be included in the biomarker system to improve in evaluating diagnosis, prognosis of disease, especially for choosing the individual tactics of treatment.

Idiopathic male infertility is strongly associated with aberrant DNA methylation of imprinted loci in sperm: a case-control study

BackgroundMale infertility is a complex disease caused by a combination of genetic, environmental, and lifestyle factors. Abnormal epigenetic programming has been proposed as a possible mechanism compromising male fertility. Recent studies suggest that aberrant imprinting in spermatozoa in a subset of infertile men is a risk factor for congenital diseases in children conceived via assisted reproduction techniques. In this study, we examined the DNA methylation status of CpG sites within the differentially methylated regions (DMRs) of three imprinted genes, H19, GNAS, and DIRAS3, using combined bisulfite PCR restriction analysis and bisulfite sequencing in sperm obtained from 135 men with idiopathic male infertility, including normozoospermia (n = 39), moderate oligozoospermia (n = 45), and severe oligozoospermia (n = 51), and fertile controls (n = 59). The percentage of global methylation was compared between fertile controls and infertile patients displaying abnormal DNA methylation status of imprinted loci. Moreover, we also analyzed whether the DNA methyltransferases (DNMTs) polymorphisms impact upon the methylation patterns of imprinted genes in idiopathic infertile males.ResultsAberrant methylation patterns of imprinted genes were more prevalent in idiopathic infertile males, especially in patients with oligozoospermia. Infertile males with aberrant methylation patterns of imprinted genes displayed a tendency of lower global methylation levels, although not reaching statistical significance (P = 0.13). In the genotype-epigenotype correlation analysis, no significant association was observed between aberrant methylation patterns of the three imprinted genes and genotypes of the four DNA methyltransferase (DNMT) genes.ConclusionWe conclude that abnormalities of DMR within imprinted genes may be associated with idiopathic male infertility. Disruption in methylation pattern of the three imprinted genes does not occur in high-risk genotypes of DNMTs.

Cloning of Phytase Genes from Pantoea Sp. 3.5.1 and Bacillus ginsengihumi M2.11 in Pichia pastoris

Phytases (myo-inositol hexakisphosphate hydrolase) catalyze the hydrolysis of phytate to inorganic phosphate and less phosphorylated myo-inositol derivatives and are widely used as feed additives in animal nutrition. Nevertheless, nowadays, there is a constant search for new phytases and new expression systems for better production of these important enzymes. In this study, we report cloning of two novel bacterial phytases belonging to the different enzyme classes and having different properties in the methylotrophic yeast Pichia pastoris. Sequences of agpP and phyC genes, encoding histidine acid phytase from Pantoea sp. 3.5.1 and β-propeller phytase from Bacillus ginsengihumi M2.11, respectively, were optimized and chemically synthesized according to the P. pastoris codon usage bias. The optimized genes were cloned into the yeast vectors pPINK-HC and pPINK-LC under the control of the inducible promoter AOX1 and two different signal peptides—signal sequence of α-amylase gene from Aspergillus niger and presequence of inulinase gene from Kluyveromyces maxianus. PCR analysis, restriction analysis, and DNA sequencing confirmed correct integration of agpP and phyC genes into P. pastoris genome. As a result, recombinant strains of P. pastoris with codon-optimized bacterial phytase genes (agpP and phyC), encoding histidine acid and β-propeller phytases, integrated into the genome under the alcohol oxidase promoter AOX1 and two different signal peptides, were obtained. Recombinant phytase AgpP was stably expressed and secreted into the culture medium of yeasts, whereas the expression of phyC gene was only confirmed at transcription level.

Association of polymorphic variants of IL-1β and IL-1RN genes in the development of Graves’ disease in Kashmiri population (North India)

PurposeGraves’ disease (GD) is a multigenic, organ specific autoimmune disorder with a strong genetic predisposition and IL-1β has been shown to be involved in its pathogenesis. The present study was aimed to determine the genetic associations between polymorphisms of IL-1β gene promoter region (−511 T>C) (rs16944), exon 5 (+3954 C>T) (rs1143634) and IL-1RN gene VNTR (rs2234663) polymorphism in patients with GD in ethnic Kashmiri population. MethodsA total of 135 Graves’ disease patients and 150 healthy individuals were included in the study. PCR and PCR-based restriction analysis methods were done for IL-1RNVNTR and IL-1β gene polymorphisms respectively. ResultsWe found statistically significant increased frequencies of the C/C + CT genotype (P = 0.001; odds ratio (OR) = 5.04, 95% confidence interval (CI) = 3.02–8.42) and the C allele (P = 0.001; OR = 3.10, 95% CI = 2.14–4.50) in IL-1β gene promoter polymorphism (rs16944) with GD patients compared to normal controls. Also in the exon 5 (rs1143634), a significant increase in frequency of the C/C homozygous genotype (P = 0.001; OR = 0.18, 95% CI = 0.11–0.30) and C allele (P = 0.001; OR = 0.31, 95% CI = 0.20–0.48) was observed in GD cases as against controls. For IL-1RNVNTR (rs2234663), we didn’t observe any significant difference in the allelic and genotypic frequencies between cases and controls. ConclusionOur findings suggest that both promoter and exon polymorphisms of IL-1β gene have a significant role in the risk of developing GD, whereas IL-1RNVNTR has no association with GD.

Genetic predisposition for chronic venous insufficiency in several genes for matrix metalloproteinases (MMP-2, MMP-9, MMP-12) and their inhibitor TIMP-2.

The project was scheduled as a case-control study to investigate the correlation between MMP-2 (rs243864), MMP-9 (3918242), MMP-12 (rs7123600) and TIMP-2 (rs8176329) polymorphisms and chronic venous disease (CVD) risk. The genotype and phenotype research envisages the testing of possible associations between MMP and TIMP-2 genotypes and phenotypes of CVD. 150 patients with CVD and 227 controls were enrolled into the study. The MMPs and TIMP-2 genotypes were identified by the PCR method and restriction analysis according to standard protocols. The G allele of MMP-2 -790 T/G was 1.85 times more frequent in men with CVD than in the control group (P = 0.008). The T allele of MMP-9 -1562 C/T was observed 2.571 times more frequently in patients with CVD than in the control individuals (both in men and women) with clinically significant specificity (P = 0.0000009). The G allele of MMP-12 rs7123600 was determined 2.082 times more frequently in female patients with CVD than in the control group with clinically significant specificity (P = 0.02). No significant result in TIMP-2 rs8176329 polymorphism in the case-control study was observed. CVD women with G allele in MMP-2 -790 T/G in the genotype-phenotype study are seen to develop ulceration 2.539 times more frequently (P = 0.003). The G allele of MMP-12 rs7123600 was detected 3.167 times more frequently in CVD women with ulceration compared with CVD women without ulceration (P = 0.007). In CVD men in C6 stage, the incidence of AG genotype in rs7123600 MMP-12 polymorphism was found to be 4.675 times higher compared to CVD women with C6 staging (P = 0.005). The AG genotype in TIMP 2 rs8176329 polymorphism was found to be associated with higher risk of tumour (P = 0.01). Studying these polymorphisms can contribute to better identification of patients at higher risk of developing CVD, while providing the most appropriate prevention and treatment strategies for limiting the progression and complications of CVD.

A study on genetic polymorphism in Matrix Metalloproteinases-3 in oral submucous fibrosis patients and in healthy individuals

BackgroundOSF is a potentially malignant condition affecting the oral cavity and oropharynx. MMP-3 also known as Stromelysin -I is a key member of the MMP family which is responsible for degradation of collagen type II,IV,V,IX and X, proteoglycans, gelatins, fibronectin, laminin and elastin. It plays an important role in activation of pro MMP-1 into the active form of MMP-1 in malignant tissues. MMP-3 expression is low in normal tissues but it is altered during tumour formation, where remodeling of ECM is required.Purpose of the studyTo assess the association of single-nucleotide polymorphisms, Adenosine (Insertion/Deletion) in -1171 5A > 6A in the MMP-3 promoter regions of patients with oral submucous fibrosis and in healthy individuals (controls).MethodsThirty cases of OSF were categorized according to Khanna et al classification into four groups and Twenty age and sex matched controls were included in the study. Blood samples were collected in EDTA coated vacutainers and PCR restriction analysis was done. A statistical analysis was done using Chi-square test and Fisher’s exact test to assess the frequency and association of the alleles in the case-control group.ResultsThe result showed a statistical significance difference between the duration of habit and disease severity with polymorphisms. The result also showed a higher frequency of the 5A allele in the study group as compared to the controls.ConclusionA long-term follow up of these patients is mandatory to see the prognosis and their susceptibility to malignancy. The positive outcome of an association of the disease with polymorphisms would result in the development of potential diagnostic and therapeutic possibilities in potentially malignant and malignant lesions.

Cutaneous infection due to Mycobacterium immunogenum: an European case report and review of the literature

(no more than 200 words): In the last few years, the incidence of cutaneous infections caused by nontuberculous mycobacteria is increasing. Since Mycobacterium immunogenum was first described in 2001, few case reports have been described, all of them in the American continent. We report a case with cutaneous infection caused by this newly discovered NTB in Europe.A 65-year-old woman presented with a 3-months history of pruritic lesions on abdomen. Examination revealed erythematous inflammatory papules, pustules, and crusts. Three weeks later, mycobacteria were cultured from the biopsy specimen. Mycobacterium immunogenum was identified based on susceptibility test results and polymerase chain reaction (PCR) restriction enzyme analysis. Treatment with clarithromycin was started. M. immunogenum is a nontuberculous mycobacterium that was first described by Wilson et al. in 2001 as a rapidly growing variety and new species in the Mycobacterium chelonae-Mycobacterium abscessus group. PCR-restriction analysis of a 439-bp segment of the hsp65 gene and/or sequencing the species-specific region of the 16S rDNA can confirm this new species. Since the description of M. immunogenum, 8 clinical case reports have been published, most involving cutaneous infections and all of them in the American continent. We present a case of cutaneous infection caused by M. immunogenum in a Spanish woman.

Infection by Mycobacterium bovis in a dog from Brazil.

Tuberculosis (TB) is a chronic disease caused by bacteria belonging to the Mycobacterium tuberculosis complex (MtbC). This disease rarely affects dogs. Canine infections are usually caused by M. tuberculosis. Mycobacterium bovis infections are rare in dogs and associated with consumption of raw milk or contaminated products. Here, we report a Boxer dog who had a M. bovis infection and was admitted to a Brazilian veterinary hospital with a presumptive diagnosis of chronic ehrlichiosis. Despite receiving treatment for chronic ehrlichiosis, it progressed to death. TB was diagnosed during post-mortem examinations using histopathological analysis. Ziehl-Neelsen staining revealed acid-fast bacilli in the kidneys, liver, mesentery, and a mass adhered to the liver. Further, PCR-restriction analysis was performed to identify mycobacteria in the samples. A restriction profile compatible with MtbC was found in the lungs. In addition, PCR-based MtbC typing deletions at different loci of chromosome 9 enabled the identification of M. bovis in the lungs. Therefore, it is very essential to perform differential diagnosis of TB in dogs with non-specific clinical signs and who do not respond to treatment, particularly those who had been in contact with TB-infected cattle or owners. Further, we highlight the use of molecular methods for the identification of bacilli, improving the diagnosis and aiding epidemiological studies.

Abstract 1095: S100A4 is post-transcriptionally inhibited by miR-505-5p and miR-520c-3p in colorectal cancer

Abstract The calcium binding protein S100A4 induces epithelial mesenchymal transition (EMT), migration, invasion, angiogenesis and metastasis. Its induced expression is reported in several cancer types and correlates with poor prognosis. S100A4 is transcriptionally regulated by Sp1, AP1 and CCAAT/enhancer binding protein β transcription factors and its regulation epigenetically controlled by CpG hyper methylation. However, apart from S100A4 functional and transcriptional regulatory aspects, post-transcriptional regulation is not yet elucidated. In this study, we show that specific microRNAs (miR) target the 3’UTR of S100A4. Luciferase-reporter assays with wild-type and mutated miR-505-5p and miR-520c-3p seed sequences of S100A4-3’UTR confirmed the specificity of targeting. Similarly, overexpression of these miRs reduced the S100A4 and inhibition of these miRs function increased the S100A4 protein significantly. Moreover, co-transfection of either S100A4 expressing vector or siRNA-S100A4 along with the miR-505-5p and miR-520c-3p significantly altered the S100A4 mediated migration and invasion in colorectal cancer (CRC) cell lines (p = 0.05). Both these miRs were down-regulated in CRC cell lines and 5 Aza treatment increased these miRs expression and reduced the S100A4 protein amounts. Moreover, in a panel of CRC resected patient tissues (n = 59), miR-520c-3p significantly down regulated (p = 0.03). miR-520c (located in Chr 19, in a big cluster) upstream region was checked for methylation status using methylation specific PCR and combined bisulfite restriction analysis (COBRA) in CRC cell lines, metastatic and non-metastatic CRC resected tissues. We found the upstream promoter region hyper methylated irrespective of metastasis status of patients and in all CRC cell lines checked. 5 Aza treatment of S100A4 less or no expressing cells line Colo206 and Colo320 showed induced miR-520c expression and reduced S100A4 protein in Colo320. Colo206 cells did not show any alternations in expression of S100A4. Taken together, our findings demonstrate that S100A4 is post-transcriptionally regulated by tumor suppressor genes miR-505c and miR-520c and especially miR-520c expression is epigenetically silenced in CRC. Citation Format: Giridhar Mudduluru, Katharina Ilm, Steffen Fuchs, Ulrike Stein. S100A4 is post-transcriptionally inhibited by miR-505-5p and miR-520c-3p in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1095.

Mycobacterium saopaulense sp. nov., a rapidly growing mycobacterium closely related to members of the Mycobacterium chelonae--Mycobacterium abscessus group.

Five isolates of non-pigmented, rapidly growing mycobacteria were isolated from three patients and,in an earlier study, from zebrafish. Phenotypic and molecular tests confirmed that these isolates belong to the Mycobacterium chelonae-Mycobacterium abscessus group, but they could not be confidently assigned to any known species of this group. Phenotypic analysis and biochemical tests were not helpful for distinguishing these isolates from other members of the M. chelonae–M.abscessus group. The isolates presented higher drug resistance in comparison with other members of the group, showing susceptibility only to clarithromycin. The five isolates showed a unique PCR restriction analysis pattern of the hsp65 gene, 100 % similarity in 16S rRNA gene and hsp65 sequences and 1-2 nt differences in rpoB and internal transcribed spacer (ITS) sequences.Phylogenetic analysis of a concatenated dataset including 16S rRNA gene, hsp65, and rpoB sequences from type strains of more closely related species placed the five isolates together, as a distinct lineage from previously described species, suggesting a sister relationship to a group consisting of M. chelonae, Mycobacterium salmoniphilum, Mycobacterium franklinii and Mycobacterium immunogenum. DNA–DNA hybridization values .70 % confirmed that the five isolates belong to the same species, while values ,70 % between one of the isolates and the type strains of M. chelonae and M. abscessus confirmed that the isolates belong to a distinct species. The polyphasic characterization of these isolates, supported by DNA–DNA hybridization results,demonstrated that they share characteristics with M. chelonae–M. abscessus members, butconstitute a different species, for which the name Mycobacterium saopaulense sp. nov. is proposed. The type strain is EPM10906T (5CCUG 66554T5LMG 28586T5INCQS 0733T).

ITS-2 sequences-based identification of Trichogramma species in South America.

ITS2 (Internal transcribed spacer 2) sequences have been used in systematic studies and proved to be useful in providing a reliable identification of Trichogramma species. DNAr sequences ranged in size from 379 to 632 bp. In eleven T. pretiosum lines Wolbachia-induced parthenogenesis was found for the first time. These thelytokous lines were collected in Peru (9), Colombia (1) and USA (1). A dichotomous key for species identification was built based on the size of the ITS2 PCR product and restriction analysis using three endonucleases (EcoRI, MseI and MaeI). This molecular technique was successfully used to distinguish among seventeen native/introduced Trichogramma species collected in South America.

Conservation of imprinting of Neuronatin (Nnat) in rabbits.

Although the expression and epigenetic status of imprinted genes have been extensively studied in a number of species, less is known about the genomic imprinting in rabbits. Neuronatin (Nnat) plays significant roles in the brain development and metabolic regulation and has been identified to be imprinted and paternally expressed in humans, mice and pigs; however, it has not yet been investigated in rabbits. In this study, we confirmed the expression of two isoforms of the rabbit Nnat (Nnat-a and Nnat-β) identified in Genbank and Ensembl by quantitative real-time PCR. In addition, we also determined the methylation profile of the CpG island in the promoter region of the rabbit Nnat using bisulfite sequencing PCR and combined bisulfite restriction analysis. Here, we provide the first evidence that Nnat has two transcripts in rabbit. Additionally, the CpG island located in the promoter region shows oocyte-specific methylation and may be the differentially methylated region of Nnat in rabbits.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1054-z) contains supplementary material, which is available to authorized users.

Algorithms of Rickettsiae’s Detection and Laboratory Diagnosis of Rickettsioses of Spotted Fever Group in Russia

An analysis of modern trends of Rickettsiae’s detection and laboratory diagnostics for spotted fever group rickettsioses is present. Due to the sharp decline in the range of manufactured products and increase the spectrum of rickettsial species identified in Russia, new approaches required to laboratory verification of diagnoses. IFA and ELISA with antigens of relevant species of Rickettsia can be recommended to detect antibodies to the SFG rickettsiae. PCR-restriction analysis and sequencing of PCR products most appropriate for the detection and identification of SFG Rickettsia, biological methods are necessary for studying of pathogenic species of Rickettsia.

Identificación de Mycobacterium spp a través de métodos moleculares en el Laboratorio de Salud Pública de Bogotá, Colombia

IntroductionGlobal epidemiology of non-tuberculous mycobacteria (NTM) is unknown due to the fact that notification is not required in many countries, however the number of infection reports and outbreaks caused by NTM suggest a significant increase in the last years. Traditionally, mycobacteria identification is made through biochemical profiles which allow to differentiate M. tuberculosis from NTM, and in some cases the mycobacteria species. Nevertheless, these methods are technically cumbersome and time consuming. On the other hand, the introduction of methods based on molecular biology has improved the laboratory diagnosis of NTM. ObjectiveTo establish the NTM frequency in positive cultures for acid-fast bacilli (AAFB) which were sent to Laboratorio de Salud Pública de Bogotá over a 12 month period. Materials and methodsA total of 100 positive cultures for acid-fast bacilli from public and private hospitals from Bogotá were identified by both biochemical methods and the molecular methods PRA (PCR-restriction enzyme analysis) and multiplex-PCR. Furthermore, low prevalence mycobacteria species and non-interpretable results were confirmed by 16SrDNA sequentiation analysis. ResultsIdentification using the PRA method showed NMT occurrence in 11% of cultures. In addition, this molecular methodology allowed to detect the occurrence of more than one mycobacteria in 4% of the cultures. Interestingly, a new M. kubicae pattern of PCR-restriction analysis is reported in our study. ConclusionUsing a mycobacteria identification algorithm, which includes the molecular method PRA, improves the diagnostic power of conventional methods and could help to advance both NTM epidemiology knowledge and mycobacteriosis control.

Identification of Clinical Corynebacterium striatum Strains by PCR-Restriction Analysis Using the RNA Polymerase β-subunit gene (rpob)

Corynebacterium striatum is frequently encountered in the routine clinical microbiology laboratory. It is widely disseminated in the environment and constitutes part of the normal micro-biota of the skin and mucous membrane. Identification of this species by biochemical methods remains difficult and several misidentifications have been reported previously. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for the identification of this microorganism was designed based on the hypervariable region of the polymorphic RNA polymerase β-subunit gene (rpoB). All available Corynebacterium rpoB sequences were analyzed by computerassisted restriction analysis. The rpoB PCR-RFLP pattern predicted by using endonucleases MseI and NlaIV clearly differentiated C. striatum from all other Corynebacterium species. This method was successfully applied for the reliable identification of 67 C. striatum clinical isolates and can be used for the timely detection of infected patients or for epidemiological studies.

BAX G(-248)A Gene Polymorphism and Its Association with Risk of Non-Small Cell Lung Cancer—A Case Control Study

Pro-apoptotic Bcl-2 protein BAX is an important member of mitochondrial dependent apoptosis regulation and ultimately plays a pivotal role in malignancies. A promoter G(-248)A polymorphism in the TP53 binding region of BAX results in differential binding capacity of TP53 protein there by regulating its expression, which has been found to be associated with different clinical outcomes in various malignancies. Presently we aimed to analyze the possible impact of the BAX G(-248)A polymorphism on the risk and other clinical features of non-small cell lung cancer in Indian population. The BAX promoter polymorphism was analyzed in blood samples of 320 subjects with 1:1 case/control ratio by primer-introduced restriction analysis PCR and survival curves were plotted using Kaplan-Meier analysis. It was observed that more than 3-fold increased risk of developing non-small cell lung cancer was associated with homozygous AA genotype of BAX G(-248)A promoter polymorphism in Indian population, with more predominant in smokers with pack-year > 45 (heavy) and using cigarette or huka as their smoking source than homozygous GG genotype. Significant associations was observed between TNM stage (p = 0.037) and histological type (0.02), of non-small cell lung cancer patients with the polymorphism. Patients homozygous for A allele exhibited a significant poor overall survival compared with patients displaying GA + AA or GA or GG genotype [median survival 6.0 vs 9.0, 11.0, and 30.0 months, respectively (p < 0.0001)]. Adenocarcinoma and advanced stage patients with AA genotype showed lower median survival time than squamous cell carcinoma and early stage non-small cell lung cancer patients (median 3.0 and 5.0 vs 8.0 and 9.0 months, respectively). We conclude that the genetic polymorphism G(-248)A in the TP53 binding promoter region of pro-apoptotic genes BAX may contribute to the risk of developing non-small cell lung cancer in Indian population and also may be an important factor for adverse clinical outcome for patients with non-small cell lung cancer.