Optimized molecular resolution of cross-contamination alerts in clinical mycobacteriology laboratories

BACKGROUND. The phenomenon of misdiagnosing tuberculosis (TB) by laboratory cross-contamination when culturing Mycobacterium tuberculosis (MTB) has been widely reported and it has an obvious clinical, therapeutic and social impact. The final confirmation of a cross-contamination event requires the molecular identification of the same MTB strain cultured from both the potential source of the contamination and from the false-positive candidate. The molecular tool usually applied in this context is IS6110-RFLP which takes a long time to provide an answer, usually longer than is acceptable for microbiologists and clinicians to make decisions. Our purpose in this study is to evaluate a novel PCR-based method, MIRU-VNTR as an alternative to assure a rapid and optimized analysis of cross-contamination alerts. RESULTS. MIRU-VNTR was prospectively compared with IS6110-RFLP for clarifying 19 alerts of false positivity from other laboratories. MIRU-VNTR highly correlated with IS6110-RFLP, reduced the response time by 27 days and clarified six alerts unresolved by RFLP. Additionally, MIRU-VNTR revealed complex situations such as contamination events involving polyclonal isolates and a false-positive case due to the simultaneous cross-contamination from two independent sources. CONCLUSION. Unlike standard RFLP-based genotyping, MIRU-VNTR i) could help reduce the impact of a false positive diagnosis of TB, ii) increased the number of events that could be solved and iii) revealed the complexity of some cross-contamination events that could not be dissected by IS6110-RFLP.

The L162V polymorphism of the PPARA gene is associated with stage C of heart failure.

In human heart failure (HF) peroxisome proliferator-activated receptor alpha (PPAR alpha) is downregulated and consequently, the expression of genes involved in fatty acid oxidation repressed. The L162V (rs1800206) is a functional polymorphism of the human PPAR alpha gene (PPARA). In the present study we have investigated whether this polymorphism is associated with the development of stage C of HF.

Human genetic selection on the MTHFR 677C>T polymorphism.

BACKGROUND The prevalence of genotypes of the 677C>T polymorphism for the MTHFR gene varies among humans. In previous studies, we found changes in the genotypic frequencies of this polymorphism in populations of different ages, suggesting that this could be caused by an increase in the intake of folate and multivitamins by women during the periconceptional period. The aim was to analyze changes in the allelic frequencies of this polymorphism in a Spanish population, including samples from spontaneous abortions (SA). METHODS A total of 1305 subjects born in the 20th century were genotyped for the 677C>T polymorphism using allele specific real-time PCR with Taqman probes. A section of our population (n = 276) born in 1980-1989 was compared with fetal samples (n = 344) from SA of unknown etiology from the same period. RESULTS An increase in the frequency of the T allele (0.38 vs 0.47; p < 0.001) and of the TT genotype (0.14 vs 0.24; p < 0.001) in subjects born in the last quarter of the century was observed. In the 1980-1989 period, the results show that the frequency of the wild type genotype (CC) is about tenfold lower in the SA samples than in the controls (0.03 vs 0.33; p < 0.001) and that the frequency of the TT genotype increases in the controls (0.19 to 0.27) and in the SA samples (0.20 to 0.33 (p < 0.01)); r = 0.98. CONCLUSION Selection in favor of the T allele has been detected. This selection could be due to the increased fetal viability in early stages of embryonic development, as is deduced by the increase of mutants in both living and SA populations.

The TT genotype of the STAT4 rs7574865 polymorphism is associated with high disease activity and disability in patients with early arthritis

BACKGROUND The number of copies of the HLA-DRB1 shared epitope, and the minor alleles of the STAT4 rs7574865 and the PTPN22 rs2476601 polymorphisms have all been linked with an increased risk of developing rheumatoid arthritis. In the present study, we investigated the effects of these genetic variants on disease activity and disability in patients with early arthritis. METHODOLOGY AND RESULTS We studied 640 patients with early arthritis (76% women; median age, 52 years), recording disease-related variables every 6 months during a 2-year follow-up. HLA-DRB1 alleles were determined by PCR-SSO, while rs7574865 and rs2476601 were genotyped with the Taqman 5' allelic discrimination assay. Multivariate analysis was performed using generalized estimating equations for repeated measures. After adjusting for confounding variables such as gender, age and ACPA, the TT genotype of rs7574865 in STAT4 was associated with increased disease activity (DAS28) as compared with the GG genotype (β coefficient [95% confidence interval] = 0.42 [0.01-0.83], p = 0.044). Conversely, the presence of the T allele of rs2476601 in PTPN22 was associated with diminished disease activity during follow-up in a dose-dependent manner (CT genotype = -0.27 [-0.56- -0.01], p = 0.042; TT genotype = -0.68 [-1.64- -0.27], p = 0.162). After adjustment for gender, age and disease activity, homozygosity for the T allele of rs7574865 in STAT4 was associated with greater disability as compared with the GG genotype. CONCLUSIONS Our data suggest that patients with early arthritis who are homozygous for the T allele of rs7574865 in STAT4 may develop a more severe form of the disease with increased disease activity and disability.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Polymerase chain reaction detection of LeishmaniaDNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detectLeishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmaniagenus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

Complement Factor B Polymorphism and the Phenotype of Early Age-related Macular Degeneration.

Abstract Purpose: Age-related macular degeneration (AMD) has been associated with a number of polymorphisms in genes in the complement pathway. We examined the potential genotype-phenotype correlation of complement factor B (CFB) (R32Q) polymorphisms in Caucasian patients with AMD. Methods: Data from a Central European cohort of 349 patients with early AMD in at least one eye were analyzed for potential associations of the CFB (R32Q/rs641153) polymorphism with phenotypic features of early AMD. Early AMD was classified according to the International Classification and Grading System into predominant drusen size, largest drusen, drusen covered surface, central or ring-like location, peripheral drusen, and pigmentary changes. The potential association with single nucleotide polymorphisms on CFB (R32Q/rs641153) was evaluated for all patients, corrected for age, sex, and the polymorphisms of CFH (Y402H) and ARMS2 (A69S). Results: CFB (R32Q) polymorphisms showed a significant association with smaller drusen size (largest drusen ≤250 µm, p = 0.021, predominant drusen ≤125 µm, p = 0.016), with smaller surface covered by drusen (≤10%; p = 0.02), and with more frequent occurrence of peripheral drusen (p = 0.007). No association was found for pigmentary changes. Conclusions: The CFB (R32Q) polymorphism was associated with AMD characterized by small drusen only, and appeared to be protective of large drusen (OR 0.48/0.45) and of larger drusen covered area (OR 0.34). Furthermore, peripheral drusen were more frequently found (OR 2.27). This result supports the role of complement components and their polymorphisms in drusen formation and may enable a better understanding of AMD pathogenesis.

Interaction of GAG trinucleotide repeat and C-129T polymorphisms impairs expression of the glutamate-cysteine ligase catalytic subunit gene.

Glutamate cysteine ligase (GCL) catalyzes the rate-limiting step in the de novo synthesis of glutathione (GSH). The catalytic subunit (GCLC) of GCL contains a GAG trinucleotide-repeat (TNR) polymorphism within the 5'-untranslated region (5'-UTR) that has been associated with various human disorders. Although several studies suggest that this variation influences GSH content, its implication for GCLC expression remains unknown. To better characterize its functional significance, we performed reporter gene assays with constructs containing the complete GCLC 5'-UTR upstream of a luciferase gene. Transfection of these vectors into various human cell lines did not reveal any significant differences between 7, 8, 9, or 10 GAG repeats, under either basal or oxidative stress conditions. To correlate these results with the previously described down-regulation induced by the C-129T GCLC promoter polymorphism, combinations of both variations were tested. Interestingly, the -129T allele down-regulates gene expression when combined with 7 GAG but not with 8, 9, or 10 GAG TNRs. This observation was confirmed in primary fibroblast cells, in which the combination of GAG TNR 7/7 and -129C/T genotypes decreased the GCLC protein level. These results provide evidence that interaction of the two variations can efficiently impair GCLC expression and thus suggest its involvement in the pathogenesis of diseases related to GSH metabolism.

Skin fibroblast model to study an impaired glutathione synthesis: consequences of a genetic polymorphism on the proteome.

An impaired glutathione (GSH) synthesis was observed in several multifactorial diseases, including schizophrenia and myocardial infarction. Genetic studies revealed an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL). Disease-associated genotypes of this polymorphism correlated with a decrease in GCLC protein expression, GCL activity and GSH content. To clarify consequences of a decreased GCL activity at the proteome level, three schizophrenia patients and three controls have been selected based on the GCLC GAG TNR polymorphism. Fibroblast cultures were obtained by skin biopsy and were challenged with tert-butylhydroquinone (t-BHQ), a substance known to induce oxidative stress. Proteome changes were analyzed by two dimensional gel electrophoresis (2-DE) and results revealed 10 spots that were upregulated in patients following t-BHQ treatment, but not in controls. Nine corresponding proteins could be identified by MALDI mass spectrometry and these proteins are involved in various cellular functions, including energy metabolism, oxidative stress response, and cytoskeletal reorganization. In conclusion, skin fibroblasts of subjects with an impaired GSH synthesis showed an altered proteome reaction in response to oxidative stress. Furthermore, the study corroborates the use of fibroblasts as an additional mean to study vulnerability factors of psychiatric diseases.

Mspl restriction fragment length polymorphism near exon 10 of cystic fibrosis (CFTR) gene.

Source/Description: The probe used is a 98 bp fragment amplified by PCR from a cDNA clone of the CFTR gene or from genomic DNA corresponding to exon 10, using two primers from this exon (1)...

Plasminogen activator inhibitor-1 activity and 4G/5G polymorphism in hemodialysis

Introduction: Chronic insufficiency alters homeostasis, in part due to endothelial inflammation. Plasminogen activator inhibitor-1 (PAI-1) is increased in renal disease, contributing to vascular damage. We assessed PAI-1 activity and PAI-1 4G/5G polymorphism in hemodialysis (HD) subjects and any association between thrombotic vascular access (VA) events and PAI-1 polymorphism. Methods: Prospective, observational study in 36 HD patients: mean age: 66.6 +/- 12.5 yr, males n=26 (72%), time on HD: 28.71 +/- 22.45 months. Vascular accesses: 10 polytetrafluoroethylene grafts (PTFEG), 22 arteriovenous fistulae (AVF), four dual lumen catheters (CAT). Control group (CG): 40 subjects; mean age: 60.0 +/- 15 yrs, males n=30 (75%). Group A (GA): thrombotic events (n=12), and group B (GB): No events (n=24). Groups were no different according to age (69.2 +/- 9.12 vs. 65.3 +/- 14.5 yrs), gender (males: 7; 58.3% vs. 18; 81.8%), time on HD (26.1 +/- 14.7 vs. 30.1 +/- 38.7 months), causes of renal failure. Time to follow-up, for access thrombosis: 12 months. Results: PAI-1 levels in HD: 7.21 +/- 2.13 vs. CG: 0.42 +/- 0.27 U/ml (p < 0.000 1). PAI-1 4G/5G polymorphic variant distribution in HD: 5G/5G: 6 (17%),4G/5G: 23 (64%); 4G/4G: 7 (19%) and in CG: 5G/5G: 14 (35%); 4G/5G: 18 (45%); 4G/4G: 8 (20%). C-reactive protein (CRP) in HD: 24.5 +/- 15.2 mg/L vs. in CG 2.3 +/- 0.2 mg/L (p < 0.0001). PAI-1 4G/5G variants: GA: 5G/5G: 3; 4G/5G: 8; 4G/4G: 1; GB: 5G/5G: 3; 4G/5G: 15; 4G/4G: 6. Thrombosis occurred in 8/10 patients (80%) with PTFEG, 3/22 (9%) in AVF, and 1/4 (25%) in CAT. Among the eight PTFEG patients with thrombosis, seven were PAI 4G/5G. Conclusions: PAI-1 levels were elevated in HD patients, independent of their polymorphic variants, 4G/5G being the most prevalent variant. Our data suggest that in patients with PTFEG the 4G/5G variant might be associated with an increased thrombosis risk.

Leishmania major: histone H1 gene expression from the sw3 locus.

Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by the sw3 gene. Here we report that histone H1 variants exist in different Leishmania species and strains of L. major and that they are encoded by polymorphic genes. Amplification of the sw3 gene from the genome of three strains of L. major gave rise to different products in each strain, suggesting the presence of a multicopy gene family. In L. major, these genes were all restricted to a 50-kb Bg/II fragment found on a chromosomal band of 1.3 Mb (chromosome 27). The detection of RFLPs in this locus demonstrated its heterogeneity within several species and strains of Leishmania. Two different copies of sw3 (sw3.0 and sw3.1) were identified after screening a cosmid library containing L. major strain Friedlin genomic DNA. They were identical in their 5' UTRs and open reading frames, but differed in their 3' UTRs. With respect to the originally cloned copy of sw3 from L. major strain LV39, their open reading frames lacked a repeat unit of 9 amino acids. Immunoblots of L. guyanensis parasites transfected with these cosmids revealed that both copies could give rise to the histone H1 protein. The characterization of this locus will now make possible a detailed analysis of the function of histone H1 in Leishmania, as well as permit the dissection of the molecular mechanisms governing the developmental regulation of the sw3 gene.

Study of simple sequence repeat markers from coffee expressed sequences associated to leaf miner resistance

The objective of this work was to identify expressed simple sequence repeats (SSR) markers associated to leaf miner resistance in coffee progenies. Identification of SSR markers was accomplished by directed searches on the Brazilian Coffee Expressed Sequence Tags (EST) database. Sequence analysis of 32 selected SSR loci showed that 65% repeats are of tetra-, 21% of tri- and 14% of dinucleotides. Also, expressed SSR are localized frequently in the 5'-UTR of gene transcript. Moreover, most of the genes containing SSR are associated with defense mechanisms. Polymorphisms were analyzed in progenies segregating for resistance to the leaf miner and corresponding to advanced generations of a Coffea arabica x Coffea racemosa hybrid. Frequency of SSR alleles was 2.1 per locus. However, no polymorphism associated with leaf miner resistance was identified. These results suggest that marker-assisted selection in coffee breeding should be performed on the initial cross, in which genetic variability is still significant.

Ecology and life history affect different aspects of the population structure of 27 high-alpine plants.

A plant species' genetic population structure is the result of a complex combination of its life history, ecological preferences, position in the ecosystem and historical factors. As a result, many different statistical methods exist that measure different aspects of species' genetic structure. However, little is known about how these methods are interrelated and how they are related to a species' ecology and life history. In this study, we used the IntraBioDiv amplified fragment length polymorphisms data set from 27 high-alpine species to calculate eight genetic summary statistics that we jointly correlate to a set of six ecological and life-history traits. We found that there is a large amount of redundancy among the calculated summary statistics and that there is a significant association with the matrix of species traits. In a multivariate analysis, two main aspects of population structure were visible among the 27 species. The first aspect is related to the species' dispersal capacities and the second is most likely related to the species' postglacial recolonization of the Alps. Furthermore, we found that some summary statistics, most importantly Mantel's r and Jost's D, show different behaviour than expected based on theory. We therefore advise caution in drawing too strong conclusions from these statistics.