An MLSA-based online scheme for the rapid identification of Stenotrophomonas isolates

An online scheme to assign Stenotrophomonas isolates to genomic groups was developed using the multilocus sequence analysis (MLSA), which is based on the DNA sequencing of selected fragments of the housekeeping genes ATP synthase alpha subunit (atpA), the recombination repair protein (recA), the RNA polymerase alpha subunit (rpoA) and the excision repair beta subunit (uvrB). This MLSA-based scheme was validated using eight of the 10 Stenotrophomonas species that have been previously described. The environmental and nosocomial Stenotrophomonas strains were characterised using MLSA, 16S rRNA sequencing and DNA-DNA hybridisation (DDH) analyses. Strains of the same species were found to have greater than 95% concatenated sequence similarity and specific strains formed cohesive readily recognisable phylogenetic groups. Therefore, MLSA appeared to be an effective alternative methodology to amplified fragment length polymorphism fingerprint and DDH techniques. Strains of Stenotrophomonas can be readily assigned through the open database resource that was developed in the current study (www.steno.lncc.br/).

Surfactant protein B gene polymorphism in preterm babies with respiratory distress syndrome

The etiology of respiratory distress syndrome (RDS) is multifactorial and multigenic. Studies have suggested that polymorphisms and mutations in the surfactant protein B (SP-B) gene are associated with the pathogenesis of RDS. The objectives of this study were to determine and compare the frequencies of SP-B gene polymorphisms in preterm babies with and without RDS. We studied 151 neonates: 79 preterm babies without RDS and 72 preterm newborns with RDS. The following four SP-B gene polymorphisms were analyzed: A/C at -18, C/T at 1580, A/G at 9306, and G/C at nucleotide 8714. The polymorphisms were detected by PCR amplification of genomic DNA and genotyping. The genotypes were determined using PCR-based converted restriction fragment length polymorphisms. The control group consisted of 42 (53%) girls and 37 (47%) boys. Weight ranged from 1170 to 3260 g and mean gestational age (GA) was 33.9 weeks (range: 29 to 35 weeks and 6 days). The RDS group consisted of 31 (43%) girls and 41 (57%) boys. Weight ranged from 614 to 2410 g and mean GA was 32 weeks (range: 26 to 35 weeks). The logistic regression model showed that GA was the variable that most contributed to the occurrence of RDS. The AG genotype of the A/G polymorphism at position 9306 of the SP-B gene was a protective factor in this population (OR = 0.1681; 95%CI = 0.0426-0.6629). We did not detect differences in the frequencies of the other polymorphisms between the two groups of newborns.

Polymorphisms of toll-like receptor 2 and 4 genes in Chagas disease

The aim of this study was to test the possible implication of toll-like receptor 2 (TLR2) and TLR4 gene polymorphisms in determining the susceptibility to Chagas' disease. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism in 475 individuals from Colombia, 143 seropositive with chagasic cardiomyopathy, 132 seropositive asymptomatic and 200 seronegative. The TLR2 arginine to glutamine substitution at residue 753(Arg753Gln) polymorphism was absent in the groups analyzed. The TLR4 Asp299Gly and Thr399Ile polymorphisms are in linkage disequilibrium and we observed a very low frequency of these polymorphisms in our study population (2.6% and 1.8% respectively). The overall TLR2 and TLR4 alleles and genotype distribution in seronegative and seropositive were not significantly different. We compared the frequencies between asymptomatic patients and those with chagasic cardiomyopathy and we did not observe any significant differences in the distribution of alleles or genotypes. In summary, this study corroborates the low frequency of TLR2 and TLR4 polymorphisms observed in other populations and suggest that these do not play an important role in Chagas' disease. The validation of these findings in independent cohorts is needed to firmly establish a role for TLR2 and TLR4 variants in Chagas' disease.

The effect of combined polymorphisms in chemokines and chemokine receptors on the clinical course of HIV-1 infection in a Brazilian population

Polymorphisms in genes that encode chemokines or their receptors can modulate susceptibility to human immunodeficiency virus (HIV) infection and disease progression. The objective of this study was to assess the frequency of polymorphisms CCR5-Δ32, CCR2-64I, CCR5-59029A and SDF1-3'A and their role in the course of HIV infection in a Southern Brazilian population. Clinical data were obtained from 249 patients for an average period of 6.4 years and genotypes were determined by standard polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism. Survival analyses were conducted for three outcomes: CD4+ T-cell counts below 200 cells/µL, acquired immune deficiency syndrome (AIDS) or death. The frequency of the polymorphisms CCR5-Δ32, CCR2-64I, CCR5-59029A and SDF1-3'A were 0.024, 0.113, 0.487 and 0.207, respectively. CCR5-Δ32 was associated with a reduction in the risk for CD4+ T-cell depletion and with an increased risk for death after AIDS diagnosis. CCR2-64I was associated with a reduction in the risk for developing AIDS. SDF1-3'A was also associated with decreased risk for AIDS, but its effect was only evident when CCR2-64I was present as well. These results highlight the possibility of using these markers as indicators for the prognosis of disease progression and provide evidence for the importance of analysing the effects of gene polymorphisms in a combined fashion.

CCR2 and CCR5 genes polymorphisms in women with cervical lesions from Pernambuco, Northeast Region of Brazil: a case-control study

Polymorphisms in chemokine receptors play an important role in the progression of cervical intraepithelial neoplasia (CIN) to cervical cancer (CC). Our study examined the association of CCR2-64I (rs1799864) andCCR5-Δ32 (rs333) polymorphisms with susceptibility to develop cervical lesion (CIN and CC) in a Brazilian population. The genotyping of 139 women with cervical lesions and 151 women without cervical lesions for the CCR2-64I and CCR5-Δ32 polymorphisms were performed using polymerase chain reaction-restriction fragment length polymorphism. The individuals carrying heterozygous or homozygous genotypes (GA+AA) for CCR2-64I polymorphisms seem to be at lower risk for cervical lesion [odds ratio (OR) = 0.37, p = 0.0008)]. The same was observed for the A allele (OR = 0.39, p = 0.0002), while no association was detected (p > 0.05) with CCR5-Δ32 polymorphism. Regarding the human papillomavirus (HPV) type, patients carrying the CCR2-64Ipolymorphism were protected against infection by HPV type 16 (OR = 0.35, p = 0.0184). In summary, our study showed a protective effect ofCCR2-64I rs1799864 polymorphism against the development of cervical lesions (CIN and CC) and in the susceptibility of HPV 16 infection.

ApoE polymorphisms and diarrheal outcomes in Brazilian shanty town children

A series of studies have shown that the heavy burdens of diarrheal diseases in the first 2 formative years of life in children living in urban shanty towns have negative effects on physical and cognitive development lasting into later childhood. We have shown that APOE4 is relatively common in shanty town children living in Brazil (13.4%) and suggest that APOE4 has a protective role in cognitive development as well as weight-for-height in children with heavy burdens of diarrhea in early childhood (64/123; 52%), despite being a marker for cognitive decline with Alzheimer’s and cardiovascular diseases later in life. APOE2 frequency was higher among children with heaviest diarrhea burdens during the first 2 years of life, as detected by PCR using the restriction fragment length polymorphism method, raising the possibility that ApoE-cholesterol balance might be critical for growth and cognitive development under the stress of heavy diarrhea burdens and when an enriched fat diet is insufficient. These findings provide a potential explanation for the survival advantage in evolution of genes, which might raise cholesterol levels during heavy stress of diarrhea burdens and malnutrition early in life.

Impact of β-2 Thr164Ile and combined β-adrenergic receptor polymorphisms on prognosis in a cohort of heart failure outpatients

Genetic polymorphisms of adrenergic receptors (ARs) have been associated with the development, progression, and prognosis of patients with heart failure (HF), with few data for the Brazilian population. We evaluated the role of the β2-AR Thr164Ile polymorphism at codon 164 on prognosis in a prospective study on 315 adult Brazilian HF patients, predominantly middle-aged Caucasian men in functional class I-II, with severe left ventricular systolic dysfunction. Genomic DNA was extracted from peripheral blood and β2-AR164 genotypes were detected by PCR followed by restriction fragment length analysis. During a median follow-up of 3 years, 95 deaths occurred and 57 (60%) were HF-related. Unexpectedly, Ile164 carriers (N = 12) had no HF-related events (log-rank P value = 0.13). Analysis using genotype combination with β1-AR polymorphisms at codons 49 and 389 identified patients with favorable genotypes (Thr164Ile of β2-AR, Gly49Gly of β1-AR and/or Gly389Gly of β1-AR), who had lower HF-related mortality (P = 0.01). In a Cox proportional hazard model adjusted for other clinical characteristics, having any of the favorable genotypes remained as independent predictor of all-cause (hazard ratio (HR): 0.41, 95%CI: 0.17-0.95) and HF-related mortality (HR: 0.12, 95%CI: 0.02-0.90). These data show that the β2-AR Thr164Ile polymorphism had an impact on prognosis in a Brazilian cohort of HF patients. When combined with common β1-AR polymorphisms, a group of patients with a combination of favorable genotypes could be identified.

Relationship of IL-1 and TNF-α polymorphisms with Helicobacter pylori in gastric diseases in a Brazilian population

It is well known that the risk of development of gastric cancer (GC) in Helicobacter pylori-infected patients depends on several factors. Thus, the aim of this study was to investigate the effect of proinflammatory cytokine gene polymorphisms for IL-1β, IL-1RN and TNF-α on the development of GC in a Brazilian population. A total of 202 biopsies obtained from Brazilian patients with chronic gastritis and GC were included in the study. Infection with H. pylori cagA+ was determined by the polymerase chain reaction (PCR) as previously described. IL-1β, IL-1RN and TNF-α polymorphism genotyping was performed by restriction fragment length polymorphism PCR. Associations between gene polymorphisms, clinical diseases and virulence markers were evaluated using either the χ² test or the Fisher exact test. Our results demonstrated that the IL-1β -511 C/C and IL-1β -511 C/T alleles were associated with chronic gastritis in H. pylori-positive patients (P = 0.04 and P = 0.05, respectively) and the IL-1β -511 C/C genotype was associated with GC (P = 0.03). The frequency of IL-1RN alleles from patients with chronic gastritis and GC indicated that there was no difference between the genotypes of the groups studied. Similar results were found for TNF-α -308 gene polymorphisms. Our results indicate that the IL-1β -511 C/C and C/T gene polymorphisms are associated with chronic gastritis and GC development in H. pylori-infected individuals.

Association of MDR1 gene polymorphisms with the risk of hepatocellular carcinoma in the Chinese Han population

The multidrug resistance 1 gene (MDR1) is an important candidate gene for influencing susceptibility to hepatocellular carcinoma (HCC). The objective of the present study was to evaluate the association ofMDR1 polymorphisms with the risk of HCC in the Chinese Han population. A total of 353 HCC patients and 335 healthy subjects were enrolled in the study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), created restriction site-PCR (CRS-PCR) and DNA sequencing methods were used to identify MDR1 gene polymorphisms. Two allelic variants (c.335T>C and c.3073A>C) were detected. The CC genotype of the c.335T>C polymorphism was associated with an increased risk of developing HCC compared to the TT genotype (OR = 2.161, 95%CI = 1.350-3.459, χ2 = 10.55, P = 0.0011). The risk of HCC was significantly higher for the CC genotype in the c.3073A>C polymorphism compared to the AA genotype in the studied populations (CCvs AA: OR = 2.575, 95%CI = 1.646-4.028, χ2 = 17.64, P < 0.0001). The C allele of the c.335T>C and c.3073A>C variants may contribute to the risk of HCC (Cvs T of c.335T>C: OR = 1.512, 95%CI = 1.208-1.893, χ2 = 13.07, P = 0.0003, and Cvs A of c.3073A>C: OR = 1.646, 95%CI = 1.322-2.049, χ2 = 20.03, P < 0.0001). The c.335T>C and c.3073A>C polymorphisms of the MDR1 gene were associated with the risk of occurrence of HCC in the Chinese Han population. Further investigations are needed to confirm these results in larger different populations.

Single nucleotide polymorphisms predisposing to asthma in children of Mauritian Indian and Chinese Han ethnicity

Our objective was to investigate the distributions of six single nucleotide polymorphisms (SNPs) MS4A2 E237G, MS4A2 C-109T, ADRB2 R16G, IL4RA I75V,IL4 C-590T, and IL13 C1923T in Mauritian Indian and Chinese Han children with asthma. This case-control association study enrolled 382 unrelated Mauritian Indian children, 193 with asthma and 189 healthy controls, and 384 unrelated Chinese Han children, 192 with asthma and 192 healthy controls. The SNP loci were genotyped using polymerase chain reaction (PCR)-restriction fragment length polymorphism for the Chinese Han samples and TaqMan real-time quantitative PCR for the Mauritian Indian samples. In the Mauritian Indian children, there was a significant difference in the distribution of IL13 C1923T between the asthma and control groups (P=0.033). The frequency of IL13 C1923T T/T in the Mauritian Indian asthma group was significantly higher than in the control group [odds ratio (OR)=2.119, 95% confidence interval=1.048-4.285]. The Chinese Han children with asthma had significantly higher frequencies ofMS4A2 C-109T T/T (OR=1.961, P=0.001) and ADRB2 R16G A/A (OR=2.575, P=0.000) than the control group. The IL13 C1923T locus predisposed to asthma in Mauritian Indian children, which represents an ethnic difference from the Chinese Han population. TheMS4A2 C-109T T/T and ADRB2 R16G A/Agenotypes were associated with asthma in the Chinese Han children.

Associations between CD36 gene polymorphisms and susceptibility to coronary artery heart disease

Associations between polymorphisms of the CD36 gene and susceptibility to coronary artery heart disease (CHD) are not clear. We assessed allele frequencies and genotype distributions of CD36 gene polymorphisms in 112 CHD patients and 129 control patients using semi-quantitative polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Additionally, we detected CD36 mRNA expression by real-time quantitative PCR, and we quantified plasma levels of oxidized low-density lipoprotein (ox-LDL) using an enzyme-linked immunosorbent assay (ELISA). There were no significant differences between the two groups (P>0.05) in allele frequencies of rs1761667 or in genotype distribution and allele frequencies of rs3173798. The genotype distribution of rs1761667 significantly differed between CHD patients and controls (P=0.034), with a significantly higher frequency of the AG genotype in the CHD group compared to the control group (P=0.011). The plasma levels of ox-LDL in patients with the AG genotype were remarkably higher than those with the GG and AA genotypes (P=0.010). In a randomized sample taken from patients in the two groups, the CD36 mRNA expression of the CHD patients was higher than that of the controls. In CHD patients, the CD36 mRNA expression in AG genotype patients was remarkably higher than in those with an AA genotype (P=0.005). After adjusted logistic regression analysis, the AG genotype of rs1761667 was associated with an increased risk of CHD (OR=2.337, 95% CI=1.336-4.087, P=0.003). In conclusion, the rs1761667 polymorphism may be closely associated with developing CHD in the Chongqing Han population of China, and an AG genotype may be a genetic susceptibility factor for CHD.

Genotypic identification of Cryptosporidium spp. isolated from hiv-infected patients and immunocompetent children of São Paulo, Brazil

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

The existence of only one haplotype of Leishmania major in the main and potential reservoir hosts of zoonotic cutaneous leishmaniasis using different molecular markers in a focal area in Iran

IntroductionLeishmania major is the causative agent of zoonotic cutaneous leishmaniasis (ZCL), and great gerbils are the main reservoir hosts in Iran. Abarkouh in central Iran is an emerging focal point for which the reservoir hosts of ZCL are unclear. This research project was designed to detect any Leishmania parasites in different wild rodent species.MethodsAll rodents captured in 2011 and 2012 from Abarkouh district were identified based on morphological characteristics and by amplification of the rodent cytochrome b (Cyt b) gene. To detect Leishmania infection in rodents, deoxyribonucleic acid (DNA) of each ear was extracted. Internal transcribed spacer-ribosomal deoxyribonucleic acid (ITS-rDNA), microsatellites, kinetoplast deoxyribonucleic acid (kDNA) and cytochrome b genes of Leishmania parasites were amplified by polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) and sequencing were employed to confirm the Leishmania identification.ResultsOf 68 captured rodents in the region, 55 Rhombomys opimus were identified and nine Leishmaniainfections (9/55) were found. In addition, eight Meriones libycus and two Tatera indicawere sampled, and one of each was confirmed to be infected. Two Meriones persicus and one Mus musculuswere sampled with no infection.ConclusionsThe results showed that all 11 unambiguously positive Leishmania infections were Leishmania major. Only one haplotype of L. major(GenBank access No. EF413075) was found and at least three rodents R. opimus, M. libycus and T. indica—appear to be the main and potential reservoir hosts in this ZCL focus. The reservoir hosts are variable and versatile in small ZCL focal locations.

A Laboratory-based Approach to Biological Control of Snails

Development of Schistosoma mansoni in the intermediate host Biomphalaria glabrata is influenced by a number of parasite and snail genes. Understanding the genetics involved in this complex host/parasite relationship may lead to an often discussed approach of introducing resistant B. glabrata into the field as a means of biological control for the parasite. For the snail, juvenile susceptibility to the parasite is controlled by at least four genes, whereas one gene seems to be responsible for adult nonsusceptibility. Obtaining DNA from F2 progeny snails from crosses between parasite-resistant and-susceptible snails, we have searched for molecular markers that show linkage to either the resistant or susceptible phenotype. Both restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) approaches have been used. To date, using a variety of snail and heterologous species probes, no RFLP marker has been found that segregates with either the resistant or susceptible phenotype in F2 progeny snails. More promising results however have been found with the RAPD approach, where a 1.3 kb marker appears in nearly all resistant progeny, and a 1.1 kb marker appears in all susceptible progeny

Analysis of genetic diversity of Trypanosoma cruzi: an application of riboprinting and gradient gel electrophoresis methods

Analysis of restriction fragment length polymorphism (RFLP) profiles derived from digestion of polymerase chain reaction (PCR) products of the ribosomal 18S from Trypanosoma cruzi yields a typical `riboprint' profile that can vary intraspecifically. A selection of 21 stocks of T. cruzi and three outgroup taxa: T. rangeli, T. conorhini and Leishmania braziliensis were analysed by riboprinting to assess divergence within and between taxa. T. rangeli, T. conorhini and L. braziliensis could be easily differentiated from each other and from T. cruzi. Phenetic analysis of PCR-RFLP profiles indicated that, with one or two exceptions, stocks of T. cruzi could be broadly partitioned into two groups that formally corresponded to T. cruzi I and T. cruzi II respectively. To test if ribosomal 18S sequences were homogeneous within each taxon, gradient gel electrophoresis methods were employed utilising either chemical or temperature gradients. Upon interpretation of the melting profiles of riboprints and a section of the 18S independently amplified by PCR, there would appear to be at least two divergent 18S types present within T. cruzi. Heterogeneity within copies of the ribosomal 18S within a single genome has therefore been demonstrated and interestingly, this dimorphic arrangement was also present in the outgroup taxa. Presumably the ancestral duplicative event that led to the divergent 18S types preceded that of speciation within this group. These divergent 18S paralogues may have, or had, different functional pressures or rates of molecular evolution. Whether or not these divergent types are equally transcriptionally active throughout the life cycle, remain to be assessed.