The non-palindromic adaptor-PCR method for the identification of the T-cell receptor genes of an interferon-gamma-secreting T-cell hybridomaspecific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen

Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vß genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable ß chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice.

Prevalence of hepatitis C virus (HCV) infection and HCV genotypes of hemodialysis patients in Salvador, Northeastern Brazil

Hepatitis C virus (HCV) infection has been identified as the major cause of chronic liver disease among patients on chronic hemodialysis (HD), despite the important reduction in risks obtained by testing candidate blood donors for anti-HCV antibodies and the use of recombinant erythropoietin to treat anemia. A cross-sectional study was performed to estimate the prevalence of HCV infection and genotypes among HD patients in Salvador, Northeastern Brazil. Anti-HCV seroprevalence was determined by ELISA in 1243 HD patients from all ten different dialysis centers of the city. HCV infection was confirmed by RT-PCR and genotyping was performed by restriction fragment length polymorphism. Anti-HCV seroprevalence among HD patients was 10.5% (95% CI: 8.8-12.3) (Murex anti-HCV, Abbott Murex, Chicago, IL, USA). Blood samples for qualitative HCV detection and genotyping were collected from 125/130 seropositive HD patients (96.2%). HCV-RNA was detected in 92/125 (73.6%) of the anti-HCV-positive patients. HCV genotype 1 (77.9%) was the most prevalent, followed by genotype 3 (10.5%) and genotype 2 (4.6%). Mixed infections of genotypes 1 and 3 were found in 7.0% of the total number of patients. The present results indicate a significant decrease in anti-HCV prevalence from 23.8% detected in a study carried out in 1994 to 10.5% in the present study. The HCV genotype distribution was closely similar to that observed in other hemodialysis populations in Brazil, in local candidate blood donors and in other groups at risk of transfusion-transmitted infection.

Detection of multiple mycoplasma infection in cell cultures by PCR

A total of 301 cell cultures from 15 laboratories were monitored for mycoplasma (Mollicutes) using PCR and culture methodology. The infection was detected in the cell culture collection of 12 laboratories. PCR for Mollicutes detected these bacteria in 93 (30.9%) samples. Although the infection was confirmed by culture for 69 (22.9%) samples, PCR with generic primers did not detect the infection in five (5.4%). Mycoplasma species were identified with specific primers in 91 (30.2%) of the 98 samples (32.6%) considered to be infected. Mycoplasma hyorhinis was detected in 63.3% of the infected samples, M. arginini in 59.2%, Acholeplasma laidlawii in 20.4%, M. fermentans in 14.3%, M. orale in 11.2%, and M. salivarium in 8.2%. Sixty (61.2%) samples were co-infected with more than one mycoplasma species. M. hyorhinis and M. arginini were the microorganisms most frequently found in combination, having been detected in 30 (30.6%) samples and other associations including up to four species were detected in 30 other samples. Failure of the treatments used to eliminate mycoplasmas from cell cultures might be explained by the occurrence of these multiple infections. The present results indicate that the sharing of non-certified cells among laboratories may disseminate mycoplasma in cell cultures.

PCR identification of Mycobacterium tuberculosis complex in a clinical sample from a patient with symptoms of tuberculous spondylodiscitis

A 42-year-old male complaining of thoracic spine pain was admitted to the hospital for evaluation. An X-ray and computer tomography of the thoracic spine showed spondylodiscitis of the L3 lumbar and L2-L3 intervertebral disk. The tuberculin skin test (PPD) was strongly positive. A radioscopy-guided fine needle aspirate of the affected area was cultured but did not reveal the cause of the disease. Two biopsy attempts failed to reveal the cause of the disease by culturing or by acid-fast-resistant staining (Ziehl Neelsen) of the specimens. A third biopsy also failed to detect the infectious agent by using microbiological procedures, but revealed the presence of a 245-bp amplicon characteristic of the Mycobacterium tuberculosis complex after PCR of the sample. The result demonstrates the efficacy of PCR for the identification of M. tuberculosis in situations in which conventional diagnosis by culturing techniques or direct microscopy is unable to detect the microorganism. Following this result the patient was treated with the antituberculous cocktail composed by rifampicin, pirazinamide and isoniazid during a six-month period. At the end of the treatment the dorsalgia symptoms had disappeared.

Epidemiological characterization of resistance and PCR typing of Shigella flexneri and Shigella sonnei strains isolated from bacillary dysentery cases in Southeast Brazil

Shigella spp are Gram-negative, anaerobic facultative, non-motile, and non-sporulated bacilli of the Enterobacteriaceae family responsible for "Shigellosis" or bacillary dysentery, an important cause of worldwide morbidity and mortality. However, despite this, there are very few epidemiological studies about this bacterium in Brazil. We studied the antibiotic resistance profiles and the clonal structure of 60 Shigella strains (30 S. flexneri and 30 S. sonnei) isolated from shigellosis cases in different cities within the metropolitan area of Campinas, State of São Paulo, Brazil. We used the following well-characterized molecular techniques: enterobacterial repetitive intergenic consensus, repetitive extragenic palindromic, and double-repetitive element-polymerase chain reaction to characterize the bacteria. Also, the antibiotic resistance of the strains was determined by the diffusion disk method. Many strains of S. flexneri and S. sonnei were found to be multi-resistant. S. flexneri strains were resistant to ampicillin in 83.3% of cases, chloramphenicol in 70.0%, streptomycin in 86.7%, sulfamethoxazole in 80.0%, and tetracycline in 80.0%, while a smaller number of strains were resistant to cephalothin (3.3%) and sulfazotrim (10.0%). S. sonnei strains were mainly resistant to sulfamethoxazole (100.0%) and tetracycline (96.7%) and, to a lesser extent, to ampicillin (6.7%) and streptomycin (26.7%). Polymerase chain reaction-based typing supported the existence of specific clones responsible for the shigellosis cases in the different cities and there was evidence of transmission between cities. This clonal structure would probably be the result of selection for virulence and resistance phenotypes. These data indicate that the human sanitary conditions of the cities investigated should be improved.

Comparison of the diagnosis of malaria by microscopy, immunochromatography and PCR in endemic areas of Venezuela

Whole blood samples (N = 295) were obtained from different locations in Amazonas and Sucre States, in Venezuela. Malaria was diagnosed by microscopy, OptiMAL™ and polymerase chain reaction (PCR), with Plasmodium vivax, P. falciparum, and P. malariae being detected when possible. We identified 93 infections, 66 of which were caused by P. vivax, 26 by P. falciparum, and 1 was a mixed infection. No infection caused by P. malariae was detected. The sensitivity and specificity of each diagnostic method were high: 95.7 and 97.9% for microscopy, 87.0 and 97.9% for OptiMAL, and 98.0 and 100% for PCR, respectively. Most samples (72.2%) showed more than 5000 parasites/µL blood. The sensitivity of the diagnosis by microscopy and OptiMAL decreased with lower parasitemia. All samples showing disagreement among the methods were reevaluated, but the first result was used for the calculations. Parasites were detected in the 6 false-negative samples by microscopy after the second examination. The mixed infection was only detected by PCR, while the other methods diagnosed it as P. falciparum (microscopy) or P. vivax (OptiMAL) infection. Most of the false results obtained with the OptiMAL strip were related to the P. falciparum-specific band, including 3 species misdiagnoses, which could be related to the test itself or to genetic variation of the Venezuelan strains. The use of the microscopic method for malaria detection is recommended for its low cost but is very difficult to implement in large scale, population-based studies; thus, we report here more efficient methods suitable for this purpose.

Cloning, sequence analysis, and expression of cDNA coding for the major house dust mite allergen, Der f 1, in Escherichia coli

Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1) allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+), expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87% identity in amino acid sequence with Eur m 1 but only 80% with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG), 267-277 (NYHAVNIVGYG) and 284-303 (YWIVRNSWDTTWGDSGYGYF). Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96%), an extended strand (17.13%), a ß turn (5.61%), and a random coil (43.30%). A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.

Simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene using quenched-FRET real-time PCR

Hereditary hemochromatosis (HH) is a common autosomal disorder of iron metabolism mainly affecting Caucasian populations. Three recurrent disease-associated mutations have been detected in the hemochromatosis gene (HFE): C282Y, H63D, and S65C. Although HH phenotype has been associated with all three mutations, C282Y is considered the most relevant mutation responsible for hemochromatosis. Clinical complications of HH include cirrhosis of the liver, congestive cardiac failure and cardiac arrhythmias, endocrine pancreatic disease, which can be prevented by early diagnosis and treatment. Therefore, a reliable genotyping method is required for presymptomatic diagnosis. We describe the simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene by real-time PCR followed by melting curve analysis using fluorescence resonance energy transfer (FRET) probes. The acceptor fluorophore may be replaced by a quencher, increasing multiplex possibilities. Real-time PCR results were compared to the results of sequencing and conventional PCR followed by restriction digestion and detection by agarose gel electrophoresis (PCR-RFLP). Genotypes from 80 individuals obtained both by the conventional PCR-RFLP method and quenched-FRET real-time PCR were in full agreement. Sequencing also confirmed the results obtained by the new method, which proved to be an accurate, rapid and cost-effective diagnostic assay. Our findings demonstrate the usefulness of real-time PCR for the simultaneous detection of mutations in the HFE gene, which allows a reduction of a significant amount of time in sample processing compared to the PCR-RFLP method, eliminates the use of toxic reagents, reduces the risk of contamination in the laboratory, and enables full process automation.

The performance of semi-quantitative differential PCR is similar to that of real-time PCR for the detection of the MYCN gene in neuroblastomas

Amplification of the MYCN gene in neuroblastomas is a potent biological marker of highly aggressive tumors, which are invariably fatal unless sound clinical management is applied. To determine the usefulness of semi-quantitative differential PCR (SQ-PCR) for accurate quantification of MYCN gene copy number, we evaluated the analytical performance of this method by comparing the results obtained with it for 101 tumor samples of neuroblastoma to that obtained by absolute and relative real-time PCR. Similar results were obtained for 100 (99%) samples, no significant difference was detected between the median log10 MYCN copy number (1.53 by SQ-PCR versus 1.55 by absolute real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). In the comparison of SQ-PCR and relative real-time PCR, SQ-PCR versus relative real-time PCR concordant results were found in 100 (99%) samples, no significant difference was found in median log10 MYCN copy number (1.53 by SQ-PCR versus 1.27 by relative real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). These findings indicate that the performance of SQ-PCR was comparable to that of real-time PCR for the amplification and quantification of MYCN copy number. Thus, SQ-PCR can be reliably used as an alternative assay in laboratories without facilities for real-time PCR.

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.

The use of polymerase chain reaction for early diagnosis of tuberculosis in Mycobacterium tuberculosis culture

Early diagnosis plays a vital role in controlling tuberculosis. The conventional methodology is slow, with results taking several weeks, in addition to having low sensitivity, especially in clinical paucibacillary samples. The objective of this study was to evaluate the use of polymerase chain reaction (PCR) on solid medium culture for a rapid diagnosis of tuberculosis, mainly in cases of negative sputum smears. Forty sputum samples were collected from inpatients with tuberculosis treated for less than 2 days. Bacilloscopy, PCR for sputum, culture on Löwestein-Jensen (LJ) solid medium, and daily PCR from culture were performed on each sample. DNA extracted from the BCG vaccine, which contains attenuated bacillus Calmette-Guérin, was used as the positive control. Smear microscopy showed 68.6% sensitivity, 80% specificity, 96% positive predictive value, and 26.7% negative predictive value, with culture on LJ medium as the gold standard. Culture at day 28 showed 74.3% sensitivity and 100% specificity. PCR of DNA extracted from sputum amplified a 1027-bp fragment of the 16s RNA gene, showing 22.9% sensitivity and 60% specificity. PCR performed with DNA extracted from daily culture showed that, from the 17th to the 40th day, the sensitivity (85.7%) and specificity (60%) were constant. We conclude that a 17-day culture is a good choice for rapid diagnosis and to interfere with the transmission chain of tuberculosis.

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.

Human papillomavirus detection and p16 methylation pattern in a case of esophageal papilloma

Esophageal cancer is a prevalent cancer worldwide. Some studies have reported the possible etiology of human papillomavirus (HPV) in benign and malignant papillomas of the esophagus but the conclusions are controversial. In the present study, we investigated an esophageal papilloma from a 30-year-old male patient presenting aphasia. HPV DNA was detected by generic PCR using MY09/11 primers, and restriction fragment length polymorphism revealed the presence of HPV54, usually associated with benign genital lesions. Hypermethylation of the pINK4A gene was also investigated due to its relation to malignant transformation, but no modification was detected in the host gene. Except for an incipient reflux, no risk factors such as cigarette smoking, alcohol abuse or an infected sexual partner were recorded. Since esophageal lesions may have a malignant potential, HPV detection and typing are useful tools for patient follow-up.

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.

Protective effect of the APOE-e3 allele in Alzheimer’s disease

Although several alleles of susceptibility to Alzheimer’s disease (AD) have been studied in the last decades, few polymorphisms have been considered as risk factors for the disease. Among them, the APOE-e4 allele appears to be the major genetic risk factor for the onset of the disease. However, it is important to confirm the potential susceptibility of these genetic variants in different populations in order to establish a genetic profile for the disease in specific communities. This study analyzed the APOE polymorphisms regarding susceptibility to AD in a sample of 264 individuals (primarily Caucasians; 82 cases and 182 controls) in the population from Vitória, ES, Brazil, by PCR restriction fragment length polymorphism (PCR-RFLP) methods. The patients were selected according to clinical criteria for probable AD. Whereas the e4 allele showed statistically significant positive association with susceptibility to AD (OR = 3.01, 95%CI = 1.96-4.61; P < 0.0001), the e2 allele did not. The results of the e4 allele confirm the role of this polymorphism as a risk factor for AD in the sample studied as observed in other populations. Although the e3 allele has been considered neutral in several studies, our results suggest that it acts as a protective factor against AD in the population studied (OR = 0.46, 95%CI = 0.30-0.67; P < 0.0001). This study may provide a new insight into the role of the APOE-e3 allele in the etiology of AD and might help to estabilish a profile of risk for AD in the population from Vitória, ES.