PCR-RFLP of 16S ribosomal DNA to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples

INTRODUCTION: This study aimed to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples by PCR-RFLP. METHODS: Fifty-two strains identified by conventional biochemical exams were submitted to PCR amplification and digested with HinfI. Only 20 (38.5%) of the 52 strains showed a DNA pattern expected for E. gallinarum and E. casseliflavus. RESULTS: Analysis of the results of this study showed that E. gallinarum and E. casseliflavus are occasionally erroneously identified and confirmed the potential application of 16S rDNA analysis for accurate identification of these species. CONCLUSIONS: A correct identification is important to distinguish between intrinsic and acquired vancomycin resistance.

Linfócitos CD4, CD8 e células NK no estroma da cérvice uterina de mulheres infectadas pelo papilomavírus humano

INTRODUÇÃO: A resposta imune pode ser um elemento chave para a progressão ou remissão da infecção pelo papilomavírus humano (HPV) no estroma da cérvice uterina. Este estudo objetivou quantificar no estroma cervical a presença de linfócitos T CD4, CD8 e células NK, por imunohistoquímica, em lesões de alto e baixo grau em pacientes infectadas por HPV MÉTODOS: Utilizou-se 56 amostras de biópsia da estroma cervical, sendo 43 amostras positivas para DNA de HPV de alto risco oncogênico e com diagnóstico histopatológico de neoplasia intraepitelial cervical (NIC) de alto e baixo grau, ou negativa para lesão intraepitelial e malignidade (NILM), e 13 amostras de pacientes negativas para DNA de HPV com diagnóstico histopatológico NILM RESULTADOS: Maior quantidade de linfócitos T CD4 foi observada em amostras NIC II/III, carcinoma e NILM (p=0,04) e naquelas cuja carga viral esteve entre 10 e 1,000 RLU/PCB. O predomínio de linfócitos T CD8 ocorreu em maior proporção nas amostras NIC II/III (p=0,02) e em amostras com carga viral entre 100 e 1.000 RLU/PCB. As células NK prevaleceram nas amostras com lesões de baixo grau e com baixa carga viral CONCLUSÕES: Este estudo comprovou que nas fases iniciais da infecção, onde não há ainda alterações celulares de alto grau, não temos a presença de células que possam desencadear a fase efetora da resposta imune.

PCR detection of multiple human herpesvirus DNA in saliva from HIV-infected individuals in Teresina, State of Piauí, Brazil

INTRODUCTION: Human herpesviruses are frequently associated with orofacial diseases in humans (HSV-1, EBV, CMV and HHV-8), some can also cause systemic disease (CMV and HHV-8). The transmission of these viruses occurs by contact with infected secretions, especially saliva. Human immunodeficiency virus infection is associated with an increased risk of HHVs and related diseases. METHODS: This work aimed to detect HSV-1, EBV, CMV and HHV-8 DNA in saliva of HIV-infected patients from Teresina, northeast Brazil, by PCR and compare these findings with age and sex matched HIV-seronegative individuals. RESULTS: No difference in prevalence was verified between HHV detection in the saliva of HIV-seropositive individuals and controls. The individual frequencies of these viruses in these two populations were different. HIV seropositivity correlated positively with the presence of CMV (OR: 18.2, p= 0.00032) and EBV (OR: 3.44, p= 0.0081). No association between CD4 counts and the prevalence of HHVs in the saliva was observed; however, a strong association was determined between seropositivity and the presence of multiple HHV DNAs in saliva (OR: 4.83, p = 0.0028). CONCLUSIONS: These findings suggest the asymptomatic salivary shedding of HHVs is a common event between HIV-seropositive and seronegative individuals from Teresina, Piauí, Brazil, and, especially for HIV-seropositive patients, saliva is a risk factor for the acquisition/transmission of multiple HHVs.

Comparison among three polymerase chain reaction assays on detection of DNA from Leishmania in biological samples from patients with american cutaneous leishmaniasis

INTRODUCTION: The study analyzed positivity of polymerase chain reaction (PCR) on detection of DNA from Leishmania in patients' samples. METHODS: Extracted DNA was submitted to L150/L152, 13Y/13Z, and seminested PCR (snPCR). RESULTS: Results were evidenced by bands of approximately 120, 720, and 670 bp for L150/L152, 13Y/13Z, and snPCR, respectively. L150/L152, 13Y/13Z, and snPCR positivity indexes were 76.9, 56.4, and 9.2 (p>0.05), respectively, for suspected and 93.7, 68.7, and 84.4 (p<0.05), respectively, for confirmed. CONCLUSIONS: Preliminary results showed that these assays, mainly L150/L152 and snPCR, can detect Leishmania DNA and carry potential on laboratory diagnosis of leishmaniasis.

Evidence of a higher prevalence of HPV infection in HTLV-1-infected women: a cross-sectional study

INTRODUCTION:HTLV-1 infection increases susceptibility to other infections. Few studies have addressed the co-infection between HPV and HTLV-1 and the immune response involved in this interaction. The aim of this study was to determine the prevalence of cervical HPV infection in HTLV-1-infected women and to establish the risk factors involved in this co-infection. METHODS: A cross-sectional study was carried out in Salvador, Brazil, between September 2005 and December 2008, involving 50 HTLV-1-infected women from the HTLV Reference Center and 40 uninfected patients from gynecological clinic, both at the Bahiana School of Medicine. HPV infection was assessed using hybrid capture. HTLV-1 proviral load was quantified using real-time polymerase chain reaction (PCR). RESULTS: The mean age of HTLV-1-infected women (38 ± 10 years) was similar to that of the control group (36 ± 13 years). The prevalence of HPV infection was 44% in the HTLV-1-infected group and 22.5% in uninfected women (p = 0.03). HTLV-1-infected women had lower mean age at onset of sexual life (17 ± 3 years versus 19 ± 3 years; p = 0.03) and greater number of lifetime partners compared with the control group (4 ± 3 versus 2 ± 1; p < 0.01). In the group of HTLV-1-infected patients, there was neither difference in HTLV-1 proviral load between HPV-infected women and the uninfected. CONCLUSIONS: The prevalence of HPV infection was higher in HTLV-1-infected women. Further studies should be performed to evaluate the progression of this co-infection.

Glucocorticoid-induced tumor necrosis factor receptor expression in patients with cervical human papillomavirus infection

Introduction The progression of human papillomavirus (HPV) infection in the anogenital tract has been associated with the involvement of cells with regulatory properties. Evidence has shown that glucocorticoid-induced tumor necrosis factor receptor (GITR) is an important surface molecule for the characterization of these cells and proposes that GITR ligand may constitute a rational treatment for many cancer types. We aimed to detect the presence of GITR and CD25 in cervical stroma cells with and without pathological changes or HPV infection to better understand the immune response in the infected tissue microenvironment. Methods We subjected 49 paraffin-embedded cervical tissue samples to HPV DNA detection and histopathological analysis, and subsequently immunohistochemistry to detect GITR and CD25 in lymphocytes. Results We observed that 76.9% of all samples with high GITR expression were HPV-positive regardless of histopathological findings. High GITR expression (77.8%) was predominant in samples with ≥1,000 RLU/PCB. Of the HPV-positive samples negative for intraepithelial lesion and malignancy, 62.5% had high GITR expression. High GITR expression was observed in both carcinoma and high-grade squamous intraepithelial lesion (HSIL) samples (p = 0.16). CD25 was present in great quantities in all samples. Conclusions The predominance of high GITR expression in samples with high viral load that were classified as HSIL and carcinoma suggests that GITR+ cells can exhibit regulatory properties and may contribute to the progression of HPV-induced cervical neoplasia, emphasizing the importance of GITR as a potential target for immune therapy of cervical cancer and as a disease evolution biomarker.

A comparison of four DNA extraction protocols for the analysis of urine from patients with visceral leishmaniasis

Introduction Polymerase chain reaction (PCR) may offer an alternative diagnostic option when clinical signs and symptoms suggest visceral leishmaniasis (VL) but microscopic scanning and serological tests provide negative results. PCR using urine is sensitive enough to diagnose human visceral leishmaniasis (VL). However, DNA quality is a crucial factor for successful amplification. Methods A comparative performance evaluation of DNA extraction methods from the urine of patients with VL using two commercially available extraction kits and two phenol-chloroform protocols was conducted to determine which method produces the highest quality DNA suitable for PCR amplification, as well as the most sensitive, fast and inexpensive method. All commercially available kits were able to shorten the duration of DNA extraction. Results With regard to detection limits, both phenol: chloroform extraction and the QIAamp DNA Mini Kit provided good results (0.1 pg of DNA) for the extraction of DNA from a parasite smaller than Leishmania (Leishmania) infantum (< 100fg of DNA). However, among 11 urine samples from subjects with VL, better performance was achieved with the phenol:chloroform method (8/11) relative to the QIAamp DNA Mini Kit (4/11), with a greater number of positive samples detected at a lower cost using PCR. Conclusion Our results demonstrate that phenol:chloroform with an ethanol precipitation prior to extraction is the most efficient method in terms of yield and cost, using urine as a non-invasive source of DNA and providing an alternative diagnostic method at a low cost.

Detection of Leishmania (Viannia) DNA in leucocytes from the blood of patients with cutaneous leishmaniasis

ABSTRACTINTRODUCTION:Cutaneous leishmaniasis (CL) is a serious and global public health issue, with the potential of developing a mucosal form, occurring as subclinical cases, and showing recurrence despite previous treatment.METHODS:Polymorphonuclear and mononuclear DNA obtained from 49 patients was subjected to polymerase chain reaction for detection of Leishmania (Viannia).RESULTS:DNA was detected in mononuclear cells from two patients with active primary lesions positive for CL, with infection periods of 3 and 6 months, respectively.CONCLUSIONS:The DNA of Leishmania (Viannia) indicates probable parasite dissemination possibly explaining subclinical case emergence, lesion recurrence, and mucosal lesion appearance.

Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Abstract: INTRODUCTION : Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

Extraction of Trypanosoma cruzi DNA from food: a contribution to the elucidation of acute Chagas disease outbreaks

Abstract: INTRODUCTION: Before 2004, the occurrence of acute Chagas disease (ACD) by oral transmission associated with food was scarcely known or investigated. Originally sporadic and circumstantial, ACD occurrences have now become frequent in the Amazon region, with recently related outbreaks spreading to several Brazilian states. These cases are associated with the consumption of açai juice by waste reservoir animals or insect vectors infected with Trypanosoma cruzi in endemic areas. Although guidelines for processing the fruit to minimize contamination through microorganisms and parasites exist, açai-based products must be assessed for quality, for which the demand for appropriate methodologies must be met. METHODS: Dilutions ranging from 5 to 1,000 T. cruzi CL Brener cells were mixed with 2mL of acai juice. Four Extraction of T. cruzi DNA methods were used on the fruit, and the cetyltrimethyl ammonium bromide (CTAB) method was selected according to JRC, 2005. RESULTS: DNA extraction by the CTAB method yielded satisfactory results with regard to purity and concentration for use in PCR. Overall, the methods employed proved that not only extraction efficiency but also high sensitivity in amplification was important. CONCLUSIONS: The method for T. cruzi detection in food is a powerful tool in the epidemiological investigation of outbreaks as it turns epidemiological evidence into supporting data that serve to confirm T. cruzi infection in the foods. It also facilitates food quality control and assessment of good manufacturing practices involving acai-based products.

Prevalence and genotype distribution of human papillomavirus: implications for cancer screening and vaccination in Henan province, China

Abstract: INTRODUCTION: To provide information for cervical cancer screening and vaccination in Henan province, China, the distribution of human papillomavirus (HPV) was analyzed. METHODS: The HPV genotypes were detected using gene array and flow-through hybridization. RESULTS: Overall, 38.1% (1,536/4,033) of the women were human papillomavirus deoxyribonucleic acid (HPV DNA) positive. The prevalence of high-risk HPV types was 32.4%. HPV 16 was the most prevalent genotype (8.9%), followed by HPV 52 (5.8%) and HPV 58 (4.4%). CONCLUSIONS: The data support close surveillance of women for cervical cancer screening, and HPV prophylactic vaccines including HPV16, HPV 52, and HPV 58 might offer greater protection in this area.

Human identification and analysis of DNA in bones

The introduction of molecular biology techniques, especially of DNA analysis, for human identification is a recent advance in legal medicine. Substantial effort has continuously been made in an attempt to identify cadavers and human remains after wars, socio-political problems and mass disasters. In addition, because of the social dynamics of large cities, there are always cases of missing people, as well as unidentified cadavers and human remains that are found. In the last few years, there has also been an increase in requests for exhumation of human remains in order to determine genetic relationships in civil suits and court action. The authors provide an extensive review of the literature regarding the use of this new methodology for human identification of ancient or recent bones.

A descoberta da Dupla Hélice do DNA: contributo para possíveis narrativas

Até ao final dos anos 40, o DNA não era reconhecido como portador da informação genética. Era uma molécula demasiado simples, difícil de isolar e incompatível com os métodos de análise da química orgânica e da biologia. Quando alguns cientistas começam a acreditar na importância do DNA, percebem que são incapazes, tecnicamente, de determinar a sua estrutura. É nesse espírito que James Watson vai para a Europa e, na primavera de 1951, ao assistir à conferência de Maurice Wilkins, da King’s College, onde vê uma fotografia do padrão de difração de raios X, percebe que será esta a técnica chave para a determinação da estrutura do DNA e, subsequentemente, dos segredos da vida. É este o início de uma das possíveis narrativas sobre uma das principais descobertas científicas do séc. XX que muitas vezes se reduz a: “A dupla hélice do DNA foi descoberta em 1953 por Watson e Crick”. Esta dissertação propõe-se a demonstrar que, apesar de em termos estritos, se tratar de uma afirmação verdadeira, não é suficiente para garantir uma experiência pedagógica significativa, nem fazer jus ao que é o funcionamento da ciência, com todas as implicações humanas, contextuais, éticas, consequências e impacto.

Integrating data from heterogeneous DNA microarray platforms

DNA microarrays are one of the most used technologies for gene expression measurement. However, there are several distinct microarray platforms, from different manufacturers, each with its own measurement protocol, resulting in data that can hardly be compared or directly integrated. Data integration from multiple sources aims to improve the assertiveness of statistical tests, reducing the data dimensionality problem. The integration of heterogeneous DNA microarray platforms comprehends a set of tasks that range from the re-annotation of the features used on gene expression, to data normalization and batch effect elimination. In this work, a complete methodology for gene expression data integration and application is proposed, which comprehends a transcript-based re-annotation process and several methods for batch effect attenuation. The integrated data will be used to select the best feature set and learning algorithm for a brain tumor classification case study. The integration will consider data from heterogeneous Agilent and Affymetrix platforms, collected from public gene expression databases, such as The Cancer Genome Atlas and Gene Expression Omnibus.

Microscopy- or DNA-based analyses: Which methodology gives a truer picture of stream-dwelling decomposer fungal diversity?

We assessed aquatic hyphomycete diversity in autumn and spring on oak leaves decomposing in five streams along a gradient of eutrophication in the Northwest of Portugal. Diversity was assessed through microscopy-based (identification by spore morphology) and DNA-based techniques (Denaturing Gradient Gel Electrophoresis and 454 pyrosequencing). Pyrosequencing revealed five times greater diversity than DGGE. About 21% of all aquatic hyphomycete species were exclusively detected by pyrosequencing and 26% exclusively by spore identification. In some streams, more than half of the recorded species would have remained undetected if we had relied only on spore identification. Nevertheless, in spring aquatic hyphomycete diversity was higher based on spore identification, probably because many species occurring in this season are not yet connected to ITS barcodes in genetic databases. Pyrosequencing was a powerful tool for revealing aquatic hyphomycete diversity on decomposing plant litter in streams and we strongly encourage researchers to continue the effort in barcoding fungal species.