Fábio Lopes de Melo

Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA

This report describes the development of a SYBR Green I based real time polymerase chain reaction (PCR) protocol for detection on the ABI Prism 7000 instrument. Primers targeting the gene encoding the SSU rRNA were designed to amplify with high specificity DNA from Schistosoma mansoni, in a real time quantitative PCR system. The limit of detection of parasite DNA for the system was 10 fg of purified genomic DNA, that means less than the equivalent to one parasite cell (genome ~580 fg DNA). The efficiency was 0.99 and the correlation coefficient (R(2)) was 0.97. When different copy numbers of the target amplicon were used as standards, the assay could detect at least 10 copies of the specific target. The primers used were designed to amplify a 106 bp DNA fragment (Tm 83 degrees C). The assay was highly specific for S. mansoni, and did not recognize DNA from closely related non-schistosome trematodes. The real time PCR allowed for accurate quantification of S. mansoni DNA and no time-consuming post-PCR detection of amplification products by gel electrophoresis was required. The assay is potentially able to quantify S. mansoni DNA (and indirectly parasite burden) in a number of samples, such as snail tissue, serum and feces from patients, and cercaria infested water. Thus, these PCR protocols have potential to be used as tools for monitoring of schistosome transmission and quantitative diagnosis of human infection.

Current epidemiological status of schistosomiasis in the state of Pernambuco, Brazil

Uncontrolled peripheral urbanisation coupled with environmental degradation has affected the status of schistosomiasis in Pernambuco (PE), Brazil. This endemic disease continues to perpetuate its transmission in rural areas and has also become a cause for concern in coastal towns of the state. The lack of basic infrastructure (sanitation and health programmes) to support the new urban areas leads to faecal contamination of natural aquatic environments, resulting in consequent infection of vector snails and the emergence of new sources of schistosomiasis transmission. In the present paper, we discuss the current epidemiological status of schistosomiasis in PE. We have consolidated and analysed information from parasitological, malacological and morbidity surveys undertaken by the group of researchers at the Laboratory of Schistosomiasis, Centro de Pesquisas Aggeu Magalhães-Fiocruz. The results of our analysis show: (i) the maintenance of the levels of schistosomiasis in the rural Zona da Mata, PE, (ii) the record of the human cases of schistosomiasis and the foci of infected snails detected along the coast of PE through 2007, (iii) the high record of the severe clinical form of schistosomiasis in the metropolitan region of Recife (RMR) and (iv) new breeding sites of schistosomiasis vector snails that were identified in a 2008 survey covering the RMR and the coastal localities of PE.

Molecular approaches for the detection of Schistosoma mansoni: possible applications in the detection of snail infection, monitoring of transmission sites, and diagnosis of human infection

The detection of specific DNA sequences by polymerase chain reaction (PCR) has proved extremely valuable for the analysis of genetic disorders and the diagnosis of a variety of infectious disease pathogens. However, the application to the detection of Schistosoma mansoni is rare, despite a recommendation of the World Health Organization that a major focus of research on schistosomiasis should be on the development and evaluation of new strategies and tools for control of the disease. In this context, a few studies were published for the detection of the parasite in snails, monitoring of cercariae in water bodies, and diagnosis of human infection. The present minireview describes sensitive and specific PCR based systems to detect S. mansoni, indicating possible applications in the detection of snail infection, monitoring of transmission sites, and diagnosis of human infection.

Prevalence and risk factors for Staphylococcus aureus in health care workers at a University Hospital of Recife-PE

Staphylococcus aureus is the main human pathogen that colonizes individuals in general population. The objective of the study was evaluate the epidemiological and sensitivity profile of S. aureus lineage, isolated in health care workers (HCW) of a University Hospital in Pernambuco state, Brazil. Biological samples of hands and nasal cavities were sown in agar sheep blood. Colonies under suspicion of being S. aureus were identified using Gram staining, catalase test and coagulase, mannitol-salty agar fermentation and DNAse agar. The resistance to mupirocin was analyzed through the Kirby Bauer technique. In relation to methicillin and vancomycin the determination was by the minimum inhibitory concentration method (E-test). From the 202 HCW evaluated, 52 were colonized by S. aureus (25,7%). The factors associated to the colonization by S. aureus were: age-group, professional category, use of individual protection equipments (frequency and numbers). All S. aureus isolate lineages were sensitive to mupirocin and vancomycin, and three of them were identified as methicillin-resistant. The prevalence of MSSA and MRSA among HCW was considered low and was below the results described in the literature. The isolate S. aureus lineages have shown low resistance profile.

Correlation of biological serum markers with the degree of hepatic fibrosis and necroinflammatory activity in hepatitis C and schistosomiasis patients

Liver biopsy is the gold-standard method to stage fibrosis; however, it is an invasive procedure and is potentially dangerous. The main objective of this study was to evaluate biological markers, such as cytokines IL-13, IFN-gamma, TNF-alpha and TGF-beta, platelets, bilirubins (Bil), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), total proteins, gamma-glutamil transferase (gamma-GT) and alkaline phosphatase (AP), that could be used to predict the severity of hepatic fibrosis in schistosomiasis and hepatitis C (HC) as isolated diseases or co-infections. The following patient groups were selected: HC (n = 39), HC/hepatosplenic schistosomiasis (HSS) (n = 19), HSS (n = 22) and a control group (n = 13). ANOVA and ROC curves were used for statistical analysis. P < 0.05 was considered significant. With HC patients we showed that TNF-alpha (p = 0.020) and AP (p = 0.005) could differentiate mild and severe fibrosis. With regard to necroinflammatory activity, AST (p = 0.002), gamma-GT (p = 0.034) and AP (p = 0.001) were the best markers to differentiate mild and severe activity. In HC + HSS patients, total Bil (p = 0.008) was capable of differentiating between mild and severe fibrosis. In conclusion, our study was able to suggest biological markers that are non-invasive candidates to evaluate fibrosis and necroinflammatory activity in HC and HC + HSS.

Self-assembled monolayers of mercaptobenzoic acid and magnetite nanoparticles as an efficient support for development of tuberculosis genosensor

In this work, a genosensor for the electrochemical detection of genomic DNA from Mycobacterium tuberculosis was developed. The biosensor is based on self-assembled monolayers of mercaptobenzoic acid (MBA) and magnetite nanoparticles (Fe3O4Nps) on bare gold electrode for immobilization of DNA probe. The aim of this work was the development of a platform based on cysteine-coated magnetic Fe3O4Nps linked via the carboxylate group from MBA to the work electrode surface and subsequently to the DNA probe. The probe-genome interaction was evaluated using a [Fe(CN)6](4-)/[Fe(CN)6](3-) redox pair. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to evaluate the bioelectrochemical behavior of the sensor. Atomic force microscopy images showed Fe3O4Nps immobilized across the electrode surface. The interaction of the sensor with different genome DNA concentrations resulted in changes in the charge transfer resistance, indicating a possible use for tuberculosis detection at low concentrations (detection limit of 6ngμL(-1)).

Colonização pelo Staphylococcus aureus em profissionais de enfermagem de um hospital escola de Pernambuco

Estudio realizado para identificar prevalencia de colonizacion por Staphylococcus aureus en profesionales de enfermeria de hospital universitario de Pernambuco, asi como evaluar el perfil de resistencia de la bacteria aislada. Se realizo un estudio transversal en el que se recolectaron muestras biologicas de manos y cavidad nasal. La identificacion del S. aureus se realizo mediante cultivo en agar-sangre, agar-manitol salado y mediante pruebas de catalasa y coagulasa. El perfil de sensibilidad se determino por tecnica de Kirby Bauer y para la determinacion de resistencia a meticilina se realizo screening en placa con oxalacina, con adicion de 4% de NaCl. De 150 profesionales evaluados, 39 estaban colonizados, lo que demostro prevalencia de 25,8%. Entre las variables estudiadas, faja etaria y cantidad de EPI se presentaron asociadas con la colonizacion por la bacteria. De todas las cepas aisladas, apenas cinco presentaron resistencia a meticilina.This study was performed with the objective to identify the prevalence of colonization by Staphylococcus aureus in nursing professionals from a teaching hospital in Pernambuco, and evaluate the resistance profile of these isolates. To do this, we performed a cross-sectional study where biological samples were collected from the hands and nasal cavities of the subjects. S. aureus was identified using agar (blood agar and mannitol salt) via catalase and coagulase tests. The sensitivity profile was determined by Kirby Bauer technique and determination of methicillin resistance was performed with oxacillin screening with sodium chloride (NaCl) addition. Of the 151 professionals evaluated, 39 were colonized which showed a prevalence of 25.8%. Among the variables studied, age and use of PPE were associated with colonization by the organism. Of all the isolates, only five were resistant to methicillin.

Detection of Wuchereria bancrofti DNA in paired serum and urine samples using polymerase chain reaction-based systems

The Global Program for the Elimination of Lymphatic Filariasis (GPELF) aims to eliminate this disease by the year 2020. However, the development of more specific and sensitive tests is important for the success of the GPELF. The present study aimed to standardise polymerase chain reaction (PCR)-based systems for the diagnosis of filariasis in serum and urine. Twenty paired biological urine and serum samples from individuals already known to be positive for Wuchereria bancrofti were collected during the day. Conventional PCR and semi-nested PCR assays were optimised. The detection limit of the technique for purified W. bancrofti DNA extracted from adult worms was 10 fg for the internal systems (WbF/Wb2) and 0.1 fg by using semi-nested PCR. The specificity of the primers was confirmed experimentally by amplification of 1 ng of purified genomic DNA from other species of parasites. Evaluation of the paired urine and serum samples by the semi-nested PCR technique indicated only two of the 20 tested individuals were positive, whereas the simple internal PCR system (WbF/Wb2), which has highly promising performance, revealed that all the patients were positive using both samples. This study successfully demonstrated the possibility of using the PCR technique on urine for the diagnosis of W. bancrofti infection.

Optimization of single-tube nested PCR for the diagnosis of visceral leishmaniasis

Conventional nested PCR is a very sensitive and specific method for the diagnosis of visceral leishmaniasis. However, this type of PCR is notorious for contamination problems related to the processing of the product between the first and the second PCR steps. In order to have a PCR method that is just as efficient but without the risk of contamination, we attempted the optimization of a single-tube nested PCR (STNPCR) method. During the first and the second PCR steps, we used the small subunit of ribosomal RNA (ssu rRNA) and the ribosomal internal transcribed spacer (ITS) as targets, respectively. The performances of STNPCR and nested PCR in detecting the DNA of Leishmania chagasi were compared. In the case of STNPCR, the inner primers were immobilized on the interior of the tube cap by means of adsorption microtubes and then were solubilized before the second reaction. This procedure eliminated the need to open the microtube, which could have led to false-positive results through cross-contamination. The detection limit for the purified L. chagasi DNA was 1 fg by using nested PCR and 10 fg by using STNPCR. We also tested the specificity of the system against other parasites, and observed that Trypanosoma cruzi DNA was amplified with a detection limit of up to 1 pg. This study not only presents a promising tool for the diagnosis of visceral leishmaniasis, but also provides a new tool for the diagnosis of Chagas disease, either in mono-infection by T. cruzi or in co-infection with Leishmania spp.

Colonization by Staphylococcus aureus among the nursing staff of a teaching hospital in Pernambuco

Estudio realizado para identificar prevalencia de colonizacion por Staphylococcus aureus en profesionales de enfermeria de hospital universitario de Pernambuco, asi como evaluar el perfil de resistencia de la bacteria aislada. Se realizo un estudio transversal en el que se recolectaron muestras biologicas de manos y cavidad nasal. La identificacion del S. aureus se realizo mediante cultivo en agar-sangre, agar-manitol salado y mediante pruebas de catalasa y coagulasa. El perfil de sensibilidad se determino por tecnica de Kirby Bauer y para la determinacion de resistencia a meticilina se realizo screening en placa con oxalacina, con adicion de 4% de NaCl. De 150 profesionales evaluados, 39 estaban colonizados, lo que demostro prevalencia de 25,8%. Entre las variables estudiadas, faja etaria y cantidad de EPI se presentaron asociadas con la colonizacion por la bacteria. De todas las cepas aisladas, apenas cinco presentaron resistencia a meticilina.This study was performed with the objective to identify the prevalence of colonization by Staphylococcus aureus in nursing professionals from a teaching hospital in Pernambuco, and evaluate the resistance profile of these isolates. To do this, we performed a cross-sectional study where biological samples were collected from the hands and nasal cavities of the subjects. S. aureus was identified using agar (blood agar and mannitol salt) via catalase and coagulase tests. The sensitivity profile was determined by Kirby Bauer technique and determination of methicillin resistance was performed with oxacillin screening with sodium chloride (NaCl) addition. Of the 151 professionals evaluated, 39 were colonized which showed a prevalence of 25.8%. Among the variables studied, age and use of PPE were associated with colonization by the organism. Of all the isolates, only five were resistant to methicillin.