Strain differentiation of Trichophyton rubrum by random amplification of polymorphic DNA (RAPD)

Trichophyton rubrum is an important cause of dermatomycoses. Molecular strain typing methods have recently been developed to address questions about epidemiology and source of relapse following treatment. This report describes the application of RAPD for molecular strain differentiation of this fungus utilizing the primers 1- (5'-d[GGTGCGGGAA]-3') and 6- (5'-d[CCCGTCAGCA]-3'). A total of five RAPD patterns were observed among 10 strains of T. rubrum, with each of the primers used. We conclude that RAPD analysis using primers 1 and 6 can be used in epidemiological studies.

Screening for pulmonary tuberculosis in Teresópolis, RJ, Brazil: the search for respiratory symptomatic patients in emergency service of "Hospital das Clínicas de Teresópolis Costantino Ottaviano, Fundação Educacional Serra dos Órgãos"

The aim of the present study was to investigate the detection percentage of tuberculosis among patients that are respiratory symptomatic (TB suspects). In this work, we present the preliminary results of research carried out at "Hospital das Clínicas de Teresópolis Costantino Ottaviano da Fundação Educacional Serra dos Órgãos (FESO)" from November 2003 to April 2004. Among the 40 respiratory symptomatic individuals identified and referred to the Tuberculosis Control Program in Teresópolis, two (5.0%) were characterized as smear-positive. These results confirm reports in the literature and underscore the need for and importance of this strategy.

Analysis of the genetic polymorphism of Paracoccidioides brasiliensis and Paracoccidioides cerebriformis "Moore" by random amplified polymorphic DNA (RAPD) and 28S ribosomal DNA sequencing: Paracoccidioides cerebriformis revisited

Our purpose was to compare the genetic polymorphism of six samples of P. brasiliensis (113, 339, BAT, T1F1, T3B6, T5LN1), with four samples of P. cerebriformis (735, 741, 750, 361) from the Mycological Laboratory of the Instituto de Medicina Tropical de São Paulo, using Random Amplified Polymorphic DNA Analysis (RAPD). RAPD profiles clearly segregated P. brasiliensis and P. cerebriformis isolates. However, the variation on band patterns among P. cerebriformis isolates was high. Sequencing of the 28S rDNA gene showed nucleotide conservancy among P. cerebriformis isolates, providing basis for taxonomical grouping, and disclosing high divergence to P. brasiliensis supporting that they are in fact two distinct species. Moreover, DNA sequence suggests that P. cerebriformis belongs in fact to the Aspergillus genus.

Optimization of random amplified polymorphic DNA techniques for use in genetic studies of Cuban triatominae

Random amplified polymorphic DNA (RAPD) technique is a simple and reliable method to detect DNA polymorphism. Several factors can affect the amplification profiles, thereby causing false bands and non-reproducibility of assay. In this study, we analyzed the effect of changing the concentration of primer, magnesium chloride, template DNA and Taq DNA polymerase with the objective of determining their optimum concentration for the standardization of RAPD technique for genetic studies of Cuban Triatominae. Reproducible amplification patterns were obtained using 5 pmoL of primer, 2.5 mM of MgCl2, 25 ng of template DNA and 2 U of Taq DNA polymerase in 25 µL of the reaction. A panel of five random primers was used to evaluate the genetic variability of T. flavida. Three of these (OPA-1, OPA-2 and OPA-4) generated reproducible and distinguishable fingerprinting patterns of Triatominae. Numerical analysis of 52 RAPD amplified bands generated for all five primers was carried out with unweighted pair group method analysis (UPGMA). Jaccard's Similarity Coefficient data were used to construct a dendrogram. Two groups could be distinguished by RAPD data and these groups coincided with geographic origin, i.e. the populations captured in areas from east and west of Guanahacabibes, Pinar del Río. T. flavida present low interpopulation variability that could result in greater susceptibility to pesticides in control programs. The RAPD protocol and the selected primers are useful for molecular characterization of Cuban Triatominae.

Active search for leprosy cases in Midwestern Brazil: a serological evaluation of asymptomatic household contacts before and after prophylaxis with bacillus Calmette-Guérin

Leprosy is a disease caused by Mycobacterium leprae that carries a high risk of disability, making early diagnosis mandatory. This study aimed to determine the applicability of anti-PGL-1 IgM antibody detection, using the ML FLOW technique, as an assistant tool for the detection of leprosy infection in asymptomatic household contacts (AHHC) of multibacillary leprosy index cases from Midwest Brazil. Serological changes induced by the prophylaxis of these household contacts with Bacillus Calmette-Guérin (BCG) were also verified. A total of 91 AHHC were assessed, among which, 18.68% (n = 17) presented both positive bacilloscopy and positive anti-PGL-1 IgM serology. Positivity concordance between these two laboratorial exams (Kappa Index = 1; p < 0.001) was indicated, however, one case did not demonstrate concordance between the semiquantitative assessment of anti-PGL-1 IgM and the bacilloscopy index (Kappa Index = 0.96; p < 0.001). Among the 17 AHHC with positive bacilloscopy, eight were reassessed after prophylaxis with BCG and two of them presented negative anti-PGL-1 IgM serology, being these patients who had presented a bacilloscopy index of < 2[+] in the initial assessment. This study shows that anti-PGL-1 IgM detection may be used as a tool to determine the bacillary load in AHHC and to detect immune changes related to prophylaxis by nonspecific vaccination.

Problems and perspectives for Chagas disease control: in search of a realistic analysis

Chagas disease was an important medical and social problem in almost all of Latin America throughout the twentieth century. It has been combated over a broad swath of this continent over recent decades, with very satisfactory results in terms of vector and transfusional transmission. Today, a surveillance stage still remains to be consolidated, in parallel with appropriate care required for some millions of infected individuals who are today living in endemic and non-endemic areas. Contradictorily, the good results attained have generated excessive optimism and even disregard among health authorities, in relation to this disease and its control. The loss of visibility and priority may be a logical consequence, particularly in Latin American healthcare systems that are still disorganized and overburdened due to insufficiencies of financial and human resources. Consolidation of the victories against Chagas disease is attainable but depends on political will and continual attention from the most consequential protagonists in this struggle, especially the Latin American scientific community.

Identification and differentiation of Candida species from pediatric patients by random amplified polymorphic DNA

Thirty-four Candida isolates were analyzed by random amplified polymorphic DNA using the primer OPG-10:24 Candida albicans; 4 Candida tropicalis; 2 Candida parapsilosis; 2 Candida dubliniensis; 1 Candida glabrata and 1 Candida krusei. The UPGMA-Pearson correlation coefficient was used to calculate the genetic distance between the different Candida groupings. Samples were classified as identical (correlation of 100%); highly related samples (90%); moderately related samples (80%) and unrelated samples (< 70%). The results showed that the RAPD proposed was capable of classifying the isolates coherently (such that same species were in the same dendrogram), except for two isolates of Candida parapsilosis and the positive control (Netherlands, 1973), probably because they are now recognized as three different species. Concerning the only fluconazole-resistant Candida tropicalis isolate with a genotype that was different to the others, the data were insufficient to affirm that the only difference was the sensitivity to fluconazole. We concluded that the Random Amplified Polymorphic DNA proposed might be used to confirm Candida species identified by microbiological methods.

Leprosy in Buriticupu, state of Maranhão: active search in the general population

INTRODUCTION: This study was developed to evaluate the situation of leprosy in the general population of the municipality of Buriticupu, State of Maranhão, Brazil. METHODS: We used the method of active search to identify new cases from 2008 to 2010. Bacilloscopy of intradermal scrapings was performed in all patients with skin lesions compatible with leprosy, and histopathological examination in those who had doubts on the definition of the clinical form. RESULTS: The study included 19,104 individuals, with 42 patients diagnosed with leprosy after clinical examination, representing a detection rate of 219.84 per 100,000 inhabitants. The predominant clinical presentation was tuberculoid with 24 (57.1%) cases, followed by borderline with 11, indeterminate with four, and lepromatous with three cases. The study also allowed the identification of 81 patients with a history of leprosy and other skin diseases, such as pityriasis versicolor, dermatophytosis, scabies, vitiligo, and skin carcinoma. The binomial test showed that the proportion of cases in the headquarters was significantly higher than that in the villages (p = 0.04), and the generalized exact test showed that there was no association between age and clinical form (p = 0.438) and between age and gender (p = 0.083). CONCLUSIONS: The elevated detection rate defines the city as hyperendemic for leprosy; the active search for cases, as well as the organization of health services, is an important method for disease control.

Triatomines in dwellings and outbuildings in an endemic area of Chagas disease in northeastern Brazil

INTRODUCTION: The present study identified the triatomines collected in intra and peri-domestic environments, observed the occurrence of Trypanosoma cruzi infection in triatomines and correlated this information with housing conditions and the fauna associated with the rural areas of the City of Itabaianinha, located in the State of Sergipe, Brazil. METHODS: Quarterly visits were conducted between March 2009 and March 2010, and the homes to be visited for the active search of insects were determined by random selection. In each housing unit, the insects were collected by a manual search with a metal clip and flashlight to inspect openings and cavities, with a collection time of one hour/home/individual. The Pirisa® dislodge chemical was used to force the insects to leave their ecotopes. Analysis of the intestinal contents of triatomines was performed in the laboratory to establish the presence of Trypanosomatidae. RESULTS: Of the 103 dwellings surveyed, 17.5% were infested with Panstrongylus megistus. The village of Mutuca exhibited the highest infestation rate (38.1%). All the villages with relevant infestation rates were situated in the northern area of the city. The highest percentage of vector infection was found in the village of Água Boa (56.5%). The rural dwellings were found to be primarily brick or wooden house with or without roughcast or plastered walls, and the outbuilding most frequently associated with triatomines was the chicken run. CONCLUSIONS: These results emphasise the need for broader vector control and surveillance and for educational campaigns in the context of the Chagas Disease Control Program.

Long-term Outcomes of Drug-eluting versus Bare-metal stent for ST-elevation Myocardial Infarction

Background:Long-term outcomes of drug-eluting stents (DES) versus bare-metal stents (BMS) in patients with ST-segment elevation myocardial infarction (STEMI) remain uncertain.Objective:To investigate long-term outcomes of drug-eluting stents (DES) versus bare-metal stents (BMS) in patients with ST-segment elevation myocardial infarction (STEMI).Methods:We performed search of MEDLINE, EMBASE, the Cochrane library, and ISI Web of Science (until February 2013) for randomized trials comparing more than 12-month efficacy or safety of DES with BMS in patients with STEMI. Pooled estimate was presented with risk ratio (RR) and its 95% confidence interval (CI) using random-effects model.Results:Ten trials with 7,592 participants with STEMI were included. The overall results showed that there was no significant difference in the incidence of all-cause death and definite/probable stent thrombosis between DES and BMS at long-term follow-up. Patients receiving DES implantation appeared to have a lower 1-year incidence of recurrent myocardial infarction than those receiving BMS (RR = 0.75, 95% CI 0.56 to 1.00, p= 0.05). Moreover, the risk of target vessel revascularization (TVR) after receiving DES was consistently lowered during long-term observation (all p< 0.01). In subgroup analysis, the use of everolimus-eluting stents (EES) was associated with reduced risk of stent thrombosis in STEMI patients (RR = 0.37, p=0.02).Conclusions:DES did not increase the risk of stent thrombosis in patients with STEMI compared with BMS. Moreover, the use of DES did lower long-term risk of repeat revascularization and might decrease the occurrence of reinfarction.

Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease. 1 - Comparative xenodiagnosis with nine triatomine species of animals with acute infections by Trypanosoma cruzi

In search of a suitable vector species for xenodiagnosis of humans and animals with chronic Chagas' disease we first investigated the reactions of different vector species to acute infection with Trypanosoma cruzi. Vector species utilized in this study were: Triatoma infestans, Rhodnius prolixus and Triatoma dimidiata, all well adapted to human habitats; Triatoma rubrovaria and Rhodnius neglectus both considered totally wild species; Panstrongylus megistus, Triatoma sordida, Triatoma pseudomaculata and Triatoma brasiliensis, all essentially sylvatic but some with domiciliary tendencies and others restricted to peridomestic biotopes with incipient colonization of human houses after successful eradication of T. infestans. Results summarized in Table IV suggest the following order of infectivity among the 9 studied vector species: P. megistus with 97.8% of infected bugs, T. rubrovaria with 95% of positive bugs a close second followed by T. Pseudomaculata with 94.3% and R. neglectus with 93.8% of infected bugs, almost identical thirds. R. prolixus, T. infestans and T. dimidiata exhibited low infection rates of 53.1%, 51.6% and 38.2% respectively, coupled with sharp decreases occuring with aging of infection (Fig. 1). The situation was intermediate in T. brasiliensis and T. sordida infection rates being 76.9% and 80% respectively. Results also point to the existence of a close correlation between prevalence and intensity of infection in that, species with high infection rates ranging from 93.8% to 97.8% exhibited relatively large proportions of insects (27.3% - 33.5%) harbouring very dense populations of T. cruzi. In species with low infection rates ranging from 38.2% to 53.1% the proportion of bugs demonstrating comparable parasite densities was at most 6%. No differences attributable to blood-meal size or to greater susceptibility of indigenous vector species to parasites of their own geographical area, as suggested in earlier...

Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease: 2 - Attempts to upgrade the reliability and the efficacy of xenodiagnosis in chronic Chagas' disease

In order to upgrade the reliability of xenodiagnosis, attention has been directed towards population dynamics of the parasite, with particular interest for the following factors: 1. Parasite density which by itself is not a research objective, but by giving an accurate portrayal of parasite development and multiplication, has been incorporated in screening of bugs for xenodiagnosis. 2. On the assumption that food availability might increase parasite density, bugs from xenodiagnosis have been refed at biweekly intervals on chicken blood. 3. Infectivity rates and positives harbouring large parasite yields were based on gut infections, in which the parasite population comprised of all developmental forms was more abundant and easier to detect than in fecal infections, thus minimizing the probability of recording false negatives. 4. Since parasite density, low in the first 15 days of infection, increases rapidly in the following 30 days, the interval of 45 days has been adopted for routine examination of bugs from xenodiagnosis. By following the enumerated measures, all aiming to reduce false negative cases, we are getting closer to a reliable xenodiagnostic procedure. Upgrading the efficacy of xenodiagnosis is also dependent on the xenodiagnostic agent. Of 9 investigated vector species, Panstrongylus megistus deserves top priority as a xenodiagnostic agent. Its extraordinary capability to support fast development and vigorous multiplication of the few parasites, ingested from the host with chronic Chagas' disease, has been revealed by the strikingly close infectivity rates of 91.2% vs. 96.4% among bugs engorged from the same host in the chronic and acute phase of the disease respectively (Table V), the latter comporting an estimated number of 12.3 x 10[raised to the power of 3] parasites in the circulation at the time of xenodiagnosis, as reported previously by the authors (1982).

Schistosomiasis mansoni: immunodiagnosis aspects and search for an immunological marker related to therapeutic efficacy

The recent findings on immunodiagnosis of schistosomiasis mansoni have shown that purified Schistosoma mansoni antigens do not provide maximum positivity. Therefore, the authors suggest the use of semi-purified antigens for diagnostic purposes. So far, no serological marker for cured patients as shown by negative stool examination was found. However, a tendency of IgG antibody titre decrease was observed, when egg antigen was used.