Development of duplex-PCR for identification of Aeromonas species

Introduction The number of reports of intestinal infections caused by Aeromonas spp. has increased significantly in recent years. In most clinical laboratories, identification of these bacteria is carried out by general phenotypic tests that sometimes do not accurately differentiate Aeromonas and Vibrio. Methods A duplex-polymerase chain reaction (PCR) was developed directed to 2 targets identifying Aeromonas spp. pathogenic to humans. Results The duplex-PCR results were reproducible and specific for Aeromonas spp. pathogenic to humans. Conclusions This method will allow differentiation between Vibrio and Aeromonas spp. in patients with in cholera-like symptoms and can also be used in water quality monitoring.

Leprosy and pregnancy in the State of Para: an epidemiological perspective

Introduction A few older publications describe leprosy associated with pregnancy, a situation that has been linked to leprosy exacerbation. This study aimed to describe the detection rate of this association in the State of Pará by county and Integration Region (IR) from 2007 to 2009 via an analysis of sociodemographic, epidemiological and operational indices. Methods This was a descriptive study using information generated by the SINAN. The Detection Coefficient of the Leprosy and Pregnancy Association (DCLP) epidemiological index was constructed to help interpret the endemicity parameters. The disease was considered hyperendemic when greater than two cases per 10,000 inhabitants were identified. Results During the study period, 149 associations were detected, with 14 hyperendemic counties: seven in 2007, five in 2008 and two in 2009. The Carajás Integrated Region displayed the highest DCLP index in the period. Eldorado dos Carajás had the single highest DCLP index (5.7/10,000 inhabitants, 2008), whereas the DCLP index in Conceição do Araguaia was very high in all three years. However, most counties displayed low or medium DCLP indices. The annual averages were 0.31 DCLP (2007), bass; 0.30 (2008), bass and 0.19 (2009), bass. The average DCLP index was 0.26, which is considered low. Three clusters of medium endemicity were identified by the average DCLP in the study period. Conclusions The analyses indicated that the surveillance program is still unsatisfactory in Pará. The interpretation of the endemicity parameters enabled qualitative and quantitative analyses to determine the epidemiological panorama of this association. The identification of high endemicity requires further clarification.

Environmental isolation, biochemical identification, and antifungal drug susceptibility of Cryptococcus species

Introduction The incidence of opportunistic fungal infections has increased in recent years and is considered an important public health problem. Among systemic and opportunistic mycoses, cryptococcosis is distinguished by its clinical importance due to the increased risk of infection in individuals infected by human immunodeficiency virus. Methods To determine the occurrence of pathogenic Cryptococcus in pigeon excrement in the City of Araraquara, samples were collected from nine environments, including state and municipal schools, abandoned buildings, parks, and a hospital. The isolates were identified using classical tests, and susceptibility testing for the antifungal drugs (fluconazole, itraconazole, voriconazole, and amphotericin B) independently was also performed. After collection, the excrement samples were plated on Niger agar and incubated at room temperature. Results A total of 87 bird dropping samples were collected, and 66.6% were positive for the genus Cryptococcus. The following species were identified: Cryptococcus neoformans (17.2%), Cryptococcus gattii (5.2%), Cryptococcus ater (3.5%), Cryptococcus laurentti (1.7%), and Cryptococcus luteolus (1.7%). A total of 70.7% of the isolates were not identified to the species level and are referred to as Cryptococcus spp. throughout the manuscript. Conclusions Although none of the isolates demonstrated resistance to antifungal drugs, the identification of infested areas, the proper control of birds, and the disinfection of these environments are essential for the epidemiological control of cryptococcosis.

Molecular identification of the agent of Q fever – Coxiella burnetii – in domestic animals in State of Rio de Janeiro, Brazil

Introduction Over the last recent years, the number of Q fever cases have has increased throughout the world. An epidemiological investigation was performed in the area in which the first molecular documentation of Q fever in Brazil was previously reported. Methods Indirect immunofluorescence assay (IFA) and PCR of Coxiella burnetii targeting the htpAB gene were performed in samples from 14 dogs (blood); 1 cat (blood); 10 goats (blood, milk, vaginal swab and anal swab); 3 sheep (blood); and 2 horses (blood). Results Two dogs, two sheep and five goats were seroreactive. DNA was amplified from 6 milk and 2 blood samples from goats and from dogs, respectively. The sequence of the amplicons exhibited 99% sequence similarity with the homologous sequence of the htpAB gene of C. burnetii RSA 331 (GenBank - CP000890). Conclusions The results confirm C. burnetii infection in animals in Rio de Janeiro and reinforce the need for the surveillance of Q fever in Brazil.

Performance of the Vitek 2 system software version 5.03 in the bacterial identification and antimicrobial susceptibility test: evaluation study of clinical and reference strains of Gram-positive cocci

Introduction. The genera Enterococcus, Staphylococcus and Streptococcus are recognized as important Gram-positive human pathogens. The aim of this study was to evaluate the performance of Vitek 2 in identifying Gram-positive cocci and their antimicrobial susceptibilities. Methods. One hundred four isolates were analyzed to determine the accuracy of the automated system for identifying the bacteria and their susceptibility to oxacillin and vancomycin. Results. The system correctly identified 77.9% and 97.1% of the isolates at the species and genus levels, respectively. Additionally, 81.8% of the Vitek 2 results agreed with the known antimicrobial susceptibility profiles. Conclusion. Vitek 2 correctly identified the commonly isolated strains; however, the limitations of the method may lead to ambiguous findings.

Molecular identification and antifungal susceptibility profiles of Candida parapsilosis complex species isolated from culture collection of clinical samples

AbstractINTRODUCTION:Candida parapsilosis is a common yeast species found in cases of onychomycosis and candidemia associated with infected intravascular devices. In this study, we differentiated Candida parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis from a culture collection containing blood and subungual scraping samples. Furthermore, we assessed the in vitro antifungal susceptibility of these species to fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin.METHODS:Differentiation of C. parapsilosis complex species was performed by amplification of the secondary alcohol dehydrogenase (SADH) gene and digestion by the restriction enzyme Ban I. All isolates were evaluated for the determination of minimal inhibitory concentrations using Etest, a method for antifungal susceptibility testing.RESULTS:Among the 87 isolates, 78 (89.7%) were identified as C. parapsilosis sensu stricto , five (5.7%) were identified as C. orthopsilosis , and four (4.6%) were identified as C. metapsilosis . Analysis of antifungal susceptibility showed that C. parapsilosis sensu strictoisolates were less susceptible to amphotericin B and itraconazole. One C. parapsilosis sensu stricto isolate was resistant to amphotericin B and itraconazole. Moreover, 10.2% of C. parapsilosis sensu stricto isolates were resistant to caspofungin. Two C. parapsilosis sensu strictoisolates and one C. metapsilosis isolate were susceptible to fluconazole in a dose-dependent manner.CONCLUSIONS:We reported the first molecular identification of C. parapsilosiscomplex species in State of Goiás, Brazil. Additionally, we showed that although the three species exhibited differences in antifungal susceptibility profiles, the primary susceptibility of this species was to caspofungin.

Polymerase chain reaction-based method for the identification of Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in mucosal tissues conserved in paraffin

ABSTRACTINTRODUCTION: In the Americas, mucosal leishmaniasis is primarily associated with infection by Leishmania (Viannia) braziliensis. However, Leishmania (Viannia) guyanensis is another important cause of this disease in the Brazilian Amazon. In this study, we aimed at detecting Leishmaniadeoxyribonucleic acid (DNA) within paraffin-embedded fragments of mucosal tissues, and characterizing the infecting parasite species.METHODS: We evaluated samples collected from 114 patients treated at a reference center in the Brazilian Amazon by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses.RESULTS: Direct examination of biopsy imprints detected parasites in 10 of the 114 samples, while evaluation of hematoxylin and eosin-stained slides detected amastigotes in an additional 17 samples. Meanwhile, 31/114 samples (27.2%) were positive for Leishmania spp. kinetoplast deoxyribonucleic acid (kDNA) by PCR analysis. Of these, 17 (54.8%) yielded amplification of the mini-exon PCR target, thereby allowing for PCR-RFLP-based identification. Six of the samples were identified as L. (V.) braziliensis, while the remaining 11 were identified as L. (V.) guyanensis.CONCLUSIONS: The results of this study demonstrate the feasibility of applying molecular techniques for the diagnosis of human parasites within paraffin-embedded tissues. Moreover, our findings confirm that L. (V.) guyanensisis a relevant causative agent of mucosal leishmaniasis in the Brazilian Amazon.

Venomous and poisonous arthropods: identification, clinical manifestations of envenomation, and treatments used in human injuries

Abstract This review presents the main species of venomous and poisonous arthropods, with commentary on the clinical manifestations provoked by the toxins and therapeutic measures used to treat human envenomations. The groups of arthopods discussed include the class Arachnida (spiders and scorpions, which are responsible for many injuries reported worldwide, including Brazil); the subphylum Myriapoda, with the classes Chilopoda and Diplopoda (centipedes and millipedes); and the subphylum Hexapoda, with the class Insecta and the orders Coleoptera (beetles), Hemiptera (stink bugs, giant water bugs, and cicadas), Hymenoptera (ants, wasps, and bees), and Lepidoptera (butterflies and moths).

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 pictorial field guide for the rapid identification of sandflies (DIPTERA: PSYCHODIDAE), commonly encountered in Pará state, Brazil.

A pictorial field guide to the 30 species of sandfly most commonly encountered in Pará State is presented, based on the easily recognised external characters of the length of the 5th palpal segment, thoracic infuscation, abdominal colour and head and body size. In most cases this allows identification to the species. In others, especially with females, it gives an indication of the species, which may then be confirmed with data from more detailed taxanomix studies. This type of field guide helps in teaching, rapid sorting of flies prior to dissection and in acquainting visitors with the variety of species present in a given area.A rapid technique for the taxonomic sorting of unmounted, freshly killed female sandflies is required, prior to the dissection of large numbers of a particular species. Such a method is useful in areas where numerous species occur in studies on natural flagellate infections, age determination and for ecological studies. With the above points in mind a pictorial field guide has been designed that enables the identification of unmounted, unmacerated specimens of the 30 more commonly encountered species of phleboto-mine sandflies (***) in Pará State, North Brazil. It is based on the easily recognised external characters of the length of the 5th palpal segment, thoracic infuscation, ad-dominal colour and proboscis and body size.Taxonomy of male phlebotomine sandflies is based on the structure of the genitalia and, as most of this is external, a wholly external character key is readily made. Female taxonomy, however, is based on the internal character of the cibarium, pharynx and sperma thecae. In order to produce an external character key we therefore return to an unso phisticated "phlebotometry" (see Martins et al., 1978 p. 3 for review), using relative lengths of the proboscis, palpal segments and body, along with the degree of infuscation. Ihis idea is not new; indeed many sandfly specialists presently use external characters to separate certain species (H. Fraiha, R. P. Lane, P. D. Ready, D. G. Young and R. D. Ward personal communications 1983 & 1984).A key used to separate five anthropophillic sandflies by Biagi (1966), in Mexico, was based mainly on palpal segment length and infuscation. Floch and Abonnenc (1952) stressed the use of relative lengths of palpal segments in their keys to the sandflies of French Guiana, and four members of the shannoni group have been similarly separated according to the degree of infuscation by Morales et al. (1982). The use of thoracic infuscation as a reliable character seems to be gaining favour, having been used by young & Fairchild (1974) and Ready & Fraiha (1981). Indeed Chariotis 1974) showed the usefulness of thoracic infuscation to sepenate 7 anthropophillic species, during studies onvesicular stomatitis in Panama. Identification using external characters is essential for work on viral isolations from sandflies, where bulk samples of whole sandflies are used.Perhaps the major advantage of a simple visual guide is for teaching purposes. Technical staff in this lnstitute are able to identify most of the species they encounter without having to use the standard, more unwieldly (and in many cases unavailable) internal character keys, and the guides presented below have allowed rapid species sorting prior to the dissection of sandflies in our leismaniasis study areas (Ryan et at. ,1985).

The relationship between stem vessel parameters and the development of strata in the early stages of secondary forest succession in Amazonia

In order to compare the development of strata in the early stages of secondary forest succession with vessel parameters of the tree species, a forest inventory was carried out in 4-year (Q1: 48 m2), 11-year (Q2: 400 m2) and 20-year (Q3: 400 m2) forests and vessel parameters were investigated from stem cross sections of 18 species obtained in Q2. Thirty three species (21 families), 77 species (35 families), 39 species (20 families) were found in Ql, Q2, Q3, respectively. The percentage of dead individuals, dead stems and the percentage of individuals with multiple stems increased with time after clear cutting. Also, the total D2H of Q3 was 26.1 times that of Q1, and the development of strata started in Q2 and Q3. The image analysis of vessel size, area and number of vessels revealed that species which reach the forest canopy had a large D2H value, vessel diameter and area, while species which remain near the forest floor had smaller ones. Poecilanthe effusa (Huber) Ducke is an example of the latter case, with a large number of individuals and abundant sprouting of new stems from stumps, but with high mortality.

The influence of climatic parameters in the haematophagic daily activity of Cerqueirellum argentiscutum (Shelley & Luna Dias) (Diptera: Simuliidae) in Amazonas, Brazil

We studied the influence of climatic parameters in the daily haematophagic activity of Cerqueirellum argentiscutum from September/1999 to August/2000. The bite activity observed was different according to the annual rain precipitation (dry and rainy seasons). Humidity and temperature were the factors that most influenced it in both periods. During the dry season, it was greater in the beginning of the morning, showing a positive association with the humidity. However, during the rainy season, it was negatively related to that same factor. When wind speed was higher than 10 Km.h-1, it was reduced abruptly. Light intensity, atmospheric pressure and cloudiness seemed to act as secondary factors in the daily abundance of C. argentiscutum.

Comparative study on hematological parameters of farmed matrinxã, Brycon amazonicus Spix and Agassiz, 1829 (Characidae: Bryconinae) with others Bryconinae species

The aim of this study was not only to determine the red blood cells parameters, thrombocyte and leukocyte counts in farmed Brycon amazonicus (matrinxã), to compare these parameters among Bryconinae species from literature, and also to investigate the presence of special granulocytic cells in these fish. The results of the blood cells parameters here established for farmed B. amazonicus, a species of great economic importance in Brazilian aquaculture, could help a better understanding of the blood features in natural populations of this Amazon species. Blood parameters varied between Bryconinae species investigated, mainly the red blood cell counts, hemoglobin, hematocrit and mean corpuscular volume (MCV). The presence of the blood granulocytes, neutrophils and heterophils in matrinxã suggest that both leukocytes can be a characteristic for Bryconinae family. Furthermore, it indicates that the existence of special granulocytic cells in the blood of Bryconinae species from literature is an artifact, and this was herein discussed.

Forest structure of artificial islands in the Tucuruí dam reservoir in northern Brazil: a test core-area model

Construction of hydroelectric dams in tropical regions has been contributing significantly to forest fragmentation. Alterations at edges of forest fragments impact plant communities that suffer increases in tree damage and dead, and decreases in seedling recruitment. This study aimed to test the core-area model in a fragmented landscape caused by construction of a hydroelectric power plant in the Brazilian Amazon. We studied variations in forest structure between the margin and interiors of 17 islands of 8-100 hectares in the Tucuruí dam reservoir, in two plots (30 and >100m from the margin) per island. Mean tree density, basal area, seedling density and forest cover did not significantly differ between marginal and interior island plots. Also, no significant differences were found in liana density, dead tree or damage for margin and interior plots. The peculiar topographic conditions associated with the matrix habitat and shapes of the island seem to extend edge effects to the islands' centers independently of the island size, giving the interior similar physical microclimatic conditions as at the edges. We propose a protocol for assessing the ecological impacts of edge effects in fragments of natural habitat surrounded by induced (artificial) edges. The protocol involves three steps: (1) identification of focal taxa of particular conservation or management interest, (2) measurement of an "edge function" that describes the response of these taxa to induced edges, and (3) use of a "Core-Area Model" to extrapolate edge function parameters to existing or novel situations.

Identification of duplicates of cassava accessions sampled on the North Region of Brazil using microsatellite markers

Duplicates are common in germplasm banks and their identification is needed to facilitate germplasm bank management and to reduce maintenance costs. The aim of this work was to identify duplicates of cassava from a germplasm bank in Eastern Amazon, which had been previously characterized both morphological and agronomically. In order to be genotyped with 15 microsatellite loci, 36 accessions were selected. These accessions were classified into 13 groups of similar morpho-agronomical characteristics. All loci were polymorphic, and 75 alleles were identified, with an average of five alleles per loci and H E = 0.66. There were determined 34 pairs of genotypes with identical multiloci profiles and the probability of genetic identity was 1.1x10-12 with probability of exclusion of 99.9999%. Among these duplicates, there are accessions sampled on different years and places, but with different names and accessions with the same name sampled in different places and years. The study identified genotypes that are grown in different places and that have been maintained over the years by local farmers.