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.

SPATIAL DISTRIBUTION OF PHLEBOTOMINAE IN PUERTO IGUAZU-MISIONES, ARGENTINA-BRAZIL-PARAGUAY BORDER AREA

SUMMARY The first Argentinian autochthonous human case of visceral leishmaniasis (VL) was confirmed in Posadas (Misiones) in 2006. Since then, the disease has increased its incidence and geographical distribution. In the 2006-2012 period, 107 human cases were detected (11 deaths). The presence of Lutzomyia longipalpis was detected in peridomiciles in Puerto Iguazú urban area in 2010; some of these findings were associated with households where cases of canine VL had already been reported. The objective of this study was to ascertain the abundance and spatial distribution of Lu. longipalpis in Puerto Iguazú City, on the Argentina-Brazil-Paraguay border. Lu. longipalpis proved to be exclusively urban and was found in 31% of the households sampled (n = 53), 67% of which belonged to areas of low abundance, 20% to areas of moderate abundance and 13% to areas of high abundance. Nyssomyia whitmani was the only species found both in urban and peri-urban environments, and Migonemyia migonei was registered only on the outskirts of the city. Due to the fact that Puerto Iguazú is considered to be at moderate risk at the moment, it is necessary to intensify human and canine case controls, as well as take integrated prevention and control measures regarding the environment, vectors and reservoirs on the Argentina-Brazil-Paraguay border area.

AN UPWARD TREND IN DNA P16INK4A METHYLATION PATTERN AND HIGH RISK HPV INFECTION ACCORDING TO THE SEVERITY OF THE CERVICAL LESION

SUMMARY High-risk human papillomavirus (hr-HPV) infection is necessary but not sufficient for cervical cancer development. Recently, P16INK4A gene silencing through hypermethylation has been proposed as an important cofactor in cervical carcinogenesis due to its tumor suppressor function. We aimed to investigate P16INK4A methylation status in normal and neoplastic epithelia and evaluate an association with HPV infection and genotype. This cross-sectional study was performed with 141 cervical samples from patients attending Hospital Moncorvo Filho, Rio de Janeiro. HPV detection and genotyping were performed through PCR and P16INK4A methylation by nested-methylation specific PCR (MSP). HPV frequency was 62.4% (88/141). The most common HPV were HPV16 (37%), HPV18 (16.3%) and HPV33/45(15.2%). An upward trend was observed concerning P16INK4A methylation and lesion degree: normal epithelia (10.7%), low grade lesions (22.9%), high grade (57.1%) and carcinoma (93.1%) (p < 0.0001). A multivariate analysis was performed to evaluate an association between methylation, age, tobacco exposure, HPV infection and genotyping. A correlation was found concerning methylation with HPV infection (p < 0.0001), hr-HPV (p = 0.01), HSIL (p < 0.0007) and malignant lesions (p < 0.0001). Since viral infection and epigenetic alterations are related to cervical carcinoma, we suggest that P16INK4A methylation profile maybe thoroughly investigated as a biomarker to identify patients at risk of cancer.

MOLECULAR TYPING OF Candida albicans ISOLATES FROM HOSPITALIZED PATIENTS

SUMMARYIntroduction: The majority of nosocomial fungal infections are caused by Candida spp. where C. albicans is the species most commonly identified. Molecular methods are important tools for assessing the origin of the yeasts isolated in hospitals.Methods: This is a study on the genetic profifiles of 39 nosocomial clinical isolates of C. albicans using two typing methods: random amplifified polymorphic DNA (RAPD) and microsatellite, two different primers for each technique were used.Results: RAPD provided 10 and 11 different profiles with values for SAB of 0.84 ± 0.126 and 0.88 ± 0.08 for primers M2 and P4, respectively. Microsatellite using two markers, CDC3 and HIS3, allowed the observation of six and seven different alleles, respectively, with combined discriminatory power of 0.91.Conclusions: Although genetic variability is clear, it was possible to identify high similarity, suggesting a common origin for at least a part of isolates. It is important to emphasize that common origin was proven from yeasts isolated from colonization (urine, catheter or endotracheal secretions) and blood culture from the same patient, indicating that the candidemia must have started from a site of colonization. The combination of RAPD and microsatellite provides a quick and efficient analysis for investigation of similarity among nosocomial isolates of C. albicans.

Presence of anti-Toxoplasma antibodies in humans and their cats in the urban zone of Guadalajara

Cats are the definitive hosts of Toxoplasma gondii. Infected cats excrete oocysts in their feces, infecting humans and other animals. The objective of the present study was to determine the presence of anti-Toxoplasma antibodies in cat owners and their pets, and determine if there was a relationship between Toxoplasma infection and humans who live with infected cats. IgG anti-Toxoplasma antibodies in sera of 59 cat owners were determined by enzyme-linked immunosorbent assay (ELISA), in 24 sera from their cats, IgG, IgM, and IgA antibodies were found using Burney's ELISA. Thirty-eight (64%) of 59 cat owners were positive to IgG anti-Toxoplasma. Seropositivity for cats was 70.8% IgG, 8.3% IgM, and 62.5% IgA. Cohabitation with cats infected by T. gondii, feeding with leftovers or raw viscera, and lack of control over how their feces were handled are risk factors conducive for humans to become infected by T. gondii.

Enfermedad de Chagas-Mazza congenita en Salta

Se estudió la infección por T. cruzi en mujeres embarazadas en la localidad de General Güemes, provincia de Salta. La misma fue del 12,3 %. El 8,8% de los recién nacidos estudiados tuvieron diagnóstico de Enfermedad de Chagas utilizando la técnica directa (microhematocrito). Todos fueron tratados con benznidazol a razón de 5mg/kg/día durante 30 dias. Todos presentaron anemia, que fue interpretada como reacción adversa medicamentosa. Se estima que la técnica directa representa la mejor opción para llevar a cabo el diagnóstico de esta enfermedad en el recién nacido. Se ha propuesto un flujograma para el seguimiento de la infección por T. cruzi en el recién nacido.

Gastrópodes límnicos do Campus de Manguinhos, Fundação Oswaldo Cruz, Rio de Janeiro, RJ

Um levantamento da malacofauna límnina do Campus de Manguinhos, Fundação Oswaldo Cruz, Rio de Janeiro, foi realizado nos últimos dois anos visando comparar as espécies hoje existentes com aquelas encontradas no início deste século. Foram pesquisadas 18 coleções hídricas numa extensão de 880.000m², sendo encontradas em 13 delas as seguintes espécies: Antillorbis nordestensis, Biomphalaria glabrata, Biomphalaria straminea, Lymnaea columella, Melanoides tuberculatus, Physa cubensis, Pomacea glauca e Pomacea lineata. Destacam-se o desaparecimento de Biomphalaria tenagophila, registrada por Lutz em 1918, a introdução de B. straminea e da espécie asiática M. tuberculatus. Nenhum molusco apresentou infecção por Schistosoma mansoni.

New evidence of spontaneous cure in human Chagas' disease

A new case of spontaneous cure of human Chagas' disease is described in Uruguay. An 87-year-old man who had a typical acute phase of Trypanosoma cruzi infection in 1947 and never received specific treatment against the disease, when examined in 1998 revealed several completely negative parasitological and serological tests, including traditional serology, PCR and flow cytometry. As a whole, such findings fulfill the current criteria to define the cure of Chagas' disease. Clinical data suggest the possibility of a benign evolution of Chagas' disease in this case, but the basic findings (slight cardiac and esophageal impairment) could also be due to the advanced age of the patient.

Recommendations for the detection of Leptospira in urine by PCR

In the present study PCR was applied to detect leptospires in human urine. Several approaches for sample processing were evaluated to optimize the detection of leptospires in urine mixed with this bacterium. Furthermore, some changes in the composition of the reaction mix were studied. No amplification was observed in acidic urine, therefore neutralization of the sample immediately after collection is strongly recommended. PBS gave better results than Tris or NaOH as neutralizing reagents. Freezing and thawing of samples before processing yielded negative results. Elimination of epithelial cells, leukocytes and crystals by centrifugation at 3,000 rpm at room temperature increased sensitivity. In addition, both the washing step after collecting leptospires by centrifugation and the inclusion of 0.1% bovine serum albumin in the reaction mix minimized the interference of other inhibitory compounds. These modifications were useful to improve the detection of Leptospira in urine by PCR.

Hydatidosis cases in one of Mar del Plata City hospitals, Buenos Aires, Argentina

Hydatidosis is a zoonosis of worldwide distribution produced mainly by the metacestode Echinococcus granulosus. In Argentina, its distribution reaches endemic levels. The aims of this investigation were to contribute to the knowledge of hydatidosis in the southeast of Buenos Aires province, study its evolution at the Interzonal General Hospital for Acute Diseases between 1992 and 2002 and discuss its importance. Clinical records of operated and/or diagnosed patients were reviewed with regard to this disease. One hundred and twenty cases were analyzed. Among the patients, 56.7% were women. The average age was 42.2+16.8 years. 68.3% lived in urban areas. In 75% of the cases, ultrasonography was used. Hepatic location was most frequently seen. 89.2% received surgical treatment. Albendazole was used for 19 patients. The mean length of hospital stay was 16 days. We conclude that this zoonosis is a disease that generates high costs in medical care and for this reason more epidemiological studies should be carried out and public health authorities should implement control and prevention strategies in the region.

New focus of active transmission of Chagas disease in indigenous populations in the Peruvian Amazon basin

Introduction Several cases of acute Chagas disease (ACD) have been reported in the Peruvian Amazon basin. Methods The objective was to describe and investigate 6 ACD cases in children from indigenous Amazon communities in the province of Datem del Marañón in Loreto department (2006-2010). Results The mean age was 3.6 years. All patients had fever, 4/6 hepatomegaly, 2/6 splenomegaly, and 5/6 had trypomastigotes of Trypanosoma cruzi on thick smears. The fatality rate was 33.3%. Rhodnius pictipes and Rhodnius robustus adults were found inside the homes and in the peri-domiciles. Conclusions All cases reported were isolated cases. We report a new focus of ACD in indigenous populations.

Freshwater gastropods of the Baixada Maranhense Microregion, an endemic area for schistosomiasis in the State of Maranhão, Brazil: I - qualitative study

Introduction: The Baixada Maranhense Microregion currently has the highest prevalence of schistosomiasis in the State of Maranhão, likely because this parasitosis is characterized as an occupational disease, and increased contact with water increases the risk of infection by Schistosoma mansoni. This paper reports the results of the first comprehensive freshwater malacological survey performed in the Baixada Maranhense Microregion. Methods: Freshwater mollusks were collected from the twenty-one municipalities of the Baixada Maranhense Microregion and from Bacurituba and Cajapió and were evaluated for infection by trematodes. Results: A total of 9,129 mollusks were collected (sixteen species), which included the first records of six species in the State of Maranhão: Gundlachia radiata, G. ticaga, Hebetancylus moricandi, Plesiophysa guadeloupensis, Pomacea bridgesii diffusa and Omalonyx sp. Biomphalaria glabrata was found in five municipalities, whereas B. straminea was found in nine. Biomphalaria glabrata and B. straminea were observed in syntopy in Pinheiro and São Bento. Of the 990 specimens of B. glabrata and the 2,109 specimens of B. straminea that were exposed to and/or analyzed for the presence of larval trematodes, only a single specimen of B. glabrata (0.1%) from São Bento shed S. mansoni. Other larval trematodes were first observed in mollusks from the State of Maranhão. Conclusions: These results indicate that the study area is epidemiologically important due to the presence of two natural vectors of schistosomiasis and the active transmission of schistosomiasis, which was confirmed in the infected specimen that was collected in this study.

A malacological survey in the Manso Power Plant, State of Mato Grosso, Brazil: new records of freshwater snails, including transmitters of schistosomiasis and exotic species

Introduction Schistosomiasis is a parasitic disease of public health concern in Brazil, and the construction of hydroelectric dams, in addition to increasing permanent human settlement and tourism, has created conditions suitable for the establishment of mollusks that can transmit schistosomiasis. Such areas require a number of actions to prevent the establishment of schistosomiasis. This paper reports on a freshwater malacological survey carried out in the geographical area of the Manso Power Plant. Methods Mollusks were collected in 18 municipalities in the State of Mato Grosso between February 2002 and February 2004 (qualitative study) and from April 2009 to February 2011 (quantitative study). Results Thirty-one species of mollusks were collected, including newly recorded species (Antillorbis nordestensis and Burnupia ingae). In addition, the geographic distributions of known species, including Biomphalaria straminea, a snail vector of Schistosoma mansoni, were expanded. A total of 4,507 specimens were collected in the APM Manso reservoir (Usina Hidrelétrica de Aproveitamento Múltiplo de Manso) during the quantitative study, and Biomphalaria amazonica was found in six of the 10 localities analyzed. The Afroasiatic species Melanoides tuberculata, introduced after February 2009, was the dominant species (relative abundance 94.96%). Conclusions The study area is epidemiologically important due to the occurrence of B. straminea and B. amazonica, which are vectors of schistosomiasis, and M. tuberculata, a snail host of Centrocestus formosanus, which is responsible for centrocestiasis transmission. Observations of M. tuberculata and the exotic freshwater clams Corbicula fluminea and Corbicula largillierti raise concerns about biodiversity.

Assessment of schistosomiasis in the semi-arid Northeast region of Brazil: the São Francisco River large-scale water transposition project

Abstract: INTRODUCTION Risk of schistosomiasis expansion to semi-arid northeastern Brazil under the influence of the Integration Project of the São Francisco River (IPSFR) was assessed. METHODS: Stool examinations of schoolchildren, epidemiological investigation, and survey of the local host snail Biomphalaria straminea were performed in five IPSFR municipalities. RESULTS Six of 4,770 examined schoolchildren were egg-positive for Schistosoma mansoni. Biomphalaria straminea was widespread, but not naturally infected with S. mansoni. Snails experimentally exposed to two laboratory S. mansoni strains yielded infection indices of 1-4.5%. CONCLUSIONS: There is evidence of active schistosomiasis transmission in the area; thus, intensive surveillance actions are required.