Drug resistance in the sexually transmitted protozoan Trichomonas vaginalis

Trichomoniasis is the most common, sexually transmitted infection. It is caused by the flagellated protozoan parasite Trichomonas vaginalis. Symptoms include vaginitis and infections have been associated with preterm delivery, low birth weight and increased infant mortality, as well as predisposing to HIV/AIDS and cervical cancer. Trichomoniasis has the highest prevalence and incidence of any sexually transmitted infection. The 5-nitroimidazole drugs, of which metronidazole is the most prescribed, are the only approved, effective drugs to treat trichomoniasis. Resistance against metronidazole is frequently reported and cross-resistance among the family of 5-nitroimidazole drugs is common, leaving no alternative for treatment, with some cases remaining unresolved. The mechanism of metronidazole resistance in T. vaginalis from treatment failures is not well understood, unlike resistance which is developed in the laboratory under increasing metronidazole pressure. In the latter situation, hydrogenosomal function which is involved in activation of the prodrug, metronidazole, is down-regulated. Reversion to sensitivity is incomplete after removal of drug pressure in the highly resistant parasites while clinically resistant strains, so far analysed, maintain their resistance levels in the absence of drug pressure. Although anaerobic resistance has been regarded as a laboratory induced phenomenon, it clearly has been demonstrated in clinical isolates. Pursuit of both approaches will allow dissection of the underlying mechanisms. Many alternative drugs and treatments have been tested in vivo in cases of refractory trichomoniasis, as well as in vitro with some successes including the broad spectrum anti-parasitic drug nitazoxanide. Drug resistance incidence in T. vaginalis appears to be on the increase and improved surveillance of treatment failures is urged.

Cross-protective and Infection-Enhancing Immunity in Mice Vaccinated Against Flaviviruses Belonging to the Japanese Encephalitis Virus Serocomplex

The Japanese encephalitis virus serocomplex is a group of mosquito-borne flaviviruses that cause severe encephalitic disease in humans. The recent emergence of several members of this serocomplex in geographic regions where other closely related flaviviruses are endemic has raised urgent human health issues. Thus, the impact of vaccination against one of these neurotropic virus on the outcome of infection with a second, serologically related virus is unknown. We show here that immunity against Murray Valley encephalitis virus in vaccinated mice can cross-protect but also augment disease severity following challenge with Japanese encephalitis virus. Immunepotentiation of heterologous flavivirus disease was apparent in animals immunized with a 'killed' virus preparation when humoral antiviral immunty of low magnitude was elicited. (C) 2002 Elsevier Science Ltd. All rights reserved.

Synthesis, stability, antiviral activity, and protease-bound structures of substrate-mimicking constrained macrocyclic inhibitors of HIV-1 protease

Three new peptidomimetics (1-3) have been developed with highly stable and conformationally constrained macrocyclic components that replace tripeptide segments of protease substrates. Each compound inhibits both HIV-1 protease and viral replication (HIV-I, HIV-2) at nanomolar concentrations without cytotoxicity to uninfected cells below 10 mu M. Their activities against HIV-1 protease (K-i 1.7 nM (1), 0.6 nM (2), 0.3 nM (3)) are 1-2 orders of magnitude greater than their antiviral potencies against HIV-1-infected primary peripheral blood mononuclear cells (IC50 45 nM (1), 56 nM (2), 95 nM (3)) or HIV-1-infected MT2 cells (IC50 90 nM (1), 60 nM (2)), suggesting suboptimal cellular uptake. However their antiviral potencies are similar to those of indinavir and amprenavir under identical conditions. There were significant differences in their capacities to inhibit the replication of HIV-1 and HIV-2 in infected MT2 cells, 1 being ineffective against HIV-2 while 2 was equally effective against both virus types. Evidence is presented that 1 and 2 inhibit cleavage of the HIV-1 structural protein precursor Pr55(gag) to p24 in virions derived from chronically infected cells, consistent with inhibition of the viral protease in cells. Crystal structures refined to 1.75 Angstrom (1) and 1.85 Angstrom (2) for two of the macrocyclic inhibitors bound to HIV-1 protease establish structural mimicry of the tripeptides that the cycles were designed to imitate. Structural comparisons between protease-bound macrocyclic inhibitors, VX478 (amprenavir), and L-735,524 (indinavir) show that their common acyclic components share the same space in the active site of the enzyme and make identical interactions with enzyme residues. This substrate-mimicking minimalist approach to drug design could have benefits in the context of viral resistance, since mutations which induce inhibitor resistance may also be those which prevent substrate processing.

Lipid rafts and caveolae as portals for endocytosis: New insights and common mechanisms

Clathrin-coated pits and caveolae are two of the most recognizable features of the plasma membrane of mammalian cells. While our understanding of the machinery regulating and driving clathrin-coated pit-mediated endocytosis has progressed dramatically, including the elucidation of the structure of individual components and partial in vitro reconstitution, the role of caveolae as alternative endocytic carriers still remains elusive 50 years after their discovery. However, recent work has started to provide new insights into endocytosis by caveolae and into apparently related pathways involving lipid raft domains. These pathways, distinguished by their exquisite sensitivity to cholesterol-sequestering agents, can involve caveolae but also exist in cells devoid of caveolins and caveolae. This review examines the current evidence for the involvement of rafts and caveolae in endocytosis and the molecular players involved in their regulation.

Detection of West Nile Virus Infection in birds in the United States by blocking ELISA and immunohistochemistry

A blocking ELISA targeting an immunodominant West Nile epitope on the West Nile Virus NS1 protein was assessed for the detection of West Nile-specific antibodies in blood samples collected from 584 sentinel chickens and 238 wild birds collected in-New Jersey from May-December 2000. Ten mallard ducks (Anas platyrhynchos) experimentally infected with West Nile virus and six uninfected controls were also tested. The ELISA proved specific in detecting WNV antibodies in 9/10 chickens and 4/4 wild birds previously confirmed as positive by Plaque Reduction Neutralization test (PRNT) at the Center for Disease Control, Division of Vector Borne Diseases, Fort Collins, CO, USA (CDC). Nine out of the ten experimentally infected mallard ducks also tested positive for WN antibodies in the blocking ELISA, while 6/6 uninfected controls did not. Additionally, 1705 wild birds, collected in New Jersey from December 2000-November 2001 and Long Island, New York between November 1999 and August 2001 were also tested for WN antibodies by the blocking ELISA. These tests identified 30 positive specimens, 12 of which had formalin-fixed tissues available to allow detection of WN specific viral antigen in various tissues by WNV-specific immunohistochemistry. Our results indicate that rapid and specific detection of antibodies to WN virus in sera from a range of avian species by blocking ELISA is an effective strategy for WN Virus surveillance in avian hosts. In combination with detection of WN-specific antigens in tissues by immunohistochemistry (IHC) the blocking ELISA will also be useful for confirming WN infection in diseased birds.

Caracterização molecular de papilomavírus humano (HPV) e vírus adeno-associado (AAV) em lesões intraepiteliais de colo uterino: um estudo de seguimento

O câncer de colo uterino (CCU), cujo agente etiológico é o papilomavírus humano (HPV), é um dos tipos de câncer mais frequentes em mulheres em todo o mundo, não só em incidência como também em mortalidade. Alguns genótipos de HPV, denominados de alto risco (HR-HPV), e suas variantes gênicas, estão mais associados à indução de lesões malignas, sendo HPV16 e 18 os mais frequentes. Algumas infecções do trato genital podem atuar como cofatores da progressão carcinogênica do CCU, porém a infecção por vírus adeno-associado (AAV) parece estar inversamente relacionada, o que pode refletir em um papel protetor no desenvolvimento do CCU induzido pelo HPV. Portanto, este estudo objetivou investigar o papel da infecção mista AAV-HPV e das variantes oncogênicas de HPV na progressão das lesões intraepiteliais de colo de útero e acompanhar a eliminação / persistência viral em relação à progressão / regressão das lesões cervicouterinas. Exames citológicos foram realizados em amostras de espécime cervical, coletadas em dois momentos, de mulheres atendidas no Hospital Universitário Cassiano Antonio Moraes – HUCAM e seguiram para tratamento conforme preconizado. DNA foi extraído pelo kit comercial QIAamp® DNA Mini Kit, seguindo instruções do fabricante. DNA de AAV foi investigado por PCR e nPCR e, de HPV, por PCR e Captura Híbrida® (CH). Genotipagem de AAV e HPV foram realizadas por RFLP e RLB, respectivamente. Dos casos encaminhados ao ambulatório de colposcopia, 57,3% tiveram citologia normal, 23,1% lesões de baixo grau e 19,6% lesões de alto grau. Dos casos com citologia normal, 78% permaneceram normais, enquanto 22% progrediram à lesão; dos casos com lesão de baixo grau, 74% regrediram para citologia normal, enquanto 78,6% dos casos com lesão de alto grau apresentaram lesão de baixo grau ou citologia normal na segunda coleta. Foram positivas para HPV, 56% e 36,5% das amostras da primeira e segunda coletas, respectivamente. Foi observada boa correlação (kappa= 0,66) entre os testes de PCR e CH para detecção de HPV. Os HR-HPV foram detectados em mais de 90% das amostras de ambas as coletas, sendo os mais frequentes os HPV16, 58, 51, 52 e 53. Variante não-europeia esteve associada ao desenvolvimento de lesão cervical de alto grau, enquanto a presença de AAV foi inversamente relacionada à progressão da lesão cervical induzida por HPV.

Molybdenum mixed with glyphosate and alone via foliar spray in no-tillage common bean grown on corn stover

The effect of molybdenum (Mo) on common bean grown in desiccated corn stover in a no-tillage system was evaluated under two application modes: Mo mixed with the desiccant glyphosate and Mo direct spray to the bean leaves. The treatments (four replicates) were assigned to a completely randomized block design in a split-plot arrangement with the application of Mo (0, 100, 200, 400 and 800 g ha-1) mixed with glyphosate in the main plots and Mo foliar spray (0 and 100 g ha-1) in the sub-plots. The field experiments were carried out in 2009 and 2010 in the municipality of Coimbra, Minas Gerais State, with the common bean cultivar Ouro Vermelho. Mo mixed with glyphosate had neither an effect on common bean yield nor on the Mo and N contents in leaves, however it increased the Mo and N contents in seeds. Application of Mo via foliar spray increased Mo content in leaves and Mo and N contents in seeds. The reapplication of molybdenum with glyphosate for desiccation in subsequent crops caused a cumulative effect of Mo content in bean seeds.

Selection of common bean lines with high grain yield and high grain calcium and iron concentrations

Genetic improvement of common bean nutritional quality has advantages in marketing and can contribute to society as a food source. The objective of this study was to evaluate the genetic variability for grain yield, calcium and iron concentrations in grains of inbred common bean lines obtained by different breeding methods. For this, 136 F7 inbred lines were obtained using the Pedigree method and 136 F7 inbred lines were obtained using the Single-Seed Descent (SSD) method. The lines showed genetic variability for grain yield, and concentrations of calcium and iron independently of the method of advancing segregating populations. The Pedigree method allows obtaining a greater number of lines with high grain yield. Selection using the SSD method allows the identification of a larger number of lines with high concentrations of calcium and iron in grains. Weak negative correlations were found between grain yield and calcium concentration (r = -0.0994) and grain yield and iron concentration (r = -0.3926). Several lines show genetic superiority for grain yield and concentrations of calcium and iron in grains and their selection can result in new common bean cultivars with high nutritional quality.

Selection of anthracnose resistant common beans using detached leaves in partially controlled environment

The objectives of this study were to evaluate the possibility of selecting anthracnose resistant common bean plants using detached primary leaves in partially controlled environment of a greenhouse and identify differences in the reaction of genotypes to anthracnose. The common bean cultivars Ouro Negro, OuroVermelho, ManteigãoFosco 11, Rudá, Rudá-R, VP8, BRSMG Madrepérola, Pérola, MeiaNoite and BRSMG Talismãwere characterizedfor resistance to the races 65, 81 and 453 of Colletotrichum lindemuthianum and the method of detached primary leaves was compared to the method with the traditional inoculation of plants at the phenological stage V2. The lines Rudá, Rudá-R and Pérola were inoculated with the races 65 and 453 of C. lindemuthianum, aiming to assess the rate of coincidence of anthracnose severity by both inoculation methods. In general, the two methods presented similar results for the reaction of the cultivars. The use of detached primary leaves of common bean plants in the partially controlled environment was feasible for selection of plants resistant to anthracnose and has the advantages of low-needed infrastructure and reduction of resources, space and time.

Agronomic performance of common bean in straw mulch systems and topdressing nitrogen rates in no-tillage

ABSTRACTIn no-tillage systems, straw coverage on soil surface is the key to success, and the choice of crops for rotation is crucial to achieve the sustainability and quality that conservation agriculture requires. The objective of this study was to evaluate the agronomic performance of the common bean cultivar IAC Formoso sown in succession to three straw mulch systems (corn alone, corn/Urochloa ruziziensisintercrop and U. ruziziensisalone) and topdress nitrogen rates (0; 40; 80; 120 and 160 kg ha-1N), at the four-leaf stage, three years after the implementation of no-tillage. The experiment was arranged in a randomized block split plot design, with three replications. Common bean highest yields were achieved in succession to U. ruziziensisalone and intercropped with corn. The corn/U. ruziziensisintercrop provided both straw and seed production, allowing for quality no-tillage. Topdressed nitrogen influenced the common bean yield when in succession to corn alone, U. ruziziensisalone and corn/U. ruziziensisintercrop in no-tillage.

Co-inoculation of Rhizobium tropici and Azospirillum brasilense in common beans grown under two irrigation depths

ABSTRACT The alternative technique of co-inoculation or mixed inoculation with symbiotic and non-symbiotic bacteria has been studied in leguminous plants. However, there are few field studies with common beans and under the influence of the amount of irrigated water. Thus, the objective of this study was to evaluate the efficiency of inoculation and co-inoculation of common beans with Rhizobium tropici and Azospirillum brasilense under two irrigation depths. The experiment was carried out in the winter of 2012 and 2013, in Selvíria, state of Mato Grosso do Sul. The experimental design was composed of randomized blocks in split-plot scheme with two irrigation depths in the plots (recommended for common beans and 75% of the recommended) and five forms of nitrogen (N) supply in the split-plots (control non-inoculated with 40 kg ha- 1 of N in topdressing, 80 kg ha- 1 of N in topdressing, A. brasilense inoculation with 40 kg ha-1 of N in topdressing, R. tropici inoculation with 40 kg ha-1 of N in topdressing, and co-inoculation of A. brasilense and R. tropici with 40 kg ha- 1 of N in topdressing) with four repetitions. Co-inoculation increased nodulation in the second year of cultivation. None of the evaluated treatments increased the grain yield in relation to non-inoculated control with 40 kg ha-1 of nitrogen in topdressing, which presented average yield of 2,200 kg ha-1. The use of 75% of the recommended irrigation depth provides similar grain yield to the recommended irrigation depth in common beans cropped in winter.

Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease: 3 - On the interaction of vector species and parasite strain in the reaction of bugs to infection by Trypanosoma cruzi

The reaction of nine vector species of Chagas' disease to infection by seven different Trypanosoma cruzi strains; Berenice, Y, FL, CL, S. Felipe, Colombiana and Gávea, are examined and compared. On the basis of the insects' ability to establish and maintain the infection, vector species could be divided into two distinct groups which differ in their reaction to an acute infection by T. cruzi. While the proportion of positive bugs was found to be low in Triatoma infestans and Triatoma dimidiata it was high, ranging from 96.9% to 100% in the group of wild (Rhodnius neglectus, Triatoma rubrovaria)and essentially sylvatic vectors in process of adaptation to human dwellings, maintained under control following successful insecticidal elimination of Triatoma infestans (Panstrongylus megistus, Triatoma sordida and Triatoma pseudomaculata). An intermediate position is held by Triatoma brasiliensis and Rhodnius prolixus. This latter has been found to interchange between domestic and sylvatic environments. The most important finding is the strikingly good reaction between each species of the sylvatic bugs and practically all T. cruzi strains herein studied, thus indicating that the factors responsible for the excellent reaction of P.megistus to infection by Y strain, as previously reported also come into operation in the reaction of the same vector species to acute infections by five of the remaining T.cruzi strains. Comparison or data reported by other investigators with those herein described form the basis of the discussion of Dipetalogaster maximus as regards its superiority as a xenodiagnostic agent.

Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease: 4 - The reflection of parasite stock in the responsiveness of different vector species to chronic infection with different Trypanosoma cruzi stocks

Previous studies (1982,1987) have emphasized the superiority of sylvatic vector species over domestic species as xenodiagnostic agents in testing hosts with acute or chronic infections by T. cruzi "Y" stock. The present study, which is unique in that it contains data on both infectivity rates produced by the same stock in 11 different vector species and also the reaction of the same vector species to seven different parasite stocks, establishes the general validity of linking efficiency of xenodiagnosis to the biotope of its agent. For example, infectivity rates produced by "São Felipe" stock varied from 82.5% to 98.3% in sylvatic vectors but decreased to 42.5% to 71.3% in domestic species. "Colombiana" stock produced in the same sylvatic vectors infectivity rates ranging from 12.5% to 45%. These shrank to 5%-22.5% in domestic bugs. The functional role of the biotope in the vector-parasite interaction has not been eluddated. But since this phenomenon has been observed to be stable and easy to reproduce, it leads us to believe that the results obtained are valid. Data presented also provide increasing evidence that the infectivity rates exhibited by bugs from xenodiagnosis in chronic hosts, are parasite stock specific. For example, infectivity rates produced by "Berenice", "Y", "FL" and "CL" varied in R. neglectus from 26.3% to 75%; in P. megistus from 56.3% to 83.8%; in T. sordida from 28.8% to 58.8% in T. pseudomaculata from 41.3% to 66.3% and in T. rubrovaria from 48.8% to 85%. Data from xenodiagnosis in the same hosts, carrying acute infections by the same parasite stocks, gave the five sylvatic vectors a positive rating of approximately 100%, thus suggesting that the heavy loads of parasites circulating in the acute hosts obscured the characteristic interspecific differences for the parasite stock. Nonetheless these latter were revealed in the same hosts with chronic infections stimulated by very low numbers of the same parasite stocks. Certain observations here described lead us to speculate as to the possibility of further results from other parasite stocks, allowing the association of the infectivity rates produced in bugs by different parasite stocks with the isoenzymic patterns revealed by these stocks.