A high efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors

Lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) are emerging as the vectors of choice for in vitro and in vivo gene therapy studies. However, the current method for harvesting lentivectors relies upon ultracentrifugation at 50 000 g for 2 h. At this ultra-high speed, rotors currently in use generally have small volume capacity. Therefore, preparations of large volumes of high-titre vectors are time-consuming and laborious to perform. In the present study, viral vector supernatant harvests from vector-producing cells (VPCs) were pre-treated with various amounts of poly-L-lysine (PLL) and concentrated by low speed centrifugation. Optimal conditions were established when 0.005% of PLL (w/v) was added to vector supernatant harvests, followed by incubation for 30 min and centrifugation at 10 000 g for 2 h at 4 degreesC. Direct comparison with ultracentrifugation demonstrated that the new method consistently produced larger volumes (6 ml) of high-titre viral vector at 1 x 10(8) transduction unit (TU)/ml (from about 3000 ml of supernatant) in one round of concentration. Electron microscopic analysis showed that PLL/viral vector formed complexes, which probably facilitated easy precipitation at low-speed concentration (10 000 g), a speed which does not usually precipitate viral particles efficiently. Transfection of several cell lines in vitro and transduction in vivo in the liver with the lentivector/PLL complexes demonstrated efficient gene transfer without any significant signs of toxicity. These results suggest that the new method provides a convenient means for harvesting large volumes of high-titre lentivectors, facilitate gene therapy experiments in large animal or human gene therapy trials, in which large amounts of lentiviral vectors are a prerequisite.

Evaluation of liquid Bacillus thuringiensis var. israelensis products for control of Australian Aedes arbovirus vectors

Laboratory bioassay studies were conducted in southeast Queensland, Australia,: on the efficacy of Teknar (R), VectoBac (R) 12AS, and Cybate (R) (active ingredient: 1,200 international toxic units Bacillus thuringiensis var, israelensis [Bti]) against 3rd instars of the arbovirus vectors Aedes aegypti. Ae. notoscriptus, Ae. vigilax, and Ae. camptorhynchus. Probit analyses were then used to determine LD,, (median lethal dose), LD95, and lethal dose ratios (LDR). Aedes aegypti and Ae. notoscriptus, both container-habitat species, tolerated the highest Bti concentrations compared with saltmarsh Ae. vigilax and Ae. camptorhynchus. For example, the LDR for Ae. vigilax versus Ae. notoscriptus exposed to Cybate was 0.14 (95% confidence limit [CL] 0.03-0.61). Similarly, the Cybate LDR for Ae. camptorhynchus versus Ae. notoscriptus was 0.22 (95% CL 0.07-0.70). Teknar produced similar results with an LDR of 0.21 (95% CL 0.04-1.10) for Aedes vigilax versus Aedes notoscriptus. Differences in product efficacy were found when tested against the 2 container-breeding species. Cybate was less effective than Teknar with LDRs of 1.55 (95% CL 0.65-3.67) and 1.87 (95% CL 0.68-5.15) for Aedes aegypti and Ae. notoscriptus, respectively. The significant differences in susceptibility between mosquito species and varying efficacy between products highlight the importance of evaluating concentration-response data prior to contracting with distributors of mosquito control products. This information is crucial to resistance management strategies.

Malaria vectors on Buka and Bougainville islands, Papua New Guinea

Anophelines were sampled from 82 locations oil Buka and Bougainville islands in Papua New Guinea by larval collections, carbon dioxide-baited Mosquito traps, and human biting catches. Anopheles farauti s.s. was collected in larval Surveys but infrequently in mosquito traps on both islands; on Buka Island this species was readily collected in human biting catches. Anopheles faraunti 2 was commonly collected in larval surveys on both islands however. it was not collected in either mosquito traps or human biting catches. Anopheles punctulatus was found only on Buka Island, where it was commonly collected as larvae, but rarely in human biting catches and mosquito traps. Anopheles lungae was collected Lis larvae from only I site on Bougainville. Anopheles farauti s.s. led consistently throughout the night (1900-0600 h): small peaks at midnight and dawn were not statistically significant. Of 1,156 An. farauti s.s. specimens examined by enzyme-linked immunosorbent assay for malaria sporozoites. 20 were found to be positive: 12 were positive for Plasmodium falciparum and 8 were positive for P. vivax (247 variant = 5: 210 variant = 3). Anopheles farauti s.s. seems to be the major malaria vector on these islands, whereas An. punctulatus may play a minor role on Buka Island. Anophele farauti 2 is unlikely to be involved in malaria transmission on Buka or Bougainville islands.

Spatial distribution of vectors of Ross River virus and Barmah Forest virus on Russell Island, Moreton Bay, Queensland

We used a network of 20 carbon dioxide- and octenol-supplemented light traps to sample adult mosquitoes throughout Russell Island in southern Moreton Bay, south-east Queensland. Between February and April 2001, an estimated 1365 564 adult female mosquitoes were collected. In contrast to an average catch of 9754 female mosquitoes per trap night on Russell Island, reference traps set on Macleay Island and on the mainland returned average catches of 3172 and 222, respectively. On Russell Island, Ochlerotatus vigilax (Skuse), Coquillettidia linealis (Skuse), Culex annulirostris Skuse and Verrallina funerea (Theobald), known or suspected vectors of Ross River (RR) and/or Barmah Forest (BF) viruses, comprised 89.6% of the 25 taxa collected. When the spatial distributions of the above species were mapped and analysed using local spatial statistics, all were found to be present in highest numbers towards the southern end of the island during most of the 7 weeks. This indicated the presence of more suitable adult harbourage sites and/or suboptimal larval control efficacy. As immature stages and the breeding habitat of Cq. linealis are as yet undescribed, this species in particular presents a considerable impediment to proposed development scenarios. The method presented here of mapping the numbers of mosquitoes throughout a local government area allows specific areas that have high vector numbers to be defined.

Control of Aedes vectors of dengue in three provinces of Vietnam by use of Mesocyclops (Copepoda) and community-based methods validated by entomologic, clinical, and serological surveillance

We describe remarkable success in controlling dengue vectors, Aedes aegypti (L.) and Aedes albopictus (Skuse), in 6 communes with 11,675 households and 49,647 people in the northern provinces of Haiphong, Hung Yen, and Nam Dinh in Vietnam. The communes were selected for high-frequency use of large outdoor concrete tanks and wells. These were found to be the source of 49.6-98.4% of Ae. aegypti larvae, which were amenable to treatment with local Mesocyclops, mainly M. woutersi Van der Velde, M. aspericornis (Daday) and M. thermocyclopoides Harada. Knowledge, attitude, and practice surveys were performed to determine whether the communities viewed dengue and dengue hemorrhagic fever as a serious health threat; to determine their knowledge of the etiology, attitudes, and practices regarding control methods including Mesocyclops; and to determine their receptivity to various information methods. On the basis of the knowledge, attitude, and practice data, the community-based dengue control program comprised a system of local leaders, health volunteer teachers, and schoolchildren, supported by health professionals. Recycling of discards for economic gain was enhanced, where appropriate, and this, plus 37 clean-up campaigns, removed small containers unsuitable for Mesocyclops treatment. A previously successful eradication at Phan Boi village (Hung Yen province) was extended to 7 other villages forming Di Su commune (1,750 households) in the current study. Complete control was also achieved in Nghia Hiep (Hung Yen province) and in Xuan Phong (Nam Dinh province); control efficacy was greater than or equal to 99.7% in the other 3 communes (Lac Vien in Haiphong, Nghia Dong, and Xuan Kien in Nam Dinh). Although tanks and wells were the key container types of Ae. aegypti productivity, discarded materials were the source of 51% of the standing crop of Ae. albopictus. Aedes albopictus larvae were eliminated from the 3 Nam Dinh communes, and 86-98% control was achieved in the other 3 communes. Variable dengue attack rates made the clinical and serological comparison of control and untreated communes problematic, but these data indicate that clinical surveillance by itself is inadequate to monitor dengue transmission.

Kunjin virus replicon vectors for human immunodeficiency virus vaccine development

We have previously demonstrated the ability of the vaccine vectors based on replicon RNA of the Australian flavivirus Kunjin (KUN) to induce protective antiviral and anticancer CD8(+) T-cell responses using murine polyepitope as a model immunogen (I. Anraku, T. J. Harvey, R. Linedale, J. Gardner, D. Harrich, A. Suhrbier, and A. A. Khromykh, J. Virol. 76:3791-3799, 2002). Here we showed that immunization of BALB/c mice with KUN replicons encoding HIV-1 Gag antigen resulted in induction of both Gag-specific antibody and protective Gag-specific CD8(+) T-cell responses. Two immunizations with KUNgag replicons in the form of virus-like particles (VLPs) induced anti-Gag antibodies with titers of greater than or equal to1:10,000. Immunization with KUNgag replicons delivered as plasmid DNA, naked RNA, or VLPs induced potent Gag-specific CD8(+) T-cell responses, with one immunization of KUNgag VLPs inducing 4.5-fold-more CD8(+) T cells than the number induced after immunization with recombinant vaccinia virus carrying the gag gene (rVVgag). Two immunizations with KUNgag VLPs also provided significant protection against challenge with rVVgag. Importantly, KUN replicon VLP vaccinations induced long-lasting immune responses with CD8(+) T cells able to secrete gamma interferon and to mediate protection 6 to 10 months after immunization. These results illustrate the potential value of the KUN replicon vectors for human immunodeficiency virus vaccine design.

Potential role of head lice, Pediculus humanus capitis, as vectors of Rickettsia prowazekii

Since the pioneering work of Charles Nicolle in 1909 [see Gross (1996) Proc Natl Acad Sci USA 93:10539-10540] most medical officers and scientists have assumed that body lice are the sole vectors of Rickettsia prowazekii, the aetiological agent of louse-borne epidemic typhus (LBET). Here we review the evidence for the axiom that head lice are not involved in epidemics of LBET. Laboratory experiments demonstrate the ability of head lice to transmit R. prowazekii, but evidence for this in the field has not been reported. However, the assumption that head lice do not transmit R. prowazekii has meant that head lice have not been examined for R. prowazekii during epidemics of LBET. The strong association between obvious (high) infestations of body lice and LBET has contributed to this perception, but this association does not preclude head lice as vectors of R. prowazekii. Indeed, where the prevalence and intensity of body louse infections may be high (e.g. during epidemics of LBET), the prevalence and intensity of head louse infestations is generally high as well. This review of the epidemiology of head louse and body louse infestations, and of LBET, indicates that head lice are potential vectors of R. prowazekii in the field. Simple observations in the field would reveal whether or not head lice are natural vectors of this major human pathogen.

Rinossinusites em crianças infectadas pelo HIV sob terapia anti-retroviral

A associação dos inibidores de protease (IP) à terapia anti-retroviral provocou mudanças importantes na morbidade e mortalidade de pacientes infectados pelo HIV. OBJETIVOS: Avaliar o impacto desta associação na prevalência de rinossinusite (RS) e na contagem sérica de linfócitos CD4 em crianças infectadas pelo HIV. CASUÍSTICA E MÉTODOS: A forma de estudo foi cross-sectional com 471 crianças infectadas pelo HIV. Em 1996, inibidores de protease foram liberados para terapia anti-retroviral. Desta forma, dois grupos de crianças foram formados: as que não fizeram uso de IP e as que fizeram uso desta droga após 1996. A prevalência de RS e a contagem sérica de linfócitos CD4 foram comparadas entre estes grupos. RESULTADOS: 14,4% das crianças infectadas pelo HIV apresentaram RS. A RS crônica foi mais prevalente que a RS aguda em ambos os grupos. Crianças menores de 6 anos tratadas com a associação de IP apresentaram maior prevalência de RS aguda. A associação de IP esteve associada à maior contagem de linfócitos CD4 séricos com menor prevalência de RS crônica. CONCLUSÕES: A terapia com IP esteve associada ao aumento na contagem de linfócitos CD4. Crianças abaixo dos 6 anos em uso de IP apresentaram menor tendência à cronificação da doença.

Tendências da epidemia de Aids no Brasil após a terapia anti-retroviral

OBJETIVO: A terapia anti-retroviral disponível no Brasil a partir de 1996, modificou o curso da epidemia de Aids, alterando sua evolução e tendências. Nesse sentido, o estudo teve por objetivo avaliar a epidemia da Aids no Brasil, nos seus aspectos epidemiológicos. MÉTODOS: Estudo realizado a partir de bases de dados do Ministério da Saúde que caracterizavam a evolução temporal da Aids nas macrorregiões brasileiras, de 1990 a 2003. Foram utilizados modelos de regressão exponencial, ajustados à série temporal de 1990 a 1996 e estimados valores esperados para toda a série. RESULTADOS: O percentual de internações não se modificou no tempo, mas ocorreu diminuição de hospitalizações entre os usuários de terapia anti-retrovial. Houve um incremento de 2,7 vezes no número de indivíduos em uso da terapia, de 1997 a 2003. Incidência e mortalidade apresentaram crescimentos uniformes até 1995, em todas as regiões. A partir de 1996, verificou-se uma redução progressiva da mortalidade, embora a incidência continue crescendo. Em todas as regiões, exceto a Norte, as incidências esperadas foram maiores do que as observadas nos últimos anos, embora as diferenças somente tenham atingido níveis de significância estatística nas regiões Sudeste e Centro-Oeste. CONCLUSÕES: As mudanças observadas no perfil de morbi-mortalidade da epidemia de Aids no Brasil poderiam ser explicadas pelo amplo acesso a terapia anti-retroviral. Tal fato representou um impacto importante sobre a mortalidade por HIV/Aids, porém, outros fatores devem ser considerados, como idade da epidemia, medidas de prevenção, conhecimento sobre HIV/Aids e anos de escolaridade.

Tendência da transmissão vertical de Aids após terapia anti-retroviral no Brasil

OBJETIVO: O crescimento de casos de Aids entre mulheres teve como conseqüência o aumento da transmissão vertical da infecção pelo vírus da imunodeficiência adquirida. Medidas de controle dessa modalidade de transmissão foram implementadas a partir de 1996. O objetivo do estudo foi analisar a tendência temporal da transmissão vertical de Aids em crianças brasileiras. MÉTODOS: Foram incluídas no estudo crianças nascidas entre 1990 e 2001 no Brasil. Utilizou-se o banco de casos notificados como Aids em menores de 13 anos, no período de 1990 a 2004. Modelos de regressão exponencial, ajustados à série temporal, forneceram as taxas de variação anual e os valores observados e esperados para todo o período. RESULTADOS: Observou-se tendência significativamente crescente para os casos com ano de nascimento no período anterior à introdução da terapia anti-retroviral, com taxa de crescimento em torno de 12% (t<0,003) ao ano, e com diferenciais entre os Estados entre 5,9% e 31%. A análise dos casos observados e esperados, para cada uma das macrorregiões, mostrou uma redução dos casos para as crianças nascidas a partir de 1997, atingindo cifras consistentemente menores a cada ano. O número de casos notificados para crianças nascidas em 2001 representou menos de 90% dos casos esperados. CONCLUSÕES: Os resultados obtidos sugerem uma resposta favorável à implementação das políticas de intervenção na prevenção da transmissão vertical do HIV, no Brasil, como ocorreu em outras partes do mundo.

Eventos adversos relacionados à profilaxia anti-retroviral em acidentes ocupacionais

O objetivo do estudo foi descrever os eventos adversos clínicos e laboratoriais secundários ao uso dos agentes anti-retrovirais em indivíduos submetidos à quimioprofilaxia. Foram avaliados 37 funcionários de um hospital universitário submetidos à quimioprofilaxia com quatro esquemas de medicação anti-retroviral após exposição ocupacional a fluidos de pacientes contaminados com infecção pelo vírus da imunodeficiência humana. Trinta e dois (86,5%) desenvolveram eventos adversos clínicos ou laboratoriais. A profilaxia teve que ser suspensa em dois profissionais (5,4%) em virtude das reações ocorridas. Os eventos adversos relacionados à quimioprofilaxia para infecção por HIV em funcionários de saúde, vítimas de acidente ocupacional foram freqüentes. Porém, raramente foi necessário retirar a medicação anti-retroviral.

Usuários de drogas injetáveis e terapia anti-retroviral: percepções das equipes de farmácia

OBJETIVO: Compreender as percepções das equipes de farmácia sobre seu papel nos desafios assistenciais e adesão aos anti-retrovirais de usuários de drogas injetáveis vivendo com HIV/Aids. MÉTODOS: Estudo qualitativo por grupos focais e análise temática das falas produzidas com farmacêuticos, técnicos e auxiliares com experiência superior a seis meses na dispensação de medicamentos, de 15 unidades assistenciais de DST/Aids do município de São Paulo, em 2002. RESULTADOS: Formaram-se três grupos, totalizando 29 participantes, provenientes de 12 dos 15 serviços existentes, com 12 universitários e 17 profissionais de nível médio. Os grupos concluíram que a farmácia tem importante papel na dispensação de anti-retrovirais, o que se reflete na adesão ao tratamento, pois por meio de seus procedimentos podem ser construídas relações de confiança. Avaliam, porém, que tal construção não ocorra pela excessiva burocratização de suas atividades. Isso repercute negativamente para todos os pacientes, em especial sobre usuários de drogas injetáveis, concebidos como "pessoas difíceis". Tal concepção essencializa seus comportamentos: seriam confusos, com limites de compreensão e sem possibilidades de adesão ao tratamento. Os profissionais, porém, afirmam que tratam esses pacientes tal como os demais, igualando-os. Não percebem neste proceder, uma invisibilização das necessidades específicas dos usuários de drogas injetáveis no serviço, bem como a possibilidade de gerarem estereótipos estigmatizantes com barreira adicional ao trabalho da adesão. CONCLUSÕES: Embora a farmácia seja indicada como espaço potencialmente favorável para escuta e construção de vínculos com os usuários, os resultados mostram obstáculos objetivos e subjetivos para torná-la propícia para trabalhar a adesão.