41 research outputs found
Transmission phenotype of mycobacterium tuberculosis strains is mechanistically linked to induction of distinct pulmonary pathology
In a study of household contacts (HHC), households were categorized into High (HT) and Low (LT) transmission groups based on the proportion of HHC with a positive tuberculin skin test. The Mycobacterium tuberculosis (Mtb) strains from HT and LT index cases of the households were designated Mtb-HT and Mtb-LT, respectively. We found that C3HeB/FeJ mice infected with Mtb-LT strains exhibited significantly higher bacterial burden compared to Mtb-HT strains and also developed diffused inflammatory lung pathology. In stark contrast, a significant number of mice infected with Mtb-HT strains developed caseating granulomas, a lesion type with high potential to cavitate. None of the Mtb-HT infected animals developed diffused inflammatory lung pathology. A link was observed between increased in vitro replication of Mtb-LT strains and their ability to induce significantly high lipid droplet formation in macrophages. These results support that distinct early interactions of Mtb-HT and Mtb-LT strains with macrophages and subsequent differential trajectories in pathological disease may be the mechanism underlying their transmission potential.publishersversionpublishe
Mycobacterium tuberculosis progresses through two phases of latent infection in humans
Little is known about the physiology of latent Mycobacterium tuberculosis infection. We studied the mutational rates of 24 index tuberculosis (TB) cases and their latently infected household contacts who developed active TB up to 5.25 years later, as an indication of bacterial physiological state and possible generation times during latent TB infection in humans. Here we report that the rate of new mutations in the M. tuberculosis genome decline dramatically after two years of latent infection (two-sided p < 0.001, assuming an 18 h generation time equal to log phase M. tuberculosis, with latency period modeled as a continuous variable). Alternatively, assuming a fixed mutation rate, the generation time increases over the latency duration. Mutations indicative of oxidative stress do not increase with increasing latency duration suggesting a lack of host or bacterial derived mutational stress. These results suggest that M. tuberculosis enters a quiescent state during latency, decreasing the risk for mutational drug resistance and increasing generation time, but potentially increasing bacterial tolerance to drugs that target actively growing bacteria.publishersversionpublishe
Estudo sobre caracterÃsticas genéticas de Mycobacterium tuberculosis isolados de pacientes com e sem lesões cavitárias
Background: Based on the hypothesis that genetic variability of Mycobacterium tuberculosis (MTB) could influence virulence and immunopathology we analyzed genetic profiles of different MTB strains in order to detect relatedness between genetic diversity and presence of cavity (disease severity). Methods: We conducted a retrospective molecular study in Vitória ES, based on TB strains (2003 to 2006, n = 214) from patients with pulmonary cavitary and non-cavitary TB using IS6110-RFLP, Spoligotyping and MIRU-VNTR methodologies. RESULTS:
Initially, we compared the association of the demographic and clinical characteristics of patients with the presence of cavities. After logistic regression the variables that most contributed to explain the model of the disease were smear
positive (ORajust = 5.96; IC= 2.58-13.73) and sputum production (ORajust = 4.55; IC= 1.28-16.12), there was no statistically significant association with the remaining
variables. The LAM family was the most frequent within the samples of the two groups analyzed, representing 65 (62%) of the isolates in the cavitary group and 40 isolates (38%) of the non-cavitary. After comparing the proportions of LAM and
other spoligotyping families there was no statistically significant difference between the groups (p=0.17). In relation to deletions RDRio (p=0.65) and RD174 (p=0.65) there were no statistically significant difference between the groups. Amongst the 205 isolates analyzed, 25 (12%) belonging to the non-cavitary group and 43 (21%) belonging to the cavitary group, were grouped in clusters. The statistical analysis of
the association of the occurence of clusters with the presence of cavity showed no statistically significant difference between the quantity of clusters and the groups that were analyzed, (p= 0.4). Conclusion: The genotipic profile for the isolates from patients with cavitary and non-cavitary disease was determined. Our data showed that LAM9 was the most frequent among the strains between cavitary and noncavitary
groups, corroborating findings that this family is the most frequent in Brasil. There were no statistical differences that could show association among the variables analyzed related to presence of cavity or disease severityIntrodução: Baseado na hipótese de que a variabilidade genética de Mycobacterium tuberculosis (MTB) pode influenciar a virulência e a gravidade da doença os perfis genéticos de isolados clÃnicos de MTB foram avaliados para
detectar associação entre diversidade genética e gravidade da doença. Objetivos: Analisar caracterÃsticas genéticas de isolados de MTB e verificar sua possÃvel associação com a gravidade da TB pulmonar. Métodos: Estudo retrospectivo, caso controle, conduzido em Vitória-ES, utilizando isolados de MTB (2003 a 2006, n=214) de pacientes com TB pulmonar, cavitária (127) e não cavitária (87). Realizou-se genotipagem por meio de RFLP-IS6110, Spoligotyping, MIRU-VNTR 24 loci, e a análise de deleções e inserções, como RDRio, RD174 utilizando PCR multiplex, bem como a detecção do Ag85C103. Realizou-se análise estatÃstica, para verificação dos padrões de distribuição das variáveis, seguida de análises bivariadas para verificação de associações entre elas, empregando-se os teste exato de Fisher ou Chi-quadrado, ambos com 95% de intervalo de confiança e nÃvel de significância () < 0,05. Resultados: Após a regressão logÃstica, as variáveis que contribuÃram no modelo explicativo da doença foram baciloscopia (ORajust = 5,96; IC= 2,58-13,73) e produção de escarro (ORajust = 4,55; IC= 1,28- 16,12). Não houve associação estatisticamente significativa com o restante das
variáveis.A famÃlia LAM foi a mais frequente entre os dois grupos analisados, representando 65 (62%) dos isolados no grupo cavitário e 40 isolados (38%) do grupo não cavitário. Não houve diferença estatisticamente significativa entre os
grupos em relação à deleção RDRio (p=0,65) e com relação à deleção RD174 (p=0,65). Dentre os 205 isolados analisados, 25 (12%) isolados do grupo não cavitário e 43 (21%) do grupo cavitário, estavam em cluster. não houve diferença
estatisticamente significativa entre a quantidade de clusters e os grupos analisados (p= 0,4). Conclusões: Foi determinado o perfil genotÃpico dos isolados de pacientes com doença pulmonar, cavitária e não cavitária. Não houve associação
entre a presença de cavidade e os genótipos encontrados. Não houve associação do genótipo com nenhum dos marcadores moleculares avaliado
Avaliação de segurança in vitro de extratos de plantas: potencial citotóxico e genotóxico
A sociedade atual tem se preocupado cada vez mais com a sustentabilidade,
principalmente a nÃvel ambiental, o que se tem refletido no aumento da procura de
produtos naturais por parte dos consumidores. Aliado a isso, a aplicação de extratos de
plantas em produtos consumidos na vida quotidiana da população tem sido um dos
grandes focos das diversas indústrias, em todo o mundo.
Dentro dos diversos extratos de plantas, desde há vários séculos, os óleos essenciais
encontram-se num lugar de destaque, devido às suas propriedades biológicas
extensamente estudadas. Contudo, mais recentemente, os subprodutos do seu processo
de destilação, designados por hidrolatos, têm suscitado interesse nas indústrias, por
serem misturas diluÃdas com uma menor complexidade e, por isso, teoricamente, com
uma maior tolerância a nÃvel da segurança para o consumidor. No entanto, os estudos
por parte da comunidade cientÃfica acerca destes últimos extratos ainda são escassos,
pelo que se torna necessário promover o aumento do conhecimento relativamente à sua
composição e respetivas propriedades biológicas, bem como ao seu nÃvel de segurança.
As plantas, embora tenham inúmeros benefÃcios para a saúde humana, podem promover
toxicidade, o que levou a uma significativa consciencialização quanto à necessidade de
se avaliar a sua eficácia e segurança, bem como dos seus extratos, antes da sua possÃvel
aplicação em produtos de consumo humano. Nesse sentido, o presente trabalho visa,
então, caracterizar quimicamente cinco extratos de plantas, dentro dos quais se
encontravam dois tipos de extratos, nomeadamente: o hidrolato de Hamamelis
virginiana, o hidrolato de Matricaria chamomilla, o hidrolato e o óleo essencial de
Ocimum basilicum e o óleo essencial de Cupressus lusitanica.
Para além disso, procedeu-se ao estudo do respetivo potencial genotóxico e citotóxico
dos cinco extratos de plantas, com o objetivo de poderem vir a ser incorporados em
formulações farmacêuticas e cosméticas, em especial de aplicação tópica.
Assim, no decorrer deste trabalho, realizou-se o teste de Ames, com o intuito de se avaliar
o potencial mutagénico. Além disso, avaliou-se a citotoxicidade dos extratos, através do
ensaio MTT com uma linha celular de queratinócitos humanos (HaCaT). Os resultados
obtidos neste ensaio bacteriano foram classificados, com base nos três seguintes
critérios: relação dose-resposta, rácio de mutagenicidade e aumento fora do histórico de
controlo. Os resultados obtidos permitiram concluir que os três hidrolatos podem ser
considerados seguros em baixas concentrações, o que, em conjunto com as suas
atividades biológicas continuamente em estudo, lhes confere um lugar de valorização e
destaque quanto à potencialidade de serem utilizados em produtos farmacêuticos e
cosméticos.
Dentro dos óleos essenciais em estudo, ainda que não tenham apresentado atividade
mutagénica nas condições de teste utilizadas, o óleo essencial de Ocimum basilicum, em
comparação com o de Cupressus lusitanica, demonstrou uma citotoxicidade superior,
sendo apenas biocompatÃvel em concentrações baixas.
Este estudo constitui uma avaliação primária da segurança deste extratos, pelo que,
futuramente, outros parâmetros toxicológicos devam ser avaliados, para uma
determinação completa do perfil de segurança dos extratos de plantas.Today's society has been increasingly concerned with sustainability, especially at an
environmental level, which has been reflected in the increased demand for natural
products by consumers. Allied to this, the application of plant extracts in products
consumed in the daily life of the population has been one of the major focuses of various
industries around the world.
Within the different plant extracts, essential oils have been in a prominent place for
several centuries, due to their extensively studied biological properties. However, more
recently, the by-products of its distillation process, known as hydrolates, have attracted
interest in industries, as they are diluted mixtures with a lower complexity and, therefore,
theoretically, with a greater tolerance in terms of safety for the consumer. However,
studies by the scientific community about these last extracts are still scarce, so it is
necessary to promote the increase of knowledge regarding their composition and
respective biological properties, as well as their level of safety.
Plants, although they have numerous benefits for human health, can promote toxicity,
which has led to a significant awareness of the need to evaluate their efficacy and safety,
as well as their extracts, before their possible application in human consumer products.
In this sense, the present work aims to chemically characterize five plant extracts, within
which two types of extracts were found, namely: Hamamelis virginiana hydrolate,
Matricaria chamomilla hydrolate, Ocimum basilicum hydrolate and essential oil and
Cupressus lusitanica essential oil.
In addition, the respective genotoxic and cytotoxic potential of the five plant extracts was
studied, with the aim of being able to incorporate them into pharmaceutical and cosmetic
formulations, especially for topical application.
In the course of this work, the Ames test was performed in order to assess the mutagenic
potential. In addition, the cytotoxicity of the extracts was evaluated through the MTT
assay with a human keratinocyte cell line (HaCaT). The results obtained in this bacterial
assay were classified based on the following three criteria: dose-response relationship,
mutagenicity ratio and increase outside the control history.
The results obtained allowed us to conclude that the three hydrolates can be considered
safe in low concentrations, which, together with their biological activities, which are continuously being studied, gives them a place of appreciation and prominence
regarding their potential to be used in pharmaceuticals and cosmetics products.
Among the essential oils under study, although they did not show mutagenic activity
under the test conditions used, the essential oil of Ocimum basilicum, in comparison
with that of Cupressus lusitanica, showed superior cytotoxicity, being only
biocompatible at low concentrations.
This study constitutes a primary evaluation of the safety of these extracts, therefore, in
the future, other toxicological parameters must be evaluated, for a complete
determination of the safety profile of the plant extracts
The Small Membrane Filter Method of Microscopy to Diagnose Pulmonary Tuberculosis
We modified microscopy for acid-fast bacilli to diagnose tuberculosis (TB) using small membrane filters (SMFs) after special processing and prefiltration. With the first specimen obtained from each of 335 persons suspected of having TB, the sensitivity of the new SMF method using fluorescence microscopy (FM) was 89% (95% confidence interval [CI]: 80%, 95%). This was significantly better (P = 0.0001) than the sensitivity of routine FM of centrifuged specimens of 60% (95% CI: 49%, 71%) or that of direct sputum smears of 56% (95% CI: 40%, 72%)
In vitro activity of amphotericin B cochleates against Leishmania chagasi
Cochleate delivery vehicles are a novel lipid-based system with
potential for delivery of amphotericin B (AmB). In this study, the
efficacy of cochleates was evaluated by examining the in vitro activity
of AmB cochleates (CAMB) against Leishmania chagasi in a macrophage
model of infection. We demonstrate that CAMB is nontoxic to macrophages
at concentrations as high as 2.5 μg/mL, whereas the conventional
formulation, AmB deoxycholate, showed high toxicity at this
concentration. The in vitro activity of CAMB against L. chagasi was
found to be similar to that of the reference drug AmB deoxycholate,
with ED50s of 0.017 μg/mL and 0.021 μg/mL, respectively.
Considering that L. chagasi affects organs amenable to
cochleate-mediated delivery of AmB, we hypothesize that CAMB will be an
effective lipid system for the treatment of visceral leishmaniasis
Acurácia do lavado gástrico realizado em ambiente hospitalar e ambulatorial no diagnóstico da tuberculose pulmonar em crianças
OBJETIVO: Comparar a acurácia do lavado gástrico (LG) realizado em ambiente hospitalar e ambulatorial no diagnóstico da tuberculose (TB) pulmonar em crianças. MÉTODOS: Estudo prospectivo realizado no Estado do EspÃrito Santo, Brasil, de 1999 a 2003. Um total de 230 crianças com suspeita de TB foi selecionado para realizar exame de LG em ambiente hospitalar (n = 103) ou em ambiente ambulatorial (n = 127). Desse total, 53 foram diagnosticadas como casos de TB e divididas em dois grupos: LG hospitalar (n = 30) e LG ambulatorial (n = 23). Todas as 53 crianças foram monitoradas por 6 meses para avaliação da acurácia do diagnóstico. A acurácia foi determinada com base na mudança do diagnóstico, na taxa de cura e no percentual de culturas positivas nos dois grupos estudados. RESULTADOS: A taxa de cura foi de 100% nos dois grupos, e não houve mudança de diagnóstico nas 53 crianças estudadas. Nenhuma diferença significativa foi encontrada entre os dois grupos estudados em relação ao achado do Mycobacterium tuberculosis (RR = 1,47; IC95%: 0,95-2,27; p = 0,095), apesar de o grupo LG ambulatorial ter apresentado o maior Ãndice de cultura positivas. CONCLUSÕES: Nossos resultados mostram que a acurácia do LG realizado em ambiente hospitalar é semelhante à do realizado em ambiente ambulatorial, o que indica que a internação é necessária apenas em casos mais graves nos quais não se pode realizar o procedimento em ambiente ambulatorial
Use of in-house PCR for identification of Mycobacterium tuberculosis in BACTEC broth cultures of respiratory specimens
We evaluated the ability of a PCR assay to identify Mycobacterium
tuberculosis complex (MTBC) from positive BACTEC® 12B broth
cultures. A total of 107 sputum samples were processed and inoculated
into Ogawa slants and BACTEC® 12B vials. At a growth index (GI)
> 30, 1.0 ml of the 12B broth was removed, stored, and assayed with
PCR. Molecular results were compared to those obtained by phenotypic
identification methods, including the BACTEC® NAP method. The
average times required to perform PCR and NAP were compared. Of the 107
broth cultures evaluated, 90 were NAP positive, while 91 were PCR
positive for MTBC. Of particular interest were three contaminated
BACTEC® 12B broth cultures yielding microorganisms other than
acid-fast bacilli growth with a MTBC that were successfully identified
by PCR, resulting in a mean time of 14 days to identify MTBC before NAP
identification. These results suggest that PCR could be used as an
alternative to the NAP test for the rapid identification of MTBC in
BACTEC® 12B cultures, particularly in those that contained both
MTBC and nontuberculous mycobacteria
Risk factors associated with cluster size of Mycobacterium tuberculosis (Mtb) of different RFLP lineages in Brazil
Abstract Background Tuberculosis (TB) transmission is influenced by patient-related risk, environment and bacteriological factors. We determined the risk factors associated with cluster size of IS6110 RFLP based genotypes of Mycobacterium tuberculosis (Mtb) isolates from Vitoria, Espirito Santo, Brazil. Methods Cross-sectional study of new TB cases identified in the metropolitan area of Vitoria, Brazil between 2000 and 2010. Mtb isolates were genotyped by the IS6110 RFLP, spoligotyping and RDRio. The isolates were classified according to genotype cluster sizes by three genotyping methods and associated patient epidemiologic characteristics. Regression Model was performed to identify factors associated with cluster size. Results Among 959 Mtb isolates, 461 (48%) cases had an isolate that belonged to an RFLP cluster, and six clusters with ten or more isolates were identified. Of the isolates spoligotyped, 448 (52%) were classified as LAM and 412 (48%) as non-LAM. Our regression model found that 6–9 isolates/RFLP cluster were more likely belong to the LAM family, having the RDRio genotype and to be smear-positive (adjusted OR = 1.17, 95% CI 1.08–1.26; adjusted OR = 1.25, 95% CI 1.14–1.37; crude OR = 2.68, 95% IC 1.13–6.34; respectively) and living in a Serra city neighborhood decrease the risk of being in the 6–9 isolates/RFLP cluster (adjusted OR = 0.29, 95% CI, 0.10–0.84), than in the others groups. Individuals aged 21 to 30, 31 to 40 and > 50 years were less likely of belonging the 2–5 isolates/RFLP cluster than unique patterns compared to individuals < 20 years of age (adjusted OR = 0.49, 95% CI 0.28–0.85, OR = 0.43 95% CI 0.24–0.77and OR = 0. 49, 95% CI 0.26–0.91), respectively. The extrapulmonary disease was less likely to occur in those infected with strains in the 2–5 isolates/cluster group (adjustment OR = 0.45, 95% CI 0.24–0.85) than unique patterns. Conclusions We found that a large proportion of new TB infections in Vitoria is caused by prevalent Mtb genotypes belonging to the LAM family and RDRio genotypes. Such information demonstrates that some genotypes are more likely to cause recent transmission. Targeting interventions such as screening in specific areas and social risk groups, should be a priority for reducing transmission