Perfil genético por sequenciamento e genotipagem de cepas multirresistentes do Mycobacterium tuberculosis provenientes de diversos estados brasileiros

A tuberculose multirresistente (MR) a drogas é uma séria ameaça à saúde pública devido à maiores complexidade, custo e efeitos colaterais do tratamento. Poucos estudos descreveram a epidemiologia molecular de isolados de Mycobacterium tuberculosis MR no Brasil. Neste trabalho foi investigada a diversidade genética e mutações associadas à resistência a drogas de 99 isolados MR e 7 não MR coletados em um período de 8 anos e provenientes de 12 estados brasileiros. Esta investigação foi feita através da análise do polimorfismo de fragmentos de restrição do elemento de inserção IS6110 (IS6110-RFLP), spoligotyping e sequenciamento de regiões dos genes rpoB e katG que conferem resistência aos antibióticos rifampicina e isoniazida, respectivamente. Mutações nos genes katG e rpoB foram encontradas em 90,9% e 93% dos isolados MR analisados, respectivamente. Para o gene rpoB, 91,9% das mutações estavam contidas na região RRDR de 81-pb. Um total de 51 (51.5%), 23 (23.3%) e 11 (11.1%) isolados MR apresentaram mutações nos códons 531, 526 e 516, respectivamente. Com relação ao gene katG, foram encontradas mutações em 93% dos isolados MR analisados, sendo que 7 apresentaram mutações apenas na primeira região analisada (katG1). O codon 315 da segunda região analisada do gene katG (katG2) apresentou mutações em 82.8% dos isolados MR, sendo a maioria Ser315Thr. A região katG1 apresentou mutações em 30.3% dos isolados MR sendo a maioria deleção do códon 4. Pelo spoligotyping foi possível determinar que os isolados MR deste estudo pertencem a 5 diferentes famílias (com suas subfamílias) de M. tuberculosis circulantes no Brasil, onde as mais frequentemente encontradas foram: LAM (46%), T (17%) e H (12%). Nós observamos que uma das famílias, a EAI5, carrega mutações no códon 463 do gene katG, o que não ocorre para as demais. Além disso, entre nossos isolados foi identificada um isolado pertencente à cepa Beijing (extremamente virulenta), mas este fato não é alarmante já que se tratou de apenas um caso. Através de nossos dados foram descritos novos alelos mutados para os genes rpoB e katG. Com exceção da família X2, foi identificada uma região inicial do gene katG com alta frequência de mutações nos isolados MR. A análise por IS6110-RFLP revelou que 25 isolados formaram 11 grupos genotípicos enquanto 74 mostraram um padrão único de bandas. Esta alta taxa de polimorfismo indica aquisição independente de resistência entre nossos isolados. Para a família H, foi identificada uma inversão na freqüência de ocorrência de mutações no gene rpoB, sendo o códon 516 o mais mutado, seguido pelo 526 e 531. Os resultados deste estudo fornecem informações úteis para um melhor entendimento do espectro de mutações dos isolados MR de pacientes no Brasil. Nossos resultados também se tornam úteis no desenvolvimento de testes diagnósticos de tuberculose MR e para auxiliar no rastreamento da transmissão global desta doença.

Avian comparative genomics: reciprocal chromosome painting between domestic chicken (Gallus gallus) and the stone curlew (Burhinus oedicnemus, Charadriiformes) - An atypical species with low diploid number

The chicken is the most extensively studied species in birds and thus constitutes an ideal reference for comparative genomics in birds. Comparative cytogenetic studies indicate that the chicken has retained many chromosome characters of the ancestral avia

Detection of usual and atypical aldehyde dehydrogenase alleles by mismatch amplification mutation assay

The genotypes of liver mitochondrial high-affinity aldehyde dehydrogenase-2 (ALDH2) are strongly associated with the drinking behavior and the alcohol liver diseases, since the individuals with atypical ALDH(2)(2) allele have higher levels of acetaldehyde in their plasma. The atypical ALDH(2)(2) allele has a nucleotide base transition (G-->A) in its exon 12. Based on this point mutation, we developed a rapid, reliable and inexpensive method, mismatch amplification mutation assay (MAMA), for the determination of human ALDH2 usual and atypical alleles. Two pairs of primers were designed for the amplification of the usual ALDH(2)(1) allele and the atypical ALDH(2)(2) allele, respectively. If the sample for the detection was heterozygous, it could be amplified by both of the primers. The product of polymerase chain reaction (PCR) of ALDH2 exon 12 could be easily screened by electrophoresis on a 2% agarose gel. The results of the MAMA method were further confirmed by sequencing. In the total of fifty samples from unrelated healthy Chinese Han people from Wuhan, China, the frequency of atypical ALDH(2)(2) allele was found to be 12%.

Origin and dispersal of atypical aldehyde dehydrogenase ALDH2*487Lys

The East Asian respond with a marked facial flushing and mild to moderate symptoms of intoxication after drinking the amounts of alcohol that has no detectable effect on European. The alcohol sensitivity in Orientals is due to a delayed oxidation of aceta

In vitro activities of small peptides from snake venom against clinical isolates of drug-resistant Mycobacterium tuberculosis

The recent re-emergence of tuberculosis, especially the multidrug-resistant cases, has highlighted the importance of screening effective novel drugs against Mycobacterium tuberculosis. In this study, the in vitro activities of small peptides isolated from snake venom were investigated against multidrug-resistant M. tuberculosis. Minimum inhibitory concentrations (MICs) were determined by the Bactec TB-460 radiometric method. A small peptide with the amino acid sequence ECYRKSDIVTCEPWQKFCYREVTFFPNHPVYLSGCASECTETNSKWCCTTDKCNRARGG (designated as vgf-1) from Naja atra (isolated from Yunnan province of China) venom had in vitro activity against clinically isolated multidrug-resistant strains of M. tuberculosis. The MIC was 8.5 mg/l. The antimycobacterial domain of this 60aa peptide is under investigation. (C) 2003 Elsevier Science B.V. and the International Society of Chemotherapy. All rights reserved.

Simulation in user-centred design: Helping designers to empathise with atypical users

Time and budget constraints frequently prevent designers from consulting with end-users while assessing the ease of use of the products they create. This has resulted in solutions that are difficult to use by a wide range of users, especially the growing older adult population and people with different types of impairments. To help designers with this problem, capability-loss simulators have been developed with the aim of temporarily representing users who are otherwise difficult to access. This paper questions the reliability of existing tools in providing designers with meaningful information about the users' capabilities. Consequently, a new capability-loss simulation toolkit is presented, followed by its empirical evaluation. The new toolkit proved to be significantly helpful for a group of designers identifying real usability problems with everyday devices. © 2012 Copyright Taylor and Francis Group, LLC.

Multiplex detection of mutations in clinical isolates of rifampin-resistant Mycobacterium tuberculosis by short oligonucleotide Ligation assay on DNA chips

A new approach, short-oligonucleotide-ligation assay on DNA chip (SOLAC), is developed to detect mutations in rifampin-resistant Mycobacterium tuberculosis. The method needs only four common probes to detect 15 mutational variants of the rpoB gene within 12 h. Fifty-five rifampin-resistant M. tuberculosis isolates were analyzed, resulting in 87.3% accuracy and 83.6% concordance relative to DNA sequencing.

Produção e caracterização de MPB70 de Mycobacterium bovis para uso em imunodiagnóstico.

Demonstrou-se a produção e caracterização de MPB70 recombinante de M. bovis.

Identification of major factors influencing ELISpot-based monitoring of cellular responses to antigens from Mycobacterium tuberculosis.

A number of different interferon-gamma ELISpot protocols are in use in laboratories studying antigen-specific immune responses. It is therefore unclear how results from different assays compare, and what factors most significantly influence assay outcome. One such difference is that some laboratories use a short in vitro stimulation period of cells before they are transferred to the ELISpot plate; this is commonly done in the case of frozen cells, in order to enhance assay sensitivity. Other differences that may be significant include antibody coating of plates, the use of media with or without serum, the serum source and the number of cells added to the wells. The aim of this paper was to identify which components of the different ELISpot protocols influenced assay sensitivity and inter-laboratory variation. Four laboratories provided protocols for quantifying numbers of interferon-gamma spot forming cells in human peripheral blood mononuclear cells stimulated with Mycobacterium tuberculosis derived antigens. The differences in the protocols were compared directly. We found that several sources of variation in assay protocols can be eliminated, for example by avoiding serum supplementation and using AIM-V serum free medium. In addition, the number of cells added to ELISpot wells should also be standardised. Importantly, delays in peripheral blood mononuclear cell processing before stimulation had a marked effect on the number of detectable spot forming cells; processing delay thus should be minimised as well as standardised. Finally, a pre-stimulation culture period improved the sensitivity of the assay, however this effect may be both antigen and donor dependent. In conclusion, small differences in ELISpot protocols in routine use can affect the results obtained and care should be given to conditions selected for use in a given study. A pre-stimulation step may improve the sensitivity of the assay, particularly when cells have been previously frozen.

Characterization of the morphonuclear features and DNA ploidy of typical and atypical carcinoids and small cell carcinomas of the lung

The authors analyzed several cytomorphonuclear parameters related to chromatin distribution and DNA ploidy in typical and atypical carcinoids and in small cell lung cancers. Nuclear measurements and analysis were performed with a SAMBA 200 (TITN, Grenoble, France) cell image processor with software allowing the discrimination of parameters computed on cytospin preparations of Feulgen-stained nuclei extracted from deparaffinized tumor tissues. The authors' results indicate a significant increase in DNA content--assessed by integrated optical density (IOD)--from typical carcinoids to small cell lung carcinomas, with atypical carcinoids showing an intermediate value. Parameters related to hyperchromatism (short and long run length and variance of optical density) also characterize the atypical carcinoids as being intermediate between typical carcinoids and small cell lung cancers. The systematic measurement of these cytomorphonuclear parameters seems to define an objective, reproducible "scale" of differentiation that helps to define the atypical carcinoid and may be of value in establishing cytologic criteria for differential diagnosis.

Long- incubation time- interferon-gamma release assays in response to PPD-, ESAT-6- and/or CFP-10 for the diagnosis of Mycobacterium tuberculosis infection in children.

Background:Diagnosis of childhood active tuberculosis (aTB) or latent Mycobacterium tuberculosis (Mtb) infection (LTBI) remains a challenge, and replacement of tuberculin skin tests (TST) by commercialized interferon-gamma release assays (IGRA) is not currently recommended.Methods:266 children between 1 month and 15 years of age, 214 being at risk of recent Mtb infection and 51 being included as controls, were prospectively enrolled. According results of clinical evaluation, TST, chest X-Ray and microbiology, children were classified as non-infected, LTBI or aTB. Long-incubation time PPD-, ESAT-6-, and CFP-10-IGRA were performed and evaluated for their accuracy to correctly classify the children.Results:Whereas both TST and PPD-IGRA were suboptimal to detect aTB, combining CFP-10-IGRA with TST or with PPD-IGRA allowed us to detect all the children with aTB, with 96% specificity for children who were positive for CFP-10-IGRA. Moreover, combination of CFP-10- and PPD-IGRA also detected 96% of children classified as LTBI, but a strong IFN-γ response to CFP-10 (>500 pg/ml) was highly suggestive of aTB at least among children less than 3 years old.Conclusions:Long-incubation time CFP-10- and PPD-IGRA should help the clinicians to identify quickly aTB or LTBI in young children.

Atypical Fibroxanthoma of the Bulbar Conjunctiva.

PURPOSE: To report a rare case of atypical fibroxanthoma (AFX) of the bulbar conjunctiva, and to compare it with previously published cases of conjunctival AFX. METHODS: A 37-year-old woman developed a growth on the bulbar conjunctiva of her left eye that increased in size and redness over 4 months and was associated with blurry vision in the left eye, occasional diplopia, irritation of the eye, and increasing tearing. The mass was surgically excised. RESULTS: Slit-lamp examination disclosed a highly vascularized conjunctival lesion with intact lustrous epithelium and a raised nodular edge encroaching on the nasal corneal limbus of the left eye. Pathological examination and immunohistochemistry were diagnostic of AFX. CONCLUSIONS: AFX of the conjunctiva is rare, with this being only the fifth example of this neoplasm reported at this site. Complete surgical excision is the most appropriate treatment option.

Whole genome sequencing identifies circulating Beijing-lineage Mycobacterium tuberculosis strains in Guatemala and an associated urban outbreak.

Limited data are available regarding the molecular epidemiology of Mycobacterium tuberculosis (Mtb) strains circulating in Guatemala. Beijing-lineage Mtb strains have gained prevalence worldwide and are associated with increased virulence and drug resistance, but there have been only a few cases reported in Central America. Here we report the first whole genome sequencing of Central American Beijing-lineage strains of Mtb. We find that multiple Beijing-lineage strains, derived from independent founding events, are currently circulating in Guatemala, but overall still represent a relatively small proportion of disease burden. Finally, we identify a specific Beijing-lineage outbreak centered on a poor neighborhood in Guatemala City.