RESUMEN
Leprosy is a prevalent disease in Brazil, which ranks as the country with the second highest number of cases in the world. The disease manifests in a spectrum of forms, and genetic differences in the host can help to elucidate the immunopathogenesis. For a better understanding of MICA association with leprosy, we performed a case-control and a family-based study in two endemic populations in Brazil. MICA and HLA-B alleles were evaluated in 409 leprosy patients and in 419 healthy contacts by PCR-SSOP-Luminex-based technology. In the familial study, analysis of 46 families was completed by direct sequencing of all exons and 3'/5'untranslated regions, using the Ilumina MiSeq platform. All data were collected between 2006 and 2009. Statistical analysis was performed using the Chi-square or Fisher's exact test together with a multivariate analysis. Family-based association was assessed by transmission disequilibrium test (TDT) software FBAT 2.0.4. We found associations between the haplotype MICA*002-HLA-B*35 with leprosy in both the per se and the multibacillary (MB) forms when compared to healthy contacts. The MICA allele *008 was associated with the clinical forms of paucibacillary (PB). Additionally, MICA*029 was associated with the clinical forms of MB. The association of MICA*029 allele (MICA-A4 variant) with the susceptibility to the MB form suggests this variant for the transmembrane domain of the MICA molecule may be a risk factor for leprosy. Two MICA and nine HLA-B variants were found associated with leprosy per se in the Colônia do Prata population. Linkage disequilibrium analysis revealed perfect linkage disequilibrium (LD) between HLA-B markers rs2596498 and rs2507992, and high LD (R2 = .92) between these and the marker rs2442718. This familial study demonstrates that MICA association signals are not independent from those observed for HLA-B. Our findings contribute the knowledge pool of the immunogenetics of Hansen's disease and reveals a new association of the MICA*029 allele.
Asunto(s)
Antígenos HLA-B/genética , Antígenos de Histocompatibilidad Clase I/genética , Lepra/inmunología , Regiones no Traducidas 3'/genética , Regiones no Traducidas 5'/genética , Adolescente , Adulto , Alelos , Brasil/epidemiología , Estudios de Casos y Controles , Niño , Enfermedades Endémicas , Etnicidad/genética , Exones/genética , Salud de la Familia , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Haplotipos/genética , Humanos , Lepra/epidemiología , Lepra/genética , Desequilibrio de Ligamiento , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Dominios Proteicos , Adulto JovenRESUMEN
Leprosy is a prevalent disease in Brazil, which ranks as the country with the second highest number of cases in the world. The disease manifests in a spectrum of forms, and genetic differences in the host can help to elucidate the immunopathogenesis. For a better understanding of MICA association with leprosy, we performed a casecontrol and a familybased study in two endemic populations in Brazil. MICA and HLAB alleles were evaluated in 409 leprosy patients and in 419 healthy contacts by PCRSSOPLuminexbased technology. In the familial study, analysis of 46 families was completed by direct sequencing of all exons and 3'/5'untranslated regions, using the Ilumina MiSeq platform. All data were collected between 2006 and 2009. Statistical analysis was performed using the Chisquare or Fisher's exact test together with a multivariate analysis. Familybased association was assessed by transmission disequilibrium test (TDT) software FBAT 2.0.4. We found associations between the haplotype MICA*002HLAB*35 with leprosy in both the per se and the multibacillary (MB) forms when compared to healthy contacts. The MICA allele *008 was associated with the clinical forms of paucibacillary (PB). Additionally, MICA*029 was associated with the clinical forms of MB. The association of MICA*029 allele (MICAA4 variant) with the susceptibility to the MB form suggests this variant for the transmembrane domain of the MICA molecule may be a risk factor for leprosy. Two MICA and nine HLAB variants were found associated with leprosy per se in the Colônia do Prata population. Linkage disequilibrium analysis revealed perfect linkage disequilibrium (LD) between HLAB markers rs2596498 and rs2507992, and high LD (R2 = .92) between these and the marker rs2442718. This familial study demonstrates that MICA association signals are not independent from those observed for HLAB. Our findings contribute the knowledge pool of the immunogenetics of Hansen's disease and reveals a new association of the MICA*029 allele(AU).
Asunto(s)
Humanos , Masculino , Femenino , Antígenos de Histocompatibilidad Clase I , Antígenos HLA-B , Lepra/genética , Lepra/inmunología , Polimorfismo Genético , Desequilibrio de Ligamiento , Factores de Riesgo , Predisposición Genética a la Enfermedad , Alelos , Lepra/transmisiónRESUMEN
BACKGROUND: Leprosy has been treated with multidrug therapy (MDT) distributed for free across the globe and regarded as highly efficient. However, the impossibility to grow M. leprae in axenic media has historically impaired assessment of M. leprae resistance, a parameter only recently detectable through molecular methods. METHODS: A systematic, population-based search for M. leprae resistance in suspected leprosy relapse cases and contacts was performed in Prata Village, an isolated, hyper-endemic former leprosy colony located in the Brazilian Amazon. Results led to an extended active search involving the entire Prata population. Confirmed leprosy cases were investigated for bacterial resistance using a combination of in vivo testing and direct sequencing of resistance genes folP1, rpoB and gyrA. Molecular epidemiology analysis was performed using data from 17 variable number tandem repeats (VNTR). RESULTS: M. leprae was obtained from biopsies of 37 leprosy cases (18 relapses and 19 new); 16 (43.24%) displayed drug-resistance variants. Multi-drug resistance to rifampicin and dapsone was observed in 8 relapses and 4 new cases. Single resistance to rifampicin was detected in one new case. Resistance to dapsone was present in two relapses and one new case. Combined molecular resistance and VNTR data revealed evidence of intra-familial primary transmission of resistant M. leprae. CONCLUSIONS: A comprehensive, population-based systematic approach to investigate M. leprae resistance in a unique population revealed an alarming scenario of emergence and transmission of resistant strains. These findings may be used for the development of new strategies for surveillance of drug resistance in other populations.
Asunto(s)
Farmacorresistencia Bacteriana/genética , Lepra/transmisión , Mycobacterium leprae/efectos de los fármacos , BrasilRESUMEN
There is evidence that in southern US, leprosy is a zoonosis infecting wild Dasypus novemcinctus armadillos but the extent of this finding is unknown. This ecological study investigated leprosy in rural communities and in wild armadillos from the Brazilian Amazon. The study area was the Mamia´ Lake of Coari municipality, Amazonas State, Northern region, a hyper endemic leprosy area where residents live on subsistence farming, fishing and armadillo hunting and its meat intake are frequent. The leprosy survey was conducted in sixteen communities by a visiting team of specialists. Local partakers provided wild armadillos to investigate M. leprae infection. Volunteers had complete dermato-neurological examination by a dermatologist with expertise in leprosy diagnosis, suspect skin lesions were biopsied for histopathology (Hematoxylin-eosin/HE, Fite-Faraco/FF staining); slit skin smears were collected. Armadillos' tissue fragments (skins, spleens, livers, lymph nodes, adrenal glands, others) were prepared for histopathology (HE/FF) and for M. leprae repetitive elementRLEP-qPCR. Among 176 volunteers, six new indeterminate leprosy cases were identified (incidence = 3.4%). Suspect skin sections and slit skin smears were negative for bacilli. Twelve wild D. novemcinctus were investigated (48 specimens/96 slides) and histopathological features of M. leprae infection were not found, except for one skin presenting unspecific inflammatory infiltrate suggestive of indeterminate leprosy. Possible traumatic neuroma, granuloma with epithelioid and Langhans cells, foreign-body granuloma were also identified. Granulomatous/non-granulomatous dermatitides were periodic-acid-Schiff/ PAS negative for fungus. M. leprae-RLEP-qPCR was negative in all armadillos' tissues; no bacillus was found in histopathology. Our survey in rural communities confirmed the high endemicity for leprosy while one armadillo was compatible with paucibacillary M. leprae infection. At least in the highly endemic rural area of Coari, in the Brazilian Amazon region where infectious sources from untreated multibacillary leprosy are abundant, M. leprae infected armadillos may not represent a major source of infection nor a significant public health concern.
Asunto(s)
Humanos , Animales , Masculino , Femenino , Adolescente , Adulto , Adulto Joven , Armadillos/microbiología , Población Rural , Reservorios de Enfermedades/microbiología , Zoonosis , Ecosistema , Lepra Paucibacilar/veterinaria , Lepra Paucibacilar/epidemiología , Lepra Paucibacilar/virología , Lepra/microbiología , Lepra/veterinaria , Lepra/epidemiología , Mycobacterium leprae/aislamiento & purificación , Mycobacterium leprae/genética , PielAsunto(s)
Humanos , Masculino , Adolescente , Adulto , Adulto Joven , Recurrencia , ADN Bacteriano/aislamiento & purificación , Ensayos Clínicos Controlados Aleatorios como Asunto , Genoma Bacteriano , Análisis de Secuencia de ADN/métodos , Biología Computacional/métodos , Tipificación Molecular/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Lepra/diagnóstico , Mycobacterium leprae/aislamiento & purificación , Mycobacterium leprae/clasificación , Mycobacterium leprae/genética , América del Sur , BrasilRESUMEN
Leprosy is an important cause of disability in the developing world. Early diagnosis is essential to allow for cure and to interrupt transmission of this infection. MicroRNAs (miRNAs) are important factors for host-pathogen interaction and they have been identified as biomarkers for various infectious diseases. The expression profile of 377 microRNAs were analyzed by TaqMan low-density array (TLDA) in skin lesions of tuberculoid and lepromatous leprosy patients as well as skin specimens from healthy controls. In a second step, 16 microRNAs were selected for validation experiments with reverse transcription-quantitative PCR (qRT-PCR) in skin samples from new individuals. Principal-component analysis followed by logistic regression model and receiver operating characteristic (ROC) curve analyses were performed to evaluate the diagnostic potential of selected miRNAs. Four patterns of differential expression were identified in the TLDA experiment, suggesting a diagnostic potential of miRNAs in leprosy. After validation experiments, a combination of four miRNAs (miR-101, miR-196b, miR-27b, and miR-29c) was revealed as able to discriminate between healthy control and leprosy patients with 80% sensitivity and 91% specificity. This set of miRNAs was also able to discriminate between lepromatous and tuberculoid patients with a sensitivity of 83% and 80% specificity. In this work, it was possible to identify a set of miRNAs with good diagnostic potential for leprosy.
Asunto(s)
Humanos , Adulto , Persona de Mediana Edad , Adulto Joven , Marcadores Genéticos/genética , Curva ROC , Sensibilidad y Especificidad , MicroARNs/genética , Diagnóstico Precoz , Reacción en Cadena en Tiempo Real de la Polimerasa , Lepra/diagnóstico , Lepra/inmunología , Lepra/microbiología , Mycobacterium leprae/genética , Mycobacterium leprae/inmunologíaRESUMEN
BACKGROUND: The objective of this study was to investigate the association between KIR genes and the immunopathogenesis of leprosy. METHODS: The types of KIR and HLA genes were evaluated by PCR-SSOP-Luminex in 408 patients with leprosy and 413 healthy individuals. Statistical analysis was performed using the Chi-square or Fisher's exact test and stepwise multivariate analysis. RESULTS: There was a higher frequency of activating KIR genes (KIR2DS1, 2DS2 and 3DS1) together with their HLA ligands in the tuberculoid (TT) group as compared to the lepromatous leprosy (LL) group. KIR2DL2/2DL2-C1 was more frequent in the patient, TT and LL groups than in the control group. Borderline patients presented a higher frequency of inhibitory pairs when compared to the control group, and a higher frequency of activating pairs as compared to the LL group. Multivariate analysis confirmed the associations and demonstrated that being a female is a protective factor against the development of the disease per se and the more severe clinical form. CONCLUSIONS: This study showed that activating and inhibitory KIR genes may influence the development of leprosy - in particular, activating genes may protect against the more aggressive form of the disease - thereby demonstrating the role of NK cells in the immunopathology of the disease.
Asunto(s)
Genes MHC Clase I , Lepra/genética , Lepra/patología , Receptores KIR/genética , Adulto , Brasil , Estudios de Casos y Controles , Femenino , Genotipo , Humanos , Células Asesinas Naturales/citología , Ligandos , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
BACKGROUND: The objective of this study was to investigate the association between KIR genes and the immunopathogenesis of leprosy. METHODS: The types of KIR and HLA genes were evaluated by PCR-SSOP-Luminex in 408 patients with leprosy and 413 healthy individuals. Statistical analysis was performed using the Chi-square or Fisher's exact test and stepwise multivariate analysis. RESULTS: There was a higher frequency of activating KIR genes (KIR2DS1, 2DS2 and 3DS1) together with their HLA ligands in the tuberculoid (TT) group as compared to the lepromatous leprosy (LL) group. KIR2DL2/2DL2-C1 was more frequent in the patient, TT and LL groups than in the control group. Borderline patients presented a higher frequency of inhibitory pairs when compared to the control group, and a higher frequency of activating pairs as compared to the LL group. Multivariate analysis confirmed the associations and demonstrated that being a female is a protective factor against the development of the disease per se and the more severe clinical form. CONCLUSIONS: This study showed that activating and inhibitory KIR genes may influence the development of leprosy - in particular, activating genes may protect against the more aggressive form of the disease - thereby demonstrating the role of NK cells in the immunopathology of the disease.
Asunto(s)
Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Fenotipo , Brasil , Genes MHC Clase I , Células Asesinas Naturales/citología , Estudios de Casos y Controles , Receptores KIR/genética , Genotipo , Lepra/genética , Lepra/patología , LigandosRESUMEN
Leprosy is a complex disease with phenotypes strongly influenced by genetic variation. A Chinese genome-wide association study (GWAS) depicted novel genes and pathways associated with leprosy susceptibility, only partially replicated by independent studies in different ethnicities. Here, we describe the results of a validation and replication study of the Chinese GWAS in Brazilians, using a stepwise strategy that involved two family-based and three independent case-control samples, resulting in 3,614 individuals enrolled. First, we genotyped a family-based sample for 36 tag single-nucleotide polymorphisms (SNPs) of five genes located in four different candidate loci: CCDC122-LACC1, NOD2, TNFSF15 and RIPK2. Association between leprosy and tag SNPs at NOD2 (rs8057431) and CCDC122-LACC1 (rs4942254) was then replicated in three additional, independent samples (combined OR(AA) = 0.49, P = 1.39e-06; OR(CC) = 0.72, P = 0.003, respectively). These results clearly implicate the NOD2 pathway in the regulation of leprosy susceptibility across diverse populations.
Asunto(s)
Humanos , Masculino , Femenino , Niño , Adolescente , Adulto , Persona de Mediana Edad , Anciano , Adulto Joven , Brasil , Desequilibrio de Ligamiento , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Proteína Adaptadora de Señalización NOD2/genética , Estudios de Asociación Genética , Frecuencia de los Genes , Lepra/genéticaRESUMEN
Conflicting findings about the association between leprosy and TLR1 variants N248S and I602S have been reported. Here, we performed case-control and family based studies, followed by replication in 2 case-control populations from Brazil, involving 3162 individuals. Results indicated an association between TLR1 248S and leprosy in the case-control study (SS genotype odds ratio [OR], 1.81; P = .004) and the family based study (z = 2.02; P = .05). This association was consistently replicated in other populations (combined OR, 1.51; P < .001), corroborating the finding that 248S is a susceptibility factor for leprosy. Additionally, we demonstrated that peripheral blood mononuclear cells (PBMCs) carrying 248S produce a lower tumor necrosis factor/interleukin-10 ratio when stimulated with Mycobacterium leprae but not with lipopolysaccharide or PAM3cysK4. The same effect was observed after infection of PBMCs with the Moreau strain of bacillus Calmette-Guerin but not after infection with other strains. Finally, molecular dynamics simulations indicated that the Toll-like receptor 1 structure containing 248S amino acid is different from the structure containing 248N. Our results suggest that TLR1 248S is associated with an increased risk for leprosy, consistent with its hypoimmune regulatory function.
Asunto(s)
Humanos , Femenino , Adulto , Persona de Mediana Edad , Haplotipos , Ensayo de Inmunoadsorción Enzimática , Leucocitos Mononucleares/inmunología , Estudios de Casos y Controles , Factores de Riesgo , Polimorfismo de Nucleótido Simple/fisiología , Polimorfismo de Nucleótido Simple/genética , Receptor Toll-Like 1/fisiología , Receptor Toll-Like 1/genética , Citometría de Flujo , Frecuencia de los Genes/genética , Genotipo , Heterocigoto , Inmunidad/genética , Lepra/genética , Lepra/inmunología , Mycobacterium leprae/inmunologíaRESUMEN
Leprosy is a chronic infectious disease caused by Mycobacterium leprae, a low virulence mycobacterium, and the outcome of disease is dependent on the host genetics for either susceptibility per se or severity. The IFNG gene codes for interferon-γ (IFN-γ), a cytokine that plays a key role in host defense against intracellular pathogens. Indeed, single nucleotide polymorphisms (SNPs) in IFNG have been evaluated in several genetic epidemiological studies, and the SNP +874T>A, the +874T allele, more specifically, has been associated with protection against infectious diseases, especially tuberculosis. Here, we evaluated the association of the IFNG locus with leprosy enrolling 2,125 Brazilian subjects. First, we conducted a case-control study with subjects recruited from the state of São Paulo, using the +874 T>A (rs2430561), +2109 A>G (rs1861494) and rs2069727 SNPs. Then, a second study including 1,370 individuals from Rio de Janeiro was conducted. Results of the case-control studies have shown a protective effect for +874T carriers (OR(adjusted) = 0.75; p = 0.005 for both studies combined), which was corroborated when these studies were compared with literature data. No association was found between the SNP +874T>A and the quantitative Mitsuda response. Nevertheless, the spontaneous IFN-γ release by peripheral blood mononuclear cells was higher among +874T carriers. The results shown here along with a previously reported meta-analysis of tuberculosis studies indicate that the SNP +874T>A plays a role in resistance to mycobacterial diseases(AU)
Asunto(s)
Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Interferón gamma/genética , Lepra/genética , Brasil/epidemiología , Distribución de Chi-Cuadrado , Oportunidad Relativa , Factores de Riesgo , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Lepra/epidemiología , Mycobacterium leprae/aislamiento & purificaciónRESUMEN
A poliquimioterapia/Organização Mundial da Saúde foi implantada efetivamente no Brasil em 1991, contribuindo drasticamente para redução da taxa de prevalência e cura da hanseníase. No entanto, a sua comprovada eficácia não tem impedido a ocorrência de recidiva da doença. Falha no tratamento, persistência bacilar ou resistência a drogas são fatores que podem ou não estarem associados a ela. O objetivo deste estudo foi verificar a ocorrência de recidiva e associá-la com a presença de cepas resistentes do Mycobacterium leprae entre 28 indivíduos que apresentaram suspeita clínica de recidiva após tratamento por monoterapia sulfônica, esquema da Divisão Nacional de Dermatologia Sanitária ou poliquimioterapia. Biópsias das lesões de pacientes multibacilares, com diagnóstico clínico de recidiva, atendidos por demanda espontânea, foram coletadas para avaliar resistência a drogas por meio da técnica de inoculação em pata de camundongo. Dentre as amostras avaliadas 42,8% apresentaram bacilos sensíveis à dapsona e rifampicina e 10,7% apresentaram resistência à dapsona; não foram isolados bacilos resistentes à rifampicina. A emergência de bacilos resistentes, especialmente à rifampicina, é um alerta para os programas de controle da hanseníase. Monitorar a disseminação destas cepas é importante, pois elas apresentam um sério obstáculo para a eliminação da doença, principalmente em países onde a hanseníase ainda é endêmica.
The multidrugtherapy proposed by the World Health Organization has been effectively implemented in Brazil in 1991. It helped reduce the prevalence and achieve the cure of leprosy. However, its proven efficacy has not prevented the occurrence of relapses in some leprosy patients. Irregular treatment, bacillary persistence or resistance of Mycobacterium leprae to drugs are factors that may be associated with relapse. The objective of this study was assess the occurrence of relapse and associate it with the presence of Mycobacterium leprae resistant strains. In order to do that, 28 individuals who were clinically diagnosed as relapse after treatment with sulphone monotherapy, the National Division of Sanitary Dermatology scheme or multidrugtherapy. Biopsies from lesions of multibacillary patients attended by spontaneous demand were collected to verify resistance to drugs through the mouse foot pad inoculation technique. Among the samples evaluated 42.8% had bacilli susceptible to dapsone and rifampicin and 10.7% showed resistance to dapsone. No rifampicin resistant bacilli were isolated. The emergence of resistant strains, especially to rifampicin, is a threat to leprosy control programs, therefore, monitoring the spread of these strains is important because resistance pose a serious obstacle to the elimination of disease, particularly in countries where the disease is endemic.
Asunto(s)
Humanos , Animales , Masculino , Femenino , Ratones , Lepra/tratamiento farmacológico , Quimioterapia Combinada , Cumplimiento de la Medicación , Brasil , Farmacorresistencia Bacteriana , Colonias de Leprosos , Recurrencia , Resistencia a Medicamentos , Sistema Único de SaludRESUMEN
Leprosy is a complex infectious disease influenced by genetic and environmental factors. The genetic contributing factors are considered heterogeneous and several genes have been consistently associated with susceptibility like PARK2, tumor necrosis factor (TNF), lymphotoxin-α (LTA) and vitamin-D receptor (VDR). Here, we combined a casecontrol study (374 patients and 380 controls), with meta-analysis (5 studies; 2702 individuals) and biological study to test the epidemiological and physiological relevance of the interleukin-10 (IL-10) genetic markers in leprosy. We observed that the −819T allele is associated with leprosy susceptibility either in the casecontrol or in the meta-analysis studies. Haplotypes combining promoter single-nucleotide polymorphisms also implicated a haplotype carrying the −819T allele in leprosy susceptibility (odds ratio (OR)=1.40; P=0.01). Finally, we tested IL-10 production in peripheral blood mononuclear cells stimulated with Mycobacterium leprae antigens and found that −819T carriers produced lower levels of IL-10 when compared with non-carriers. Taken together, these data suggest that low levels of IL-10 during the disease outcome can drive patients to a chronic and unprotective response that culminates with leprosy
Asunto(s)
Humanos , Masculino , Femenino , Antígenos Bacterianos , Lepra , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Regulación de la Expresión Génica , Enfermedad Crónica , Estudios de Casos y Controles , Polimorfismo de Nucleótido Simple , Regiones Promotoras GenéticasRESUMEN
Multidrug-resistant tuberculosis (MDR-TB) is an increasing global problem. The extent and burden of MDR-TB varies significantly from country to country and region to region. Globally, about three per cent of all newly diagnosed patients have MDR-TB and the proportion is higher in patients who had previously received anti-tuberculosis (anti-TB) treatment reflecting the failure of programs designed to ensure complete cure of patients with tuberculosis. The management of MDR-TB is a challenge that should be undertaken by experienced clinicians at centers equipped with reliable laboratory services and implementation of DOTS-Plus strategy.
Asunto(s)
Humanos , Genes MDR , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/virología , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología , Tuberculosis/epidemiologíaRESUMEN
Biópsias de pele oriundas dos serviços de diagnóstico da hanseníase podem ser grande fonte de material para estudos retrospectivos em genética humana e do Mycobacterium leprae. No entanto, os procedimentos de fixação e inclusão em parafina podem dificultar a obtenção de DNA de qualidade para amplificação por PCR. Assim, estas amostras requerem protocolos especiais para a extração do material genético. O objetivo deste trabalho é apresentar um método alternativo para extração de DNA com base na combinação de calor e digestão enzimática. Para tanto, os cortes foram aquecidos a 120º C em solução tampão de pH 9,0, submetidos à digestão enzimática com proteinase K e o DNA foi extraído por meio de solução de fenol:clorofórmio:álcool isoamílico. A amplificação por PCR para as regiões dos genes humanos TNF e LTA foi bem sucedida para 85,4% dos espécimes. Considerando o DNA do M. leprae, obtivemos amplificação em 67,6%, 48,5%, 36,7% e 64,8% para os marcadores TA18, GTA9, TTC e RLEP, respectivamente. Concluímos que este é um método de baixo custo que proporcionou um rendimento satisfatório de DNA de boa qualidade para emprego em PCR a partir de biópsias parafinadas de pele.
Skin biopsies from leprosy diagnostic services can be great sources of material for retrospective studies concerning human and Mycobacterium leprae genetic. However, fixation and paraffin embedding procedures make difficult to obtain good quality DNA to PCR amplification. Thus, paraffin-embedded samples require special protocols to DNA extraction. The aim of this paper is to present an alternative method for DNA extraction based on the combination of heat and enzymatic digestion. The sections were heated at 120ºC at pH 9.0, submitted to enzymatic digestion with proteinase K and the DNA was extracted by using phenol:chlorophorm:isoamilic alcohol. PCR amplification for regions in TNF and LTA human genes was successful for 85.4 % of specimens. Regarding M. lepraeDNA, we obtained amplification in 67.6%, 48.5%, 36.7% and 64.8% for the TA18, GTA9, TTC and RLEP markers, respectively. We conclude that this is an inexpensive method which provided a satisfactory yield of a good quality DNA for PCR from paraffin- embedded skin biopsies.
Asunto(s)
Humanos , Bases de Datos de Ácidos Nucleicos , Epidemiología Molecular , Lepra/patología , Piel/patología , Biopsia , Colonias de Leprosos , Reacción en Cadena de la Polimerasa , Sistema Único de SaludRESUMEN
A comunicação e a cultura estão inter-relacionadas aos sistemas de participação e cidadania e constituem um papel de integração e de incentivo a promoção da saúde. Nessa perspectiva, um dos entraves está direcionado para a função da tecnologia e pesquisa; ambas correm o risco de desagregação da sociedade quando os gestores não garantem a transição e implementação do conhecimento e a efetiva promoção da saúde coletiva. A gestão da saúde não pode ser um procedimento puramente tecnicista, ela deve agregar a multiplicidade de saberes, engendrar informações e garantir a transformação em comunicações efetivas em co-dependência com as características culturais. Regiões, cidades, bairros e vilarejos possuem concepções distintas em relação a elementos como as linguagens, imagens, tabus e símbolos que podem interferir na assimilação dos projetos que estejam desconectados dessas peculiaridades.