Automatic bright-field smear microscopy for diagnosis of pulmonary tuberculosis.

In recent decades, many studies have been published on the use of automatic smear microscopy for diagnosing pulmonary tuberculosis (TB). Most of them deal with a preliminary step of the diagnosis, the bacilli detection, whereas sputum smear microscopy for diagnosis of pulmonary TB comprises detecting and reporting the number of bacilli found in at least 100 microscopic fields, according to the 5 grading scales (negative, scanty, 1+, 2+ and 3+) endorsed by the World Health Organization (WHO). Pulmonary TB diagnosis in bright-field smear microscopy, however, depends upon the attention of a trained and motivated technician, while the automated TB diagnosis requires little or no interpretation by a technician. As far as we know, this work proposes the first automatic method for pulmonary TB diagnosis in bright-field smear microscopy, according to the WHO recommendations. The proposed method comprises a semantic segmentation step, using a deep neural network, followed by a filtering step aiming to reduce the number of false positives (false bacilli): color and shape filtering. In semantic segmentation, different configurations of encoders are evaluated, using depth-wise separable convolution layers and channel attention mechanism. The proposed method was evaluated with a large, robust, and annotated image dataset designed for this purpose, consisting of 250 testing sets, 50 sets for each of the 5 TB diagnostic classes. The following performance metrics were obtained for automatic pulmonary TB diagnosis by smear microscopy: mean precision of 0.894, mean recall of 0.896, and mean F1-score of 0.895.

Histoplasmosis in HIV/AIDS patients in Amazonas, Northern Brazil.

Histoplasmosis is commonly observed in AIDS patients as a neglected opportunistic disease that has an important relationship with environmental factors. The present study described the clinical characteristics of HIV/AIDS patients diagnosed with disseminated histoplasmosis in a tertiary healthcare facility in Manaus, Amazonas, Brazil, and evaluated the patients' homes and urban environmental samples as a source of exposure to Histoplasma capsulatum. A review of medical records from 2017 to 2019 of patients with HIV/AIDS associated with histoplasmosis was carried out, as well as the collection of environmental samples in the homes of these patients. These samples were subjected to DNA extraction and then subjected to qPCR. A total of 62 patients diagnosed with HIV/AIDS and histoplasmosis were identified, which corresponds to 4.5% (n = 62/1372) of the HIV/AIDS cases detected in the period. Of these, 68% (n = 42/62) were male, with a mean age of 36 years and low education. In 47% (n = 29/62) of the cases, the diagnosis of HIV/AIDS and histoplasmosis occurred simultaneously. Mortality was 45% (n = 28/62), and 68% (n = 42/62) of these patients did not regularly use highly active antiretroviral therapy. The main symptoms found were respiratory, gastrointestinal, and weight loss, and in 81% (n = 50/62), the place of residence was in an urban area. A total of 57 environmental samples were analyzed, and the presence of Histoplasma capsulatum was not detected in any of the analyzed samples. There was a high mortality rate in the studied group of patients with AIDS and histoplasmosis. Most patients reported residing in urban areas of Manaus, with no history of travel to other areas previously known as being high risk for histoplasmosis.

Design and optimization of an improved qPCR assay for the detection of Histoplasma capsulatum.

Background: This study aimed at improving a real-time polymerase-chain-reaction (qPCR) assay for the detection of Histoplasma capsulatum, a fungal pathogen that can cause severe respiratory infections in humans, in clinical and soil samples. Methods: Primer and probes were in-silico designed, in-silico and in-vitro evaluated including clinical biopsy materials and finally subjected to a real-world application with collected soil samples. Results: Applying the qPCR assay with liver and lung biopsies from 71 patients each, including 59 patients infected with human immunodeficiency virus (HIV), as well as with Sabouraud (SAB) agar culture as the diagnostic reference standard, diagnostic accuracy of the qPCR assay of 100% (5/5) sensitivity and 96% (63/66) specificity for liver samples and 100% (4/4) sensitivity and 94% (63/67) specificity for the lung samples was recorded. When applying the assay with soil samples from caves near of Presidente Figueiredo city, Amazonas, Brazil, one sample from the Maroaga cave was confirmed as positive. Conclusions: The improved qPCR assessed in this study was successful in detecting H. capsulatum with high efficiency and accuracy in in-vitro evaluation, including the identification of the target pathogen in both clinical and environmental samples.

A genetic variant in the TRAF1/C5 gene lead susceptibility to active pulmonary tuberculosis by decreased TNF-α levels.

Host genetics are important to consider in the role of resistance or susceptibility for developing active pulmonary tuberculosis (TB). Several association studies have reported the role of variants in STAT4 and TRAF1/C5 as risk factors to autoimmune diseases. Nevertheless, more data is needed to elucidate the role of these gene variants in infectious disease. Our data reports for the first time, variant rs10818488 in the TRAF1/C5 gene (found 47% of the population worldwide), is associated with susceptibility (OR = 1.51) to development TB. Multivariate analysis evidenced association between rs10818488 TRAF1/C5 and risk to multibacillary TB (OR = 4.18), confers increased bacteria load in the lung, indicates a decreased ability to control pathogen levels in the lung, and spread of the pathogen to new hosts. We showed that the "loss-of-function" variant in TRAF1/C5 led to susceptibility for TB by decreased production of TNF-α. Our results suggest the role of variant TRAF1/C5 in susceptibility to TB as well as in clinical presentation of multibacillary TB.

Inflammasome genes polymorphisms may influence the development of hepatitis C in the Amazonas, Brazil.

Hepatitis C is considered a major public health problem caused by the hepatitis C virus (HCV). Viral infections are known to induce production of IL1ß through the signaling pathway of inflammasomes. Emerging evidences suggest that Inflammasome genes may influence the immune response against HCV as the host genetic background may contribute to the balance between acute and chronic inflammation. We investigated in 151 patients with chronic hepatitis C and 206 healthy blood donors' individuals (HD). Polymorphisms in the IL1B and IL18 genes were genotyped by PCR-RFLP, while NLRP3, CARD8, CTSB and AIM2 by RT- PCR. Serum assay of IL-1ß cytokine was performed by ELISA. 84 patients presented mild fibrosis (

Genetic polymorphisms of inflammasome genes associated with pediatric acute lymphoblastic leukemia and clinical prognosis in the Brazilian Amazon.

The immune system plays an important role in the control of cancer development. To investigate the possible association of inflammasome genes to childhood leukemia we performed a case-control study with 158 patients with acute lymphoblastic leukemia and 192 healthy individuals. The IL1B and IL18 genetic polymorphisms were genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and NLRP1, NLRP3 and P2RX7 were genotyped using Real Time quantitative PCR (qPCR). The IL1B C/T rs19644 genotype was associated with the risk of developing ALL (C/C vs. C/T + T/T OR: 2.48 [95% CI: 1.26-4.88, p = 0.006]; C/C vs C/T OR: 2.74 [95% CI: 1.37-5.51, p = 0.003]) and the NLRP1 A/T rs12150220 (OR: 0.37 [95% CI: 0.16-0.87, p = 0.023]) was associated with protection against infectious comorbidities. It was not found association between NLRP3 and P2RX7 polymorphisms and acute lymphoblastic leukemia in our study. Our results suggest that the inflammasome single-variant polymorphisms (SNVs) may play a role in the development and prognostic of childhood leukemia. However, this finds requires further study within a larger population in order to prove it.

Single-Nucleotide Variants in the AIM2 - Absent in Melanoma 2 Gene (rs1103577) Associated With Protection for Tuberculosis.

Tuberculosis (TB) remains a serious public health burden worldwide. TB is an infectious disease caused by the Mycobacterium tuberculosis Complex. Innate immune response is critical for controlling mycobacterial infection. NOD-like receptor pyrin domain containing 3/ absent in melanoma 2 (NLRP3/AIM2) inflammasomes are suggested to play an important role in TB. NLRP3/AIM2 mediate the release of pro-inflammatory cytokines IL-1ß and IL-18 to control M. tuberculosis infection. Variants of genes involved in inflammasomes may contribute to elucidation of host immune responses to TB infection. The present study evaluated single-nucleotide variants (SNVs) in inflammasome genes AIM2 (rs1103577), CARD8 (rs2009373), and CTSB (rs1692816) in 401 patients with pulmonary TB (PTB), 133 patients with extrapulmonary TB (EPTB), and 366 healthy control (HC) subjects with no history of TB residing in the Amazonas state. Quantitative Real Time PCR was performed for allelic discrimination. The SNV of AIM2 (rs1103577) is associated with protection for PTB (padj: 0.033, ORadj: 0.69, 95% CI: 0.49-0.97). CTSB (rs1692816) is associated with reduced risk for EPTB when compared with PTB (padj: 0.034, ORadj: 0.50, 95% CI: 0.27-0.94). Serum IL-1ß concentrations were higher in patients with PTB than those in HCs (p = 0,0003). The SNV rs1103577 of AIM2 appeared to influence IL-1ß release. In a dominant model, individuals with the CC genotype (mean 3.78 ± SD 0.81) appeared to have a higher level of IL-1ß compared to carriers of the T allele (mean 3.45 ± SD 0.84) among the patients with PTB (p = 0,0040). We found that SNVs of AIM2 and CTSB were associated with TB, and the mechanisms involved in this process require further study.

Toll-Like Receptor-1 Single-Nucleotide Polymorphism 1805T/G Is Associated With Predisposition to Multibacillary Tuberculosis.

Tuberculosis (TB), caused by mycobacterial species of the Mycobacterium tuberculosis complex, is a serious global health issue. Brazil is among the 22 countries with the highest number of TB cases, and the state of Amazonas has the highest incidence of TB cases in the country. Toll-like receptors (TLRs) are important pattern recognition receptors of the innate immunity and play a key role in orchestrating an effective immune response. We investigated whether the single-nucleotide polymorphisms (SNPs) 1805T/G TLR1, 2258G/A TLR2, 896A/G and 1196C/T of TLR4, 745T/C TLR6, and -1237A/G and -1486A/G of TLR9 are associated with the predisposition to TB and/or bacillary load. The SNPs genotyping was performed by nucleotide sequencing in 263 TB patients and 232 healthy controls residing in the state of Amazonas. Alleles and genotypes frequencies were similar between patients and healthy individuals for most of the investigated SNPs. Stratification of the TB patients according to their bacillary load showed that the genotype 1805TT TLR1 (rs5743618) was prevalent among paucibacillary patients [odds ratio (OR) = 0.38; 95% confidence interval (CI) = 0.19-0.76; p = 0.009] while the genotype 1805TG was common among multibacillary patients (OR = 3.72; CI = 1.65-8.4; p = 0.004). Comparison of demographic characteristics of patients to controls showed that TB is strongly associated with smoking (OR = 6.55; 95% CI = 3.2-13.6; p < 0.0001); alcohol use disorder (OR = 7.14; 95% CI = 3.7-13.9; p < 0.0001); and male gender (OR = 3.66; 95% CI = 2.52-5.3; p < 0.0001). Multivariate logistic regression demonstrated that alcoholism (OR = 2.93; 95% CI = 1.05-8.16; p = 0.03) and the 1805G allele (OR = 2.75; 95% CI = 1.33-5.7; p = 0.006) are predictive variables for multibacillary TB. Altogether, we suggest that the TLR1 1805G allele may be a relevant immunogenetic factor for the epidemiology of TB together with environmental, sociodemographic, and behavioral factors.

Metabolites from endophytic Aspergillus fumigatus and their in vitro effect against the causal agent of tuberculosis / Metabolitos de Aspergillus fumigatus endofítico e seu efeito in vitro contra o agente causal da tuberculose

ABSTRACT Tuberculosis (TB) remains one of the most deadly communicable infectious diseases, causing 1.4 million deaths in 2015 worldwide due to many conditions, including the inadequate treatment and the emergence of multidrug-resistant strains of the causal agent, Mycobacterium tuberculosis. Therefore, drugs developed from natural sources, as microorganisms and plant extracts, are a frequent target for the research and discovery of antimicrobial compounds. The current study started the characterization of compounds produced by an Aspergillus fumigatus isolated from copaíba (Copaifera multijuga) that efficiently inhibits M. tuberculosis by releasing the compounds into the fermentation broth under specific culture conditions. A preliminary assay was carried out with a correlate species, M. smegmatis, aiming to detect an antimicrobial effect related to A. fumigatus fermentation broth. The direct use of this substrate in antibiosis assays againstM. tuberculosis H37Rv strain (ATCC 27294) allowed the detection of antimicrobial activity with a minimal inhibitory concentration of 256 μg mL-1, demonstrating that purification processes developed by the Biotage Flash Chromatography System are robust and reliable techniques for purification of compounds from natural sources. Also, this chromatographic system can be used in combination with specific biochemical tests, improving the search for reliable results. We conclude that this fraction can express a broad action range, inhibiting both Mycobacterium species used as target organisms.

RESUMO A tuberculose continua a ser uma das doenças infecciosas transmissíveis mais mortais, causando 1,4 milhão de mortes em 2015 em todo o mundo devido a vários fatores, incluindo o tratamento inadequado e o surgimento de cepas multirresistentes do agente causal, Mycobacterium tuberculosis. Portanto, as drogas desenvolvidas a partir de fontes naturais, como micro-organismos e extratos de plantas, são um alvo freqüente para a pesquisa e descoberta de compostos antimicrobianos. O presente estudo foi um ponto de partida para caracterizar compostos produzidos por um Aspergillus fumigatus isolado de copaíba (Copaifera multijuga) que inibe eficientemente M. tuberculosis, liberando os compostos no caldo de fermentação em condições de cultura específicas. Realizou-se um ensaio preliminar com uma espécie correlata, M. smegmatis, com o objetivo de detectar um efeito antimicrobiano relacionado ao caldo de fermentação de A. fumigatus. O uso direto deste substrato em ensaios de antibiose contra a estirpe H37Rv de M. tuberculosis (ATCC 27294) permitiu a detecção de atividade antimicrobiana com uma concentração inibitória mínima de 256 μg mL-1, demonstrando que os processos de purificação desenvolvidos pelo Biotage Flash Chromatography System são técnicas robustas e confiáveis para purificar compostos de fontes naturais. Além disso, este sistema cromatográfico pode ser usado em combinação com testes bioquímicos específicos, melhorando a busca de resultados confiáveis. Concluímos que esta fração pode expressar uma ampla gama de ação, inibindo ambas as espécies de Mycobacterium utilizadas como organismos-alvo.

Association between the PTPN22 1858C/T gene polymorphism and tuberculosis resistance.

Previous studies identified the functional polymorphism 1858C/T in the gene PTPN22 in association with several autoimmune diseases and with resistance to tuberculosis (TB). This study is the first to investigate the association between pulmonary TB and the PTPN22 1858C/T polymorphism in the Brazilian Amazon. We conducted a case-control study involving a group of 413 individuals, comprised of 208TB carriers and 205 controls. No significant association between the PTPN22 1858T allele frequency in controls (2.4%) and TB carriers (2.7%, p=0.982, odds ratio (OR)=0.89, 95% confidence interval=0.37-2.13) was identified in the Brazilian Amazon population. An additional evaluation by meta-analysis, however, suggested a protective role of the T allele in relation to TB (pooled OR=0.44, p=0.011). These results suggest that the PTPN22 1858T allele serves as a protective genetic factor for TB in those individuals who carry this minor allele.

The influence of a TNF gene polymorphism on the severity of rheumatoid arthritis in the Brazilian Amazon.

PURPOSE: The aim of this study was to investigate the influence of the TNF -308 G/A polymorphism in the promoter region of the tumor necrosis factor-α gene on the susceptibility and severity of rheumatoid arthritis (RA) in individuals from the Brazilian Amazon. METHODS: A total of 323 individuals-192 healthy controls without arthritis and 131 individuals suffering from arthritis-were genotyped for this polymorphism using a methodology based on PCR-RFLP. RESULTS: The frequency of the A allele (TNF2) in rheumatoid arthritis sufferers was not significantly higher than in the controls (p=0.926; OR=0.97; confidence interval 0.54-1.76). However, using a logistic regression model, when the patients were stratified according to whether the manifestations were preponderantly articular or systemic, there was a strong association between the TNF2 allele and systemic arthritis (p=0.001; OR=5.89; confidence interval=1.98-17.5) as well as the use of anti-TNF immunotherapy (p=0.023; OR=1.10; confidence interval=1.00-1.14). The main factors that were found to influence the risk of extra-articular disease were age greater than or equal to 60 years (p=0.008; OR=4.06; confidence interval=1.45-11.38), disease duration greater than 10 years (p=0.031; OR=3.10; confidence interval=1.11-8.63) and positive rheumatoid factor (p=0.035; OR=2.07; confidence interval=1.05-4.09). CONCLUSIONS: These results suggest that the TNF2 allele is associated with the more serious forms of the disease in individuals from the Brazilian Amazon but not with a risk for developing RA.

Evaluation of new strategies for the diagnosis of tuberculosis among pediatric contacts of tuberculosis patients.

BACKGROUND: In young children, underdiagnosis and diagnostic delay have an adverse effect on morbidity and mortality of tuberculosis (TB). This study evaluated new strategies for early TB diagnosis using an outpatient protocol in children between 0 and 5 years of age, with a recent household TB contact. METHODS: Case recruitment was performed in Manaus, Amazonas, Brazil, from 2008 to 2009. Epidemiologic and clinical data, tuberculin test, chest radiograph and 2 induced sputum respiratory samples from each participant were obtained. Laboratory diagnosis was based on Lowenstein-Jensen (LJ) culture, mycobacteria growth indicator tube (MGIT) and polymerase chain reaction. We conducted a study of comparison of diagnostic tests and a study of cases and controls to identify the clinical characteristics of the population with positive culture and polymerase chain reaction results. RESULTS: A total of 102 children were evaluated. Thirty-two fulfilled criteria of suspicion of TB. MGIT was more sensitive (P = 0.035) and faster (P < 0.001) than LJ. Clinical score, MGIT, LJ and polymerase chain reaction presented no concordance or slight concordance. A positive MGIT culture was only associated with a strong tuberculin test reaction (P = 0.026). The combination of MGIT with the clinical score allowed the diagnosis of 33% more cases with little or no symptomatology compared with the exclusive use of the clinical classification. CONCLUSIONS: The sensitivity and speed of MGIT demonstrate the utility of liquid cultures for the diagnosis in children. Furthermore, these results suggest that the use of MGIT in children presenting recent household TB contact and a strong tuberculin test reaction may be a strategy to improve early TB diagnosis.

Molecular characterisation of the causative agents of Cryptococcosis in patients of a tertiary healthcare facility in the state of Amazonas-Brazil.

As there are four major molecular types of Cryptococcus neoformans (VNI, VNII, VNIII and VNIV) and four molecular types of Cryptococcus gattii (VGI, VGII, VGIII and VGIV), it is important to identify the specific groups causing cryptococcosis in different geographical regions. Here, we investigated the molecular types of 57 cryptococcal isolates from patients in a tertiary care hospital in the state of Amazonas, Brazil, between 2006 and 2010. The isolates were characterised by PCR fingerprinting using the M13 minisatellite and confirmed by URA5-RFLP analysis, and the presence of specific genes from the mating type locus (MATα and MATa) of these species was analysed by PCR. Most of the patients were male (66.7%), between 16 and 30 years of age (51.7%), and HIV-positive (75.0%). Most isolates were collected from cerebrospinal fluid samples (71.7%). Most of the C. neoformans isolates (n=40) were characterised as members of the VNI molecular group (n=39), a unique isolate was characterised as VNII whereas all isolates of C. gattii (n=17) were members of the VGII molecular group. With regard to mating types, 55 isolates were type 'α', and only two were type 'a'. This study revealed the prevalence of the VNI molecular group and provides the first reported observation of the VNII molecular group in the northern region of Brazil.

Avaliação do protocolo PCR4 de Marchetti em tecidos parafinizados para o diagnóstico da tuberculose cutânea e ganglionar / Evaluation of Marchetti PCR4 amplification assay to the diagnosis of cutaneous and lymph node tuberculosis from formalin-fixed paraffin-embedded tissue

INTRODUÇÃO: A tuberculose cutaneoganglionar (TbCG) corresponde a 25,4 por cento dos casos de tuberculose (Tb) extrapulmonar no estado do Amazonas. Os métodos tradicionais, bacteriológicos e histopatológicos envolvem algumas dificuldades diagnósticas, e a reação em cadeia da polimerase (PCR) surge como método alternativo, podendo propiciar resultados específicos e em menor tempo. Nesse sentido, verificou-se a acurácia do protocolo PCR4 de Marchetti et al. no diagnóstico da TbCG comparativamente aos métodos bacteriológicos e histopatológicos. MATERIAIS E MÉTODOS: Realizou-se o nested-PCR com oligonucleotídeos para a IS6110 do complexo do M. tuberculosis em 83 amostras parafinizadas, sendo 52 cutâneas e 31 ganglionares, de pacientes clinicamente suspeitos de TbCG. Todos os casos foram avaliados pelos métodos bacteriológicos e histopatológicos. Foi realizada análise da acurácia entre os resultados obtidos na PCR em relação ao cultivo e à histopatologia. RESULTADOS E DISCUSSÃO: A positividade da PCR em todos os casos estudados foi de 50,6 por cento (42/83), sendo de 59,6 por cento (31/52) em amostras cutâneas e de 35,5 por cento (11/31) nas ganglionares. Em ambos os grupos foram observados resultados falso-positivos e falso-negativos. Algumas hipóteses que podem justificar estes resultados estão relacionadas à presença da IS6110 em micobactérias ambientais da região amazônica e à não-padronização da amostra de DNA amplificado. CONCLUSÃO: O protocolo em avaliação apresentou positividade em percentual semelhante a diferentes protocolos existentes na literatura. Sugere-se o uso da PCR em tecidos parafinizados associada com o cultivo ou com a histopatologia para o diagnóstico definitivo de Tb ganglionar. Para as lesões cutâneas continua sendo necessária a busca de protocolo que amplie a acurácia do método.

BACKGROUND: Cutaneous lymph node tuberculosis (CLTb) represents 25.4 percent of all cases of extra-pulmonary Tb in the state of Amazonas. The current methods of diagnose including bacteriological and histopathological assays involve some technical difficulties, and the polymerase chain reaction - PCR arise as an alternative method allowing specific results faster than the others. In this context the accuracy of PCR4 Marchetti et al. protocol was compared with traditional methods. MATERIAL AND METHOD: Nested-PCR for IS6110 (123 pb) were applied on 83 CLTb suspicious formalin fixed and paraffin embedded samples of tissues (52 cutaneous and 31 lymph node), obtained from 1997 to 2002. All cases were evaluated by bacteriological and histopathological methods. Accuracy analyses were carried out between the PCR amplification results and those related on bacteriological and histopathological methods. RESULTS AND DISCUSSIONS: Positive results of PCR4 were about 50.6 percent (59.6 percent in cutaneous samples and of 35.5 percent in lymph nodes samples). In both groups were observed false-negative and false-positive results. Some hypotheses that explain those results are related to the presence of IS6110 in environmental mycobacterias in the Amazon region and the absence of standardized DNA concentration to amplification assays. CONCLUSIONS: The proposed protocol was as positive as others ones available in the literature. Definitive Tb diagnostic can be obtained on lymph node paraffin embedded PCR in association with bacteriological or histopathological method. A better accuracy of an amplification assay applied to cutaneous Tb suspicious lesions has to be still under research.

PKO: alternative method for isolating mycobacteria from sputum

We elaborated an alternative culture method, which we denominated PKO (initials in tribute of respect to Petroff, Kudoh and Ogawa), for isolating Mycobacterium tuberculosis from sputum for diagnosis of pulmonary tuberculosis (TB), and to compare its performance with the Swab and Petroff methods. For the technique validation, sputum samples from patients suspected of pulmonary TB cases were examined by acid-fast microscopy (direct and concentrated smear), PKO, Swab and Petroff methods. We found that Petroff and PKO methods have parity in the effectiveness of M. tuberculosis isolation. However, by the PKO method, 65 percent of isolated strains were detected in a period of £15 days, while by the Petroff method the best detection was in an interval of 16-29 days (71 percent). In positive smear samples, the average time of PKO isolation is only superior to the one related for Bactec 460TB. In conclusion, the exclusion of the neutralization stage of pH in the PKO reduces the manipulation of the samples, diminishes the execution time of the culture according to the Petroff method and facilitates the qualification of professionals involved in the laboratorial diagnosis of Tuberculosis.

Foi elaborado um método de cultivo alternativo, denominado por nós PKO (iniciais referentes à Petroff, Kudoh e Ogawa), para o isolamento do Mycobacterium tuberculosis em amostras de escarro para o diagnóstico da tuberculose pulmonar (TB). Para validação da técnica, amostras de escarro de pacientes suspeitos de TB foram submetidas aos métodos de baciloscopia (direta e pós-concentração), PKO, Swab e Petroff. A análise comparativa entre o método de Petroff e o PKO mostrou paridade de resultados em relação ao isolamento e número de colônias de M. tuberculosis. Porém, pelo método PKO, 65 por cento das cepas isoladas foi detectada em um período £15 dias, enquanto que pelo método de Petroff a melhor detecção ocorreu em um intervalo de 16-29 dias (71 por cento). O tempo médio de isolamento pelo PKO é somente superior ao sistema comercial Bactec 460TB em amostras positivas na baciloscopia. A exclusão da etapa de neutralização de pH no método PKO reduz a manipulação das amostras, diminui o tempo de execução do cultivo em relação ao de Petroff e facilita o treinamento de profissionais que realizam o diagnóstico laboratorial da TB.

Avaliação da reação em cadeia da polimerase no diagnóstico da tuberculose pulmonar em pacientes indígenas e não indígenas / Evaluation of polymerase chain reaction in the diagnosis of pulmonary tuberculosis in indigenous and non-indigenous patients

OBJETIVO: Avaliar a acurácia dos métodos bacteriológicos e da reação em cadeia da polimerase com oligonucleotídeos específicos para a IS6110 do complexo Mycobacterium tuberculosis, em amostras de escarro de indígenas e não indígenas. MÉTODOS: Analisaram-se 214 amostras de escarro (154 de indígenas e 60 de não indígenas) quanto à acurácia da baciloscopia direta e pós-concentração, cultivo e reação em cadeia da polimerase. RESULTADOS: Ambos os métodos baciloscópicos, quando comparados com o cultivo ou a reação em cadeia da polimerase foram de baixa sensibilidade. A especificidade variou de 91 por cento a 100 por cento, sendo a baciloscopia pós-concentração menos específica. Nas amostras indígenas constataram-se três vezes mais isolamentos de micobactérias não tuberculosas do que nas não indígenas. Resultados da reação em cadeia da polimerase aparentemente falsos-positivos e negativos foram encontrados com maior freqüência na população indígena. CONCLUSÃO: Baciloscopias positivas para bacilos álcool-acidorresistentes com isolamento de micobactérias não tuberculosas e reação em cadeia da polimerase positiva estabelecem as hipóteses de: existência na Amazônia de espécies de micobactérias não tuberculosas com regiões do DNA homólogas à IS6110 ou ainda que possuam a IS6110, até então só descrita no complexo M. tuberculosis; impossibilidade de isolamento do M. tuberculosis pelo crescimento mais rápido de micobactérias não tuberculosas presentes nas amostras de escarro, por colonização da orofaringe ou da lesão tuberculosa; presença de DNA de M. tuberculosis devida a antecedente de tuberculose. A ausência de positividade em resultados bacteriológicos com reação em cadeia da polimerase positiva sugere questões técnicas inerentes aos métodos bacteriológicos ou precedentes de tuberculose.

OBJECTIVE: To evaluate the accuracy of bacteriological methods and of polymerase chain reaction (with primers specific for IS6110 of the Mycobacterium tuberculosis complex) in testing sputum samples from indigenous (Amerindian) and non-indigenous patients. METHODS: A total of 214 sputum samples (154 from indigenous patients and 60 from non-indigenous patients) were analyzed in order to determine the accuracy of smear microscopy (direct and concentrated versions) for acid-fast bacilli, culture, and polymerase chain reaction. RESULTS: Both microscopy methods presented low sensitivity in comparison with culture and polymerase chain reaction. Specificity ranged from 91 percent to 100 percent, the concentrated acid-fast smear technique being the least specific. Nontuberculous mycobacteria were isolated three times more frequently in samples from indigenous patients than in those from non-indigenous patients. False-positive and false-negative polymerase chain reaction results were more common in the indigenous population. CONCLUSION: Positivity and isolation of nontuberculous mycobacteria in the acid-fast smear in conjunction with polymerase chain reaction positivity raise the following hypotheses: nontuberculous mycobacteria species with DNA regions homologous to, or even still possessing, the M. tuberculosis IS6110 exist in the Amazon; colonization of the oropharynx or of a tuberculous lesion accelerates the growth of the nontuberculous mycobacteria present in the sputum samples, making it impossible to isolate M. tuberculosis; A history of tuberculosis results in positivity for M. tuberculosis DNA. The absence of bacteriological positivity in the presence of polymerase chain reaction positivity raises questions regarding the inherent technical characteristics of the bacteriological methods or regarding patient history of tuberculosis.

Evaluation of polymerase chain reaction in the diagnosis of pulmonary tuberculosis in indigenous and non-indigenous patients.

OBJECTIVE: To evaluate the accuracy of bacteriological methods and of polymerase chain reaction (with primers specific for IS6110 of the Mycobacterium tuberculosis complex) in testing sputum samples from indigenous (Amerindian) and non-indigenous patients. METHODS: A total of 214 sputum samples (154 from indigenous patients and 60 from non-indigenous patients) were analyzed in order to determine the accuracy of smear microscopy (direct and concentrated versions) for acid-fast bacilli, culture, and polymerase chain reaction. RESULTS: Both microscopy methods presented low sensitivity in comparison with culture and polymerase chain reaction. Specificity ranged from 91% to 100%, the concentrated acid-fast smear technique being the least specific. Nontuberculous mycobacteria were isolated three times more frequently in samples from indigenous patients than in those from non-indigenous patients. False-positive and false-negative polymerase chain reaction results were more common in the indigenous population. CONCLUSION: Positivity and isolation of nontuberculous mycobacteria in the acid-fast smear in conjunction with polymerase chain reaction positivity raise the following hypotheses: nontuberculous mycobacteria species with DNA regions homologous to, or even still possessing, the M. tuberculosis IS6110 exist in the Amazon; colonization of the oropharynx or of a tuberculous lesion accelerates the growth of the nontuberculous mycobacteria present in the sputum samples, making it impossible to isolate M. tuberculosis; A history of tuberculosis results in positivity for M. tuberculosis DNA. The absence of bacteriological positivity in the presence of polymerase chain reaction positivity raises questions regarding the inherent technical characteristics of the bacteriological methods or regarding patient history of tuberculosis.

Análise de diferentes primers utilizados na PCR visando ao diagnóstico da tuberculose no Estado do Amazonas / Analysis of different primers used in the PCR method: diagnosis of tuberculosis in the State of Amazonas, Brazil

INTRODUÇAO: Há diferentes primers sendo testados para a detecção do DNA do Mycobacterium tuberculosis. A acuidade da reação em cadeia da polimerase (PCR) depende da existência da seqüência alvo no bacilo e de os testes serem realizados em cepas isoladas ou em amostras clínicas. OBJETIVO: Verificar a presença das seqüências de DNA alvo mais relatadas na literatura para o diagnóstico da tuberculose em amostras clínicas usando como controle positivo as respectivas cepas de M. tuberculosis isoladas. MÉTODO: Oitenta e uma amostras clínicas de pacientes com suspeita de tuberculose foram submetidas à baciloscopia e cultivo. A técnica de PCR foi realizada nas amostras clínicas e cepas isoladas com primers específicos para os seguintes alvos: IS6110, 65 kDa, 38 kDa e MPB64. RESULTADOS: Em 24 amostras com baciloscopia e cultivo negativos, a PCR também foi negativa com todos os primers testados. Em 19 amostras com baciloscopia positiva e nas cepas isoladas obteve-se 100 por cento de resultados positivos nas PCR, exceto nas PCR em amostras clínicas com os primers para a seqüência MPB64 (89,4 por cento). Em 38 amostras com baciloscopia negativa e cultivo positivo, as PCR tiveram resultados variáveis, sendo que os primers específicos que amplificam o fragmento de 123 pb da seqüência IS6110 foram os que forneceram os maiores percentuais de positividade (92,1 por cento), concordância diagnóstica (0,9143), co-positividade (94,7 por cento) e co-negatividade (100 por cento). CONCLUSAO: As seqüências IS6110, 38 kDa, MPB64 e 65 kDa foram encontradas no genoma de todas as cepas de M. tuberculosis isoladas desses pacientes do Estado do Amazonas. O protocolo utilizado no processamento das amostras clínicas e os primers específicos utilizados para amplificação do fragmento de 123 pb da seqüência IS6110 demonstraram maior eficiência no diagnóstico da tuberculose pulmonar (paucibacilar) em comparação com a literatura.

HLA in Brazilian Ashkenazic Jews with chronic dermatophytosis caused by Trichophyton rubrum

A freqüência dos HLA foi analisada em 25 Judeus Ashkenazitas, não consangüíneos, residentes em São Paulo, Brasil, com dermatofitose crônica causada por T. rubrum e em 25 indivíduos sadios, pertencentes ao mesmo grupo étnico dos pacientes. Observou-se valor estatisticamente significante (p<0,05) para HLA-B14 associado a resistência à dermatofitose crônica enquanto HLA-DQB1*06 (p=0,05) possivelmente relacionado a susceptibilidade. Estes achados indicam que o desenvolvimento da dermatofitose crônica pode ser influenciado por genes localizados no cromossomo 6, na região do complexo principal de histocompatibilidade.

PCR in the diagnosis of cutaneous tuberculosis

Seeking to improve the laboratory diagnosis of Cutaneous Tuberculosis, a study was carried out on the application of PCR technique in macerated, decontaminated(with 4 percent H2SO4 for elimination of normal microbiot), neutralized (with 4 percent NaOH)biopsies tissues samples stored at -20liC. Of the 37 samples submitted for study, 16.22 percent were positive by microscopy for acid-fast bacilli (concentrated method) and in 43.24 percent the Mycobacterium tuberculosis was isolated in L que wenstein-Jensen medium. Using a M. tuberculosis complex specific primer set (gene sequence for 16S rDNA), the mycobacterial DNA was detected in 24.32 percent of the biopsies. The sensitivity and specificity of PCR were 43.7 percent and 90.4 percent, respectively. Due to low sensitivity and discrepant results between bacteriological techniques and PCR methodology, the samples were repeated in a new PCR with primers for the IS6110 target. The sensitivity and specificity of PCR for the IS6110 target obtained 100 percent in comparison with the culture method. The results confirm the effectiveness of PCR methodology using primers for the IS6110 gene sequence and permit the PCR method to be applied to frozen cutaneous biopsies sent by services that do not identify the M. tuberculosis by the biology molecular method.