PCR identification of Mycobacterium tuberculosis complex in a clinical sample from a patient with symptoms of tuberculous spondylodiscitis

A 42-year-old male complaining of thoracic spine pain was admitted to the hospital for evaluation. An X-ray and computer tomography of the thoracic spine showed spondylodiscitis of the L3 lumbar and L2-L3 intervertebral disk. The tuberculin skin test (PPD) was strongly positive. A radioscopy-guided fine needle aspirate of the affected area was cultured but did not reveal the cause of the disease. Two biopsy attempts failed to reveal the cause of the disease by culturing or by acid-fast-resistant staining (Ziehl Neelsen) of the specimens. A third biopsy also failed to detect the infectious agent by using microbiological procedures, but revealed the presence of a 245-bp amplicon characteristic of the Mycobacterium tuberculosis complex after PCR of the sample. The result demonstrates the efficacy of PCR for the identification of M. tuberculosis in situations in which conventional diagnosis by culturing techniques or direct microscopy is unable to detect the microorganism. Following this result the patient was treated with the antituberculous cocktail composed by rifampicin, pirazinamide and isoniazid during a six-month period. At the end of the treatment the dorsalgia symptoms had disappeared.

Preliminary epidemiological study of latent tuberculosis in Mexican agricultural workers in the Niagara region, Canada

It is well documented that the majority of Tuberculosis (TB) cases diagnosed in Canada are related to foreign-bom persons from TB high-burden countries. The Canadian seasonal agricultural workers program (SAWP) operating with Mexico allows migrant workers to enter the country with a temporary work permit for up to 8 months. Preiimnigration screening of these workers by both clinical examination and chest X-ray (CXR) reduces the risk of introducing cases of active pulmonary TB to Canada, but screening for latent TB (LTBI) is not routinely done. Studies carried out in industrialized nations with high immigration from TBendemic countries provide data of lifetime LTBI reactivation of around 10% but little is known about reactivation rates within TB-endemic countries where new infections (or reinfections) may be impossible to distinguish from reactivation. Migrant populations like the SAWP workers who spend considerable amounts of time in both Canada and TBendemic rural areas in Mexico are a unique population in terms of TB epidemiology. However, to our knowledge no studies have been undertaken to explore either the existence of LTBI among Mexican workers, the probability of reactivation or the workers' exposure to TB cases while back in their communities before returning the following season. Being aware of their LTBI status may help workers to exercise healthy behaviours to avoid TB reactivation and therefore continue to access the SAWP. In order to assess the prevalence of LTBI and associated risk factors among Mexican migrant workers a preliminary cross sectional study was designed to involve a convenience sample of the Niagara Region's Mexican workers in 2007. Research ethics clearance was granted by Brock University. Individual questionnaires were administered to collect socio-demographic and TB-related epidemiological data as well as TB knowledge and awareness levels. Cellular immunity to M tuberculosis was assessed by both an Interferon-y release assay (lGRA), QuantiFERON -TB Gold In-Tube (QFf™) and by the tuberculin skin test (TSn using Mantoux. A total of 82 Mexican workers (out of 125 invited) completed the study. Most participants were male (80%) and their age ranged from 22 to 65 years (mean 38.5). The prevalence of LTBI was 34% using TST and 18% using QFTTM. As previously reported, TST (using ~lOmm cut-off) showed a sensitivity of 93.3% and a specificity of 79.1 %. These findings at the moment cannot predict the probability of progression to active TB; only longitudinal cohort studies of this population can ascertain this outcome. However, based on recent publications, lORA positive individuals may have up to 14% probability of reactivation within the next two years. Although according to the SA WP guidelines, all workers undergo TB screening before entering or re-entering Canada, CXR examination requirements showed to be inconsistent for this population: whereas 100% of the workers coming to Canada for the first time reported having the procedure done, only 31 % of returning participants reported having had a CXR in the past year. None of the participants reported ever having a CXR compatible with TB which was consistent with the fact that none had ever been diagnosed with active pulmonary TB and with only 3.6% reporting close contact with a person with active TB in their lifetime. Although Mexico reports that 99% of popUlation is fully immunized against TB within the first year of age, only 85.3% of participants reported receiving BOC vaccine in childhood. Conversely, even when TST is not part of the routine TB screening in endemic countries, a suqDrisingly high 25.6% reported receiving a TST in the past. In regards to TB knowledge and awareness, 74% of the studied population had previous knowledge about (active) TB, 42% correctly identified active TB symptomatology, 4.8% identified the correct route of transmission, 4.8% knew about the existence of LTBI, 3.6% knew that latent TB could reactivate and 48% recognized TB as treatable and curable. Of all variables explored as potential risk factors for LTBI, age was the only one which showed statistical significance. Significant associations could not be proven for other known variables (such as sex, TB contact, history of TB) probably because of the small sample size and the homogeneity of the sample. Screening for LTBI by TST (high sensitivity) followed by confirmation with QFT''"'^ (high specificity) suggests to be a good strategy especially for immigrants from TB high-burden countries. After educational sessions, workers positive for LTBI gained greater knowledge about the signs and symptoms of TB reactivation as well as the risk factors commonly associated with reactivation. Additionally, they were more likely to attend their annual health check up and request a CXR exam to monitor for TB reactivation.

DNA damage in peripheral blood mononuclear cells of patients undergoing anti-tuberculosis treatment

Tuberculosis (TB), a chronic infectious disease, is a major cause of morbidity and mortality worldwide. Expression of iNOS and consequent production of NO during the inflammatory process is an important defense mechanism against TB bacteria. We have tested whether pulmonary TB patients undergoing anti-tuberculosis treatment present DNA damage, and whether this damage is related to oxidative stress, by evaluating total hydrophilic antioxidant capacity and iNOS expression. DNA damage in peripheral blood mononuclear cells from patients and healthy tuberculin test (PPD) positive controls was evaluated by single-cell gel electrophoresis (comet assay), and iNOS expression was measured by qPCR. We also evaluated total hydrophilic antioxidant capacity in plasma from patients and controls. Compared to controls, pulmonary TB patients under treatment presented increased DNA damage, which diminished during treatment. Also, the antioxidant capacity of these individuals was increased at the start of treatment, and reduced during treatment. TB patients showed lower iNOS expression, but expression tended to increase during treatment. Our results indicate that pulmonary TB patients under anti-TB treatment exhibit elevated DNA damage in peripheral blood mononuclear cells. This damage was not related to nitric oxide but may be due to other free radicals. (C) 2012 Elsevier B.V. All rights reserved.

Teste tuberculínico em indivíduos com infecção pelo vírus da imunodeficiência humana: relação com número de linfócitos T periféricos e atividade tuberculosa

OBJETIVO: Avaliar os resultados do teste tuberculínico e relacioná-los com a presença ou não de tuberculose em atividade e com a contagem de linfócitos T CD4+/CD8+. MÉTODOS: Foram revisados 802 prontuários de pacientes com síndrome da imunodeficiência adquirida atendidos no período de agosto de 1985 a março de 2003. Cento e oitenta e cinco pacientes realizaram o teste tuberculínico (23,1%) e, destes, 107 eram do sexo masculino (57,8%). A média de idade no grupo de reatores ao teste tuberculínico foi de 30,6 anos, com desvio-padrão de 6,62 anos, e entre os não reatores de 34,45 anos com desvio-padrão de 10,32 anos. Foram constituídos dois grupos de estudo: reatores ao teste tuberculínico, com 28 pacientes, e não reatores ao teste tuberculínico, com 157 pacientes. RESULTADOS: Grande parte dos indivíduos foi pouco responsiva ao teste tuberculínico. Constatou-se, no grupo de reatores, maior porcentagem de indivíduos com tuberculose ativa à época da realização do teste, quando se comparou com os não reatores. Dez pacientes entre os reatores e onze entre os não reatores apresentavam alguma forma clínica de tuberculose em atividade à época da realização do teste, sendo que seis do primeiro grupo e oito do segundo tinham contagem de linfócitos T CD4+ menor que 200 células/mm³. CONCLUSÃO: Indurações maiores do que 5 mm não se relacionaram com contagens absolutas mais altas de células T CD4+.

Aspectos epidemiológicos da tuberculose pulmonar nos municípios de Botucatu, Conchas, São Manuel e Avaré, Estado de São Paulo, Brasil, de 1963 a 1972

O diagnóstico da situação da tuberculose pulmonar nos Municípios de Botucatu, Conchas, São Manuel e Avaré, SP, Brasil, baseou-se na prevalência e risco de infecção em escolares de lª série, em 1972, e em dados de morbidade e mortalidade específica, de 1963 a 1972. Realizou-se inquérito tuberculínico em 2.913 escolares, com PPD 23 RT-2UT; foram analisados os prontuários de 718 casos inscritos no CSI de Botucatu e os atestados de óbito de residentes, incluindo-se os ocorridos em hospitais especializados. As taxas de prevalência de infecção para os quatro Municípios foram respectivamente 2,4%; 6,8%; 1,9% e 4,5%. Para a idade de 7,5 anos, os riscos de infecção foram: 0,27%; 0,32; 0,20% e 0,34%. O nível de infecção apurado caracterizou o conjunto como área de média prevalência da tuberculose pulmonar. Os índices de prevalência de infecção relacionaram-se diretamente à incidência de casos bacilíferos de cada Município. em 530 casos com baciloscopia, houve 62,0% de positividade. As formas radiológicas moderada e avançada predominaram principalmente em adultos jovens do sexo masculino. Foram registradas alta taxa de abandono (39,5%}, baixo percentual de cura (17,9%) e 3,4% de óbitos; ao redor de 32,0% estava em tratamento. Não houve diferenças estatisticamente significativas das proporções de abandono, segundo procedência por Município ou formas da doença. Invocaram-se razões técnico-administrativas do sistema vigente para explicar esses achados. A incidência média de casos confirmados, no período, foi de 35,4 em Conchas, 33,1 em Avaré, 23,7 em Botucatu e 18,5 em São Manuel (por 100.000). Com casos confirmados e suspeitos os índices foram: 57,6; 48,8; 43,8 e 35,3. Os índices médio-anuais de mortalidade, foram: 7,6 em Botucatu, 12,14 em Conchas, 5,0 em São Manuel e 18,7 em Avaré (por 100.000). Apenas em Botucatu registrou-se declínio das taxas de mortalidade de 1963 a 1972. A mortalidade específica em adultos jovens ainda era elevada, predominando no sexo masculino, principalmente em Conchas. Sublinhou-se a necessidade de descentralizar as atividades antituberculose para atingir um controle eficiente.

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Murine alpha-2-macroglobulin increase during inflammatory responses and tumor growth

Objective and Design: To determine the alpha-2-macroglobulin (alpha2M) levels in mice during acute and chronic inflammatory responses. Materials and Methods: Inflammation was induced by one of the following stimuli: carrageenin, zymosan, lipopolysacharide, thioglycollate, bacilli Calmette Guerin, PPD (in pre-immunized and non-immunized animals) and tumor cells. The concentration of alpha2M was determined in plasma or peritoneal liquid by electroimmunoassay. Results: In all the treatments employed, the plasma levels of alpha2M were higher than in untreated animals. This increase varied from 9%, 24 h after injection up a maximum of 66% 72 h post-injection. When compared to animals injected only with saline, the increases were significant 48 h after treatment with either zymosan or LPS, and 72 h after treatment with either thioglycollate or carrageenin. Treatment with BCG triggers an increase in alpha2M levels after 24 h (18.60%) and 48 h (27.90%). Immunized mice presented higher levels of this protein than non-immunized animals after challenge with PPD. The growth of Ehrlich tumor cells in the peritoneal cavity was directly correlated with the local levels of alpha2M which increased 3.5 fold, 10 days after injection. Conclusions: These results strongly indicate that in mice, the concentration of alpha2M can increase during acute and chronic inflammatory reactions with kinetics dependent on the particular kind of inflammatory agent.

Prevalência de reagentes ao teste de tuberculinização no rebanho bovino leiteiro da região de São Carlos - SP

Tuberculosis (TB), an infectious disease readily transmitted between animals and humans, is an great threat to public and animal health a like and has been declared an international emergency. According to the WHO, animal TB is a source of much concern, especially in the poorer countries where there is little awareness of the problem. Thus, improvements in veterinary health are essential, with regard to infection by Mycobacterium bovis, particularly in areas where the public is at risk. The prevalence of infected animals in Brazil is about 1%, whereas, in the dairy cattle region of São Carlos, the average proportion of positive tests was found to be 3. 1% from 1987 to 1996. The current research was carried out to investigate the prevalence, in the region, of dairy cattle reacting positively to the tuberculin test, from 1997 to the first half of 2001. The results were obtained by searching through the data collected by the Federal Inspection Authority, and they indicated an average prevalence of 1.3% over the period, similar to the official national average. It was concluded that bovine TB continues to be present in the region, posing a potential threat of infection to the human population.

Ocorrência de tuberculose em um hospital psiquiátrico do interior de Goiás

Objective: To investigate the prevalence of infection, disease and eventual institutional outbreak of tuberculosis in a psychiatric hospital using the PPD test, as well as testing for mycobacteria in material collected from the respiratory tree and using molecular tracking technique based on insertion sequence 6110 (IS6110). Methods: Between February and August of 2002, PPD tests were given to 74 inpatients and 31 staff members at a psychiatric hospital in the city of Rio Verde, located in the state of Goiás, Brazil. In addition, respiratory tree material collected from the inpatients was submitted to testing for Mycobacterium tuberculosis. Results: Among the patients analyzed, mycobacteria were isolated from five (6.8%): four identified as M. tuberculosis and one as M. chelonae. The M. tuberculosis isolates were sensitive to isoniazid and rifampicin, and, when submitted to the restriction fragment length polymorphism/IS6110 technique, presented unique genetic profiles, totally distinct from one another, suggesting that all of the tuberculosis cases were due to endogenous reactivation. It was not possible to characterize this group of cases as an institutional outbreak. Performing the two-step tuberculin test in the patients, the infection rates were 23% and 31%, compared with 42% among staff members, who were submitted to the one-step test. Conclusion: The results indicate a high incidence of tuberculosis infection among inpatients and hospital staff, as well as a high occurrence of the disease among inpatients.

Tendência da tuberculose em pacientes internados no hospital Nestor Goulart Reis de Américo Brasiliense-SP no período de 1994 a 2004 e prevalência da infecção entre os funcionários deste hospital em 2005

Pós-graduação em Biociências e Biotecnologia Aplicadas à Farmácia - FCFAR

Perfil epidemiológico da tuberculose urogenital no Distrito Federal, Brasil, em nove anos (2001 a 2009)

Pós-graduação em Ginecologia, Obstetrícia e Mastologia - FMB

Adenopatia cervical tuberculosa em crianças: aspectos epidemiológicos, clínicos e laboratoriais

A tuberculose constitui um sério problema de saúde pública, sendo o M. tuberculosis o principal agente da doença no Brasil. Entre as formas extra pulmonares, a ganglionar periférica é uma das mais freqüentes na infância, apesar de pouco estudada. Com o objetivo de avaliar a incidência e conhecer os aspectos epidemiológicos, clínicos e laboratoriais da tuberculose ganglionar foram atendidas no ambulatório do Hospital Ofir Loiola, no período de janeiro de 1995 a dezembro de 1996, 73 crianças entre 0-14 anos, de ambos os sexos, portadoras de linfadenopatia cervical. A amostra foi dividida em dois grupos: o primeiro constituído por 61 crianças com adenopatias de outras etiologias e o segundo formado de 12 pacientes com etiologia tuberculosa. Nesse período, para as adenopatias tuberculosas, a taxa anual de incidência na área metropolitana de Belém, por 100 mil habitantes, foi de 1,03 e para o grupo de outras etiologias a incidência foi de 4,27 e 6,15 para os anos de 1995 e 1996, respectivamente. Verificou-se que a maioria das adenopatias na infância foram inespecíficas (64,4%), entretanto, quando consideradas somente as de etiologia conhecida, o risco relativo de ser tuberculose foi de 1,17. A análise comparativa entre os dois grupos não revelou diferença estatisticamente significativa no que tange à faixa etária, sexo, estado nutricional, apresentação clínica inicial, cadeias ganglionares comprometidas e características dos linfonodos. Em ambos foi observado maior incidência em pré-escolares e no sexo masculino. O comprometimento do estado nutricional pode ter contribuído para o aumento da morbidade em 41,7% dos casos de tuberculose. A presença de massa cervical constitui a queixa principal nos dois grupos embora durante o exame tenha sido constatado comprometimento ganglionar generalizado em 75,1% das crianças com adenopatia tuberculosa. Quanto à duração dos sintomas, os casos de adenopatia tuberculosa foram atendidos a partir do primeiro mês de doença e tiveram como manifestação clínica abscesso frio em 25% dos casos. A fonte de infecção foi identificada em 1/3 dos pacientes. A reação tuberculínica com leitura >10mm foi positiva em 63,6% das crianças tuberculosas. Entre os exames bacteriológicos realizados, a cultura constituiu o elemento fundamental para o diagnóstico de tuberculose, obtendo-se 100% de positividade nos exames realizados; a baciloscopia foi de menor importância. O exame histopatológico com lesão granulomatosa compatível com tuberculose foi observado em 88,9% dos casos. O comprometimento pulmonar associado esteve presente em 27, 3% das crianças tuberculosas. A resposta à terapêutica com esquema padronizado pelo Ministério da Saúde foi satisfatória, não tendo sido observados efeitos colaterais aos medicamentos utilizados.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Tuberculosis prevalence and risk factors for water buffalo in Para, Brazil

The prevalence of and possible risk factors for tuberculosis were studied in water buffalo from Para, Brazil. In this study, 3,917 pregnant and nonpregnant female Murrah and Mediterranean buffaloes were studied; 2,089 originated from Marajo Island, and 1,108 were from the mainland. The comparative cervical tuberculin test was used as a diagnostic test for tuberculosis in these animals. The prevalence of positive buffaloes was 3.5 % (100/2,809) on Marajo Island and 7.2% (80/1,108) on the mainland. The municipalities with the highest tuberculosis prevalence rates in animals were Ipixuna do Para (10.1 %), Marapanim (9.8 %), Chaves (9.4 %), Paragominas (8.6 %), and Cachoeira do Arari (6.7 %). The tuberculosis prevalence was not significantly different between the Murrah (4.3 %) and Mediterranean (4.8 %) breeds or between pregnant (5%) and nonpregnant (4.3%) buffaloes. Tuberculosis was detected in water buffaloes from Para, Brazil; the mainland buffalo exhibited the highest tuberculosis prevalence. These results indicate that this disease is dangerous to public health and buffalo farming in Para.

Advances in Bovine Tuberculosis Diagnosis and Pathogenesis: What Policy Makers Need to Know

The mainstay of tuberculosis diagnosis in cattle and deer has been the tuberculin skin test. Recent advances have allowed the incorporation of blood based assays to the diagnostic arsenal for both cattle and deer. Use of defined and specific antigens has allowed for improved specificity of cell mediated assays in both cattle and deer and advances in antibody tests for tuberculosis have potential for use in free-ranging and captive cervid populations. Combined use of blood-based assays with skin testing will require further understanding of the effect of skin testing on the accuracy of blood based assays. Models of experimental infection of cattle have allowed for increased understanding of natural disease pathogenesis. Differences likely exist; however, between cattle and deer in both disease distribution and primary route of inoculation in naturally infected animals.