Rapid detection of Mycobacterium tuberculosis in children using blood and urine specimens

Abstract INTRODUCTION: Laboratory and clinical features of childhood tuberculosis (TB) are non-specific and establishing an accurate diagnosis remains a challenge. This study evaluated a Single tube nested-PCR (STNPCR) to detect genomic DNA of Mycobacterium tuberculosis complex in blood and urine. METHODS: Biological samples were obtained from children (<15 years old) with clinical suspicion of pulmonary and extrapulmonary TB at public hospitals in Recife-Pernambuco, Brazil. Cultures yielded negative results in a majority of childhood TB cases, which are generally paucibacillary. A set of clinical, epidemiological, radiological, and laboratory criteria with evident clinical improvement after anti-TB treatment were frequently used to define childhood TB cases. RESULTS: Ninety children with clinical suspicion were enrolled in this study (44 with TB and 46 without TB). The pulmonary TB group had 20 confirmed cases and 46 negative controls, while the extrapulmonary TB group had 24 confirmed cases. The STNPCR showed sensitivities to pulmonary and extrapulmonary TB of 47.4% and 52.2% (blood) and 38.8% and 20% (urine), respectively. Considering the low performance of STNPCR on separate samples, we decided to perform a combined analysis (parallel sensitivity analysis) of the results from blood and urine samples. The parallel sensitivity increased to 65% in blood and 62.5% in urine. The specificity in both samples ranged from 93.5-97.8%. CONCLUSIONS: Although STNPCR showed moderate sensitivity, the specificity is high; therefore, the test can be used as an auxiliary tool to diagnose TB in children. It is a rapid test that demonstrated better performance than other diagnostic tests in paucibacillary samples as it does in childhood tuberculosis.

Characteristic profile of antibody responses to PPD, ESAT-6, and CFP-10 of Mycobacterium tuberculosis in pulmonary tuberculosis suspected cases in Surabaya, Indonesia

Abstract Accurate and rapid diagnostic tools are important aspects of managing tuberculosis (TB) cases appropriately. However, the sensitivity and specificity of diagnostic kits based on immune response such as the tuberculin skin test (TST) and interferon gamma release assay (IGRA) are still debated. Thus, the exploration and assessment of specific biomarker-targeted antibodies are needed for the development of an accurate and rapid diagnostic tool. The present study was conducted in patients with a respiratory problem suspected to be TB at Dr. Soetomo Hospital, Surabaya, Indonesia. Among 102 patients tested by GeneXpert and AFB, 59 serum samples were from cases retrospectively determined to have active TB. A total of 102 serum of healthy controls (HC) was also collected. The PPD antigen and the recombinant CFP-10 and ESAT-6 proteins were prepared. Antibody responses against these proteins were evaluated by ELISA. All samples were also screened for the possibility of Mycobacterium avium-intracellulare complex (MAC) infection using Capilla MaC kit. The results showed that TB patients had a significantly higher concentration of IgG antibody in response to PPD than the HC. In addition, the receiver operating characteristic (ROC) curve analysis showed that PPD was acceptable for diagnostic purposes with an AUC value of 0.835 (95% CI 0.770-0.900, p < 0.0001). However, ESAT-6 and CFP-10 had low AUCs, and 32 samples from both groups showed a low concentration of IgA antibody against all antigens. The MAC detection results also showed that the concentration of IgA in the HC group was the highest. The current results indicate that PPD is a better antigen for antibody-based detection of TB than ESAT-6 and CFP-10. Based on the MAC detection assay, 53 people in the HC group were probably infected with rapidly growing nontuberculous mycobacteria (NTM), although antibody response to PPD was low.

Concentrations of rifampicin in pre-dose samples in patients with pulmonary tuberculosis

ABSTRACT Rifampicin is used in both phases of treatment for tuberculosis. In chronic use, the short half-life and the self-induction of metabolism can decrease the levels of the drug below the minimal inhibitory concentration. The aim of the study was to investigate whether plasma concentrations of rifampicin are sustained above 0.5 µg/mL in patients with tuberculosis using 600 mg/day. Rifampicin was measured in plasma by high-performance liquid chromatography and a sputum smear microscopy was performed in all days of the study. A total of 44 male patients completed the study. On days 31, 61 and 91, the mean plasma concentrations of rifampicin were 0.6 (0.5) µg/mL, 0.55 (0.5) µg/mL and 0.46 (0.4) µg/mL. There was a high variation of rifampicin levels leading to a high percentage of samples with concentrations below 0.5 µg/mL. There was no significant association between the frequency of samples with drug levels below 0.5 µg/mL with the conversion of the sputum microscopy. These data suggest that pre-doses samples offer limited information on the exposure of M. tuberculosis to rifampicin.

Protective effect of metformin against tuberculosis infections in diabetic patients: an observational study of south Indian tertiary healthcare facility

ABSTRACT Background: World Health Organization estimated that people with diabetes (DM) are at 2-3 times higher risk for tuberculosis (TB). Studies have shown that DM not only increases the risk of active TB, but also puts co-affected persons at increased risk of poor outcomes. Objectives: To determine the protective effect of metformin against TB in DM patients and also, to investigate the relationship between poor glycemic control and TB. Methods: A case-control study was conducted over 8 months, where cases and controls were selected based on the inclusion and exclusion criteria of the study. The diabetics diagnosed with TB were selected as study group (SG = 152) and without TB were as control group (CG = 299). Exposure status of metformin in both groups were analyzed. Results: The mean (SD) age of both CG and SG were 55.54 ± 11.82 and 52.80 ± 11.75, respectively. Majority of the subjects in the study were males. The mean hospital stay of SG and CG were 7 days and 6 days, respectively. Poor glycemic control (HbA1c > 8) observed in SG (51.7%) vs CG (31.4%). HbA1c value <7 is associated protective factor for TB occurrence [OR = 0.52 (95% CI 0.29-0.93)]. The protective effect of metformin against TB was 3.9-fold in diabetics (OR = 0.256, 0.16-0.40). Conclusion: Poor glycemic control among diabetics is a risk factor for TB occurrence. The result shows metformin use is a protective agent against TB infection in diabetics. Hence, incorporation of metformin into standard clinical care would offer a therapeutic option for the prevention of TB.

Evaluation of four molecular methods for the diagnosis of tuberculosis in pulmonary and blood samples from immunocompromised patients

The present study analysed the concordance among four different molecular diagnostic methods for tuberculosis (TB) in pulmonary and blood samples from immunocompromised patients. A total of 165 blood and 194 sputum samples were collected from 181 human immunodeficiency virus (HIV)-infected patients with upper respiratory complaints, regardless of suspicious for TB. The samples were submitted for smear microscopy, culture and molecular tests: a laboratory-developed conventional polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) and the Gen-Probe and Detect-TB Ampligenix kits. The samples were handled blindly by all the technicians involved, from sample processing to results analysis. For sputum, the sensitivity and specificity were 100% and 96.7% for qPCR, 81.8% and 94.5% for Gen-Probe and 100% and 66.3% for Detect-TB, respectively. qPCR presented the best concordance with sputum culture [kappa (k) = 0.864)], followed by Gen-Probe (k = 0.682). For blood samples, qPCR showed 100% sensitivity and 92.3% specificity, with a substantial correlation with sputum culture (k = 0.754) and with the qPCR results obtained from sputum of the corresponding patient (k = 0.630). Conventional PCR demonstrated the worst results for sputa and blood, with a sensitivity of 100% vs. 88.9% and a specificity of 46.3% vs. 32%, respectively. Commercial or laboratory-developed molecular assays can overcome the difficulties in the diagnosis of TB in paucibacillary patients using conventional methods available in most laboratories.

Association between serum selenium level and conversion of bacteriological tests during antituberculosis treatment / Associações entre níveis de selênio sérico e conversão de testes bacteriológicos durante o tratamento antituberculose

Objective: To determine whether serum selenium levels are associated with the conversion of bacteriological tests in patients diagnosed with active pulmonary tuberculosis after eight weeks of standard treatment. Methods: We evaluated 35 healthy male controls and 35 male patients with pulmonary tuberculosis, the latter being evaluated at baseline, as well as at 30 and 60 days of antituberculosis treatment. For all participants, we measured anthropometric indices, as well as determining serum levels of albumin, C-reactive protein (CRP) and selenium. Because there are no reference values for the Brazilian population, we used the median of the serum selenium level of the controls as the cut-off point. At 30 and 60 days of antituberculosis treatment, we repeated the biochemical tests, as well as collecting sputum for smear microscopy and culture from the patients. Results: The mean age of the patients was 38.4 ± 11.4 years. Of the 35 patients, 25 (71%) described themselves as alcoholic; 20 (57.0%) were smokers; and 21 (60.0%) and 32 (91.4%) presented with muscle mass depletion as determined by measuring the triceps skinfold thickness and arm muscle area, respectively. Of 24 patients, 12 (39.2%) were classified as moderately or severely emaciated, and 15 (62.5%) had lost > 10% of their body weight by six months before diagnosis. At baseline, the tuberculosis group had lower serum selenium levels than did the control group. The conversion of bacteriological tests was associated with the CRP/albumin ratio and serum selenium levels 60 days after treatment initiation. Conclusions: Higher serum selenium levels after 60 days of treatment were associated with the conversion of bacteriological tests in pulmonary tuberculosis patients. .

Objetivo: Determinar se os níveis séricos de selênio estão associados à conversão dos testes bacteriológicos em pacientes diagnosticados com tuberculose pulmonar ativa após oito semanas de tratamento-padrão. Métodos: No início do estudo, avaliamos 35 controles saudáveis, do sexo masculino, e 35 pacientes do sexo masculino com tuberculose pulmonar. Estes foram também avaliados após 30 e 60 dias de tratamento antituberculose. Todos os participantes submeteram-se a medições antropométricas e quantificação dos níveis séricos de albumina, proteína C reativa (PCR) e selênio. Como não há valores de referência para a população brasileira, usamos a mediana dos resultados de selênio sérico dos controles como ponto de corte. Aos 30 e 60 dias do tratamento antituberculose, todos os testes bioquímicos foram repetidos, e foram coletadas amostras de escarro para baciloscopia e cultura. Resultados: A média de idade dos pacientes foi de 38,4 ± 11.4 anos. Dos 35 pacientes, 25 (71,0%) referiram alcoolismo, 20 (57,0%) eram fumantes, e 21 (60,0%) e 32 (91,4%) apresentavam depleção muscular pela medição da dobra cutânea tricipital e da área muscular do braço, respectivamente. De 24 pacientes, 12 (39,2%) foram classificados em moderadamente ou gravemente magros, e 15 (62,5%) apresentaram perda de peso > 10% em até seis meses antes do diagnóstico. No início do estudo, o grupo com tuberculose apresentou menores níveis de selênio sérico que os controles. A conversão dos testes bacteriológicos associou-se à relação PCR/albumina e aos níveis de selênio sérico 60 dias após o início do tratamento. Conclusões: Níveis maiores de selênio sérico após 60 ...

Whole Blood Interferon-gamma Release Assay Is Insufficient for the Diagnosis of Sputum Smear Negative Pulmonary Tuberculosis

PURPOSE: We investigated the value of an interferon-gamma release assay (IGRA) for the diagnosis of active pulmonary tuberculosis (PTB) among sputum smear negative PTB suspects in an environment with intermediate burden of PTB and high Bacillus Calmette-Guerin (BCG) vaccination rate. MATERIALS AND METHODS: We retrospectively reviewed IGRA, medical records, chest PA and CT scan of PTB suspects seen at Gangnam Severance Hospital, Seoul, Korea from Oct. 2007 to Apr. 2013. "Active PTB" was diagnosed when 1) M. tuberculosis culture positive, 2) confirmation by pathologic examination; or 3) clinical findings compatible with TB. RESULTS: Of 224 sputum smear negative PTB suspects, 94 were confirmed as having active PTB. There were no statistically significant differences in the diagnostic yield of IGRA between immunocompromised and immunocompetent sputum smear negative PTB suspects. IGRA did show superior sensitivity [81.9%, 95% confidence interval (CI); 74.13-89.70%] in the diagnosis of sputum smear negative PTB when compared with chest high-resolution computed tomography (HRCT), tuberculin skin test (TST), and chest X-ray (p<0.001). Also, IGRA showed highest negative predictive value (82.7%, 95% CI; 75.16-90.15%) when compared with HRCT, TST and chest X-ray (p=0.023). However, combining the results of IGRA with those of HRCT, TST, or both did not increase any diagnostic parameters. CONCLUSION: Failure to increase diagnostic yields by combination with other diagnostic modalities suggests that additional enforcement with IGRA may be insufficient to exclude other diagnoses in sputum smear negative PTB suspects and to screen active PTB in an environment with intermediate TB prevalence and a high BCG vaccination rate.

Inflammatory and immunogenetic markers in correlation with pulmonary tuberculosis / Marcadores inflamatorios e imunogeneticos e sua relacao com tuberculose pulmonar

OBJECTIVE: To describe serum levels of the cytokines IL-10, TNF-α, and IFN-γ, as well as polymorphisms in the genes involved in their transcription, and their association with markers of the acute inflammatory response in patients with pulmonary tuberculosis. METHODS: This was a descriptive, longitudinal study involving 81 patients with pulmonary tuberculosis treated at two referral hospitals. We collected data on sociodemographic variables and evaluated bacteriological conversion at the eighth week of antituberculosis treatment, gene polymorphisms related to the cytokines studied, and serum levels of those cytokines, as well as those of C-reactive protein (CRP). We also determined the ESR and CD4+ counts. RESULTS: The median age of the patients was 43 years; 67 patients (82.7%) were male; and 8 patients (9.9%) were infected with HIV. The ESR was highest in the patients with high IFN-γ levels and low IL-10 levels. IFN-γ and TNF-α gene polymorphisms at positions +874 and −238, respectively, showed no correlations with the corresponding cytokine serum levels. Low IL-10 levels were associated with IL-10 gene polymorphisms at positions −592 and −819 (but not −1082). There was a negative association between bacteriological conversion at the eighth week of treatment and CRP levels. CONCLUSIONS: Our results suggest that genetic markers and markers of acute inflammatory response are useful in predicting the response to antituberculosis treatment. .

OBJETIVO: Descrever os níveis séricos das citocinas IL-10, TNF-α e IFN-γ, assim como polimorfismos presentes em genes envolvidos na sua transcrição, e sua associação com marcadores de resposta inflamatória aguda em pacientes com tuberculose. MÉTODOS: Estudo descritivo e longitudinal realizado em 81 pacientes com tuberculose pulmonar atendidos em dois hospitais de referência. Foram coletadas informações sociodemográficas, conversão bacteriológica na oitava semana de tratamento antituberculose, polimorfismos relacionados às citocinas estudadas, níveis séricos dessas citocinas, assim como de proteína C reativa (PCR). Também foram avaliados VHS e contagem de CD4+. RESULTADOS: A mediana de idade dos pacientes era de 43 anos, sendo 67 (82,7%) do sexo masculino e 8 (9,9%) infectados por HIV. Os pacientes com níveis elevados de IFN-γ e baixos níveis de IL-10 apresentaram valores mais elevados de VHS. Não houve associação dos polimorfismos do gene IFN-γ na posição +874 e do gene TNF-α na posição −238 com os níveis das citocinas correspondentes. Houve uma associação entre polimorfismos do gene IL-10 nas posições −592 e −819 (mas não −1082) e baixos níveis de IL-10. Houve uma associação negativa entre a taxa de conversão bacteriológica na oitava semana de tratamento e níveis de PCR. CONCLUSÕES: Nossos resultados sugerem que marcadores genéticos e de resposta inflamatória aguda podem ser úteis na predição da resposta ao tratamento antituberculose. .

Manifestações clínicas, radiológicas e laboratoriais em indivíduos com tuberculose pulmonar: estudo comparativo entre indivíduos HIV positivos e HIV negativos internados em um hospital de referência / Clinical, radiological, and laboratory characteristics in pulmonary tuberculosis patients: comparative study of HIV-positive and HIV-negative inpatients at a referral hospital

OBJETIVO: Comparar as manifestações clínicas, radiológicas e laboratoriais de indivíduos com tuberculose pulmonar coinfectados com HIV com aqueles sem a coinfecção. MÉTODOS: Estudo transversal, no qual sinais e sintomas foram analisados por meio de anamnese e exame físico em pacientes internados com tuberculose pulmonar. A baciloscopia, a cultura para Mycobacterium tuberculosis, a dosagem de hemoglobina e a contagem de células T CD4+ foram obtidas de registros dos prontuários, assim como os laudos das radiografias de tórax. RESULTADOS: Foram incluídos 50 pacientes com tuberculose pulmonar, que foram divididos em dois grupos (HIV positivo e HIV negativo; n = 25 por grupo). A média de idade dos participantes foi de 38,4 ± 10,5 anos, 46 (92 por cento) eram do sexo masculino, e 27 (54 por cento) eram caucasianos. Apresentaram expectoração 21 (84 por cento) e 13 (52 por cento) dos pacientes nos grupos HIV negativo e HIV positivo, respectivamente (p = 0,016). Achados radiológicos de cavitação estavam presentes em 10 (43 por cento) e 2 (10 por cento) dos pacientes nos grupos HIV negativo e HIV positivo, respectivamente (p = 0,016), ao passo que padrão intersticial estava presente em 18 (78 por cento) e 8 (40 por cento) dos pacientes nesses grupos (p = 0,012). O nível médio de hemoglobina foi de 11,1 ± 2,9 g/dL e 9,3 ± 2,2 g/dL nos grupos HIV negativo e HIV positivo, respectivamente (p = 0,015). CONCLUSÕES: Entre pacientes coinfectados com tuberculose e HIV desta amostra, houve menor prevalência de expectoração, foram menos frequentes os achados radiológicos de cavitação e de padrão intersticial, e os níveis de hemoglobina foram mais baixos do que naqueles sem essa coinfecção.

OBJECTIVE: To compare clinical, radiological, and laboratory characteristics of individuals with pulmonary tuberculosis co-infected or not with HIV. METHODS: A cross-sectional study, in which signs and symptoms were assessed by anamnesis and physical examination in patients hospitalized with pulmonary tuberculosis. The results of sputum smear microscopy and culture for Mycobacterium tuberculosis, as well as hemoglobin levels and CD4+ T-cell counts, were obtained from medical records, and chest X-ray reports were consulted. RESULTS: We included 50 pulmonary tuberculosis patients, who were divided into two groups (HIV-positive and HIV-negative; n = 25 per group). The mean age of the participants was 38.4 ± 10.5 years; 46 (92 percent) were males; and 27 (54 percent) were White. Expectoration was presented by 21 (84 percent) and 13 (52 percent) of the patients in the HIV-negative and HIV-positive groups, respectively (p = 0.016). Radiological findings of cavitation were present in 10 (43 percent) and 2 (10 percent) of the patients in the HIV-negative and HIV-positive groups, respectively (p = 0.016), whereas an interstitial pattern was observed in 18 (78 percent) and 8 (40 percent), respectively (p = 0.012). The mean hemoglobin level was 11.1 ± 2.9 g/dL and 9.3 ± 2.2 g/dL in the HIV-negative and HIV-positive groups, respectively (p = 0.015). CONCLUSIONS: In our sample of tuberculosis patients, expectoration was less prevalent, hemoglobin levels were lower, and cavitation was less common, as was an interstitial pattern, among those co-infected with HIV than among those without HIV co-infection.

Radiografia torácica e bacteriologia na fase inicial de tratamento de 800 pacientes masculinos com tuberculose pulmonar / Chest X-ray and bacteriology in the initial phase of treatment of 800 male patients with pulmonary tuberculosis

OBJETIVO: Avaliar radiografias de tórax de pacientes com tuberculose pulmonar e determinar se a extensão das lesões radiográficas correlaciona-se com os parâmetros bacteriológicos. MÉTODOS: Neste estudo descritivo e retrospectivo; foram avaliadas radiografias de tórax, baciloscopias para BAAR e culturas de escarro para Mycobacterium tuberculosis no momento basal e durante os dois primeiros meses de tratamento. A amostra foi composta por 800 pacientes masculinos internados entre 1995 até o presente em um hospital com 250 leitos no noroeste da Turquia. RESULTADOS: A VHS média inicial foi de 58 ± 37 mm/h. Inicialmente, a baciloscopia e as culturas de escarro tiveram resultado positivo em 83,8 por cento e em 89,5 por cento dos pacientes, respectivamente. Após o primeiro mês do tratamento, a proporção de culturas positivas foi maior nos pacientes com doença cavitária do que naqueles sem doença cavitária (53,7 por cento vs. 37,7 por cento; p < 0,001). Não houve correlação do número de zonas afetadas com idade, duração de sintomas, contato com paciente com tuberculose ativa, diabetes concomitante (p > 0.05 para todos), mas houve correlação positiva com VHS (r = 0,23, p < 0,001). Durante o primeiro e o segundo mês de tratamento, a negativação da baciloscopia foi menos frequente nos pacientes com comprometimento bilateral do que naqueles com comprometimento unilateral (p < 0,001 e p = 0,002 para os meses 1 e 2, respectivamente). A extensão da doença não se correlacionou com idade, duração dos sintomas, contato com paciente com tuberculose ativa e diabetes concomitante, mas sim com a recuperação bacteriológica atrasada. CONCLUSÕES: Radiografias de tórax e bacteriologia são ferramentas valiosas na avaliação de tuberculose pulmonar.

OBJECTIVE: To evaluate chest X-rays of patients with pulmonary tuberculosis and to determine whether the extent of radiographic lesions correlates with bacteriological parameters. METHODS: In this retrospective, descriptive study, we evaluated chest X-rays, as well as AFB detection by smear microscopy and culture for Mycobacterium tuberculosis, initially and during the first two months of treatment, in 800 male patients hospitalized between 1995 and the present at a 250-bed hospital in northwestern Turkey. RESULTS: The initial mean ESR was 58 ± 37 mm/h. Initial sputum smears and cultures were positive in 83.8 percent and 89.5 percent of the patients, respectively. After the first month of treatment, the proportion of patients with positive sputum culture was higher among those with cavitary tuberculosis than among those with non-cavitary tuberculosis (53.7 percent vs. 37.7 percent, p < 0.001). The number of affected zones was not correlated with age, symptom duration, contact with an active tuberculosis patient, or concomitant diabetes (p > 0.05 for all) but was positively correlated with the ESR (r = 0.23, p < 0.001). During the first and second months of treatment, conversion to smear-negative status was less common in patients with bilateral involvement than in those with unilateral involvement (p < 0.001 and p = 0.002 for months 1 and 2, respectively). Disease extent did not correlate with age, symptom duration, contact with an active tuberculosis patient, or concomitant diabetes but did correlate with delayed bacteriological recovery. CONCLUSIONS: Chest X-ray and bacteriology are valuable tools for the evaluation of pulmonary tuberculosis.

Anti-mycobacterial treatment reduces high plasma levels of CXC-chemokines detected in active tuberculosis by cytometric bead array

Chemokines recruit and activate leukocytes, assisting granuloma formation. Herein, we evaluated plasma chemokines in patients with active tuberculosis (ATB) and after completing treatment (TTB) and compared them to BCG-vaccinated healthy controls (HC). Levels of chemokines were measured by cytometric bead array. Levels of CXCL8, CXCL9 and CXCL10 were higher in ATB patients compared to HC, but they decreased in TTB. Levels of CCL2 and CCL5 in ATB patients were similar to those observed in HC. Thus, the high levels of CXC-chemokines detected during ATB, which can modulate the trafficking of immune cells from the periphery to the site of infection, were reversed by anti-mycobacterial treatment.

Desempenho da técnica nested PCR na detecção específica do complexo Mycobacterium tuberculosis em amostras sanguíneas de pacientes pediátricos / Performance of nested PCR in the specific detection of Mycobacterium tuberculosis complex in blood samples of pediatric patients

Objetivo: Avaliar o desempenho da técnica nested PCR (nPCR) para detectar o complexo Mycobacterium tuberculosis em amostras de sangue de pacientes com suspeita de TB para sua possível utilização como uma ferramenta auxiliar no diagnóstico laboratorial da doença em crianças. Métodos: Detecção do complexo M. tuberculosis em amostras de sangue usando como alvo a sequência de inserção IS6110 do DNA genômico do bacilo. Foram avaliados 120 pacientes, menores de 15 anos de idade, de ambos os sexos, provenientes de hospitais públicos do Recife (PE), no período entre janeiro de 2003 e agosto de 2005. O diagnóstico de TB foi realizado pelo médico assistente do serviço de saúde de acordo com os critérios da Sociedade Torácica Americana. A nPCR amplificou um fragmento de 123 pb com oligonucleotídeos externos (IS1/IS2) e, na reação subsequente, com oligonucleotídeos internos (IS3/IS4), gerando um amplicon de 81 pb. Resultados: A TB ativa ou latente esteve presente em 65 pacientes, foi descartada em 28 suspeitos e 27 não tinham a doença (controles). A sensibilidade da nPCR foi de 26,15%, sendo significativamente maior na forma extrapulmonar (55,56%) em relação à pulmonar (18,18%), e a especificidade foi de 92,73%. Conclusões: Diante das dificuldades diagnósticas da TB infantil e do baixo número de casos estudados,a nPCR em sangue demonstrou ser uma técnica rápida e específica, mas com baixa sensibilidade. Para saber a suareal utilidade no diagnóstico de formas paucibacilares, sobretudo as extrapulmonares, novas pesquisas devem ser desenvolvidas com uma casuística maior de crianças e com outros espécimes biológicos além do sangue.

Objective: To evaluate the performance of nested PCR (nPCR) in detecting the Mycobacterium tuberculosis complex in blood samples of patients suspected of having TB, in order to determine its potential for use as an auxiliary tool in the laboratory diagnosis of TB in children. Methods: Detection of the M. tuberculosis complex in blood samples using as a target the insertion sequence IS6110 of the genomic DNA of the bacillus. Blood samples of 120 patients were evaluated. All of the patients were under 15 years of age at the time of their treatment at public hospitals in the city of Recife, Brazil (between January of 2003 and August of 2005). Attending physicians at the hospitals diagnosed TB based on the criteria recommended by the American Thoracic Society. The nPCRamplified a 123-bp fragment with outer oligonucleotides (IS1/IS2) and, in the subsequent reaction, using inner oligonucleotides (IS3/IS4), generating an 81-bp amplicon. Results: Active or latent TB was found in 65 patients,TB was ruled out in 28 suspected cases, and 27 patients were TB-free (controls). The sensitivity of nPCR was26.15% and was significantly higher for the extrapulmonary form of the disease (55.56%) than for the pulmonary form (18.18%). The specificity was 92.73%. Conclusions: Despite the difficulties in diagnosing TB in children and the low number of cases evaluated in the present study, nPCR in blood samples proved to be a rapid and specifictechnique, albeit one with low sensitivity. In order to establish its true usefulness in the diagnosis of paucibacillary forms, especially extrapulmonary TB, further studies need to be carried out with a larger sample of children and analyzing biological specimens other than blood.

Intestinal barrier function and serum concentrations of rifampin, isoniazid and pyrazinamide in patients with pulmonary tuberculosis

Intestinal barrier function and serum concentrations of rifampin, isoniazid and pyrazinamide were studied in healthy controls and patients with active pulmonary tuberculosis. A case-control study of 29 controls and 30 cases attending at the Health Center, July, 2004 to December, 2005 was conducted. The body mass index was significantly reduced in cases compared to controls (p < 0.001). The intestinal paracellular transport of lactulose was significantly (p = 0.019) reduced in cases compared to controls. The transcellular transport of mannitol and the lactulose:mannitol ratio were not significantly (p = 0.0698) reduced in cases compared to controls. Low serum concentrations of rifampin, isoniazid and pyrazinamide were observed in 81 percent (48/59), 92 percent (54/59) and 28 percent (12/59), respectively, in all individuals. The results demonstrated a marked decrease on intestinal paracellular transport in patients with active pulmonary tuberculosis and reduced serum concentrations of rifampin and isoniazid in both groups.

¿Es suficiente la prueba tuberculínica para el diagnóstico de la infección tuberculosa? / Is the tuberculin skin test still suitable to diagnose tuberculosis infection?

La infección tuberculosa (TB) se determina por la prueba tuberculínica (PTC) con PPD, un extracto de proteínas/péptidos de Mycobacterium tuberculosis, algunos compartidos con otras micobacterias como BCG, lo cual origina falsos resultados positivos en vacunados/no infectados. La nuevas pruebas ex vivo miden el interferón ? (IFN- ?) liberado en sangre, o la cantidad de células que lo producen, en presencia de los péptidos ESAT-6 y CFP-10 de M. tuberculosis. Como estos antígenos no existirían en BCG, las pruebas IFN-? diferenciarían infección TB de vacunación. Numerosos estudios han comparado estas pruebas con la PTC con resultados aún no concluyentes. Las pruebas IFN-? tendrían menor sensibilidad que la PTC, aunque su menor positividad en poblaciones vacunadas podría interpretarse como mayor especificidad. Por otra parte, la vacunación BCG, si no es reciente, no es causa de falsos positivos a la PTC: reacciones =10 mm o =15 mm indican infección TB con altísima probabilidad. Donde la incidencia de TB es mediana o alta, la PTC aventaja en costo-eficiencia a las pruebas IFN-?, siempre que se emplee PPD de calidad garantizada, disponible en todos los centros de salud del país, con aplicación, lectura e interpretación estandarizadas. Como existen en la Argentina problemas de abastecimiento de PPD importado, es preciso producirlo localmente y asegurar su control de calidad. También es necesaria la investigación aplicada al desarrollo de nuevos métodos y la evaluación de su capacidad de predecir la evolución de infección a TB activa, es decir, de identificar las personas que más se beneficiarían con quimioprofilaxis.

Tuberculosis (TB) infection is currently being diagnosed by the tuberculin skin test (TST) with PPD. Some Mycobacterium tuberculosis PPD components are present in BCG, which can be the cause of false positive TST results in BCG vaccinated persons. New IFN-? release assays (IGRAs) are based on the ex vivo release of IFN-? by peripheral blood cells in presence of M. tuberculosis antigens ESAT-6 and CFP-10, which should be absent in BCG. These assays consist in either quantifying released IFN-? or enumerating IFN-? producing cells. In principle, IGRAs should differentiate true TB infection from vaccination and results of several studies suggest that these assays display lower positivity than TST. Whether the lower positivity could be attributed to higher specificity or to lower sensitivity as compared with PPD is still unclear. BCG vaccination, if not recently applied, cannot be blamed for false positive TST reactions. Strong TST reactions (=10 mm or =15 mm) are highly correlated with TB infection. In settings where TB continues being a serious health problem, cost-effectiveness evaluations would privilege TST under certain conditions: supply of quality-assured PPD reagent, standardized criteria for TST application, reading and interpretation, and regular availability in health centers countrywide. In view of current limitations in the supply of imported PPD in Argentina, its production/quality assurance should be considered a public health priority. Still, key questions remain to be addressed concerning the role of IGRAs and TST in predicting risk of TB disease, in other words, in identifying persons who will benefit most from chemoprophylaxis.

Role of C-Reactive Protein and Procalcitonin in Differentiation of Tuberculosis from Bacterial Community Acquired Pneumonia

BACKGROUND/AIMS: We investigated the utility of serum C-reactive protein (CRP) and procalcitonin (PCT) for differentiating pulmonary tuberculosis (TB) from bacterial community-acquired pneumonia (CAP) in South Korea, a country with an intermediate TB burden. METHODS: We conducted a prospective study, enrolling 87 participants with suspected CAP in a community-based referral hospital. A clinical assessment was performed before treatment, and serum CRP and PCT were measured. The test results were compared to the final diagnoses. RESULTS: Of the 87 patients, 57 had bacterial CAP and 30 had pulmonary TB. The median CRP concentration was 14.58 mg/dL (range, 0.30 to 36.61) in patients with bacterial CAP and 5.27 mg/dL (range, 0.24 to 13.22) in those with pulmonary TB (p<0.001). The median PCT level was 0.514 ng/mL (range, 0.01 to 27.75) with bacterial CAP and 0.029 ng/mL (range, 0.01 to 0.87) with pulmonary TB (p<0.001). No difference was detected in the discriminative values of CRP and PCT (p=0.733). CONCLUSIONS: The concentrations of CRP and PCT differed significantly in patients with pulmonary TB and bacterial CAP. The high sensitivity and negative predictive value for differentiating pulmonary TB from bacterial CAP suggest a supplementary role of CRP and PCT in the diagnostic exclusion of pulmonary TB from bacterial CAP in areas with an intermediate prevalence of pulmonary TB.

Citocinas e proteínas de fase aguda do soro como marcadores de regressão da resposta inflamatória ao tratamento da tuberculose pulmonar / Cytokines and acute phase serum proteins as markers of inflammatory regression during the treatment of pulmonary tuberculosis

OBJETIVO: Analisar o padrão de citocinas pró- e antiinflamatórias e da resposta de fase aguda (RFA) como marcadores de resposta ao tratamento da tuberculose pulmonar. MÉTODOS: Determinação dos níveis de interferon-gama (IFN-γ), tumor necrosis factor-alpha (TNF-α, fator de necrose tumoral-alfa), interleucina-10 (IL-10) e transforming growth factor-beta (TGF-β, fator transformador de crescimento-beta), pelo método ELISA, em sobrenadante de cultura de células mononucleares do sangue periférico e monócitos, assim como dos níveis de proteínas totais, albumina, globulinas, alfa-1-glicoproteína ácida (AGA), proteína C reativa (PCR) e velocidade de hemossedimentação (VHS) em 28 doentes com tuberculose pulmonar, em três tempos: antes (T0), aos três meses (T3) e aos seis meses (T6) de tratamento, em relação aos controles saudáveis, em um único tempo. RESULTADOS: Os pacientes apresentaram valores maiores de citocinas e RFA que os controles em T0, com diminuição em T3 e diminuição (TNF-α, IL-10, TGF-β, AGA e VHS) ou normalização (IFN-γ e PCR) em T6. CONCLUSÕES: PCR, AGA e VHS são possíveis marcadores para auxiliar no diagnóstico de tuberculose pulmonar e na indicação de tratamento de indivíduos com baciloscopia negativa; PCR (T0 > T3 > T6 = referência) pode também ser marcador de resposta ao tratamento. Antes do tratamento, o perfil Th0 (IFN-γ, IL-10, TNF-α e TGF-β), indutor de e protetor contra inflamação, prevaleceu nos pacientes; em T6, prevaleceu o perfil Th2 (IL-10, TNF-α e TGF-β), protetor contra efeito nocivo pró-inflamatório do TNF-α ainda presente. O comportamento do IFN-γ (T0 > T3 > T6 = controle) sugere sua utilização como marcador de resposta ao tratamento.

OBJECTIVE: To evaluate the pattern of pro-inflammatory cytokines, anti-inflammatory cytokines and the acute phase response (APR) as markers of the response to treatment of pulmonary tuberculosis. METHODS: Twenty-eight patients with pulmonary tuberculosis were evaluated at three time points: pretreatment (T0), treatment month 3 (T3) and treatment month 6 (T6). Levels of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukine-10 (IL-10) and transforming growth factor-beta (TGF-β) were determined using ELISA in the supernatant of peripheral blood mononuclear cell and monocyte culture. Levels of total protein, albumin, globulins, C-reactive protein (CRP), alpha-1-acid glycoprotein (AAG) and erythrocyte sedimentation rate (ESR) were also determined. All of these parameters were also evaluated, only once, in a group of healthy controls. RESULTS: In relation to controls, patients presented cytokine levels and APR that were higher at T0, lower at T3 and either lower (TNF-α, IL-10, TGF-β, AAG and ESR) or normal (IFN-γ and CRP) at T6. CONCLUSIONS: For individuals with negative smear sputum microscopy, CRP, AAG and ESR are potential markers of pulmonary tuberculosis and of the need for treatment; CRP (T0 > T3 > T6 = reference) can also be a marker of treatment response. In the patients, the Th0 profile (IFN-γ, IL-10, TNF-α and TGF-β), inducer of and protector against inflammation, predominated at T0, whereas the Th2 profile (IL-10, TNF-α and TGF-β), protecting against the harmful pro-inflammatory effect of the remaining TNF-α, predominated at T6. The behavior of IFN-γ (T0 > T3 > T6 = controls) suggests its use as a marker of treatment response.

Tuberculosis associated hemophagocytic syndrome in infancy.

An infant presented with prolonged fever, generalized lymphadenopathy, splenohepatomegaly, anemia and seborrheic dermatitis. Investigations including bone marrow findings confirmed the diagnosis of hemo-phagocytic syndrome (HPS) and the infant succumbed. Liver biopsy features of epithelioid granuloma and positive AFB culture of gastric aspirate confirmed the diagnosis of tuberculosis (TB). This rare association of HPS and tuberculosis in infancy is reported.

Detection of in vitro and in vivo released antigens of diagnostic interest in Mycobacterium tuberculosis by immunoblotting.

Identification of in vitro and in vivo released mycobacterial antigens are of considerable interest in diagnosis of Mycobacterium tuberculosis. Isolation of in vitro released antigen from M. tb excretory-secretory culture filtrate protein and in vivo released circulating tuberculous antigen from smear positive pulmonary tuberculosis sera by ammonium sulphate precipitation is reported. The antigens were resolved by SDS-PAGE and immunoblotting was performed using pooled serum of smear positive, smear negative pulmonary tuberculosis sera and normal sera to identify reactive tuberculous antigens. In vitro and in vivo released mycobacterial antigens showed reactivity at 100, 31, 43 and 20 kDa with smear positive and smear negative pulmonary tuberculosis patients. Further, the in vitro released antigen showed strong reactivity exclusively at 55 kDa antigen with smear positive and 24 kDa antigen with smear negative pulmonary tuberculosis sera. In vivo released antigen reacted exclusively at 170 and 16 kDa with smear positive and 19 kDa antigen with smear negative pulmonary tuberculosis patients. Antigens of 24 and 19 kDa which are reactive with sputum negative sera will be of diagnostic interest and need further study in patients with low bacillary load. The in vitro and in vivo released mycobacterial 100, 31,43 and 20 kDa antigens, reactive with patients sera are of diagnostic interest in tuberculosis.

Clinical Investigation of Cavitary Tuberculosis and Tuberculous Pneumonia

BACKGROUND: The radiographic characteristics of tuberculous pneumonia in adults are similar to primary tuberculosis that occurs in childhood, and upper lobe cavitary tuberculosis is the hallmark of postprimary tuberculosis. The purpose of this study was to investigate the factors associated with tuberculous pneumonia by making comparison with cavitary tuberculosis. METHODS: The medical records and radiographic findings of patients with cavitary tuberculosis and tuberculous pneumonia, and who were diagnosed between March 2003 and February 2006, were analyzed retrospectively. RESULTS: Forty patients had cavitary tuberculosis and sixteen patients had tuberculous pneumonia. Fever was more frequent for tuberculous pneumonia, whereas hemoptysis was more frequent for cavitary tuberculosis. The duration of symptoms before visiting the hospital was shorter, but the diagnosis after admission was more delayed for tuberculous pneumonia patients than for cavitary tuberculosis patients. The prevalence of underlying comorbidities such cancer, diabetes, alcoholism and long-term steroid use was not different between the two groups. The patients with tuberculous pneumonia were older and they had lower levels of serum albumin and hemoglobin than those with cavitary tuberculosis. The patients with tuberculous pneumonia showed a tendency to have more frequent endobronchial lesion. Tuberculous pneumonia occurred in any lobe, whereas the majority of cavitary tuberculosis patients had upper lung lesion, but the prevalence of lymphadenopathy, pleural effusion and previous tuberculosis scar was not different between the two groups. CONCLUSIONS: Older age, a lower level of serum albumin and hemoglobin and a random distribution of lesion were associated with tuberculosis pneumonia as compared with cavitary tuberculosis. These findings suggest that the pathogenesis of tuberculous pneumonia might be different from that of cavitary tuberculosis.