Expresión diferencial de micro-ARN circulantes en pacientes con tuberculosis activa y latente / Differential expression of circulating micro-RNAs in patients with active and latent tuberculosis

RESUMEN Objetivo: El objetivo del estudio fue analizar la expresión diferencial de miR-21, miR-29a, miR-99b y miR-155 en muestras de suero de pacientes con tuberculosis (TB) latente y TB activa respecto a contro les sanos. Materiales y métodos: Se utilizaron 28 muestras de suero (nueve con TB activa, diez con TB latente y nueve controles sanos) para el análisis de expresión génica mediante RT-qPCR con Primers y sondas TaqMan. Se calculó la expresión diferencial por el método de Livak utilizando un gen norma lizador (RNU-48). Resultados: Se halló una sobreexpresión de miR-155 en personas con tuberculosis latente, respecto a los controles sanos (0,63 vs. 0,01; valor de p=0,032). Conclusión: El miR-155 podría ser considerado un biomarcador para diferenciar TB latente de enfermedad activa. Se requieren estudios con mayores tamaños muestrales para corroborar nuestros hallazgos.

ABSTRACT Objectives: To analyze the differential expression of miR-21, miR-29a, miR-99b and miR-155 in serum samples from patients with latent tuberculosis (TB) and active TB compared to healthy controls. Mate rials and Methods: We used 28 serum samples (9 with active TB, 10 with latent TB and 9 healthy con trols) for the analysis of gene expression by RT-qPCR with Primers and TaqMan probes. The differential expression was calculated by the Livak method using a normalizing gene (RNU-48). Results: Overex pression of miR-155 was found in people with latent tuberculosis, compared to healthy controls (0.63 vs. 0.01; p value = 0.032). Conclusion: The miR-155 could be considered a biomarker to differentiate latent TB from active disease. Studies with larger sample sizes are required to corroborate the findings.

Evaluation of the transcriptional immune biomarkers in peripheral blood from warao indigenous associate with the infection by mycobacterium tuberculosis

Abstract INTRODUCTION: Biomarkers are critical tools for finding new approaches for controlling the spread of tuberculosis (TB), including for predicting the development of TB therapeutics, vaccines, and diagnostic tools. METHODS: Expression of immune biomarkers was analyzed in peripheral blood cells stimulated and non-stimulated with M. tuberculosis antigens ESAT-6, CFP10 and TB7.7. in Warao indigenous individuals. These biomarkers may be able to differentiate TB states, such as active tuberculosis (ATB) cases and latent tuberculosis infection (LTBI) from non-infected controls (NIC). A real-time reverse transcription polymerase chain reaction (RT-qPCR) assay was performed on 100 blood samples under non-stimulation or direct ex vivo conditions (NS=50) and stimulation conditions (S=50). RESULTS: The findings are shown as the median and interquartile range (IQR) of relative gene expression levels of IFN-γ, CD14, MMP9, CCR5, CCL11, CXCL9/MIG, and uPAR/PLAUR immune biomarkers. MMP9 levels were significantly higher in the LTBI-NS and LTBI-S groups compared with the NIC-NS and NIC-S groups. However, CCR5 levels were significantly lower in the LTBI-S group compared with both NIC-NS and NIC-S groups. CCL11 levels were significantly lower in the LTBI-S group compared with the NIC-NS group. CONCLUSIONS: Preliminary findings showed that MMP9 immune biomarkers separated LTBI indigenous individuals from NIC indigenous individuals, while CCR5, CCL11, CD14, and IFN-γ did not differentiate TB states from NIC. MMP9 may be useful as a potential biomarker for LTBI and new infected case detection among Warao indigenous individuals at high risk of developing the disease. It may also be used to halt the epidemic, which will require further validation in larger studies.

Follow-up Testing of Interferon-Gamma Release Assays Are Useful in Ankylosing Spondylitis Patients Receiving Anti-Tumor Necrosis Factor Alpha for Latent Tuberculosis Infection

We evaluated the utility of follow-up interferon-gamma release assays (IGRAs) for the diagnosis of reactivation of latent tuberculosis infection (LTBI) or new tuberculosis in ankylosing spondylitis (AS) patients receiving anti-tumor necrosis factor alpha (anti-TNFalpha). The study participants (n=127) had a negative IGRA screening before receiving anti-TNFalpha and were evaluated by follow-up IGRA. We retrospectively examined data of the subjects according to age, gender, tuberculosis prophylaxis, concomitant medications, IGRA conversion and anti-TNFalpha, including type and treatment duration. The median duration of anti-TNFalpha was 21.5 months, and the median age was 35.3 yr. Of the 127 patients, IGRA conversion was found in 10 patients (7.9%). There was no significant variation between IGRA conversion rate and any risk factors except for age. IGRA conversion rate was not significantly different between AS and rheumatoid arthritis (P=0.12). IGRA conversion was observed in AS patients receiving anti-TNFalpha in Korea. A follow-up IGRA test can be helpful for identifying LTBI or new tuberculosis in AS patients receiving anti-TNFalpha.

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.