Leukotrienes are not essential for the efficacy of a heterologous vaccine against Mycobacterium tuberculosis infection

Leukotrienes are reported to be potent proinflammatory mediators that play a role in the development of several inflammatory diseases such as asthma, rheumatoid arthritis and periodontal disease. Leukotrienes have also been associated with protection against infectious diseases. However, the role of leukotrienes in Mycobacterium tuberculosis infection is not understood. To answer this question, we studied the role of leukotrienes in the protective immune response conferred by prime-boost heterologous immunization against tuberculosis. We immunized BALB/c mice (4-11/group) with subcutaneous BCG vaccine (1 x 10(5) M. bovis BCG) (prime) followed by intramuscular DNA-HSP65 vaccine (100 µg) (boost). During the 30 days following the challenge, the animals were treated by gavage daily with MK-886 (5 mg·kg-1·day-1) to inhibit leukotriene synthesis. We showed that MK-886-treated mice were more susceptible to M. tuberculosis infection by counting the number of M. tuberculosis colony-forming units in lungs. The histopathological analysis showed an impaired influx of leukocytes to the lungs of MK-886-treated mice after infection, confirming the involvement of leukotrienes in the protective immune response against experimental tuberculosis. However, prime-boost-immunized mice treated with MK-886 remained protected after challenge with M. tuberculosis, suggesting that leukotrienes are not required for the protective effect elicited by immunization. Protection against M. tuberculosis challenge achieved by prime-boost immunization in the absence of leukotrienes was accompanied by an increase in IL-17 production in the lungs of these animals, as measured by ELISA. Therefore, these data suggest that the production of IL-17 in MK-886-treated, immunized mice could contribute to the generation of a protective immune response after infection with M. tuberculosis.

Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

Fluorescent quantitative PCR of Mycobacterium tuberculosis for differentiating intestinal tuberculosis from Crohn's disease

Intestinal tuberculosis (ITB) and Crohn's disease (CD) are granulomatous disorders with similar clinical manifestations and pathological features that are often difficult to differentiate. This study evaluated the value of fluorescent quantitative polymerase chain reaction (FQ-PCR) for Mycobacterium tuberculosis (MTB) in fecal samples and biopsy specimens to differentiate ITB from CD. From June 2010 to March 2013, 86 consecutive patients (38 females and 48 males, median age 31.3 years) with provisional diagnoses of ITB and CD were recruited for the study. The patients' clinical, endoscopic, and histological features were monitored until the final definite diagnoses were made. DNA was extracted from 250 mg fecal samples and biopsy tissues from each patient. The extracted DNA was amplified using FQ-PCR for the specific MTB sequence. A total of 29 ITB cases and 36 CD cases were included in the analysis. Perianal disease and longitudinal ulcers were significantly more common in the CD patients (P<0.05), whereas night sweats, ascites, and circumferential ulcers were significantly more common in the ITB patients (P<0.05). Fecal FQ-PCR for MTB was positive in 24 (82.8%) ITB patients and 3 (8.3%) CD patients. Tissue PCR was positive for MTB in 16 (55.2%) ITB patients and 2 (5.6%) CD patients. Compared with tissue FQ-PCR, fecal FQ-PCR was more sensitive (X2=5.16, P=0.02). We conclude that FQ-PCR for MTB on fecal and tissue samples is a valuable assay for differentiating ITB from CD, and fecal FQ-PCR has greater sensitivity for ITB than tissue FQ-PCR.

Use of conventional PCR and smear microscopy to diagnose pulmonary tuberculosis in the Amazonian rainforest area

The diagnostic usefulness of Ziehl-Neelsen (ZN)-stained sputum smears combined with conventional polymerase chain reaction (ZN/PCR) to amplify IS6110 region DNA extracted from ZN slides was evaluated. The objective was to verify if this association could improve tuberculosis (TB) diagnosis in patients at remote sites. The study was carried out in 89 patients with culture-confirmed pulmonary TB as defined by the Brazilian Manual for TB Treatment. The participants were recruited in a reference unit for TB treatment in Rondônia, a state in the Amazonian area in northern Brazil. ZN, PCR, and culture performed in the sputum samples from these patients were analyzed in different combinations (i.e., ZN plus PCR and ZN plus culture). The prevalence rates of pulmonary TB in these patients were 32.6 and 28.1% considering culture and ZN/PCR, respectively. The sensitivity and specificity of ZN/PCR were 86 and 93%, respectively. ZN/PCR was able to detect more TB cases than ZN alone. This method could offer a new approach for accurate tuberculosis diagnosis, especially in remote regions of the world where culture is not available.

Role of a GenoType MTBDRplus line probe assay in early detection of multidrug-resistant tuberculosis at a Brazilian reference center

Resistance to Mycobacterium tuberculosis is a reality worldwide, and its diagnosis continues to be difficult and time consuming. To face this challenge, the World Health Organization has recommended the use of rapid molecular tests. We evaluated the routine use (once a week) of a line probe assay (Genotype MTBDRplus) for early diagnosis of resistance and for assessment of the main related risk factors over 2 years. A total of 170 samples were tested: 15 (8.8%) were resistant, and multidrug resistance was detected in 10 (5.9%). The sensitivity profile took 3 weeks (2 weeks for culture and 1 week for rapid testing). Previous treatment for tuberculosis and the persistence of positive acid-fast smears after 4 months of supervised treatment were the major risk factors observed. The use of molecular tests enabled early diagnosis of drug-resistant bacilli and led to appropriate treatment of the disease. This information has the potential to interrupt the transmission chain of resistant M. tuberculosis.

Reconstructive dosimetry for cutaneous radiation syndrome

According to the International Atomic Energy Agency (IAEA), a relatively significant number of radiological accidents have occurred in recent years mainly because of the practices referred to as potentially high-risk activities, such as radiotherapy, large irradiators and industrial radiography, especially in gammagraphy assays. In some instances, severe injuries have occurred in exposed persons due to high radiation doses. In industrial radiography, 80 cases involving a total of 120 radiation workers, 110 members of the public including 12 deaths have been recorded up to 2014. Radiological accidents in industrial practices in Brazil have mainly resulted in development of cutaneous radiation syndrome (CRS) in hands and fingers. Brazilian data include 5 serious cases related to industrial gammagraphy, affecting 7 radiation workers and 19 members of the public; however, none of them were fatal. Some methods of reconstructive dosimetry have been used to estimate the radiation dose to assist in prescribing medical treatment. The type and development of cutaneous manifestations in the exposed areas of a person is the first achievable gross dose estimation. This review article presents the state-of-the-art reconstructive dosimetry methods enabling estimation of local radiation doses and provides guidelines for medical handling of the exposed individuals. The review also presents the Chilean and Brazilian radiological accident cases to highlight the importance of reconstructive dosimetry.

Tuberculosis in renal transplant patients: The experience of a single center in Medellín-Colombia, 2005-2013

Introduction: Tuberculosis is a common opportunistic infection in renal transplant patients. Objective: To obtain a clinical and laboratory description of transplant patients diagnosed with tuberculosis and their response to treatment during a period ranging from 2005 to 2013 at the Pablo Tobón Uribe Hospital. Methods: Retrospective and descriptive study. Results: In 641 renal transplants, tuberculosis was confirmed in 12 cases. Of these, 25% had a history of acute rejection, and 50% had creatinine levels greater than 1.5 mg/dl prior to infection. The disease typically presented as pulmonary (50%) and disseminated (33.3%). The first phase of treatment consisted of 3 months of HZRE (isoniazid, pyrazinamide, rifampicin and ethambutol) in 75% of the cases and HZME (isoniazid, pyrazinamide, moxifloxacin and ethambutol) in 25% of the cases. During the second phase of the treatment, 75% of the cases received isoniazid and rifampicin, and 25% of the cases received isoniazid and ethambutol. The length of treatment varied between 6 and 18 months. In 41.7% of patients, hepatotoxicity was associated with the beginning of anti-tuberculosis therapy. During a year-long follow-up, renal function remained stable, and the mortality rate was 16.7%. Conclusion: Tuberculosis in the renal transplant population studied caused diverse nonspecific symptoms. Pulmonary and disseminated tuberculosis were the most frequent forms and required prolonged treatment. Antituberculosis medications had a high toxicity and mortality. This infection must be considered when patients present with a febrile syndrome of unknown origin, especially during the first year after renal transplant.