Nested-PCR using MPB64 fragment improves the diagnosis of pleural and meningeal tuberculosis

Fluids in which Mycobacterium tuberculosis are seldom found, such as pleural and cerebrospinal liquids, are good candidates to be studied using PCR techniques. We detail our experience with a PCR assay applied to pleural and cerebrospinal fluids using the primer MPB64. Seventy three specimens were analyzed: 30 pleural fluids (PF), 26 pleural biopsies (PB) and 17 cerebrospinal fluids (CSF). The gold standard for the diagnosis of tuberculous meningitis was the positive culture for M. tuberculosis in CSF. Tuberculous pleural effusion was diagnosed when cultures of PF and/or PB were positive for M. tuberculosis, or the PB histology showed granulomas. Our results, compared to the gold standards employed, showed a sensitivity of 70%, specificity of 88%, positive predictive value of 82% and negative predictive value of 80%. The high specificity of the MPB64 fragment while still retaining a good sensitivity makes it very well suited for pleural and cerebrospinal tuberculosis diagnosis.

Intestinal nematodes and pulmonary tuberculosis

We report a significantly higher prevalence of intestinal nematodes in patients with pulmonary tuberculosis (TB) compared to a matched control group: 33/57 (57.8%) in patients with TB and 18/86 (20.9%) in the control group; OR=5.19; 95% CI= 2.33-11.69; p=0.000). When TB patients eosinophilia was also significantly higher among those with intestinal parasites (69.8%) compared to those without this condition (45.6%). We hypothesized that the immune modulation induced by nematodes is a factor that enhances TB infection/progression and that eosinophilia seen in TB patients is a consequence of helminth infection.

Characteristics of pulmonary tuberculosis in HIV seropositive and seronegative patients in a Northeastern region of Brazil

The aim of this study was to analyse the clinical, epidemiological and bacteriological features present in 60 pulmonary tuberculosis patients who were also infected with human immunodeficiency virus (HIV) and to compare these with 120 TB patients who were not infected with HIV. The patients with pulmonary tuberculosis and HIV coinfection were mostly male (p = 0.001), showed a higher frequency of weight loss >10 kilos (p <0.001), had a higher rate of non-reaction result to the tuberculin skin test (p <0.001), a higher frequency of negative sputum smear examination for acid-fast bacilli (p = 0.001) and negative sputum culture for Mycobacterium tuberculosis (p = 0.001). Treatment failure was more common in those who were HIV positive (p <0.000). No higher frequency of resistance to antituberculosis drugs was found to be associated with TB/HIV coinfection (p = 0.407). Association between extrapulmonary and pulmonary tuberculosis was more frequent in those seropositive to HIV than those without HIV virus, 30% and 1.6% respectively. These findings showed a predominance of atypical clinical laboratory features in co-infected patients, and suggest that health care personnel should consider the possibility this diagnosis.

Clinical and radiographic features of HIV-related pulmonary tuberculosis according to the level of immunosuppression

Medical charts and radiographs from 38 HIV-infected patients with positive cultures for Mycobacterium tuberculosis from sputum or bronchoalveolar lavage were reviewed in order to compare the clinical, radiographic, and sputum bacilloscopy characteristics of HIV-infected patients with pulmonary tuberculosis according to CD4+ lymphocyte count (CD4). The mean age of the patients was 32 years and 76% were male. The median CD4 was 106 cells/mm³ and 71% had CD4 < 200 cells/mm³. Sputum bacilloscopy was positive in 45% of the patients. Patients with CD4 < 200 cells/mm³ showed significantly less post-primary pattern (7% vs. 63%; p = 0.02) and more frequently reported weight loss (p = 0.04). Although not statistically significant, patients with lower CD4 showed lower positivity of sputum bacilloscopy (37% vs. 64%; p = 0.18). HIV-infected patients with culture-confirmed pulmonary tuberculosis had a high proportion of non-post-primary pattern in thoracic radiographs. Patients with CD4 lower than 200 cells/mm³ showed post-primary patterns less frequently and reported weight loss more frequently.

Improving tuberculosis control through the partnership between university and the health system

INTRODUCTION: Tuberculosis (TB) control is linked to the availability of qualified methods for microbiological diagnostics; however, microscopy with limited sensitivity is the only method available in many locations. The objective of this study was to evaluate the introduction of culture, drug susceptibility testing (DST), and genotyping in the routine of a Municipal Program of Tuberculosis Control. METHODS: Direct microscopy of sputum and culture in Ogawa-Kudoh were performed on 1,636 samples from 787 patients. DST of positive cultures was performed by resazurin microtiter assay and genotyping by mycobacterial interspersed repetitive units-variable number tandem repeat. RESULTS: A total 91 patients with TB were identified. The culture increased case detection by 32% compared with the microscopy; acquired resistance was 3.3% and the genotyping showed high genetic diversity. CONCLUSIONS: Ogawa-Kudoh contributed significantly to the increase in case detection and is suitable for implementation in poor-resource locations. The acquired resistance rate was lower than that reported in a recent Brazilian survey. The high genetic diversity is possibly related to the high TB prevalence in the population, as well as to early detection and suitable treatment of patients. The interaction between research and health care is important for reorienting the practice, transferring technology, and improving TB control.

Bone tuberculosis: a case report on child

The authors report a case of a 12-year-old child with a complaint of pain and deformity in the lower thoracic region that had lasted for two years. Clinical, epidemiological and laboratory characteristics associated with images of apparent damage in the T9-T10 and T11-T12 vertebrae taken by radiography of the thoracic spine and nuclear magnetic resonance in addition to the positivity of the molecular test based on the polymerase chain reaction, led to tuberculous spondylitis being diagnosed and specific therapy was started. Culture of vertebral biopsy was positive for Mycobacterium tuberculosis after thirty days.

The performance of an in-house nested-PCR technique for pleural tuberculosis diagnoses

Introduction This study evaluated the performance of an in-house nested-PCR system for the detection of the Mycobacterium tuberculosis complex in pleural fluid, blood and urine samples from pleural effusion tuberculosis patients by health services physicians in Pernambuco, Brazil. Methods A prospective double-blind study with 37 hospitalized patients of both sexes, aged over 15, was used to investigate the diagnosis of pleural effusion. The criteria used to define the cases included the demonstration of bacillus in biological samples by smear or culture or by a granulomatous finding in the histopathological examination, associated with an evident response to specific treatments to each clinical situation. Pleural fluid, blood and urine samples were collected and subjected to routine tests and the nested PCR technique to assess for M. tuberculosis amplification. Results In total, 37 pleural effusion patients took part in the study, of whom 19 (51.3%) had tubercular etiologies and 18 (48.7%) had etiologies from other causes. When the pleural fluid, blood and/or urine sample in-house nested-PCR sensitivities were evaluated simultaneously, the results were positive regardless of the biological specimen (the sensitivity was 84.2%); however, when the blood and/or urine samples were analyzed together, the sensitivity was 72.2%. When the pleural fluid samples were evaluated alone, the sensitivity was only 33.3%. Conclusions The performance of the diagnostic pleural tuberculosis nested-PCR was directly related to the diversity of the samples collected from the same patient. Additionally, this study may identify a need to prioritize non-invasive blood and urine collection for this diagnosis.

Diagnosing lymphoma in a setting with a high burden of infection: a pediatric case of Epstein-Barr virus-associated aggressive B-cell lymphoma with t(8;14) (q23;q32) and extensive necrosis mimicking tuberculosis

The association of lymphoma with necrotic granuloma can pose diagnostic challenges and delay treatment, especially in settings with a high burden of infection. In these settings, the timely use of cytogenetic and molecular methods is most relevant. Here, we report a case of B-cell lymphoma with t (8;14) in a 5-year-old male child. The lymphoma was associated with necrotic granuloma and was initially misdiagnosed as tuberculosis. Polymerase chain reaction was used to detect clonal lymphoproliferation and to rule out Mycobacterium tuberculosis infection. Tumor cells harbored Epstein-Barr virus and expressed CD20, CD10, BCL6, and Ki67 (30%), leading to the diagnosis of B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma.

Bile duct-duodenal fistula caused by AIDS/HIV-associated tuberculosis

Allthough infrequent, digestive fistulae in HIV/AIDS patients have been reported throughout the digestive tract from the esophagus to the anus, with predominance of esophageal fistulae. AIDS/HIV-associated opportunistic infections may invade the digestive system and lead to fistula formation. Tuberculosis is the most common infection associated with these esophageal fistulae. We report here one case of bile duct-duodenal fistula in a female AIDS patient with associated abdominal Mycobacterium tuberculosis infection compromising lymphnodes of the hepatic pedicle where the fistula was found. According to the reviewed literature, this is the third case of bile duct-duodenal fistula associated with abdominal tuberculosis in AIDS patient, and the first where both the fistula and the tuberculosis infection were diagnosed at laparotomy for acute abdomen. Whether the AIDS patient with abdominal pain needs or not a laparotomy to treat an infectious disease is often a difficult matter for the surgeon to decide, as most of the times appropriate medical treatment will bring more benefit.

LAY USE OF AMAZONIAN PLANTS FOR THE TREATMENT OF TUBERCULOSIS

The Brazilian State of Amazonas has a high incidence of Tuberculosis, 91.4 in 10,000 habitants (SESAU, 1994) and resistant strains of Mycobacterium tuberculosis are frequently being found in the region (SALEM et.al, 1990). These problems have been associated with side effects caused by the antibiotics used to treat Tuberculosis, which have in rum been associated with treatment non-compliance (PATTISAPU, 1984). To resolve this problem a cost effective alternative treatment for Tuberculosis with few or no side effects, needs to be found. Amazonas has an abundance of plants, many of which are used by the lay population for medicinal purposes. A survey was carried out in five towns of the region, interviewing patients receiving treatment for Tuberculosis, to find out whether and which plants have been used to treat Tuberculosis. Results showed that the majority of patients in the sample had used medicinal plants before or after diagnosis of Tuberculoses. Thirteen different plants were recorded for this purpose. Chenopodium ambrosioides L, popularly known as Mastruz, was the most commonly used, followed by Caesalpinia ferrea Mart. Jucá and Spilanthes acmella DC. Jambu. This study concentrates on Mastruz as it was used more frequently than the other medicinal plants. No significant effects on baciloscopy test results were found when Mastruz was used before diagnosis. ln-vitro laboratory tests have also not shown any tuberculocidal effects for Mastruz. Further tests are being carried out on the other medicinal plants.

Phenotypes of lung mononuclear phagocytes in HIV seronegative tuberculosis patients: evidence for new recruitment and cell activation

Mycobacterium tuberculosis preferentially resides in mononuclear phagocytes. The mechanisms by which mononuclear phagocytes keep M. tuberculosis in check or by which the microbe evades control to cause disease remain poorly understood. As an initial effort to delineate these mechanisms, we examined by immunostaining the phenotype of mononuclear phagocytes obtained from lungs of patients with active tuberculosis. From August 1994 to March 1995, consecutive patients who had an abnormal chest X-ray, no demostrable acid-fast bacilli in sputum specimens and required a diagnostic bronchoalveolar lavage (BAL) were enrolled. Of the 39 patients enrolled, 21 had microbiologically diagnosed tuberculosis. Thirteen of the 21 tuberculosis patients were either HIV seronegative (n = 12) or had no risk factor for HIV and constituted the tuberculosis group. For comparison, M. tuberculosis negative patients who had BAL samples taken during this time (n = 9) or normal healthy volunteers (n = 3) served as control group. Compared to the control group, the tuberculosis group had significantly higher proportion of cells expressing markers of young monocytes (UCHM1) and RFD7, a marker for phagocytic cells, and increased expression of HLA-DR, a marker of cell activation. In addition, tuberculosis group had significantly higher proportion of cells expressing dendritic cell marker (RFD1) and epithelioid cell marker (RFD9). These data suggest that despite recruitment of monocytes probably from the peripheral blood and local cell activation, host defense of the resident lung cells is insufficient to control M. tuberculosis.

The Changing Face of the Epidemiology of Tuberculosis due to Molecular Strain Typing: A Review

About one third of the world population is infected with tubercle bacilli, causing eight million new cases of tuberculosis (TB) and three million deaths each year. After years of lack of interest in the disease, World Health Organization recently declared TB a global emergency and it is clear that there is need for more efficient national TB programs and newly defined research priorities. A more complete epidemiology of tuberculosis will lead to a better identification of index cases and to a more efficient treatment of the disease. Recently, new molecular tools became available for the identification of strains of Mycobacterium tuberculosis (M. tuberculosis), allowing a better recognition of transmission routes of defined strains. Both a standardized restriction-fragment-length-polymorphism-based methodology for epidemiological studies on a large scale and deoxyribonucleic acids (DNA) amplification-based methods that allow rapid detection of outbreaks with multidrug-resistant (MDR) strains, often characterized by high mortality rates, have been developed. This review comments on the existing methods of DNA-based recognition of M. tuberculosis strains and their peculiarities. It also summarizes literature data on the application of molecular fingerprinting for detection of outbreaks of M. tuberculosis, for identification of index cases, for study of interaction between TB and infection with the human immunodeficiency virus, for analysis of the behavior of MDR strains, for a better understanding of risk factors for transmission of TB within communities and for population-based studies of TB transmission within and between countries

Mechanisms of cell accumulation induced by Mycobacterium bovis BCG

Mycobacteria, specially Mycobacterium tuberculosis are among the micro-organisms that are increasing dramatically the number of infections with death, all over the world. A great number of animal experimental models have been proposed to investigate the mechanisms involved in the host response against these intracellular parasites. Studies of airway infection in guinea-pigs and rabbits, as well as, in mice intravenously infected with BCG have made an important contribution to our understanding of the virulence, pathogenesis and the immunology of mycobacterial infections. Although, there are few models to study the mechanisms of the initial inflammatory process induced by the first contact with the Mycobacteria, and the relevance of the acute generation of inflammatory mediators, cytokines and leukocyte infiltration to the development of the mycobacterial infection. In this work we reviewed our results obtained with a model of M. bovis BCG-induced pleurisy in mice, describing the mechanisms involved in the leukocyte influx induced by BCG at 24 hr. Different mechanisms appear to be related with the influx of neutrophils, eosinophils and mononuclear cells and distinct inflammatory mediators, cytokines and adhesion molecules are involved in the BCG-induced cell accumulation.

Transmission of tuberculosis in Havana, Cuba: a molecular epidemiological study by IS6110 restriction fragment length polymorphism typing

The combination of molecular and conventional epidemiological methods has improved the knowledge about the transmission of tuberculosis in urban populations. To examine transmission of tuberculosis in Havana, Cuba, with DNA fingerprinting, we studied 51 out of 92 Mycobacterium tuberculosis strains isolated from tuberculosis patients who resided in Havana and whose infection was culture-confirmed in the period from September 1997 to March 1998. Isolates from 28 patients (55%) had unique IS6110 restriction fragment length polymorphism (RFLP) patterns, while isolates from 23 others (45%) had identical patterns and belonged to 7 clusters. Three clusters consisting of six, five and two cases were each related to small outbreaks that occurred in a closed setting. Three other clustered cases were linked to a large outbreak that occurred in another institution. Younger patients were more correlated to clustering than older ones. The finding that 45% of the isolates had clustered RFLP patterns suggests that recent transmission is a key factor in the tuberculosis cases in Havana. The IS6110 RFLP typing made it possible to define the occurrence of outbreaks in two closed institutions.

Mycobacterium bovis: polymerase chain reaction identification in bovine Lymphonode biopsies and genotyping in isolates from Southeast Brazil by spolygotyping and restriction fragment length polymorphism

Diagnosis of the Mycobacterium tuberculosis complex by direct PCR of mediastinal lymphnode DNA and microbiological tests were compared in cattle suspicious of bearing tuberculous-like lesions detected during slaughter. The PCR procedure applied on DNA samples (n=54) obtained by adding alpha -casein into the thiocyanate extraction mix was positive in 70% of the samples. PCR confirmed the identification of 23 samples (100%) that grew in culture, 9 samples (60%) that failed to grow in culture, plus 6 (37.5%) samples that resulted in growth of bacterial contaminants. Genotyping by IS6110-RFLP and DR-spoligotyping analysis of seven samples revealed the presence of several polimorphisms. Seven of the isolates contained multiple copies of IS6110, thus defining the existence of five singular genotypes.