Heparin-Binding Haemagglutinin, a New Tool for the Detection of Latent Mycobacterium tuberculosis Infection in Hemodialysis Patients

Background:Patients with end-stage renal disease (ESRD) and latently infected with Mycobacterium tuberculosis (LTBI) are at higher risk to develop tuberculosis (TB) than healthy subjects. Interferon-gamma release assays (IGRAs) were reported to be more sensitive than tuberculin skin tests for the detection of infected individuals in dialysis patients.Methods:On 143 dialysis patients prospectively enrolled, we compared the results from the QuantiFERON®-TB Gold assay (QFT), to those of an IGRA in response to in vitro stimulation of circulating mononuclear cells with the mycobacterial latency antigen Heparin-Binding Haemagglutinin purified from Mycobacterium bovis BCG (native HBHA, nHBHA).Results:Seven patients had a past history of active TB and 1 had an undetermined result with both IGRAs. Among the other 135 patients, 94 had concordant results with the QFT and nHBHA-IGRA, 40.0% being negative and therefore not latently infected, and 29.6% being positive and thus LTBI. Discrepant results between these tests were found for 36 patients positive only with the nHBHA-IGRA and 5 only with the QFT.Conclusions:The nHBHA-IGRA is more sensitive than the QFT for the detection of LTBI dialysis patients, and follow-up of the patients will allow us to define the clinical significance of discrepant results between the nHBHA-IGRA and the QFT. © 2013 Dessein et al.

Contribution of a heparin-binding haemagglutinin interferon-gamma release assay to the detection of Mycobacterium tuberculosis infection in HIV-infected patients: comparison with the tuberculin skin test and the QuantiFERON-TB Gold In-tube.

The screening and treatment of latent tuberculosis (TB) infection reduces the risk of progression to active disease and is currently recommended for HIV-infected patients. The aim of this study is to evaluate, in a low TB incidence setting, the potential contribution of an interferon-gamma release assay in response to the mycobacterial latency antigen Heparin-Binding Haemagglutinin (HBHA-IGRA), to the detection of Mycobacterium tuberculosis infection in HIV-infected patients.

Immunological Signatures Identifying Different Stages of Latent Mycobacterium tuberculosis Infection and Discriminating Latent from Active Tuberculosis in Humans

Objectives: One third of the world population is considered latently infected with Mycobacterium tuberculosis(LTBI) and sterilizing this reservoir of bacteria that may reactivate is required for tuberculosis (TB) elimination. Thegroup of individuals with LTBI is heterogeneous with some of them being more at risk to develop TB disease thanothers. Improved diagnosis of subjects with LTBI is needed, allowing to differentiate subjects with LTBI from thosewith active TB, and to select among LTBI subjects those who are more at risk to develop active TB. We havecharacterized at the cellular level both the quantitative and qualitative T cell responses to different mycobacterialantigens in selected populations of infected subjects in order to identify new biomarkers that could help to identify M.tuberculosis-infected subjects and to stratify them in risk groups for reactivation of the infection.Methods: Lymphoblast frequencies and cytokine production (IFN-γ, TNF-α, IL-2) among CD4+ and CD8+ T cellswere analyzed by flow cytometry after in vitro stimulation with the latency antigen heparin-binding haemagglutinin(HBHA) or early-secreted antigen Target-6 (ESAT-6) of peripheral blood mononuclear cells from clinically wellcharacterized M. tuberculosis-infected humans (28 LTBI, 22 TB disease,12 controls). The LTBI group definedaccording to the Center for Disease Control guidelines was subdivided into QuantiFERON-TB Gold in-Tube (QFT)positive and negative subgroups.Results: Similar to TB patients, QFT+ LTBI subjects had higher proportions of HBHA-induced TNF-αsingle+ CD4+lymphocytes than QFT- LTBI subjects (p<0.05). Compared to LTBI subjects, TB patients had higher frequencies ofESAT-6-induced CD8+ lymphoblasts (p<0.001), higher proportions of ESAT-6-induced IFN-γ+TNF-α+ CD4+ Tlymphocytes (p<0.05), and lower proportions of HBHA-induced IFN-γ+TNF-α+IL-2+ (p<0.05) CD4+ T lymphocytes.Conclusions: These data provide new biomarkers to discriminate active TB from LTBI, and more interestingly,help to identify LTBI subjects with increased likelihood to develop TB disease.

Contribution of PPD/HBHA Interferon-gamma Release Assay for the Detection of Mycobacterium tuberculosis Infection in HIV Infected Patients

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Improved Detection of Mycobacterium bovis Infection in Bovine Lymph Node Tissue Using Immunomagnetic Separation (IMS)-Based Methods

Immunomagnetic separation (IMS) can selectively isolate and concentrate Mycobacterium bovis cells from lymph node tissue to facilitate subsequent detection by PCR (IMS-PCR) or culture (IMS-MGIT). This study describes application of these novel IMS-based methods to test for M. bovis in a survey of 280 bovine lymph nodes (206 visibly lesioned (VL), 74 non-visibly lesioned (NVL)) collected at slaughter as part of the Northern Ireland bovine TB eradication programme. Their performance was evaluated relative to culture. Overall, 174 (62.1%) lymph node samples tested positive by culture, 162 (57.8%) by IMS-PCR (targeting IS6110), and 196 (70.0%) by IMS-MGIT culture. Twelve (6.9%) of the 174 culture positive lymph node samples were not detected by either of the IMS-based methods. However, an additional 78 M. bovis positive lymph node samples (26 (12.6%) VL and 54 (73.0%) NVL) were detected by the IMS-based methods and not by culture. When low numbers of viable M. bovis are present in lymph nodes (e.g. in NVLs of skin test reactor cattle) decontamination prior to culture may adversely affect viability, leading to false negative culture results. In contrast, IMS specifically captures whole M. bovis cells (live, dead or potentially dormant) which are not subject to any deleterious treatment before detection by PCR or MGIT culture. During this study only 2.7% of NVL lymph nodes tested culture positive, whereas 73% of the same samples tested M. bovis positive by the IMS-based tests. Results clearly demonstrate that not only are the IMS-based methods more rapid but they have greater detection sensitivity than the culture approach currently used for the detection of M. bovis infection in cattle.. Adoption of the IMS-based methods for lymph node testing would have the potential to improve M. bovis detection in clinical samples.

Mycobacterium tuberculosis aortic graft infection with recurrent hemoptysis: a case report.

INTRODUCTION: Mycobacterium tuberculosis may cause a large variety of clinical presentations due to its ability to disseminate by contiguity or hematogenously. Tuberculosis may remain undiagnosed for years due to the chronic course of the disease, with potentially life-threatening long-term complications. CASE PRESENTATION: In this case report, we describe a tuberculous aortic graft infection in a 72-year-old man documented by polymerase chain reaction and cultures. The patient presented with three episodes of hemoptysis following a remote history of miliary tuberculosis. The infection was treated by graft replacement and prolonged antimycobacterial therapy. CONCLUSION: Tuberculous infection of a vascular graft is an uncommon complication, but should be considered in patients with an intravascular device and a history of previous tuberculosis, especially when hematogenous spread may have occurred a few months after surgery, or when an active mycobacterial infection is present in close proximity to the graft.

Apoptosis of macrophages during pulmonary Mycobacterium bovis infection: correlation with intracellular bacillary load and cytokine levels

P>Apoptosis of macrophages infected with pathogenic mycobacteria is an alternative host defence capable of removing the environment supporting bacterial growth. In this work the influence of virulence and bacterial load on apoptosis of alveolar macrophages during the initial phase of infection by Mycobacterium bovis was investigated. BALB/c mice were infected intratracheally with high or low doses of the virulent (ATCC19274) or attenuated (bacillus Calmette-Guerin Moreau) strains of M. bovis. The frequency of macrophage apoptosis, the growth of mycobacteria in macrophages, and the in situ levels of the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10) and IL-12 and of the anti-apoptotic protein Bcl-2 were measured at day 3 and day 7 post-infection. An increase of macrophage apoptosis was observed after infection with both strains but the virulent strain induced less apoptosis than the attenuated strain. On the 3rd day after infection with the virulent strain macrophage apoptosis was reduced in the high-dose group, while on the 7th day post-infection macrophage apoptosis was reduced in the low-dose group. Inhibition of apoptosis was correlated with increased production of IL-10, reduced production of TNF-alpha and increased production of Bcl-2. In addition, the production of IL-12 was reduced at points where the lowest levels of macrophage apoptosis were observed. Our results indicate that virulent mycobacteria are able to modulate macrophage apoptosis to an extent dependent on the intracellular bacterial burden, which benefits its intracellular growth and dissemination to adjacent cells.

Risk factors for Mycobacterium ulcerans infection, southeastern Australia

Buruli/Bairnsdale ulcer (BU) is a severe skin and soft tissue disease caused by Mycobacterium ulcerans. To better understand how BU is acquired, we conducted a case-control study during a sustained outbreak in temperate southeastern Australia. We recruited 49 adult patients with BU and 609 control participants from a newly recognized BU-endemic area in southeastern Australia. Participants were asked about their lifestyle and insect exposure. Odds ratios were calculated by using logistic regression and were adjusted for age and location of residence. Odds of having BU were at least halved for those who frequently used insect repellent, wore long trousers outdoors, and immediately washed minor skin wounds; odds were at least doubled for those who received mosquito bites on the lower legs or lower arms. This study provides new circumstantial evidence that implicates mosquitoes in the transmission of M. ulcerans in southeastern Australia.

Mycobacterium ulcerans infection : factors influencing diagnostic delay

Objective: To document the epidemiology, clinical characteristics and diagnosis of an outbreak of Mycobacterium ulcerans infection (Bairnsdale or Buruli ulcer [BU]) during the period 1998–2006, and compare delays in diagnosis between residents of endemic and non-endemic regions.

Design and setting: Retrospective case study of patients identified through infectious disease physicians on the Bellarine Peninsula and the Victorian Department of Human Services notifiable diseases database.

Main outcome measures: Description of events leading to diagnosis of BU.

Results: Eighty-five BU patients recalled their experience. Fifty-three patients were older than 60 years, and 61 permanently resided on the Bellarine Peninsula. The onset of symptoms occurred most frequently in mid winter. Twenty-eight patients had lesions on the arm and 51 on the leg. The median time between onset of symptoms and first medical contact was shorter for those living in the endemic area (3.0 weeks; interquartile range [IQR], 1.0–5.0 weeks) compared with non-endemic areas (5.3 weeks; IQR, 2.0–9.5 weeks) (P = 0.05). Patients who resided in the endemic area had a shorter median time from their first medical appointment to diagnosis (1.0 week; IQR, 0.0–3.9 weeks) than those who resided in non-endemic areas (5.0 weeks; IQR, 1.3–8.0 weeks) (P = 0.001).

Conclusion: Delay in presentation and time to diagnosis of BU are longer in non-endemic than endemic areas. Measures should be taken to raise awareness of the disease in non-endemic areas.

Epitope-specific CD4+, but not CD8+, T-cell responses induced by recombinant influenza A viruses protect against Mycobacterium tuberculosis infection

Tuberculosis remains a global health problem, in part due to failure of the currently available vaccine, BCG, to protect adults against pulmonary forms of the disease. We explored the impact of pulmonary delivery of recombinant influenza A viruses (rIAVs) on the induction of Mycobacterium tuberculosis (M. tuberculosis)-specific CD4(+) and CD8(+) T-cell responses and the resultant protection against M. tuberculosis infection in C57BL/6 mice. Intranasal infection with rIAVs expressing a CD4(+) T-cell epitope from the Ag85B protein (PR8.p25) or CD8(+) T-cell epitope from the TB10.4 protein (PR8.TB10.4) generated strong T-cell responses to the M. tuberculosis-specific epitopes in the lung that persisted long after the rIAVs were cleared. Infection with PR8.p25 conferred protection against subsequent M. tuberculosis challenge in the lung, and this was associated with increased levels of poly-functional CD4(+) T cells at the time of challenge. By contrast, infection with PR8.TB10.4 did not induce protection despite the presence of IFN-γ-producing M. tuberculosis-specific CD8(+) T cells in the lung at the time of challenge and during infection. Therefore, the induction of pulmonary M. tuberculosis epitope-specific CD4(+), but not CD8(+) T cells, is essential for protection against acute M. tuberculosis infection in the lung.

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

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Case report: Disseminated Mycobacterium tuberculosis infection in a dog

An uncommon disseminated Mycobacterium tuberculosis infection is described in a 12-year-old female dog presenting with fever, dyspnea, cough, weight loss, lymphadenopathy, melena, epistaxis, and emesis. The dog had a history of close contact with its owner, who died of pulmonary tuberculosis. Radiographic examination revealed diffuse radio-opaque images in both lung lobes, diffuse visible masses in abdominal organs, and hilar and mesenteric lymphadenopathy. Bronchial washing samples and feces were negative for acid-fast organisms. Polymerase chain reaction (PCR)-based species identification of bronchial washing samples, feces, and urine revealed M. tuberculosis using PCR-restriction enzyme pattern analysis-PRA. Because of public health concerns, which were worsened by the physical condition of the dog, euthanasia of the animal was recommended. Rough and tough colonies suggestive of M. tuberculosis were observed after microbiological culture of lung, liver, spleen, heart, and lymph node fragments in Löwenstein-Jensen and Stonebrink media. The PRA analysis enabled diagnosis of M. tuberculosis strains isolated from organs. Copyright © 2013 by The American Society of Tropical Medicine and Hygiene.

Mycobacterium marinum infection: a case report

The infection by Mycobacterium marinum in humans is relatively uncommon. When it occurs, it mainly affects the skin, usually with a chronic, indolent and benign evolution. The diagnosis requires a high index of suspicion, and a significant delay may be observed between the first symptoms to the final diagnosis. This present case reports a M. marinum infection in an immunocompetent patient that had a chronic undiagnosed injury on the dominant hand for at least five years. The patient had several medical consultations, without proper suspicion, hampering adequate diagnostic investigation. Histopathology detected tuberculoid granulomas, but showed no acid-fast bacilli. The culture in appropriate medium and the polymerase chain reaction-restriction enzyme analysis (PRA)-hsp65 confirmed the diagnosis. Treatment with clarithromycin (1 g/day) for three months was effective. Although uncommon, this infection is a contact zoonosis. Therefore, it is important for clinicians to be aware of this diagnosis and properly guide preventable measures to professionals that are in risk group.

Tuberculosis

Tuberculosis is a term that encompasses various diseases caused by bacteria of the Mycobacterium tuberculosis complex, including M tuberculosis, M bovis, M africanum, and other mycobacterial species. Whereas M tuberculosis infection is largely spread from human to human, M bovis infection has been identified as a zoonotic disease with most cases of human infection attributable to animal sources. The mycobacteria other than tuberculosis complex (MOTT), which includes M avium subsp avium and M avium subsp intracellulare isolated from animals, has been isolated from immune-compromised humans (ie, those with human immunodeficiency virus [HIV] infection), but seldom from immunocompetent humans. Recently, there has been increased interest among public health officials in drug-resistant strains of M tuberculosis, M bovis, and M avium because several have been isolated from HIV-infected and nonimmuno-compromised humans.

Development of a Model of Natural Infection with Mycobacterium bovis in White-Tailed Deer

The objective of this study was to develop a suitable experimental model of natural Mycobacterium bovis infection in white-tailed deer (Odocoileus virginianus), describe the distribution and character of tuberculous lesions, and to examine possible routes of disease transmission. In October 1997, 10 mature female white-tailed deer were inoculated by intratonsilar instillation of 2 3 103 (low dose) or 2 3 105 (high dose) colony forming units (CFU) of M. bovis. In January 1998, deer were euthanatized, examined, and tissues were collected 84 to 87 days post inoculation. Possible routes of disease transmission were evaluated by culture of nasal, oral, tonsilar, and rectal swabs at various times during the study. Gross and microscopic lesions consistent with tuberculosis were most commonly seen in medial retropharyngeal lymph nodes and lung in both dosage groups. Other tissues containing tuberculous lesions included tonsil, trachea, liver, and kidney as well as lateral retropharyngeal, mandibular, parotid, tracheobronchial, mediastinal, hepatic, mesenteric, superficial cervical, and iliac lymph nodes. Mycobacterium bovis was isolated from tonsilar swabs from 8 of 9 deer from both dosage groups at least once 14 to 87 days after inoculation. Mycobacterium bovis was isolated from oral swabs 63 and 80 days after inoculation from one of three deer in the low dose group and none of four deer in the high dose group. Similarly, M. bovis was isolated from nasal swabs 80 and 85 days after inoculation in one of three deer from the low dose group and 63 and 80 days after inoculation from two of four deer in the high dose group. Intratonsilar inoculation with M. bovis results in lesions similar to those seen in naturally infected white-tailed deer; therefore, it represents a suitable model of natural infection. These results also indicate that M. bovis persists in tonsilar crypts for prolonged periods and can be shed in saliva and nasal secretions. These infected fluids represent a likely route of disease transmission to other animals or humans.