Tuberculosis

Editorial Board

2012-05-01

Tuberculosis and advances in the online delivery of scientific research

Ash Allan — 2012-05-01

Catching a train to work, taking a stroll through the park, or waiting to meet someone in a café, there is no need to be without access to Tuberculosis. These days you can read Tuberculosis and search for the most relevant research on the move and not only that, the content you are able to retrieve is becoming more sophisticated.

Mycobacterium tuberculosis – Heterogeneity revealed through whole genome sequencing

2012-01-05

Summary: The emergence of whole genome sequencing (WGS) technologies as primary research tools has allowed for the detection of genetic diversity in Mycobacterium tuberculosis (Mtb) with unprecedented resolution. WGS has been used to address a broad range of topics, including the dynamics of evolution, transmission and treatment. Here, we have analyzed 55 publically available genomes to reconstruct the phylogeny of Mtb, and we have addressed complications that arise during the analysis of publically available WGS data. Additionally, we have reviewed the application of WGS to the study of Mtb and discuss those areas still to be addressed, moving from global (phylogeography), to local (transmission chains and circulating strain diversity), to the single patient (clonal heterogeneity) and to the bacterium itself (evolutionary studies). Finally, we discuss the current WGS approaches, their strengths and limitations.

Resistance mechanisms of Mycobacterium tuberculosis against phagosomal copper overload

Jennifer L. Rowland, Michael Niederweis — 2012-02-24

Summary: Mycobacterium tuberculosis is an important bacterial pathogen with an extremely slow growth rate, an unusual outer membrane of very low permeability and a cunning ability to survive inside the human host despite a potent immune response. A key trait of M. tuberculosis is to acquire essential nutrients while still preserving its natural resistance to toxic compounds. In this regard, copper homeostasis mechanisms are particularly interesting, because copper is an important element for bacterial growth, but copper overload is toxic. In M. tuberculosis at least two enzymes require copper as a cofactor: the Cu/Zn-superoxide dismutase SodC and the cytochrome c oxidase which is essential for growth in vitro. Mutants of M. tuberculosis lacking the copper metallothionein MymT, the efflux pump CtpV and the membrane protein MctB are more susceptible to copper indicating that these proteins are part of a multipronged system to balance intracellular copper levels. Recent evidence showed that part of copper toxicity is a reversible damage of Fe–S clusters of dehydratases and the displacement of other divalent cations such as zinc and manganese as cofactors in proteins. There is accumulating evidence that macrophages use copper to poison bacteria trapped inside phagosomes.Here, we review the rapidly increasing knowledge about copper homeostasis in M. tuberculosis and contrast those with similar mechanisms in Escherichia coli. These findings reveal an intricate interplay between the host which aims to overload the phagosome with copper and M. tuberculosis which utilizes several mechanisms to reduce the toxic effects of excess copper.

A mouse model of tuberculosis reinfection

2012-03-19

Summary: Recent clinical observations shows that individuals treated with chemotherapy for tuberculosis who live in endemic areas are four times more likely to develop secondary disease, often as not caused by exogenous reinfection. In a mouse model described here, we show that mice infected with the virulent W-Beijing Mycobacterium tuberculosis strain HN878, then given chemotherapy to clear the infection, were resistant to re-challenge with the same organism thereafter. This resistance, which was mediated by rapid expression of CD4 T cells expressing markers consistent with both central and effector memory immunity, was only transient however. After 20–30 days of the reinfection the numbers of these cells steadily declined, the bacterial load in the lungs surged up, and the lung tissues became increasingly consolidated. No evidence was found for a regulatory T cell response in these mice, but many T cells harvested from the lungs showed evidence of increased PD-1 expression, indicating exhaustion. These data indicate that the memory T cell response to reinfection may not be as stable and long lived as previously thought, a finding with obvious implications for vaccine development.

Mycobacterium tuberculosis-induced neutrophil ectosomes decrease macrophage activation

2012-03-05

Summary: Background: The existence of ectosome-like microvesicles released by neutrophils was proposed a few decades ago. Other studies revealed that the innate immune response during mycobacterial infection is accompanied by an intense migration of neutrophils to the site of infection, which may be important during the acute phase of tuberculosis. We found that the ectosomes derived from infected neutrophils are biologically active and can influence the survival of Mycobacterium tuberculosis within macrophages.Methods: Mycobacteria were cultured on supplemented Middlebrook-7H9 broth. All strains were grown to the exponential phase and quantitated by serial dilution. Human neutrophils and macrophages were infected with mycobacteria. Ectosomes from neutrophils were isolated post-infection and characterized by transmission electron microscopy and flow cytometry. To determine whether these microvesicles influenced mycobactericidal activity, mycobacteria-infected macrophages were treated with isolated ectosomes.Results: Ectosomes were released from neutrophils infected with mycobacteria. These ectosomes were derived from neutrophil plasma membrane and a small proportion stained with PKH26. These microvesicles, when incubated with infected macrophages, influenced antimycobacterial activity.Conclusions: This is the first study to demonstrate that ectosomes that are shed from infected neutrophils influence mycobactericidal activity in macrophages in vitro, suggesting that these microvesicles have biological significance. Nevertheless, major gaps in our knowledge of microvesicle biology remain.

Characteristics of non-clustered tuberculosis in a low burden country

2012-03-14

Summary: Molecular genotyping studies often focus on clustered tuberculosis and recent transmission. Less attention has been paid to non-clustered tuberculosis. However, non-clustered cases also contribute significantly to the tuberculosis burden, especially in low-incidence countries. The objective of this study is to characterize non-clustered tuberculosis cases in Denmark and point out potential implications for tuberculosis control. The study is based on nationwide IS6110-RFLP genotyping of tuberculosis cases from 1992 through 2004, corresponding to 98% of culture verified cases.Of 3988 cases, 45% were non-clustered. Both Danes and immigrants had a peak incidence of non-clustered tuberculosis at older ages, 80–89 years (4.3 cases/105 population/year) and 60–69 years (28.8 cases/105 population/year), respectively. In addition, immigrants had a peak at 20–29 years (43.2 cases/105 inhabitants/year). In Danes, the incidence of non-clustered tuberculosis decreased during the study period and was predominantly found in elderly persons, presumably reactivating infection acquired during 1910–40, when tuberculosis incidence was high. In immigrants, the incidence was high at all ages, presumably reflecting reactivation of imported infections.In the future, the number of non-clustered tuberculosis cases will decrease, as older Danes die, and as time since primary infection increases for immigrants residing in Denmark. TB control should include focus on non-clustered cases.

Dynamic and complex Mycobacterium tuberculosis microevolution unrevealed by standard genotyping

Laura Pérez-Lago, Marta Herranz, Emilio Bouza, Darío García de Viedma — 2012-02-20

Summary: We performed an in-depth analysis of a large Mycobacterium tuberculosis cluster involving nine isolates with identical or highly similar RFLP types. The strain involved in this transmission cluster microevolved and accumulated up to five differential IS6110 transposition events, several of them not revealed by standard genotyping approaches. Some of these events probably generated extended deletions. In two cases of tuberculosis within the cluster, we observed the simultaneous presence of both the reference and microevolved variants. A longitudinal genotypical survey of M. tuberculosis in a real clinical evolutionary scenario can reveal dynamic and complex microevolution events.

Multiplex allele specific PCR for rapid detection of extensively drug resistant tuberculosis

Viral Vadwai, Anjali Shetty, Camilla Rodrigues — 2012-02-20

Summary: Effective tuberculosis (TB) control is hindered by lack of rapid diagnostic tests for detection of drug-resistant TB (DR-TB). Use of molecular tools for rapid detection of multi-and extensively- DR-TB, could facilitate early initiation of appropriate anti-tubercular treatment (ATT) regimen thereby interrupting transmission. Understanding the urgent situation, we standardized and evaluated 4 individual multiplex allele specific PCR (MAS-PCR) assays on 450 sputum specimens for Mycobacterium tuberculosis (MTB) detection and determination of drug resistance by targeting katG315, rpoB531, gyrA 94, rrs 1401 codon mutations for determination of resistance to Isoniazid (INH), Rifampicin (RIF), Fluoroquinolones (FQ) and Aminoglycosides (AG) respectively. Using a single sputum specimen, MAS-PCR correctly identified 97.2% (281/289; 95% CI:95–99) culture positive patients as MTB positive, 100% (271/271; 95% CI:99–100) for smear positive cases and 55.5% (10/18; 95% CI:34–75) for smear negative cases; and correctly identified 93.6% (104/111; 95% CI:87–97) of culture negative patients. Individual MAS-PCR assays reported variable diagnostic accuracy for determination of drug resistance. On comparison with phenotypic drug susceptibility testing, MAS-PCR assays correctly identified 89.2% (191/214; 95% CI:84–93), 94.9% (187/197; 95% CI:91–97), 72.5% (98/135; 95% CI:65–79) and 92.3% (24/26; 95% CI:75–99) of INH resistant, RIF resistant, FQ resistant and AG resistant specimens respectively; and correctly identified 94% (63/67; 95% CI:85–98), 86.9% (73/84; 95% CI:78–93), 93.1% (136/146; 95% CI:88–96) and 99.2% (253/255; 95% CI:97–100) of INH sensitive, RIF sensitive, FQ sensitive and AG sensitive specimens respectively. Thus, use of MAS-PCR assays for rapid detection of DR-TB is recommended, enabling early initiation of appropriate ATT.

Anti-phospholipid antibody levels as biomarker for monitoring tuberculosis treatment response

2012-03-14

Summary: Standard methods to monitor tuberculosis (TB) treatment response rely on sputum microscopy and culture conversion. Alternatives to these methods are needed for those patients whose sputum tests are smear or culture negative. Here, we examine anti-phospholipid IgM antibody level changes as a biomarker for treatment response in smear positive TB patients. Serum samples were obtained from 40 pulmonary TB patients at the start and end of the intensive phase treatment (IPT) from the CDC-TB Trials Consortium randomized clinical trial in Kampala, Uganda. Samples were screened by ELISA for IgM levels against five phospholipids found in Mycobacterium tuberculosis and host cells. Lipid antigens included cardiolipin (CL), phosphatidyl inositol (PI), phosphatidyl ethanolamine (PE), phosphatidyl choline (PTC), and sphingolipid (SL). Levels of IgM against all phospholipids significantly decreased (p = 0.034, 0.001, 0.008 0.008, 0.040, respectively) following anti-TB drug treatment in patients without lung cavitary disease at baseline. The mean sensitivity of this test in these patients was 83% when the IgM response to a single lipid antigen was used; it was >90% when responses to 2 or more lipids were assessed. In contrast, cavitary TB patients showed an overall IgM increase, with a significant rise against PE (p = 0.025). There was no significant difference in the change in antibody levels between patients who remained culture-positive and those who culture-converted after 40 doses of drug therapy. The measurement of IgM anti-phospholipid antibodies may be a useful biomarker to monitor treatment response in non-cavitary TB patients.

CXCL12 as a biological marker for the diagnosis of tuberculous pleurisy

2012-02-01

Summary: Although a chemokine CXCL12 is implicated in some infectious diseases, especially those in which T cell-mediated immunity plays critical roles, the relevance of CXCL12 to tuberculosis has never been elucidated. To determine the clinical efficacy of CXCL12 as a diagnostic marker for tuberculous (TB) pleurisy, we measured CXCL12 concentration in pleural fluid and serum from patients with various etiologies.Of 60 patients with pleural fluid, the median age of TB patients was 52 which was significantly lower than 71 of non-TB patients (P < 0.01). CXCL12 level in TB effusion (4456 ± 1013 pg/mL, n = 15) was significantly higher than non-TB effusion (2851 ± 1229 pg/mL, n = 45) (P < 0.01). On the other hand, serum CXCL12 level showed no significant differences among TB pleurisy, non-TB pleurisy, and normal healthy subjects. The sensitivity and specificity of CXCL12 in pleural fluid for the diagnosis of TB pleurisy was 60.0% and 93.2% (cut-off value = 4600 pg/mL), respectively. Area under the receiver operating characteristic (ROC) curve (AUC) for CXCL12 was 0.84. As the source of CXCL12, pleural mesothelium, endothelium of pulmonary vessels, bronchial epithelium, multinucleated giant epithelioid cells, and macrophages were positive for CXCL12 staining.Increased CXCL12 level in pleural fluid could be an informative diagnostic marker for differentiating TB pleurisy from other etiologies.

Genetic variants in antioxidant pathway: Risk factors for hepatotoxicity in tuberculosis patients

2012-02-17

Summary: Tuberculosis (TB) treatment can cause serious sequelae including adverse effects such as anti-TB drug-induced hepatotoxicity (ATDH). We performed a candidate gene-based association study between single nucleotide polymorphisms (SNPs) in 10 genes in the antioxidant pathway and ATDH susceptibility. The subjects comprised 100 Japanese patients with pulmonary TB who received a treatment regimen including isoniazid and rifampicin. Out of them, 18 patients had ATDH. Thirty-four tag SNPs in 10 genes were analyzed by PCR-restriction fragment length polymorphism or PCR-direct DNA sequencing. The frequencies of alleles and genotypes between patients with and without ATDH were compared in three different genetic models. Statistical analyses revealed that a C/C genotype at rs11080344 in NOS2A, a C/C genotype at rs2070401 in BACH1, and a G/A or A/A genotype at rs4720833 in MAFK independently conferred ATDH susceptibility. Remarkably, the association of the latter two tag SNPs with ATDH susceptibility was highly statistically significant (P = 0.0006) with an odds ratio of 9.730. This study is the first report to demonstrate that NOS2A, BACH1, and MAFK appear to be genetic determinants of ATDH in Japanese patients with TB. Furthermore, a combination of BACH1 and MAFK polymorphisms may be useful as new biomarkers to identify high-risk Japanese TB patients for ATDH.

Synergistic effect of two combinations of antituberculous drugs against Mycobacterium tuberculosis

Emma Rey-Jurado, Griselda Tudó, José Antonio Martínez, Julian González-Martín — 2012-02-20

Summary: Fluoroquinolones such as ofloxacin are promising drugs to treat drug-resistant tuberculosis (TB) and have been proposed for shortening the treatment of TB. The objectives were to study the synergistic effect of the combinations of three drugs and to evaluate the in vitro interactions of the following combinations against Mycobacterium tuberculosis: A) isoniazid, rifampicin, and ethambutol and B) ofloxacin, rifampicin, and ethambutol using an adaptation of the two-dimensional chequerboard assay. A total of 12 isolates resistant to isoniazid or to isoniazid-streptomycin and 11 drug-susceptible isolates were tested. The fractional inhibitory concentration (FICI) was calculated as follows: FICI = FICA + FICB + FICC = A/MICA + B/MICB + C/MICC where A, B and C were the MICs of each antibiotic in combination and MICA, MICB and MICC were the individual MICs. The FICI was interpreted as synergism when the value was ≤0.75. In combination A, 11 drug-susceptible isolates decreased the individual MIC one to three dilutions, showing indifferent activity in 81.8% (FICI = 0.88–1.6) and synergistic activity in 18.1% (FICI = 0.6). In combination B, 21 out of the 23 isolates studied (91.3%) showed synergism (FICI = 0.31–0.62). In conclusion, adaptation of the two-dimensional chequerboard assay is a reliable method to study in vitro three-drug combinations. Both three-drug combinations tested may be useful against drug-resistant isolates, although the combination including ofloxacin showed better efficacy, being of potential use in drug-susceptible and isoniazid-resistant isolates.

Evaluation of 24-locus MIRU-VNTR in extrapulmonary specimens: Study from a tertiary centre in Mumbai

Viral Vadwai, Anjali Shetty, Philip Supply, Camilla Rodrigues — 2012-02-10

Summary: Genotyping of Mycobacterium tuberculosis isolates is a useful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. There is a lack of information on the discriminatory power of standard 24-locus mycobacterial interspersed repetitive unit (MIRU) – variable number tandem repeats (VNTR) in India, which has the highest tuberculosis (TB) burden worldwide. Therefore, we assessed its utility on 69 M. tuberculosis (MTB) isolates from patients with extrapulmonary tuberculosis, in comparison to standard insertion sequence (IS) 6110-Restriction fragment length polymorphism (RFLP) fingerprinting and spoligotyping. IS6110-RFLP (HGDI, 0.9987) identified a single cluster of 3 (4.3%) single-copy IS6110 isolates. Spoligotyping showed 69.5% clustering (HGDI, 0.8857). In contrast, MIRU-VNTR analysis identified 69 (100%) unique strains (HGDI, 1.0000). Within the study limits, this observed high discriminatory power suggests that 24-locus MIRU-VNTR genotyping could potentially be used to study long-term transmission of MTB infection in Mumbai. Moreover, high congruence between the MIRU-VNTR-based and spoligotyping-based strain groupings suggests that CAS, EAI and Beijing are the predominant strain lineages in the Mumbai TB patient population. The Beijing lineage isolates were found to be more significantly associated with multi-drug resistance (p < 0.01) than CAS and EAI lineages.

Identifying multidrug resistant tuberculosis transmission hotspots using routinely collected data

2012-03-09

Summary: In most countries with large drug resistant tuberculosis epidemics, only those cases that are at highest risk of having MDRTB receive a drug sensitivity test (DST) at the time of diagnosis. Because of this prioritized testing, identification of MDRTB transmission hotspots in communities where TB cases do not receive DST is challenging, as any observed aggregation of MDRTB may reflect systematic differences in how testing is distributed in communities. We introduce a new disease mapping method, which estimates this missing information through probability-weighted locations, to identify geographic areas of increased risk of MDRTB transmission. We apply this method to routinely collected data from two districts in Lima, Peru over three consecutive years. This method identifies an area in the eastern part of Lima where previously untreated cases have increased risk of MDRTB. This may indicate an area of increased transmission of drug resistant disease, a finding that may otherwise have been missed by routine analysis of programmatic data. The risk of MDR among retreatment cases is also highest in these probable transmission hotspots, though a high level of MDR among retreatment cases is present throughout the study area. Identifying potential multidrug resistant tuberculosis (MDRTB) transmission hotspots may allow for targeted investigation and deployment of resources.

Surfactant preparations for tuberculosis and other diseases beyond infancy: A letter from Russia

Sergei V. Jargin — 2012-03-14

Preparations of pulmonary surfactant (Sf) are used for treatment of infant respiratory distress syndrome (RDS). The role of Sf in conditions without its primary deficiency, particularly in lung diseases beyond the neonatal period, is less straightforward. Some publications from Russia have reported about successful use of Sf preparations for treatment of ARDS in children and adults, including ARDS developing after surgical operations; for viral and bacterial pneumonia in children and the newborn, bronchopneumonia complicated by atelectasis and for pulmonary tuberculosis (Tb). In particular, it was reported that inhalations of Sf improve effectiveness of Tb chemotherapy, significantly accelerating healing, dissolution of infiltrates and closing of caverns, while mycobacteria disappear from sputum at an early date. In the international literature, it was reported that Sf participates in host resistance against Tb and that Sf preparations may enhance effectiveness of antibiotics and the immune system response in the process of Tb treatment; but no reports on the above-mentioned or similar effects in cavernous Tb have been found in the literature. The data on the decrease of mortality in adults with ARDS after the Sf-therapy are, in general, not confirmed in the international literature, although improved survival for ARDS due to pneumonia and aspiration has been reported. Improvement of lung histopathology and ultrastructure after Sf instillation in rats with bleomycin- and radiation-induced pulmonary fibrosis and in dogs with lung injury induced by lavage was reported in; but the illustrations, displaying singular cells with unspecific changes, appear to be non-representative. Although perspectives of Sf-therapy of ARDS have been discussed, recent reviews concluded that its use for ARDS is not recommended at least as a routine therapy for adults. Accordingly, no recommendations for the use of Sf preparations beyond the infancy have been found in the documents issued by the U.S. Food and Drug Administration (FDA), European regulating authorities, and the drug register. In particular, no recommendations for treatment of pulmonary Tb by Sf preparations were found in the official documents; while with regard to the ARDS it was stated that “in a meta-analysis of nine trials randomizing 1441 adult patients, surfactant showed no effect on early mortality compared to control”. Moreover, inhalations by means of an inhaler–nebulizer (http://www.inter-eton.ru/catalog.php?tovar_id=2) used in the studies as a treatment of Tb is probably an inadequate method of the Sf administration: in neonates it is performed by endotracheal intubation; while intratracheal delivery, aerosolisation in ventilator gas, or direct bronchoscopic instillation have been used in ARDS. However, Lovacheva et al. mentioned that they did perform bronchoscopies (for the purpose of bronchoalveolar lavage) within the scope of their study in humans, which included Sf treatment of destructive pulmonary Tb.

BCG: Myths, realities, and the need for alternative vaccine strategies

2012-02-20

This document is a synopsis of a workshop held on August 2nd, 2011, at the Bill and Melinda Gates Foundation in Seattle, WA. The purpose of the meeting was to re-evaluate exactly what BCG does, what it doesn’t do, and perhaps more importantly, what we think it does but it doesn’t. The basic conclusions arising from the meeting reflected the fact that many questions can still be raised about BCG, and it is important for the field to fully understand the strengths and limitations of BCG to ensure that lessons are learned in the design and evaluation of new vaccine strategies.