Marcel A. Behr

Transmission of Mycobacterium tuberculosis from patients smear-negative for acid-fast bacilli

BACKGROUND The microscopic examination of sputum for acid-fast bacilli, is a simple and rapid test that is used to provide a presumptive diagnosis of infectious tuberculosis. While patients with tuberculosis with sputum smears negative for acid-fast bacilli are less infectious than those with positive smears, both theoretical and empirical evidence suggest that they can still transmit Mycobacterium tuberculosis. We aimed to estimate the risk of transmission from smear-negative individuals. METHODS As part of an ongoing study of the molecular epidemiology of tuberculosis in San Francisco, patients with tuberculosis with mycobacterial isolates with the same DNA fingerprint were assigned to clusters that were assumed to have involved recent transmission. Secondary cases with tuberculosis, whose mycobacterial isolates had the same DNA, were linked to their presumed source case to estimate transmission from smear-negative patients. Sensitivity analyses were done to assess potential bias due to misclassification of source cases, unidentified source cases, and HIV-1 co-infection. FINDINGS 1574 patients with culture-positive tuberculosis were reported and DNA fingerprints were available for 1359 (86%) of these patients. Of the 71 clusters of patients infected with strains that had matching fingerprints, 28 (39% [95% CI 28-52]) had a smear-negative putative source. There were 183 secondary cases in these 71 clusters, of whom a minimum of 32 were attributed to infection by smear-negative patients (17% [12-24]). The relative transmission rate of smear-negative compared with smear-positive patients was calculated as 0.22 (95% CI 0.16-0.32). Sensitivity analyses and stratification for HIV-1 status had no impact on these estimates. INTERPRETATION In San Francisco, the acid-fast-bacilli smear identifies the most infectious patients, but patients with smear-negative culture-positive tuberculosis appear responsible for about 17% of tuberculosis transmission.

Deletion of RD1 from Mycobacterium tuberculosis Mimics Bacille Calmette-Guérin Attenuation

The tuberculosis (TB) vaccine bacille Calmette-Guérin (BCG) is a live attenuated organism, but the mutation responsible for its attenuation has never been defined. Recent genetic studies identified a single DNA region of difference, RD1, which is absent in all BCG strains and present in all Mycobacterium tuberculosis (MTB) strains. The 9 open-reading frames predicted within this 9.5-kb region are of unknown function, although they include the TB-specific immunodominant antigens ESAT-6 and CFP-10. In this study, RD1 was deleted from MTB strain H37Rv, and virulence of H37Rv:DeltaRD1 was assessed after infections of the human macrophage-like cell line THP-1, human peripheral blood monocyte-derived macrophages, and C57BL/6 mice. In each of these systems, the H37Rv:DeltaRD1 strain was strikingly less virulent than MTB and was very similar to BCG controls. Therefore, it was concluded that genes within or controlled by RD1 are essential for MTB virulence and that loss of RD1 was important in BCG attenuation.

Genome plasticity of BCG and impact on vaccine efficacy.

To understand the evolution, attenuation, and variable protective efficacy of bacillus Calmette–Guérin (BCG) vaccines, Mycobacterium bovis BCG Pasteur 1173P2 has been subjected to comparative genome and transcriptome analysis. The 4,374,522-bp genome contains 3,954 protein-coding genes, 58 of which are present in two copies as a result of two independent tandem duplications, DU1 and DU2. DU1 is restricted to BCG Pasteur, although four forms of DU2 exist; DU2-I is confined to early BCG vaccines, like BCG Japan, whereas DU2-III and DU2-IV occur in the late vaccines. The glycerol-3-phosphate dehydrogenase gene, glpD2, is one of only three genes common to all four DU2 variants, implying that BCG requires higher levels of this enzyme to grow on glycerol. Further amplification of the DU2 region is ongoing, even within vaccine preparations used to immunize humans. An evolutionary scheme for BCG vaccines was established by analyzing DU2 and other markers. Lesions in genes encoding σ-factors and pleiotropic transcriptional regulators, like PhoR and Crp, were also uncovered in various BCG strains; together with gene amplification, these affect gene expression levels, immunogenicity, and, possibly, protection against tuberculosis. Furthermore, the combined findings suggest that early BCG vaccines may even be superior to the later ones that are more widely used.

Mycobacterial disease and impaired IFN-γ immunity in humans with inherited ISG15 deficiency.

Tuberculosis Vaccine Conundrum Some children experience severe clinical disease when they are vaccinated against tuberculosis, an attenuated live vaccine that is normally innocuous in humans. Several germline mutations have been identified that account for this susceptibility, and now Bogunovic et al. (p. 1684, published online 2 August) add another to the list—ISG15. Uncovering this mutation, which is inherited in an autosomal recessive manner, was a surprise because studies with mice deficient in ISG15 showed enhanced susceptibility to some viral, but not bacterial, infections. Nevertheless, patients lacking ISG15 were not able to produce adequate amounts of interferon-γ, a cytokine critical for clearance of the bacteria. A mutation that accounts for adverse reactions to the Bacille Calmette-Guérin vaccine against tuberculosis is identified. ISG15 is an interferon (IFN)-α/β–inducible, ubiquitin-like intracellular protein. Its conjugation to various proteins (ISGylation) contributes to antiviral immunity in mice. Here, we describe human patients with inherited ISG15 deficiency and mycobacterial, but not viral, diseases. The lack of intracellular ISG15 production and protein ISGylation was not associated with cellular susceptibility to any viruses that we tested, consistent with the lack of viral diseases in these patients. By contrast, the lack of mycobacterium-induced ISG15 secretion by leukocytes—granulocyte, in particular—reduced the production of IFN-γ by lymphocytes, including natural killer cells, probably accounting for the enhanced susceptibility to mycobacterial disease. This experiment of nature shows that human ISGylation is largely redundant for antiviral immunity, but that ISG15 plays an essential role as an IFN-γ–inducing secreted molecule for optimal antimycobacterial immunity.

Direct and Indirect Induction by 1,25-Dihydroxyvitamin D3 of the NOD2/CARD15-Defensin β2 Innate Immune Pathway Defective in Crohn Disease

Vitamin D signaling through its nuclear vitamin D receptor has emerged as a key regulator of innate immunity in humans. Here we show that hormonal vitamin D, 1,25-dihydroxyvitamin D3, robustly stimulates expression of pattern recognition receptor NOD2/CARD15/IBD1 gene and protein in primary human monocytic and epithelial cells. The vitamin D receptor signals through distal enhancers in the NOD2 gene, whose function was validated by chromatin immunoprecipitation and chromatin conformation capture assays. A key downstream signaling consequence of NOD2 activation by agonist muramyl dipeptide is stimulation of NF-κB transcription factor function, which induces expression of the gene encoding antimicrobial peptide defensin β2 (DEFB2/HBD2). Pretreatment with 1,25-dihydroxyvitamin D3 synergistically induced NF-κB function and expression of genes encoding DEFB2/HBD2 and antimicrobial peptide cathelicidin in the presence of muramyl dipeptide. Importantly, this synergistic response was also seen in macrophages from a donor wild type for NOD2 but was absent in macrophages from patients with Crohn disease homozygous for non-functional NOD2 variants. These studies provide strong molecular links between vitamin D deficiency and the genetics of Crohn disease, a chronic incurable inflammatory bowel condition, as Crohns pathogenesis is associated with attenuated NOD2 or DEFB2/HBD2 function.

Genomic Deletions Suggest a Phylogeny for the Mycobacterium tuberculosis Complex

To better understand the evolution of the Mycobacterium tuberculosis complex, subspecies were tested for large sequence polymorphisms. Samples with greater numbers of deletions, without exception, were missing all the same regions that were deleted from samples with lesser numbers of deletions. Principal genetic groups based on single-nucleotide polymorphisms were restricted to one of the deletion-based groups, and isolates that shared genotypes based on molecular epidemiological markers were assigned almost exclusively to the same deletion type. The data provide compelling evidence that human tuberculosis did not originate from the present-day bovine form. Genomic deletions present themselves as an attractive modality to study the evolution of the M. tuberculosis complex.

NOD2, RIP2 and IRF5 play a critical role in the type I interferon response to Mycobacterium tuberculosis

While the recognition of microbial infection often occurs at the cell surface via Toll-like receptors, the cytosol of the cell is also under surveillance for microbial products that breach the cell membrane. An important outcome of cytosolic recognition is the induction of IFNα and IFNβ, which are critical mediators of immunity against both bacteria and viruses. Like many intracellular pathogens, a significant fraction of the transcriptional response to Mycobacterium tuberculosis infection depends on these type I interferons, but the recognition pathways responsible remain elusive. In this work, we demonstrate that intraphagosomal M. tuberculosis stimulates the cytosolic Nod2 pathway that responds to bacterial peptidoglycan, and this event requires membrane damage that is actively inflicted by the bacterium. Unexpectedly, this recognition triggers the expression of type I interferons in a Tbk1- and Irf5-dependent manner. This response is only partially impaired by the loss of Irf3 and therefore, differs fundamentally from those stimulated by bacterial DNA, which depend entirely on this transcription factor. This difference appears to result from the unusual peptidoglycan produced by mycobacteria, which we show is a uniquely potent agonist of the Nod2/Rip2/Irf5 pathway. Thus, the Nod2 system is specialized to recognize bacteria that actively perturb host membranes and is remarkably sensitive to mycobacteria, perhaps reflecting the strong evolutionary pressure exerted by these pathogens on the mammalian immune system.

Proportion of tuberculosis transmission that takes place in households in a high-incidence area

The prevalence of infection among household contacts of people with tuberculosis is high. This information frequently guides active case finding. We analysed DNA fingerprints of Mycobacterium tuberculosis from 765 tuberculosis patients in Ravensmead and Uitsig, adjacent suburbs of Cape Town, South Africa. In 129 households in which DNA fingerprints were available for more than one patient, we identified 313 patients, of whom 145 (46%) had a fingerprint pattern matching that of another member of the household. The proportion of transmission in the community that took place in the household was 19%, and therefore, in this high-incidence area, tuberculosis transmission occurs mainly outside the household.

The BCG World Atlas: A Database of Global BCG Vaccination Policies and Practices

Madhu Pai and colleagues introduce the BCG World Atlas, an open access, user friendly Web site for TB clinicians to discern global BCG vaccination policies and practices and improve the care of their patients.

BCG--different strains, different vaccines?

After nearly a century of use, BCG vaccines continue to generate controversy and confusion. Their ability to prevent tuberculosis in studies has been inconsistent. When they have been protective, no clear mechanism of action has been established. Furthermore, the existence of different BCG strains has been described since the 1940s. These strains vary according to several laboratory properties, which may or may not translate into a discernible effect on vaccination. With recent genomic comparisons, it is now clear that different BCG vaccine strains have evolved and differ from each other and from the original BCG first used in 1921. Some of these genetic alterations explain certain variations in laboratory properties of BCG. However, these mutations in BCG strains have yet to be shown to affect BCG-associated protection and/or adverse effects. Continuing research is attempting to assess the effect of these genetic alterations on properties of BCG strains, with the goals of suggesting the ideal BCG for vaccination and providing avenues for improvement on existing BCG vaccines.