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The causative agents of tuberculosis, grouped in the Mycobacterium tuberculosis complex (MTBC), have infected one-third of the present human population and a wide range of other mammals. However, questions, such as why, where and when the disease began and expanded, have largely remained unanswered. A new study provides genetic evidence indicating that the common ancestor of the tuberculosis complex emerged some 40,000 years ago in East Africa, the region from where modern human populations disseminated around the same period. This initial step was followed 10,000 to 20,000 years later by the radiation of two major lineages, one of which spread from human to animals. In more recent years (approximately 180 years ago), coinciding with the human population explosion and the industrial revolution, the human-associated pathogen lineages have strongly expanded. These results thus reveal the strikingly parallel demographic evolution between humans and one of their primary pathogens.

Using mycobacterial tandem repeat sequences as genetic markers, the authors show that the MTBC consists of two independent clades, one composed exclusively of M. tuberculosis lineages from humans and the other composed of both animal and human isolates. The latter also likely derived from a human pathogenic lineage, supporting the hypothesis of an original human host. These findings unveil the dynamic dimension of the association between human host and pathogen populations.

Origin, Spread and Demography of the Mycobacterium tuberculosis Complex. 2008 PLoS Pathog 4(9): e1000160

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Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is responsible for dramatic health problems globally. It is estimated that this pathogen infects one third of the human population and causes three million deaths annually. Most individuals remain asymptomatic for years before developing an active disease. In such individuals, the bacilli are not cleared but rather persist in a dormant state. Major goals of TB research are to (i) understand how the bacilli remain alive for years within infected individuals, and (ii) find how to prevent their reactivation and hence clinical disease. During dormancy, most of the bacilli are confined to granulomas that consist of well-defined aggregates of different host immune cells. Granulomas prevent spreading of bacilli. Researchers analyzed the role of a particular cell population found within granulomas, the ‘‘foamy macrophages’’. These cells are filled with droplets of lipids, a well-known nutrient for persistent bacilli. They found that within these cells, the bacilli do not replicate, but remain alive and seem to internalize host lipids. The foamy macrophages might thus constitute a reservoir for persisting bacilli within their human host, and could provide a relevant model for screening of new antimicrobials against non-replicating persistent mycobacteria.

Foamy Macrophages from Tuberculous Patients’ Granulomas Constitute a Nutrient-Rich Reservoir for M. tuberculosis Persistence. PLoS Pathog 4(11): e1000204
Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis–infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria.

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The discovery of the earliest known cases of human tuberculosis (TB) in bones found submerged off the coast of Israel shows that the disease is 3000 years older than previously thought. Direct examination of this ancient DNA confirms the latest theory that bovine TB evolved later than human TB. The new research sheds light on how the TB bacterium has evolved over the millennia and increases our understanding of how it may change in the future.

The bones, thought to be of a mother and baby, were excavated from Alit-Yam, a 9000 year-old Neolithic village, which has been submerged off the coast of Haifa, Israel for thousands of years. Scientists found characteristic bone lesions that are signs of TB in skeletons from the settlement, one of the earliest with evidence of domesticated cattle. An international team conducted detailed analyses of the bones using scientific techniques that revealed DNA and cell wall lipids from Mycobacterium tuberculosis, the principal agent of human TB. The DNA was sufficiently well-preserved for molecular typing to be carried out and the analysis of the bacterial cell wall lipids by high performance liquid chromatography provided direct, confirmatory evidence of tuberculosis.

This is the best evidence yet that in a community with domesticated animals but before dairying, the infecting strain was actually a human pathogen. The presence of large numbers of animal bones shows that animals were an important food source, and this probably led to an increase in the human population that helped the TB to be maintained and spread. The DNA of the strain of TB in these skeletons had lost a particular piece of DNA which is characteristic of a common family of strains present in the world today. The fact that this deletion had occurred 9000 years ago gives us a much better idea of the rate of change of the bacterium over time, and indicates an extremely long association with humans.

Examining ancient human remains for the markers of TB is very important because it helps to aid our understanding of prehistoric tuberculosis and how it evolved. This then helps us improve our understanding of modern TB and how we might develop more effective treatments.

Detection and Molecular Characterization of 9000-Year-Old Mycobacterium tuberculosis from a Neolithic Settlement in the Eastern Mediterranean. PLoS ONE 3(10): e3426
Mycobacterium tuberculosis is the principal etiologic agent of human tuberculosis. It has no environmental reservoir and is believed to have co-evolved with its host over millennia. This is supported by skeletal evidence of the disease in early humans, and inferred from M. tuberculosis genomic analysis. Direct examination of ancient human remains for M. tuberculosis biomarkers should aid our understanding of the nature of prehistoric tuberculosis and the host/pathogen relationship. We used conventional PCR to examine bone samples with typical tuberculosis lesions from a woman and infant, who were buried together in the now submerged site of Atlit-Yam in the Eastern Mediterranean, dating from 9250-8160 years ago. Rigorous precautions were taken to prevent contamination, and independent centers were used to confirmauthenticity of findings. DNA from five M tuberculosis genetic loci was detected and had characteristics consistent with extant genetic lineages. High performance liquid chromatography was used as an independent method of verification and it directly detected mycolic acid lipid biomarkers, specific for the M. tuberculosis complex. Human tuberculosis was confirmed by morphological and molecular methods in a population living in one of the first villages with evidence of agriculture and animal domestication. The widespread use of animals was not a source of infection but may have supported a denser human population that facilitated transmission of the tubercle bacillus. The similarity of the M. tuberculosis genetic signature with those of today gives support to the theory of a long-term co-existence of host and pathogen.

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According to a new analysis, neglected tropical diseases (NTDs) as a group may have surpassed HIV/AIDS, tuberculosis and malaria as the most prevalent infectious diseases in Latin America and the Caribbean. The work found that NTDs are the most common infections of approximately 200 million of the poorest people in the region. They include tens of millions of cases of intestinal worm infections, and almost 10 million cases of Chagas disease, as well as schistosomiasis, trachoma, dengue fever, leishmaniasis, lymphatic filariasis (LF), and onchocerciasis. NTDs produce extreme poverty by adversely impacting child development, pregnancy outcomes and worker productivity. In some cases in Latin America and the Caribbean, NTDs also represent a living legacy of slavery, because they were first introduced into the region through the global slave trade, and even today they predominantly affect people of African descent and indigenous groups, as well as other vulnerable groups such as women and children.

In the coming years, schistosomiasis transmission could be eliminated in the Caribbean, and that transmission of lymphatic filariasis and onchocerciasis could be eliminated in Latin America and the Caribbean with proven successful, cost effective and low-cost treatments. The most burdensome NTDs, such as Chagas disease, intestinal worm infections, and schistosomiasis may first require scale-up of existing resources and/or the development of new tools in order to achieve wider control and/or elimination. Ultimately, successful wide-scale efforts for NTD elimination will require an inter-sectoral approach that bridges public health with social services and environmental interventions.

Neglected diseases impose a huge burden on developing countries, constituting a serious obstacle for socioeconomic development and quality of life. They mostly affect people living either in shantytowns, indigenous communities or poor rural and agricultural areas. Last week, UK government officials announced that they will be contributing £50 million over the next five years toward the control and elimination of NTDs, including Guinea worm. In addition, the World Health Organziation announced that in 2007 alone, 546 million of the world’s poorest people received treatment for lymphatic filariasis at a cost of 10 cents per person, enabling them to live healthier more productive lives. After rainfall-induced disasters like Hurricane Ike, respiratory and intestinal infections usually increase and there is increased risk of breeding of the mosquito that transmits lymphatic filarisis in Haiti. While around three million people will be treated in Haiti in 2008 for lymphatic filariasis, additional resources are needed to step up and maintain treatment coverage in Haiti with its population of 9.5 million people, particularly in the wake of the Hurricane.

The Neglected Tropical Diseases of Latin America and the Caribbean: A Review of Disease Burden and Distribution and a Roadmap for Control and Elimination. 2008 PLoS Negl Trop Dis 2(9): e300

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Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, kills over 2 million people each year. It is estimated that approximately one-third of the world population is infected with M. tuberculosis, though the majority will never develop active disease. Almost 1 in 20 cases of tuberculosis worldwide is resistant to multiple drugs (known as multidrug-resistant TB or MDR-TB).

Tuberculosis can be a difficult disease to diagnose, mainly due to the difficulty in culturing this slow-growing organism in the laboratory. It takes 4–12 weeks for a laboratory culture to be clearly positive. While microscopy and culture are still the major backbone for laboratory diagnosis of tuberculosis, rapid diagnosis still relies on a medical evaluation including a medical history, a chest X-ray and a physical examination, and may also include a tuberculin skin test or serological tests. These methods are used in rich countries but are probably not available in much of the developing world where the majority of tuberculosis carriers live.

New TB tests are being developed that offer the hope of offering cheaper, faster and more accurate TB testing. The majority of new molecular tests use polymerase chain reaction (PCR)-based detection of nucleic acids, including both DNA and RNA, which are specific to M. tuberculosis, or mutations in mycobacterial genes which are associated with resistance to anti-tuberculosis drugs such as isoniazid and rifampicin. Other new diagnostic tests use PCR or antibody assays to detect the release of interferon-gamma in response to mycobacterial infection. Recently, microarray techniques have been employed extensively for the detection of drug resistance-related mutations.

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While genetic assays are useful for rapid detection of drug resistance compared to the time taken by culture-based methods, not all drug-resistant isolates have mutations in the “hot spots” of genes commonly associated with drug resistance. Another drawback of some tests is their inability to detect minor populations of drug-resistant organisms in cultures from clinical samples. In defining drug resistance, the presence of 1–10% of drug-resistant organisms in culture can make it functionally drug resistant. However, routine nucleic acid hybridization assays require about 15–20% of a drug-resistant strain in a culture to qualify as a mutant isolate.

Interferon-gamma tests have been quite successful in detecting latent TB infection in areas with concurrent vaccination programs, since these tests do not get “confused” by the presence of an immunogen. Currently, two types of test for the detection of interferon-gamma production by T lymphocytes are available, ELISA and ELISPOT. These tests seem to reflect the degree of exposure to M. tuberculosis in household contacts of TB patients and occupational contact in TB hospitals. The major obstacles in implementing interferon-gamma tests include their high costs, requirement of highly trained personnel and fresh blood samples, all formidable obstacles in most areas with high TB prevalance.

Mycobacterium tuberculosis has no significant animal or environmental reservoirs and shows limited genetic diversity. In spite of this, TB continues to be a widespread and devastating disease. The need for new faster-acting diagnostic tests and better drugs is clear.

Tuberculosis: diagnostics. Tuberculosis (Edinb). 2007 87 Suppl 1: S14-7 doi:10.1016/j.tube.2007.05.001

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Almost 1 in 20 cases of tuberculosis worldwide is resistant to multiple drugs (known as multidrug-resistant TB or MDR-TB) and the World Health Organization has called for a massive scale up in public health efforts to tackle these cases. A group of MDR-TB experts has outlined its recommendations on conducting research that would help in the scale up. MDR-TB can be effectively treated using second-line TB drugs, though these drugs are more expensive, less potent, and less well tolerated than first-line drugs. Fewer than 2% of all patients with MDR-TB are receiving appropriate second-line treatment. The WHO has therefore called for a dramatic scale up of MDR-TB treatment as a routine component of TB control, setting a target of treating 1.6 million patients with MDR-TB by 2015. Pilot projects of MDR-TB management (known as programmatic management of drug-resistant TB or PMDT) in five low income settings showed treatment success rates of 59%-83%. The new agenda identifies the most important barriers to scaling up the treatment of MDR-TB and prioritizes the research questions to be addressed to overcome these barriers. The research priorities include new and improved tools for testing patients to see if they have drug-resistant TB clinical trials of simplified and shorter second-line treatments for MDR-TB new and improved strategies for diagnosis of drug-resistant TB, for helping patients complete the whole course of drug treatment, and for controlling the spread of the infection understanding geographic variations in the occurrence of drug resistance clinical trials to test whether giving TB drugs to people who came into contact with patients with drug-resistant TB prevents them from developing resistant TB. With increasing recognition of drug-resistant TB worldwide, the time has come to move PMDT in resource-limited settings beyond the limited, pilot project phase.

Scaling up programmatic management of drug-resistant tuberculosis: A prioritized research agenda. 2008 PLoS Med 5(7): e150
Summary:
The World Health Organization calls for massive scale-up of programmatic management of drug-resistant tuberculosis in resource-limited settings. Several technical and operational barriers impede the achievement of this scale-up. A research agenda, developed by the Stop TB Partnership, identifies the most important barriers and prioritizes the research questions to be addressed in order to overcome these barriers. Research priorities include:

• new and improved tools for drug resistance testing
• clinical trials of simplified and shorter second-line treatment regimens
• new and improved strategies for diagnosis of drug-resistant tuberculosis, treatment adherence, and infection control
• understanding of the geographic variations in occurrence of drug resistance
• clinical trials of prophylactic treatment of contacts of patients with drug-resistant tuberculosis

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Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, kills over 2 million people each year. It is estimated that approximately one-third of the world population is infected with M. tuberculosis, though the majority will never develop active disease. The most severe form of tuberculosis occurs when the bacterium spreads to the brain to cause meningitis. A recent paper examined whether the genetic variation of the person and the bacteria influenced the type of disease a person develops. The authors had previously shown that certain mutations in genes of the human immune system can predispose adults in Vietnam to developing tuberculous meningitis. In this study they show that some strains of M. tuberculosis commonly found in Europe and America are less likely to cause tuberculous meningitis in Vietnamese adults than strains predominantly found in Asia. They then looked at the interaction between M. tuberculosis strains and mutations in human immune genes and show that a particular mutation is more commonly found in patients infected with the East-Asian/Beijing strains of M. tuberculosis. This is the first study to look at both the host and pathogen genotypes together in tuberculosis infection, and the findings suggest that the outcome of exposure to M. tuberculosis can depend on both the human genotype and the bacterial genotype.

The Influence of Host and Bacterial Genotype on the Development of Disseminated Disease with Mycobacterium tuberculosis. PLoS Pathog 2008 4(3): e1000034
The factors that govern the development of tuberculosis disease are incompletely understood. We hypothesized that some strains of Mycobacterium tuberculosis (M. tuberculosis) are more capable of causing disseminated disease than others and may be associated with polymorphisms in host genes responsible for the innate immune response to infection. We compared the host and bacterial genotype in 187 Vietnamese adults with tuberculous meningitis (TBM) and 237 Vietnamese adults with uncomplicated pulmonary tuberculosis. The host genotype of tuberculosis cases was also compared with the genotype of 392 cord blood controls from the same population. Isolates of M. tuberculosis were genotyped by large sequence polymorphisms. The hosts were defined by polymorphisms in genes encoding Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and Toll-like receptor-2 (TLR-2). We found a significant protective association between the Euro-American lineage of M. tuberculosis and pulmonary rather than meningeal tuberculosis, suggesting these strains are less capable of extra-pulmonary dissemination than others in the study population. We also found that individuals with the C allele of TLR-2 T597C allele were more likely to have tuberculosis caused by the East-Asian/Beijing genotype than other individuals. The study provides evidence that M. tuberculosis genotype influences clinical disease phenotype and demonstrates, for the first time, a significant interaction between host and bacterial genotypes and the development of tuberculosis.

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Tuberculosis has had many names, including consumption, scrofula and the great white plague, but whatever you call it, this disease still claims one life every 10 seconds and global mortality rates are increasing despite the use of chemotherapy (Drugs versus bugs: in pursuit of the persistent predator Mycobacterium tuberculosis. 2008 Nature Reviews Microbiology 6: 41-52). Why have we not progressed further towards the eradication of this disease? There are many answers, including politics and poverty, and some less shameful excuses such as HIV and drug resistance. Whatever the reason, without new weapons in the armory against TB, the disease will continue to make ground.

Two factors, persistence and resistance, make the treatment of Mycobacterium tuberculosis infections particularly difficult. The term persistence describes the survival of the causative organism despite the use of antibiotics. The local concentration of antibiotics in lesions such as granulomas might not be adequate to kill the cells, or some bacteria might adopt a physiological state that renders them less susceptible to antibiotics. For these reasons, drug treatments must be extended. Currently, even the most effective regimes require a combination of at least 3 drugs and last for six months. Because patients feel better within 1 2 weeks, they have little motivation to continue with therapy, so the current World Health Organization guidelines call for treatment to be directly observed (DOTS). This can be difficult to provide in much of the world, including the areas where tuberculosis rates are highest.

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There is an excellent chance that patients who have tuberculosis can be cured using currently available drugs if they complete the required course of therapy. But what characteristics should new drugs have to improve on current treatments?

• Oral bioavailability: to avoid the need for injections.
• Good tolerance: to avoid unwanted side-effects might cause treatment to be abandoned.
• Widespread usability: including AIDS patients, young children and pregnant women.
• Compatibility with anti-retroviral drugs: because co-infection with HIV and TB is common.
• Infrequent dosing: once a day drugs improve treatment compliance.
• Activity against drug-resistant TB strains: possibly the most important factor with the rise of MDR and XDR-TB.
• Rapid clearance of chronic infection: so that treatment times can be shortened.
• Affordability: so they can be used in the areas of the world where TB is most prevalent.

Mycobacterium tuberculosis has no significant animal or environmental reservoirs and shows limited genetic diversity. In spite of this, TB continues to be a widespread and devastating disease. The need for new faster-acting drugs is clear. Recent work by my colleague Dr Mark Carr from the School of Biological Sciences at the University of Leicester might help in future drug development. The M. tuberculosis ESAT-6/CFP-10 complex consists of two proteins which, together, allow the bacteria to survive inside white blood cells. Removal of the genes for these proteins from the TB genome renders the bacteria unable to cause disease. Similarly, studies of the structure of the protein complex have shown that removal of a “long arm” from the molecule prevents the complex s ability to bind to the outer surface of human white blood cells. In the structure of the ESAT-6/CFP-10 complex above, the “long arm” is in red on the right side of CFP-10. When this is intact, it allows the complex to attach to the outside of host white blood cells. When the long arm is cleaved off, the complex shows greatly reduced attachment. This data provides an insight into the important components of this complex. Mark Carr says: “Current work is attempting to identify the exact components of the human white blood cells that this complex is targeting. Once found, this should give us a greater knowledge of the action of these molecular weapons of TB and give us the edge in the war against an ancient, reawakened foe.”

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