Category Archives: Tuberculosis

War as a Driver in Tuberculosis Evolution

by Michelle Ziegler

Russia has been all over the news lately. Beyond our recent election, increased Russian activity on the world stage has public health consequences for Europe and farther afield. It has been known for a long time that post-Soviet Russia had and continues to have serious public health problems. One of their particular problems that they have shared with the world is their alarmingly high rate of antibiotic resistant tuberculosis. There is no mystery over the root cause of their antibiotic resistance woes — poor antibiotic stewardship (Garrett, 2000; Bernard et al 2013).

A study by Vegard Eldholm and colleagues that came out this fall sheds light on the origins of particularly virulent tuberculosis strains with high rates of antibiotic resistance that recently entered Europe.  A large outbreak among Afghan refugees and Norwegians in Oslo, Norway, provided a core set of 26 specimens for this study that could be compared with results generated elsewhere in Europe (Eldholm et al, 2010). The Oslo outbreak clearly fits within the Russian clade A group that is concentrated to the east of the Volga River in countries of the former Soviet Union. They name this cluster the Central Asian Clade, noting that it co-localizes with region of origin of migrants carrying the MDR strains of tuberculosis reported in Europe.

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Figure 5. Phylogeny of the Afghan Strain Family (ASF). Colored boxes represent the country of origin: Afghanistan is orange; other countries are gray. (Eldholm et al, 2016)

When the Oslo samples are added to the family tree, phylogeny, of recent tuberculosis isolates from elsewhere in Europe a distinctive pattern emerges. The branches on the family tree are short and dense, suggesting that this is recent diversity, that they calculate to have occurred within approximately the last twenty years (Eldholm et al, 2016).

The Central Asian Clade spread into Afghanistan before drug resistance began to develop, probably during the Soviet-Afghan war (1979-1989) producing the Afghan Strain Diversity clade. Slightly later, the Central Asian Clade still in the former Soviet states begins to accumulate antibiotic resistance as the public health infrastructure crumbles in the wake of the dissolution of the USSR. The invasion of Afghanistan by the US and its allies in 2002 toppled the Afghan state, crippling infrastructure and spurring refugee movements within and out of Afghanistan. The lack of modern public health standards in Afghanistan since their war with the introduction of these strains by the Soviets in the 1980s provided fertile ground for the establishment and diversity of tuberculosis in the country. Instability has been pervasive throughout the entire region sending refugees and economic migrants from both Afghanistan and the former Soviet states into Europe.

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Movements of the Central Asian Clade (CAC) since c. 1960 and the subsequent Afghan Strain Family (ASF). (Eldholm et al, 2016)

Their dating of the last common ancestor for the Central Asian Clade to c. 1961 is significantly younger than the previous dating of 4,415 years before present for the Russian clade A (CC1) of the Beijing lineage of Mycobacteria tuberculosis. They account for this difference by noting differences in their methods of assessing sequence differences and note that their method is in line with other recent evolutionary rates for other tuberculosis clades.  The diagnosis dates and length of the arms on their reconstructed phylogeny suggests that there were multiple, independent introductions of the cases from Afghanistan and the former Soviet republics. This is consistent with a repeated periods of refugee movements from central Asia into Europe.

The rapid proliferation and diversification of the Afghan Strain Family may be explained by a known syndemic between tuberculosis and war (Ostrach & Singer, 2013). Conditions of war everywhere disrupt food systems, destroy critical infrastructures such as electricity and water systems, interrupts medical supplies, and the human public health infrastructure of the country. Malnutrition and stress are known contributors to immune suppression. Many pathogens flourish simultaneously in these conditions increasing the infectious challenges the population must fend off. Diarrheal diseases are the most acute and demanding of rapid attention, allowing longer-term diseases like tuberculosis to slip through the overburdened healthcare system. Afghanistan has experienced nearly forty years of war, political instability, and repeated infrastructure destruction. Thus, they were primed for both the establishment of new tuberculosis strains during the Afghan-Soviet war in the 1980s along with the proliferation and diversification of tuberculosis during the Afghan-American war of the last sixteen years.

Established syndemics between tuberculosis and war have been made retrospectively following the Vietnam war and the Persian Gulf war of 1991 (Ostrach & Singer, 2013). In Vietnam, prolonged malnutrition caused an eruption of tuberculosis along with malaria, leprosy, typhoid, cholera, plague, and parasitic diseases.  A WHO survey in 1976 found that Vietnam had twice the incidence of tuberculosis over all of its neighboring countries (Ostrach & Singer, 2013). When the military intentionally targets water infrastructure as it did in Vietnam and Iraq, the production of civilian infectious disease is a tactic of war. In both Vietnam and post-Gulf war Iraq, more civilians died of malnutrition and infectious disease than enemy soldiers died of all causes (Ostrach & Singler, 2013).

It seems likely that this is just one of the first studies to establish a link between serious infectious disease developments and the Afghan wars. The current war zones throughout central Asia and the Middle East already have ramifications for the public health of the entire world that walls along borders will not be able to stop. Most of the cases in the Oslo outbreak were Norwegians, not Afghan immigrants. Diseases will spread beyond the migrants so country of origin screening will be of little use before long.


Reference

Eldholm, V., Pettersson, J. H. O., Brynildsrud, O. B., Kitchen, A., Rasmussen, E. M., Lillebaek, T., et al. (2016). Armed conflict and population displacement as drivers of the evolution and dispersal of Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences of the United States of America, 201611283–16. http://doi.org/10.1073/pnas.1611283113

Ostrach, B., & Singer, M. C. (2013). Syndemics of War: Malnutrition-Infectious Disease Interactions and the Unintended Health Consequences of Intentional War Policies. Annals of Anthropological Practice, 36(2), 257–273. http://doi.org/10.1111/napa.12003

Bernard, C., Brossier, F., Sougakoff, W., Veziris, N., Frechet-Jachym, M., Metivier, N., et al. (2013). A surge of MDR and XDR tuberculosis in France among patients born in the Former Soviet Union. Euro Surveillance: Bulletin Européen Sur Les Maladies Transmissibles = European Communicable Disease Bulletin, 18(33), 20555.

Multi-Drug Resistant Tuberculosis in Former Soviet States

It has been known for some time that the former Soviet Union had a huge tuberculosis problem. The problem was so big that no one really knew how bad it was in the Soviet Union or is now in its successor states. Over the last couple months, three reports have appeared in Euro Surveillance and Emerging Infectious Diseases that begin to quantitate the problem.

In the first report, France sent out a warning to states accepting immigrants from the former Soviet Union. Over the last two complete years 2010 and 2011, France has experienced a surge in multi-drug resistant (MDR) tuberculosis.

MDR-TB in France 2006-2012 [1]
MDR-TB in France 2006-2012 [1]
XDR-TB by country of birth in France 2006-2012
XDR-TB in France 2006-2012 [1]
 When they examined the country of birth for these cases, almost all of the surge came from countries of the former Soviet Union (fig. 1) [1]. When they looked for more regionalism, they discovered that the majority of the increase came from the country of Georgia and the Russian Federation.[1] For the more worrisome extensively drug resistant (XDR) tuberculosis, the vast majority of the French cases have come from the former Soviet Union going back to 2008; 14 of 17 cases in 2012 came from Georgia (fig. 3) [1]. Genetic analysis of the MDR-TB and XDR-TB strains in France showed variation indicating that transmission did not occur in France but was brought into France by immigration [1].

A survey of MDR-TB in Uzbekistan yielded even more grim results. In the first national TB survey, 23% of all newly diagnosed cased of TB and 62% of previously treated cases were resistant to at least two antibiotics; only 3.8% of MDR-TB cases were co-infected with HIV [2].  The XDR-TB rate was 5.3% with no HIV co-infections [2].  Demographics analysis yielded three primary risk factors or groups: adults under age 45, institutionalization in prisons or previous anti-TB treatment centers, and not owning their own home [2].

The news out of Siberia is no better. A survey published last month showed MDR-TB rates in Siberia are over 25% of primary TB cases with a a mean age of 33 [3]. The two regions, Irkutsk and Yakutia had strains of different origins. The Irkutsk MDR-TB were primarily a common Beijing lineage. On the other hand, the more isolated community of Yakutia had the MDR-TB S256 strain that has been linked with a strain only found among Canadian aboriginal population [3]. The linkage between these the Siberian and Canadian strains have not yet been fully investigated. While these strains are related they are not identical so it is possible that these are a previously undetected ancient lineage that has developed antibiotic resistance in Russia. This was the first isolation of this strain in Russia. The Siberian strains had uncommon mutations in the resistance genes that would not have been picked up well by commercial tests. Zhdanova and co-authors stress the importance of investigating regional strains and developing tests that will adequately detect local strains.

MDR-TB rates from the former Soviet Union are higher than anywhere else in the world [5]. These surveys show that it is even worse than the WHO estimated in 2010. It is far worse than any survey coming out of South Africa, a country often mentioned as being a particular concern for MDR-TB. For comparison, the MDR-TB rate for the United States in 2011 was 1% for MDR-TB and far less than 1% for XDR-TB [4].  Given the vast size and population of the former Soviet Union, migration out of former Soviet states could jump-start a new white plague, strains of TB that even the best medical care will have difficulty keeping under control.

References:

  1. Bernard, C., Brossier, F., Sougakoff, W., Veziris, N., Frechet-Jachym, M., Metivier, N., et al. (2013). A surge of MDR and XDR tuberculosis in France among patients born in the Former Soviet Union. Euro Surveillance : bulletin européen sur les maladies transmissibles = European communicable disease bulletin, 18(33).
  2. Ulmasova, D. J., Uzakova, G., Tillyashayhov, M. N., Turaev, L., van Gemert, W., Hoffmann, H., et al. (2013). Multidrug-resistant tuberculosis in Uzbekistan: results of a nationwide survey, 2010 to 2011. Euro Surveilliance : bulletin européen sur les maladies transmissibles = European communicable disease bulletin, 18(42).
  3. Zhdanova, S., Heysell, S. K., Ogarkov, O., Boyarinova, G., Alexeeva, G., Pholwat, S., et al. (2013). Primary Multidrug-Resistant Mycobacterium tuberculosis in 2 Regions, Eastern Siberia, Russian Federation. Emerging Infectious Diseases, 19(10), 1649–1652. doi:10.3201/eid1910.121108
  4. Centers for Disease Control and Prevention. (2013). Antibiotic Resistance Threats in the United States, 2013 (pp. 1–114). Department of Health and Human Services.
  5. World Health Organization. (2010)  Multidrug and extensively drug-resistant TB (M/XDR-TB): 2010 Global Report on Surveillance and Response.   http://whqlibdoc.who.int/publications/2010/9789241599191_eng.pdf

Ancient Remnants: Biomolecules in Paleomicrobiology

Ancient DNA is not the only method of detecting and identifying ancient pathogens. Survival challenges for ancient DNA place very real limitations on its usefulness and sensitivity as a detection method. The main advantage of aDNA is that it can be genotyped to compare with modern species. For archaeological purposes, other biomolecules may be detected better and cheaper than aDNA.

Sources of Ancient Biomolecules

Proteins and lipids can be extracted from a variety of materials. Bone tissue and especially teeth are still the primary source for non-nucleic acid biomolecules. Hair and mummified skin, muscle, and internal organs have all been successfully used to extract usable proteins. Both proteins and DNA can be extracted old paraffin embedded human tissues previously used for past histological examination. Frozen tissue can yield protein, lipid and DNA.

Materials and methods for nonnucleic acid biomolecule detection in ancient material (Fig 2, Tran, Aboudharam, Raoult, and Drancourt, 2011)

Methods of Detection

  • Immunohistochemistry: Immunohistochemistry combines antibody detection of microbial components with cellular morphology. These methods are similar, if not identical to, standard immunohistochemical assays done on modern patient tissue. Although protein and tissue degradation can be a problem, immunohistochemistry has been done successfully on mummified tissue, detecting 16th century smallpox and syphilis.
  • ELISA and Immunochromography: Extracted protein can be assayed by immunochromatography (potentially as a dipstick) or ELISA (enzyme linked immunosorbent assay). Both immunochromatography and ELISA has been used to detect Yersinia pestis‘ F1 antigen in late medieval and early modern specimens. Pusch et al (2004) does a good comparison of the sensitivity of Y. pestis F1 antigen immunochromography versus ancient DNA amplification on the same specimens.  Immunodetection methods have been used to detect Plasmodium falciparum  (malaria) in natural mummies from ancient Nubia and Egypt, as well as in skeletal tissue from the 16th century Medici family of Italy.
  • High Performance Liquid Chromatography (HPLC) can be used to differentiate ancient lipids extracted from specimens. It has been used primarily for the detection of mycolic acids and mycocerosic acids found in the cell wall of Mycobacterium tuberculosis. Mycolic acids have been used to confirm tuberculosis in remains as old as 9000 years before present.
  • Mass Spectrometry: Mass spectrometry has been successfully used on ancient proteins and peptides. Studies on collagen proteins have been able to distinguish ancient animal species and tuberculosis. Mass spectrometry can also identify microbial products like tetracycline identified and analyzed in Late Antique Nubian bones (Nelson et al, 2010).  Their analysis was able to prove that the tetracycline was deposited in the living bone tissue rather than infiltration from soil bacteria.
  • Late Serology: Active immunoglobulins (IgG) have been extracted from ancient bones. When exposed to “pathogen-specific antigen” the antibody still reacts. Antibodies against syphilis reacted from extracts of ancient bones that also showed osteological lesions of syphilis and PCR confirmation of Treponema pallidum (Tran et al, 2011). I haven’t read any studies using this technique yet.
  • ImmunoPCR: ImmunoPCR is a somewhat odd technique that detects protein, not DNA. The DNA is essentially an amplified reporter of the antigen-antibody reaction monitored by real-time PCR. I haven’t read any studies using immunoPCR yet either.

Any of these methods can be used in conjunction with aDNA analysis to confirm a diagnosis on an ancient specimen. A developing consensus for diagnosing ancient infectious disease calls for the identification of species specific aDNA and some species specific non-nucleic acid biomolecule. Like ancient DNA studies, consensus standards for non-nucleic acid biomolecules have yet to be agreed upon. Determining what the appropriate positive and negative controls are is vital.The entire field is still rapidly evolving and it is likely to take years to develop. The one thing we know for sure is that these new molecular techniques will revolutionize archaeology over the next generation.

ResearchBlogging.org
Tran TN, Aboudharam G, Raoult D, & Drancourt M (2011). Beyond ancient microbial DNA: nonnucleotidic biomolecules for paleomicrobiology. BioTechniques, 50 (6), 370-80 PMID: 21781037.
[Review article] This review article is available free online.

Pusch CM, Rahalison L, Blin N, Nicholson GJ, & Czarnetzki A (2004). Yersinial F1 antigen and the cause of Black Death. The Lancet infectious diseases, 4 (8), 484-5 PMID: 15288817

Nelson ML, Dinardo A, Hochberg J, & Armelagos GJ (2010). Brief communication: Mass spectroscopic characterization of tetracycline in the skeletal remains of an ancient population from Sudanese Nubia 350-550 CE. American journal of physical anthropology, 143 (1), 151-4 PMID: 20564518