Category Archives: Asia

Keeping Bronze Age Yersinia pestis in Perspective

Graphic abstract from ____
Graphic abstract from Rasmussen et al, 2015.

The latest plague news to splash across headlines is the discovery of Yersinia pestis aDNA in seven Bronze Age remains from Eurasia.  The most important findings in this new study are not anthropological; they are evolutionary. This paper allows us to drop a couple more evolutionary mile markers. Finding  7% of the tested remains  (7 out of 101) positive for plague is surprising, but I’m not yet ready to believe that it was endemic over such a huge area scattered over 2000 years. Not yet anyway.

The new phylogenetic tree places Y. pestis in humans since the Bronze Age and the origin of the species as far back 50,000 years ago.  It also opens up questions on the original reservoir species and the location of the birth of the species, although central Asia is still the most likely location.

Figure6
Stretching out the Yersinia pestis tree. Blue arrows are gains and red arrows are losses. (Rasmussen et al, 2015)

 

So let’s look at the genetic results in three areas highlighted by Rasmussen et al: flea transmission, Pla activity, and suppression of the immune response stimulating flagellin production. These traits are critical to producing bubonic plague as we know today.

Late phase flea transmission of modern Yersinia pestis is dependent on the ability to survive in and colonize the flea. The Bronze Age strains have all of the plasmids and virulence genes of modern strains except one, the ymt gene that encodes the murine toxin. The basic tool set of modern strains also have deactivated or knocked-out the protein products of three ancestral genes that hinder Yersinia pestis biofilm formation. Remnants of these genes persist as pseudogenes in modern strains. (A pseudogene is the corpse of a former gene.) This genetic combination allows Y. pestis to survive in the mid-gut of the flea, persist longer and form a biofilm; a necessity for late phase flea transmission. However, as Monica Green reminded me,  ymt is not required for early phase flea transmission, dirty-needle style (Johnson et al, 2014). In fact, since Y. pestis does not need to persist long or multiply at all, there are no known genes needed to be present or absent for early phase transmission.  As I recently reviewed, early phase transmission is very common and effective (see Eisen, Dennis & Gage, 2015). Based on the dates of their samples, they estimate that ymt was gained in about 1000 BC. In the RISE509 strain from Afanasievo Gora in southern Siberia, the pde3 is inactive but the other two, pde2 and rcsA, are still functional. Taken together this genetic combination should allow early phase flea transmission but not late phase flea transmission that requires biofilm formation. They are still mid-way in developing late phase flea transmission. This makes sense for a microbe being transmitted dirty-needle style, providing the opportunity for natural selection to develop late phase transmission bit by bit. While early phase transmission can support regional epizootics and epidemics,  late phase flea transmission is probably important for long distance transmission by fleas in grain or textiles, or by sea.

The recent discovery of the Pla gene in Citrobacter koseri and Escherichia coli, other enteric opportunistic flora, but not found in Yersinia pseudotuberculosis, suggests that lateral gene transfer  brought the plasmid to the young Y. pestis while still in the enteric environment (Hänsch et al, 2015) . This is consistent with Y. pestis Pla and Salmonella enterica PgeE both evolving from the same ancestral omptin ancestor in an enteric environment (Haiko et al, 2009).  This suggests that Y. pestis may have remained an enteric organism for some time after it split from Y. pseudotuberculosis.

Six of seven Bronze Age Y. pestis strains contain the Pla gene required for deep tissue invasion and bubo formation. Rasmussen et al (2015) suggest that the strain lacking the Pla gene has lost it and that this gene has been lost more than once in the phylogenetic tree. In other words, Pla was present in the common ancestral strain. However, to support development of bubonic plague Pla needed to gain a mutation at position 259 that these strains lack (Haiko et al, 2009). So the Pla gene without the mutation at position 259 can support pneumonic plague but not bubonic in the Pestoides F strain (0.PE2) of Y. pestis (Zimbler et al, 2015).  On the other hand, Sebbane et al (2006) showed that strains completely lacking Pla can still develop primary septicemic plague following flea transmission. They can envision an “evolutionary scenario in which plague emerged as a flea-borne septicemic disease of limited transmissibility”(Sebbane et al, 2006).  Without the polymorphism at position 259, bubo formation should be retarded, if not suppressed.

A third genetic difference of possible significance is the apparent ability to produce flagellin, a major activator of the human innate immune system. Modern Y. pestis strains have deactivated the production of flagellin by a frameshift mutation in the regulatory gene flhD. The Bronze Age strains lack this frameshift and so presumably had normal flagellin production. However, Y. pseudotuberculosis and  Y. enterocolitica down regulate production at mammalian body temperatures. If the ancestral Y. pestis did also then its possible that it wasn’t a factor in human infections.  Experimentally recreating the regulatory environment from  Y. pseudotuberculosis would be much more difficult than simply inserting an intact copy of the gene in a modern strain of Y. pestis.

Predicting the impact of these ancestral genes is highly conjectural. This combination of genes has never been studied together. Since these strains were isolated from human remains we can assume that there is a path for transmission and pathogenesis. The reliance on early phase flea transmission, the less virulent pla allele and the possible production of flagellin suggest that Bronze Age local (dermal) infections from flea bites would be less virulent (more survivable). Interestingly, these milder local infections may have been immunogenic.

As Y. pestis moved away from an enteric lifestyle, producing a septicemia was necessary for either flea transmission or development of a secondary pneumonia with aerosol transmission. I find it hard to believe that Bronze Age Siberia or Estonia had a large enough population for sustained pneumonic transmission. Since Pulex irritans can transmit Y pestis without development of a biofilm, there is no reason to see humans as a dead-end to flea transmission even as early as the Bronze Age.

Humans could have also contracted septicemic plague by ingesting infected meat. Although natural ingestion infections are very rare today, this mode remains effective. A village size outbreak could easily occur from sharing a large infected animal as happened in Afghanistan in 2007. In that outbreak a single infected camel shared among two villages produced 83 probable cases of plague with 17 deaths, a case fatality rate of 20.5%. (Leslie et al, 2011).  Last but certainly not least, the further back we go in Yersinia pestis‘ evolution the more likely ingestion is to be a mode of transmission like its ancestor Yersinia pseudotuberculosis.

Its takes more than good transmission to cause a demographic changing epidemic over large areas like the Eurasian continent. It also requires a fairly high human density and good trade or communication routes. Humans play the the most important role in transmitting plague of pandemic size. I can’t say if the cultural factors that make such large epidemics possible were in place in Bronze Age Eurasia.

Let’s keep things in perspective before we conjure up the specter of virgin soil epidemics of plague in the Bronze Age. Yersinia pestis is the kind of over achiever that may have been a player in Bronze Age demographics but it would be nice to have a lot more evidence before jumping to that conclusion.

References:

Rasmussen, S., Allentoft, M. E., Nielsen, K., Orlando, L., Sikora, M., Sjögren, K.-G., et al. (2015). Early Divergent Strains of Yersinia pestis in Eurasia 5,000 Years Ago. Cell, 163(3), 571–582. http://doi.org/10.1016/j.cell.2015.10.009

Eisen, R. J., Dennis, D. T., & Gage, K. L. (2015). The Role of Early-Phase Transmission in the Spread of Yersinia pestis. Journal of Medical Entomology, tjv128–10. http://doi.org/10.1093/jme/tjv128

Johnson, T. L., Hinnebusch, B. J., Boegler, K. A., Graham, C. B., MacMillan, K., Montenieri, J. A., et al. (2014). Yersinia murine toxin is not required for early-phase transmission of Yersinia pestis by Oropsylla montana (Siphonaptera: Ceratophyllidae) or Xenopsylla cheopis (Siphonaptera: Pulicidae). Microbiology, 160(Pt_11), 2517–2525. http://doi.org/10.1099/mic.0.082123-0

LESLIE, T., WHITEHOUSE, C. A., YINGST, S., BALDWIN, C., KAKAR, F., MOFLEH, J., et al. (2011). Outbreak of gastroenteritis caused by Yersinia pestis in Afghanistan. Epidemiology and Infection, 139(5), 728–735. http://doi.org/10.1017/S0950268810001792

Sebbane, F., Jarrett, C. O., Gardner, D., Long, D., & Hinnebusch, B. J. (2006). Role of the Yersinia pestis plasminogen activator in the incidence of distinct septicemic and bubonic forms of flea-borne plague. Proceedings of the National Academy of Sciences of the United States of America, 103(14), 5526–5530. http://doi.org/10.1073/pnas.0509544103

Zimbler, D. L., Schroeder, J. A., Eddy, J. L., & Lathem, W. W. (2015). Early emergence of Yersinia pestis as a severe respiratory pathogen. Nature Communications, 6, 1–10. http://doi.org/10.1038/ncomms8487

Hänsch, S., Cilli, E., Catalano, G., Gruppioni, G., Bianucci, R., Stenseth, N. C., et al. (2015). The pla gene, encoding plasminogen activator, is not specific to Yersinia pestis. BMC Research Notes, 1–3. http://doi.org/10.1186/s13104-015-1525-x

Haiko, J., Kukkonen, M., Ravantti, J. J., Westerlund-Wikstrom, B., & Korhonen, T. K. (2009). The Single Substitution I259T, Conserved in the Plasminogen Activator Pla of Pandemic Yersinia pestis Branches, Enhances Fibrinolytic Activity. Journal of Bacteriology, 191(15), 4758–4766. http://doi.org/10.1128/JB.00489-09

Plasmodium knowlesi: A New Ancient Malaria Parasite

There are over a hundred different species of the malaria-causing Plasmodium parasites in reptiles, birds and mammals. Being so widespread among terrestrial vertebrates, zoonotic transfer of Plasmodium has come at humans from multiple different sources. Plasmodium knowlesi had been known for some time as a parasite of long-tailed macaques but was not considered a significant human parasite until 2004 when a large number of human infections were identified in Borneo. Molecular analysis implies that Plasmodium knowlesi is as old as Plasmodium vivax and Plasmodium falciparum.

Cover image the phases of Plasmodium knowlesi from the April 2013 issue of Clinical Microbiology Reviews.

Diagnosis is complicated by the histological similarity between Plasmodium knowlesi and Plasmodium malariae. They can’t be distinguished in blood smears like those shown here, so infections were most often misdiagnosed as P. malariae even though they cause a quotidian (daily) fever. The WHO recommends that microscopic detection in areas where P. knowlesi is found report positive results as “P. malariae/P. knowlesi”.  It can only be securely diagnosed by molecular methods  that can distinguish all five human malarial species. PCR based detection methods have shown promise but no one method has been clinically tested with a large enough number of cases to become the standard of care. Antibody-based Rapid Diagnostic Tests (RDT dipstick tests) for malaria do not reliably detect knowlesi malaria which was discovered in humans after the RDT tests were developed. For now in resource poor areas, microscopic analysis followed by molecular testing where available is the only way to detect knowlesi malaria. Clinical research continues for a RDT test that can be employed areas with poor laboratory resources.

Infections have now been confirmed in all of the countries of southeast Asia. Between 2000 and 2011, 881 cases of local P. knowlesi local transmission have been identified in Borneo, with only 8 cases of P. malariae.  It is now suspected that past diagnoses of P. malariae in the region were actually P. knowlesi. Unlike other forms of malaria, P. knowlesi infects more adults than children, although actual infection rates are still not known.

Long-tailed and pig-tailed macaques are the reservoirs for P. knowlesi. In some areas of Malaysia the macaques are around 90% seropositive for malaria, in one study 87% were P. knowlesi. The malaria vector for P. knowlesi and other malarial parasites is Anopheles leucosphyrus group which is also concentrated in southeastern Asia.  Anopheles balabacensis is the most efficient vector, capable of transmitting P. knowlesi from monkey-to-human, human-to-human and human-to-monkey. A. latens, on the other hand, has been most commonly indicated as the vector to humans in Borneo, where it feeds in the high elevation canopy.  As the map below shows, the macacque reservoir and the mosquito vectors are limited to  the islands and peninsulas south-east Asia. It has been hypothesized, based on genetic diversity, that P. knowlesi has caused human malaria as long as  humans, macaques and the Anopheles vectors have all been on the islands of south-east Asia.

Source:
Source: Singh, B., & Daneshvar, C. (2013). Human Infections and Detection of Plasmodium knowlesi. Clinical Microbiology Reviews, 26(2), 165–184. doi:10.1128/CMR.00079-12

Difficulty in diagnosis has made it made it challenging to study the full spectrum of knowlesi malaria across the population. What studies have been done show that it produces a full spectrum of malarial disease from mild to fatal. Most cases reported to-date are in adult males, making an occupational exposure a significant possibility.

Symptoms are representative of other malarial infections: fever, chills and rigor, headache, along with a cough, abdominal pain and diarrhea. Gastrointestinal symptoms correlate with high levels of the parasite in the blood. Thrombocytopenia (low platelet counts) is the most common clinical finding and more severe than in either vivax or falciparum malaria, while anemia appears to be mild in knowlesi malaria. In the few pediatric cases that have been observed, they all responded to anti-malarial therapy. In the few cases of severe disease reported, abdominal symptoms have been so severe in some that malaria was not initially suspected. Acute Respiratory Distress Syndrome (ARDS) has been reported in about 50% of severe cases and acute renal failure in approximately 40%. There have not yet been enough confirmed cases of knowlesi malaria to accurately determine the case fatality rate. Although it appears to respond to a wide range of anti-malarial drugs, an optimized treatment based on a sufficient number of cases was not yet available in 2013.

The discovery of Plasmodium knowlesi in humans comes in the context of increasingly successful control of vivax and falciparum malaria in southeastern Asia. Some of the latest epidemiology from Malaysia suggest that 50-60% of the cases of malaria are now knowlesi. There are fears that knowlesi will jeopardize regional malaria elimination efforts. Is the rate really increasing or is it only apparent as levels of falciparum and vivax decrease? Does a real increase represent an opening niche for knowlesi as vivax and falciparum decrease? Only time and more data will answer our questions.

Primary Reference:

Singh, B., & Daneshvar, C. (2013). Human Infections and Detection of Plasmodium knowlesi. Clinical Microbiology Reviews, 26(2), 165–184. doi:10.1128/CMR.00079-12

For additional epidemiology from Malaysia:

Yusof, R., Lau, Y. L., Mahmud, R., Fong, M. Y., Jelip, J., Ngian, H. U., et al. (2014). High proportion of knowlesi malaria in recent malaria cases in Malaysia, Malaria Journal 13(1), 1–9. doi:10.1186/1475-2875-13-168

William, T., Jelip, J., Menon, J., Anderios, F., Mohammad, R., Mohammad, T. A. A., et al. (2014). Changing epidemiology of malaria in Sabah, Malaysia: increasing incidence of Plasmodium knowlesi, Malaria Journal 13(1), 1–11. doi:10.1186/1475-2875-13-390

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