Category Archives: aDNA

The Paleomicrobiology of Malaria Detection

Malaria is arguably one of the most influential infectious diseases in human history. Its been with us as long as we have been human, but as Teddi Setzer shows us in her recent review of detection methods, our abilities to find it in the past leaves a lot to be desired.

The standard method of looking for malaria involves searching for signs of anemia on the skeleton on the hypothesis that the anemia caused by malaria leaves these marks. This is not as clear as it might seem. There have been very few skeletal studies of modern people who have been diagnosed with malaria. There is no medical need; there are much more reliable methods of diagnosing malaria in a living person (or recent cadaver). So, it is unclear how often these lesions form in malaria patients. Other causes of anemia and even scurvy can cause the same or very similar lesions as well.  The number of malarial infections and/or relapses also effect bone changes. Plasmodium falciparium produces a short, virulent disease that may kill before bone changes develop. On the other extreme, a single P. malariae infection can relapse for life, although the anemia is not as severe.  Osteology must be correlated with other information to support the diagnosis. 

Cribra Orbitalia from Jess Beck’s blog Bone Broke

Cribra orbitalia and porotic hyperostosis are the two main indicators sought. Both are caused by bone marrow expansion in an attempt to compensate for the loss of red blood cells. Cribra orbitalia is pitting and extra bone growth in the orbits of the eyes, as seen in the photo.  Porotic hyperostosis causes pitting and thinning of the compact bone ‘shell’ that covers the cranial bones. A  correlation of nutritionally informed osteology with later epidemiology and mosquito incidence in England reviewed in a previous post shows that a convincing case can be made for malaria in ancient remains.

Detection of human genetic traits selected for by malaria such as the Duffy blood group, sickle cell trait, thalassemias, and glucose-6-phosphatase deficiency (G6PD) can with supporting information suggests that the population was once under selection by malaria. Balanced polymorphisms like sickle cell trait can remain in a population for centuries after the selection is gone (by either ecological change or by migration away from the malarious region). While there are some skeletal indicators of some hemoglobinopathies, human ancient DNA analysis would be a more secure method of diagnosis. Care has to be taken to distinguish skeletal changes made by malaria’s hemolytic anemia and the hemoglobinopathy anemias.

Ancient DNA detection of the malaria Plasmodium parasite has been disappointing. To date, only the tropical Plasmodium falciparium, that causes the most severe disease, has been detected by PCR. It is believed that attempts of detect the historically more common Plasmodium vivax have been stymied by the low parasite load in the blood.  The difficulty in finding vivax aDNA is a reminder that pathogens really do need to be in high concentrations within the sample to overcome degradation and be detected by PCR or sequencing technology. As far as I know, there have not been attempts to detect the other three human malarial parasites– Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi — by aDNA analysis.

Hemozoin crystals in the liver (Source: KMU Pathology Lab)

Modern medicine is devising an ever expanding array of tests for malaria diagnostics and prognostics. However, most of these tests all require fresh (soft) tissue or blood. Immunological methods have not been applied to malaria in archaeological material yet. The most promising detection method for malaria among the newer diagnostics is the detection of the iron containing waste product of the Plasmodium parasite hemozoin. When the parasite feeds on hemoglobin in the red blood cell, toxic iron waste products are processed into the biocrystal hemozoin and excreted into the tissues. In patients with reoccurring or multiple malaria infections, hemozoin will stain their bone marrow black and can be found in liver, spleen, brain and lungs. It can be detected microscopically (as seen above) or by mass spectrometer. Although some other blood parasites also excrete hemozoin, they can be distinguished from the malarial product. 

Despite the advances in diagnosis for existing malaria patients taking advantage of new methods and technologies, archaeological detection has not enjoyed the same success. Building a case for malaria in the past, must rely on an array of data with knowledge of ecology, vectors, and nutritional status of the population in addition to osteological markers of anemia. Hopefully, the detection of hemozoin will eventually be the key to opening up biological studies of malaria in the past. If hemozoin can identify malaria victims, then perhaps focusing the ancient DNA work on hemozoin positive remains will be more successful breaking through the firewall to malaria’s evolution and historical epidemiology. 

 Source:

Setzer, T. J. (2014). Malaria detection in the field of paleopathology: A meta-analysis of the state of the art. Acta Tropica, 140, 97–104. doi:10.1016/j.actatropica.2014.08.010 (open access early editionfinal edition)

See also Jess Brek “Porotic Hyperostosis and Cribra OrbitaliaBone Broke, March 2014. 

Historians Chronicling Plague Genetic Discoveries

After my last post critiquing Cohn’s scientific interpretations, I think its only fair to write about all the historians who are actively engaging and incorporating scientific findings in their work. I’ve communicated with a lot of historians who are following the scientific work on the plague and I know there will be some articles and books coming out over the next year or so that incorporate some of new genetics in historical analysis.

So for science folks, these two articles give us some insight into how historians see plague genetics unfolding. Little concentrates on the early drama over plague genetics. Bolton covers that material also, but also looks at newer information on transmission dynamics too.

Little, L. K. (2011). Plague Historians in Lab Coats. Past & Present, 213(1), 267–290. doi:10.1093/pastj/gtr014

Bolton, J.L. ‘Looking for Yersinia pestis: scientists, historians and the Black Death’ in L. Clark and C. Rawcliffe (eds.), Society in an Age of Plague, The Fifteenth Century XII (Woodbridge: Boydell, 2013), publication date 15 August 2013, ISBN 9781843838753. (In the same book/issue as Cohn’s paper discussed in the last post.)

Overall, I am really optimistic about the interdisciplinary work that can be done on the plague.

Academic Plague Identity Wars Continue

Just when you think the academic wars over the identity of the medieval plague are over, another volley is cast by Samuel Cohn. In the past I haven’t mustered the energy to respond to his papers and books because there are just so many scientific misunderstandings, but its time to respond. Obviously, scientific studies that cover all the bases aren’t enough, so for now I’ll to correct some of his misinformation (leaving most of his historical analysis to the historians to critique).

Samuel K. Cohn, Jr. (2013) “The Historian and the Laboratory: The Black Death Disease” pp. 195- 212 in Society in the Age of Plague. The Fifteenth Century XII. Clark, L and Rawcliffe, C. Eds. Boydell Press.

Cohn portrays the discovery of Yersinia pestis at the turn of the 20th century as yet another French vs British competition in a partisan British manner. While trying to undercut the importance of French findings, he does not include the political context of Victorian India and particularly English trade interests in his discussion of the Indian outbreak. He does blame harsh British tactics in India on their recognition that Yersinia pestis was the pathogen (which he questions). This whole section needs a good going over by an modern historian.

In his criticism of the aDNA work, he claims that most researchers had “negative findings” but Gilbert et al is the only paper he cites that really failed to find plague. If he is referring to the number of negative specimens, then he doesn’t understand how rare the survival of aDNA is in general and the importance of the more sensitive protein methods. Like most of the press, he makes way too much out of the observation that the East Smithfield isolates represent an ‘extinct’ clade. It would be far more surprising if a Black Death isolate were identical to a modern strain. Evolution does not stop, especially not in the accumulation of polymorphisms (neutral mutations). He ignores the fact that the Third Pandemic strains are descendants of the Black Death isolates. He doesn’t seem to understand that genetic diversity is produced in every epidemic and that most of it is lost at the end of the epidemic. This is especially true of Yersinia pestis diversity generated in humans because it must be transmitted to a reservoir species to be preserved.

Not for the first time, I wonder if he understands what natural immunity is and he uses references from the 1950s or before on human immunity to the plague. All medieval immunity is natural. They did not have the means to generate artificial immunity. Natural immunity can be passive (mother to child) or active (generated after exposure).   He misrepresents Li et al (2012) as an indication that immunity is short-lived when in fact the study shows the antibody response is strong (69.5% at 10+ years) and correlated with the strength of their response at the time of the initial infection. Just because we are having problems generating a vaccine that can repel pneumonic plague doesn’t mean that a response generated against an active infection couldn’t repel bubonic plague.  It takes a much stronger response to cope with an aerosol exposure.  When plague epidemics are coming about every 10-15 years, an immunity that lasts 10-20 years would be enough to produce an age differential in the mortality rate. I will leave his analysis of the mortality from historic sources to historians to comment upon. While childhood mortality rates in plague epidemics are clues toward immunity, they are only one variable in comparing the epidemics. We also have to look at what else is occurring among the children such as normal childhood mortality, co-infection and other co-morbidity.

He makes the leap of logic that decreases in total mortality rates equals changes in human immunity. There are many variables that effect the intensity of an epidemic. Decreases in human mortality may suggest changes in the rodent population and/or rodent immunity. If epidemics occur too closely spaced the rodent population will not have recovered enough to generate a large outbreak. Of course, other environmental changes can alter the rodent population and exposure of humans to rodents.

He makes assertions on vector transmission that are not referenced and uses a reference from 1913 (!) to assert that the septicemia in humans is not high enough to allow human-to-human flea transmission. He seems to be assuming that transmission would need to be accomplished by a single flea or louse, which is unlikely. He gives no reference for his assertion that the bacterial load in plague is lower than insect vector transmitted typhus or Lyme disease. He seems to think that only one vector could be at work in the second pandemic rather than rat fleas, human fleas, and lice all transmitting Y. pestis in the same epidemic. Pathogens will take any opportunity available to transmit.

He starts reaching for straws in the conclusions:

There are statements like this: “The ancestor of this family, Yersinia psuedotuberculosis, which geneticists argue gave birth to this new strain of Yersinia, perhaps as late as the eve of the Black Death” (p. 210) Yersinia pestis is not a new strain of Yersinia pseudotuberculosis! It is a species in its own right. A strain is a distinctive subpopulation of a species.  Emerged as late as the eve of the Black Death? Nonsense. There is the little thing of the Plague of Justinian about 800 years earlier, with aDNA and protein evidence. This emergence involving genome rearrangement, loss of genes, gain of chromosomal genes and plasmids would likely have taken at a minimum centuries before 541.

“Could an earlier variety of the ancestor Yersinia suddenly have developed pathogenic factors such as plasmids or, on the level of protein biosynthesis, abilities form a capsule or to release endotoxin, thus suddenly transforming the benign pseudotuberculosis into a new and vicious pathogen, but without diminishing its ability to spread effectively from person to person?” (p. 211)

This one is easy…. NO! Bacteria do not suddenly develop plasmids; they acquire them from other species. In Y. pestis’s case, all of these plasmids are significantly modified from the ancestral plasmids they received. It also takes more than one gene or even plasmid to produce Y. pestis virulence from Y. pseudotuberculosis. He seems to also be implying that a change to increased virulence in humans is the species differentiating event for a primarily rodent pathogen.  Then he strangely follows this (a few sentences down) with the speculation that the “modern bacillus may actually be more toxic than that of the pathogen of the historic plague.”(p. 211) Huh? What happened to his speculation above that “a new and vicious pathogen” was at work?

“As regards Black Death and the ‘Third Pandemic, when and by what criteria does ‘a strain’ of a pathogen come to be reckoned as the causal agent of another ‘disease’, which has to be classified differently from that caused by a related pathogen of the same genetic family, as is currently recognized in the case of Yersinia pestis and its older relative, Yersinia pseudotuberculosis? Even if scientists thought that a pathogen is the equivalent of the disease it in part causes, that is the only pertinent defining feature? Even if scientists thought that the pathogens of the ‘Second’ and ‘Third’ Pandemics were identical (and now they do not), should we then return to the strict reductionism of Koch circa 1890, that a pathogen is the equivalent of the disease it in part causes, that it is the only pertinent feature?” (p. 212)

What? Now we have to reargue germ theory? Pathogens can have different presentations and different epidemic dynamics; some transmit by a variety of means. Co-infections and other co-morbidities certainly matter, but you don’t have the disease without the pathogen.  This is not a type of disease like pneumonia where multiple pathogens cause similar effects. Cohn is grasping at straws and bending scientific concepts to suit his purposes.