Plague Detection by Immuno-PCR

Once again the Marseille research group is pushing the bounds of plague detection. This time their target is looking for a more sensitive method of detecting non-nucleic acid biomolecules from Yersinia pestis, ‘the plague’. We have now moved into an era where PCR is being used in the mechanics of testing, rather than amplifying the ultimate target of the test.

Immuno-PCR

The immuno-PCR (iPCR) method is outlined in the figure. The selective component of the assay is the mouse polyclonal anti-Yersinia pestis antibody. Polyclonal antibodies are the products of several different B lymphocytes reacting to the same antigen, protein in this case. This means the antibodies in preparation will bind to different parts (epitopes) of the same protein. This should be an advantage in working with badly degraded material.

In iPCR the selective reagent is the non-human antibody generated against the microbial target. The second antibody is against the non-human antibody (mouse, rabbit, etc) and carries biotin as a marker. These biotinylated antibodies are a very common and widely available immunology reagent. The third antibody is against the biotin and carries a reporter sequence of DNA. Quantitative real-time PCR is then done on the reporter DNA sequence attached to the antibody. This amplified reporter DNA can easily measure very tiny amounts of DNA. The iPCR system utilizes advances in PCR technology to measure specific protein levels.

Malou et al (2012) took  known positive and negative teeth, and subjected them to iPCR, PCR and the standard ELISA antibody assay.   They first determined the detection limit for ELISA and iPCR, and set a threshold for a positive iPCR result with 10 known negative teeth (5 ancient and 5 modern). They then coded and randomized  34 historically known positive teeth, the 10 negative teeth (5 ancient and 5 modern) and two blanks for testing with ELISA, PCR, and iPCR. The results and how they compare are shown in the diagram below. The ELISA only picked up four teeth with one being a known negative resulting in a sensitivity of 9% and specificity of 90%. Of the three ELISA true positives, two were from a 17th century site at Lariey and one from a 6th century site at Sens, both in France. For the iPCR, 14 of 34 exceeded the threshold and were considered positive for a 41% sensitivity with a 100% specificity (all positives were historically positive, no false positives). These teeth came from Lariey, Bourges, Sens, Bondy, and Venice with a date range from the 6th to 17th century. Traditional PCR identified 10 out of 34 historically positive teeth fora  sensitivity of 29% with 8 of 10 also being identified by iPCR.

Venn diagram of positive teeth detected by ELISA, PCR, and iPCR.

Immuno-PCR compares well with the efficiency and specificity of standard PCR for Yersinia pestis DNA and is more sensitive than the standard ELISA antibody assay. A standard ELISA produced the worst results. Although not quantitative, it would have been interesting if they had also done the Rapid Diagnostic Dipstick Test (RDT), another antibody based protein detection method, that has been used in several studies.  They are suggesting that iPCR be added to traditional PCR as a method of further confirmation of Yersinia pestis at a site. Immuno-PCR can be done on the same teeth as traditional PCR and should be easily doable with the expertise and equipment in labs that conduct traditional aDNA PCR.  By identifying both aDNA and protein, the confirmation of Yersinia pestis in the ancient remains should become quite strong.

Reference:

Malou, N., Tran, T., Nappez, C., Signoli, M., Le Forestier, C., Castex, D., Drancourt, M., & Raoult, D. (2012). Immuno-PCR – A New Tool for Paleomicrobiology: The Plague Paradigm PLoS ONE, 7 (2) DOI: 10.1371/journal.pone.0031744

 

Holly Tucker’s Blood Work

Holly Tucker, Blood Work: A Tale of Murder and Medicine in the ‘Scientific Revolution’. Norton, 2011.

I picked up this book at just the right time. I read it a few weeks ago just before I started teaching my summer anatomy course. Its always a good to be reminded of all of the work and drama that went into what seems like dull facts and standard procedures today. In this case there really was a lot of blood, sweat and tears by the medical establishments of two countries intensely throughout the 1660s before the work was banned for over a century. Holly Tucker’s Blood Work is centered on the century when the quest to understand blood went from counter-productive procedures based on medieval philosophy to the beginning of scientific understanding and experimentally based medicine.

Pride and glory drove the main protagonists of the story. For Jean-Baptiste Denis, the physician at the center of Blood Work,  the need to overcome his humble origins by proving his worth and more to the gentlemen physicians of France was a driving force. His benefactor Henri-Louis Habert de Montmor wanted to be the patron of the most brilliant, the most significant scientists of France, to be the Lorenzo de’ Medici to a scientific Michaelangelo. Add in an Anglo-French medical society rivalry and some pirates for good measure, and you have a page-turner of a history. I have to admit chuckling every few pages. Some of their experiments were like watching a train-wreck in slow motion. Holly Tucker is no doubt right that they were saved from causing much more damage by their inefficiency at transfusion.

I won’t give away any of the main plot or the murder, but there were some memorable sidelines. This description of ‘heroic medicine’ will stay with me for a while.

Claude arranged for a barber-surgeon to administer numerous bleedings to his brothers arms and legs. When bleeding seemed no longer to have any effect, they tried to place leeches behind Jean’s ears, but blistering there from other treatments with warming salves kept the leeches from doing their work. Bouillons, enemas, and purgings accompanied each bleeding in a desperate attempt to save Jean’s life. And to these were added chest rubs with concoctions of ground pearls mixed with extracts of hyacinth bulbs to warm Jean’s blood, as well as placement of gutted pigeons on his scalp to create heat to stave off the shivering. Despite Claude’s heroic attempts to save his brother (or perhaps because of them), Jean died a few weeks later. (p. 105-6)

Claude Perrault was one of the best trained physicians in France, and  a founding member of King Louis XIV’s Academy of Science. He held a prominent position in the Paris Faculty of Medicine and he used all learning to try to save his brother Jean. The idea of keeping your brother warm with a gutted pigeon on his head is priceless.

If you think that today’s publish or perish environment is tough, you should read what it did to Henry Oldenburg, who nearly single-handed published the Philosophical Transactions for the Royal Society of London. He volunteered his own time and talent, published at his own considerable expense for printing and postage, was accused to treason for gathering scientific news from France, and landed in the Tower of London for his troubles (where he had to pay rent for his prison cell). He then got out and worked even harder for the Royal Society (still for free) to reestablish his reputation. His treatment is enough to keep freelance science writers, bloggers, and researchers up nights.

I do have to say that I’m not bothered by the fact that the transfusion experiments were stopped. While Holly Tucker is no doubt right that other equally dangerous experiments and treatments were being done at the same time, the paradigm shift from blood letting to blood transfusion was too great, too fast. They didn’t understand even the basics of what they were transfusing and they were doing it for the wrong scientific and philosophical reasons. Simply stopping the blood letting would have improved survival significantly!

As memorable as the information and stories in Blood Work will be, perhaps more importantly it has made me think about the importance of narrative in books on history. So many of the history books I read are organized and written like textbooks, academic in their boredom as much as their content. I read them for their information rather than for entertainment. Its nice to have both!

Lice, Ancient DNA, and Napoleon’s Grand Army

Identification of human body lice. (Raoult et al, 2006)

Life in Napoleon’s Grand Army wasn’t always so grand. The Russian campaign was a disaster, recently most tangibly manifest in the mass grave found at Vilnius, Lithuania, in 2001. Local records suggested that the remains belonged to Napoleon’s soldiers who paused at Vilnius during their retreat from Moscow in 1812. The densely packed bodies were buried at the same time leaving behind buttons, buckles and gear of 40 regiments of Napoleon’s army. The initial trench revealed 717 skeletons at 7 corpses per meter squared, predicting 2000-3000 corpses at the site. Winter weather may have claimed most of the soldiers, but they were also known to have been plagued by lice and fevers.

For Raoult et al (2006) this was the perfect test case for detecting lice and louse-borne diseases in archaeological remains. After perfecting their technique of isolating cultured, dried human lice (Pediculus humanus humanus) in the laboratory*, they obtained soil from the mass grave at Vilnius. They isolated fragments of five lice from two kilograms of soil containing remains of bones and clothing, and confirmed the identification by “binocular magnification” (right), scanning electron microscopy and isolated P. humanus humanus DNA to confirm the species. Three of the lice produced Bartonella quintana DNA (trench fever) but none of the lice yielded Rickettsia powazekii DNA (typhus) or Borellia recurrentis DNA (relapsing fever).

They extracted DNA from 72 unerupted teeth from 35 skeletons for ‘suicide’ PCR pathogen detection. They found R. powazekii (typhus) in teeth from three skeletons and B. quintana (trench fever) teeth from seven skeletons. Overall, louse-transmitted disease was identified in 10 out of 35 skeletons (28.6%). B. quintana is believed to be the most common louse-transmitted pathogen in Antiquity and the Middle Ages. Trench fever alone is usually not fatal but when combined with the harsh conditions on the retreat from Russia it could have been a significant contributing factor. Epidemic typhus on the other hand is a more virulent organism and is more likely to be the cause of death of the three individuals in whose teeth R. powazekii was discovered.

The ability to identify pathogen vectors, in this case the human louse, and amplify pathogen DNA from both ancient vectors and humans is a big advance in our understanding of infectious disease in pre-modern populations.

* I don’t envy the research assistants or students who had to maintain the colonies of human lice in the lab, or roast them incubators to simulate conditions in soil.

Raoult D, Dutour O, Houhamdi L, Jankauskas R, Fournier PE, Ardagna Y, Drancourt M, Signoli M, La VD, Macia Y, & Aboudharam G (2006). Evidence for louse-transmitted diseases in soldiers of Napoleon’s Grand Army in Vilnius. The Journal of infectious diseases, 193 (1), 112-20 PMID: 16323139

Trench Fever and Plague in 14th Century France

Five Skeletons from Bondy, France. (Source: Tran et al, 2011a)

The Marseille plague group has been suggesting for some time now that human lice could be a major vector of medieval plague. To test their hypothesis the group devised a multiplex PCR screening method to rapidly screen many aDNA samples for seven pathogens that could cause medieval epidemics, including relapsing fever and trench fever transmitted by human lice. Their method was previously published for a  screening of mass burials from medieval Venice (Tran et al, 2011b).

In their most recent publication, they have examined 14 teeth from five skeletons found in Bondy, near Paris, France (Tran et al, 2011a). The remains are from people of varied ages and both genders, radiocarbon dated to the 14th century (1297-1373 with 70% probability). They were also screened for the pathogens that cause anthrax, trench fever, relapsing fever, typhus, typhoid fever, smallpox and plague. As in Venice, they only found two of these, trench fever (Bartonella quintana) and plague (Yersinia pestis) (Tran et al, 2011a; Tran et al, 2011b). Overall, B. quintana was found in three individuals and Y. pestis in two individuals, with one individual (#73, far left skeleton half revealed in the picture) positive for both. Trench fever is transmitted by the human louse and Tran et al  (2011a) see this as supporting their hypothesis that lice transmitted the plague as well. While one unfortunate co-infected individual is not proof, it does add one more piece of evidence to support their hypothesis. Plague was confirmed by sequencing and identified as the orientalis biovar. This group has previously asserted that the orientalis biovar is transmitted much more efficiently by lice than other Y. pestis biovars (Avyadurai et al, 2010).

They are citing one more paper as ‘in press’ in Tran et al, 2011a. It looks like there is a summary paper coming up that will lay out all their arguments for lice as transmitters of medieval plague, so stay tuned!

Tran TN, Forestier CL, Drancourt M, Raoult D, & Aboudharam G (2011a). Brief communication: Co-detection of Bartonella quintana and Yersinia pestis in an 11th-15th burial site in Bondy, France. American journal of physical anthropology PMID: 21541920

Tran TN, Signoli M, Fozzati L, Aboudharam G, Raoult D, & Drancourt M (2011b). High throughput, multiplexed pathogen detection authenticates plague waves in medieval Venice, Italy. PloS one, 6 (3) PMID: 21423736

Ayyadurai S, Sebbane F, Raoult D, & Drancourt M (2010). Body lice, yersinia pestis orientalis, and black death. Emerging infectious diseases, 16 (5), 892-3 PMID: 20409400