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

 

Mapping Malaria in Anglo-Saxon England

Guthlac at Croyland in the marshes of the Wash.

England once looked very different. Much of southern Britain was marshland for most of the island’s occupied history. These bogs, fens, and marshes ensured that areas of virtual wilderness persisted  from before Roman Britain through the Norman period and beyond. Despite the difficulties of using fenlands, these areas were not only occupied throughout the Anglo-Saxon period, but important centers like Croyland, Bardney, and Ely eventually developed in the marsh.

The largest fenland region was known as ‘the Wash’.  This low-lying region drained four rivers into  a square bay of the North Sea that forms the corner between Lincolnshire and Norfolk. In Anglo-Saxon times, this tidal marsh and bog was a vast border region between the region of Lindsey and East Anglia.  Places like Croyland and Ely were islands in the wetlands.  The eighth century Life of Guthlac describes the environment of Croyland when Guthlac arrived:

There is in the Midland district of Britain a most dismal fen of immense size, which begins at the banks of the river Granta not far from the camp which is called Gronte (Cambridge) and stretches from the south as far north as the sea. It a very long tract, now consisting of marshes, now of bogs, sometimes with black waters overhung by fog, sometimes studded with woodland islands and traversed by the windings of tortuous streams. (Hill, 1981:11 cited in Gowland & Western, 2011).

These marshes are ideal for malaria, but evidence of malaria in Anglo-Saxon England has been lacking. It is supposed that malaria would have been brought to Britain with the Romans (1). Unfortunately, there is no evidence that I know of that malaria became endemic in Roman Britain much less lasted into the early medieval (Anglo-Saxon) period. It has also been speculated that ‘spring fever’ (lecten adl) found in Anglo-Saxon leechbooks is the spring manifestation of tertian malaria (1) caused by Plasmodium vivax. This would fit the pattern of malaria in cool or cold climates like that found in Finland discussed in a recent post. Indoor transmission in Anglo-Saxon earthen floored, open structured wooden homes with thatched roofs would be an ideal way to concentrate malaria in a thinly populated marsh.  (Without chimneys homes had to open enough to allow smoke to escape from a central hearth.)

Incidence of Malaria in England, 1840-1910 (2)

It has long been known that Britain can environmentally support endemic malaria. Malaria was fairly wide-spread in 19th century Britain when it was first mapped (figure to left) (2). The upper black area on the map includes much of ‘the Wash’. However, proof of malaria is more tenuous for the medieval period.  Together with the unhealthy reputation of the brackish marshlands there is at least enough evidence to suggest that endemic malaria reached back into the late medieval period.

Malaria went by a variety of local names before the early modern period. Malaria-like fevers are mentioned in literature from Geoffrey Chaucer to William Shakespeare (2, 3). Terminology for malaria was not settled upon the Italian ‘malaria’ until the early modern period. Before then, it went by a variety of terms the most universal being ‘ague’, meaning the shakes, and sometimes  ‘fever and ague’ referring to the cyclic breaking of a fever.

Gowland and Western (2011) took a new approach to finding evidence of malaria in Anglo-Saxon England (400-1100 AD) (4). Malaria caused by Plasmodium vivax causes chronic hemolytic anemia that may result in cribra orbitalia due to expansion of the bone marrow in the cranium. Gowland and Western correlated the presence of cribra orbitalia in Anglo-Saxon skeletal remains with the presence of the malarial vector Anopheles atroparavus and reports of ‘ague’ in 19th century England.

The Anglo-Saxon cemeteries used in their study are mapped in the figure below on the left. Note that not many cemeteries are located near the modern coastline of ‘the Wash’. This area would have likely been too wet for settlement.

Anglo-Saxon cemeteries (4)

Map of A. atroparvus with 19th century "ague" records. (4)

Gowland and Western  determined areas capable of sustaining malaria by mapping the presence of A. atroparvus from a 1900 AD British Museum survey (shown above on the right) (4).  The darker the shading the more reports of mosquitoes. This survey was reported to not have been systematic, so they augmented it with 19th century ‘ague’ reports (triangles).  There are some notable areas with high levels of mosquitoes that lack ague reports. This map was use to determine malarial regions for correlation with either cribra orbitalia or the poor nutrition control enamel hypoplasia. It also roughly correlates with the 1840-1910 malaria incidence in the color map above by Kuhn et al (2).

An inverse distance map showing A. atroparus incidence vs. hot and cold spots for cribra orbitalia. (4)

In this last map, malarial areas are plotted with hot and cold spots for cribra orbitalia.  Purple and blue areas on the map indicate the highest areas of A. atroparvus in 1900, while red and orange circles indicate the cribra orbitalia ‘hot’ spots. Areas of cribra orbitalia correlate very well with malarial areas around the Wash.  Cribra orbitalia ‘cold’ spots (blue circles) correlate with areas of low A. atroparvus. They found no correlation between enamel hypoplasia with either ‘malarial’ or ‘non-malarial’ areas (4).

If this cribra orbitalia is due to malaria, it is likely an underestimate of the amount of malaria in the English wetlands. Cribra orbitalia forms in children so it will not indicate adults who contract malaria. Communities like Ely, Croyland and Peterborough were large monasteries who probably drew many into the marsh as adults.

Confirmation of malaria in Anglo-Saxon England will have to wait for molecular evidence, but this skeletal evidence strengthens the hypothesis that it was endemic in early medieval Britain. It also should be informative for the areas to concentrate efforts to find molecular evidence.

References:

(1) Cameron, M.L. (1993, repr. 2006) Anglo-Saxon Medicine. Cambridge University Press.

(2) Kuhn, K., Campbell-Lendrum, D., Armstrong, B., & Davies, C. (2003). Malaria in Britain: Past, present, and future Proceedings of the National Academy of Sciences, 100 (17), 9997-10001 DOI: 10.1073/pnas.1233687100

(3) Reiter P (2000). From Shakespeare to Defoe: malaria in England in the Little Ice Age. Emerging infectious diseases, 6 (1), 1-11 PMID: 10653562

(4) Gowland RL, & Western AG (2011). Morbidity in the marshes: Using spatial epidemiology to investigate skeletal evidence for malaria in Anglo-Saxon England (AD 410-1050). American journal of physical anthropology PMID: 22183814

Health and Healing Sessions at Kalamazoo 2012

Regular readers might remember that last fall I was regularly posting and tweeting a call for papers for a session on health and healing in early medieval Europe for the International Congress on Medieval Studies at Kalamazoo in 2012. The schedule for the Congress is now out so I can tell everyone all about it. As you can see I got a great response to my CFP and the Congress committee let me put together two sessions. So without further ado, here are the sessions co-sponsored by The Heroic Age and Medica: The Society for the Study of Healing in the Middle Ages.

Session 264 (Friday 1:30)
Schneider 1255

Health and Healing in Early Medieval Britain and Ireland
Presider: Deanna Forsman, North Hennepin Community College

• Famine and Pestilence in the Irish Sea Region, 500–800 AD: Michelle Ziegler
• Regional Patterns of Health in Early Medieval Ireland: Distributions of Non-specific Stress Indicators: Mara Tesorieri, Univ. College Cork
• The Experience and Practice of Medicine by the Laity in Anglo-Saxon England: Julia Bolotina, Univ. of Cambridge
• By Rome, or By Spain? Possible Mediterranean Origins of Irish Holy Well Veneration: Silas J. Mallery, North Hennepin Community College

Session 319 (Friday 3:30)
Schneider 1255

Medical Texts of the Early Medieval Mediterranean
Presider: Michelle Ziegler

• Animal-Derived Medicines in Early Medieval Pharmacy: Jayna Brett, Centre for Medieval Studies, Univ. of Toronto
• A Medieval Hippocrates? The construction of the Articella during the eleventh century.: Marco A. Viniegra, Harvard Univ.
• Book-Learning and Medicine in Medieval Byzantium: Theory and Practice in the Alexiad of Anna Comnena: Glen M. Cooper, Brigham Young Univ.

Contagions Round-up 20: Past is Prologue in Science too!

Congratulations to Wonders & Marvels for winning the Cliopatria Award for Best History Group Blog of 2011 and to Lindsey Fitzharris of The Chirurgeon’s Apprentice who won the Cliopatria Award for Best History Individual Blog of 2011. What a year for history of medicine!

Starting with Lindsey’s latest post, let’s get this round-up rolling. The Chirurgenon’s Apprentice started the new year with a post on Cold Like the Dead: Learning Dispassion through Dissection.

Marri Lynn of Wonders and Marvels writes about the many medicinal uses Nicolas of Poland found for snakes.  Elizabeth Fix also of Wonders & Marvels writes about the search for immortality and the Philosopher’s Stone. Adrienne Mayor of Wonders & Marvels tells us that the quest for stem-cell like rejuvenation is ancient indeed!

Eric Michael Johnson of Primate Diaries discusses uses of the past by scientists and science writers.

Caroline Rance of The Quack Doctor reviews Moore’s Shropshire Doctors and Quacks.

Here at Contagions, I looked at a study of malaria in 18th century Finland and the process of retrospective diagnosis today.

Katy Meyers of Bones Don’t Lie looks at malaria trends in the UK and the discovery of brucellosis in medieval Albanian remains.

Kristina Killgrove of Powered by Osteons takes on a recent paper on the plague of Athens in the play Oedipus Rex, shares some material on ethics in bioarchaeology (part 1: Americas), and skeletal evidence of lead poisoning in ancient to medieval Rome.

Maryn McKenna of Superbug has been on the trail of completely resistant TB in India and in Italy.

On the other hand, Vincent Racaniello of The Virology Blog celebrates India’s polio free anniversary, and reassessed the mortality rate of ebolavirus, and discusses the recent moratorium on influenza H5N1 transmission research.

Zoonotica writes about the importance of bovine TB that is causing the planned cull of badgers in Britain.

Tara Smith of Aetiology writes about some of her recent research on MRSA in pork products.

Jennifer Frazer of The Artful Amoeba writes a scary finding on the durability of noravirus (Norwalk virus).

Michael Walsh of Infection Landscapes writes on our ancient passengers the Helminths (worms).

Small Things Considered took a look at what’s so scary about restroom microbiota.

Retrospective Diagnosis in the 21st Century

Black Death at Tourinai, 1349

The way we make and think about retrospective diagnosis is changing. Over the last decade, laboratory results have become the preferred (maybe even mandatory) method of making a retrospective diagnosis [1]. To extrapolate a few positive laboratory results to cover an entire epidemic, it must correlate with reported signs and symptoms and ideally epidemiology. There are pitfalls at every step of the process.

Today, laboratory results focus on identifying species specific biomolecules, aDNA and non-DNA molecules such as specific capsule lipids. I previously posted more detail on the non-DNA biomolecules used in paleomicrobiology. Each of these methods has its critics and proponents. Laboratory results should always be logical with the osteological analysis of the skeletal remains and with at least the majority of the written record. We have to remember that just because an infection was detected, it doesn’t necessarily mean that they died from it. If a skull has a sword wounds, it is unlikely that they died from tuberculosis. There is a significant difference between dying with a disease and dying from it.

For these science reports to be helpful to historians and anthropologists, they need to provide better context on the skeletal remains including how the remains were dated and associated with a particular epidemic. To give one example, a recent letter to Emerging Infectious Diseases reports Yersinia pestis in unerupted teeth from tombs under Sant’ Andrea church in Barletta, Italy [4]. They suggest this identifies the 1656-1657 epidemic as the plague. Although the tombs were said to have been used “since the 14th century” and have been hypothesized to have been used for the 1656-1657 epidemic, they do not give any indication how these five youths were selected for study. They report that their negative control from the same tomb complex dated to before the epidemic, so presumably this could be easily corrected by giving the rationale an archaeologist used to select these youths.

Assessing signs and symptoms and the epidemiology of past disease requires correctly using ancient and medieval documents. Medical treatises are not necessarily as helpful as they may seem. Medieval authors often copied classical texts that were centuries old and their new observations were heavily influenced by contemporary philosophical paradigms [2]. Instead, a variety of documents that can be broadly classified under government records (tax and manor rolls, wills, court records, etc); biographies and hagiographies; chronicles, annals and medieval histories can all be very useful in part because the disease is often not the main focus of the account [2].

Using such an array of ancient and medieval works has plenty of pitfalls even for historians, much less those from other disciplines. Mitchell has outlined a number of pitfalls to retrospective diagnosis from medieval texts that I will try to summarize with a few notes of my own [2].

1. Not enough information in the source for an accurate diagnosis.
2. An apparent eye-witness account is actually copied from an older source.
3. Translation is not representative enough of the original language for diagnosis.
4. Inadequate knowledge (of the researcher) of disease signs and symptoms and how they are represented in this cultural context.
5. Cherry picking symptoms to fit a theory.
6. Failure to understand that a source is not representative of the body of contemporary sources.
7. Not realizing that more than one disease is present. Likewise, assuming that two or more diseases are present when they are not.
8. Assumption that the antique or medieval disease is one that still exists today.
9. Assumption that the characteristics of the disease have not changed from then until now, including transmission methods. Likewise, assuming that characteristics of the microbe must have significantly changed over time.
10. Failure to understand how the environment and living conditions change the epidemiological characteristics of the epidemic.

Epidemiology is the most difficult parameter to assess. For ancient and medieval epidemics, I find it very difficult to consider epidemiology part of the retrospective diagnostic process. There are simply too many variables in the environment and human cultures of the past. Even diseases like malaria and influenza that we think we know so well still throw us surprises today [3]. Not even seasonality is set in stone as the 2009 H1N1 influenza pandemic recently showed us. After now centuries of study, we still can be hard pressed to explain the rise and fall of malaria. By it’s very definition, a pandemic is an extraordinary situation compounded by historical distance for study of historic outbreaks.

Even removing it from the diagnostic process, epidemiology is still one of the most important fields of study for past epidemics. If we can answer the important epidemiological questions on past epidemics then I think we can say that we understand at least the science of the epidemic. For many well-known pathogens like influenza and plague, we have had many outbreaks but very few pandemics to learn from to prepare for future threats.

[1] Little, L. (2011). Plague Historians in Lab Coats Past & Present, 213 (1), 267-290 DOI: 10.1093/pastj/gtr014

[2] Mitchell, P. (2011). Retrospective diagnosis and the use of historical texts for investigating disease in the past International Journal of Paleopathology, 1 (2), 81-88 DOI: 10.1016/j.ijpp.2011.04.002

[3] Raoult D (2011). Molecular, epidemiological, and clinical complexities of predicting patterns of infectious diseases. Frontiers in microbiology, 2 PMID: 21687417

[4] Scasciamacchia S, Serrecchia L, Giangrossi L, Garofolo G, Balestrucci A, Sammartino G, et al. Plague epidemic in the Kingdom of Naples, 1656–1658. Emerging Infectious Disease. 2012 Jan . http://dx.doi.org/10.3201/eid1801.110597

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Malaria Near the Arctic Circle

Malaria study area (Hulden et al, 2005)

When I think of Finland, malaria just doesn’t normally come to mind. Although northern climes often have swarms of mosquitoes, its hard to imagine mosquito-borne infections gaining much traction in the short summer season. Yet defying imagination, malaria has thrived in northern Finland, Sweden and Russia near the arctic circle in the past. In the late 19th and early 20th century, Plasmodium falciparum and Plasmodium vivax caused outbreaks in northern Europe. Despite the outbreak of P. falciparium at Archangelsk in the 1930s, P. vivax is believed to be the primary malarial species in northern Europe.

Finnish researchers Lena Huldén, Larry Huldén, and Kari Heliövaara focused on the 1800-1870 period in southern Finland as having the ideal demographic, medical and temperature records before the advent of quinine to study malaria transmission in cold climates.

Medical records are available for Finland from annual reports of ‘district physicians’ and local ministers for most of the 19th century. Doctors were stretched thin across Finland but in the fifty years between 1826 to 1870 there were 542 reports of malaria. Ministers were required to record the cause of death of their parishioners from 1749.  Digitization of parish records by the Finnish Genealogical Society has made this data available online for 1800-1850. Terms used for malaria were specific enough that general fever terms in the records did not correlate with malaria outbreaks or temperatures.

Malaria isn’t recorded in Finland until the 17th century, probably brought by migrant workers and gained traction among people gathered for summer infrastructure projects in southern Finland in the 18th century. Death records and physician reports indicate that during mid-19th century epidemics the mortality rate reached as high as 3% of the population with 7-20% infected. The worst epidemic occurred in 1862.

There are three Anopheles mosquito species found in Finland. All are believed to have been present in Finland since prehistory.  It had been thought summer temperatures of 16 C (60.8 F) were required to maintain endemic malaria, but malaria has been recorded areas in of northern Sweden and Finland that don’t reach 16C in the summer. Males die shortly after mating and female Anopheles must hibernate from late summer until well into spring. Therefore, the female spends most of its life indoors hibernating with humans and sheltered domestic livestock. The female will take sporadic nocturnal blood meals over the winter but won’t lay her eggs until spring.

Huldén,  Huldén, and Heliövaara correlated malarial deaths with annual, seasonal and monthly temperatures. The only significant correlation occurred between summer temperatures of the previous year, but not at all with annual or seasonal temperatures of the same year. Malarial deaths peaked in the spring rather than the usual late summer or autumn. So how does this work with a temperature correlation to the previous summer? Winter infections. The previous summer temperatures effect how many mosquitoes will be hibernating over the winter in homes. Sporadic blood meals over the winter in the confined space of the home spreads the infection to most of the humans and other hibernating mosquitoes causing infections that peak in the spring. Humans are the primary reservoir of infection in cold climates. It doesn’t matter that the malarial sporozoites won’t mature outdoors during the cool summer because they will mature in the cozy confines of winter homes. Fatal spring infections in 40-50 infants born in the winters from 1750 to 1850 supports the theory that the female mosquitoes were capable of transmitting malaria for the entire winter.

Age distribution among the malarial deaths was very similar to the total population indicating that all ages groups were equally vulnerable to infection. Huldén,  Huldén, and Heliövaara interpret this as indicating infection occurred at a time when the entire family would be together in heated buildings, in the winter rather than in summer when  occupations cause families to live apart by age and gender often in unheated buildings.

Epidemiological data can usually be explained but not necessarily predicted. They have provided another example of why epidemiology can’t always be definitive in ruling in or ruling out the diagnosis of a historic epidemic. Based on outdoor temperatures malaria should have never been endemic in Finland at all. This study highlights the importance of the indoor environment for malaria (and other zoonoses).

Reference

Huldén L, Huldén L, & Heliövaara K (2005). Endemic malaria: an ‘indoor’ disease in northern Europe. Historical data analysed. Malaria journal, 4 (1) PMID: 15847704

Top 11 in 2011

As the year comes to a close, I thought I would share the top 11 posts here at Contagions for this past year. I’m excluding round-ups and the educational chain of infection posts. These chain of infection posts account for over a third of all page views on this blog over the year and all time! So without further ado, here are the top 11 regular posts based on page view stats as of December 29, 2011.

1. Pandemic Influenza: 1510-2010
2. Beyond Pelusium
3. Did India and China Escape the Black Death?
4. Vampire Prevention in Eighth Century Ireland
5. Rinderpest, Measles and Medieval Emerging Infectious Diseases
6. The Vampire in the Plague Pit
7. Hunting Pathogens in the Siberian Permafrost
8. Plague in 18th century Egypt
9. DNA of the Black Death at East Smithfield, London
10. Plague DNA from Late Antique Bavaria
11. Epidemiology of the Russian Flu, 1889-1890

Happy New Year!!