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Transactions of the Royal Society of Tropical Medicine and Hygiene 104 (2010) 583–587

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Transactions of the Royal Society ofTropical Medicine and Hygiene

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lasmodium falciparum immunodetection in bone remains of membersf the Renaissance Medici family (Florence, Italy, sixteenth century)

ino Fornaciari a,∗, Valentina Giuffraa, Ezio Ferrogliob, Sarah Ginoc, Raffaella Bianuccid

Division of Paleopathology, History of Medicine and Bioethics, Department of Oncology, Transplants and Advanced Technologies in Medicine, University ofisa, Via Roma 57, 56126 Pisa, ItalyLaboratory of Parasitology and Parasitic Diseases, Department of Animal Production, Epidemiology and Ecology, University of Turin, Via Leonardo da Vinci4, 10095 Grugliasco, Turin, ItalyLaboratory of Crimininalistic Sciences, Department of Anatomy, Pharmacology and Legal Medicine, University of Turin, C.so Galileo Galilei 22, 10126 Turin,

talyLaboratory of Crimininalistic Sciences, Department of Anatomy, Pharmacology and Legal Medicine, University of Turin, C.so Galileo Galilei 22, 10126 Turin,

taly & UMR 6578 CNRS-EFS (Biocultural Anthropology), University of Marseille, Boulevard Pierre Dramard, 13916 Marseille Cedex 20, France

r t i c l e i n f o

rticle history:eceived 7 December 2009eceived in revised form 22 June 2010ccepted 22 June 2010

eywords:enaissanceone remains

alciparum malariaouble antibody immunoassays

a b s t r a c t

Medical accounts and ancient autopsy reports imply that tertian malarial fevers caused thedeath of four members of the Medici family of Florence: Eleonora of Toledo (1522–1562),Cardinal Giovanni (1543–1562), don Garzia (1547–1562) and Grand Duke Francesco I(1531–1587).

All members of the Medici family hunted in the endemic malarial areas of Tuscany, suchas the marshy areas surrounding their villas and along the swampy Maremma and were,therefore, highly exposed to the risk of being infected by Falciparum malaria. To determineif the original death certificates issued by the court physicians were correct, we carried outimmunological investigations and then compared the biological results to the historicalsources.

Bone samples were examined for the presence of Plasmodium falciparum histidine-rich-protein-2 (PfHRP2) and P. falciparum lactate dehydrogenase (PfLDH) using two differentqualitative double–antibody immunoassays.

Our findings provide the first modern laboratory evidence of the presence of P. falciparum

ancient proteins in the skeletal remains of four members of the Medici family. We confirmthe clinical diagnosis of the court physicians, using modern methods.

Finally, this study demonstrates that immunodetection can be successfully applied notonly to mummified tissues but also to skeletal remains, thus opening new paths of inves-tigation for large archaeological series.

Society

© 2010 Royal

. Introduction

Medical accounts and autopsy reports imply that ter-ian malarial fevers caused the death of four members ofhe Medici family: Eleonora of Toledo (1522–1562), Car-

∗ Corresponding author. Tel.: +39 050992894; fax: +039 050992706.E-mail address: g.fornaciari@med.unipi.it (G. Fornaciari).

035-9203/$ – see front matter © 2010 Royal Society of Tropical Medicine and Hoi:10.1016/j.trstmh.2010.06.007

of Tropical Medicine and Hygiene. Published by Elsevier Ltd. Allrights reserved.

dinal Giovanni (1543–1562), don Garzia (1547–1562) andFrancesco I (1531–1587).1

Malaria was endemic in central Italy along the coastsof Tuscany and the Maremma lowlands from the third cen-

tury BC until the end of World War II and had severe effectsin terms of both morbidity and mortality on human popu-lations living in this region.2

All members of the Medici family hunted in the endemicmalarial areas of Tuscany such as the marshy areas

ygiene. Published by Elsevier Ltd. All rights reserved.

ety of Tr

584 G. Fornaciari et al. / Transactions of the Royal Soci

surrounding their villas and along the swampy Maremma1

and were highly exposed to the risk of being infected byprotozoan parasites.

In October 1562, Cosimo I de’ Medici (1519–1574),first Grand Duke of Tuscany, his wife Eleonora of Toledoand their sons, Cardinal Giovanni, don Garzia and donFerdinando (1549–1609) visited the malarial Maremmacountry near Grosseto (southern Tuscany). During theirstay, Cosimo and his family enjoyed hunting in the lowerArno Valley and along the marine swamp.

On their way back to Florence, Cardinal Giovanni, donGarzia and Eleonora suffered from sudden irregular boutsof fever and died in a time span of three weeks. Histor-ical and medical accounts state that severe malaria wasthe cause of their deaths.1 Fifteen years later, in Octo-ber 1587, Francesco I de’ Medici, son of Cosimo I andEleonora of Toledo, second Grand Duke of Tuscany, and hissecond wife, Bianca Cappello (1548–1587), died suddenlywithin 24 hours of each other. Again, medical accounts andautopsy reports imply that severe malaria was the cause oftheir deaths.1,3

Recent studies on ancient biomolecules suggestedthe possibility to identify Plasmodium falciparum anti-gens in ancient skeletal remains using modern laboratorytechniques.4–9

To determine if the original death certificates issuedby the court physicians were correct, immunologicalinvestigations were carried out to determine whether P.falciparum malaria might have been the cause of death ofthose personages, and then compared the biological resultsto the historical sources.

2. Materials and Methods

2.1. Sampling

The skeletal remains of Eleonora, Giovanni, Garzia andFrancesco I were unearthed from the topsoil of the MediciChapels in San Lorenzo Church in 2004.10,11 Bianca Cap-pello’s remains have not been recovered: her burial siteremains unknown.12

Cancellous bone was harvested from the vertebrae ofEleonora of Toledo (sample code number: MED5), CardinalGiovanni (sample code number: MED3), don Garzia (sam-ple code number: MED4) and Francesco I (sample codenumber: MED11).

Bone samples of Cosimo I de’ Medici (sample codenumber: MED6), who died of pneumonia, and of hisdaughter-in-law, Joan of Austria, who died in childbirth(sample code number: MED8) were used as negativecontrols.1

Additionally, six medieval bone samples from twosites known to be free from malaria (Briancon, France,seventeenth century and Augsburg, Germany, fourteenthcentury) were used as negative controls.13

2.2. Immunochromatographic assays

Bone samples were tested for malaria immunodetectionby using two different dipstick assays which target specific

opical Medicine and Hygiene 104 (2010) 583–587

immunoreactive plasmodial molecules. Extracts preparedfrom spongy bone samples were examined for the pres-ence of P. falciparum histidine rich-protein-2 (PfHRP2) andPlasmodium lactate dehydrogenase (PfLDH) using two dif-ferent qualitative double-antibody immunoassays: MalariaAntigen RAPYDTEST® and MalariaDetectTM RAPYDTEST®(DiaSys, Connecticut, U.S.A).14

Malaria Antigen RAPYDTEST® targets P. falciparumspecific histidine rich-protein-2, a water-soluble proteinexcreted by asexual and young gametocytes of P. falciparumand is not present in the other three human pathogenicPlasmodium spp.15,16,20

Several studies on living patients reported that the mon-oclonal IgG antibody used in certain tests (i.e. ParaSightF-test) cross-reacted with rheumatoid factor (RF) in bloodthus resulting in false positive tests for malaria. Replace-ment of IgG with IgM in recent products has drasticallyreduced this problem.16,17

MalariaDetectTM RAPYDTEST® targets Plasmodium lac-tate dehydrogenase (pLDH), which is a soluble enzymefound in the glycolitic pathways of the malaria parasite andis produced by sexual and asexual stages of the parasite.This test is aimed at the differential diagnosis between P.falciparum and the three other Plasmodium species by usingtwo monoclonal antibodies (a P. falciparum anti-pLDHand a pan specific anti-pLDH). The monoclonal antibodiesemployed in rapid examinations detecting pLDH have beenexhaustively tested for cross-reactivity from other bloodprotozoa such as Leishmania, Babesia and pathogenic bac-teria and fungi. No evidence of such cross-reactivity hasbeen found.18,19

2.3. Protocol

Bone samples were cleaned with dry brushes anddecontaminated by UV light. The external bone surface wasremoved with a drill (Kavo Intramatic Lux 2, KaVo Den-tal GmbH, Biberach/Riss, Germany) while mounted on amicromotor turning at 9000 rpm. Next, the cancellous bonewas powdered by hand in sterile conditions using a mortarand pestle. Powder from each sample was stored in 15 mlsterile vials until use.

Tests were performed on sterile physiologic solution,extracts prepared from 50 mg of bony material in 200 �lof physiologic solution. These were subjected to fourfreeze/thaw cycles (10 seconds each cycle). The suspen-sions were incubated at 4 ◦C for 24 h followed by anincubation at 37 ◦C for 30 minutes to solubilize the remain-ing antigens. The extracts were then centrifuged at roomtemperature at 10 000 rpm for 10 minutes and an aliquotof the supernatant used in the test.

3. Results

Our findings provide the first biological evidenceof the presence of PfHRP2 and PfLDH in the skele-

tal remains of Cardinal Giovanni (MED3), don Garzia(MED4), Eleonora of Toledo (MED5) and Francesco Ide’ Medici (MED11). No mixed falciparum infectionsnor non-falciparum infections were identified using theMalariaDetectTM RAPYDTEST® (Figure 1). Bone samples

G. Fornaciari et al. / Transactions of the Royal Society of Tro

Figure 1. Results of the immunological investigation.(A) Immunological response to P. falciparum malaria by theMalariaDetectTM RAPYDTEST®: from left to right: positive identifi-cation of PfLDH in the skeletal remains of Cardinal Giovanni (MED3), donGarzia (MED4), Eleonora of Toledo (MED5) and Francesco I (MED11).(B) Immunological response to P. falciparum malaria by the MalariaAntigen RAPYDTEST®: from left to right, positive line in a modernfalciparum malaria serum and positive identification of PfHRP2 in theskeletal remains of Francesco I (MED 11), negative results from Joan ofAustria (MED8) and from a Briancon control sample.(C) Immunological response to P. falciparum malaria by the MalariaAntigen RAPYDTEST®: from left to right, positive line in a modernfalciparum malaria serum and positive identification of PfHRP2 in theskeletal remains of Cardinal Giovanni (MED3), don Garzia (MED4),Eleonora of Toledo (MED5) and from a Briancon control sample.

pical Medicine and Hygiene 104 (2010) 583–587 585

from Cosimo I, Joan of Austria and eight control sampleswere all negative, as expected.

Simple and rapid immunochromatographic tests (ICTs)had been initially developed and evaluated under fieldconditions in tropical countries and had been applied forrapid post-mortem diagnosis of P. falciparum malaria incorpses in a forensic setting.18 Immunochromatographyhas also been applied to diagnosis of falciparum malaria inancient human remains.4,21,22 The diagnosis of P. malaria inEgyptian mummies has been proposed by different studiescarried on specimens belonging to different periods of theancient Egyptian history.21,22 However, until recently, thereproducibility of some of these results was not achievedby further investigations.4,8

Until now only one report using immunological tools8

and few molecular genetics studies have clearly identifiedP. falciparum in ancient specimens.4–7,9

In a previous report, we provided evidence of malariaantigen preservation in natural Egyptian mummified skele-tal muscles dating back to the Pre-Dynastic and EarlyDynastic periods. Positive results were confirmed indepen-dently by immunofluorescence microscopy.8 Muscle hasbeen, hitherto, considered the best tissue for the detectionof P. falciparum malaria because of its abundant red cellcontent.8 We now provide evidence that malaria antigenscan also be detected in ancient bone samples.

Even if the molecular approach is preferred, we ratheruse an alternative protein-based method, immunochro-matography, which has shown to be a reliable tool indetecting proteins of protozoan and bacterial pathogensin ancient human remains (i.e. P. falciparum and Yersiniapestis). This method is well suited to the task since proteinsare known to be more resistant to environmental degrada-tion than aDNA.8,13,23,24

4. Discussion

Malaria seems not to have reached mainland Italy untilthe second century BC25,26 For a time, the marsh-riddencountryside around Rome, the Pontine marshes and Tus-cany lowlands became a virtual desert, and by the earlyImperial times Sicily and Sardinia had become notoriousfor summer and autumnal fevers.26

However, under the prosperity of the Roman Empire(circa 50 BC to 400 AD), by drainage, husbandry andbuilding development, malaria was excluded for severalcenturies from the Roman countryside itself.25,26 Then, asthe Empire declined and fell into ruin, the Pontine marshesand Tuscany lowlands became surrounded, once more, bythe dangerous and largely uninhabitable marshes knownand feared by later generations both in Dark Age, MedievalPeriod and Renaissance.25,26

The Medici and the Lorena families were responsible forthe first drainages of the main marshy areas in Tuscany.1

4.1. Fall 1562: the sudden deaths of Eleonora, Giovanni

and Garzia

In October 1562 Cosimo I de’ Medici’s official visit wastimed to coincide with the start of the drainage of Gros-seto marshes, to allow the cultivation of the land and the

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building of new settlements. In the meantime, Cosimo Iand his family enjoyed hunting in the lower Arno Valleyand along the marine swamps. Because of the high risk ofcatching tertian fevers, the court physicians strongly dis-couraged the Medici from travelling and hunting in theendemic malarial areas of Tuscany during the autumn, buttheir recommendations were unobserved.2 The journey ofCosimo I and his family across the Maremma of Grosseto isthoroughly documented.5

On 14 October 1562 the family reached the Fucecchiomarshes, and five days later they moved to the Maremmaswamps. It was not until 15 November, that cardinal Gio-vanni was suddenly affected by a fever. In the followingdays, he displayed the typical symptomatology of cerebralmalaria: abnormal behaviour, impairment of conscious-ness and coma; he died on 21 November. Don Garzia fell illon 18 November and developed undulating fevers followedby delirium, until death occurred on 12 December. Eleonorafrom Toledo presented the same symptomatology and diedon 17 December. Our positive biological findings were thencompared to the medical documentary sources and to theautopsy reports left by the Medici’s court physicians whodescribed the different stages of the sudden illnesses of theGrand Dukes until their deaths.

In malarial infections, the prepatent period is the lapseof time from the bite of the Anopheles mosquito to theappearance of the trophozoites in the circulating erythro-cytes. This period of time is constant for each speciesand ranges from 9–10 days in P. falciparum infection. Theprepatent period is then followed by the incubation periodwhich is the lapse of time between the mosquito bite andappearance of the first symptoms of the disease.

In P. falciparum infection, the incubation time rangesfrom 9–14 days. Considering the entire period between thefirst contact of the Medici family with the malarial envi-ronment and the dates of their deaths, it perfectly fits theprepatent and incubation periods of falciparum malaria.The autopsy reports support the initial diagnosis of tertianmalaria as the cause of those premature and sudden deaths.Autopsy was not a routine procedure at the time and wasreserved only for relevant personages, such as the GrandDukes and other members of the Medici family.27

While the clinical history of Cardinal Giovanni and donGarzia indicated that they were both healthy and fit menuntil they visited the swampy Maremma, Eleonora fromToledo was an immunodeficient individual, as she washeavily affected by pulmonary tuberculosis.

Necroscopy carried out on the corpse of Giovannirevealed the absence of alterations of the internal organsapart from the brain and the spleen. Marked changeswere observed in brain which was grossly congested andleaden in colour. The spleen appeared slightly enlarged andtense with a dark red colour.1 The lungs were perfectlypreserved and no signs of pulmonary oedema were iden-tified. Bronchial pneumonia due to ‘catarrhal fevers’ wasexcluded.

While no autopsy reports are available for Garzia,Eleonora’s necroscopic exam revealed that the lungs weredamaged and had been for a long time. Hepatomegaly andsplenomegaly were also described.1 While lungs lesionscan be considered consistent with the pulmonary infec-

opical Medicine and Hygiene 104 (2010) 583–587

tion that afflicted Eleonora for several years, hepato- andsplenomegaly have to refer to a tertian malarial infection.28

A severe illness, tuberculosis, undoubtedly accelerated thetime of death when she was infected by P. falciparum.

Ferdinando I was the only member of the family who gotill and recovered fairly well after two months of persistentfever attacks.

4.2. Fall 1587: the sudden death of Francesco I

We also have detailed medical documentary sources ofthe court physicians who assisted Grand Duke Francesco Iuntil his death. On 6–8 October 1587, Francesco I was hunt-ing near his villa at Poggio a Caiano, a known unhealthyrice-field area, typical for endemic malaria. On 8 October,he began to feel ill with intermittent high fevers which ledthe court physicians to diagnose a malarial tertian fever. Hisdeath took place on 19 October 1587.1,3 Necroscopic examshowed enlarged and hard-to-touch liver, congestion of thelungs along with multiple bruises and extensive oedema.1,3

All these findings are consistent with acute malaria.A symptomatology characterized by high and undulated

fever together with gastrointestinal symptoms (i.e. violentvomiting, dryness of mouth, epigastralgia, splenomegalyand hepatomegaly) is specific of severe falciparum malariaand is common in both children and adults living in an areaof malarial endemicity.29–31 The aetiology of the diseasewhich caused the sudden deaths of four members of theMedici family has been unabated for four centuries. Com-parison between historical accounts, autopsy reports andmodern biological findings has finally lead to confirmationof the infectious nature of their deaths.

5. Conclusions

We provide first evidence of the presence of P. falci-parum ancient proteins in the skeletal remains of four of sixmembers of the Medici family we analyzed and, therefore,we confirm that they were affected by falciparum malariaat the time of their death.

We also provide further evidence that immunodetec-tion can be successfully applied to the palaeodiagnosis ofseveral different infectious diseases in skeletal remains,thus opening new paths of investigation for large skeletalseries.

Authors’ contributions: GF and RB conceived the study.GF coordinated the study, provided biological samples, par-ticipated in the interpretation of the data and contributedto the preparation of the manuscript. RB performedRDT analysis, interpreted the results and prepared themanuscript. VG carried out the historical investigations,participated in the interpretation of the data and con-tributed to the preparation of the manuscript. EF and SGparticipated in the analysis and interpretation of the data,

and contributed to the preparation of the manuscript. Allthe authors read and approved the final manuscript. GF isguarantor of the paper.

Funding: None.

ty of Tro

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onflicts of interest: None declared.

thical approval: Not required.

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