Gino Fornaciari (a), Valentina Giuffra (a), Ezio Ferroglio (b), Sarah Gino (c), Raffaella Bianucci (d)

  1. Division of Paleopathology, History of Medicine and Bioethics, Department of Oncology, Transplants and Advanced Technologies in Medicine, University of Pisa, Via Roma 57, 56126 Pisa, Italy
  2. Laboratory of Parasitology and Parasitic Diseases, Department of Animal Production, Epidemiology and Ecology, University of Turin, Via Leonardo da Vinci 44, 10095 Grugliasco, Turin, Italy
  3. Laboratory of Crimininalistic Sciences, Department of Anatomy, Pharmacology and Legal Medicine, University of Turin, Galileo Galilei 22, 10126 Turin, Italy
  4. Laboratory of Crimininalistic Sciences, Department of Anatomy, Pharmacology and Legal Medicine, University of Turin, Galileo Galilei 22, 10126 Turin, Italy & UMR 6578 CNRS-EFS (Biocultural Anthropology), University of Marseille, Boulevard Pierre Dramard, 13916 Marseille Cedex 20, France

From: Transactions of the Royal Society of Tropical Medicine and Hygiene
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1. Introduction

Medical accounts and autopsy reports imply that tertian malarial fevers caused the death of four members of the Medici family: Eleonora of Toledo (1522–1562), Cardinal Giovanni (1543–1562), don Garzia (1547–1562) and Francesco I (1531–1587).1
Malaria was endemic in central Italy along the coasts of Tuscany and the Maremma lowlands from the third century BC until the end of World War II and had severe effects in terms of both morbidity and mortality on human populations living in this region.2
All members of the Medici family hunted in the endemic malarial areas of Tuscany such as the marshy areas surrounding their villas and along the swampy Maremma1 and were highly exposed to the risk of being infected by protozoan parasites.
In October 1562, Cosimo I de’ Medici (1519–1574), first Grand Duke of Tuscany, his wife Eleonora of Toledo and their sons, Cardinal Giovanni, don Garzia and don Ferdinando (1549–1609) visited the malarial Maremma country near Grosseto (southern Tuscany). During their stay, Cosimo and his family enjoyed hunting in the lower Arno Valley and along the marine swamp.
On their way back to Florence, Cardinal Giovanni, don Garzia and Eleonora suffered from sudden irregular bouts of fever and died in a time span of three weeks. Historical and medical accounts state that severe malaria was the cause of their deaths.1 Fifteen years later, in October 1587, Francesco I de’ Medici, son of Cosimo I and Eleonora of Toledo, second Grand Duke of Tuscany, and his second wife, Bianca Cappello (1548–1587), died suddenly within 24 hours of each other. Again, medical accounts and autopsy reports imply that severe malaria was the cause of their deaths.1,3
Recent studies on ancient biomolecules suggested the possibility to identify Plasmodium falciparum antigens in ancient skeletal remains using modern laboratory techniques.4–9
To determine if the original death certificates issued by the court physicians were correct, immunological investigations were carried out to determine whether P. falciparum malaria might have been the cause of death of those personages, and then compared the biological results to the historical sources.

2. Materials and Methods

2.1. Sampling
The skeletal remains of Eleonora, Giovanni, Garzia and Francesco I were unearthed from the topsoil of the Medici Chapels in San Lorenzo Church in 2004.10,11 Bianca Cappello’s remains have not been recovered: her burial site remains unknown.12
Cancellous bone was harvested from the vertebrae of Eleonora of Toledo (sample code number: MED5), Cardinal Giovanni (sample code number: MED3), don Garzia (sample code number: MED4) and Francesco I (sample code number: MED11).
Bone samples of Cosimo I de’ Medici (sample code number: MED6), who died of pneumonia, and of his daughter-in-law, Joan of Austria, who died in childbirth (sample code number: MED8) were used as negative controls.1
Additionally, six medieval bone samples from two sites known to be free from malaria (Brianc¸ on, France, seventeenth century and Augsburg, Germany, fourteenth century) were used as negative controls.13

2.2. Immunochromatographic assays
Bone samples were tested for malaria immunodetection by using two different dipstick assays which target specificimmunoreactive plasmodial molecules. Extracts prepared from spongy bone samples were examined for the presence of P. falciparum histidine rich-protein-2 (PfHRP2) and Plasmodium lactate dehydrogenase (PfLDH) using two different qualitative double-antibody immunoassays: Malaria Antigen RAPYDTEST® and MalariaDetectTM RAPYDTEST® (DiaSys, Connecticut, U.S.A).14 Malaria Antigen RAPYDTEST® targets P. falciparum specific histidine rich-protein-2, a water-soluble protein excreted by asexual and young gametocytes of P. falciparum and is not present in the other three human pathogenic Plasmodium spp.15,16,20
Several studies on living patients reported that themonoclonal IgG antibody used in certain tests (i.e. ParaSight F-test) cross-reacted with rheumatoid factor (RF) in blood thus resulting in false positive tests for malaria. Replacement of IgG with IgM in recent products has drastically reduced this problem.16,17
MalariaDetectTM RAPYDTEST® targets Plasmodium lactate dehydrogenase (pLDH), which is a soluble enzyme found in the glycolitic pathways of the malaria parasite and is 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 using two monoclonal antibodies (a P. falciparum anti-pLDH and a pan specific anti-pLDH). The monoclonal antibodies employed in rapid examinations detecting pLDH have been exhaustively tested for cross-reactivity from other blood protozoa such as Leishmania, Babesia and pathogenic bacteria and fungi. No evidence of such cross-reactivity has been found.18,19

2.3. Protocol
Bone samples were cleaned with dry brushes and decontaminated by UV light. The external bone surface was removed with a drill (Kavo Intramatic Lux 2, KaVo Dental GmbH, Biberach/Riss, Germany) while mounted on a micromotor turning at 9000 rpm. Next, the cancellous bone was powdered by hand in sterile conditions using a mortar and pestle. Powder from each sample was stored in 15 ml sterile vials until use.
Tests were performed on sterile physiologic solution, extracts prepared from 50mg of bony material in 200_l of physiologic solution. These were subjected to four freeze/thaw cycles (10 seconds each cycle). The suspensions were incubated at 4 ◦C for 24 h followed by an incubation at 37 ◦C for 30 minutes to solubilize the remaining antigens. The extracts were then centrifuged at room temperature at 10 000rpm for 10 minutes and an aliquot of the supernatant used in the test.

Figure 1. Results of the immunological investigation.
(A) Immunological response to P. falciparum malaria by the MalariaDetectTM RAPYDTEST®: from left to right: positive identification of PfLDH in the skeletal remains of Cardinal Giovanni (MED3), don Garzia (MED4), Eleonora of Toledo (MED5) and Francesco I (MED11).
(B) Immunological response to P. falciparum malaria by the Malaria Antigen RAPYDTEST®: from left to right, positive line in a modern falciparum malaria serum and positive identification of PfHRP2 in the skeletal remains of Francesco I (MED 11), negative results from Joan of Austria (MED8) and from a Brianc¸ on control sample.
(C) Immunological response to P. falciparum malaria by the Malaria Antigen RAPYDTEST®: from left to right, positive line in a modern falciparum malaria serum and positive identification of PfHRP2 in the skeletal remains of Cardinal Giovanni (MED3), don Garzia (MED4), Eleonora of Toledo (MED5) and from a Brianc¸ on control sample.

3. Results

Our findings provide the first biological evidence of the presence of PfHRP2 and PfLDH in the skeletal remains of Cardinal Giovanni (MED3), don Garzia (MED4), Eleonora of Toledo (MED5) and Francesco I de’ Medici (MED11). No mixed falciparum infections nor non-falciparum infections were identified using the MalariaDetectTM RAPYDTEST® (Figure 1). Bone samples
from Cosimo I, Joan of Austria and eight control samples were all negative, as expected.
Simple and rapid immunochromatographic tests (ICTs) had been initially developed and evaluated under field conditions in tropical countries and had been applied for rapid post-mortem diagnosis of P. falciparum malaria in corpses in a forensic setting.18 Immunochromatography has also been applied to diagnosis of falciparum malaria in ancient human remains.4,21,22 The diagnosis of P. malaria in Egyptian mummies has been proposed by different studies carried on specimens belonging to different periods of the ancient Egyptian history.21,22 However, until recently, the reproducibility of some of these results was not achieved by further investigations.4,8
Until now only one report using immunological tools8 and few molecular genetics studies have clearly identified P. falciparum in ancient specimens.4–7,9
In a previous report, we provided evidence of malaria antigen preservation in natural Egyptianmummifiedskeletal muscles dating back to the Pre-Dynastic and Early Dynastic periods. Positive results were confirmed independently by immunofluorescence microscopy.8 Muscle has been, hitherto, considered the best tissue for the detection of P. falciparum malaria because of its abundant red cell content.8 We now provide evidence that malaria antigens can also be detected in ancient bone samples. Even if the molecular approach is preferred, we rather use an alternative protein-based method, immunochromatography, which has shown to be a reliable tool in detecting proteins of protozoan and bacterial pathogens in ancient human remains (i.e. P. falciparum and Yersinia pestis). This method is well suited to the task since proteins are known to bemore resistant to environmental degradation than aDNA.8,13,23,24

4. Discussion

Malaria seems not to have reached mainland Italy until the second century BC25,26 For a time, the marsh-ridden countryside around Rome, the Pontine marshes and Tuscany lowlands became a virtual desert, and by the early Imperial times Sicily and Sardinia had become notorious for summer and autumnal fevers.26
However, under the prosperity of the Roman Empire (circa 50 BC to 400 AD), by drainage, husbandry and building development, malaria was excluded for several centuries from the Roman countryside itself.25,26 Then, as the Empire declined and fell into ruin, the Pontine marshes and Tuscany lowlands became surrounded, once more, by the dangerous and largely uninhabitable marshes known and feared by later generations both in Dark Age, Medieval Period and Renaissance.25,26
The Medici and the Lorena families were responsible for the 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 was timed to coincide with the start of the drainage of Grosseto marshes, to allow the cultivation of the land and the building of new settlements. In the meantime, Cosimo I and his family enjoyed hunting in the lower Arno Valley and along the marine swamps. Because of the high risk of catching tertian fevers, the court physicians strongly discouraged the Medici from travelling and hunting in the endemic malarial areas of Tuscany during the autumn, but their recommendations were unobserved.2 The journey of Cosimo I and his family across the Maremma of Grosseto is thoroughly documented.5
On 14 October 1562 the family reached the Fucecchio marshes, and five days later they moved to the Maremma swamps. It was not until 15 November, that cardinal Giovanni was suddenly affected by a fever. In the following days, he displayed the typical symptomatology of cerebral malaria: abnormal behaviour, impairment of consciousness and coma; he died on 21 November. Don Garzia fell ill on 18 November and developed undulating fevers followed by delirium, until death occurred on 12 December. Eleonora from Toledo presented the same symptomatology and died on 17 December. Our positive biological findings were then compared to the medical documentary sources and to the autopsy reports left by the Medici’s court physicians who described the different stages of the sudden illnesses of the Grand Dukes until their deaths.
In malarial infections, the prepatent period is the lapse of time from the bite of the Anopheles mosquito to the appearance of the trophozoites in the circulating erythrocytes. This period of time is constant for each species and ranges from 9–10 days in P. falciparum infection. The prepatent period is then followed by the incubation period which is the lapse of time between the mosquito bite and appearance of the first symptoms of the disease. In P. falciparum infection, the incubation time ranges from 9–14 days. Considering the entire period between the first contact of the Medici family with the malarial environment and the dates of their deaths, it perfectly fits the prepatent and incubation periods of falciparum malaria. The autopsy reports support the initial diagnosis of tertian malaria as the cause of those premature and sudden deaths. Autopsy was not a routine procedure at the time and was reserved only for relevant personages, such as the Grand Dukes and other members of the Medici family.27
While the clinical history of Cardinal Giovanni and don Garzia indicated that they were both healthy and fit men until they visited the swampy Maremma, Eleonora from Toledo was an immunodeficient individual, as she was heavily affected by pulmonary tuberculosis.
Necroscopy carried out on the corpse of Giovanni revealed the absence of alterations of the internal organs apart from the brain and the spleen. Marked changes were observed in brain which was grossly congested and leaden in colour. The spleen appeared slightly enlarged and tense with a dark red colour.1 The lungs were perfectly preserved and no signs of pulmonary oedema were identified. Bronchial pneumonia due to ‘catarrhal fevers’ was excluded.
While no autopsy reports are available for Garzia, Eleonora’s necroscopic exam revealed that the lungs were damaged and had been for a long time. Hepatomegaly and splenomegaly were also described.1 While lungs lesions can be considered consistent with the pulmonary infection that afflicted Eleonora for several years, hepato- and splenomegaly have to refer to a tertian malarial infection.28 A severe illness, tuberculosis, undoubtedly accelerated the time of death when she was infected by P. falciparum. Ferdinando I was the onlymemberof the familywhogot ill and recovered fairly well after two months of persistent fever attacks.

4.2. Fall 1587: the sudden death of Francesco I
We also have detailed medical documentary sources of the court physicians who assisted Grand Duke Francesco I until his death. On 6–8 October 1587, Francesco I was hunting near his villa at Poggio a Caiano, a known unhealthy rice-field area, typical for endemic malaria. On 8 October, he began to feel ill with intermittent high fevers which led the court physicians to diagnose amalarial tertian fever. His death took place on 19 October 1587.1,3 Necroscopic exam showed enlarged and hard-to-touch liver, congestion of the lungs along with multiple bruises and extensive oedema.1,3 All these findings are consistent with acute malaria. Asymptomatology characterized by high and undulated fever together with gastrointestinal symptoms (i.e. violent vomiting, dryness of mouth, epigastralgia, splenomegaly and hepatomegaly) is specific of severe falciparum malaria and is common in both children and adults living in an area of malarial endemicity.29–31 The aetiology of the disease which caused the sudden deaths of four members of the Medici family has been unabated for four centuries. Comparison between historical accounts, autopsy reports and modern biological findings has finally lead to confirmation of the infectious nature of their deaths.

5. Conclusions

We provide first evidence of the presence of P. falciparum ancient proteins in the skeletal remains of four of six members of the Medici family we analyzed and, therefore, we confirm that they were affected by falciparum malaria at the time of their death.
We also provide further evidence that immunodetection can be successfully applied to the palaeodiagnosis of several different infectious diseases in skeletal remains, thus opening new paths of investigation for large skeletal series.

Authors’ contributions:

GF and RB conceived the study.
GF coordinated the study, provided biological samples, participated in the interpretation of the data and contributed to the preparation of the manuscript. RB performed RDT analysis, interpreted the results and prepared the manuscript. VG carried out the historical investigations, participated in the interpretation of the data and contributed to the preparation of the manuscript. EF and SG participated in the analysis and interpretation of the data, and contributed to the preparation of the manuscript. All the authors read and approved the final manuscript. GF is guarantor of the paper.

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