Molecular Identification of Falciparum Malaria and Human Tuberculosis Co-Infections in Mummies from the Fayum Depression (Lower Egypt)

et al. (2013) Molecular Identification of Falciparum Malaria and Human Tuberculosis Co-Infections in Mummies from the Fayum Depression (Lower Egypt). PLoS ONE 8(4): e60307. doi:10.1371/journal.pone.0060307 Molecular Identification of Falciparum Malaria and Human Tuberculosis Co-Infections in Mummies from the Fayum Depression (Lower Egypt) Albert Lalremruata 0 Markus Ball 0 Raffaella Bianucci 0 Beatrix Welte 0 Andreas G. Nerlich 0 Ju rgen F. J. Kun 0 Carsten M. Pusch 0 Igor Mokrousov, St. Petersburg Pasteur Institute, Russian Federation 0 1 Institute of Tropical Medicine, University of Tu bingen , Tu bingen, Germany , 2 Institute of Human Genetics, Department of Medical Genetics, University of Tu bingen , Tu bingen, Germany , 3 Laboratory of Physical Anthropology, Department of Public Health and Paediatric Sciences, University of Turin , Turin , Italy , 4 Division of Paleopathology, History of Medicine and Bioethics, Department of Oncology, Transplants and Advanced Technologies in Medicine, University of Pisa , Pisa , Italy , 5 Institute of Pre- and Protohistory and Medieval Archaeology, Department of Early Prehistory and Quaternary Ecology, University of Tu bingen , Tu bingen, Germany , 6 Division of Paleopathology, Institute of Pathology, Academic Clinic Mu nchen- Bogenhausen , Mu nchen , Germany Due to the presence of the lake Quarun and to the particular nature of its irrigation system, it has been speculated that the Fayum, a large depression 80 kilometers south- west of modern Cairo, was exposed to the hazards of malaria in historic times. Similarly, it has been speculated that, in the same area, also human tuberculosis might have been far more widespread in the antiquity than in its recent past. If these hypotheses were confirmed, it would imply that frequent cases of co-infection between the two pathogens might have occurred in ancient populations. To substantiate those speculations, molecular analyses were carried out on sixteen mummified heads recovered from the necropolis of Abusir el Meleq (Fayum) dating from the 3rd Intermediate Period (1064- 656 BC) to the Roman Period (30 BC- 300 AD). Soft tissue biopsies were used for DNA extractions and PCR amplifications using well-suited protocols. A partial 196-bp fragment of Plasmodium falciparum apical membrane antigen 1 gene and a 123-bp fragment of the Mycobacterium tuberculosis complex insertion sequence IS6110 were amplified and sequenced in six and five of the sixteen specimens, respectively. A 100% concordance rates between our sequences and those of P. falciparum and M. tuberculosis complex ones were obtained. Lastly, concomitant PCR amplification of P. falciparum and M. tuberculosis complex DNA specific fragments was obtained in four mummies, three of which are 14 C dated to the Late and Graeco-Roman Periods. Our data confirm that the hydrography of Fayum was extremely conducive to the spread of malaria. They also support the notion that the agricultural boom and dense crowding occurred in this region, especially under the Ptolemies, highly increased the probability for the manifestation and spread of tuberculosis. Here we extend back-wards to ca. 800 BC new evidence for malaria tropica and human tuberculosis co-occurrence in ancient Lower Egypt. - Funding: The study was supported by Landesgraduiertenfo derung, University of Tu bingen, and The German Academic Exchange Service (DAAD). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Tuberculosis (TB) and malaria, two of the most ancient and deadly diseases of mankind, have ravaged human communities since the beginning of civilization and remain a major global health problem in the 21st century [1,2]. TB causes ill-health among millions of people each year and ranks as the second leading cause of death of adults from an infectious disease worldwide, after the human immuno- deficiency virus (HIV). The latest World Health Organization report indicate that there were almost 9 million new cases in 2011 and 1.4 million TB deaths [1]. Malaria is the 5th cause of death from infectious diseases worldwide after respiratory infections, HIV/AIDS, diarrheal diseases and tuberculosis. In 2011, there were 1.2 million malaria deaths globally and malaria is recognized as the 2nd leading cause of death from infectious diseases in Africa, after HIV/AIDS [23]. In many parts of sub-Saharian Africa, the geographic overlap between TB and malaria is extensive and co-infection with TB and malaria is likely to be common [45]. Several investigations aimed at tracing the origins and frequencies of malaria and TB back have been carried out on Egyptian mummified remains over the past 25 years [615]. Tuberculosis DNA has long been recognized in mummies from Upper Egypt dating to different historical periods [713]. Mycobacterium tuberculosis (MTB) complex DNA was successfully amplified and sequenced in populations from Thebes-West (Upper Egypt) dating to the New Kingdom (c.a. 1550-1000 BC) [7,912]. Where additional characterization was possible, it was shown that human lineages of MTB complex were present: M. tuberculosis and possibly M. africanum but not M. bovis [12]. Molecular signatures of TB infection were found in individuals with and without macromorphological typical signs of tubercular spondylitis and belonging to different age classes [9]. It was speculated that, in the affected populations, there was a relatively low life expectancy and that this may have resulted from a considerable proportion of chronic infections by various pathogenic organisms such as tuberculosis and other parasitoses (i.e. malaria, leishmaniasis, schistosomiasis and other worm infections) [9,12]. Similarly, but to a lesser extent, also Plasmodium falciparum ancient DNA was identified in mummified skeletons from ThebesWest dating from the New Kingdom to the Late Period (1500-500 BC) [14] and in 18th Dynasty royal mummies [15]. However, no cases of malaria and tuberculosis co-infections were reported. Furthermore, most of the studies carried out till the present day have focused on the impact of infectious diseases on ancient populations from Upper Egypt whereas less attention has been paid to populations from Lower Egypt. Stimulated by the notion that malaria and TB were rampant in 19th and early 20th century Fayum [16], the present studys aim was to detect and characterize P. falciparum and MTB complex DNAs in sixteen mummified heads from Lower Egypt and, eventually, to verify, the existence of co-infections. Here we not only extend back-wards to ca. 800 BC evidence for single malarial and mycobacterial infections but provide new evidence of falciparum malaria/tuberculosis co-occurrence in individuals from ancient Fayum (Table 1). Partial 196-bp AMA1 gene was amplified in six (...truncated)