Molecular identification of falciparum malaria and human tuberculosis co-infections in mummies from the Fayum depression (Lower Egypt)

Abstract

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 3(rd) 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.

Figure 1. Identification of plasmodial DNA in Fayum mummies.

(a) Amplification of partial apical membrane antigen 1 gene (AMA1). Lane M, 100- bp DNA ladder (NEB); lane 1, 1543; lane 6, 1554; lane 9, 1564; lane 11, 1622; lane 14, 1643; lane 15, PCR negative control; (b) Amplification of MSP1 K1 allele. Lane M, 100- bp DNA ladder (NEB); lane 1, 1543; lane 2, 1554; lane 3, 1554; lane 4, 1622; lane 5, 1622; lane 6, 1564; lane 7, 1611; lane 8, 1643; lane 9, PCR negative control; (c) Nucleotide alignment of Plasmodium MSP1 K1 alleles amplified from mummy 1564 in comparison with Plasmodium falciparum isolate (accession no. AF061126). The variable central region of K1 allele types differ by the number and arrangement of repeat motifs. A total of 8 K1 type alleles were identified. Dashes represent gaps introduced to maximize the alignment.

Figure 2. Identification of Mycobacterium tuberculosis complex DNA in Fayum mummies.

a. M. tuberculosis complex specific IS6110 fragment (92-bp) amplified using nested primers (IS-3 and IS-4). Lane M, 100-bp DNA ladder (NEB); lane 1, 1554; lane 2, 1564; lane 3, 1622; lane 4, 1643; lane 5, PCR no template control; lane 6, extraction blank control; b. M. tuberculosis complex IS6110 (92-bp) sequence electropherogram and sequence similarity search by BLAST; c. Best hits include Mycobacterium tuberculosis GenBank ac. no. CP001642.1 showing 100% identity with an E-value of 4e-26.