Identification of Mycobacterium tuberculosis DNA in a pre-Columbian Peruvian mummy

Abstract

The existence of tuberculosis in the preColumbian Americas is controversial because the morphology of the lesion is not specific, the organism is culturally nonviable in ancient tissues, and nonpathogenic soil mycobacteria can contaminate buried bodies. We report the recovery of DNA unique to Mycobacterium tuberculosis from a lung lesion of a spontaneously mummified, 1000-year-old adult female body in southern Peru. This provides the most specific evidence possible for the pre-Columbian presence of human tuberculosis in the New World. even those of some other genera such as Nocardia) demonstrate acid-fast staining characteristics. Furthermore, many of the species of Mycobacterium are free-living soil organisms, and one recent observation indicates these can gain access to interred human tissues (9). While support for the pre-Columbian American existence of tuberculosis is growing, its acceptance is by no means universal and commonly Reconstructing evolutionary patterns of infectious diseases such as tuberculosis, malaria, AIDS, and others carries an enormous potential for understanding the nature of our present encounters with these conditions as well as guidance to rational policy decisions for their control. However, our source of knowledge about diseases in ancient populations is limited largely to archaeological findings-principally the bodies themselves. Recognition of infectious diseases in ancient human remains is seriously constrained by the fact that only rarely do such illnesses produce an etiologically specific morphological change in bone or preserved soft tissues. Reliable reconstruction of infectious disease patterns in antiquity, then, is dependent upon recovery and identification of the infectious agent directly from the human remains. Unfortunately, modern methods of isolation by culture techniques are ineffective in archaeological tissues (1, 2). The alternative approach, identification of specific antibodies, has progressed but little beyond the detection of trace quantities of nondiagnostic immunoglobulin G (3) or rare examples of antibody induction. However, it is conceivable that DNA fragments diagnostic of the pathogen persist in a lesion and could be retrievable by the methods of molecular biology. This report documents the recovery of a DNA segment unique to Mycobacterium tuberculosis from the lung of a 1000-year-old spontaneously mummified Peruvian body showing a gross morphology suggestive of primary pulmonary tuberculosis. We selected tuberculosis as the target disease to test our hypothesis because the pre-Columbian presence of this condition in the New World remains controversial (4-6) because of the ambiguous nature of most direct morphological diagnostic criteria available in the archaeological record. The kyphotic, "hunchback," spinal deformity (Pott's disease) secondary to tuberculous vertebral involvement that typically has been used to identify skeletal tuberculosis in the New World (4) dating to the millennium prior to 1492 (5) can be caused, however, by a variety of fungi such as Coccidioides immitis (7) and other organisms. Demonstration of acid-fast bacilli within a paravertebral abscess in an 800-yearold Peruvian child with Pott's disease made the diagnosis of pre-Columbian tuberculosis in the New World much more probable (8). However, all species of Mycobacterium (and Mummified Human Remains. We chose to examine a right hilar lymph node and a lung lesion from a 40- to 45-year-old spontaneously mummified female body. The tomb (Cemetery 1, T-30) from which this body was exhumed was in a burial site (Chiribaya Alta) used by the Chiribaya, an archaeological, largely agricultural population that occupied the lower Osmore Valley near the southern Peruvian coastal community of Ilo about A.D. 1000-1300. A sample of liver tissue yielded a 14C radiocarbon-determined age of 1040 ± 44 years before present (B.P.), corrected for 13C (sample no. GX18799-AMS; Krueger Enterprises, Cambridge, MA). Skeletal lesions typical of tuberculosis-like pathology have been identified in other skeletons from Chiribaya Alta and other late prehistoric sites (5). Removal of the chest wall revealed intact, collapsed lungs bilaterally. The left lung and the middle and lower lobes of the right lung were not abnormal. The right upper lobe was diffusely adherent to the chest wall, and a largely calcified, discrete nodule 1.2 cm in diameter was present in a lateral, subpleural position (L in Fig. 1). At its point of adhesion to the parietal pleura overlaying the lateral part of the fourth rib, a 1-mm aperture was apparent in the calcified shell, communicating with a central 3-mm diameter cavity within the nodule. In the right hilum, two partly calcified masses, each about 1.5 x 0.8 x 0.5 cm, were found in peribronchial positions (N1 and N2 in Fig. 1). No evidence of skeletal change could be identified in the spine, ribs, or elsewhere, and no abnormalities were found in the liver, heart, bowel, skin, trachea, or other soft tissues. After rehydration, fixation, decalcification, and preparation of histologic slides from selected parts of the abnormal areas, the lung lesion revealed only a largely calcified wall whose central, noncalcified area apparently had represented caseous, necrotic tissue but was now occupied only by a fibrous-lined defect. The hilar nodules were acellular, partly anthracotic, peribronchial lymph nodes demonstrating focally calcified, partly fibrotic replacement of node tissue. No acid-fast bacilli were demonstrable in any of the lesions. DNA Extraction. The DNA was extracted from samples of the lesions as described (10, 11). PCR. The extracted DNA was subjected to PCR (12) targeting a segment of DNA unique to Mycobacterium tuberculosis. This target was that described by Eisenach et al. (13) for a 123-bp segment of DNA in M. tuberculosis and is particularly suited for this work because it exists as 10-16 copies per cell. This target was later reported to be part of a larger repetitive insertion sequence (IS)-like element of 1361 remains characterized by ambivalence (6).