Reader Comments

Post a new comment on this article

Comments from group that supplied sequence data

Posted by knharper on 21 Jan 2010 at 00:18 GMT

As members of the research group that produced the sequence data used in the recent article by de Melo et al [1], we read these findings with great interest and are gratified that the authors felt our data worthy of further study. There are a few important points dealing with our data and the methods used in this paper, however, that we felt the need to highlight. As a group that contains both geneticists and paleopathologists, it is probably not surprising that we too, are interested in integrating data from both of these fields. After producing the sequence data published in our 2008 PLoS NTDs paper on the phylogeny of the treponemes [2], though, it was clear to us that an approach similar to the one employed here was not feasible. The following highlights some of the reasons underlying our position.
First, our data cannot be used to estimate divergence times using the molecular clock. As we stated in our article, a number of the regions we sequenced were chosen “either because of their previously demonstrated polymorphism or because they were thought likely to contain variation. This may have weighted the regions sampled towards exceptionally polymorphic areas.” As a result, the number of substitutions per basepair indicated by our data “is likely to significantly overestimate the amount of polymorphism typical of the genome” [2: 6]. When de Melo et al used these data to estimate a substitution rate for T. pallidum, they were estimating a rate for a sampling of the genome purposefully enriched for genetic variation, in which regions subject to recombination were intentionally avoided. This rate cannot be applied to the levels of whole-genome polymorphism found between two T. pallidum subsp. pallidum strains [3], which were not enriched for variation and from which recombining areas were not removed. This latter factor is not insignificant, since a high proportion of intergenomic polymorphism falls within the tpr gene family [3], in which recombination occurs frequently [4]. We regret that we did not have the opportunity to discuss these limitations of our dataset with the authors, but all of these observations were present in our paper.

Our second major concern is that the authors did not independently evaluate the dating and diagnosis of the paleopathological evidence they used to calibrate the molecular clock. The paleopathological datapoints on which their analysis was based relied on “reported diagnoses of treponemal diseases… as published in the peer reviewed articles” [1: 3]. Many of these publications, however, employ different methods and sets of diagnostic criteria to establish the presence of treponemal disease. The authors acknowledge this problem and that consequent interpretations of the skeletal evidence might be flawed, but they do not seem to believe that it cripples their analysis. We disagree. Even in the peer-reviewed literature, many diagnoses of treponemal disease do not meet established standards in paleopathology. In their paper, de Melo et al express the desire for a diagnostic “gold standard.” We would like to draw their attention to the work of Cecil J. Hackett [5], who rigorously evaluated the specificity of various skeletal lesions to treponemal disease. Using skeletons from clinically diagnosed cases of treponemal disease and other disorders, Hackett demonstrated that only caries sicca, a sequence of pits and radial scars on the crania, and pitted bony expansions on the postcrania, are diagnostic of treponemal disease. The great majority of lesions commonly attributed to treponemal disease, such as periostitis and tibial bowing, are non-specific and can be caused by a variety of infectious and non-infectious insults. This finding has been replicated in numerous studies [6,7] and incorporated into the more rigorous diagnostic criteria [8,9]. Nonetheless, non-specific indicators have frequently been used to diagnose cases of Pre-Columbian treponemal disease, resulting in many false positive cases. For example, the lesions on the tibiae shown in this paper’s Figure 1 are insufficient for a diagnosis of treponemal disease; they could have been caused by other conditions, ranging from trauma to cancer [10-13].

The third concern is even more serious. The consensus among paleopathologists is that it is not possible to differentiate between lesions caused by syphilis, yaws, or bejel in isolated skeletal remains [5,7,8,14]. In describing their literature review, the authors note that “in many cases, the authors did not distinguish between the different treponematoses and thus this broader term [treponematoses] was used” [1: 3]. This is because, other than using the average age of affected individuals in a skeletal sample, which requires a large number of skeletons to estimate and is hampered by bone remodeling when infection is contracted early in life [15], and more tentatively, the prevalence of lesions in a sample, there is no established method for differentiating between sexually and non-sexually transmitted forms of treponemal disease in the skeleton. Distinguishing between yaws and bejel, both of which are non-sexually transmitted and contracted most often by children, appears even more hopeless. Although one method for differential diagnosis has been proposed [16], it has been met with substantial criticism [15,17] and has yet to be independently tested. Where does this state of affairs leave the datapoints used for calibration in this study?

The authors used the oldest case of treponemal disease documented in the genus Homo and the oldest probable cases of bejel, congenital syphilis, and venereal syphilis from different parts of the world to calibrate their molecular clock. The first case, an H. erectus specimen, displays only periostitis. This has been attributed to yaws because of the involvement of multiple skeletal elements [18], but also to hypervitaminosis A, a non-infectious condition [19]. Because the lesions are non-specific it is impossible to arrive at a certain diagnosis. The cases of bejel from the Sudan, dated to 15,000 YBP, were diagnosed based solely on having a pattern of periostitis similar to that observed in another sample known to have been afflicted with the disease [20]. In both cases, a diagnosis of treponemal disease is questionable, and the assignment to a particular treponematosis unfounded. Likewise, the reported case of congenital syphilis from France dated to 1,600 YBP [21] lacks specific indicators for the condition, and diagnoses of trauma [22] or lithopedion [23] have also been suggested. Lastly, de Melo et al describe evidence from the Indian Knoll site, KY, dated to around 5300 YBP [24], as the “oldest and ‘safest’ osseous evidences of venereal syphilis” [1: 9]. Crania from Indian Knoll do display radial scarring, but since there is no reliable method for distinguishing between the lesions of syphilis, yaws, or bejel, a diagnosis of syphilis cannot be confirmed. Snow, the investigator who gave a diagnosis of syphilis [25], did so in 1948, before the complexities involved in differentiating between the treponematoses were fully understood. More recently, the prevalence of treponemal lesions in this particular skeletal sample and the absence of signs of congenital syphilis have led investigators to believe that a diagnosis of non-venereal treponemal disease is more likely [26,27].

The authors state that the drawbacks limiting their analysis include a scarcity of accessible reports of Pre-Columbian treponemal disease and, in those reports, the absence of standardized diagnostic criteria for treponemal disease. Indeed, cases of treponemal disease that can be definitively diagnosed are relatively infrequent in the published literature. This makes suitable datapoints exceedingly rare and in consequence, accurate calibration based on diagnosed cases all but impossible. Further, cases of treponemal disease that can be definitively diagnosed and assigned to a specific treponemal disease are nonexistent. This precludes this method of calibration. Incorporating cases of treponemal disease whose diagnostic certainty has not been independently evaluated introduces false positive datapoints into the analysis. As the authors note, “since the diagnostic criteria differ between the reports on treponematoses in ancient bones, one cannot exclude that reassessing all possible cases with standardized methods would lead to different results ” [1: 9]. We could not agree more and question the utility of an analysis based on evidence that does not meet standardized criteria.

We applaud the spirit of the approach used in this paper and hope that in future such an integration will yield valuable results. However, with the data available at present, we conclude that it is not currently possible to use the molecular clock, calibrated with paleopathological data, to estimate the time at which the various treponemes emerged. Despite this, we agree with many of the recommendations for future research made by the authors. We believe that characterizing the polymorphism present in the entire genome of many T. pallidum strains, including simian strains, may produce new datasets suitable for molecular clock analyses. We also agree that careful assessments of paleopathological evidence from around the world, as performed for the Americas in a recent volume by Powell and Cook [28] will help to clarify where and when treponemal disease existed in the past. We are less optimistic, however, about the possibility of differentiating between the different treponematoses using skeletal evidence, though the ability to do so would allow great strides to be made in discerning the origins and antiquity of the treponemes. We would certainly welcome the presentation of such a system, backed by good evidence. Though we do not believe that the conclusions drawn in this paper were warranted by the data, we do believe that cross-fertilization between paleopathology and phylogenetics is likely to bear fruit, and we look forward to the insight into the history of this fascinating family of diseases that the future will bring.

Kristin Harper
Robert Wood Johnson Health & Society Scholars Program
Columbia University

Molly Zuckerman
Department of Anthropology
Emory University

Works Cited
1. de Melo F, de Mello J, Fraga A, Nunes K, Eggers S (2010) Syphilis at the Crossroad of Phylogenetics and Paleopathology. PLoS Neglected Tropical Diseases 4: e575.
2. Harper K, Ocampo P, Steiner B, George R, Silverman M, et al. (2008) On the Origin of the Treponematoses: A Phylogenetic Approach. PLoS Neglected Tropical Diseases 1: e148.
3. Matejková P, Strouhal M, Smajs D, Norris S, Palzkill T, et al. (2008) Complete genome sequence of Treponema pallidum ssp pallidum strain SS14 determined with oligonucleotide arrays. BMC Microbiology 8: 76.
4. Gray R, Mulligan C, Molini B, Sun E, Giacani L, et al. (2006) Molecular Evolution of the tprC, D, I, K, G, and J Genes in the Pathogenic Genus Treponema. Molecular Biology and Evolution 23: 2220-2233.
5. Hackett C (1976) Diagnostic Criteria of Syphilis, Yaws and Treponarid (Treponematoses) and of Some Other Diseases in Dry Bones (for Use in Osteo-Archaeology). Berlin: Springer-Verlag.
6. Weston D (2008) Investigating the Specificity of Periosteal Reactions in Pathology Museum Specimens. American Journal of Physical Anthropology 137: 48-59.
7. Webb S (1995) Paleopathology of Australian Aboriginals: Health and Disease across a Hunter-Gatherer Continent. Cambridge: Cambridge University Press.
8. Ortner D (2003) Identification of Pathological Conditions in Human Skeletal Remains. Amsterdam: Academic Press.
9. Waldron T (2009) Paleopathology. Cambridge: Cambridge University Press.
10. Adler C-P (2000) Bone Diseases. Berlin: Springer Verlag.
11. Ragsdale B (1993) Morphologic analysis of skeletal lesions: correlation of imaging studies and pathologic findings. Advances in Pathologic and Laboratory Medicine 6: 445-490.
12. Ragsdale B, Madewell J, Sweet D (1981) Radiologic and pathologic analysis of solitary bone lesions, part II: periosteal reactions. Radiological Clinics of North America 19: 749-783.
13. Richardson M (2001) Approaches to differential diagnosis in musculoskeletal imaging. Seattle: University of Washington.
14. Meyer C, Jung C, Kohl T, Poenicke A, Poppe A, et al. (2002) Syphilis 2001 - A palaeopathological reappraisal. Homo 53: 39-58.
15. Cook D, Powell M (2005) Piecing the Puzzle Together: North American Treponematosis in Overview. In: Powell M, Cook D, editors. The Myth of Syphilis: the Natural History of Treponematosis in North America. Gainesville, FL: University Press of Florida/ Florida Museum of Natural History. pp. 442-477.
16. Rothschild B, Rothschild C (1995) Treponemal disease revisited: skeletal discriminators for yaws, bejel, and venereal syphilis. Clinical Infectious Diseases 20: 1402-1408.
17. Heathcote G, Stodder A, Buckley H, Hanson D, Douglas M, et al. (1998) On Treponemal Disease in the Western Pacific: Corrections and Critique. Current Anthropology 39: 359-368.
18. Rothschild B, Hershkovitz I, Rothschild C (1995) Origin of Yaws in the Pleistocene. Nature 378: 343-344.
19. Walker A, Zimmerman M, Leakey R (1982) A possible case of hypervitaminosis A in Homo erectus. Nature 296: 248-250.
20. Rothschild B, Rothschild C (1996) Analysis of Treponemal Disease in North Africa: The Case for Bejel in the Sudan, but Absence in West North Africa. Journal of Human Evolution 11: 11-15.
21. Pálfi G, Borreani M, Brun J-P, Berato J, Dutour O (1992) Pre-Columbian congenital syphilis from the late antiquity in France. International Journal of Osteoarchaeology 2: 245-261.
22. Rothschild B (2005) History of Syphilis. Clinical Infectious Diseases 40: 1454-1463.
23. Rothschild B, Rothschild C, Naples V, Billard M, Panero B (2006) Bejel: Acquirable only in childhood? Acta Tropica 99: 160-164.
24. El-Najjar M (1979) Human treponematosis and tuberculosis: Evidence from the New World. American Journal of Physical Anthropology 51: 599-618.
25. Snow C (1948) Indian Knoll skeletons of site Oh 2, Ohio County, Kentucky. University of Kentucky Reports in Anthropology 4: 371-554.
26. Brothwell D (1970) The Real History of Syphilis. Science Journal 6: 27-32.
27. Kelley M (1980) Disease and Environment: A Comparative Analysis of Three Early american Indian Skeletal Collections. Cleveland: Case Western Reserve University.
28. Powell M, Cook D, editors (2005) The Myth of Syphilis: the Natural History of Treponematosis in North America. Gainesville, FL: University Press of Florida/ Florida Museum of Natural History.

No competing interests declared.