Tuberculosis in the New World: a study of ribs from the Schild Mississippian population, West-Central Illinois

Vertebral lesions have been the main evidence for infection by the Mycobacterium tuberculosis complex (MTC) in paleopathology. Skeletal involvement is expected in a small percentage of infected individuals. Recently, several authors report a correlation between rib lesions and tuberculosis (TB) complex infection. This study tests the hypothesis that rib lesions can serve as a useful marker for MTC infection within the Mississippian Schild skeletal collection from West-Central Illinois. Ribs from 221 adults and juveniles were examined, and affected individuals were tested for TB complex infection. DNA from rib samples of affected individuals was amplified with primers targeting the IS6110 insertion element, which is common to all members of the TB complex. Although it cannot allow discrimination between different species of TB, IS6110 is present in many copies within their genomes, and its presence is thus an indication of MTC infection. The results support the use of rib lesions as a marker for TB infection. Additionally, we demonstrate that MTC DNA can be recovered from ribs that lack lesions in individuals who have lesions of other bones. We recommend that an examination of ribs be incorporated into investigations for TB.     

On the trail of pulmonary tuberculosis based on rib lesions: results from the Human Identified Skeletal Collection from the Museu Bocage (Lisbon, Portugal)

In the last 20 years, studies on human identified skeletal collections have revealed a significant relationship between new bone formation on the visceral surface of ribs and pulmonary tuberculosis (TB). To improve methods of differential diagnosis of respiratory diseases in archaeological skeletons, an investigation was conducted on 197 individuals from the Human Identified Skeletal Collection of the Museu Bocage (Lisbon, Portugal). This sample included 109 males and 88 females who lived during the 19th-20th centuries, with ages at death ranging from 13-88 years. The skeletons were grouped according to cause of death: 1) pulmonary TB (N = 84); 2) pulmonary non-TB diseases (N = 49); and 3) a control group (N = 64) composed of individuals randomly selected among the extrapulmonary non-TB causes of death. The ribs, sterna, scapulae, and clavicles were macroscopically observed. New bone formation on the visceral surface of ribs was recorded in 90.5% (76/84) of individuals who died from pulmonary TB, in 36.7% (18/49) with a pulmonary non-TB disease as cause of death, and in 25.0% (16/64) of the control group. These differences were statistically significant (P < 0.001). Furthermore, in individuals with pulmonary TB, the bony lesions presented mainly as lamellar bone on the vertebral end of the upper and middle thoracic rib cage. Proliferative alterations also occurred on one sternum and in nine clavicles and eight scapulae. This work strongly supports the results of similar studies performed on other documented collections, suggesting that new bone formation on ribs, although not pathognomonic, is a useful criterion for the differential diagnosis of pulmonary TB.     

Broken ribs: Paleopathological analysis of costal fractures in the human identified skeletal collection from the Museu Bocage,Lisbon, Portugal (late 19th to middle 20th centuries)

Although rarely reported in the anthropological literature, rib fractures are commonly found during the analysis of human skeletal remains of past and modern populations. This lack of published data precludes comparison between studies and restricts an accurate understanding either of the mechanisms involved in thoracic injuries or their impact on past societies. The present study aimed: 1) to report rib fracture prevalence in 197 individuals, 109 males, and 88 females, with ages at death ranging from 13 to 88 years old, from the Human Identified Skeletal Collection, Museu Bocage, Portugal (late 19th‐middle 20th centuries); 2) to test the hypothesis that a higher prevalence of rib stress fractures existed in the 133 individuals who died from respiratory diseases, in a period before antibiotics. The macroscopic analysis revealed 23.9% (n = 47) of individuals with broken ribs. 2.6% (n = 124) out of 4,726 ribs observed were affected. Males presented more rib fractures, and a significantly higher prevalence was noted for older individuals. Fractures were more frequently unilateral (n = 34), left sided (n = 19) and mainly located on the shaft of ribs from the middle thoracic wall. Nineteen individuals presented adjacent fractured ribs. Individuals who died from pulmonary diseases were not preferentially affected. However, a higher mean rate of fractures was found in those who died from pneumonia, a scenario still common nowadays. Since rib involvement in chest wall injury and its related outcomes are important issues both for paleopathology and forensic anthropology, further investigations are warranted. Am J Phys Anthropol 2009. © 2009 Wiley‐Liss, Inc.     

Skeletal evidence of tuberculosis in a modern identified human skeletal collection (Certosa cemetery,Bologna, Italy)

The diagnosis of tuberculosis (TB) in osteoarcheological series relies on the identification of osseous lesions caused by the disease. The study of identified skeletal collections provides the opportunity to investigate the distribution of skeletal lesions in relation to this disease. The aim of this study was to examine the skeletal evidence for TB in late adolescent and adult individuals from the identified human collection of the Certosa cemetery of Bologna (Italy, 19th–20th c.). The sample group consists of 244 individuals (138 males, 106 females) ranging from 17 to 88 years of age. The sample was divided into three groups on the basis of the recorded cause of death: TB (N = 64), pulmonary non‐TB (N = 29), and other diseases (N = 151). Skeletal lesions reported to be related to TB were analyzed. The vertebral lesions were classified into three types: enlarged foramina (EnF, vascular foramina with diameter of 3–5 mm), erosions (ER), and other foramina (OtF, cavities of various shapes > 3 mm). A CT scan analysis was also performed on vertebral bodies. Some lesions were seldom present in our sample (e.g., tuberculous arthritis). OtF (23.7%) and subperiosteal new bone formation on ribs (54.2%) are significantly more frequent in the TB group with respect to the other groups. The CT scan analysis showed that the vertebrae of individuals who have died of TB may have internal cavities in the absence of external lesions. These traits represent useful elements in the paleopathological diagnosis of TB. Am J Phys Anthropol 157:389–401, 2015. © 2015 Wiley Periodicals, Inc.     

Analysis of the effect of immunization in rabies time series

The authors analysed time series of rabies cases diagnosed in Hungary between 1967 and 2001. In Transdanubia (West Hungary), an oral immunization program started in 1992 and in East Hungary in 2001. Both long term and seasonal trends were identified in the time series of rabies cases. In order to characterize the underlying processes governing the behaviour of the epidemic, the fluctuations around the trend were analysed before and after immunization separately. It turned out that the tail of the complementary cumulative distribution functions differ. The tail of the distribution follows an inverse power law (IPL) function and describes the distribution of extreme events. The significant difference in the IPL exponents before and after immunization can be explained by the theory of Highly Optimized Tolerance (HOT).     

Hyperostosis frontalis interna - A marker of social status? Evidence from the Bronze-Age “high society” of Qatna, Syria

In 1719 Morgagni described a condition, today known as hyperostosis frontalis interna (HFI), as one sign within a triad consisting of HFI, virilism, and obesity. Today, HFI is predominantly found in older women. Although the etiology of HFI has not yet been determined precisely, the condition has been linked to metabolic disorders. HFI is reported to be rare in the archaeological record and the frequency of the condition is thought to have increased during the 19th and 20th centuries. We present preliminary results on the occurrence of HFI in the commingled human bone assemblage from “tomb VII” discovered underneath the Bronze Age royal palace of the ancient city of Qatna, Syria. A preliminary minimal number of individuals of 70 has been estimated for the as yet not fully analyzed skeletal remains. Skull fragments of nine individuals exhibit endocranial bone formations consistent with HFI. Rarity of stress indicators in the skeletons, the rich grave goods, and the burial place within the area of the Royal palace are suggestive of a high social status and an economically favorable situation of the buried individuals. Assuming that their life style included a high calorie diet in combination with little physical activity, acquired metabolic disorders may have been present in many individuals. The comparatively high number of individuals presenting HFI in the studied sample might therefore be viewed as being related to their high social status. Multiple occurrences of HFI in archaeological skeletal assemblages might serve as a proxy for social status.     

Evolution of Bombina bombina and Bombina variegata (Anura: Discoglossidae) in the Carpathian Basin: a history of repeated mt-DNA introgression across species

The structure and geographic location of hybrid zones change through time. Current patterns result from present and historical population-environment interactions that act on each of the hybridizing taxa. This is particularly evident for species involved in complex hybrid zones, such as that formed by the toad species Bombina bombina and Bombina variegata (Anura: Discoglossidae), which interact along extensive areas in Central Europe. We used data on external morphology and partial sequences of the cytochrome oxidase I (cox1) and nicotinamide adenine dinucleotid dehydrogenase subunit 4 (nad4) mitochondrial DNA (mt-DNA) genes to analyze the current patterns of genetic structure shown by both species of Bombina along their contact zone in Hungary. Phylogenetic, phylogeographic, and historical demography analyses were applied to 1.5kb mt-DNA obtained from 119 individuals representing 24 populations from Hungary and additional specimens from Slovakia, Albania, and Bosnia-Herzegovina. We use these data to infer the evolutionary history of the isolated populations of B. variegata in Hungary and to discriminate between competing biogeographic scenarios accounting for the historical interactions between species in this region. Results from the inferred phylogenetic branching pattern and sequence divergence among species and populations support the following: (i) recent population expansion has occurred in Hungarian populations of B. bombina, which are genetically very homogeneous; (ii) the Hungarian populations of B. variegata correspond to two distinct mitochondrial lineages (Carpathian and Alpine, respectively); average maximum-likelihood-corrected sequence divergence between these lineages is 8.96% for cox1 and 10.85% for nad4; (iii) mt-DNA divergence among the three isolated western populations of B. variegata from Transdanubia is low, with four closely related haplotypes, which suggests that the isolation between these populations is the result of a recent process, possibly mediated by the invasion of B. bombina; and (iv) we have detected discordances between morphology and mt-DNA data in the Transdanubia region (Bakony Mountains, Mecsek Mountains, Orség area), suggesting mt-DNA introgression across species in this regions. These results are discussed with reference to previous biogeographic hypotheses.     

Self-clearance of Mycobacterium tuberculosis infection: implications for lifetime risk and population at-risk of tuberculosis disease

Background: it is widely assumed that individuals with Mycobacterium tuberculosis (Mtb) infection remain at lifelong risk of tuberculosis (TB) disease. However, there is substantial evidence that self-clearance of Mtb infection can occur. We infer a curve of self-clearance by time since infection and explore its implications for TB epidemiology. Methods and findings: data for self-clearance were inferred using post-mortem and tuberculin-skin-test reversion studies. A cohort model allowing for self-clearance was fitted in a Bayesian framework before estimating the lifetime risk of TB disease and the population infected with Mtb in India, China and Japan in 2019. We estimated that 24.4% (17.8–32.6%, 95% uncertainty interval (UI)) of individuals self-clear within 10 years of infection, and 73.1% (64.6–81.7%) over a lifetime. The lifetime risk of TB disease was 17.0% (10.9–22.5%), compared to 12.6% (10.1–15.0%) assuming lifelong infection. The population at risk of TB disease in India, China and Japan was 35–80% (95% UI) smaller in the self-clearance scenario. Conclusions: the population with a viable Mtb infection may be markedly smaller than generally assumed, with such individuals at greater risk of TB disease. The ability to identify these individuals could dramatically improve the targeting of preventive programmes and inform TB vaccine development, bringing TB elimination within reach of feasibility.     

Widespread occurrence of Mycobacterium tuberculosis DNA from 18th-19th century Hungarians

A large number (265) of burials from 1731-1838 were discovered in sealed crypts of the Dominican Church, Vác, Hungary in 1994. Many bodies were naturally mummified, so that both soft tissues and bones were available. Contemporary archives enabled the determination of age at death, and the identification of family groups. In some cases, symptoms before death were described and, occasionally, occupation. Initial radiological examination of a small number of individuals had indicated calcified lung lesions and demonstrable acid-fast bacteria suggestive of tuberculosis infection. Tuberculosis was endemic in 18th-19th century Europe, so human remains should contain detectable Mycobacterium tuberculosis complex (MTB) DNA, enabling comparisons with modern isolates. Therefore, a comprehensive examination of 168 individuals for the presence of MTB DNA was undertaken. Specific DNA amplification methods for MTB showed that 55% of individuals were positive and that the incidence varied according to age at death and sampling site in the body. Radiographs were obtained from 27 individuals and revealed an association between gross pathology and the presence of MTB DNA. There was an inverse relationship between PCR positivity and MTB target sequence size. In some cases, the preservation of MTB DNA was excellent, and several target gene sequences could be detected from the same sample. This information, combined with MTB DNA sequencing data and molecular typing techniques, will enable us to study the past epidemiology of TB infection, and extends the timeframe for studying changes in molecular fingerprints.     

A picture of tuberculosis in young Portuguese people in the early 20th century: a multidisciplinary study of the skeletal and historical evidence

The aim of this study was to examine the evidence, and consider the differential diagnosis, for tuberculosis (TB) in juvenile individuals from early 20th century documented skeletons. There are 66 male and female juvenile individuals in the Coimbra Identified Skeletal Collection (CISC) with an age at death ranging from 7-21 years. The individuals died between 1904-1936 in different areas of Coimbra, Portugal. Eighteen of these individuals died from TB affecting different parts of the body. Thirteen (72.2%) showed skeletal lesions that may be related to this infection. Of the 48 individuals with a non-tuberculous cause of death, only 2 (4.2%) had skeletal changes that could be attributed to TB. The distribution of skeletal manifestations caused by the types of TB under study, based on macroscopic and radiological findings, is described and discussed. In addition, the medical records from 6 tuberculous individuals who died in Coimbra University Hospital (CUH) were analysed, and the information, including their diet and access to treatment, is presented. This work, based on data arising before antibiotics became available for treatment, can contribute to the future diagnosis of TB in non-documented skeletal material, and will facilitate a more reliable diagnosis of TB in juvenile individuals.     

Serpens endocrania symmetrica (SES): a new term and a possible clue for identifying intrathoracic disease in skeletal populations

This paper describes a phenomenon in the endocranial plate, which we have termed "serpens endocrania symmetrica" (SES), and discusses its value as a diagnostic tool. The affected discolored bone area exhibits disruption of the endocranial surface, lending it a maze-like appearance. Histological sections demonstrate that the process is limited to the most superficial portion of the endocranium, with no diploic and ectocranial involvement (sinus areas excepted). Adult skulls (n = 1,884) from the Hamann-Todd collection (HTH), housed at the Cleveland Museum of Natural History, were utilized for the present study. SES was recognized in 32 of the 1,884 skulls studied (1.7%). The frequency of SES among individuals reported to have died from tuberculosis (TB) was 4.4%. The rate of SES in the non-TB sample was only 0.53%. The locations were as follows: limited to sinus area, 28.1%; calvarium (excluding the sinuses), 46.9%; sinus + calvarium, 25.0%. SES was bilateral in 90.9% of cases. Twenty-five of the 32 individuals (78.1%) with SES in the HTH collection had tuberculosis specifically listed as the cause of death. Six of the other 7 individuals had infections other than TB. In 29 of the 32 individuals with SES, infection involved structures within the thorax. As SES was also associated with another osteological phenomenon known to represent pulmonary disease, i.e., hypertrophic osteoarthropathy (HOA; 68.0% of SES individuals also had HOA), SES may be of diagnostic value in paleopathology for the recognition of intrathoracic disease, and perhaps tuberculosis.     

Effects of age and occupation on cortical bone in a group of 18th-19th century British men

The effects of age and occupation on cortical bone in a group of adult males from the 18th-19th century AD skeletal collection from Christ Church Spitalfields, London, were investigated. Cortical bone was monitored using metacarpal radiogrammetry. Individual age at death was known exactly from coffin plates. Occupation for individuals was known from historical sources. Results showed that continued periosteal apposition was evident throughout adult life, but from middle age onwards this was outstripped by about 2:1 by endosteal resorption, so that there was net thinning of cortical bone. The rate of cortical thinning resembled that seen in modern European males. Cross-sectional properties, as measured by second moments of area, bore no relationship to occupation. The results may suggest that, firstly, patterns of loss of cortical bone have remained unchanged in males for at least two centuries in Britain, and secondly, that biomechanical analyses of metacarpal cortical bone may be rather insensitive indicators of intensity of manual activity.     

Recombinant antigen production for assays of intradermoreaction for diagnosis and surveillance of tuberculosis

The goal of the present work was to develop reagents with potential for tuberculosis diagnosis. Genetic sequences of Mycobacterium tuberculosis secretion antigens were amplified by PCR, cloned into the Gateway^® system, and expressed in Escherichia coli. The recombinant M. tuberculosis proteins were purified by metal affinity chromatography and preparative gel SDS-PAGE electrophoresis followed by electroelution and removal of endotoxins using Triton X-114. In total, seven recombinant proteins were obtained (ESAT-6, CFP10, TB10.3, TB10.4, MTSP11, MPT70, and MPT83). Delayed hypersensitivity reactions (DHR) was evaluated in Cavia porcellus and compared to the response using a standard purified protein derivative (PPD). All seven recombinant proteins produced a positive induration reaction in an intradermal test in guinea pigs previously sensitized with M. tuberculosis. When applied together, at a concentration of each recombinant protein 0.04 mg/mL, the intradermoreaction in C. porcellus was significantly higher than that obtained by standard PPD (p-value = 0.00386).     

Tumor necrosis factor alpha − 308G>A, − 863C>A, − 857C>T gene polymorphisms and tuberculosis susceptibility: A meta-analysis

Background/aims

A large number of studies have shown that polymorphisms in the tumor necrosis factor-α (TNF-α, TNFA) gene are implicated in susceptibility to tuberculosis (TB). However, the results are inconsistent. We performed this meta-analysis to estimate the association between polymorphisms in the TNFA gene and TB susceptibility.

Methods

Relevant studies published before March 2012 were identified by searching PubMed, ISI web of knowledge, EBSCO and CNKI. The strength of relationship between the TNFA gene and TB susceptibility was assessed using odds ratios (ORs).

Results

A total number of twenty-three case–control studies including 3630 cases and 4055 controls were identified referring to three previously chosen single-nucleotide polymorphisms (SNPs): − 308G>A, − 863C>A and − 857C>T. No association was found between − 308G>A, − 863C>A and TB susceptibility: − 308G>A (GG + GA vs. AA): OR 0.85, 95%CI: 0.55–1.30, P = 0.44; − 863C>A (CC + CA vs. AA): OR 0.93, 95%CI: 0.84–1.81, P = 0.83. Increased risk of TB was associated with − 857C>T in the dominant genetic model (CC + CT vs. TT: OR 2.13, 95%CI: 1.25–3.63, P = 0.01), the heterozygote comparison (CT vs. TT: OR 2.69, 95%CI: 1.44–5.02, P = 0.00) and the homozygote comparison (CC vs. TT: OR 2.08, 95%CI: 1.22–3.53, P = 0.01) in Asian subjects.

Conclusion

There is an increased association between TNFA − 857C>T polymorphism and TB risk among Asian subjects. No association was found between − 308G>A and − 863C>A with TB risk. Due to several limitations in the present study, well-designed epidemiological studies with large sample size among different ethnicities should be performed in the future.     

Carabelli's trait in contemporary Slovenes and inhabitants of a medieval settlement (Sredisce by the Drava River)

The objectives of this study were to determine the total frequency, expression and asymmetry of Carabelli's trait in permanent dentitions of contemporary Slovenes and a medieval skeletal population from northeastern Slovenia. A total of 254 dental casts from contemporary Slovene children were examined. The population of a medieval settlement (10th-15th centuries), was represented by 94 skeletons. A modification of the method of Alvesalo and associates was used to classify Carabelli's trait on a five-grade scale. The trait was expressed on the upper first molars of 79.7% of the contemporary subjects and 75.8% of the medieval sample. Positive expressions of the trait were found in 10.1% of the contemporary subjects and 15.2% of the medieval sample. While the observed total frequency of the trait in both samples is characteristic of Europeans, the rates of positive expressions are surprisingly low but consistent with data from a recently published worldwide literature survey. Both populations showed a low rate of left-right fluctuating asymmetry of the trait. This finding might reflect a pronounced ability of individuals in the medieval population to buffer unfavourable influences from the environment and a relatively low level of environmental stress in the contemporary population.     

Tuberculosis and leprosy in Italy. New skeletal evidence

Tuberculosis (TB) and leprosy are infections caused by Mycobacteria. This paper documents new skeletal evidence in Italy from the Iron Age site of Corvaro (Central Italy; 5th century BCE) and the Roman site of Palombara (Central Italy; 4th–5th century CE), and briefly reviews the extant evidence for these infections in Italy. The skeletal evidence for TB in Italy is more ancient than for leprosy, and is more common. The oldest evidence for both mycobacterial diseases is in the North of Italy, but this could be by chance, even if biomolecular models suggest a land route from the East to central Europe, especially for leprosy.     

Osteological and molecular identification of Brucellosis in ancient Butrint, Albania

Ancient skeletal remains can harbor unique information about past civilizations at both the morphological and molecular levels. For instance, a number of diseases manifest in bone, some of which have been confirmed through DNA analysis, verifying their presence in ancient populations. In this study, anthropological analysis of skeletal remains from the ancient Albanian city of Butrint identified individuals with severe circular lytic lesions on their thoracic and lumbar vertebrae. Differential diagnosis suggested that the lesions resulted from pathologies known to affect these skeletal regions, such as tuberculosis (TB) or brucellosis. Relevant bones of two adolescent males from the 10th to 13th century AD that displayed the lesions, along with unaffected individuals, were collected in the field. Genetic screening of the skeletal samples for TB was repeatedly negative, thus additional testing for Brucella spp.-bacteria of livestock and the causative agent of brucellosis in humans-was conducted. Two Brucella DNA markers, the IS6501 insertion element and Bcsp31 gene, amplified from the affected vertebrae and/or ribs, whereas all unaffected individuals and control samples were negative. Subsequent DNA sequencing confirmed the presence of the brucellar IS6501 insertion element. On the basis of the skeletal lesions, negative tests for TB, and positive Brucella findings, we report a confirmed occurrence of brucellosis in archaeologically recovered human bone. These findings suggest that brucellosis has been endemic to the area since at least the Middle Ages.     

Anatomy of a serial killer: differential diagnosis of tuberculosis based on rib lesions of adult individuals from the Coimbra Identified Skeletal Collection, Portugal

The role of new bone formation on visceral surfaces of ribs in the diagnosis of tuberculosis (TB) in past human populations has been explored by many researchers, using both skeletal remains with known causes of death and archaeological samples. This study focuses, firstly, on adult skeletons from the Coimbra Identified Skeletal Collection in Portugal and investigates the skeletal manifestations of individuals known to have died from TB; secondly, this study focuses on the role of rib lesions in the diagnostic criteria for TB. One hundred and fifty-seven males and 106 females aged between 22-87 years were examined; causes of death were assigned as pulmonary TB, extrapulmonary TB, and pulmonary non-TB; a control group, extrapulmonary non-TB, was selected from the remaining individuals. Of individuals with rib lesions, 85.7% (69/81) had pulmonary or extrapulmonary TB as an assigned cause of death, while 17.8% (16/90) of individuals with rib lesions had a non-TB cause of death. Rib lesions were significantly more common in individuals who had died from TB, although the lesions cannot be considered pathognomonic for TB. In individuals dying from pulmonary TB, ribs in the central part of the rib cage were most affected, at their vertebral ends. The lower part of the rib cage may be a marker for peritoneal TB, and "coral-like" new bone formation on ribs may be an indicator of neoplastic disease. Further work on rib involvement in TB in clinical contexts, and the study of further documented skeletal collections, are recommended.     

Polymorphic Variation in TIRAP Is Not Associated with Susceptibility to Childhood TB but May Determine Susceptibility to TBM in Some Ethnic Groups

Host recognition of mycobacterial surface molecules occurs through toll like receptors (TLR) 2 and 6. The adaptor protein TIRAP mediates down stream signalling of TLR2 and 4, and polymorphisms in the TIRAP gene (TIRAP) have been associated with susceptibility and resistance to tuberculosis (TB) in adults. In order to investigate the role of polymorphic variation in TIRAP in childhood TB in South Africa, which has one of the highest TB incidence rates in the world, we screened the entire open reading frame of TIRAP for sequence variation in two cohorts of childhood TB from different ethnic groups (Xhosa and mixed ancestry). We identified 13 SNPs, including seven previously unreported, in the two cohorts, and found significant differences in frequency of the variants between the two ethnic groups. No differences in frequency between individual SNPs or combinations were found between TB cases and controls in either cohort. However the 558C→T SNP previously associated with TB meningitis (TBM) in a Vietnamese population was found to be associated with TBM in the mixed ancestry group. Polymorphisms in TIRAP do not appear to be involved in childhood TB susceptibility in South Africa, but may play a role in determining occurrence of TBM.     

Epidemiological approach to the paleopathological diagnosis of leprosy

In paleopathology it is usually assumed that modern diagnostic criteria can be applied to infectious diseases in the past. However, as both the human species and populations of pathogenic microorganisms undergo evolutionary changes, this assumption is not always well-founded. To get valid estimates of the frequency (the point prevalence at death) of leprosy in skeletal samples, sensitivity, specificity, and sample frequency must be estimated simultaneously. It is shown that more than three symptoms must be evaluated in at least three samples in order to reach estimates with well-described properties. The method is applied to three skeletal samples from Medieval Denmark; the samples were scored for the presence of seven osteological conditions indicating leprosy. For the osteological conditions, sensitivity varied from 0.36-0.80, and specificity from 0.58-0.98. The frequency of leprosy in the three samples was: Odense (a lepers' institution), 0.98, 95% CI 0.64-1.00; Malm? (urban cemetery), 0.02, 95% CI 0.00-0.07; and Tirup (rural cemetery), 0.36, 95% CI 0.23-0.46. It is concluded that it is indeed possible to estimate disease frequencies without reference to modern standards, and that leprosy occurred with widely differing frequencies in different segments of the Medieval population in southern Scandinavia.