Review
Tuberculosis: from prehistory to Robert Koch, as
revealed by ancient DNA
Helen D Donoghue, Mark Spigelman, Charles L Greenblatt, Galit Lev-Maor, Gila Kahila Bar-Gal, Carney
Matheson, Kim Vernon, Andreas G Nerlich, and Albert R Zink
During the past 10 years palaeomicrobiology, a new
scientific discipline, has developed. The study of ancient
pathogens by direct detection of their DNA has answered
several historical questions and shown changes to
pathogens over time. However, ancient DNA (aDNA)
continues to be controversial and great care is needed to
provide valid data. Here we review the most successful
application of the technology, which is the study of
tuberculosis. This has provided direct support for the current
theory of Mycobacterium tuberculosis evolution, and
suggests areas of investigation for the interaction of
M tuberculosis with its host.
Lancet Infect Dis 2004; 4: 584–92
Ancient DNA (aDNA) has been controversial since it
was retrieved from an extinct animal, the quagga, in 1984.1
Originally, cloning was used to obtain the DNA sequence
but the preferred method has become DNA amplification
by PCR.2 There are many anthropological and epide-
miological reasons for the appeal of aDNA analysis—eg, it
provides information on sequence changes in real time,
rather than relying on calculations based on a molecular
clock. In addition to phylogenetics and population
genetics, animal and plant remains can tell us about
early social and agricultural practices, and coprolites
(ancient faeces) give information about health status
and diet. Nuclear DNA from human beings can reveal
their sex, which may not be determined from skeletal
morphometrics or inferences from grave goods. Analysis
of mitochondrial DNA provides information about
familial or kinship relatedness and the data to test
hypotheses on population migrations in prehistory. Some
reviews of the use of aDNA in palaeontology3 and
anthropology4 include comprehensive overviews of its
applications.
DNA is not a stable molecule, and oxidation and
hydrolysis damage DNA over time.5 As a result, the DNA
becomes fragmented, especially during the strand separation
stage of PCR. Therefore, the best amplifications are obtained
using a small DNA target size, preferably below 200 bp. It is
not the age of the DNA but the environmental conditions
which are critical in preservation.6,7 A stable environment is
best for DNA preservation. Cool temperatures have allowed
the recovery of Neanderthal DNA,7,8 but hot, dry
environments such as that of Egypt9,10 have also yielded
aDNA (figure 1). The upper limit for recovery of amplifiable
DNA is believed to be around 100 000 years.7
584
For personal use. Only reproduce with permission from Elsevier Ltd.
Tuberculosis and ancient DNA
Figure 1. Use of endoscopy to obtain samples from an ancient Egyptian
mummy.
Unfortunately, some of the earliest work on ancient
DNA—from prehistoric human remains—led to concerns
about contamination from modern DNA, including that of
the investigators.2,11 This problem resulted in a series of
recommendations for good practice: physical separation of
activities before and after PCR, strict protocols to prevent
and monitor the introduction of modern DNA, the use of
negative controls, observation of an inverse relationship
between target sequence size and PCR efficiency, replication
of samples (preferably) in different laboratories to confirm
results, and assessment of sequence data to check
phylogenetics.5,12 Cloning has been used for aDNA analyses,
especially of human remains, since it can identify DNA
damage, PCR error, or the presence of mixtures in the
sample. However, where there is good DNA preservation
and specific DNA sequences are detected, cloning is rarely
needed.
HDD and MS are at the Centre for Infectious Diseases and
International Health, University College London, London, UK; CLG,
GLM, MS, and KV are at the Kuvin Centre for the Study of Infectious
and Tropical Diseases, The Hebrew University, Hadassah Medical
School, Jerusalem, Israel; GKBG is at the Laboratory of Genomic
Diversity, National Cancer Institute, Frederick, MD, USA; CM is at
the Paleo-DNA Laboratory, Faculty of Science and Environmental
Studies, Lakehead University, Thunder Bay, ON, Canada; KV is at
the Department of Zoology, The University of Queensland, Brisbane,
Queensland, Australia; AGN and ARZ are at the Division of
Palaeopathology, Institute of Pathology, Academic Teaching
Hospital München-Bogenhausen, Munich, Germany.
Correspondence: Dr Helen D Donoghue, Centre for Infectious
Diseases and International Health, University College London, 46
Cleveland Street, London W1T 4JF, UK. Tel +44 207 679 9153;
fax +44 207 636 8175; email h.donoghue@ucl.ac.uk
Infectious Diseases Vol 4 September 2004 http://infection.thelancet.com
 Tuberculosis and ancient DNA
Microbial pathogens and ancient DNA
The success in obtaining mammalian aDNA soon led to the
search for microbial pathogens. Initially, interest focused on
retrospective disease diagnosis and confirmation of
diagnoses that had been inferred from pathological skeletal
changes. However, it became clear that phylogenetic studies
might be possible. The clearest example of this technology in
the field of acute infections is the characterisation of the
strain of influenza virus13,14 that was responsible for the 1918
pandemic, which killed 40 million people worldwide. This
work relied on remains preserved in the Arctic permafrost
and archival pathology samples. It is hoped that the study of
chronic diseases will clarify the interaction between host and
pathogen. Ancient pathogen DNA research has been
reviewed15,16 and Spigelman and Donoghue17 have reviewed
mycobacterial diseases.
Two strategies have been adopted to detect specific
pathogenic microbes. The most successful strategy uses
specific detection techniques that were developed in clinical
diagnostic microbiology; these techniques are applied to
ancient material. The other strategy uses non-specific
methods to amplify aDNA, which is then separated and
sequenced.18 In theory, mycobacteria are the ideal micro-
organisms for studying the aDNA of pathogens and were the
earliest to be sought. This group includes the organisms that
cause tuberculosis and leprosy, and these pathogens are
found only in an infected host. Tuberculosis was widespread
in the past and is a re-emerging global threat.19,20 The
pathology of tuberculosis is characteristic (figure 2);
localised lesions tend to contain residual microbial DNA.
Mycobacteria have DNA with a high proportion of guanine
and cytosine; this increases DNA stability and may aid its
survival. In addition, the thick cell walls of these
mycobacteria are lipid-rich,21,22 protecting DNA from the
attack of lytic enzymes after autolysis and necrosis of the
host cells, the other microflora and fauna of the host upon
death, and the first-stage decomposers that invade once a
person dies. M tuberculosis has survived after fixing in
formalin and staining,23 the death of the host,24 and has even
been transmitted from the corpse to a new host 1 year after
burial.25
Figure 2. Gross pathology of the spine in an 18th century Hungarian from
Vàc who had tuberculosis.
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For personal use. Only reproduce with permission from Elsevier Ltd.
Review
Panel 1. Advantages of studying aDNA of microbial
pathogens
Confirmation of palaeopathology
To answer historical questions
Epidemiology of the disease
Geographic range in the past
Population studies
Comparison of microbial pathogens from the past with those of today
Molecular evolution
Changes in virulence
Development of the relationship between host and pathogen relation
Ancient DNA from the M tuberculosis complex
In 1993, Spigelman and Lemma26 published the first finding
of aDNA in a human microbial pathogen (panel 1).
Archaeological specimens from human beings that had been
morphologically diagnosed with tuberculosis were used. The
insertion sequence IS6110 was found with PCR primers
specific to the M tuberculosis complex, which includes
M tuberculosis, M bovis, and M aftricanum.27 This sequence is
highly conserved and is typically present in multiple copies
in M tuberculosis, which increases detection sensitivity.
These primers also detect IS6110 in the other members of
the M tuberculosis complex.
11 specimens were used in the first report of
archaeological M tuberculosis DNA26 and four of these, bones
ranging in age from 300–1400 years, tested positive. The
bones came from Europe, Turkey, and Borneo (before
European contact) and M tuberculosis in these samples has
since been confirmed with sequence data.28 Subsequently,
Salo et al29 used identical primers to clone and sequence
amplicons from the lung tissue of a 1000-year-old Peruvian
mummy. Shortly after these publications a number of
laboratories published similar studies, the great majority of
which used the IS6110 target site for the detection of
M tuberculosis DNA.30–40 This method continues to be the
most sensitive for the detection of the M tuberculosis
complex. Other specific sequences—in addition to the
IS6110 region—have been targeted.10,41–48
Verification of findings of ancient
M tuberculosis DNA
At least two reports provide biomolecular evidence for the
preservation of the tubercle bacillus in ancient specimens:
M tuberculosis-specific mycolic acids were found together
with M tuberculosis-specific IS6110 DNA. The presence of
both types of biomolecules—amplification of the DNA and
direct detection of the cell wall mycolic with high
performance liquid chromatography—is one method of
verifying the diagnosis of tuberculosis.36,49
Pathogenic microbial DNA in a specimen has been
validated with the corresponding host DNA. For example,
using material from ancient Egyptians, Zink et al46
demonstrated host DNA by amplification of a 202 bp
segment of the human beta-actin gene. In further work,
Zink and Nerlich found a few samples in which
M tuberculosis DNA could be amplified but the human beta-
actin gene could not be amplified (unpublished observations
585
 Review
Figure 3. Microscopic appearance of chest material from a 36-year-old
man who haemorrhaged from the mouth and died in Vàc in 1808.
Samples were strongly positive for M tuberculosis DNA with Ziehl-
Neelsen stain for acid-fast bacilli.
by the authors). The reason is likely to be the differential
preservation of M tuberculosis DNA.17 Matheson and Vernon
have also observed that mycobacterial DNA can be detected
when genomic and mitochondrial host DNA is severely
degraded (unpublished observations by the authors). In
some cases the tubercle bacilli have persisted (figure 3).
Taphonomy of mammalian DNA after death commonly
damages mitochondrial DNA50 and this can lead to
interpretation errors in ancient human population genetics.
By contrast, point mutations are relatively rare in
mycobacteria51 and molecular analysis mainly uses genomic
deletions. Therefore, it is not surprising that mycobacterial
DNA seems better than human DNA in preservation and
sequence fidelity.
Independent verification
Independent verification by another laboratory, and the
detection of several different target sequences in the
M tuberculosis genome, are important strategies for
verification of aDNA data. An indication of the reliability of
data obtained from different laboratories is provided by
some research conducted at Jerusalem and University
College London (UCL). Lev-Maor et al52 used PCR to
analyse 30 suspected tuberculosis samples obtained across a
wide range of times and locations. 15 specimens tested
positive and replication in a second laboratory confirmed
this result in 12 specimens. Different quantities of bone-
powder sample were assessed in the two laboratories, which
may explain the discrepancy. Also, it should be noted that by
contrast with mitochondrial or nuclear mammalian DNA,
pathogenic prokaryotic DNA is not likely to be distributed
evenly within a sample and this will lead to variable success
in its detection.
Fletcher et al47 studied the remains of 263 individuals
from the 18th century; the remains were preserved well
because they were in a sealed church crypt in Vàc, Hungary,
where they naturally mummified (figure 4). In a study of the
reproducibility of their findings, one to four independent
586
For personal use. Only reproduce with permission from Elsevier Ltd.
Tuberculosis and ancient DNA
Figure 4. Mummified body 116 from Vàc. DNA from this woman’s chest
was M tuberculosis positive. She died aged 26 years after a caesarean
operation; her child lived for 1 day.
laboratories examined 27 samples from 26 individuals with
various target sequences for M tuberculosis. Identical results
were obtained in 23 of the samples and in the four remaining
samples the results were compatible.
The use of different target sequences to
characterise M tuberculosis aDNA
In addition to the IS6110 region, other sites on the
M tuberculosis genome are being assessed to validate
M tuberculosis and to characterise the organisms that caused
tuberculosis in the past. Initially this work aimed at finding
which species of the M tuberculosis complex was present. In
M tuberculosis a few single nucleotide polymorphisms in the
katG463 and gyrA95 sites have been used to genotype
strains.51,53 Strains can be identified as M tuberculosis or
M bovis from genotyping, amplification of the oxyR and
mtp40 loci, and spoligotyping based on the direct repeat
(DR) region spacers.48,54 Amplification of these single-copy
chromosomal loci are highly dependent on the state of
preservation of the material and, commonly only the IS6110
PCR yields data.40
More recently, investigators have begun to use deletion
analysis to characterise the M tuberculosis DNA that is
present in ancient remains. There are deletion hotspots in
the M tuberculosis genome,55 which can be used to
differentiate the members of the M tuberculosis complex and
distinguish between strains.56,57 It seems that deletions are
Infectious Diseases Vol 4 September 2004 http://infection.thelancet.com
 Tuberculosis and ancient DNA
Panel 2. Questions answered by aDNA studies of
M tuberculosis
Confirmation of diagnosis from palaeopathology
M tuberculosis DNA is in bones with no pathology
Tuberculosis existed in Borneo before European contact
Tuberculosis existed in America before Columbus
Tuberculosis was widespread in ancient Egypt and Rome
Koch’s bacillus was M tuberculosis not M bovis
General absence of M bovis in human remains
M tuberculosis DNA in North American bison during Pleistocene (17 500
years BP)
Indication that M tuberculosis in North America was spread from Asia by
ungulates that crossed the Bering land bridge
Confirmation of latest theory of M tuberculosis evolution
M africanum and M tuberculosis existed over 4000 years ago
associated with some loss of virulence,58 which suggests that
deletion analysis may also throw some light on the
host–pathogen relationship.
Initial findings from ancient M tuberculosis
studies
Even the earliest experiments in this field answered
questions that have interested palaeopathologists for a long
time—such as proving that human tuberculosis existed
before European contact in both Borneo and the New World
(panel 2). It soon became clear that tuberculosis is an ancient
disease with a wide geographic distribution. Another finding
that is consistent with the course of tuberculosis infection is
that although M tuberculosis can be detected in samples
showing typical pathology—such as vertebral lesions (figure
2), rib lesions (figure 5), or pleural adhesions (figure 6)—it
can also be detected at a lower frequency in bones with no
pathological changes.10,41,46,59–61 The spread of tubercle bacilli
throughout the body, via the bloodstream in severe cases of
milliary tuberculosis and during bacteraemia after death, can
explain this finding.
aDNA techniques have identified a strain of
M tuberculosis rather than M bovis in a subculture (1901)
of the original Koch’s bacillus, increasing our knowledge
of tuberculosis during past times.62 This work was based
on a museum specimen that had probably been treated
with formalin; further molecular characterisation was
unsuccessful.
The use of spoligotyping to characterise the
M tuberculosis complex
Spoligotyping is a form of molecular typing. PCR of spacer
sequences in the DR region63 are seen by way of dot-blot
hybridisation on a membrane, giving a molecular
fingerprint. The primers are in the DR region and amplify
up to 43 unique spacer regions, which are between each DR
sequence. The function of this region is unknown and
different strains of M tuberculosis commonly show deletions,
which form the basis of an international database that is
used for epidemiological investigations. The database has
been used for evolutionary studies because loss of spacers is
unidirectional; care is needed in interpretation since
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For personal use. Only reproduce with permission from Elsevier Ltd.
Review
particular patterns can result from an accumulation of
several independent events or mixed infections.
Spoligotyping is an especially valuable technique for the
study of M tuberculosis in archaeological material since it is
possible to obtain useful data even when the DNA is highly
fragmented. The distance between adjacent DR loci is only
35–41 bp and each DR locus is 36 bp,63 so a fragment of
55–60 bp is sufficient to provide a result.
There are increasing numbers of reports of the
application of spoligotyping of aDNA for M tuberculosis. For
example, with methods described by Parwati et al,64 Fletcher
et al48 did spoligotyping of M tuberculosis DNA samples from
18th century Hungary in two different laboratories. The
technique distinguished different genotypes of
M tuberculosis in three members of the same family (figure
7). Almost complete spoligotypes were obtained from the
well-preserved remains of 12 different people; results were
reproducible on different occasions and in separate
laboratories.
Spoligotyping was also central to Zink et al’s work46 and
was done on all of the samples obtained by the
Jerusalem–UCL group.64 Of 25 positive Egyptian specimens,
12 had acceptable spoligotyping patterns. This was defined
as three separate PCR amplifications summarised as a
consensus hybridisation pattern.46 In the Jerusalem–UCL
study, 21 out of 24 positive samples had a partial
hybridisation pattern. Other studies that have presented
spoligotyping patterns of ancient samples include Taylor et
al42 who examined three mediaeval English specimens and
the Koch bacillus from the museum specimen.62 Rothschild
et al45 used spoligotyping to examine a sample of bone from
ancient bison. However, the interpretation of spoligotypes,
which are obtained by repeated typing, and the
accumulation of a consensus pattern remains controversial.
Taylor et al42 note that conclusions from spoligotypes of
archaeological material must be made cautiously; some
spacer regions may be lost due to differences in preservation.
Were ancient Egyptians from the Middle
Kingdom infected with M africanum?
Although M africanum is recognised as a separate species
within the M tuberculosis complex, some researchers dispute
Figure 5. Lesion on a rib bone with M tuberculosis DNA.39
587
 Review
Figure 6. A mummy torso of a 6–8 year old girl from a tomb in Thebes-West Dra Abu El-Naga.15 The tomb was built in the New Kingdom but used until
the Late Period; it is dated about 1500–500 BC. The arrow shows major connective tissue adhesions that extend to the left body wall, suggesting
chronic pleural infection.
this. However, the RD9 region is absent from both
M africanum types I and II,65 so they can be distinguished
from M tuberculosis. Both the German and Jerusalem–UCL
studies found with spoligotyping that some samples were
missing single spacers in numbers 38–43; the gradual
deletion of these spacers may suggest a tendency towards the
M africanum and M bovis genotypes.66,67 Several spacers
(33–36) were under-represented in Zink et al46 (figure 8) and
this also happened in the Jerusalem–UCL study,52 except for
spacer 33. The absence of these spacers is characteristic of
M tuberculosis and M africanum subtype II.67,68 The
discrepancy between the two studies may be because the
former used exclusively African samples. An exciting
preliminary result from Zink et al46 is the indication that
three spoligotypes obtained from the Middle Kingdom
(2050–1650 BC) were missing spacer 39, a characteristic of
M africanum subtype I.67,69 M africanum is believed to be
closer in evolutionary terms to their common ancestor than
M tuberculosis or M bovis. Therefore these data indicate that
this group had become differentiated from the ancestral
form because of the loss of the RD9 region by this time.
Absence of M bovis in archaeological human
remains
Almost all archaeological M tuberculosis complex aDNA
obtained from human remains and assessed with
spoligotyping consistently shows at least some of the spacers
38–43. The presence of these spacers indicates that the
organisms are not M bovis. This is consistent with the failure
to identify M bovis from archaeological material with other
genetic markers that distinguish between the two
species.44,47,48,54 However, preliminary data from a study of four
archaeological samples from a single site in Siberia indicate
that skeletal lesions were probably attributable to infection
with M bovis. Climate could have been a factor in preservation
of the M bovis DNA (GM Taylor, Imperial College London,
588
For personal use. Only reproduce with permission from Elsevier Ltd.
Tuberculosis and ancient DNA
UK, personal communication). The apparent absence of
M bovis in an overwhelming number of human archaeological
specimens suggests that either the organism was absent or that
it was present at such a low level that far more samples need to
be examined to gain a robust assessment of its distribution.
Before the introduction of any effective M bovis control
programmes in Great Britain, it is estimated that it caused 6%
of human deaths due to tuberculosis.70 Unfortunately, very
few studies have been completed on animal remains so it is
not known whether M bovis was present in wild or
domesticated animals that could have been a reservoir of
infection.
Tuberculosis in prehistory
Previously it was believed that the original source of
M tuberculosis in human beings was newly domesticated
cattle about 10 000–15 000 years ago.71,72 However, recent
work45,65,73 suggests that the relationship between bovine and
human tuberculosis may have involved co-evolution rather
than a direct transmission from bovines to human beings.
Bone samples with lesions suggestive of a tuberculosis-like
infection can now be traced back to the Pleistocene.45,74
Material came from the Natural Trap Cave in Wyoming,
USA. This is a vertical pit in the middle of a game trail in
which more than 40 000 bone samples accumulated over
100 000 years. These samples lay in the bottom of the pit at
temperatures of about 4°C, which helped preservation. One
bison metacarpal showed tubercular-like infection and dated
to about 17 500 years BP (according to stratigraphy and C14
dating), a period when the bison was unlikely to have been
domesticated. Spoligotyping of this ancient material was
difficult and only partial patterns were obtained. However,
discriminant analysis showed that the bison pattern was
more closely aligned to the M tuberculosis group than to the
M africanum and M bovis groups. Among other animals that
fell into the pit, tuberculosis-like pathology has been found
Infectious Diseases Vol 4 September 2004 http://infection.thelancet.com
 Tuberculosis and ancient DNA
Review

A
5 10 15 20 25 30 35 40

H37Rv
PCR control
Mother
Older daughter
Younger daughter

B
5 10 15 20 25 30 35 40
Mother L1
Mother L2
Mother N3
Older daughter L1
Older daughter L2
Older daughter N3
Younger daughter L1
Younger daughter L2
Younger daughter N3
Figure 7. (A) Spoligotyping of DNA from a mother and two daughters from the 18th century in Vàc.48 (Spacers 28, 29, 30, and 32 are present but
difficult to see.) (B) Schematic representation of spoligotyping results from London and Netherlands. L1=extract 1 typed in London, L2=extract 2 typed
in London, and N3=extract 3 typed in Netherlands. Reproduced with permission from the Society of General Microbiology.

in the metacarpal–phalangeal joints of sheep, bison, and
musk oxen. These are all species believed to have migrated to
America from Asia across the land bridge before the
formation of the Bering Strait during the late Pleistocene.74
Spoligotyping of this ancient material may assist in
determining how and when M bovis and M tuberculosis arose
from a progenitor species. Further work in this area is
needed.
Ancient M tuberculosis DNA and pathogen
evolution
It has been suggested that genotype 1 strains are older in
evolutionary terms than genotypes 2 and 3.51,53 Brosch et al65
used a comparative study of members of the M tuberculosis
complex to propose a phylogeny on the premise that this
group of bacteria has evolved through the unidirectional
accumulation of deletions (figure 9). The analysis of katG463
and gyrA95 and discovery of genotypes 2 and 3 by Fletcher et
al48 was used by Brosch et al65 as evidence that the TbD1
deletion of M tuberculosis took place before the 18th century.
Mostowy et al73 supported this phylogeny, suggesting that
genomic deletions should be an appealing method of
studying mycobacterial evolution. The conclusion is that
increased cases of tuberculosis in the industrial revolution
resulted from modern M tuberculosis strains and not M bovis
or ancestral strains of M tuberculosis. It is proposed that
ancestral tubercle bacilli may have originated from endemic
foci, whereas strains with the TbD1 deletion represent

5 10 15 20 25 30 35 40
MK TT196-44
MK TT196-M5
MK TT196-MW7
MK TT196-MW18
NK TT453-PC14
NK TT453-PC9
NK TT183-8
NK TT95-PC40
NK TT95-PC122
NK TT95-PC169
NK DAN 93·11
MK DAN 95·1-1
M tuberculosis H37Rv
M bovis BCG P3
M africanum I 25420
M africanum II 1567/99
Figure 8. Spoligotypes of samples from the Middle Kingdom (MK) and New Kingdom (NK).46 Tomb complexes are estimated to have been in use at
these times: TT 196=2050–1650 BC; DAN 95·1-1=2050–500 BC; DAN 93·11=1550–500 BC; TT 95=1450–500 BC; TT 453=1450–500 BC; TT
183=1250–500 BC. Spoligotypes of M africanum: M africanum subgroup I isolate ATCC 25420; M africanum subgroup II isolate 1567/99.67

Infectious Diseases Vol 4 September 2004 http://infection.thelancet.com 589

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 Review Tuberculosis and ancient DNA

Numerous sequence RDcan M canettii

polymorphisms
gr1, Ancestral M tuberculosis
Last common ancestor TbD 1 gr1, eg, Beijing
of the turbercle bacilli RD 9
katG463CTG CGG gr2, eg, CDC1551 Modern
M tuberculosis
gyrA95AGC ACC gr3, eg, H37Rv
RD 7
RD 8
RD 10
M africanum
mmplL6561AAC AAG
RDmic
M microti
RDseal Seal isolates
oxyR296G A
RD 12 Oryx isolates
RD 13

pncA57CAC GAC Goat isolates

RD 4

M bovis
Classic
RD 1

RD 2 BCG Tokyo

RD 14
BCG Pasteur

Figure 9. Diagram of the evolution of the M tuberculosis complex, from reference 65. Reproduced with permission from the National Academy of
Sciences, USA.

modern M tuberculosis epidemic strains, which are
descended from a single clone. Therefore, modern
M tuberculosis strains—which account for the vast majority
of today’s tuberculosis cases—are believed to have
undergone a genetic evolutionary bottleneck some 15 000 to
20 000 years ago,51,75 before global dissemination.
It is not yet known whether the strains of M tuberculosis
found in pre-Columbian America and other parts of the
world differ from the virulent epidemic strains associated with
18th century western Europe. The origin of these virulent
strains is of interest because the location, mechanisms, and
time-scale of their development and spread must be
understood. Archaeological material that pre-dates large-scale
international travel and antimicrobial therapy has a specific
role in the understanding of M tuberculosis.
Host resistance and susceptibility genes
While characterising the phylogeny of ancient pathogens,
any pathogen interactions with the human host can also be
addressed. Many research projects have focused on the
identification of genetic markers for host resistance or
susceptibility to M tuberculosis infection, which include
polymorphisms in CD receptors, cytokines and their
receptors, various classes of leucocytes, and MHC genes.76,77
The excellent preservation of the Vác Hungarian mummies
and the archives available for many of them should enable
the family group and state of health to be established. In
addition, it has proved feasible to distinguish individuals
with active and latent M tuberculosis disease at the time of
their death.47 These data provide an unprecedented
opportunity to analyse host susceptibility to disease. At
present there is evidence of M tuberculosis DNA in the tissues
of 62% of the samples, suggesting the possibility of
addressing past epidemiology. Preliminary research on the
correlation of NRAMP1 (SLC11A1) alleles with Hungarian
mummies that show active and latent tuberculosis has
confirmed that authentic sequences can be detected78 and do
have a susceptibility and resistance correlation.
A promising direction of research seems to be the
assessment of the M tuberculosis DNA, because different
genotypes are known to produce different immunological
and pathological host responses.79 For example, mycobacterial
cell walls contain mannose-capped lipoarabinomannans that
are key molecules in the virulence and immunopathogenesis
of tuberculosis.80,81 One of the genes involved has just been
identified82 and should be a fruitful target for studies of the
development of the host and pathogen relationship. The
exploration of genetic markers for mycobacterial virulence,
from an era before any selection pressures brought about by
antimicrobial therapy, is an exciting prospect.
The future of ancient M tuberculosis DNA
research
The genomes of both M tuberculosis and M bovis have been
sequenced83,84 and this has improved the understanding
of the evolution of these organisms and their interaction
with the host. Several target sequences can be amplified
from very old material, and as techniques improve an

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 Tuberculosis and ancient DNA
Review

“ancient M tuberculosis genome project” could be

conceived; genomics and proteomics of strains of
M tuberculosis from the past could be compared with
modern isolates. Already we have discovered the
preponderance of the human strain of the tubercle bacillus
and have verified the hypothesis made by evolutionary
microbial geneticists. The improvement in methodology
and widespread acceptance of the need for independent
verification in the study of ancient pathogens by DNA
techniques has resulted in data that can withstand external
scrutiny. Technologies identify the agents and also allow
further study of their genetics and evolution; they are now
being applied to ancient material. The study of tuberculosis
provides a reliable basis for the establishment of molecular
paleomicrobiology. Conversely, the paleomicrobiology of
tuberculosis is informing the debate on the evolution of the
M tuberculosis complex and has the potential to aid our
understanding of the development of the relation between
host and pathogen.
Search strategy and selection criteria
Most references were obtained from the authors’ extensive
files; relevant articles referenced in these were also identified.
Medline searches were done with the search term “ancient
DNA” together with the search terms “tuberculosis” and
“PCR”. Selection criteria were historical importance, scientific
relevance, and illustrations of the breadth of the field.
Acknowledgments
We are grateful to the Center for the Study of Emerging Diseases for
their generous support of this research. This work would not have
been possible without the specimens that Joel Blondiaux, Bernd
Herrmann, Antónia Marscik, Ildicó Pap, Bruce Rothschild, Douglas
Ubelaker, and Joe Zias provided. Hillel Bercovier’s enthusiasm and
support is thankfully acknowledged. Authors GKBG and CM
previously worked at the Kuvin Centre for the Study of Infectious and
Tropical Diseases, The Hebrew University, Hadassah Medical School,
Jerusalem, Israel.
Conflicts of interest
We have no conflicts of interest.

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