**3. Recognition, diagnosis and spread of ancient leprosy**

#### **3.1. Pathology and recognition of ancient leprosy**

**2.3. Nature and distribution of** *M. leprae* **genotypes**

10 Hansen's Disease - The Forgotten and Neglected Disease

whole genome sequencing (WGS) [46, 53].

armadillos acquired leprosy from human cases [45, 56].

**2.4. Transmission of leprosy**

Major collaborative studies based on single nucleotide polymorphism (SNP) typing have established that modern *M. leprae* consists of four distinct genotypes that are associated with different human populations [47]. It is believed that the ancestral precursor of *M. leprae* experienced an evolutionary bottleneck and thereafter developed independently in different human populations [26, 48]. In Europe, indigenous leprosy is now largely extinct, so a further study also looked at *M. leprae* from archaeological cases using aDNA methods [26]. This identified SNP type 3 cases from various European countries for the first time, including Denmark, Hungary, Croatia, Turkey and Britain. Some cases provided subtypes I, M or K. Genotype 3 strains were also found from Roman Egypt and by others in medieval Central Europe [30, 49]. Later studies also reported SNP type 2 strains for the first time in medieval cases from Winchester, UK [21] and from Sweden [50, 51]. Archaeological remains from Japan yielded a SNP type 1 from that country [52]. Several of the robust cases were subsequently amplified by

Monot et al. [26] also recognised sub-genotypes from extant cases, thereby enabling more precise associations between *M. leprae*, geographical location and present human populations ranging from China [54] to South America [55]. In a detailed study of modern *M. leprae* that included SNP typing, variable-number-tandem-repeat (VNTR) analysis and WGS, Truman et al. [9] examined 50 patients with leprosy and 33 wild armadillos (*Dasypus novemcinctus*) in the United States, together with reference strains from other parts of the world. Seven *M. leprae* SNP types were detected. The SNP type for some patients with possible exposure by foreign residence was typical of *M. leprae* from foreign locations. The most abundant SNP type was 3I that is generally associated with historical northwest European or American populations. The SNP sub-type 3I-1 strains, with one copy of an 11-bp indel (indel\_17915) had ancestral bases, but all other *M. leprae* strains have two copies. Type 3I-2 strains, a development of the ancestral 3I-1 strains, similarly have only one copy of indel ML\_17915 and can be identified by base C at position 1527056 instead of base G present in type 3I-1 isolates [9]. These 3I-2 strains were found in all armadillos and most of the indigenous patients so the authors concluded that armadillos act as a reservoir for *M. leprae* and that there is zoonotic spread of leprosy in the Southern United States. As the disease was not present in the New World before European contact, it is assumed that the spread of the disease was linked to human migrations and that

Recently it was realised that the enhanced hydrophobicity of tubercle bacilli is a key factor in aerosol transmission [57, 58]. Since it is becoming established that aerosol transmission is a prime mode for the spread of leprosy bacilli [33, 59], the transmissibility of the different manifestations of *M. leprae* should be considered. In a detailed study [33], it was demonstrated that MB/LL cases provided more transmissible bacilli than PB/TT patients. It would be of great interest to compare the relative cell envelope surface lipid composition of LL and TT leprosy bacilli to explore the possibility that the hydrophobicity of LL forms is enhanced or otherwise. It may also be possible to determine directly the relative hydrophobicity of *M. leprae* in biopsy Leprosy is primarily a disease of the peripheral nervous system. In the past, the disease would run its natural course, resulting in both specific and nonspecific bony changes plus paleopathology due to secondary infections following nerve damage [17, 18, 61]. Ancient leprosy is typically recognised by the presentation known as *facies leprosa* or rhinomaxillary syndrome, in which the nasopharynx is remodelled, the nasal spine and palate are resorbed, and eventually also the maxilla, leading to loss of the upper teeth. There are changes to the tubular bones of the hands and feet including osteoporosis caused by disuse, pitting and perforation. The long bones of the lower leg also show paleopathology associated with inflammatory periostitis [30, 62–64].

*M. leprae* ancient DNA (aDNA) was first detected in skeletal remains with typical leprosy paleopathology soon after the introduction of PCR [23]. Subsequently, many further paleopathological cases of leprosy were confirmed by *M. leprae* aDNA from across Europe and the Middle East [24–27, 30, 49–51, 64–69]. Specific *M. leprae* short DNA sequences were targeted as ancient DNA (aDNA) becomes highly fragmented over time [70]. *M. leprae* aDNA amplification has confirmed leprosy and enabled genotyping of isolates from Europe, Byzantine Turkey and Roman Egypt (**Table 1**). As additional methodologies were developed, different *M. leprae* strains were distinguished by microsatellite analysis based on aDNA repetitive sequences [27, 71] and now whole *M. leprae* genomes have been obtained from historical human skeletons [46, 53]. The results of aDNA amplification studies, WGS and lipid biomarker detection are summarised in **Table 1**.

#### **3.2. The potential of lipid biomarkers**

The detection of *M. leprae* in historical leprosy cases is assisted by the *M. leprae* cell envelope, which is composed of unusual lipids some of which can be used as specific biomarkers (**Figures 1**–**3**). The mycolic acids of *M. leprae* are restricted to homologous α- and ketomycolates [79, 80], whose major components are shown in **Figure 1**.

Characteristic mycocerosic acids are components of both phthiocerol dimycocerosate waxes (PDIMs) (**Figure 2**) [81–83] and so-called phenolic glycolipids (PGLs) (**Figure 3**) [82–85]. *M. leprae* mycocerosates unusually include major amounts of a C34 component, accompanied by small proportions of a C33 acid (**Figure 2**). *M. haemophilum* produces a PGL with the same two internal sugars (3-*O*-Me-rhamnose and 2,3-di-*O*-Me-rhamnose), but in reversed order and with different linkages (**Figure 3**). Besra et al. [13] concluded that this mycocerosate profile was essentially the same, thereby revealing a close phylogenetic link between *M. leprae* and *M. haemophilum* for the first time.


**Century (CE); location:** 

10th; Hungary, Hajdúdorog-Gyúlás:

10th; Hungary, Sárrétudvari-Hízóföld:

10th–11th; UK, Norwich: 11287, 11503, 11784

10th–12th; UK, Wharram

11th; Sweden, Björned:

11th; Hungary, Felgyő, Kettőshalmi-dűlő: 2467,

11th–12th; UK, Orkney:

11th; Hungary, Lászlófalva-Szentkirály:

9th–13th; UK, Winchester: Sk2, Sk7,

Sk8, Sk14, Sk27

11th–14th; Denmark, Refshale:2, 16, 26, 32, 36

12th; Spain, Seville: A43,

12th; Czech Republic, Žatec: AO9611, AO9731

12th–14th; Poland, Suraz:

10th–12th; Sweden, Sigtuna: 10, 32H, 3077, 3092V, 3093F, 3159Hsin, 3320V, 3401H, F13320,

10th–11th; Hungary, Püspökladány-Eperjesvölgy 11, 222, *M. leprae M. leprae*

+ + (503) 3K (222)

7/10+ 2F (3092 and 3077)

+ + 3I-1

**DNA Lipids**

**genotype**

+ Palate+

+ + Minnikin et al. [29];

+ 3 Watson et al. [49]

+ 3 Taylor and Donoghue

+ + MTB+ Donoghue et al. [72];

1/2+ 3658+ Donoghue et al. [30]

+ MTB+ Donoghue et al. [30]

+ Taylor et al. [66]

+ Montiel et al. [76]

+ Likovsky et al. [77]

+ Donoghue et al. [70];

WGS Sk18 (weak)

16+

2F

1/5+ + 2F (Refshale16) Refshale

3M (503)

3I (3093)

Toe−

222 and 503 MTB+

**Notes Publications**

The Distribution and Origins of Ancient Leprosy http://dx.doi.org/10.5772/intechopen.75260

Donoghue et al. [30]

13

Donoghue et al. [30]

Minnikin et al. [29]

51]; Schuenemann et al. [46]

Schuenemann et al.

Schuenemann et al.

Witas et al. [78]

Taylor et al. [21] Roffey *et al.* 2017 [75]

[46];

[46]

[71]

WGS Economou et al. [50,

Haas et al. [65]

**cases**

HG-56

S237

429, 503

A4

S10V3

3658

79

CC4

Sk19

Sk18

A120

A1

Percy: G708


**Century (CE); location:** 

12 Hansen's Disease - The Forgotten and Neglected Disease

1st; Israel, Akeldema, Himmon valley: SC1

1st–4th; Uzbekistan Devkesken 6: 5b

4th; Egypt, Dakhleh Oasis, Kellis 2: B116 and 7 other samples

4th–7th; Israel, Jerusalem: HZ

5th–6th; United Kingdom, Great Chesterfield: GC96

6th–7th; Israel, monastery on River Jordan: AR

T68, T108

T31, T144

domb: 22

20/1, 24/1

6th–8th; Italy, Morrione:

7th; Hungary, Szeged-Kiskundorozsma-Daruhalom dűlő II: KD271, KD517, KD518

7th; Italy, Vicenne: T18,

7th–8th; Hungary, Szentes-Kistőke: SK11

7th–9th; Hungary, Bélmegyer-Csömöki

7th–9th; Hungary, Szarvas Grexa, Téglagyár: SG-38

8th–9th; Turkey, Kovuklukaya: 9/1, 11/2,

8th–9th; Croatia, Radasinovci: 2A, 3A

8th–9th; Austria, Zwölfaxing: 70, 88

9th–10th; Czech Republic, Prušánky: 188 *M. leprae M. leprae*

**DNA Lipids**

**genotype**

+ Matheson et al. [69]

+ 3K/L/M (B116) Donoghue et al. [72];

+ + 3L Taylor et al. [27]

+ Spigelman and

+ + 3I-1 (variant) MTB – Inskip et al. [73]

+ Rafi et al. [23]

3/3+ KD517+ 3K (KD271) KD517

1/3+ (T18) 2/2+ (T18,

T144)

1/2 + (T108) Donoghue et al. [30]

+ Donoghue et al. [30]

+ + MTB lipid+ Donoghue et al. [30];

+ + Minnikin et al. [29];

3/4+ (11/2−) 1/3+ (24/1+) 3K (20/1) Minnikin et al. [29];

+ Watson et al. [49]

+ 3M Donoghue et al. [30]

2/2+ MTB DNA+

lipids+ and MTB+

DNAlipids+ (T144)

(88)

**Notes Publications**

Monot et al. [26]

Donoghue [67]

Minnikin et al. [29]; Lee et al. [28]; Donoghue et al. [30]

Donoghue et al. [30]

Molnár et al. [74]

Donoghue et al. [30]

Donoghue et al. [30]

Donoghue et al. [30]

**cases**


Cases are listed in a chronological order.

**Table 1.** Detection of ancient leprosy using aDNA and lipid biomarkers.

similar to that found in *M. leprae* in a previous study [88]. However, an analysis of three *M. leprae* isolates did not record unusually enhanced proportions of docosanoic acid [80], nor did an additional analysis of *M. haemophilum* fatty acids [89]. It is interesting to compare the profile of uncharacterised fatty acids from *M. haemophilum* in an older study [87] with the more recent study [86]. An unusual large peak, labelled 19A, in the first analysis [87] could possibly correspond to the minor branched C25 acid in the later analysis [86]. This unusual C25 acid is a potentially valuable biomarker for *M. haemophilum* so its structure and cellular loca-

**Figure 2.** Phthiocerol dimycocerosates (PDIMs) of *M. leprae*. The C33 and C34 mycocerosates are diagnostic components for *M. leprae* and *M. haemophilum,* but C29, C30 and C32 acids are shared with members of the *M. tuberculosis* complex [13,

The Distribution and Origins of Ancient Leprosy http://dx.doi.org/10.5772/intechopen.75260 15

The biomarker potential of *M. leprae* lipids has been harnessed by fluorescence high performance liquid chromatography (HPLC) of pyrenebutyric acid (PBA) esters of mycolic acid pentafluorobenzyl (PFB) esters [90] and negative-ion chemical-ionisation gas-chromatography mass-spectrometry (NI-CI GC-MS) of mycocerosate PFB esters [91, 92]. Mycolate HPLC is exemplified in **Figure 4** for standard *M. leprae* and an extract of a skeleton (Sk2) from a mediaeval leprosy hospital near Winchester, UK [21]. Fluorescent mycolate derivatives are recognised by reverse-phase HPLC (**Figure 4A**), collected and analysed by normal phase HPLC to separate the α- and ketomycolate classes (**Figure 4B**). Reverse-phase HPLC provides the size and overall composition of the α-mycolates (**Figure 4C**) and ketomycolates (**Figure 4D**)

tion should be investigated.

81, 82].

for comparison with standard *M. leprae.*

**Figure 1.** Mycolic acids of *M. leprae*. The main C78 α-mycolate and C83 ketomycolate are shown; additional homologous components are also present.

The lipid composition of '*M. lepromatosis*' remains to be determined, but limited information is available for *M. haemophilum*. In addition to α- and ketomycolates, *M. haemophilum* appears to have methoxymycolates, on thin-layer chromatography of extracts [86], but in a previous study, the patterns were unclear with material being degraded by acid methanolysis [87]. A gas chromatographic profile of *M. haemophilum* fatty acids [86] displayed an essentially typical mycobacterial profile, including tuberculostearic acid. The analysis was not extended to search for the unusual mycocerosic acids found previously in *M. haemophilum* (**Figure 2**) [13]. The only novel component was an incompletely characterised monounsaturated 2-methylbranched C25 fatty acid and an enhanced proportion of C22 docosanoic acid was noted as being

**Figure 2.** Phthiocerol dimycocerosates (PDIMs) of *M. leprae*. The C33 and C34 mycocerosates are diagnostic components for *M. leprae* and *M. haemophilum,* but C29, C30 and C32 acids are shared with members of the *M. tuberculosis* complex [13, 81, 82].

similar to that found in *M. leprae* in a previous study [88]. However, an analysis of three *M. leprae* isolates did not record unusually enhanced proportions of docosanoic acid [80], nor did an additional analysis of *M. haemophilum* fatty acids [89]. It is interesting to compare the profile of uncharacterised fatty acids from *M. haemophilum* in an older study [87] with the more recent study [86]. An unusual large peak, labelled 19A, in the first analysis [87] could possibly correspond to the minor branched C25 acid in the later analysis [86]. This unusual C25 acid is a potentially valuable biomarker for *M. haemophilum* so its structure and cellular location should be investigated.

The biomarker potential of *M. leprae* lipids has been harnessed by fluorescence high performance liquid chromatography (HPLC) of pyrenebutyric acid (PBA) esters of mycolic acid pentafluorobenzyl (PFB) esters [90] and negative-ion chemical-ionisation gas-chromatography mass-spectrometry (NI-CI GC-MS) of mycocerosate PFB esters [91, 92]. Mycolate HPLC is exemplified in **Figure 4** for standard *M. leprae* and an extract of a skeleton (Sk2) from a mediaeval leprosy hospital near Winchester, UK [21]. Fluorescent mycolate derivatives are recognised by reverse-phase HPLC (**Figure 4A**), collected and analysed by normal phase HPLC to separate the α- and ketomycolate classes (**Figure 4B**). Reverse-phase HPLC provides the size and overall composition of the α-mycolates (**Figure 4C**) and ketomycolates (**Figure 4D**) for comparison with standard *M. leprae.*

The lipid composition of '*M. lepromatosis*' remains to be determined, but limited information is available for *M. haemophilum*. In addition to α- and ketomycolates, *M. haemophilum* appears to have methoxymycolates, on thin-layer chromatography of extracts [86], but in a previous study, the patterns were unclear with material being degraded by acid methanolysis [87]. A gas chromatographic profile of *M. haemophilum* fatty acids [86] displayed an essentially typical mycobacterial profile, including tuberculostearic acid. The analysis was not extended to search for the unusual mycocerosic acids found previously in *M. haemophilum* (**Figure 2**) [13]. The only novel component was an incompletely characterised monounsaturated 2-methylbranched C25 fatty acid and an enhanced proportion of C22 docosanoic acid was noted as being

**Figure 1.** Mycolic acids of *M. leprae*. The main C78 α-mycolate and C83 ketomycolate are shown; additional homologous

*M. leprae M. leprae*

**DNA Lipids**

**genotype**

1/2+ + 3I (Jorgen 625) Jorgen 625+ Schuenemann et al.

+ 3I\* (variant) Taylor et al. [25, 27];

+ 3I/J Watson et al. [49]

2/2+ Haas et al. [65]

+ 1 Suzuki et al. [52]

+ + Donoghue et al. [72];

**Notes Publications**

[46]

[71]

Taylor and Donoghue

Minnikin et al. [29]

components are also present.

**Century (CE); location:** 

14 Hansen's Disease - The Forgotten and Neglected Disease

13th–14th; Denmark, Odense: Jorgen 625, 1020

13th–16th; UK, Ipswich, Blackfriars: 1914

13th–16th; Denmark, Odense: G483

15th–18th; Germany, Rain/Lech: R1788, R2208

Cases are listed in a chronological order.

**Table 1.** Detection of ancient leprosy using aDNA and lipid biomarkers.

18th–20th; Japan, Aomori: SK26

15th; Hungary, Szombathely: 10

**cases**

**Figure 3.** Phenolic glycolipids of *M. leprae* and *M. haemophilum.* The common phenolphthiocerol unit is attached to distinctive trisaccharides that share particular diagnostic sugars, 3-*O*-Me-rhamnose and 2,3-di-*O*-Me-rhamnose [13].

Selected ion monitoring NI-CI GC-MS analyses of mycocerosate PFB esters from Winchester skeleton Sk2 [21] and standard *M. leprae* are shown in **Figure 5**. There is good correspondence between the Sk2 extract and the standard; the Sk2 profile is unpublished work (O.Y-C. Lee, H.H.T. Wu, G.M. Taylor, K. Tucker, R. Butler, S. Roffey, P. Marter, D.E. Minnikin, G.S. Besra, G.R. Stewart, manuscript in preparation). In summary (**Table 1**), aDNA analysis with occasional lipid biomarker support has been successful in characterising ancient leprosy [21, 27, 29].

#### **3.3. Distribution and phylogeny of ancient leprosy**

Further, aDNA studies based on *M. leprae* sub-genotypes have given valuable information about the distribution of the disease in different human populations in the past [26]. The earliest known case of leprosy recognised by both skeletal paleopathology and aDNA, was from the early first millennium CE from the Ustyurt Plateau, Uzbekistan [93], with radiocarbon dating that suggests a date between the first and third centuries CE [94]. The *M. leprae*

aDNA from this location was found to be of sub-genotype 3L [27] and the variable number tandem repeat analysis identified a unique aDNA profile [71]. Sub-genotyping has revealed that in historical Europe, there are clear differences between the leprosy found in human

**Figure 4.** Mycolic acid profiles of Winchester skeleton Sk2. (A) Total mycolates, reverse phase HPLC; (B) collected total mycolates (MAs), normal phase; (C) collected α-mycolates, reverse phase; (D) Collected ketomycolates, reverse phase [21].

The Distribution and Origins of Ancient Leprosy http://dx.doi.org/10.5772/intechopen.75260 17

**Figure 4.** Mycolic acid profiles of Winchester skeleton Sk2. (A) Total mycolates, reverse phase HPLC; (B) collected total mycolates (MAs), normal phase; (C) collected α-mycolates, reverse phase; (D) Collected ketomycolates, reverse phase [21].

Selected ion monitoring NI-CI GC-MS analyses of mycocerosate PFB esters from Winchester skeleton Sk2 [21] and standard *M. leprae* are shown in **Figure 5**. There is good correspondence between the Sk2 extract and the standard; the Sk2 profile is unpublished work (O.Y-C. Lee, H.H.T. Wu, G.M. Taylor, K. Tucker, R. Butler, S. Roffey, P. Marter, D.E. Minnikin, G.S. Besra, G.R. Stewart, manuscript in preparation). In summary (**Table 1**), aDNA analysis with occasional lipid biomarker support has been successful in characterising ancient leprosy [21, 27, 29].

**Figure 3.** Phenolic glycolipids of *M. leprae* and *M. haemophilum.* The common phenolphthiocerol unit is attached to distinctive trisaccharides that share particular diagnostic sugars, 3-*O*-Me-rhamnose and 2,3-di-*O*-Me-rhamnose [13].

Further, aDNA studies based on *M. leprae* sub-genotypes have given valuable information about the distribution of the disease in different human populations in the past [26]. The earliest known case of leprosy recognised by both skeletal paleopathology and aDNA, was from the early first millennium CE from the Ustyurt Plateau, Uzbekistan [93], with radiocarbon dating that suggests a date between the first and third centuries CE [94]. The *M. leprae*

**3.3. Distribution and phylogeny of ancient leprosy**

16 Hansen's Disease - The Forgotten and Neglected Disease

aDNA from this location was found to be of sub-genotype 3L [27] and the variable number tandem repeat analysis identified a unique aDNA profile [71]. Sub-genotyping has revealed that in historical Europe, there are clear differences between the leprosy found in human

**Figure 5.** Mycocerosic acid profiles of Winchester skeleton Sk2. Selected ion monitoring NI-CI GC-MS of mycocerosic acid pentafluorobenzyl ester from A, Sk2 and B, *M. leprae* standard.

**3.4. Co-infection of leprosy and tuberculosis**

included where sub-typing was not determined.

*tuberculosis* co-infections are summarised in **Table 2**.

Leprosy was a significant problem in Scandinavia until a century ago, leading to the identification of the leprosy bacillus by Hansen in 1871 [95], although publication was delayed due to the inevitable unsuccessful attempts at culture. In Central Europe, however, leprosy was prevalent in the first millennium CE, but a subsequent decline appeared to coincide with the upsurge of tuberculosis. Support for a period of overlap between leprosy and tuberculosis has been provided by a number of clear archaeological examples of dual infection, from first century AD Israel, fourth to fifth century Roman Egypt, seventh to eleventh century Hungary, eighth to ninth century Austria to tenth to thirteenth century Sweden [30, 72]. In one particular case, it was possible to use quantitative lipid biomarker analysis to estimate the relative amount of leprosy and tuberculosis infection [28–30]. Mathematical modelling to explore the epidemiological consequences of dual infection concluded that the disappearance of leprosy could indeed be explained by *M. leprae*/*M. tuberculosis* co-infections [96]. This may explain the present absence of indigenous human leprosy in Europe. Currently characterised *M. leprae*/*M.* 

**Figure 6.** Geographical distribution of ancient leprosy sub-genotypes in the European area. Three type 3 strains are

The Distribution and Origins of Ancient Leprosy http://dx.doi.org/10.5772/intechopen.75260 19

populations from central and southern Europe, compared with northwest Europe (**Table 1** and **Figure 6**). In Scandinavia and the British Isles, there are examples of *M. leprae* genotypes 2F and 3I [21, 46, 53, 75]. In historical northwest Europe, 3I-1 sub-genotypes were common, but in Hungary, Byzantine Turkey and the Czech Republic, sub-genotypes 3K and 3M were found [30]. It is believed that these differences reflect past human population movements. In northwest Europe, people travelled from Siberia and the Arctic, whereas central Europe was colonised by successive migrations from central Asia *via* ancient routes, such as the so-called Silk Road. WGS of the 3K subtype shows that it belongs to the earliest lineage of extant *M. leprae*, now termed branch 0 [46], and therefore carries characteristics of the most recent common ancestor (MRCA), not found in other groups. The distribution of the various European subgenotypes is summarised in **Figure 6** and their phylogenetic relationship in **Figure 7**. It would be informative to have more data points for the Mediterranean basin and major countries, such as Spain, France and Germany.

**Figure 6.** Geographical distribution of ancient leprosy sub-genotypes in the European area. Three type 3 strains are included where sub-typing was not determined.

#### **3.4. Co-infection of leprosy and tuberculosis**

populations from central and southern Europe, compared with northwest Europe (**Table 1** and **Figure 6**). In Scandinavia and the British Isles, there are examples of *M. leprae* genotypes 2F and 3I [21, 46, 53, 75]. In historical northwest Europe, 3I-1 sub-genotypes were common, but in Hungary, Byzantine Turkey and the Czech Republic, sub-genotypes 3K and 3M were found [30]. It is believed that these differences reflect past human population movements. In northwest Europe, people travelled from Siberia and the Arctic, whereas central Europe was colonised by successive migrations from central Asia *via* ancient routes, such as the so-called Silk Road. WGS of the 3K subtype shows that it belongs to the earliest lineage of extant *M. leprae*, now termed branch 0 [46], and therefore carries characteristics of the most recent common ancestor (MRCA), not found in other groups. The distribution of the various European subgenotypes is summarised in **Figure 6** and their phylogenetic relationship in **Figure 7**. It would be informative to have more data points for the Mediterranean basin and major countries,

**Figure 5.** Mycocerosic acid profiles of Winchester skeleton Sk2. Selected ion monitoring NI-CI GC-MS of mycocerosic

such as Spain, France and Germany.

acid pentafluorobenzyl ester from A, Sk2 and B, *M. leprae* standard.

18 Hansen's Disease - The Forgotten and Neglected Disease

Leprosy was a significant problem in Scandinavia until a century ago, leading to the identification of the leprosy bacillus by Hansen in 1871 [95], although publication was delayed due to the inevitable unsuccessful attempts at culture. In Central Europe, however, leprosy was prevalent in the first millennium CE, but a subsequent decline appeared to coincide with the upsurge of tuberculosis. Support for a period of overlap between leprosy and tuberculosis has been provided by a number of clear archaeological examples of dual infection, from first century AD Israel, fourth to fifth century Roman Egypt, seventh to eleventh century Hungary, eighth to ninth century Austria to tenth to thirteenth century Sweden [30, 72]. In one particular case, it was possible to use quantitative lipid biomarker analysis to estimate the relative amount of leprosy and tuberculosis infection [28–30]. Mathematical modelling to explore the epidemiological consequences of dual infection concluded that the disappearance of leprosy could indeed be explained by *M. leprae*/*M. tuberculosis* co-infections [96]. This may explain the present absence of indigenous human leprosy in Europe. Currently characterised *M. leprae*/*M. tuberculosis* co-infections are summarised in **Table 2**.

**4. Origins and evolution of leprosy**

**4.2. Evolutionary origins of leprosy bacilli**

taxa is shown in **Figure 8**.

recent common ancestor (MRCA) about 13.9 million years ago [8].

**4.3. Animal and environmental sources of leprosy ancestors**

Whole genome sequencing has revealed large numbers of pseudogenes in both *M. leprae* and '*M. lepromatosis*' [7, 8, 98–100]. These genomic studies revealed that both *M. leprae* and '*M. lepromatosis*' have undergone a reductive evolution in which extensive recombination events have occurred between dispersed repetitive sequences, leading to less than half of their genomes containing functional genes. In a preliminary study [7], it was indicated that the genome of '*M. lepromatosis*' (~3.22 Mb) was 1.6% smaller than that (~3.27 Mb) of *M. leprae* [98, 99]. A comprehensive parallel study gave a similar genome size of ~3.21 for '*M. lepromatosis*' [8]. Functional comparisons revealed that whereas *M. leprae* has a defective *heme* pathway, '*M. lepromatosis*' lacked several genes needed for amino acid synthesis [8]. It is apparent that '*M. lepromatosis*' is the closest known mycobacterial taxon to the established species of *M. leprae*. Phylogenetic analysis indicates that '*M. lepromatosis*' and *M. leprae* diverged from a most

The Distribution and Origins of Ancient Leprosy http://dx.doi.org/10.5772/intechopen.75260 21

The deep origins of mycobacterial disease remain to be clearly defined [3, 47, 98, 99]. In contrast to tuberculosis, which appears to stretch back hundreds of thousands of years [57, 58], the earliest manifestations of human leprosy are found in skeletal remains only about 4000 years old [101]. However, the older participation of animal hosts cannot be ruled out, as it is increasingly evident that Pleistocene megafauna may have had a major involvement in tuberculosis evolution [58]. A possible ancestral organism to the organisms that cause leprosy may have been more like modern *M. haemophilum*, an emerging pathogen with a variety of possible natural reservoirs. The first significant link identified between *M. leprae* and *M. haemophilum* was established a quarter of a century ago in a study of the so-called 'phenolic glycolipids' (PGLs) [13]. As shown in **Figure 3**, the similarity in the oligosaccharide composition of the PGLs was striking and the mycocerosate profile (**Figure 2**) almost identical. This early key observation was subsequently reinforced by taxonomic studies that showed a close association of *M. leprae* and *M. haemophilum* [14, 15, 102]. Again, in studies comparing *M. leprae* and '*M. lepromatosis'*, *M. haemophilum* was the nearest neighbour [8, 39], as illustrated in **Figure 8**. The recent determination of a full genome (~4.23 Mb) for *M. haemophilum* confirmed the close link [16], as shown in **Figure 8**. *M. haemophilum* is consistently placed outside of the *M. leprae*/*M. lepromatosis* group but between *M. leprae* and other mycobacteria such as the *M. tuberculosis* complex. It was suggested that the reductive evolution of *M. leprae* and '*M. lepromatosis*' was not shared with the most recent common ancestor but started after the divergence of *M. haemophilum* from both taxa [16]. The relatedness of *M. haemophilum, M. leprae,* '*M. lepromatosis*' and related

To assess the involvement of ancient relatives of *M. haemophilum* in the evolution of leprosy bacilli, it is necessary to consider the ecological, environmental and animal host preferences of

**4.1. Genomics of modern leprosy**

**Figure 7.** A phylogeny of selected *M. leprae* strains. The phylogeny was derived from an alignment of genomic SNPs [46]; ancient strains are denoted in bold. Phylogenies were generated in MEGA7 [105], using Maximum Likelihood methods. Phylogenies based on Neighbour Joining methods generated similar dendrograms. The scale represents the number of substitutions per site. Bootstrap values were determined from 500 replicates. '*M. lepromatosis*' was used as an out-group (not shown). Subtypes are indicated in brackets.


**Table 2.** aDNA and lipid biomarker detection of ancient *M*. *leprae* and *M*. *tuberculosis* complex co-infections.
