**Author details**

Lina Fernández and Wellman Ribón\*

\*Address all correspondence to: wellmanribon@yahoo.es

Universidad Industrial de Santander, Bucaramanga, Colombia

#### **References**


**2. Conclusion**

4 Hansen's Disease - The Forgotten and Neglected Disease

of a disease considered as unattended.

Lina Fernández and Wellman Ribón\*

\*Address all correspondence to: wellmanribon@yahoo.es

[3] Pérez Y. Lepra y coleccionismo en Colombia; 2011

ncbi.nlm.nih.gov/pmc/articles/PMC3983108/

Universidad Industrial de Santander, Bucaramanga, Colombia

[1] Pastrana F, Ramírez C, Moreno E, Ramírez H, Díaz C. Impacto de la lepra en la historia;

[5] Nóbrega A, Talhari C, Ozório M, Talhari S. PCR-Based Techniques for Leprosy Diagnosis: From the Laboratory to the clinic. PLoS Neglected Tropical Diseases. 2014. https://www.

[2] Lazareto de contratación. Lepra en el mundo. 2010. https://ellazareto.wordpress.com

2012. http://www.medigraphic.com/pdfs/folia/fd-2012/fd121f.pdf

[4] Organización Panamericana de la Salud. Pensemos en Lepra; 2015

**Author details**

**References**

Although the control of leprosy in the world was achieved, it has not yet been eradicated, and the lack of an effective diagnostic method is one of the limitations in the control of the disease, since the long period of incubation of the disease and the dissemination of *M. leprae* mean that the conventional methodologies used are not conclusive and are only useful in symptomatic patients or in those with physical changes, and infected cohabitants or patients without symptoms or injuries are not diagnosed in a timely manner. Therefore, molecular methodologies are an alternative of causality and are needed for the diagnosis of the disease. The evolution of the disease and the continuous use of basic methodologies for its diagnosis highlight the importance of implementing molecular methods to achieve early diagnosis of the disease and thus diminishing the emergence of disabling forms, since methods based on PCR are capable of generating large amounts of DNA, analysis of genetic variability, typing of strains, either through the use of genetic markers, repeated sequences, genetic polymorphisms, microsatellites, and white sequences, among others, demonstrating that PCR is the method of the future for the diagnosis of leprosy, its sensitivity, specificity, diversity, and simplicity allows identifying sources of infection, patterns of transmission, monitoring treatment, and detecting resistance to drugs of the disease, which would be of great support for follow-up and timely treatment sought by health programs, and thus maintaining the control


**Chapter 2**

**Provisional chapter**

**The Distribution and Origins of Ancient Leprosy**

**The Distribution and Origins of Ancient Leprosy**

DOI: 10.5772/intechopen.75260

Human leprosy is primarily caused by *Mycobacterium leprae*, but also by the related '*M. lepromatosis'*. Ancient leprosy can be recognised in archaeological materials by the paleopathology associated with multi-bacillary or lepromatous forms of the disease. Whole *M. leprae* genomes have been obtained from human skeletons, and diagnostic aDNA fragments have been recovered. The derived *M. leprae* phylogenies, based on single nucleotide polymorphisms, mirror past human migrations, as *M. leprae* is usually an obligate pathogen. The detection of *M. leprae* in historical leprosy cases is assisted by the hydrophobic *M. leprae* cell envelope, which is composed of unusual lipids that can be used as specific biomarkers. Lipid biomarkers are more stable than aDNA and can be detected directly without amplification. Indigenous human leprosy is extinct in Western Europe, but recently, both *M. leprae* and '*M. lepromatosis*' were found in British red squirrels. Leprosy may also be found in ninebanded armadillos (*Dasypus novemcinctus*) where it can cause a zoonotic human infection. Certain leprosy-like diseases, caused by uncultivable species in cats, for example, may be related to *M. leprae*. The closest extant relatives of leprosy bacilli are probably members of the *M. haemophilum* taxon, emerging pathogens with genomic and lipid biomarker similarities.

**Keywords:** ancient DNA, lipid biomarkers, genotyping, leprosy, paleopathology,

Leprosy (Hansen's disease) is a chronic infectious disease that has been recognised over millennia. In the majority of human cases, it is caused by *Mycobacterium leprae*, but recently a

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Helen D. Donoghue, G. Michael Taylor,

Helen D. Donoghue, G. Michael Taylor,

Gurdyal S. Besra and David E. Minnikin

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Oona Y-C. Lee, Houdini H.T. Wu,

and David E. Minnikin

**Abstract**

evolution

**1. Introduction**

http://dx.doi.org/10.5772/intechopen.75260

Tom A. Mendum, Graham R. Stewart, Leen Rigouts,

Tom A. Mendum, Graham R. Stewart, Leen Rigouts, Oona Y-C. Lee, Houdini H.T. Wu, Gurdyal S. Besra

#### **The Distribution and Origins of Ancient Leprosy The Distribution and Origins of Ancient Leprosy**

DOI: 10.5772/intechopen.75260

Helen D. Donoghue, G. Michael Taylor, Tom A. Mendum, Graham R. Stewart, Leen Rigouts, Oona Y-C. Lee, Houdini H.T. Wu, Gurdyal S. Besra and David E. Minnikin Helen D. Donoghue, G. Michael Taylor, Tom A. Mendum, Graham R. Stewart, Leen Rigouts, Oona Y-C. Lee, Houdini H.T. Wu, Gurdyal S. Besra and David E. Minnikin

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75260

#### **Abstract**

Human leprosy is primarily caused by *Mycobacterium leprae*, but also by the related '*M. lepromatosis'*. Ancient leprosy can be recognised in archaeological materials by the paleopathology associated with multi-bacillary or lepromatous forms of the disease. Whole *M. leprae* genomes have been obtained from human skeletons, and diagnostic aDNA fragments have been recovered. The derived *M. leprae* phylogenies, based on single nucleotide polymorphisms, mirror past human migrations, as *M. leprae* is usually an obligate pathogen. The detection of *M. leprae* in historical leprosy cases is assisted by the hydrophobic *M. leprae* cell envelope, which is composed of unusual lipids that can be used as specific biomarkers. Lipid biomarkers are more stable than aDNA and can be detected directly without amplification. Indigenous human leprosy is extinct in Western Europe, but recently, both *M. leprae* and '*M. lepromatosis*' were found in British red squirrels. Leprosy may also be found in ninebanded armadillos (*Dasypus novemcinctus*) where it can cause a zoonotic human infection. Certain leprosy-like diseases, caused by uncultivable species in cats, for example, may be related to *M. leprae*. The closest extant relatives of leprosy bacilli are probably members of the *M. haemophilum* taxon, emerging pathogens with genomic and lipid biomarker similarities.

**Keywords:** ancient DNA, lipid biomarkers, genotyping, leprosy, paleopathology, evolution

#### **1. Introduction**

Leprosy (Hansen's disease) is a chronic infectious disease that has been recognised over millennia. In the majority of human cases, it is caused by *Mycobacterium leprae*, but recently a

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

related organism, '*M. lepromatosis*', has also been implicated [1] and appears to cause diffuse lepromatous leprosy (DLL). Both organisms are obligate pathogens that are uncultivable in cell-free growth media. Although '*M. lepromatosis*' has been the subject of many recent publications [2–5], there is still discussion about whether it is a distinct species [6]; currently, it is a name without standing in nomenclature (http://www.bacterio.net/-nonvalid.html). Leprosy is primarily a disease of peripheral nerves and skin, but it also affects bones. The genomes of *M. leprae* and '*M. lepromatosis*' have been sequenced, and it is clear that they diverged from a common ancestor many millennia ago [7, 8]. The genome of '*M. lepromatosis*' confirms a close but distinct relationship with *M. leprae*, and both organisms can also cause disease in animals, such as armadillos and squirrels [9–12]. The closest ancestors of these leprosy bacilli are probably relatives of *M. haemophilum* that has genomic and lipid biomarker similarities [13–16].

Schwann cells in nerves and macrophages in the skin [32]. The infection is transmitted by direct contact with untreated cases or healthy carriers or via infectious aerosols [33]. The clinical presentation of leprosy depends upon the cell-mediated immune (CMI) response to infection. If the host has an effective CMI response, few lesions develop, and there are only scanty bacilli in the tissues. However, some patients are anergic to *M. leprae*, so develop lepromatous leprosy with ineffective antibodies, a high bacterial load and multiple lesions. The clinical presentation of leprosy in a patient can vary over time, so there are borderline leprosy types where the immune response is unstable. It can show a wide range of clinical presentations from tuberculoid leprosy (TT) through borderline forms: borderline tuberculoid (BT), borderline borderline (BB), borderline lepromatous (BL) to lepromatous leprosy (LL) [34]. A recent World Health Organization classification scheme recognises a simplified two-category system of either paucibacillary or multibacillary forms of leprosy [35]. The histopathology of skin lesions varies from compact granulomas to diffuse infiltration of dermis, which largely depend upon the immune status of the patient and may not be in agreement with the clinical diagnosis [36, 37]. The mycobacterial antigens can activate a chronic inflammatory response that is exacerbated by pro-inflammatory cytokines. Therefore, in late stages of leprosy, there may be no *M. leprae* bacilli in the tissues, but residual mycobacterial

The Distribution and Origins of Ancient Leprosy http://dx.doi.org/10.5772/intechopen.75260 9

antigens can drive an inflammatory response that causes neurological damage [38].

'*M. lepromatosis*' appears to have a tropism for endothelial cells and can give rise to vasculitis and necrotic erythema. It seems to be less common than *M. leprae* and was initially believed to be geographically restricted to patients from Mexico and the Caribbean, where it was identified in patients suffering from diffuse lepromatous leprosy (DLL) [1, 39–41]. It was subsequently recognised in Brazil, Myanmar, Canada and Singapore and in mixed infections with *M. leprae* [3, 4]. Symptoms, characteristic of 'Lucio's phenomenon', have been associated with '*M. lepromatosis'* [1, 40, 42]. A case of two Mexican siblings infected with '*M. lepromatosis*' indicates facile transmission [5, 6]. However, '*M. lepromatosis*' has recently been found in the wild Eurasian red squirrel, *Sciurus vulgaris*, in the British Isles, from England, Scotland and Wales [11, 12]. In addition, *M. leprae* was found in red squirrels on the Isle of Wight and Brownsea Island, close to the south coast of England [43, 44]. This was very surprising, as although indigenous leprosy was prevalent in the human population of the British Isles in the first millennium (CE), it is now believed to be extinct. In these modern squirrels, the macroscopic signs and histopathology were characteristic of lepromatous leprosy, but no pathological differences were noted between infections caused by '*M. lepromatosis*' or *M. leprae* [12, 45]. The strain of '*M. lepromatosis*' in British wild squirrels is genetically distinguishable from Mexican strains found in modern day humans, and it appears that these strains diverged from a common ancestor about 26,000 years ago [12]. However, the *M. leprae* strain found in British red squirrels is similar to a strain found in human remains from a mediaeval leprosy hospital in Winchester [46], only 70 km from the Isle of Wight and Brownsea Island. One suggestion is that, in the past, humans may have been infected through direct contact with red squirrels as these were prized for their meat and fur [12]. They were also kept as pets, as is evident from various illustrated medieval manuscripts and art, for example 'A Lady with a Squirrel and a Starling' by Holbein the Younger (painted ca. 1526–1528, National Portrait Gallery, London).

**2.2. '***Mycobacterium lepromatosis***'**

Initially, ancient leprosy was recognised by the paleopathology associated with multi-bacillary or lepromatous forms of the disease [17, 18]. Leprosy causes skeletal changes in the rhinomaxillary area, including pitting and perforation in the palate, resorption of the nasal spine and the maxilla leading to loss of the upper teeth. The tubular bones of the hands and feet are frequently involved. In the tibia and fibula, inflammatory periostitis can be recognised; the metatarsals and metacarpals are often resorbed so these small bones develop a pencil shape. In sub-adult individuals afflicted with multibacillary leprosy, the development of the secondary dentition can be affected, leading to a rare condition, *leprogenic odontodysplasia* (LO), where the incisor teeth exhibit a characteristic root constriction [19]. Intriguingly, this has been seen only in archaeological cases and not in a clinical setting. Cases have been described from medieval Denmark [20] and in four individual medieval inhumations from the St. Mary Magdalen, Winchester leprosarium [21]. Subtle skeletal changes like grooving on the volar surfaces of the proximal phalanges may also accompany paucibacillary forms of leprosy that cause digital contracture or loss of pain sensation [22].

Suspected leprosy cases can be confirmed by the detection of *M. leprae* ancient DNA (aDNA) [23, 24] and further characterised by repetitive DNA sequences and genotyping [25, 26]. The aDNA detection of *M. leprae* in historical cases is probably assisted by the protective presence of unusual lipids in the *M. leprae* cell envelope. These lipids can be used as specific biomarkers; they are more stable than aDNA and can be directly detected without amplification (*vide infra*). Lipid biomarkers have been used to confirm aDNA findings [21, 27–29]. However, due to their stability, lipid biomarkers can also confirm a diagnosis of leprosy initially based on paleopathology, even in the absence of aDNA [30].

## **2. Causes and distribution of modern leprosy**

#### **2.1.** *Mycobacterium leprae*

*M. leprae*, the main cause of leprosy in humans, is a slow-growing intracellular *Mycobacterium* and the average incubation period of the disease is about 5 years, although symptoms may occur within 1 year or up to 20 years after infection [31]. Leprosy mainly affects the skin, peripheral nerves, the mucosa of the upper respiratory tract and the eyes, as *M. leprae* has a tropism for Schwann cells in nerves and macrophages in the skin [32]. The infection is transmitted by direct contact with untreated cases or healthy carriers or via infectious aerosols [33]. The clinical presentation of leprosy depends upon the cell-mediated immune (CMI) response to infection. If the host has an effective CMI response, few lesions develop, and there are only scanty bacilli in the tissues. However, some patients are anergic to *M. leprae*, so develop lepromatous leprosy with ineffective antibodies, a high bacterial load and multiple lesions. The clinical presentation of leprosy in a patient can vary over time, so there are borderline leprosy types where the immune response is unstable. It can show a wide range of clinical presentations from tuberculoid leprosy (TT) through borderline forms: borderline tuberculoid (BT), borderline borderline (BB), borderline lepromatous (BL) to lepromatous leprosy (LL) [34]. A recent World Health Organization classification scheme recognises a simplified two-category system of either paucibacillary or multibacillary forms of leprosy [35]. The histopathology of skin lesions varies from compact granulomas to diffuse infiltration of dermis, which largely depend upon the immune status of the patient and may not be in agreement with the clinical diagnosis [36, 37]. The mycobacterial antigens can activate a chronic inflammatory response that is exacerbated by pro-inflammatory cytokines. Therefore, in late stages of leprosy, there may be no *M. leprae* bacilli in the tissues, but residual mycobacterial antigens can drive an inflammatory response that causes neurological damage [38].

#### **2.2. '***Mycobacterium lepromatosis***'**

related organism, '*M. lepromatosis*', has also been implicated [1] and appears to cause diffuse lepromatous leprosy (DLL). Both organisms are obligate pathogens that are uncultivable in cell-free growth media. Although '*M. lepromatosis*' has been the subject of many recent publications [2–5], there is still discussion about whether it is a distinct species [6]; currently, it is a name without standing in nomenclature (http://www.bacterio.net/-nonvalid.html). Leprosy is primarily a disease of peripheral nerves and skin, but it also affects bones. The genomes of *M. leprae* and '*M. lepromatosis*' have been sequenced, and it is clear that they diverged from a common ancestor many millennia ago [7, 8]. The genome of '*M. lepromatosis*' confirms a close but distinct relationship with *M. leprae*, and both organisms can also cause disease in animals, such as armadillos and squirrels [9–12]. The closest ancestors of these leprosy bacilli are probably relatives of *M. haemophilum* that has genomic and lipid biomarker similarities [13–16].

Initially, ancient leprosy was recognised by the paleopathology associated with multi-bacillary or lepromatous forms of the disease [17, 18]. Leprosy causes skeletal changes in the rhinomaxillary area, including pitting and perforation in the palate, resorption of the nasal spine and the maxilla leading to loss of the upper teeth. The tubular bones of the hands and feet are frequently involved. In the tibia and fibula, inflammatory periostitis can be recognised; the metatarsals and metacarpals are often resorbed so these small bones develop a pencil shape. In sub-adult individuals afflicted with multibacillary leprosy, the development of the secondary dentition can be affected, leading to a rare condition, *leprogenic odontodysplasia* (LO), where the incisor teeth exhibit a characteristic root constriction [19]. Intriguingly, this has been seen only in archaeological cases and not in a clinical setting. Cases have been described from medieval Denmark [20] and in four individual medieval inhumations from the St. Mary Magdalen, Winchester leprosarium [21]. Subtle skeletal changes like grooving on the volar surfaces of the proximal phalanges may also accompany paucibacillary forms of leprosy that cause digital

Suspected leprosy cases can be confirmed by the detection of *M. leprae* ancient DNA (aDNA) [23, 24] and further characterised by repetitive DNA sequences and genotyping [25, 26]. The aDNA detection of *M. leprae* in historical cases is probably assisted by the protective presence of unusual lipids in the *M. leprae* cell envelope. These lipids can be used as specific biomarkers; they are more stable than aDNA and can be directly detected without amplification (*vide infra*). Lipid biomarkers have been used to confirm aDNA findings [21, 27–29]. However, due to their stability, lipid biomarkers can also confirm a diagnosis of leprosy initially based on

*M. leprae*, the main cause of leprosy in humans, is a slow-growing intracellular *Mycobacterium* and the average incubation period of the disease is about 5 years, although symptoms may occur within 1 year or up to 20 years after infection [31]. Leprosy mainly affects the skin, peripheral nerves, the mucosa of the upper respiratory tract and the eyes, as *M. leprae* has a tropism for

contracture or loss of pain sensation [22].

8 Hansen's Disease - The Forgotten and Neglected Disease

paleopathology, even in the absence of aDNA [30].

**2.1.** *Mycobacterium leprae*

**2. Causes and distribution of modern leprosy**

'*M. lepromatosis*' appears to have a tropism for endothelial cells and can give rise to vasculitis and necrotic erythema. It seems to be less common than *M. leprae* and was initially believed to be geographically restricted to patients from Mexico and the Caribbean, where it was identified in patients suffering from diffuse lepromatous leprosy (DLL) [1, 39–41]. It was subsequently recognised in Brazil, Myanmar, Canada and Singapore and in mixed infections with *M. leprae* [3, 4]. Symptoms, characteristic of 'Lucio's phenomenon', have been associated with '*M. lepromatosis'* [1, 40, 42]. A case of two Mexican siblings infected with '*M. lepromatosis*' indicates facile transmission [5, 6]. However, '*M. lepromatosis*' has recently been found in the wild Eurasian red squirrel, *Sciurus vulgaris*, in the British Isles, from England, Scotland and Wales [11, 12]. In addition, *M. leprae* was found in red squirrels on the Isle of Wight and Brownsea Island, close to the south coast of England [43, 44]. This was very surprising, as although indigenous leprosy was prevalent in the human population of the British Isles in the first millennium (CE), it is now believed to be extinct. In these modern squirrels, the macroscopic signs and histopathology were characteristic of lepromatous leprosy, but no pathological differences were noted between infections caused by '*M. lepromatosis*' or *M. leprae* [12, 45]. The strain of '*M. lepromatosis*' in British wild squirrels is genetically distinguishable from Mexican strains found in modern day humans, and it appears that these strains diverged from a common ancestor about 26,000 years ago [12]. However, the *M. leprae* strain found in British red squirrels is similar to a strain found in human remains from a mediaeval leprosy hospital in Winchester [46], only 70 km from the Isle of Wight and Brownsea Island. One suggestion is that, in the past, humans may have been infected through direct contact with red squirrels as these were prized for their meat and fur [12]. They were also kept as pets, as is evident from various illustrated medieval manuscripts and art, for example 'A Lady with a Squirrel and a Starling' by Holbein the Younger (painted ca. 1526–1528, National Portrait Gallery, London).

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

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

material, using micro fluorescence methods [60]. The evasion of airways epithelial clearance

The Distribution and Origins of Ancient Leprosy http://dx.doi.org/10.5772/intechopen.75260 11

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 perios-

*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

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 ketomyco-

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*

[33, 59] may be encouraged by enhanced hydrophobicity of infective agents.

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

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

titis [30, 62–64].

summarised in **Table 1**.

**3.2. The potential of lipid biomarkers**

and *M. haemophilum* for the first time.

lates [79, 80], whose major components are shown in **Figure 1**.

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

#### **2.4. Transmission of leprosy**

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 material, using micro fluorescence methods [60]. The evasion of airways epithelial clearance [33, 59] may be encouraged by enhanced hydrophobicity of infective agents.
