Reservoirs and transmission routes of leprosy; A systematic review

Leprosy is a chronic infectious disease caused by Mycobacterium leprae (M. leprae) and the more recently discovered Mycobacterium lepromatosis (M. lepromatosis). The two leprosy bacilli cause similar pathologic conditions. They primarily target the skin and the peripheral nervous system. Currently it is considered a Neglected Tropical Disease, being endemic in specific locations within countries of the Americas, Asia, and Africa, while in Europe it is only rarely reported. The reason for a spatial inequality in the prevalence of leprosy in so-called endemic pockets within a country is still largely unexplained. A systematic review was conducted targeting leprosy transmission research data, using PubMed and Scopus as sources. Publications between January 1, 1945 and July 1, 2019 were included. The transmission pathways of M. leprae are not fully understood. Solid evidence exists of an increased risk for individuals living in close contact with leprosy patients, most likely through infectious aerosols, created by coughing and sneezing, but possibly also through direct contact. However, this systematic review underscores that human-to-human transmission is not the only way leprosy can be acquired. The transmission of this disease is probably much more complicated than was thought before. In the Americas, the nine-banded armadillo (Dasypus novemcinctus) has been established as another natural host and reservoir of M. leprae. Anthroponotic and zoonotic transmission have both been proposed as modes of contracting the disease, based on data showing identical M. leprae strains shared between humans and armadillos. More recently, in red squirrels (Sciurus vulgaris) with leprosy-like lesions in the British Isles M. leprae and M. lepromatosis DNA was detected. This finding was unexpected, because leprosy is considered a disease of humans (with the exception of the armadillo), and because it was thought that leprosy (and M. leprae) had disappeared from the United Kingdom. Furthermore, animals can be affected by other leprosy-like diseases, caused by pathogens phylogenetically closely related to M. leprae. These mycobacteria have been proposed to be grouped as a M. leprae-complex. We argue that insights from the transmission and reservoirs of members of the M. leprae-complex might be relevant for leprosy research. A better understanding of possible animal or environmental reservoirs is needed, because transmission from such reservoirs may partly explain the steady global incidence of leprosy despite effective and widespread multidrug therapy. A reduction in transmission cannot be expected to be accomplished by actions or interventions from the human healthcare domain alone, as the mechanisms involved are complex. Therefore, to increase our understanding of the intricate picture of leprosy transmission, we propose a One Health transdisciplinary research approach.

Introduction Leprosy, also called Hansen's disease, results from infection with Mycobacterium leprae (M. leprae) or Mycobacterium lepromatosis (M. lepromatosis). It is a chronic infectious disease which primarily affects the skin and peripheral nerves. It ranges from a localized to a systemic infection. Damage of peripheral nerves can lead to serious impairment and disability. [1] Transmission pathways of M. leprae are not fully understood. Solid evidence exists of an increased risk for individuals living in close contact with leprosy patients, most likely through infectious aerosols, created by coughing and sneezing, but possibly also through skin to skin contact. [2,3] Multidrug treatment (MDT) developed in the 1980s is an effective chemotherapy and has played a critical role in the worldwide reduction of the leprosy burden, by reducing human-to-human transmission. [4] However, despite widespread application of this therapy, the World Health Organization recorded 208,619 new leprosy cases globally in 2018, only slightly less than the 219,075 cases in 2011. [5] Even though leprosy has been eliminated in most countries, in certain areas endemic leprosy persists. Hansen (1874) was the first to report rod-shaped bodies resembling bacteria in cells from leprosy patients using a light microscope. [6] Before that, leprosy was thought to be of environmental or hereditary nature. [7] After the discovery by Hansen, attempts were made to grow the pathogen on an array of artificial media and in numerous animals with the purpose of studying its characteristics, and potential treatments. With an improved tissue staining technique called Ziehl-Neelsen (ZN) it became possible to detect and visualize acid fast bacilli (AFB); and the Wade-Fite modification for the demonstration of M.leprae. In cases of leprosy, unspecified non-cultivable AFB were found.
M. leprae is an obligate intracellular pathogen that has never been cultured in vitro but can be cultivated in vivo in experimental animals. Shepard demonstrated in the early 1960s that the mouse footpad (MFP) could be infected with M. leprae. [8] Characteristic of this model are the local infection and the slow replication rate. [8,9] Armadillos had been used successfully in medical research, and their cool body temperature of 32-35˚C attracted the attention of leprosy researchers. In 1971 Kirchheimer and Storrs, working in Louisiana (USA), reported the first successful experimental infection of the nine-banded armadillo with M. leprae. [10,11] Until 2008, leprosy was thought to be caused exclusively by M. leprae. Analysis of leprosy patients in Mexico by Han et al. showed that a second species, M. lepromatosis, causes leprosy as well. [12] M. leprae and M. lepromatosis diverged from a common ancestor about 13.9 million years ago (Mya). The two pathogens are similar in genome size (*3,27 MB), and were subject to extensive reductive evolution. [12,13] Protein-coding genes share 93% nucleotide sequence identity. [14] Using genome scale comparison, the worldwide distribution and evolution of M. leprae have been studied. Phylogenetically, at least four SNP types (1)(2)(3)(4) and five branches (0-4) and 16 SNP subtypes (A to P) have been distinguished. Global distribution is thought to have coincided with human migration. [15,16] In medieval times, leprosy was endemic all over Eurasia. [17] Currently, this so-called Neglected Tropical Disease is endemic in regions within countries of the Americas, Asia, and Africa. In Europe, it is now only rarely reported, and infection is acquired outside Europe. The reasons for a spatial inequality of leprosy in endemic pockets within a country are still largely unexplained.
Further research is required to clarify transmission mechanisms and natural reservoirs of leprosy pathogens. [18] In the Americas, the nine-banded armadillo (Dasypus novemcinctus) has been established as a natural host and reservoir of M. leprae, and identical M. leprae strains are shared between humans and armadillos. [19,20] A reservoir can be defined with respect to a target population as 'one or more epidemiologically connected populations or environments in which a pathogen can be permanently maintained and from which infection is transmitted to the target population'. [21] Hence, both anthroponotic and zoonotic transmission have been proposed as modes of transmission. However, Scollard argued recently that there is a general bias towards human-to-human transmission of leprosy. [22] In 2016, both M. leprae and M. lepromatosis DNA was found in red squirrels (Sciurus vulgaris) with leprosy-like lesions in the British Isles.
[23] This was highly unexpected, primarily because leprosy was considered to be a disease of humans (with the exception of the armadillo), and secondly, because M. leprae was thought to have disappeared from the United Kingdom. The M. leprae strain isolated from red squirrels is essentially the same as the one that circulated in humans in medieval England and Denmark, and is closely related to the strains carried by armadillos in the southern United States.
[23] Transmission routes from animal reservoirs are possibly interrelated with the environment in an elaborate ecological cycle, which has yet to be unraveled. This may partly explain the steady global incidence of leprosy, as effective and widespread multidrug therapy reduces only human-to-human transmission.
This systematic review aims to provide an overview of worldwide research regarding nonhuman reservoirs and transmission. Potential natural reservoirs and additional transmission routes of leprosy are discussed.
Methods of studies and result interpretation were considered, but risk of bias was not systematically scored. A discussion of the limitations of some studies is incorporated where these could have influenced the findings or conclusions made. It was not possible to formally assess publication bias due to the variation of study methods. For the PRISMA checklist accompanying the PRISMA flow diagram of the search we refer to the online supplementary data: S1 PRISMA Checklist.

Wildlife reservoirs
The nine-banded armadillo in the southern United States (see Table 1 Blood samples (1987)(1988)(1989)1997 The prevalence varied from 0% to 29.6%, depending on the location where armadillos were caught. On average, the prevalence in Louisiana was 7.1% (n = 691) and in Texas 1.1% (n = 88). The histopathological pattern and the presence of acid fast bacilli nine-banded armadillos resembled lepromatous leprosy, and suggested that it was caused by M. leprae. [27,28] There was scientific controversy about the origin of leprosy in wild armadillos.
[29] The possibility of laboratory spillover was considered, and the absence of mycobacterial infection in armadillos from the same region was reported. [30,31] Clinical and epidemiological investigations defined the disease in these armadillos, and confirmed its identity with human leprosy.
[32,33] The concern of the possible wildlife contamination caused by experimentally infected armadillos addressed in 1986 by Truman   Armadillos in other parts of the Americas (see Table 2). The presence of M. leprae and leprosy in wild nine-banded armadillos was discovered in the southern United States, and most studies on its prevalence have been performed there. Armadillos are common throughout the southern United States nowadays, but their geographic range extends from northern Argentina up to Mexico. They migrated from Mexico into the United States.
[20] Only the nine-banded armadillo is common in the southern United States, but other armadillo species are indigenous to South America. Whereas wildlife studies almost exclusively focused on the nine-banded armadillo, susceptibility for M. leprae infection by experimental inoculation was also observed for other armadillo species like the Venezuelan or Llanos long-nosed armadillo Squirrels (see Table 3). Experiments showed that ground squirrels (Ictidomys tridecemlineatus) were susceptible to infection with M. leprae. Galletti et al. (1982) observed AFB counts in these squirrels that indicated multiplication under hibernation inducing conditions. [63] Recent studies have shown that red squirrels (Sciurus vulgaris) in England and Scotland (where the endangered red squirrel is competing with the non-native gray squirrel from North America) are infected with M. lepromatosis or M. leprae. Meredith et al. (2014) reported a novel presentation of a dermatitis in 6 red squirrels from various locations throughout Scotland. All animals showed bilateral areas of variable alopecia and cutaneous swelling of the snout area, lips, eyelids, pinnae and the distal parts of all limbs. [64] Histological examination on three squirrels showed granulomatous dermatitis, and epithelioid macrophages forming sheets, in addition to large numbers of AFB. PCR of various dermatitis causing pathogens was performed. Sequencing of the hsp65 PCR amplicons from the squirrels revealed 99% sequence homology with M. lepromatosis (FJ924). The exact nature of the mycobacterium involved and the characterization required further research. In view of these findings, another British research group re-examined four stored red squirrels with epidermal hyperplasia originating from the Isle of Wight and Brownsea Island. The presence of M. lepromatosis was confirmed by PCR in all four samples. [65] A follow-up study was conducted to map the magnitude of infection in the UK.
[23] The study also first reported neural involvement in red squirrels infected with M. leprae (n = 8) and M. lepromatosis (n = 4). Samples from 13 squirrels with and 101 animals without leprosylike symptoms were examined using differential PCR for M. leprae and M. lepromatosis. M. lepromatosis was found in red squirrels from Scotland (6/44), Ireland (2/39), and the Isle of Wight (1/1). [66] All squirrels from Brownsea Island (n = 25) were positive for M. leprae. 21 squirrels without clinical signs and all 13 animals with clinical signs were PCR positive. Anti-PGLI ELISA was positive in a significant number of the red squirrel samples (see Table 3). Gray squirrels were also tested by PCR (n = 3) and for anti-PGLI antibodies (n = 4), but all samples were negative. Phylogenetic comparison of British and Irish M. lepromatosis with two Mexican M. lepromatosis strains from humans showed that they diverged from a common ancestor around 27,000 years ago. The M. leprae strain appeared closest to the one that circulated in medieval England. It is therefore likely that the original introduction of M. leprae into red squirrels occurred when leprosy was still prevalent in the region. The medieval squirrel fur Tió-Coma et al. (2020) examined sixty-one red squirrels and one Japanese squirrel (Sciurus lis) from the Netherlands and fifty-three red squirrels from Flanders, Belgium for the presence of lesions and M. leprae and M. lepromatosis DNA. [68] No clinical signs of leprosy were observed. All samples were negative in PCR analysis.
Nonhuman primates. Cases of nonhuman primates with leprosy have been reported sporadically. All case reports described here involve imported animals. These cases were observed in highly monitored animal research facilities, not in the wild.
In 1977, leprosy-like symptoms were reported in a captive chimpanzee (Pan troglodytes) by Donham and Leininger. (1991) tested sera of these chimpanzees that had been naturally infected with leprosy and of 160 other chimpanzees housed in two primate centers. [73] An ELISA antibody assay for PGL I and the non-specific mycobacterial antigen lipoarabinomannan (LAM) was used. Seven animals were positive for anti-PGLI, and five for anti-LAM antibodies. In 2010, Suzuki et al. reported leprosy in a chimpanzee imported from Sierra Leone used for medical research in Japan. [74] SNP type of the leprosy strain was determined. The strain was SNP type 4, a strain found in West Africa, in line with the origin of the chimpanzee.
In 1979, a sooty mangabey (Cerocebus atys) monkey imported into the USA from West-Africa was observed with leprosy-like symptoms in the face. [75] The etiologic agent was identified as M. leprae by the following criteria: invasion of nerves, staining properties, electron microscopic findings, non-cultivability, lepromin reactivity, infection patterns in mice and armadillos and sensitivity to sulfone. Supposedly, the animal acquired the disease from a patient with active leprosy. [76] A second captive sooty mangabey monkey with naturally acquired leprosy was reported, that had been in direct contact with the first sooty mangabey monkey, and leprosy was believed to have been transmitted between those two monkeys. [77] Hagstad (1983) screened 5 rhesus monkeys (Macaca mulatta) and 21 bonnet monkeys (Macaca radiata) in Andhra Pradesh, India. [78] The monkeys were owned as pets (n = 3) or used for begging (n = 23). The 26 monkeys were in frequent contact with handlers in a leprosy endemic area. No AFB were observed in ear lobe tissue smear slides. Valverde et al. (1998) reported the first case of leprosy in an Asian macaque (Macaca fascicularis) imported from a leprosy endemic area in the Philippines. Diagnosis of infection was obtained by a PCR specific for M. leprae. Clinical presentation, histopathological findings, and serology of anti-PGL-I were compatible with human borderline (BB) leprosy. [79] Honap et al. (2018) sequenced M. leprae genomes from frozen tissue samples of 3 of the above infected nonhuman primates (the chimpanzee from Sierra Leone, the first sooty mangabey from West Africa and the cynomolgus macaque from The Philippines). [80] M. leprae strains from the chimpanzee and sooty mangabey monkey belong to the human M. leprae Branch 4 lineage commonly found in West Africa. Phylogenetic analysis also showed that the cynomolgus macaque M. leprae strain belongs to M. Leprae Branch 0 and is most closely related to the modern human M. leprae strain S9 from New Caledonia (southwestern Pacific). In addition, samples from wild ring-tailed lemurs (Lemur catta, n = 41) originating from Madagascar and wild chimpanzees (Pan troglodytes schweinfurthii, n = 22) from Kibale National Park, Uganda were examined. DNA was extracted from the samples for detection of M. leprae and M. tuberculosis, and a variety of mycobacteria pooled with one sequence DNA using qPCR. All samples were negative.
Screening for new animal species. The detection of leprosy infection in new species has, until now, resulted from coincidental observations. Screening of new species has been performed to find potential wildlife reservoirs. Leprosy-like disease. Leprosy-like disease, attributed to mycobacteria, has been reported in a variety of animals. Phylogenetic analysis compares the genetic sequence, or a fragment thereof, and determines the similarity between two strains. It can be visualized in a family tree, where the arms indicate the temporal distance to a previous common ancestor. Direct genetic comparison of genes can reveal overlap in sequences. This can explain overlap in characteristics of pathogens.
In 1926, a cutaneous mycobacteriosis called skin-tuberculosis was reported in Indonesian water buffaloes. [83] A large series of case studies was published under the disease name Lepra bubalorum by Lobel (1936). [84,85] In 1954, Kraneveld and Roza published the post-Second World War status of the disease. They introduced the name Lepra Bovina, as the disease had been observed in bovine species other than the water buffalo. [86] There had been no progression towards clarification of the pathogen. Similar to leprosy, the microorganism could not be cultured or grown in laboratory animals. Since a last report in 1961, no more cases have been published. [87,88] It is unclear whether the condition still exists in Indonesia. Clinical symptoms and histopathological findings indicating neural involvement were not observed.
Mycobacterium lepraemurium causes leprosy-like symptoms in rats. The given name is based on the symptoms, and rather misleading, as it suggests close relation to M. leprae. M. lepraemurium is part of the M. avium-complex, a mycobacterial sub-group different from M. leprae, as was shown by phylogenetic analysis. [89] M. lepraemurium is one of the pathogens causing feline leprosy. [90][91][92][93][94] Another feline leprosy causing pathogen was discovered, and named Candidatus "Mycobacterium lepraefelis", and this pathogen is closely related to M. leprae. [95] Canine leprosy is a disease associated with members of the Mycobacterium simiae clade, and thus unrelated to M. leprae. [96,97] A thelitis-causing pathogen in dairy goats and cows appeared to be closely related to M. leprae, and was named M. uberis. [98][99][100] Environmental reservoirs (see Table 4) The environment is defined as any location outside the host, where M. leprae resides before either dying or infecting a new host. Indications for the presence of viable M. leprae in the environment can be found from several experimental studies portraying the possibility of M. leprae to survive extracellularly under a variety of conditions. [101][102][103][104][105] These studies are shown in Table 4
M. leprae retained viability tested with mouse footpad method.   [114] Sphagnum suspension into MFP and detection with anti-PGL-I antibodies.
• Two hours of direct sunlight.
• Seven days at room temperature. Zoonotic and anthroponotic transmission Filice et al. (1977) tried to assess, in a limited study, whether armadillo exposure is a risk factor for developing leprosy. Leprosy patients in Louisiana (n = 19) did not have more exposure to armadillos than neighborhood matched controls (n = 19). [116] Deps et al. (2008) carried out the same type of study in Brazilian leprosy patients and a control group of patients with chronic diseases other than leprosy. [117] Exposure to armadillos was categorized as non-existent, indirect or direct. Physical contact was considered direct contact and living in an armadillo habitat was considered indirect contact. Direct exposure was significantly higher in leprosy patients (68%, n = 506) than in the control group (48%, n = 594) with OR 2.01 (95%CI 1.36-2.99, p = 0.0001). Schmitt et al. (2010) studied the relation between armadillo meat consumption and leprosy in a case-control study of Brazilian leprosy patients (n = 121) and patients with other skin diseases (n = 242). [118] No significant difference was observed in armadillo meat consumption between the two groups: OR 0.77-1.90 (p = 0.44). The study supported, however, an association with unfavourable socioeconomic indicators as leprosy patients differed significantly (p<0.05) from controls in access to treated water (90% vs. 96%), lower family income based on minimum wage, and more contact with other leprosy patients (35% vs. (18.5%) hunted armadillos, 96 (65.8%) handled or prepared the meat for consumption, 91 (62.3%) ate armadillo meat at least once, and 27 (18.5%) ate them more than once per month. The number of leprosy patients among armadillo hunters (4 of 27, 14.8%) was significantly higher than among non-hunters (3 of 119, 1.8%) OR 6.73 (95%CI 1.41-32.09 p = 0.02). Anti-PGL I titers were different when stratifying for exposure frequency. Eating armadillo meat more than 12 times per year was associated with an insignificant increased risk of PGL I positive antibody titers (OR 1.77 95%CI 0. 64-4.89). The median anti-PGL I titer was significantly increased in this group (n = 27), compared to never eating (n = 55, p = 0,03) and eating 1-12 times per year (n = 64, p = 0.01).
Stefani et al. (2019) studied nine-banded armadillos (n = 12) and local volunteers (n = 176) in the rural endemic area of Mamiá Lake of the Coari municipality, Brazil. [121] The armadillos were supplied locally. Volunteers were examined for clinical signs of leprosy by a dermatologist. Armadillo skin, spleen, liver, lymph, adrenal glands, ovary and fallopian tubes and human skin lesions suspect of leprosy were biopsied and examined histopathologically. All tissues from armadillos were tested with qPCR for the M. leprae specific RLEP sequence. Six new leprosy patients were identified among the local volunteers. None of the armadillo samples showed M. leprae DNA or AFB. Table 5)

Potential vectors (see
Attempts at discovering vectors for M. leprae have been reported throughout the twentieth century. In older studies, leprosy bacilli viability could not be shown by culture or the use of animal models. These studies are not discussed in depth, because of their limited impact. Three studies contained only a description of the theoretical role of arthropods in the transmission of leprosy. [122][123][124] In several studies, leprosy patients were used to test the uptake of leprosy bacilli from lesions. [125][126][127][128][129][130][131] In others, leprous material from patients was used to feed insects. [132][133][134] Insects were also collected from leprosy patient's homes. [131,[135][136][137] None of these studies were able to show possible transmission by insects. At most, insects would take up the bacteria or transmit a small number of bacilli to a subsequent feeding target, but this would not be considered a valid transmission route. M. leprae is an obligate intracellular pathogen. Intracellular microorganisms have been associated with free living amoebae (FLA) without a known benefit for either. It is currently proposed to define these microorganisms and FLA as "endocytobionts". Such a relationship was studied for M. leprae. Phagocytosis of fluorescent M. leprae by amoebae was observed by Lahiri and Krahenbuhl (2008) [141] Both are known to draw blood from armadillos. The mosquitoes, however, did not meet the essential criterion for a vector: the ability to maintain an infectious load of the pathogen until the next feed. But M. leprae remained viable in kissing bugs for 20 days. Inoculation of MFP with feces of kissing bugs proved that M. leprae had remained viable. This is a well-known transmission pathway for Trypanosoma cruzi, causing the zoonotic, vector transmitted Chagas disease. T. cruzi remains viable during the kissing bug's gastrointestinal tract passage. Kissing bugs transmit disease, not by biting, but by defecating near the bite wound. Rubbing or scratching infects the wound.
Da Silva Ferreira et al. (2018) achieved similar results with the adult female tick (Amblyomma sculptum). [142] Ticks were fed on rabbit blood containing M. leprae. Detection of M. leprae RNA in the midgut and ovaries of the ticks by qPCR analysis proved viability of the M. leprae until at least 2 days after feeding. M. leprae was visualized in gut samples with LAM-antibody immune-localization. In addition, M. leprae would, in some cases, spread to the ovaries of female ticks and subsequently infect tick eggs and larvae. Infected larvae were attached to rabbit skin for a five day maturation feeding period. Afterwards, up to 10 3 viable M. leprae could be isolated from the skin samples around the bite-mark. The study showed an ecological cycle of M. leprae in an experimental setting. The research group also performed the first successful cell culture of M. leprae in tick-IDE8 cells.  M. leprae and M. lepromatosis in British and Irish red squirrels. The diagnosis of leprosy in captive non-human primates is limited to case-reports, and it is unknown to what degree species are infected. Screening for the presence of these mycobacteriae in other species has been negative, except for the finding in 2018 of genetic material of M. leprae in nasal swabs from a variety of animal species in Brazil.

Discussion
Possible routes of transmission are schematically visualized in Fig 2. It is justified to ask whether or not we can speak of an environmental reservoir. RNA indicating the presence of viable M. leprae was found in environmental soil and water samples in Brazil and India. [143] M. lepromatosis has not yet been found in such samples, but this pathogen has only been discovered recently. In addition, it was found that amoebae are capable of taking up M. leprae by phagocytosis. Inside amoebae, M. leprae remained viable for days to weeks. This mechanism might contribute to environmental survival in the absence of a mammalian host. Moreover, M. leprae can also accumulate in amoebae. This gives amoebae a possible role as a vector in transmission. The only studies on vegetation reservoirs were on sphagnum species by Kazda (1980) (Table 4). [103,104] There are no recent studies using PCR techniques on the role of vegetation as an environmental reservoir.
Humans and armadillos in the Southern United States share a specific M. leprae strain (SNP subtype 3I-2-v15). This finding is highly suggestive of a zoonotic and/or an anthroponotic transmission pathway between humans and armadillos. Exposure of humans to armadillos can eventually lead to leprosy and/or increased anti-PGL I antibody levels. Exposure to an animal or to animals' excreta is influenced by behavior. For example, hunting and/or preparing armadillos for eating can each be expected to have a different risk of transmission, based on differences in exposure intensity and frequency. It is unclear whether transmission risk by rate of exposure to infected mammals is confounded by rate of exposure to an infected environment. This could be caused by shedding of leprosy bacteria by infected mammals.
At this moment, there is no evidence for a role for vectors in transmission. However, recent laboratory studies have shown a potential role for insects. M. leprae remains viable in the gastrointestinal tract of kissing bugs and is experimentally transmittable to the MFP through their feces, similarly to the transmission mechanism of T. cruzi. Kissing bugs can defecate after biting, and defecation near the bitten area transmits viable T. cruzi when the itchy bitten area is scratched, and feces is rubbed into the wound. In the same study, it was found that M. leprae does not remain viable within the gut of mosquitoes. It also has been shown that M. leprae can remain viable and reproduce in ticks, and spread to tick ovaria and eggs. Larvae from hatched eggs in turn are able to transmit viable M. leprae into a host during skin-bound maturation.
Animals can be affected by leprosy-like diseases, caused by pathogens phylogenetically closely related to M. leprae. Feline leprosy is caused by several pathogens, of which Candidatus "Mycobacterium lepraefelis" is closely related to M. leprae. M. uberis, the causative agent of nodular thelitis, is also closely related to M. leprae. M. haemophilum, which has been reported to infect the skin of immunocompromised human patients, was also found to be closely related to M. leprae. [144] The pathogen causing a mycobacteriosis called Lepra Bubalorum in Indonesian water buffaloes could also be related to M. leprae, but further research is needed to confirm this. These mycobacteria have been proposed to be grouped as a M. leprae-complex. [145] Such a classification connects the related mycobacteria, similar to species of the M. aviumcomplex. Insights from the transmission and reservoirs of members of the M. leprae-complex could be relevant for other pathogens in the complex. Currently, knowledge on the exact transmission mechanisms of M. leprae is poor. Using insights on related pathogens is a potentially efficient way to progress.
The studies included in this review differ in methods to determine the causative microorganism of leprosy. Techniques have developed over time from identifying unspecified non-cultivable acid-fast bacteria to specific DNA sequencing. The ability to detect M. leprae DNA with PCR techniques drastically changed the methods of modern studies. The variety of and changes in analysis methods for detection of M. leprae have resulted in poor standardization of methods, each with their particular strengths and weaknesses. This is also relevant for studies on M.leprae and M.lepromatosis in epidemiological settings. Anti-PGL I antibody detection in  blood or sera indicates past or present exposure to M. leprae but provides little information on current infection status. Studies on zoonosis and prevalence should be performed with sample sizes determined by the population sizes. Results of these studies have to be reported with confidence intervals, as this would substantiate the extrapolation value of the studies. Many studies in this review lack this method of sample size determination. Geographic origins of the animals are described clearly in most armadillo prevalence studies. It is seen that prevalence fluctuates strongly between neighboring geographic regions. This limits the value of spatially and temporally randomly acquired (frozen) samples in screening studies, which often do not represent a wildlife (sub)population. Seldom are internal control standards like those normally used in clinical laboratories incorporated. This lack of rigor contributes to the anecdotal assessment sometimes given to molecular studies reporting M. leprae in non-human sources. M.leprae rRNA has been used as a viability marker in environmental studies. [108][109][110][111] Accuracy of this method was suggested to be inadequate. [146] A reliable standard for viability is needed. Standardization in environmental studies should be increased by first developing appropriate definitions and sample criteria.
This systematic review underscores that human-to-human transmission is not the only way leprosy can be acquired. The transmission of this disease is probably much more complicated than was thought before, as indicated in Fig 2. Transmission of M. leprae involves several factors and pathways. Fig 2 shows the animal and environmental factors that might play a role in the persisting prevalence of leprosy. The proposed factors and mechanisms transgress the disciplines of human healthcare. A reduction in transmission cannot be expected to be accomplished by actions or interventions from the human healthcare domain alone, as the mechanisms involved are complex. Therefore, to increase our understanding of the intricate picture of leprosy transmission, a One Health transdisciplinary research approach is required. [138] This entails integrating human, animal, and environmental health aspects to further elucidate the transmission mechanisms and patterns of M. leprae and M. lepromatosis. In addition, geographically tailored methods-combining epidemiological, laboratory, and anthropologic data-are needed to better understand the ecological differences between leprosy pockets.