Prevalence of Mycobacterium leprae in the environment : A review

The purpose of this review was to study the presence of Mycobacterium leprae in the environment and its relation with meteorological variables such as temperature and humidity. There are reports, which provide evidence that meteorological factors such as temperature and soil humidity can influence the dynamics of M. leprae. However, leprosy cases are registered both in the rainy and dry seasons, indicating that M. leprae remains viable in different environmental conditions. Therefore, it is difficult to establish the meteorological pattern(s) required to maintain the bacilli in the environment. The extensive area of endemic countries, endemicity in the bordering countries, diversity of biomes, and lack of urban infrastructure together with weather features are possible factors that influence transmission of the disease.


INTRODUCTION
Leprosy is a chronic infectious disease caused by the bacillus Mycobacterium (M.) leprae.The disease, which is prevalent in most tropical and subtropical regions of the world (World Health Organization (WHO), 2014), can manifest itself in different clinical forms depending on the type of host immune response.
In 2011, the WHO published the Enhanced Global Strategy for minimizing the leprosy burden, in order to reduce the disease incidence and its physical, social, and economic consequences.Brazil and India are responsible for 90% of the leprosy cases in the world.In 2012, 232,857 new cases of leprosy were registered worldwide.Regions with the highest number of detected cases are Southeast Asia (71%), the Americas (15.5%),India (134,752 cases),and Brazil (33,303 cases) according to the WHO (2013).
The transmission mechanism for leprosy remains unclear, despite it being studied for centuries.For a long time, it was believed that the only source of transmission of M. leprae, the main etiologic agent, was multibacillary patients not receiving treatment.There are, however, a considerable number of epidemiological and microbiological observations indicating that environmental sources (Loughry et al., 2009) can also play an important role in transmission of the disease by indirect contact (Kadza, 2000).
Molecular biological studies have revealed the presence of bacilli in the environment.These findings strengthen the hypothesis of transmission of the disease independent of contact with patients, and/or maintenance of viable bacilli in the environment for long periods.As a corollary, meteorological conditions in the environment that favor the maintenance and viability of the bacilli must also be important to the disease transmission.To evaluate this hypothesis, we analyzed existing scientific literature on the presence of M. leprae in the environment, and its relation with meteorological variables.

LEPROSY RESEARCH: FUTURE TARGETS AND PRIORITIES
Of the 13 original articles on the association between M. leprae and the environment, eight involved relative humidity (%), and one each involved: temperature (°C) and rainfall (mm), only rainfall, temperature and humidity, and culture of Acanthamoeba castellanii and climate variations (Figure 1).
In the 1980s, studies aimed to find possible relationships between the environment and M. leprae.One of the techniques used in this period was Ziehl-Neelsen (ZN) staining, which is specific for acid-alcohol-resistant bacilli (BAAR) and non-cultivable acid-fast bacilli (NCAFB) (Salem and Fonseca, 1982;Kadza, 1981).
ZN staining is a bacilloscopic procedure that effectively stains acid-alcohol resistant mycobacteria; the staining intensity varies with the species of mycobacterium the microorganisms obtained from the soil or water samples (Wahyuni et al., 2010).
From 1980 to 1990, viability of the bacilli was tested under different environmental conditions.The specificity of the bacilli was determined using a multiplication method of M. leprae in mouse paws.Shepard in 1960 revealed their viability, the monitoring tests chemotherapeutic and levels of drug resistance using inoculation of M. leprae in the footpads of normal and immunecompromised mice (Azulay et al., 2008).
In 2000, research focusing on cultivation of the bacillus in vitro was unsuccessful, although some studies have shown evidence of metabolic activity in vitro (Levy and Ji, 2006).
Genome analyses of the mycobacterium have shown that cultivation on artificial media is not possible.This is because even less than half of the genome contains functional genes; the majority consists of inactivated or pseudo genes.Moreover, the genome has undergone progressive reduction, accompanied by genetic degradation and a decrease in size.These evolutionary changes originated with the elimination of important metabolic pathways and related ancillary functions of M. leprae, particularly those involved in catabolism (Levy and Ji, 2006).
The absence of experimental models that mimic the disease in humans, and the inability to grow M. leprae in vitro represent historically important limitations in the development of appropriate tools for the control of leprosy.However, owing to advances in molecular biology techniques, many studies on the M. leprae genome have been conducted (Silvestre, 2011).
From 2000 onwards, amplification of specific DNA sequences of the bacillus became possible by polymerase chain reaction (PCR).This technique was advantageous in that it required small numbers of the bacilli and was highly sensitive (Donoghue et al., 2001).
Recent publications on the genome sequences of M. leprae, M. tuberculosis, M. bovis and M. smegmatis, along with the almost-complete sequences of several other mycobacterial species (M.avium, M. marinum, M. paratuberculosis, and M. ulcerans) have enabled the identification of unique and specific proteins in M. leprae (Cole et al., 2001;Geluk et al., 2005).
The main method carried out in the study comprised PCR of samples from soil and water, by having high sensitivity of the bacillus, since the sequences of ribosomal RNA (rRNA) (Silvestre, 2011;Donoghue et al., 2001).
New typing methods to conclusively identify M. leprae have evolved with the technique of multiple-locus value analysis (MLVA).This technique ensures greater genetic differentiation in a wide range of samples with allelic diversity within a community, and thus, is useful in the detection of leprosy transmission (Young et al., 2004;Groathouse et al., 2004;Zhang et al., 2005).
Table 1 shows studies on M. leprae in the environment and its relation with meteorological variables published between 1980 and 2014, presented in chronological order and by the variables analyzed.In terms of temporal evolution, the highest number of studies has been published since 2000, the majority being conducted in India.
Further, analyses of soil samples have shown that M.
leprae also has non-human reservoirs such as armadillos and protozoans.Moreover, environments favorable to pathogen survival, such as water, soil, sphagnum, as well as other factors are propitious to its transmission (Desikan and Sreevatsa, 1995;Truman, 2005;Turankar et al., 2012).The presence of M. leprae in water sources reflects its association with protozoans or invertebrate hosts, as well as some free-living mycobacteria (Whan et al., 2006).
Studies on free-living amoebae have revealed an association with water consumed by the population, and in some cases, with treated water (Falkinham et al., 2001).Wheat et al. (2014) showed that M. leprae can remain viable long-term in environmental ubiquitous freeliving amoebae and retain the virulence in mouse model.
M. leprae can survive outside its main host in free-living protozoans as Acanthamoeba castellanii for 4 days without apparent difficult.These results show that freeliving terrestrial or water-borne protozoans can act as "wild macrophages," facilitating survival of the bacilli in the environment when expelled from the human host (Lahiri and Krahenbuhl, 2008).A recent experimental study verified that M. leprae remains viable for up to eight months within amoebic cysts (Wheat et al., 2014).
Multibacillary patients spread the leprosy bacilli through their nasal secretions, which in tropical regions remain viable for up to 9 days, and up to 46 days in moist soil at room temperature (Desikan, 1997).In the province of Maluku, Indonesia, where leprosy is endemic, 27% of the villagers were found to carry the bacillus within their nasal cavities (Izumi et al., 1998).
A study carried out in West Bengal, India, in 2009 analyzed 207 soil samples in areas with active cases of leprosy.M. leprae was viable in 28 of these samples.Single nucleotide polymorphism (SNP) testing of the bacilli found in both the environment and in patients revealed that they were of the same genotype.The study demonstrated the potential role of viable bacilli in the environment as a source of disease transmission (Turankar et al., 2012).However, it had limitations with regard to identifying the metabolic activity of the bacilli, as well as mechanisms of extended survival and transmission of M. leprae in different environments.Furthermore, it was observed that the proportion of samples with evidence of M. leprae was higher in humid areas (Izumi et al., 1998;Desikan, 1997).These findings indicate that humidity and rain helps the bacilli to survive for longer periods in the environment.
In a study conducted in Ghatampur, India, in 2008, 80 soil samples were collected, of which 40 were from residential areas housing leprosy patients, while the other 40 were from places with no patients identified (control).Of the 28 soil samples positive for viable M. leprae, 22 were from the residential areas, while 6 were from the control areas.Thus, the bacilli released by patients during coughing and sneezing can survive for varying periods depending on the environmental conditions.This study further showed that viable and dead organisms can be distinguished using DNA amplification (Mallika et al., 2008).
In another research conducted in Ghatampur and Jalma, known endemic areas of leprosy in India, 18 soil samples, two from each village from different locations near the residences of patients, were examined.The results revealed the presence of M. leprae DNA in 33.3% Between the months of March and April, with temperatures between 24-33°C and atmospheric humidity of 44-28%, the bacilli survived for 14 days.During the monsoon season in August and September, with atmospheric humidity between 72-80% and temperatures of 29-33°C the bacilli survived for 28 days.In September and October, with temperatures of 25-32°C and humidity between 66-44%, the bacilli remained viable in the moist soil for 46 days.The rates of recovery were consistently higher for dry season samples than for wet season samples of soil.All isolates cultured from soil appeared to be strains of M. fortuitum and not M. leprae with a complex pattern for the environmental mycobacterial flora. of the soil samples (Mallika et al., 2006).

Lahiri
Between 1998 and 1999, research was conducted in the northern and southern parts of the district of Karonga, Malawi, Africa.Soil samples from 11 villages housing 19 families with a history of leprosy were examined at the end of the dry and rainy seasons.One hundred and thirteen and 35 samples were collected at the end of the dry (1998) and rainy (1999) seasons, respectively, from 10 families.The results from a subset of 32 samples from the same locale, harvested during the dry and rainy seasons, showed the same trends with higher rates of recovery during the dry season (66%) compared with the rainy season (34%).The authors explain that the northern part of the District of Karonga has higher rainfall than the south.This result might be closely linked to climatic changes in the environment, as the bacilli can be removed from the soil and reducing the density of these bacterial population owing to the presence of the excess rainwater.The challenge in the study was the variety of mycobacteria in the soil, which might indirectly influence human health (Chilima et al., 2006).The incidence of leprosy was three times higher in the northern part of the district, which is warmer and more humid than the southern (Fine et al., 1994).
Epidemiological, microbiological, and clinical studies indicate that 50-70% of the sporadic leprosy cases in well-studied populations is reported in people who have had no known contact with other leprosy patients (Chakrabarty and Dastidar, 2002).
The environment can be an alternative transmission pathway for the spread of the disease.M. leprae thrives in soil rich in fossil fuels.In 2001, soil samples containing fossil fuels were collected from different parts of the USA, Russia, and Romania.There was a high degree of correlation between the presence of fossil fuels in the soil and leprosy in the countries surveyed.According to the authors, the disease probably occurred due to soil contamination (Chakrabarty and Dastidar, 2002).
In 1981, Kadza conducted a study across nine countries, where 729 samples were collected as follows: 273 from Norway (32.9% positive), 71 from Ivory Coast (23.9% positive), 36 from Portugal (55.6% positive), 20 from India (30.0%positive), 30 from Peru (40.0%positive), and 67 from Louisiana, USA (25.4% positive), 40 from Sweden, 77 from Scotland, and 115 from Germany, all of which were negative for the presence of the bacillus.M. leprae from positive samples was inoculated in the footpads of mice and armadillos.Through technique of isolation NCAFB it was possible to show characteristic growth in the footpads of mice and armadillos.The results suggested since more than 30 years that leprosy is transmitted not only by direct contact, but also indirectly by environmental means.However, the researchers could not culture the bacilli using the Lowenstein-Jensen and Middlebrook methods (Kadza, 1981).
A study conducted at the Institute for Leprosy in Agra, India, found important differences in viability of the bacilli in adverse conditions during dry and rainy seasons.The first experiment was carried out in dry soil in the months of March and April, at temperatures of 24-33°C and atmospheric humidity of 28%.Under these conditions, the bacilli could not survive for more than 14 days.Upon repeating the experiment during the rainy season (August and September) with an atmospheric humidity ranging between 72-80% and temperatures of 29-33°C, the bacilli survived for at least 28 days.In the months of September and October, at temperatures of 25-32°C and humidity between 66-44%, the bacilli remained viable in moist soil for 46 days.Throughout the year, M. leprae remained viable for up to five months in soil that was dry, but under the shade.When exposed to direct sunlight for 3 h/day, the bacilli survived for 7 days.Furthermore, the bacilli remained viable for 2 months when stored between 4 and −20°C but when frozen at −70°C, they remained viable for only half the time.When exposed to antiseptics such as Savlon® and alcohol, the bacilli were rapidly killed, while in saline solution at room temperature, they survived for 60 days.These results indicate different survival rates of the bacilli outside the human body under different environmental conditions in India, where the disease is endemic.The transmission by indirect contact and the occurrence of new cases in the absence of known sources is consistent with viable bacilli outside the body.However, the study presented limitations in the management of refrigeration equipment to preserve the bacilli (Desikan and Sreevatsa, 1995).

WATER
Other studies indicate that M. leprae can also survive in water.In a study conducted in Poteran Island, Sumenep, Madura, and East Java, Indonesia, 201 samples were collected and divided into three groups: 91 water samples collected from wells, 42 nasal swabs from household contacts, and 68 histological sections from leprosy patients.Upon analyses of the samples, 26.4% isolates from the water sources, 61.9% from the nasal swabs, and 35.3% from the skin biopsies tested positive.PCR results show that water used by leprosy clinics tested positive, and groups without leprosy that used this water were more susceptible to the disease.Therefore, water is considered a possible reservoir and source of infection for leprosy, because detection of M. leprae DNA was significantly higher in individuals using the water than in individuals who did not (Adriaty et al., 2010).Thus, cases of leprosy in individuals with no history of exposure to other known cases might be explained by exposure to viable M. leprae in water (Turankar et al., 2012).
Meanwhile, the research in East Java, Indonesia showed that 22 of the 90 samples of water examined were M. leprae-positive.Forty-eight samples were collected from wells used by leprosy patients; 11 of these tested positive for M. leprae.Interestingly, water samples collected from wells that were never used by leprosy patients also tested positive; M. leprae was found in freeliving aquatic amoeba-like protozoa.Therefore, existence of the bacilli in water resources used by inhabitants of endemic areas does not seem to be influenced by the presence of leprosy patients living in the same area (Wahyuni et al., 2010).
Finally, the findings of a study conducted in 2002 in an endemic area of Ceará in northeastern Brazil, in the municipalities of Juazeiro, Morada Nova, Sobral, and the state capital Fortaleza, also suggested that infections arise from contact with contaminated bodies of water.The prevalence of infection among individuals using the water for bathing was higher than that among individuals who did not.Therefore, water might be an important carrier of the disease in this region.Streams and rivers have running water only in the rainy season.Thus, when precipitation stops, stagnant pools of water remain and these might serve as potential reservoirs for the bacilli.One limitation of the survey was the small number of counties investigated (Kerr-Pontes et al., 2006).Molecular-based studies have revealed the importance of meteorological and climatic factors in the life cycle of M. leprae.The bacillus is known to remain viable as a probable source of infection leading to disease, especially under conditions of high humidity and temperature that characterize the tropical regions of the world.However, the bacilli can also survive in environments with broad variations in temperature and humidity.Therefore, basic infrastructures including sewers, water supply, and hygiene are the most important factors in protecting against the disease (Silva et al, 2010).
Besides leprosy patients without treatment, those in subclinical stages or those who exhibit spontaneous remissions may also be sources of bacillary spread, providing a transitional period of pathogen excretion via the nasal and/or oral routes (Cree and Smith, 1998).
Literature provides evidences that support the presence of M. leprae in the environment, having been found in different abiotic and biotic substrates.It was found in water (Wahyuni et al., 2010) and soil (Mallika et al., 2008) near leprosy clinics.It was also found in sphagnum (Kadza et al., 1980) and in a number of0species ranging from protozoa (Lahiri and Krahenbuhl, 2008) to more complex organisms such as mammals (Truman and Fine, 2010).
The viable bacilli found in water and soil can be an important disseminator of the disease, indicating extrahuman sources of M. leprae.Locales with moist soil and associated ambient temperatures guarantee the viability of the pathogen (Ooi and Moschella, 2001).
The finding that M. leprae can survive ingestion by amoebae suggests that protozoans can significantly improve the survival of these bacilli in the soil, and therefore be instrumental in the transmission of leprosy (Lahiri and Krahenbuhl, 2008).This shows the potential role of amoebae in the protection of M. leprae under adverse environmental conditions such as temperature and pH changes.
The handling and consumption of armadillo meat is also a possible source of M. leprae infection, chiefly in patients with no history of contact with other leprosy patients before their diagnosis (Deps et al., 2003).The mechanism of this transmission, however, has not been elucidated yet.
In 2011, a research conducted in Louisiana and Texas, in the southern region of the United States, revealed cases of leprosy in Native Americans who had never been outside the country.The exact mechanism of transmission remains unclear, but armadillos appear to be the possible reservoir, since the patients and the armadillos were shown to carry the same strain of M. leprae (Truman et al., 2011).
Before the M. leprae genome was decoded in 2001, availability of new antigens was limited mainly because the bacilli could not be grown in axenic culture.Until then, M. leprae had remained an enigma mainly due to its inability to be cultured in vitro (Cole et al., 2001).Subsequently, comparison of the genomes and proteomes of M. tuberculosis and M. leprae revealed that the latter suffers from reduced evolutionary potential.It presented a genome of 3.3 mega bases compared with 4.4 mega bases of M. tuberculosis.This reduction in the M. leprae genome has resulted in the elimination of many important metabolic pathways, explaining its intracellular habitat and inability to be cultivated in vitro (Cole et al., 2001).
Since 2000, considerable advances have been made with sequencing of the bacillus DNA.In particular, the 16S rRNA sequence has been used in viability assays, whereas detection of the M. leprae mRNAs has been proposed as a promising tool for rapid detection and measurement of viability of the bacilli in the environment (Kurabachew et al., 1998).The major advantage of PCR is its high sensitivity and specificity for detecting DNA from M. leprae, without the bacterial culture (Goulart and Goulart, 2008).The technical advances in determining the presence of M. leprae in the environment has been complemented by many new findings, such as the elucidation of its 16S rRNA sequence, facilitated by methods such as PCR and Real Time (RT)-PCR (Kadza, 1981;Opromolla, 1997;Abreu et al., 2006).
There were some limitations to the studies discussed in this review, though.First, in the 1980s, detecting acidalcohol resistant bacilli was not possible due to difficulty in cultivating the bacilli (Salem and Fonseca, 1982;Kadza, 1981).M. leprae is deficient in the transport of iron, which is required for cell division, thus making it unlikely that the bacilli can replicate by artificial means (Kato, 1994).The reduction in the M. leprae genome might also explain this difficulty (Cole et al., 2011).Secondly, the problem in experimental research in 1995 was the management of refrigeration equipment to preserve the bacilli (Desikan and Sreevatsa, 1995).Exposure to very low temperatures could cause the water to form crystals and harm the bacilli.Moreover, freezethaw cycles could also destroy the microorganisms.Thirdly, the small number of counties was an obstacle encountered during research in the state of Ceará (Kerr-Pontes et al., 2006) because of which, the results might not be similar in other parts of the state.

CONCLUSION
This review provides evidence that meteorological factors such as temperature and soil humidity can influence the dynamics of M. leprae.The occurrence of this disease is associated with variations in temperature and humidity.However, leprosy cases are registered equally in the rainy season as well as in the dry season, suggesting that M. leprae remain viable in various environmental conditions.Therefore, it is very difficult to establish the meteorological pattern to maintain the bacilli in the environment, but there are no doubts about the presence of the bacillus in water, soil as well protected by freeliving amoebas.The key aspect in the environmenthuman transmission appears to be the intensity of exposure to contaminated soil and water that differs between developed and developing countries.
The extensive land area of endemic countries, endemicity in the bordering countries, diversity of biomes, the lack of urban infrastructure, together weather features are possible factors that could influence disease transmission.

Figure 1 .
Figure 1.Flowchart of the literature review process: publications from 1980-2014 on the environmental prevalence of M. leprae and its association with meteorological variables.

Table 1 .
Studies on the presence of M. leprae in the environment and its relationship with meteorological variables, published between 1980 to 2014.

The
Acanthamoeba castellanii phagocyte showed no apparent adverse effects.The mycobacterium survived for 4 days, thus pointing to the potential role of the amoebae in the protection of M. leprae under adverse environmental conditions such as desiccation, and changes in temperature and pH.