Cohort study of the seasonal effect on nasal carriage and the presence of Mycobacterium leprae in an endemic area in the general population

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1 ORIGINAL ARTICLE BACTERIOLOGY Cohort study of the seasonal effect on nasal carriage and the presence of Mycobacterium leprae in an endemic area in the general population M. Lavania 1 *, R. P. Turankar 1 *, S. Karri 1, V. S. Chaitanya 1, U. Sengupta 1 and R. S. Jadhav 2 1) Stanley Browne Laboratory, TLM Community Hospital, Nand Nagari, Delhi, India and 2) Department of Microbiology, Government Institute of Science, Mumbai, India Abstract Leprosy continues to be a significant health problem in certain pockets in developing countries. Better understanding of the transmission and source of the infection would help to decipher the transmission link, leading to control of the spread of the disease. The nose is considered to be a portal of entry, suggesting an aerial route for transmission through droplet infection. The evidence suggests that many individuals from endemic countries carry Mycobacterium leprae in their nasal cavities without having obvious symptoms of leprosy. The objective of the present study was to assess the presence of M. leprae on the nasal mucosa in the general population from a leprosyendemic pocket. M. leprae detection was carried out using PCR targeting RLEP. Four hundred subjects from an area highly endemic for leprosy were included in the study and followed up during three different seasons winter, summer, and monsoon for evidence of nasal exposure to M. leprae. PCR positivity for M. leprae was observed in 29%, 21% and 31% of the samples collected in winter, summer and the monsoon season, respectively. Twenty-six individuals from the cohort showed amplification for M. leprae for all seasons. Our results are consistent with reports in the literature showing widespread exposure to M. leprae in the endemic community. The results also suggest possible association of the environmental conditions (climate) with the transmission pattern and levels of exposure to M. leprae. However, the present study indicated that the population from highly endemic pockets will have exposure to M. leprae irrespective of season. Keywords: Endemic, humidity, Mycobacterium leprae, PCR, seasonal Original Submission: 21 May 2012; Revised Submission: 9 August 2012; Accepted: 19 October 2012 Editor: M. Drancourt Article published online: 12 November 2012 Clin Microbiol Infect 2013; 19: / Corresponding author: R. Jadhav, Department of Microbiology, Government Institute of Science, Mumbai , India rupenjadhav@yahoo.com *These authors contributed equally to this study. Introduction Members of the genus Mycobacterium are typically found in the soil and water. The majority of mycobacteria are nonpathogenic. However, Mycobacterium leprae, the causative agent of leprosy (Hansen s disease), has affected human beings from Biblical times in the Middle East, and has been recognized in India since Vedic times. Leprosy is a chronic, infectious disease that is believed to be of low contagiousness [1]. There are many reports on the levels of exposure to the bacillus in endemic countries, but its significance in transmission and disease outcome is yet to be established. The disease primarily affects the superficial parts of the body, especially the peripheral nerves and appendages of the skin, such as sweat and sebaceous glands and mucous membranes. According to the WHO, the global registered total number of leprosy cases from 130 countries and territories at the beginning of 2011 was , whereas the number of new cases detected during 2010 was (excluding the small number of cases in Europe) [2]. In India, the year started with a total prevalence rate (PR) of 0.69/ on 1 April Up to then, out of 35 States/Union Territories, 32 had achieved leprosy elimination. A total of 510 districts Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases

2 CMI Lavania et al. Effect of seasonal variations on PCR positivity 971 (80.6%) of a total of 633 districts also achieved elimination by March 2010 [3] ( 20% pdf). The risk of transmission is related to the presence of infectious cases and, perhaps, their surrounding environmental factors. It has been shown that humidity favours the survival of M. leprae in the environment [4]. Although M. leprae primarily spreads through infectious human sources, there is evidence from the published literature indicating the presence of possible non-human sources of the organism [5 7]. One of the reports shows that naturally infected armadillos or monkeys could be a source of M. leprae infection [8]. Inanimate objects or fomites, such as articles used by infectious patients, can theoretically spread infection. Furthermore, nasal secretions [9,10], discharged into the atmosphere by coughing, sneezing, etc. by an infected person, have been shown to disperse the bacilli in dust particles and through airborne droplets falling in soil [5,11 13] and in water [14] which may act as source of infection. Experimental evidence of M. leprae infection through exposure to M. leprae aerosol in experimental susceptible mice proved beyond doubt that infection can be transmitted by the nasal route [15]. Recently, amoebae have been shown to favour the growth of M. leprae in in vitro culture [16]. Smith et al. [17] reported that 1.6% of 2552 nasal swabs from normal healthy individuals in an endemic population had evidence of the presence of M. leprae as demonstrated by PCR, and 68% of saliva samples of such a population were positive for M. leprae-reactive IgA [18 21]. The further study of endemic populations indicated that households with leprosy patients have higher attack rates of leprosy than those without such exposure. The attack rates were also higher when the index cases had a higher bacterial load [22]. Some other studies have also reported that the nasal route is a major route of exit and entry for M. leprae [23,24]. In the present study, to determine the role of the atmospheric environment, we investigated, by PCR, the nasal mucosa of a cohort population from a highly endemic area (Baligara, Purulia) for the presence of M. leprae in the peak times of three different seasons: monsoon, summer, and winter. Baligara is a small village situated 3 miles from The Leprosy Mission Community Hospital and Simonpur Leprosy Colony Purulia, a southern district in the state of West Bengal in India. The population of the village is 2451 (2011 Census), living in approximately 426 households. A total of 56 new leprosy cases were reported from Baligarain in Fifteen (26.78%) of these 56 cases were children, with an age range of 6 13 years. The PR is c. 2.2/ Materials and Methods Ethical approval Informed consent was obtained from all patients, and the study was approved by the Organization Ethical Committee of The Leprosy Mission, India. Sample details The subjects were randomly selected from the village population, and there was no known bias in the selection process. From the population, 16.31% (n = 400) was chosen as the sample (Table 1). Eight (2%) old treated cases were randomly included in the study, six of whom were treated with paucibacillary multidrug therapy, and two of whom were treated with multibacillary multidrug therapy. Collection of nasal samples Pernasal swabs (Medical Wire and Equipment, Corsham, Wiltshire, England) were used to collect nasal specimens. Swabs were dipped in normal saline immediately prior to use, and passed through the base of the turbinate until the posterior wall of the nasopharynx was encountered. The nasal swabs were obtained separately from both nostrils of the healthy individuals. Swabs were collected, chilled, maintained at 4 C, and transported at 2 8 C to the laboratory. The collected nasal swabs were kept at 20 C for further analysis. Nasal swabs were collected from a cohort of 400 subjects in three different seasons (monsoon, summer, and winter) from Baligara, a village from an area highly endemic for leprosy in the Purulia district of West Bengal. Positive controls and negative controls were added as quality controls for the authenticity of the data, and to rule out false positives and false negatives. We used known M. leprae DNA as a positive control, and plain swabs without any material as negative controls for sample processing and PCR. We analysed some of the samples randomly by using 16S rrna gene region primers to crosscheck the results. Extraction of M. leprae DNA and PCR for RLEP sequences These nasal swabs were processed for DNA extraction with a method described by Jadhav et al. [25]. Swabs from both nostrils of an individual were lysed together in one tube. PCR TABLE 1. Demographic details of the sample Male Female Adults, n (%) 160 (40) 214 (53.50) Age range (years) Children, n (%) 12 (3) 14 (3.5) Age range (years) 5 18

3 972 Clinical Microbiology and Infection, Volume 19 Number 10, October 2013 CMI targeting the RLEP gene sequence in M. leprae was performed as described by Donoghue et al. [26], with 5 - TGCATGTCATGGCCTTGAGG-3 as a forward primer and 3 -CACCGATACCAGCGGCAGAA-5 as a reverse primer, in order to amplify a fragment of 129 bp. Amplification was confirmed by 2% agarose gel electrophoresis. Statistics Graphpad software was used to analyse the data. Comparisons were made within and between all of the seasons by using the Fischer exact test. All comparisons were considered to be significant at p Results Detection of M. leprae by PCR targeting RLEP PCR targeting RLEP gene sequences in M. leprae was performed for a cohort of 400 subjects in three different seasons. Samples were collected in each season from the same group of 400 subjects, and PCR was performed. Out of 400 subjects, 124 were positive in the monsoon, 84 were positive in summer, and 117 were positive in winter. The RLEP was amplified and confirmed by 2% agarose gel electrophoresis (Fig. 1). We identified statistically significant differences in PCR positivity between monsoon and summer (31% vs. 21%, p <0.05) and between summer and winter (21% 129 bp FIG. 1. Detection of Mycobacterium leprae from nasal swabs targeting the RLEP region. PCR amplification of the RLEP region of Mycobacterium leprae obtained from nasal swabs. PCR product were electrophoresed on 2% agarose gel. Lane NC: negative control. Lanes 1 3: samples. Lane PC: positive control. Lane ladder: 100-bp ladder. vs. 29%, p <0.05), indicating that exposure to M. leprae is greater in the monsoon than in summer and winter (Table 2). We analysed 26 samples that were consistently positive in all three seasons by 16S rrna, and all were found to be positive (data not shown). Effect of humidity on PCR positivity of M. leprae from the nasal mucosa We compared the seasonal variation in the exposure levels in the cohort study as indicated by PCR positivity. By applying the t-test, we found that PCR positivity for the presence of M. leprae in the nasal cavity was highest during the monsoon, when humidity was highest, and significant differences were found between all three seasons (Table 2). Similar findings were also made in our previous study in an area with relatively low endemicity for leprosy, where the exposure levels were low but the seasonal variations were similar to those reported here (data not shown). Discussion The transmission patterns of an infectious disease may depend on environmental factors that directly facilitate the pathogen contacting the host for the propagation of disease, or the acquisition of protective immunity by the host. Alternatively, they may reflect non-environmental factors, such as socioeconomic conditions, behaviour, or nutritional status, each of which may vary within and between populations, and thereby produce variations in host susceptibility to such environmental exposure. Understanding the natural history of a disease, its geographical distribution and host pathogen interactions is important in order to establish a successful control programme. It is necessary to mention that, in spite of its success, the WHO leprosy elimination campaign, which is mainly based on new cases, and the treatment and follow-up of household contacts, has not reduced the incidence of leprosy in certain endemic areas of the world. Increased detection of hidden cases cannot explain the new occurrence of leprosy among young children, which is an indicator of ongoing active transmission [27]. TABLE 2. Comparative PCR positivity in different seasons Number Season No. of samples collected No. PCRpositive % PCR positivity p-value for difference in PCR positivity in different seasons Average relative humidity (%) 1 Summer Summer vs. monsoon (0.01), significant 76 2 Winter Summer vs. winter (0.04), significant 85 3 Monsoon Winter vs. monsoon (0.65), not significant 92

4 CMI Lavania et al. Effect of seasonal variations on PCR positivity 973 Although several countries those have reached the elimination target still report the existence of some pockets with clusters of leprosy cases. Leprosy must be eliminated in these high-endemicity regions, in order to achieve a sustained low level of transmission and reduced incidence rates. India also has such small pockets of areas highly endemic for leprosy in several parts of the country. M. leprae has been shown to be capable of tolerating adverse environmental conditions. In the available literature, hot and humid weather, wet soil and water have all been proposed as factors that favour survival of the bacilli for few months (Tuberculosis and Leprosy Control of Ethiopia 10th Annual Review Meeting, 2002) [28]. Sterne et al. [29] reported that, in Karonga Districts of Malawi, the leprosy incidence was more than twice as high in the northern district as in the southern district. The most obvious environmental difference between these regions is the north s higher rainfall and more fertile soil. In the south, rates are similar between southern hills and the southern lake shore, with slightly lower rates in the semi-urban area around the district capital. This may suggest that the geographical variation in leprosy incidence rates is dependent on environmental factors. Transmission of M. leprae may be more frequent in humid conditions when the secretions from the nose are more abundant [30,31]. An environmental source [11,12,32] will determine the exposure which in turn can result in infection of susceptible humans. Our study indicates that leprosy transmission may be influenced by environmental changes. The present study also shows that there is seasonal variation in exposure to M. leprae. When the temperature is low and humidity is high in the monsoon, PCR positivity in nasal swabs increases, and during summer (dry and hot weather) it declines. Argaw et al. [27] proposed that leprosy occurs most frequently when a suitable microenvironment, such as moist soil, coexists with other known or unknown predisposing factors. Future studies, including fieldcollected validation data (temperature, rivers, water sources, humidity, etc.), may shed more light on the precise factors associated with the environmental risk of leprosy. More studies, such as investigations of different types of environmental samples and patient samples, are required to establish the extent of the role of viable M. leprae in the environment or different environmental reservoirs in the transmission dynamics for leprosy. To achieve leprosy eradication, a strategy that is effective in preventing the disease is required. This can be achieved by a better understanding of the modes of transmission and potential sources of the pathogen. Control of transmission may be feasible through the identification and treatment of individuals within infection clusters, allowing progress towards the eradication of leprosy. Future studies should focus on the development of methods and tools for studying the transmission of M. leprae and on identifying direct methods for testing innovative interventions. Acknowledgements We wish to thank The Leprosy Mission, India for financial support. We are also grateful to S. Failbus, K.C. Lata and A. Roy for assisting us with sample collection in the field. We also thank the superintendent and staff of TLM, Purulia for their help and assistance during the fieldwork. We also acknowledge S. Anand (Director, TLM India) and P. S. S. Sundar Rao (Research Coordinator, TLM, India) for their guidance and encouragement. Transparency Declaration This work was supported by The Leprosy Mission. References 1. Tebb W. Leprosy and vaccination. The recrudescence of leprosy and its causation. In: Tebb W, ed.. 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