Subclinical leprosy detection in contact person of multibacillary leprosy patients

Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae and generally manifest as neurodegenerative disorder which may lead to permanent disability. Whether its prevalence tend to be decreased, new and relaps case, complication such as skin reaction, and disability remain problematic whether in much smaller magnitude. Objective: In this study, we observe the prevalence of subclinical leprosy in population who live with leprosy patients in order to determine the rate of transmission and initiate early treatment for those with subclinical disease as well as providing data for future research. Methods: A cross-sectional study was conducted in dermatology and genital clinic of Sanglah General Hospital from may 2016 to August 2016 which included all patient’ family that agreed to be enrolled. The leprosy status of the sample was evaluated using lateral flow test that detect IgM anti-PGL-1 in the serum. Results: 28 leprosy patients and 73 contact person were enrolled in this study. Among the patients, 21 (75%) were Balinese and 7 (25%) were Javanese. Lateral Flow examination found that almost all results were positive (27; 96.42%) for the patients with just 1 patient showed negative result. Meanwhile, from 73 contact person, 11 appear to be positive (15.06%). Conclusion: lateral flow test was proved to be effective method of detecting M. leprae infection and we found 15.06% of contact person had subclinical leprosy.


INTRODUCTION
Leprosy is a chronic infectious disease caused by Mycobacterium leprae and generally manifest as neurodegenerative disorder which may lead to permanent disability.So far, leprosy is one of health problem in developing countries, especially the one with leprosy epidemic. 1,2he prevalence of leprosy had been constantly decreasing globally after the establishment of multidrug therapy (MDT) protocol.However, the decreasing rate was found to be different from global case detection rate which is important for eradication process in the future. 3WHO data reveal that the prevalence of leprosy had decreased sharply from more than 5 million in 1980 to less than 200,000 in 2015.However, new and relaps case, complication such as skin reaction, and disability remain problematic whether in much smaller magnitude.
Internationally, Indonesia ranked as 3 rd in the amount of new cases of leprosy after Brazil and India with 17,025 new cases.Indonesia also had the highest number of new cases of Multibacillary leprosy with 14.213 cases which represent 83.4% of all cases. 3The highest prevalence was found in East Java Province with 4,132 cases. 4In Bali, the prevalence of leprosy based on the data from health department was founded around 0.21 per 10,000 people with 89 new cases.Meanwhile, in dermatology clinic at Sanglah Hospital, there were 44 new cases with 30 (68.18%) multibacillary leprosy and 14 (31.81%)paucybacillary leprosy. 3,4,5urrently, it is known that a person who is living together with leprosy patients pose great risk of contracting leprosy.This population has 5-10 times greater risk contracting leprosy compared with general population.Moreover, even subclinical infection is capable of transmitting the disease. 6,7However, the definitive mode of transmission is still unknown whether it is believed that inhalation is the most possible and reasonable mode of transmission, particularly because the great number of bacillus being detected in nasal secretion. 7,8n the other hand, current method of detection of M. Leprae is still based on clinical findings, the presence if acid fast bacillus in skin preparat, and histological examination which is highly subjective and operator dependent.Other modes of testing are deemed inconclusive since they yield unsatisfactory results. 9urrently, new methods of diagnosis have been developed to accurately diagnose leprosy even in subclinical stage.Lateral flow test was developed to detect the presence of IgM anti PGL-1 in the patient blood. 10,11Phenolic glycolipid (PGL-1) is specific antigen for M. Leprae which present in bacterial surface and highly antigenic.It is capable to induce immune system to produce high titer of IgM.Thus, the amount of IgM anti PGL-1 was positively correlated with the bacterial load, making it as potential diagnostic biomarker. 12Meanwhile, lateral flow test is also has proved as effective method to detect IgM anti PGL-1 even in subclinical patients. 10n this study, we observe the prevalence of subclinical leprosy in population who live with leprosy patients in order to determine the rate of transmission and initiate early treatment for those with subclinical disease as well as providing data for future research.

METHOD Study Design and Sample Collection
A cross-sectional study was conducted in dermatology and genital clinic of Sanglah General Hospital from May 2016 to August 2016.We included all patient's family that agreed to participate in this study.However, those who stay with patients for just 2 years or less, less than 5 years old, or those who refuse to be examined were excluded.

Leprosy Diagnosis
Leprosy was diagnosed by following guideline of Dermatological and Genital Division of Sanglah General Hospital by evaluating clinical signs such as hiposensitive skin spots, nerve thickening with or without disfunction of respective nerve, and the presence of acid-fast bacillus in skin preparates.Skin preparates were prepared by obtaining skin sample from patients ear pinna, then the skin then strapped until turned pale to avoid bleeding.Next, an incision was made using scalpel to obtain skin sample.The incision was made with enough depth so both epidermis and dermis region will be presented within the preparate.The sample then fixated on the object glass by moving it on bunsen lamp.The preparate then stained by using Ziehl Neelsen methods which enable the examiner to identify M. Leprae within the tissue.Histopathological examination was conducted to confrim the diagnosis.

Lateral Flow Test Examination
3 ml of blood sample was collected from each subject and subsequently centrifuged to obtain the serum.The serum could be stored in 2-8 0 C for further use.5 µl of serum was injected into the well followed by addition 130 µl diluent (sterile running fluid) that consist of phosphate saline buffer (pH 7.6), 1.66% albumin serum and 3% tween 20.The test result appear within 10 minutes. 2 parallel band indicate a positive result and one band on the control side was considered as negative.Based on the color of the band, the positive result can be classified into 4 class which signify weak reaction up to strong reaction.

Statistical Analysis
All of the data was analized descriptively to obtain the prevalence of subclinical leprosy in patients family.All analyses were conducted by SPSS.17.

RESULT Sample Baseline Characteristic
28 leprosy patients were enrolled in this study.Among them, 21 (75%) were Balinese and 7 (25%) were Javanese.The mean of age was 43.67 years with number of male exceeding female.The educational background of the subjects were varied from uneducated to higher education with elementary education predominant (46.42%) followed by bachelor/higher education (17.85%).More than half of the subjects work in private sector and most of them (64.28)never travel away from Bali.However, 25% of them reported to had contact with leprosy patient prior to their diagnosis.From all subjects, we obtain 73 contact persons (which lived for at least 2 years with patient) which were agreed to participate in this study consisted of 57 (78,08%) Balinese and 16 Javanese (21,91%).The mean of age in this subject population was 34.6 years which ranging from 6 years old to 75 years with equal proportion between male and female.Likewise, there is great variability in term of educational background in this population with Senior High School background as the dominant one (39.70%).As previous sample population, private sector worker was the dominant type of profession (42.20%).In term of family relationship, 36.61 % of subjects were the child of the leprosy patients.Based on relationship type, 41 subjects were familial type and the other 32 were nonfamilial.The detail of sample's characteristics are described in Table 1.

Lateral Flow Test Examination result
All patients and contact persons were examined using lateral flow test.From 28 patients sample, almost all results were positive (27; 96.42%) with just 1 patient showed negative result.Meanwhile, from 73 contact person, 11 appear to be positive (15.06%) for IgM anti PGL-1 with 5 patient showing 1 st degree positivity, 4 with 2 nd positivity and 1 sample for both 3 rd and 4 th degree positivity.The detail of lateral flow test are described in detail in Table 2

DISCUSSION
There are numerous factors that influence the risk factors of leprosy such as period of contact, low socio-economic status, living in leprosy endemic area, and genetic factors.For endemic factors, it had been confirmed that chromosome 10p13 had specific locus that influence leprosy susceptibility especially in india population. 1 From sample's baseline characteristics, it appear that leprosy could also affects people from good social status.
Tabel 2. Frequency Distribution of the Result of Lateral Flow Test Among Contact Person.
Even if Bali is not an endemic area for leprosy, Bali is an open area where people could enter or out as well as very close with Java which is an endemic area for leprosy with high prevalence and incidence.The susceptibility of Balinese population to leprosy was described in the result of this study where 64.28% of leprosy samples were Balinese with no history of contact and only 25% had history of contact.From contact persons data, it appears that the average period of contact was 8.36 years with 78.08% samples were Balinese and 21.91% were Javanese.
Early detection of Leprosy had become specific attention among scientists and health professionals.One effective and efficient approach to detect the presence of M. leprae is by examining anti-PGL-1 antibody.Phenolic Glycolipid-1 (PGL-1) is a specific antigen for M. leprae and had high immunoreactivity.It presence induces production of IgM, which is very useful for diagnostic approach or epidemiology study. 6,13,14Its role in leprosy diagnostic is also supported by the fact that most of patients with multibacilary leprosy showed an increase in anti-PGL-1 titer which is in conjunction with the result of this study..This method is also widely used to detect the presence of M. leprae in the patient's family.The presence of increased titer of anti-PGL-1 is sufficient to suspect that a person has a subclinical leprosy.Even in subclinical form, leprosy still highly contagious, which is the reason why several samples had leprosy without a history of contact with the person with the disease.
Lateral flow test is one method to detect anti-PGL-1 antibody with simple usage protocol and, thus, eliminates the need of specific skills.This method also had high sensitivity and specificity (97,4% and 90,2%) compared to ELISA. 48In this study, 15.06% samples had positive result which indicated them to have subclinical leprosy.Currently, no standardized treatment protocol or expert consensus about early MDT treatment for subclinical leprosy.However, the researchers were already ongoing in the leprosy endemic area and the results indicated that the intervention could effectively reduce the number of new cases in the area. 29

CONCLUSION
Lateral flow test was proved to be effective method of detecting M.leprae infection and we found 15.06% of contact person had subclinical leprosy.Whether small in number, it is important to keep the surveilance on these population and even consider the initiation of MDT as they had great potential to transmitt the disease to non-infected people.