MYCOBACTERIUM

LEPRAE IN DAILY WATER

RESOURCES OF
INHABITANS WHO LIVE IN
LEPROSY ENDEMIC AREA
OF EAST JAVA

INTRODUCTION

Leprosy remains a health problem in Indonesia ,

where many enclaves leprosy still persists ,
especially in the eastern part of the country .
Although the WHO program - Multidrug Therapy
( MDT ) regiment has been done elsewhere since
the 1980s , only the prevalence can be reduced
but not the incidence of new cases of leprosy .

GOAL OF STUDY

to find a correlation between the presence of

these bacteria in the environment with the
presence of leprosy patients living in the area, to
study its role in disease transmission .

MATERIAL AND METHOD

1.

Collection of water samples

about 300 ml of water sample was collected from

each well and stored at room temperature . Prior to
PCR , water 50 ml samples were filtered using
Millipore 0.22um membrane filter . Membrane
washed with PBST1. 5ml and vortexes 10 minutes .
The suspension is then centrifuged at 13,000 rpm ,
4 C for 20 minutes and the pellets are formed is
used to make a smear for Ziehl Neelsen ( ZN )
staining and DNA extraction.

2. DNA extraction

The Qiagen Miniprep kit ( Research Biolabs Co. )

was used for DNA extraction from the pellet ,
following the manual , to get a PCR template .

3. examination PCR
PCR

is done using a nested primer : LPF - LPR ( LPF

: 5'TATCGATGCAGGCGTGAG TGT3 ' , LPR :
5'CTAACACGATACTGCTGCAC3 ' ) and LP3 - Lp4
( LP3 : 5'TGAGGTGTCGGCGTGGTC3 ' , Lp4 :
5'CAGAAATGGTGCAAGGGA3 ' ) . PCR procedure
modifying of Donoghue and Spigelman (2001 ) for
detecting M.leprae of the specimen. Products that are
amplified electrophoresis on 3 % Agarose gel and
stained with ethidium bromide 0.1 ug / ml . The
positive results presented by the band at 99 bp , as
shown by the positive control ( M.leprae Thai 53
obtained from cultures of nude mice in Leprosy
Research Centre of Japan ) .

RESULT

All sediment from water samples showed positive

for AFB ( AFB ) after staining with ZN method .
Some of the bacilli found in the " amoeba - like"
protozoa , some of them moving microorganisms .
Positive results were found in 22 out of a total of
90 water samples ( 24 % ) , consisting of 11/48
( 23 % ) of water that is often used by leprosy
cases and 11/42 ( 26 % ) of water from wells once
used by leprosy patients .

DISCUSSION

Leprosy is still endemic in the three big

countries, namely India , Brazil and Indonesia. It
is known that M.leprae as the causative agent of
leprosy , can survive in the soil up to 40 days.
This bacterium is an obligate intra - cellular
microorganisms , which only grow if a live agent
within the host cell . special primer is needed ,
because LP1 - LP2 and LP3 - Lp4 coding
M.leprae 18 kDa antigen in the region RLEP3
recurring elements ( X17153 )

The first report about the existence M.leprae in

public water resources in North Maluku ,
reported by Matsuoka et al . (1999 ) . Using PCR
method , he found the 21/44 public resources
water contaminated by M.leprae . Agusni et al
( 2004 ) reported the detection of M.leprae in
some of the ponds that are used as the water
source of people living in the endemic area of
leprosy in the north coast of East Java .

Interestingly , positive result swere found deeper

water than the plant roots in water collected
from the center of the pond site . These findings
came to the suspicion that the bacilli living
protozoa that lives in the roots of many plants of
water . Leprosy cases are often used for both
their everyday activities can make the water
contaminated with M.leprae . Therefore, if the
bacteria came to water , PCR will be positive .
Most cases of leprosy who live in this area has
been treated with MDT regimens ; some of them
are already Release of Treatment ( RFT )

The results of this study showed that 11 of the 48

water samples ( 23 % ) of the wells used by
leprosy patient after swere positive PCR test to
detect M.leprae .But Interestingly , M.leprae also
positive in 11 of 42 ( 26 % ) water samples
collected from wells that were never used by
leprosy cases . Statistically , there was no
significant difference in the PCR positive results
for M.leprae .

CONCLUSION

M.leprae existence in source water daily

population in endemic areas where leprosy is not
affected with the presence of leprosy patients
living in the same area. More investigation is
needed to determine the role M.leprae
environment in the transmission of leprosy .