ABSTRACT In the present study, nasal mucus from patients with leprosy were analyzed by PCR using specific primers for Lsr2 gene of Mycobacterium leprae

Alexandre Saito

Antonio Luiz

Aparecida De

Custodio

Ionice Felipe

Ivete Conchon Costa

Lourdes Perim

Marla Karine Amarante

Shiduca Itow Jankevicius

Thiago Cezar Fujita

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Braz. Arch. Biol. Technol. v.55 n.3: pp. 375-379, May/June 2012 
375
Vol.55, n. 3: pp. 375-379, May-June 2012 
ISSN 1516-8913    Printed in Brazil 
 BRAZILIAN ARCHIVES OF  
BIOLOGY AND TECHNOLOGY 
  A N  I N T E R N A T I O N A L  J O U R N A L  
 
 
 
Detection of Lsr2 Gene of Mycobacterium leprae in Nasal 
Mucus 
 
Luiz Antonio Custodio1*, Alexandre Saito2, Marla Karine Amarante 1, Thiago Cezar 
Fujita 2, Aparecida de Lourdes Perim1, Ivete Conchon Costa2, Ionice Felipe2 and Shiduca 
Itow Jankevicius2 
1Departamento de Patologia Aplicada, Análise Clínicas e Toxicológicas; Centro de Ciências da Saúde; 
Universidade Estadual de Londrina – PR - Brasil. 2 Departamento de Patologia Geral; Centro de Ciências 
Biológicas; Universidade Estadual de Londrina; Rodvia Celso Garcia Cid, 86051-990; Londrina - PR - Brasil 
 
 
ABSTRACT 
 
In the present study, nasal mucus from patients with leprosy were analyzed by PCR using specific primers for Lsr2 
gene of Mycobacterium leprae. The presence of Lsr2 gene in the nasal mucus was detected in 25.80% of patients 
with paucibacillari leprosy, and 23.07% of contacts. Despite the absence of clinical features in the contact 
individuals, it was possible to detect the presence of Lsr2 gene in the nasal mucus of these individuals. Therefore, 
PCR detection of M. leprae targeting Lsr2 gene using nasal mucus samples could contribute to early diagnosis of 
leprosy.  
 
Key words: Mycobacterium leprae, leprosy, hanseniase, nasal mucus, PCR. 
 
 
                                                     
* Author for correspondence: harpialc@hotmail.com 
INTRODUCTION 
 
Although a long time has elapsed since the 
discovery of Mycobacterium leprae by Gerhard 
Armauer Hansen in 1873 (De Zubiria et al. 2003; 
Fernandes et al. 2004), the understanding of the 
biology of this microorganism is still incomplete. 
For instance, the reasons for the inability of M. 
leprae to grow in media and conditions commonly 
used for bacteria is still not known. Recent studies 
have presented interesting data about the 
relationship between M. leprae and its host 
(Zanazzi et al. 2000; Nurse 2003), but many 
relevant questions about it remain unanswered 
(Siddiqui et al. 2003). An important aspect of 
mycobacterial pathogenesis is the ability of the 
pathogen to establish long lasting latent infections 
in the host. Leprosy (or Hanses’s Disease) is a 
millenary chronic infectious granulomatous 
disease, which attacks skin, peripheral nerves and 
mucous membranes and is associated with 
immune response and secondary infections 
involving the status from the host. The World 
Health Organization estimates that more than 12 
million people are affected by leprosy in the world 
(Braga 2002). Leprosy remains highly endemic in 
several countries such as Angola, Brazil, Central 
African Republic, Democratic Republic of Congo, 
India, Madagascar, Mozambique, Nepal, and the 
United Republic of Tanzania. It has been a 
problem in the countries which received a large 
number of immigrants from the endemic areas 
(Chae et al. 2002; Jianping et al. 2004). 
(Wiwanitkit 2005) performed a database search in 
order to find the genes of M. leprae recorded with 
complete sequences and compared their homology 
Custodio, L. A. et al. 
Braz. Arch. Biol. Technol. v.55 n.3: pp. 375-379, May/June 2012 
376
to human genomes using BLAST method. It is 
known that B cell responses to specific sequences 
within the Lsr antigen have been shown to be 
associated with the immunopathological responses 
in leprosy patients with erythema nodosum 
leprosum. It has been observed that some B and T 
cell epitopes localized to the regions with amino 
acid substitutions may account for the putative 
differential responsiveness to this antigen in 
tuberculosis and leprosy (Oftung et al. 2000). 
(Misra et al. 1995) found that the patients who did 
not show acid-fast bacilli in the tissues by the 
conventional methods presented positive for M. 
leprae DNA. PCR was initially used for the 
detection this pathogen in 1989 (Hartskeerl et al. 
1989; Woods et al. 1989) and since them it has 
been used in most different clinical specimens as 
skin (De Wit et al. 1991; Yoon et al. 1993), nasal 
swab and hair bulb (Santos et al. 1995) and oral 
swab (Goulart et al. 2001). A proteomic approach 
was undertaken to identify the proteins present in 
the soluble/cytosol and membrane subcellular 
fractions obtained from the armadillo derived from 
M. leprae. Proteins from each fraction were 
separated by two-dimensional gel electrophoresis 
(2-DE) and identified by mass spectrometry. A 
total of 147 protein spots were identified from 2-
DE patterns and shown to comprise the products 
of 44 different genes, 28 of them corresponding to 
new proteins (Marques et al. 2004).  
Multidrug treatment has contributed to disease 
control, but new cases are being reported, showing 
that the development of early detection methods is 
imperative. Knowledge of the genomic sequence 
of M. leprae (Cole et al. 2001) is an important step 
for understanding this bacterium and its 
interactions with its host, as well as for developing 
the detection tests such as those based on PCR 
(Meima et al. 2004) or helicase-dependent 
amplification (HDA) (Young 2001; Vincente et al. 
2004). Several PCR-based methods for the 
detection and evaluation of leprosy have been 
reported (Zanazzi et al. 2000; Santos et al. 2001; 
Guerrero et al. 2002; Torres et al. 2003; Sakamuri 
et al. 2009). The aim of this work was to evaluate 
the PCR detection of lsr2 gene of M. leprae in 
nasal mucus. 
 
 
MATERIAL AND METHODS  
 
With the approval at the Human Ethics Committee 
 
of Londrina State University and Philantropic 
Society Humanitas, Parana, Brazil, 130 Brazilian 
patients were included in thisn study. DNA 
sequences of M. leprae were analyzed by BLAST 
(http://ncbi.nlm.nih.gov/BLAST) and the rate of 
repetition in the genome was checked searching 
for a unique sequence copy, as well as for 
similarity with the genome of other 
microorganisms. Lsr2 gene was analyzed in the 
PRIMER3 program (http://frodo.wi.mit.edu/ 
primer3/) to draw the primers and these were 
analyzed by the BLAST program, checking its 
specificity to M. leprae. The 130 patients enrolled 
in this study were grouped as following: 22 
samples (~16,9%) from multibacillary leprosy, 31 
amples (~23,8%) from paucibacillari leprosy, 52 
amples (40%) from asymptomatic household 
contacts of patients with leprosy and 25 samples 
(~19,2 %) from people without history of contact 
with leprosy. Mycobacterium fortuitum (11 
isolates), Mycobacterium scrofulaceum (1 isolate), 
Mycobacterium tuberculosis (29 isolates), 
Mycobacterium avium (4 isolates), which were 
kindly provided by Dra. Halha Ostrensky 
Saridakis, (Centro de Ciências Biológicas, 
Departamento de Microbiologia – Universidade 
Estadual de Londrina - Brasil), were used as 
controls. 
Swab samples of nasal mucus were collected using 
sterile cotton swabs wet in phosphate buffer (pH 
7.0), maintained at 4°C until   transferred to 
Eppendorf® tubes containing 1.0 mL of ultra pure 
water and stored at –20°C until DNA extraction. 
 
DNA extraction  
Genomic DNA was isolated following the method 
proposed by (Torres et al. 2003) with 
modification. Briefly, 200µL of the sample was 
centrifuged 15 minutes at 12.000 g and the pellet 
was treated at 100°C for 1 minute and freezing in 
nitrogen for 3 minutes. It was resuspended in  
100 µl of Tris- HCl 50 mM buffer (pH 8.0), 
containing lysozime 10 mg/mL, incubated at 37°C 
for30 minutes, added 10 µL of proteinase K 50 
mg/mL, followed by the addition of 1.0 µl of 
Triton X-100 (10%). The mixture was kept at 55oC 
for two hours and after the addition of absolute 
ethanol centrifuged at 10.000 g for five minutes. 
The DNA was suspended in ultra pure water and 
stored at –20°C until use. 
 
Amplification conditions  
Detection of Lsr2 Gene of Mycobacterium leprae in Nasal Mucus 
 
Braz. Arch. Biol. Technol. v.55 n.3: pp. 375-379, May/June 2012 
377
PCR products were analyzed by the 
electrophoresis on acrylamide gel (10%) and 
detected by a nonradioisotopic technique using a 
commercially available silver staining method 
(data not shown). 
 
 
RESULTS AND DISCUSSION 
 
The Lsr2 gene of M. leprae codifies a protein of 
15 kDa with capability of stimulating T 
lymphocytes. Table 1 shows the results of  
 
amplifications by PCR from 130 samples of nasal 
mucus of leprosy patients, asymptomatic 
household contacts of patients with leprosy and 
people without contact history. Samples with 
negative PCR results were tested for the presence 
of inhibitors in the reaction, by adding 
standardized DNA to the extracted sample. To test 
the specificity of PCR-probing with the primers 
for M. leprae Lsr2 region to Lsr2, the gene of 
other mycobacterias as M. avium, M. fortuitum, M. 
scrofulaceum and M. tuberculosis were tested in 
the same region. In this context, all the strains 
were negative in PCR test. 
 
Table 1 - Detection of Lsr2 gene of M. leprae in smear of nasal mucus from patients. 
Class 
PCR-probing 
Positive Negative Total 
Multibacillary 19 03*1 22 
Paucibacillari* 08 23 31 
Contact** 12 40 52 
No contact** 0 25 25 
Total 39 91 130 
1Three were discharged from treatment.  *p<0,0001.  **p<0,0227 
 
 
In Brazil, despite actions for the treatment and 
control of leprosy, the number of new cases is 
highly threatening, and considering the existence 
of infected and undiagnosed people as a true 
reservoir of M. leprae and their contacts (Braber 
2004) as infection sources, the development of 
methods for early diagnosis become imperative. 
PCR is a useful tool for the diagnosis of leprosy 
and its recent developments such as helicase 
dependent amplification point to the possibility of 
developing methods of detection with better 
sensitivity and specificity. (Bang et al. 2009) 
evaluated the sensitivity and utility of polymerase 
chain reaction (PCR) to detect M. leprae in 
comparison with other conventional methods for 
the diagnosis such as split skin smears, 
histopathology and serodiagnosis. They observed 
that the PCR detection of M. leprae targeting 16S 
ribosomal RNA was specific and more sensitive 
than the conventional methods, and could 
contribute to early and accurate diagnosis of 
leprosy. The present work standardized a PCR 
technique to amplify the single copy gene Lsr2 of 
M. leprae. This technique presented 100% of 
specificity for M. leprae relative to other 
mycobacteria, in agreement with previous reports 
(Santos et al. 2001; Guerrero et al. 2002; Torres et 
al. 2003; Willams et al. 2003; Braber 2004; 
Cortes-Herrera et al. 2008). Therefore, it seemed 
possible to use this gene as a target for large-scal  
creening of human reservoirs of M. leprae, 
Despite the small number of patients with 
multibacillary leprosy, the majority of the 
inhabitants of endemic areas showed the signs of 
exposure to M. leprae, which could be explained 
by the presence of subclinical bacilliferous 
infections in the community. (Guerrero et al. 2002) 
studied the use of a polymerase chain reaction 
(PCR) test to detect LSR/A15 gene that coded for 
the 15 kDa M. leprae antigen in the samples of 
nasal mucus from asymptomatic household 
contacts of patients with leprosy. Their finding 
demonstrated that this method could be used to 
monitor the high-risk populations and also for the 
elimination programs in the countries where the 
disease's prevalence has been significantly 
reduced. Recent advances in the molecular 
epidemiology of leprosy through the genotyping of 
variable number tandem repeats (VNTRs) and 
single nucleotide polymorphisms (SNPs) have 
been described. VNTRs with a broad range of 
diversity are useful genotyping tools for analyzing 
the transmission in community areas, and SNPs 
and VNTRs with a small degree of variation are 
favorable for investigating the global transmission 
of leprosy (Matsuoka 2009). The use of PCR for 
the early diagnosis to detect M. leprae in leprosy 
patients as well asymptomatic household contacts 
Custodio, L. A. et al. 
Braz. Arch. Biol. Technol. v.55 n.3: pp. 375-379, May/June 2012 
378
of patients with leprosy with a high risk for 
developing or transmitting the disease would be of 
clinical and predictive value. While the prevalence 
of leprosy has declined around the world, there has 
not been a corresponding decrease in its incidence, 
thus indicating that it has not been possible to 
prevent the transmission of the disease. 
 
 
ACKNOWLEDGEMENT 
 
We thank Sociedade Filantrópica Humanitas for 
their colaboration, Dr. John Spencer and Dr. Patric 
Brennan for their inestimable attention, and also 
Dr. Halha Ostrensky Saridakis for samples supply.  
 
 
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Received: November 12, 2009; 
Revised: April 28, 2010; 
Accepted: January 17, 2012. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PÁGINA 
EM  
BRANCO 
 


BRAZILIAN ARCHIVES OF BIOLOGY AND TECHNOLOGY A N I N T E R N A T I O N A L J O U R N A L Detection of Lsr2 Gene of Mycobacterium leprae in Nasal Mucus

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BRAZILIAN ARCHIVES OF BIOLOGY AND TECHNOLOGY A N I N T E R N A T I O N A L J O U R N A L Detection of Lsr2 Gene of Mycobacterium leprae in Nasal Mucus

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