Genotyping Analysis of Mycobacterium leprae isolated in Water Environment of Leprosy Endemic Places in Lamongan, East Java

Finding of Mycobacterium leprae from water of leprosy endemic areas were reported. East Java Province is ranked number eight as the endemic area of leprosy in Indonesia and Lamongan district is the local area with the highest new cases reported. To study the transmission of Mycobacterium leprae infection in endemic areas, it is important to detect the distribution of Mycobacterium leprae in the environment and population, also to analyze the genetic variation pattern. Keywords: endemic; environment; genotyping; leprosy; Mycobacterium leprae

Variation of TTC Repeat Pattern in the Dna of Mycobacterium Leprae Isolates Obtained From Archeological Bones and Leprosy Patients From East Nusa Tenggara

The existence of leprosy or kusta or Morbus Hansen or Hansens disease has been known for years, including in Indonesia. Starting from the discovery of Mycobacterium leprae isolates from ancient bone (about 1.000 years B.C), the archaeological excavations results in East Nusa Tenggara, interesting questions arise about how the development of leprosy in eastern Indonesia is. Biology molecular study would become a powerful tool to investigate the presence of leprosy bacillary whether there are similarities between the genomes of M. leprae isolates in the primeval and the present. PCR examinations were performed on mandibular bone fragments from ancient human who lived 1000 years B.C. discovered in archaeological surveys on the island of Lembata and three leprosy patients from East Nusa Tenggara. The DNA extraction was performed using a kit from Qiagen products and its TTC repeating pattern was seen with the method of direct sequencing. It turned out that the TTC profile obtained from samples of archaeological was as many as 13 copies, while the repetition of TTC in three samples of leprosy patients were 15, 17 and 26 copies. The different number of TTC repetition shows the different isolates of M. leprae between in the ancient times and the present. Further studies are needed to verify the differences in the genome that occur, for example from the study of SNPs (single nucleotide polymorphisms)

ACANTHAMOEBA SP.S-11 PHAGOCYTOTIC ACTIVITY ON MYCOBACTERIUM LEPRAE IN DIFFERENT NUTRIENT CONDITIONS

Background: Mycobacterium leprae (M. leprae) is a pathogenic bacterium that causes leprosy. The presence of M. leprae in the environment is supported by microorganisms that act as the new host for M. leprae. Acanthamoeba’s potential to be a host of M. leprae in the environment. Acanthamoeba sp. is Free Living Amoeba (FLA) that classified as holozoic, saprophytic, and saprozoic. The existence of nutrients in the environment influence Acanthamoeba ability to phagocytosis or pinocytosis. This study is aimed to determine Acanthamoeba sp.S-11 phagocytic activity to Mycobacterium leprae (M. leprae) which cultured in non-nutrient media and riched-nutrient media. Materials and Methods: This research conducted by culturing Acanthamoeba sp.S-11 and M. leprae on different nutrient media conditions. M. leprae intracellular DNA were isolated and amplified by M. leprae specific primers through Real Time PCR (Q-PCR). Result: The results showed that Acanthamoeba co-cultured on non-nutrient media were more active to phagocyte M. leprae than on rich-nutrient media. Conclusion: The use of non-nutrient media is recommended to optimize Acanthamoeba sp. phagocytic activity to M. leprae. Keywords: Acanthamoeba sp., Mycbacterium leprae, Nutrient, Phagocytosis, Real Time PCR Introduction Mycobacterium leprae (M. leprae) is a pathogeni

Distribution of Mycobacterium leprae genotypes from Surabaya and Bandung Clinical Isolates by Multiple Locus Variable Number of Tandem Repeat Analysis

Multiple Locus Variable Number of Tandem Repeat (VNTR) analysis has been proposed as a means of genotyping for tracking leprosy transmission due to tandem repeats’ potential as genetic markers to differentiate M. leprae strains. However, characteristics of polymorphism can vary depending on the population. This study aimed to compare the copy number of repeats in four genetic markers: TTC, AC8a, AC9 and 6-7 in leprosy patients from Surabaya and Bandung. Twenty three patients from Dr. Soetomo General Hospital and 21 from Hasan Sadikin Hospital were recruited. Multiple locus VNTR analysis was applied using total DNA extracts from Slit Skin Smear (SSS). From Surabaya, 7 samples showed the same copy number of four genetic markers (TTC=15; AC8a=10; AC9=10 and 6-7=6) and 2 showed another (TTC=16; AC8a=10; AC9=11 and 6-7=6); as for samples from Bandung, 2 showed the same copy number (TTC=15; AC8a=8; AC9=10 and 6-7=8) and 2 showed another (TTC=16; AC8a=10; AC9=11 and 6-7=6). The multiple locus VNTR analysis showed two identical M. leprae VNTR profiles from Bandung and Surabaya which supports the use of VNTR loci for transmission studies

A simpler diagnostic method using blood collection on filter paper to determine anti-natural octyl disaccharide-leprosy infectious disease research institute diagnostic in household contacts of leprosy patients

The high prevalence among children shows continued transmission from adult and lack of disease control by the health system. Detection of anti-NDO-LID-1 has been known to be more effective to predict the development of leprosy in household contacts than just detecting PGL-1 or LID- 1 alone. However, serodiagnosis is not available in poorer-settings area and the procedure (venepuncture) is still inconvenient to some people. These problems can be solved by using finger-prick blood sample on filter paper. This study aims to prove the effectiveness of using capillary blood samples on filter paper to detect the positivity of Ig G antibody against NDO-LID-1 antigen in asymptomatic household contacts. Seventeen samples of capillary blood on filter paper and sera were tested for IgG anti-NDO-LID-1 using ELISA. There was no significant difference between IgG level from filter paper and serum (p=0.754) and there was also a strong positive correlation (R=0.906) between the two procedures. These findings show that the use of filter paper and NDO-LID-1 is worthy of further investigations, especially for those with lower bacillary load or contacts of leprosy patients

A 5‑year evaluation of chemoprophylactic treatment in elementary school children with subclinical leprosy

Subclinical leprosy is an infectious disease in which the immune system remains infected with Mycobacterium leprae (M. leprae). The progress of subclinical leprosy to clinical cases within 1 year of infection is 1.5%, with an increase to 6% in the following 4 years. Rifampicin is frequently used for prevention of leprosy, and clarithromycin has a bactericidal effect on M. leprae. Thus, the combination of both is expected to improve disease control in patients with subclinical leprosy. The aim of the present study was to evaluate the efficacy of a chemoprophylactic treatment involving rifampicin and clarithromycin against subclinical leprosy in elementary school children from endemic areas of East Java over a 5‑year period. The study was performed between 2011 and 2015. Samples were collected from 2,548 healthy elementary school children in Nguling (Pasuruan) and Raas (Sumenep), and analysed using ELISA for anti‑PGL (phenolic glycolipid)‑1 IgM antibodies. Children who were seropositive for anti‑PGL‑1 IgM antibodies received a chemoprophylactic regimen consisting of rifampicin (300 mg/day) and clarithromycin (250 mg/day) daily for the initial 10 days, followed by the same regimen every 2 weeks for 3 months. Clinical and serological evaluations were performed annually for 5 years. Amongst the 2,548 healthy elementary school children, 200 were seropositive. The anti‑PGL‑1 IgM antibody levels significantly decreased between 2011 and 2015 in Nguling (from 1,066.7 to 137.4 U/ml) and Raas (from 773.1 to 563.4 U/ml), the levels decreased every year. In addition, the proportion of patients with decreased anti‑PGL‑1 IgM antibody levels was consistently higher than patients with increased anti‑PGL‑1 IgM antibody levels in all periods, except during 2013‑2014, in Nguling and Raas. Chemoprophylactic treatment involving rifampicin and clarithromycin may thus be effective against subclinical leprosy amongst elementary school children

Analisis Genotype mycobacterium leprae pada Lingkungan Air Daerah Endemis Kusta di Kabupaten Lamongan Jawa Timur dan Studi pola Transmisinya sebagai Salah Satu Sumber Penularan No-Manusia

Masalah kusta di Indonesia ditunjukkan dengan prevalensi cukup tinggi dan insidens yang tetap tinggi terutama di beberapa kawasan Indonesia Timur sehingga mempersulit penanggulangan. Secara teori sumber penularan basil kusta dapat terjadi melalui kontak langsung dengan sumber infeksi. maupun melalui jalur tidak langsung yaitu melalui lingkungan. Dari berbagai penelitian epidemiologi, timbul kecurigaan bahwa banyak individu yang terinfeksi basil kusta tanpa adanya sumber penularan yang jelas, terjadi antara lain disebabkan oleh penularan secara tidak langsung. Untuk mempelajari alur penularan Mleprae di lingkungan daerah endemik kusta adalah penting untuk mendeteksi Mleprae yang terdistribusi di Iingkungan dan penduduk Iingkungan tersebut serta menganalisa pola variasi gen yang terdistribusi di Iingkungan juga masyarakat setempat. Penelitian ini bertujuan untuk menganalisa Mleprae yang terdeteksi di lingkungan serta mendapatkan profil genotype isolat Mleprae di lingkungan

Leprosy Transmission in Endemic and Non-Endemic Areas Based on the Profile of Antibody Response of PGL-1 and PCR Detection of Mycobacterium leprae DNA from Nasal Swab among Healthy Children of East Java, Indonesia

Background: East Java has become one of the provinces that have higher prevalence of leprosy, especially in the coastal region. Environment has also influenced for leprosy transmission and early detection could reduce the incidence rate of new leprosy cases. Epidemiological studies of leprosy in children can give an illustration of the important aspects of the environment. Presence of Mycobacterium leprae (M. leprae) DNA in nasal swabs and seropositivity level among them can describe M. leprae exposure in that area. Objective: Analyzing PCR from nasal swab and seropositivity level among elementary school children between northern coast and southern coast of East Java province. Methods: Five hundred and thirty children in Pacitan and Lamongan were involved. Both areas are representation of northern and southern coastal region in East Java Province. After clinical examination, nasal swab and blood samples were obtained. ELISA test was performed to measure the titer of IgM anti Phenolic Glicolipid-1 (PGL-1) antibody then continued by PCR to detect M. leprae DNA. Results: From 301 students in Pacitan, 25 students (8.3%) are sero-positives and 9 students (2.9%) are PCR positives. from 229 students in Lamongan, 110 (48.3%) students are sero-positives and 49 students (21.4%) are PCR positives. Both are analyzed by Chi-Square, and from the PCR and ELISA, it concluded that there are statistically significant differences between the two regions. Conclusion: From study above shows that in the northern coast of East Java, incidence of subclinical leprosy is still high, it means that in this area still has a high risk of new cases of leprosy in the future and early detection tools need to performed as a preventative measure

ACANTHAMOEBA SP.S-11 PHAGOCYTOTIC ACTIVITY ON MYCOBACTERIUM LEPRAE IN DIFFERENT NUTRIENT CONDITIONS

Mycobacterium leprae (M. leprae) is a pathogenic bacterium that causes leprosy. The presence of M. leprae in the environment is supported by microorganisms that act as the new host for M. leprae. Acanthamoeba’s potential to be a host of M. leprae in the environment. Acanthamoeba sp. is Free Living Amoeba (FLA) that classified as holozoic, saprophytic, and saprozoic. The existence of nutrients in the environment influence Acanthamoeba ability to phagocytosis or pinocytosis. This study is aimed to determine Acanthamoeba sp.S-11 phagocytic activity to Mycobacterium leprae (M. leprae) which cultured in non-nutrient media and riched-nutrient media

Mycobacterium leprae BACILLEMIA IN BOTH TWINS, BUT ONLY MANIFEST AS LEPROSY IN ONE SIBLING

Leprosy in twins is rarely reported. A 19 years-old male student, from Lamongan district, was diagnosed as Multibacillary (MB) leprosy in the Skin and STD Clinic of Dr. Soetomo General Hospital Surabaya. Multiple anesthetic skin lesions were found, but the bacteriologic examination was negative for Acid Fast Bacilli (AFB). Histopathology examination support the diagnosis of BL type of leprosy. His twin brother that has been lived together since born until present seems healthy without any complaints of skin lesions and have no signs of leprosy. When a serologic examination for leprosy was performed, a high anti PGL-1 antibody level was found in patient (IgM anti PGL-1 2937 and IgG anti PGL-1 3080 unit/ml) while his healthy twin brother showed only low level (IgM 745 and IgG 0 unit/ml). Interestingly when a PCR study was performed to detect M.leprae in the blood, both of them showed positive results. Using the TTC method, a genomic study of for M.leprae, it is revealed that both samples were identic ( 27x TTC repeats). According to patient’s history, he had a traffic accident and got a wound in the knee seven years ago, while the skin lesions seems started from this area around three years ago before it spread to other parts of the body. The patient was treated with Multi-drug therapy (MDT) while his sibling got a prophylactic treatment for leprosy. After 6 months of treatment, the leprosy skin lesions were diminished and the serologic anti PGL 1 has been decreased. His healthy brother also showed a decrease in anti PGL 1 level and no skin signs of leprosy