Methicillin Resistant Staphylococcus Aureus (MRSA)

PROCEEDING

The 7th AIC-ICMR on Health and Life Sciences

The Annual International Conference 2017 Syiah Kuala University

“Advancing Knowledge, Research, and Technology for Humanity”

ISSN: 2089-208X

Banda Aceh, Aceh, Indonesia October 18-20, 2017

Proceedings of the 7th AIC-ICMR on Health and Life Sciences

Comparing Oviductal Expression During Oestrous Cycle of Epithelial Oviductin (OVGP1) in Peranakan Ettawah (PE) Goat

Herawati1*, Yudit Oktanella2, Aulia Firmawati2, Nurul Isnaini3 1Departement of Veterinary Public Health; 2Departement of Veterinary Reproduction, Faculty of Veterinary Medicine, Brawijaya University, Malang 65145, Indonesia 3Departement of Animal Husbandry, Animal Husbandry Faculty, Brawijaya University, Malang 65145, Indonesia *Correspondence e-mail: [email protected] / [email protected]

Abstract Over hundreds types of proteins are present in the oviductal tract, where fertilization occurs and zygotes begin to develop. Oviductin, also called Oviductal glycoprotein 1 (OVGP1), has been shown to be expressed by the oviductal epithelial cells and is suggested to play a role in fertilization and sperm–egg interaction. The expression pattern of oviductin become important since the number of studies suggested that its transcription is regulated via estrogen-dependent during oestrous cycle. The aim of this study was to identify the characteristics of oviductin expression in oviduct during oestrous cycle, especially on folicullar and luteal phase. Twenty-four fresh reproduction organ of Peranakan Ettawah (PE) goat were collected from abattoir, then divided into two groups (Group1: Follicular phase; Group2: luteal phase) based on the ovary performance macroscopically then we performed immunohistochemistry to identify the oviductin expression in the oviduct. Our results showed that the percentage of oviductin expression vary throughout the estrous cycle, which is higher in follicular phase. In both, the ampulla and infundibulum, the expression of OVGP1 was found to be higher at folicullar phase than luteal phase; but there was no significant difference of oviductin expression during luteal phase in isthmus. In conclusion, this finding suggests that the difference expression‘s patterns in both folicullar and luteal phase is important in regulating fertilization and embryogenesis on PE goat.

Key words: Oviductin, oviduct, PE goat, protein expression

INTRODUCTION

Although fertilization can be achieved in vitro without any contributions from the oviduct or oviductal proteins, some of these proteins play an important roles or yet remain unknown roles in fertilization and embryo development. In the pig, sheep, goat, cow, hamster, and rabbit, oviductal fluid is responsible for prefertilization ZP hardening (understanding ZP hardening as ZP changes that increase the ZP‘s resistance to proteolysis and reduce sperm binding. Oviductin that binds into ZP makes it more resistant to sperm attachment and decreases the risk of polyspermy in the pig and the cow (Coy et al. 2010). It undergoes numerous sequential morphological changes over the course of oestrous cycle, driven by cyclic fluctuations in several reproductive hormones.

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The 7th Annual International Conference (AIC) Syiah Kuala University and The 6th International Conference on Multidisciplinary Research (ICMR) in conjunction with the International Conference on Electrical Engineering and Informatics (ICELTICs) 2017, October 18-20, 2017, Banda Aceh, Indonesia Oviductal fluid contains amino acids, proteins, simple carbohydrates and complex ions, fats and phospholipids. These components include metabolic subtract such lactic, pyruvic acid, amino acids and glucose that the level is different in uterine liquids and serum (Hugentobler et al. 2010). Volume and components of some proteins in oviductal liquids is changing during estrous cycles. Part of the complex of proteins in oviductal liquids is produced from transudate serum, however there is also a specific protein synthesis and secretion of oviduct epithelium, and the secretion of these proteins is regulated by hormonal changes, followed by an increase in biosynthesis of periovulatory period (Buhi, 2002). During the oestrous cycles, the main function of cervical and oviduct on the reproduction process involves interaction with gametes, the estrus phase. Therefore, it can be hypothesized that the regulation of proteins secretion in cervical fluid and oviduct when the oestrous- oriented interacting with gametes. On the other hand, when the luteal phase, the amount of differential protein in the uterus can changes in the concentration and type. Oviductin (OVGP1) is estrogen-dependent glycoprotein secreted on epithelial oviduct by almost all mammals (Coy and Yanagimachi, 2015). Oviductin in swine, sheep, goats, cows and rabbits hamsters known to efect in increasing the resistance of the zona pellucida, when pre-fertilization against proteolysis and decrease the possibility of polyspermy (Mondejar et al., 2013). Oviductin is located in perivitelline space and the membrane of the embryo each species before implatation. Their densely glycosylated mucin-type domains would act as a protective shield around the oocyte and the early embryo and would be important for early stages of embryo development (Buhi 2002, Wolf et al. 2003, Gon alves et al. 2008). Oviductin in pigs known to increase the post-cleavage embryo development to the blastocyst is formed (Kouba et al. 2000, McCauley et al. 2003). In goats, oviductin increase the level of cleavage in the cleavage stage, as well as the morula and blastocyst formation (Pradeep et al., 2011). Detection and analysis of proteins, synthesized and secreted from the oviduct epithelial find their distribution temporal and spatial of macro molecules and the differences in the distribution of proteins in various species. Moreover, this analysis has allowed the identification of the major secretory products oviduct specific glycoprotein, whose secretion is dependent estrogen, and has a high molecular weight. Knowledge of the oviductin expression derived from normal histological appearance of the reproductive tract at each stage of the oestrous cycle is essential when evaluating female reproductive tissues for evidence of its regulation during fertilization and embryogenesis. This paper is mainly focus

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Proceedings of the 7th AIC-ICMR on Health and Life Sciences on oviductal tract since these two processes mainly occur in isthmus, ampulla, and infundibulum.

MATERIALS AND METHODS

Female reproductive organs derived from waste of Slaughterhouse Animal Gandang, Malang, collected a maximum of 2 hours after the slaughtering process. For the preparation of the organ, oviduct washed twice using physiological NaCl media and placed in a petri dish, then dissection. Determination of the follicular and luteal phases was seen by observing the dominant form in ovarian macroscpicly. The follicular phase marked by the formation of the dominant follicle in the ovary, meanwhile the luteal phase marked by the formation of the corpus luteum with minimum diameter of 0.3 mm. Result of grouping ovarian then placed in the organ pot containing 10% formalin for immunohistochemistry (IHC) staining. The IHC specimen made by goat-monoclonal antibody anti-oviductin (Abcam®), then the IHC specimen was observed under a microscope with magnification 400X and 1000X. The identification procedure oviductin expression by immunohistochemical methods, the oviduct specimen was made in object glass dipped in xylol twice, alcohol-rise (100%, 90%, 80%, 70% and 30%) consecutively. Then washed in PBS at pH 7.4 for 3 times each for

5 minutes. Then soaked in hydrogen peroxidase (H2O2) 3% for 5-10 minutes. Then soaked in 1% BSA in PBS for 10-30 minutes at room temperature. Added oviductin primary antibody for 1 hour at room temperature. Washed in PBS pH 7.4 for 3 times 5 minutes. SA-HRP was added for 30-60 minutes at room temperature. Washed in PBS pH 7,4 for 3x5 minutes. Added Cromogen DAB (3,3-diaminbenzidine tetrahydrochloride) for 10-20 minutes. Washed 3 times in PBS for 5 minutes at room temperature and then added with a counterstain for 3 minutes and then mounting with entellan. Observations were carried out by using a microscope at a magnification of 400x and 1000x times. Determination of the amount of oviductin expression can be seen from the percentage extensive DAB (brown color change in the lamina propria / oviduct epithelial cells) were compared among groups using software Immunoratio Oviductin expression data analysis.

RESULTS AND DISCUSSION

Identification of oviductin expression in the oviductal tract were analyzed by IHC staining by comparing the level of expression in the follicular and luteal phase. Based on it consideration that oviductin is a glycoprotein that is secreted by the oviduct epithelial cells

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Image 1. Oviductin expression in the oviduct PE goats with IHC staining. Description: Difference oviductin expression in lamina propria regions oviduct channel in the follicular phase (A) and luteal (B). The picture above shows the highest expression of oviductin seen in the follicular phase (A) and lower expression of oviductin in the luteal phase (B). Insertion of each image using 1000X magnification showing that oviductin expression in the cell nucleus (immunohistochemical staining, magnification 400X; microscope OLYMPUS; image of the scanned preparations using Software OlyVia) Oviductin expression obtained after measuring the percentage of area of DAB using software Immunoratio through 5 observation visual field in each oviduct specimen in the three areas which include: the isthmus, ampulla, and infundibulum. Table 1 shows the average percentage oviductin expression in follicular and luteal phases of the three locations the oviduct. Tabel 1. Average percentage oviductin expression in follicular and luteal phases of the third compartment oviduct tissue

Num. Oviductal area Average oviductin expression (%) ± SD 1 Isthmus Follicular 24.08 ± 3.041 Luteal 3.5 ± 0.969 2 Ampulla Follicular 46.65 ± 13.164 Luteal 5.52 ± 0.917

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3 Infundibulum Follicular 21.12 ± 2.218 Luteal 11.28 ± 1.416

During the oestrous cycle, main function of the cervix and the oviduct in the reproduction process involves interaction with gametes, which are present in the oestrous phase. These changes cause the micro environment (microenvironment) specified in the oviduct to the maturation of gametes, sperm capacitation, gamete and embryo transport, fertilization and early embryo cleavage. In the follicular phase, estrogen is important for epithelial differentiation and the development to maturation of cells and their secretory production of macro molecules. Therefore, the lumen environment consists of macromolecules and proteins derived from serum transudate or epithelial secretion. In the last decade it has been said that the oviduct and secretion oviduct plays an important role in various reproductive activities. Proteins produced to help increase the success of fertility, increased its concentration during proestrus and oestrus phase under the control of estrogen. Oviduct physiology of mammals have been studied intensively for many years. All information, whether old or new information has contributed to the understanding of fertilization and preimplantation embryo development. In mammals, the oviduct is the site where oocyte and zygote fertilized and zygot development stage. Oocytes were released from the ovarian follicles will be caught by the infundibulum. Oocytes will be forwarded to the ampulla to fertilize with spermatozoa into a zygote. Zygote will develop during the past the isthmus of the oviduct. Later, the early stages of embryo development phase (4 to 8 cells) into the uterus so that implantation to form the placenta. The whole process happens within the lumen of the oviduct and uterus, where the fluid is contained within the lumen of the oviduct and uterine (Coy and Yanagimachi, 2015). The fluid in the oviduct of mammals give biologic environment for fertilization and early cleavage stage embryos. Irregularities in the oviduct fluid composition can be detrimental for these processes. According Buhi (2002), the oviduct of mammals significant changes in physical, morphological, and biochemical induced by endocrine during estrus cycles or menstruation. These changes cause the micro environment (microenvironment) specified in the oviduct to the maturation of gametes, sperm capacitation, gamete and embryo transport, fertilization and early embryo cleavage. This complex function dependent on the activities of both the secretory epithelium and ciliated and non-ciliated that covers oviduct mucosal. The function of these cells are regulated by ovarian steroids, estrogen and progesterone. During the follicular phase, estrogen is important for epithelial differentiation

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CONCLUSIONS

In conclusion, this finding suggests that the difference expression‘s patterns in both folicullar and luteal phase could be important in regulating fertilization and embryogenesis on PE goat. Hopefully this recent study gives information of the oviductin physiology expression in oviduct of PE goat, to assist better understanding of its importance in tractus reproduction during follicular and luteal phase in order to maintain gamete transport, sperm and oocyte changes inside the oviductal environment.

REFERENCES

Buhi, W.C. 2002. Characterization and Biological Roles of Oviduct-specific, Oestrogen- dependent Glycoprotein. Reproduction 123: 355-362. Cognié, Y. G. Baril, N. Poulin, P. Mermillod. 2003. Current Status of Embryo Technologies in Sheep and Goat. Theriogenology 59: 171-188.

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Coy P., and M. Aviles. 2010. What Controls Polispermi in Mammals, the Oviduct or the Oocyte?. Biological Reviews 85: 593–605. Coy, P and R. Yanagimachi. 2015. Roles of Oviductal Proteins in Mammalian Fertilization and Embryo Development. BioScience 65: 973-984. Gardner, A.J., and J.P. Evans. 2006. Mammalian Membrane Block to Polyspermy: New Insight Into How Mammalian Eggs Prevent Fertilisation by Multiple Sperm. Reproduction, Fertility and Development 18: 53-61. Gonçalves RF, Staros AL, Killian GJ. 2008. Oviductal fluid proteins associated with the bovine zona pellucida and the effect on in vitro sperm-egg binding, fertilization and embryo development. Reprod Domest Anim. 2008 Dec;43(6):720-9. Hugentobler, A. Sasha & Diskin, Michael & J Leese, Henry & G Humpherson, Peter & Watson, Terry & M Sreenan, Joseph & Morris, Dermot. (2007). Amino acids in oviduct and uterine fluid and blood plasma during the estrous cycle in the bovine. Molecular reproduction and development. 74. 445-54. 10.1002/mrd.20607. Kouba AJ, Abeydeera LR, Alvarez IM, Day BN and Buhi WC. 2000. Effects of the porcine oviduct-specific glycoprotein on fertilization, polyspermy, and embryonic development in vitro. Biology of Reproduction 63 242–250 McCauley, T.C., W.C. Buhi, G.M. Wu, J. Mao,J.N Caamano, B.A. Didion, and B.N. Day. 2003. Oviduct-Specific Glycoprotein Modulates Sperm-Zona Binding and Improves Efficiency of Porcine Fertilization In vitro. Biology of Reproduction 69: 828–834. Mondèjar, I. M. Aviles, P. Coy, 2013. The Human Is An Exception To The Evolutionarily- Conserved Phenomenon Of Pre-Fertilization Zona Pellucida Resistance To Proteolysis Induced By Oviductal Fluid. Human Reproduction 28: 718-728. Pradeep MA, Jagadeesh J, De AK, Kaushik JK, Malakar D, Kumar S, Dang AK, Das SK, Mohanty AK. 2011. Purification, sequence characterization and effect of goat oviduct-specific glycoprotein on in vitro embryo development. Theriogenology. 2011 Apr 1;75(6):1005-15. Slavik T, Libik M, Wierzchos E, Fulka J. 2005. An attempt to reduce polyspermic penetration in lamb oocytes. Folia Biologica 51: 34-39

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Risa Nursanty1* and Irma Sari2

11Department of Biology, Faculty of Mathematics and Natural Sciences, University of Syiah Kuala, Banda Aceh 23111, Indonesia 2Department of Pharmacy, Faculty of Mathematics and Natural Sciences, University of Syiah Kuala, Banda Aceh 23111, Indonesia * e-mail: [email protected]

Abstract Knowledge of medicinal plants becomes part of the wealth of a nation. The wealth of local knowledge is still awake and generally inherited by traditional healers (Battra). Data collection on medicinal plants and herbs utilized by Battra in Aceh Province found several of medicinal plants, one of which was Geureudong pageu (Lannea coromandellica Houtt.). Several studies have demonstrated the ability of L. coromandellica plant inhibition against Staphylococcus aureus bacteria. So it was interesting to examine plants inhibition ability against resistant bacteria that was Methicillin Resistant Staphylococcus aureus (MRSA) bacteria. Extraction process was conducted by maceration method using two solvents that were n-hexane and methanol. Inhibition tests were performed using the Kirby- Bauer method. Ability to inhibit the MRSA bacteria can only showed by methanol extract from L. coromandellica leaves.

Keywords: Lannea coromandellica, medicinal plants, , MRSA bacteria, Kirby-Bauer method

INTRODUCTION

Knowledge of medicinal plants becomes part of the wealth of a nation. Particularly in the province of Aceh many known diverse plant species that have properties to treat the disease. Each tribe has data of medicinal plants and different ways of concocting. The wealth of local knowledge is still awake and generally inherited by the traditional healers (Battra). Data collection on medicinal plants and herbs utilized by Battra in Aceh province found various types of medicinal plants, one of them is Geureudong pageu (Lannea coromandellica Houtt.). The ethnic groups in Aceh generally use the leaves of the L. coromandellica plant. The use is mostly in the form of a herb instead of a single use. L. coromandellica plant leaves are used by ethnic Tamiang to treat stomach pain (stomach pain accompanied by blood out of the mouth / dirt). The Acehnese ethnic utilize the young leaves of L. coromandellica as

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Proceedings of the 7th AIC-ICMR on Health and Life Sciences a wound and hemorrhoids. The Aneuk Jamee ethnic uses its leaves as a medicine for diarrhea (Tim Pelaksana, 2012). Several studies have proven the antimicrobial ability of L. coromandellica plants. The results of Rupinder et al. (2013), showed 100% L. coromandellica leaf water extract can inhibit the growth of Staphylococcus aureus by 19.75 mm. Vedhanarayanan et al. (2015), reported the ability of L. coromandellica leaf methanol extract of 1000 μg / mL to inhibit the growth of S. aureus by 16.3 mm. The results of this study then become the based to determine the extent of inhibitory ability of plant extract L. coromandellica against resistant bacteria, such as Methicillin Resistant Staphylococcus aureus (MRSA).

MATERIALS AND METHODS

Plant Collection The leaves of L. coromandellica were collected from Kota Jantho, Kabupaten Aceh Besar.

Preparation of plant extracts The leaves of L. coromandellica collected were washed with water and air dried for 7 days at room temperature. The sample were ground into powder using electric blender (only two or three strokes) then filtered with mesh No 40. Six hundred grams of sample was macerated with n-hexane solvent for five days and protected from sun while stirring occasionally. The solvent was then filtered dan re-macerated for two days. The filtrates were then concentared by evaporation on a rotary evaporator. The same procedure was done using methanol solvent. The n-hexane and methanol extract were then made in concentrations of 5, 10, 15 and 20%.

Bacteri culture Bacteria tested in this study were MRSA obtained from Microbioloy Laboratory of University Indonesia (UI), Jakarta.

Antimicrobial assay The antimicrobial activity was carried out using Agar diffusion method. The procedure was carried as described by Andriani et al. (2016). In each disc were containing 20 µl of n-hexane extract, ciprofloxacin (antibiotic) and negative control (0%). The same test

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RESULTS AND DISCUSSION

The results of antibacterial activity were recorded as presence or absence of zones of inhibition around the disc (Figure 1.). According Fathia et al. (2015), the inhibitory zone around the disc indicated the absence of bacterial growth and it is reported as positive and absence of zone as negative.

20 20 15 % 20 % %

b a K K- K- K

Figure 1. Inhibition zone of methanol extract of L. coromandellica against MRSA; (a) diameter of inhibition zone; (b) disc

The antibacterial activities of Lannea coromandellica is shown in Figure 2. The results showed that n-hexane extract did not has antibacterial activity. The methanol extract shows the zones of inhibition that ranges from 6.9-8.2 mm. The resistance response generated by the methanol extracts include in medium category because it is in the range of 6 – 10 mm, according to Tokasaya (2010) . The positive control of ciprofloxacin has a zone of inhibition of 22.9 mm which belongs to a very strong category.

ts ts 20%

15%

10% Methanol (%) n-Hexane 5%

0 2 4 6 8 10 Concentrationofextrac Diameter of inhibition zone (mm)

Figure 2. Means inhibition zone (mm) of n-hexane and methanol extracts of L. coromandellica leaves diameter at 5, 10, 15 and 20% against MRSA

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Antibacterial potency of the L. coromandellica leaves extract is viable due to the presence of secondary metabolite compound. Reddy et al. (2014), described the chemical constituent in leaves were contain β sitosterol, ellergic acid, quercetin, leucocyanidin, and leucodelphinidin. Cowan (1999), have already demonstrated antibacterial activity from that chemical constituent. According Saefuddin (2014), secondary metabolite makes a synergistic relationship. The presence of a metabolite compound will strengthen the effects of other compounds. The present research work showed the methanol extracts of L. coromandellica leaves possess antibacterial activity against pathogens bacteria (Vedhanarayanan et al. 2015). However, these tested used a resistant bacteria type that was MRSA. Yuwono (2010), described that MRSA bacteria is resist to methicillin and all beta-lactam antibiotic group because it had mutation on Protein Binding Penicillinase 2 (PBP2) converted to PBP2a. PBP2 is a protein in the cell membrane of bacteria which plays role in the process of peptidoglycan synthesis.

CONCLUSIONS

The methanol extract of L. coromandellica leaves had potential antibacterial activity against MRSA bacteria. Hence, detail phytochemical investigations are necessary to isolate and characterize its active compounds.

ACKNOWLEDGEMENTS

Author is thankful to Ministry of Health Republic of Indonesia trough RISTOJA 2016 as research funder.

REFERENCES

Andriani CR, Oesman F, Nursanty R. 2016. Uji zona hambat ekstrak etil asetat daun alpukat (Persea americana Mill.) terhadap pertumbuhan bakteri Staphylococcus aureus. JKS 16:1-5. Cowan MM. 1999. Plant products as antimicrobial agents. Clin Microbial Rev 12:564-582. Fathia M, Nursanty R, Saidi N. 2015. Pengaruh Ekstrak Metanol Daun Kembang Sepatu (Hibiscus rosa-sinensis L.) Terhadap Bakteri Meticillin-Resistant Staphylococcus aureus. J. Biologi Edukasi 7: 22-28.

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Merril against three common pathogens. J Aryuveda Integr Med 9:224-228. Saifudin A. 2012. Senyawa Alam Metabolit Sekunder Teori, Konsep dan Teknik Pemurnian. Yogyakarta: Deepublish. Tim Pelaksana. Hasil Penelitian RISTOJA Aceh. 2012. Lembaga Penelitian Universitas Syiah Kuala. Banda Aceh. Tokasaya. P. 2010. Sponge-Associated Bacteria Producing Antimicrobial Compounds and Their Genetic Diversity Analysis [Thesis]. Graduate School. Bogor Agricultural University. Bogor Yuwono. 2010. Pandemi Resistensi Antimikroba: Belajar dari MRSA. JKK 42: 2837-2850 Vedhanarayanan P, Vaithiyanathan T, Sundaramoortthy P. 2015. Antimicrobial activity of chloroform and methanol extract of Lennea coromandelica Merr. International Letters of Natural Siences 45:67-74.

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Protection Effect of Glutamine Towards Aminotransferase Enzyme in Mice (Mus Musculus) Exposed by Lead

Dedy Syahrizal1*, Cut Mustika2, Raisha Fathimah3, Imam Maulana3, Sakdiah1, Reno Keumalazia Kamarlis4

1Biochemistry Department, Medical Faculty, Syiah Kuala University, Banda Aceh 23111, Indonesia 2Assesment Unit, Medical Faculty, Syiah Kuala University, Banda Aceh 23111, Indonesia 3 Biochemistry Study Club, Medical Faculty, Syiah Kuala University, Banda Aceh 23111, Indonesia 4Pathology Anatomy Department, Medical Faculty, Syiah Kuala University, Banda Aceh 23111, Indonesia

Abstract Lead is an ubiquitous element detected in all environmental media. Adult human beings and children receive lead the most from food, air and water intake. The majority of lead in the environment arises from burning of fossil fuels in automobiles and industrial emission. Lead occurs widely in the biosphere and is found to be a potent hepato-toxicant. The objective of this study was to screen the hepatoprotective role of glutamine against lead acetate intoxication in mice. Mice were randomized into control (aquadest, lead acetate and glutamine) and experiment group. Mice of the experimental group were administered 3,2 mg glutamine orally once in a day for 7 consecutive days. Mice were then treated with lead acetate (20 mg/kg of body weight animal intraperitoneally) on 7th day, one hour after glutamine administration. Then the mice blood were taken at 2 days to examine aminotransferase enzyme activation in quantitative technique. The results indicate that administration of 20 mg Pb/kg dose significantly increased levels of aspartate aminotransferase enzymes (AST) (p=0,045). Glutamine administration may decrease the activity of AST enzyme comparing to group which only given lead although in non-significant level (p=0,128). Comparing between group given glutamine protection before given lead and negative control show the result will be no different (p = 0.053). It was observed that glutamine treatment can restore ameliorated activation aminotransferase enzyme. This fact proves that glutamine is an antioxidant precursor which can reduce oxidative stress with activity parameter aminotranferase enzyme.

Keywords: aminotransferase , glutamine, lead, liver

INTRODUCTION

The increase of lead (plumbum) substances in an environment media is also linear with the increase of organ-damaged risk in the population of reluctantly contaminated. Liver is organ that gets the impacts the most from the contamination (Syahrizal, 2008). Liver is an organ which has the contributed role in the food, drugs, and toxic elimination metabolism process that get in to the body or organ. The liver damaging mechanism, caused by lead, is in the certain level so it may induce the formation of free radicals inside of body, and also decrease the capability of body‘s anti-oxidant system (Li, 2015). The liver damage can be

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The 7th Annual International Conference (AIC) Syiah Kuala University and The 6th International Conference on Multidisciplinary Research (ICMR) in conjunction with the International Conference on Electrical Engineering and Informatics (ICELTICs) 2017, October 18-20, 2017, Banda Aceh, Indonesia known through the biochemical examination. One of them is the aminotransferase enzyme group, which is aminotransferase aspartat aminotransferase enzyme (AST) or known as glutamate oksaloasetate transaminase (GOT) and alanine aminotransferase enzyme (ALT) or known as glutamat piruvat transaminase (GPT) . Both enzymes can be the indicator for identifying the liver function. There have been some efforts to decrease the liver damage due to free radicals, including the lead contamination. Several previous researches have also proven, the increase of glutation rate can prevent the cells damaging due to lead (Wardani, 2017). Glutamine content is the needed compound for forming the glutation together with sulphur will create sulfhidril glutation (GSH), GSH has the role in forming peroxidase glutation enzym (GPx) which is one of the body protection towards the over oxidation. Glutamine also has a role in forming colagen (through proline synthesis), nucleotide (through pirimidine synthesis, and also purine synthesis), and phospholipids. These three elements are so necessary in generating the new cells. Glutamine will also give alpha-ketoglutarate which will be in the Krebs cycle as the oxidative fuel for the fast cell regenaration. Because of that, glutamine will fasten the replacement and repair of damaged body tissues or the damage due to pain or injury, including liver (Leite, 2016; Petry, 2015) The formulation of the problem in this research is whether giving glutamine orally can protect the liver from the damaging due to lead so there won‘t be any increase Aminotransferase Enzyme (Aspartat Aminotransferase (AST) and Alanin Aminotransferase (ALT)) content in blood. The research purpose is to know protection effect of giving glutamine towards liver function, measured through the activity of aminotransferase enzym in blood for the mice exposed by lead. This research is hoped to give the benefit, glutamine is recommended as one of the hepatoprotective ingredients and can be used as one of the information for the next similar research in the future.

MATERIAL AND METHODS

The research is the laboratorial experimental method with Completely Randomized Design (CRD). As much as 28 male mice (Mus musculus L), divided in to 4 groups. Each group consists of 7 mice. The mice is male (Mus musculus) swiss strained, 6-9 weeks old by the body weight 25-40 grams. The animals for this trial are got from Mathmatics and Science Faculty, Biology Department, University of North Sumatera, Medan. Mice is kept in the plastic stall, closed ground, by using husk , and 7 mice were placed in each stall. The

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Proceedings of the 7th AIC-ICMR on Health and Life Sciences placement of the stall is in the ventilated and indirect sun-lighted room. The stall, the place for food and drink is cleaned and the husk base is changed twice a week minimally (Syahrizal, 2008) At the beginning of the acclimatization is the sample is set up to adapt with the surrounding environment aroung the stall for 7 days. Then, the sample is devided in to 4 groups,added each group has 7 mice. Group 1 or negative control group is treated by giving aquadest. Group 2 or positive control group is treated by giving lead acetate as much as 20mg/kg BW intraperitoneally (Syahrizal, 2008). Group 3 is treated by giving glutamine by the dose 3,2 mg/mice for 7 years in a row perorally (Jawi, 2006), and group 4 is given glutamine orally for 7 days in a row and continued by lead acetate as much as 20 mg/Kg BW intraperitoneally for an hour after giving glutamine on the 7th day. The blood withdrawal intracardially from each experimental group after 48 hours lead administration. The blood is added heparine anti-coagulan. Heparine blood is left for 30 minutes after centrifuged by the speed 3000 round per minute (rpm) for 10 minutes after its plasm is taken. Hence, the examination of enzym activity, AST and ALT. For AST, previously it must be treated by the quality control process, with Diacon N liquid. The spectrofotometer can be used if its result for AST is between 25,3-40,4 U/I. The next step is the examination on AST. Reagent liquid as much as 1000µl , added heparine plasm as much as 100 µl. Then, the incubation is done at 37°C for 1 minute. The mixture of plasm and reagent are poured in to spectofotometer, which is previously programmed for the examination of AST. Read the result, shown on the screen (U/I). The similar step is also done for ALT enzym activity examination which is previousy treated by implying the quality cotrol process by utilizing Diacon N liquid. The spectofotometer can be used if its result for ALT is between 17,6-28,2 U/I. The next step is the examination on ALT. Reagent liquid as much as 1000µl , added heparine plasm as much as 100 µl. Then, the incubation is done at 37°C for 1 minute. The mixture of plasm and reagent are poured in to spectofotometer, which is previously programmed for the examination of AST. Read the result, shown on the screen (U/I) (Syahrizal, 2008) The resulting data is the numerical data which is examined its normality by kolmogorov-smirnov test. By that test, the resulted normality is the data for AST enzyme activity is not distributed normally. It is continued by non-parametric statistic test for the numerical data which is mam-whitney test. On the other hand, the data for ALT after the normality test, it is resulted in the distributed data normally. So then, the parametric statistic

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RESULTS AND DISCUSSION

The examination of transaminase enzyme is basically an examination for identifying the changes on NADH coenzyme through the changes on its absorbance in spectrophotometry. This is done in such way because we realize it‘s nearly impossible to examine the enzyme levels directly because its level in plasm is too few. The enzyme level measurement is done through its decrease or increase of cofactor from he enzyme, which is called enzyme activity. (Panil, 2014) The effect of glutamine protection and lead acetate exposure towards transaminase enzyme level, whether it is for Aspartat Aminotransferase (AST) and Alanine Aminotransferase (ALT) of this research, are shown below. Table 1. Aminotransferase enzyme activity towards control and treatment group.

No Groups AST activity (U/l) ALT activity (U/l) 1 Negative control 85,00 32,00 2 Positive control 285,29 47,57 3 Pb Glutamine 152,86 45,00 4 Glutamine 98,14 38,14

Note:

Negative control: aquadest given - group Positive control : Lead acetate given - group by a single dose of 20 mg/kgBW intraperitoneally Pb Glutamine : Glutamine preventive given - group as much as 3,2 mg/mice for a week before giving Pb acetate by a single dose of 20 mg/kgBW intraperitoneally. Glutamine : Glutamine preventive given - group as much as 3,2 mg/mice for a week

From the examination of transaminase enzyme so there can be graphs that figure out the level of transaminase enzyme changes, whether Aspartat aminotransferase (AST) or Alanine aminotransferase (ALT) from each trial group. Here are the graphs that show the level changes of transaminase enzyme from each trial group.

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T Activity (U/l) TActivity 50 AS 0 Negative Control Positive Control Pb Glutamine Glutamine The Groups

Picture 2. The comparison of AST activity on all trial groups

50 45 40 35

30 25 20 15 10

T Activity (U/l) TActivity 5

AL 0 Negative Control Positive Control Pb Glutamine Glutamine The Groups

Picture 3. The comparison of ALT activity on all trial groups

Note:

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The 7th Annual International Conference (AIC) Syiah Kuala University and The 6th International Conference on Multidisciplinary Research (ICMR) in conjunction with the International Conference on Electrical Engineering and Informatics (ICELTICs) 2017, October 18-20, 2017, Banda Aceh, Indonesia From the result we can see that giving of single dose Pb 20 mg / kg of body weight significantly can increase AST enzyme level in the blood of mice (p <0,05). For AST group enzyme the activity increased by more than twice and for ALT group enzyme activity also increased even though not significantly (p = 0,216) compared to other group. Lead is one of the heavy metals that can cause severe organ damage in humans and animals. Previous research has suggested that the liver is one of the primary targets of lead toxicity (Adikwu, 2013). Accumulation of lead in the body can cause oxidative damage to the liver due to increased lipid peroxidation resulting in liver dysfunction (Haleagrahara, 2010; Wang, 2012). This is because Pb may cause an increase in ROS production and directly suppress the body's antioxidant system. Antioxidant levels are an indicator used to prove the damage that occurs in liver cells. Some studies suggest that antioxidants are an important and effective component in the treatment of lead poisoning (Hsu, 2002; Patrick, 2006). Reactive oxygen species (ROS) can react and cause damage to many molecules inside the cell. The phospholipids which is the fundamental elements of the plasma membrane and organela‘s membrane are often undergo lipid peroxidation. Lipid peroxidation is a free radical chain reaction that begins with the release of hydrogen from an unsaturated fatty acid by free radicals. An important consequence of lipid peroxidation is the increased membrane permeability and disrupting the distribution of ions that cause cell and organela damage (Aziz, 2014; Sharma, 2010). Liver liver damage due to exposure to lead is indicated by elevated liver enzyme activity which indicates cellular leakage and loss of functional integrity of the hepatic membrane. AST and ALT are the recommended serum enzymes to measure hepatocellular damage. AST and ALT are normally found in low levels in the blood, but if liver cell damage occurs then this enzyme will increase in the bloodstream (Wardani, 2017; Ozkaya, 2017). Increased activity of AST and ALT in the mice group exposed to lead in this study was caused by degeneration and necrosis process of hepatocyte. This is in accordance with previous studies which suggest that administration of lead acetate induces significant increases in AST and ALT activity (Mehana, 2010; Attia, 2013). Other studies suggest that increased activity of AST and ALT is a marker of increased free radical production and liver damage (Ibrahim, 2012). An increase in AST activity is higher than that of ALT, which proves that oxidative stress caused by Pb can reach organelles. As we have seen that AST is concentrated in organelles whereas ALT is in the cytoplasm (Wardani, 2017). So if the damage reaches organela then AST increment will be higher than ALT. This is also in accordance with

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Proceedings of the 7th AIC-ICMR on Health and Life Sciences research conducted by Syahrizal (2008) where the administration of Pb will increase activity of AST enzyme more significant than the activity of ALT enzyme. Glutamine administration may decrease the activity of AST enzyme comparing to group which only given Pb although in non-significant level (p = 0,128) but if we compare between group given glutamine protection before given Pb the result will be no different with the negative control. This is proved by a p value greater than 0.05 (p = 0.053). In addition, there were also groups that were given only glutamine (p = 0.011). This proves that glutamine is a powerful scavenger that able to breakdown the autocatalytic process of lipid peroxidation process of cell membranes to maintain cell integrity. In addition, the provision of glutamine has also been thought to improve the body's antioxidant system by increasing the reduced-glutathion level. The decreament of glutathione levels is a major cause of hepatotoxicity in the liver (Leite, 2016; Jawi, 2006). The effect of glutamine in decreasing the activity AST enzyme is because glutamine is an amino acid that can act as the carbon source needed in the process of glutamate metabolism. Glutamine administration can increase glutamate levels by reaction catalyzed by glutaminase enzymes. Glutamate will bind to cysteine to form γ-glutamyl-cysteine. This compound will then bind with glycine to form glutathione (Sappington, 2016). This situation confirms that the administration of glutamine is a useful action to increase glutathione levels so that it can repair damaged liver cells due to the oxidative stress process. For ALT groups, decrement of enzyme activity occurred but not significant (p = 0,216). This shows the effect of glutamine protection against exposure to Pb in liver organ. This explains that glutamine works in organelles rather than in the cell's cytoplasm (Syahrizal, 2008).

CONCLUSIONS

Pb administration with a single dose of 20mg / kg of body weight intraperitoneally can significantly cause liver cell damage. This is proven by the increased activity of enzyme aminotransferase in groups that are only given Pb. Glutamine administration also decreases the activity of the aminotransferase enzyme in mice when compared with the group given only Pb. Acknowledgment The researcher is grateful for all sides that have helped this research. Especially for the financial support for the research from DIPA Syiah Kuala University. The gratitude is

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REFERENCES

Adikwu E, Deo O, Geoffrey OB, Enimeya DA. 2013. Lead organ and tissue toxicity: Roles of mitigating agents (Part 1). Br J Pharm Toxicol 4:232–40 Attia AM, Ibrahim FA, Nabil GM, Aziz SW. 2013. Antioxidant effects of ginger (Zingiber officinale Roscoe) against lead acetate-induced hepatotoxicity in rats. Afr J Pharm Pharmacol 7:1213–9. Aziz FM, Maulood IM, Chawsheen MA. 2014. Effects of melatonin, Vitamin C and E alone or in combination on lead-induced injury in liver and kidney organs of rats. Pak J Zool 46:1425–31. Haleagrahara N, Jackie T, Chakravarthi S, Rao M, Kulur A. 2010. Protective effect of Etlingera elatior (torch ginger) extract on lead acetate–Induced hepatotoxicity in rats. J Toxicol Sci 35:663–71 Hsu PC, Guo YL. 2002. Antioxidant nutrients and lead toxicity. Toxicology 180:33–44 Ibrahim NM, Eweis EA, El-Beltagi HS, Abdel-Mobdy YE. 2012. Effect of lead acetate toxicity on experimental male albino rat. Asian Pac J Trop Biomed 2:41–6 Jawi IM, Manuaba IB, Sutirtayasa IWP, Muruti, G. 2006, Pemberian Glutamin Menurunkan Kadar Bilirubin Darah serta Mengurangi Nekrosis Sel-sel Hati Setelah Pemberian Aktivitas Fisik Maksimal dan Parasetamol Pada Mencit. Dexa Media 19(4): 192-5. Leite JS, Raizel R, Hypólito TM, Rosa TD, Cruzat VF, Tirapegui J, 2016. l-glutamine and l- alanine supplementation increase glutamine-glutathione axis and muscle HSP-27 in rats trained using a progressive high-intensity resistance exercise. Appl Physiol Nutr Metab 41(8):842-9. Li S, Tan HY, Wang N, Zhang ZJ, Lao L, Wong CW, Feng Y. 2015. The Role of Oxidative Stress and Antioxidants in Liver Diseases. Int J Mol Sci. 16(11):26087-124. Mehana EE, Abdel Raheim MA, Meki KM. 2010. Ameliorated effects of green tea extract on lead induced liver toxicity in rats. Exp Toxicol Pathol 13:173–80. Ozkaya A, Sahin Z, Kuzu M, Saglam YS, Ozkaraca M, Uckun M, Yologlu E, Comakli V, Demirdag R, Yologlu S. 2017. Role of geraniol against lead acetate-mediated hepatic damage and their interaction with liver carboxylesterase activity in rats (Abstract). Arch Physiol Biochem. doi: 10.1080/13813455.2017.1364772. Panil Z. 2014. Memahami Teori dan Praktik Biokimia Dasar Medis Untuk Mahasiswa Kedokteran,Keperawatan,Gizi Dan Analisis Kesehatan: EGC, Jakarta Patrick L. 2006. Lead toxicity part II: The role of free radical damage and the use of antioxidants in the pathology and treatment of lead toxicity. Altern Med Rev 11:114– 27.

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Petry ÉR, Cruzat VF, Heck TG, Homem de Bittencourt PI Jr, Tirapegui J. 2015. L-glutamine supplementations enhance liver glutamine-glutathione axis and heat shock factor-1 expression in endurance-exercise trained rats. Int J Sport Nutr Exerc Metab. 25(2):188-97. Sappington DR, Siegel ER, Hiatt G, Desai A, Penney RB, Jamshidi-Parsian A, Griffin RJ, Boysen G, 2016. Glutamine drives glutathione synthesis and contributes to radiation sensitivity of A549 and H460 lung cancer cell lines. Biochim Biophys Acta 1860(4):836-43. Sharma V, Pandey D. 2010. Protective role of Tinospora cordifolia against lead-induced hepatotoxicity. Toxicol Int 17:12–7 Syahrizal D, 2008, Pengaruh Proteksi Pemberian Vitamin C Terhadap Enzim Transaminase dan Gambaran Histoptologis Hati Mencit yang Dipapari Plumbum (Thesis). Retrieved from USU Digital Library. Wang J, Yang Z, Lin L, Zhao Z, Liu Z, Liu X. 2012. Protective effect of naringenin against lead-induced oxidative stress in rats. Biol Trace Elem Res 146:354–9. Wardani G, Farida N, Andayani R, Kuntoro M, Sudjarwo SA. 2017. The Potency of Red Seaweed (Eucheuma cottonii) Extracts as Hepatoprotector on Lead Acetate-induced Hepatotoxicity in Mice. Pharmacognosy Res. 9(3):282-286.

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Fitria Salim*, Sitti H, Wahyu L

Dermatology and Venereology Department Faculty of Medicine Syiah Kuala University/dr. zainoel Abidin Hospital, Banda Aceh, Indonesia *Corresponding author e-mail: [email protected]

Abstract Leprosy is the chronic infectious disease that although curable, continues to be a significant health problem in many parts of the world. The disease presents polar clinical forms (the multibacillary lepromatous leprosy and the paucibacillary tuberculoid leprosy), as well as other intermediate forms with hybrid characteristics based on patient‘s clinical state and immune status. Leprosy causes complication in eyes and nerve damage that lead to paralytic, deformity, and handicap that affect the patient‘s quality of life. We present a case of lepromatous leprosy with iridocyclitis and grade two disability in a 31-year-old man who presented multiple hyperpigmented, anesthetic lesions on the face and arms with both hands were claw-shaped and numbness since ten months ago. The patient has complained red eye with pain, tenderness and impaired vision for one month. Slit skin smear examination for M. leprae from lesions and earlobes revealed a bacteriological index of 3+ and 70% for the morphological index. The patient was treated by multi-drug therapy multi bacillary for 12 months and referred to ophthalmologist and physiotherapist for comprehensive management.

Keywords: disability, iridocyclitis, lepromatous leprosy

INTRODUCTION

Leprosy is a granulomatous infectious disease caused by the acid-fast bacillus Mycobacterium leprae. The mechanism of pathogen transmission is poorly understood, but most probably the respiratory tract plays a significant role in droplet infection although it may occur through the damaged skin. Leprosy cases occur at all ages in endemic populations, more commonly in males than females, which may reflect a gender difference in immune response. Although the precise genetic factors may be associated with leprosy overcrowding, prolonged contact, an absence of history BCG vaccination are known as the risk factors of disease. The number of registered leprosy patients decline from 5 million in 1985 to 0.7 million in 2001. During the period 2000-2005, the global decline in case detection was dramatic (about 58%) and much more limited (18%) during period 2006-2009. At the beginning of 2008, WHO reported a global registered prevalence of leprosy of 212,802 cases and an incidence of 254,525 cases. Only three countries (Brazil, Nepal, and Timor-Leste) still

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Proceedings of the 7th AIC-ICMR on Health and Life Sciences report prevalence figures above the target of one per 10.000 Population. In the united states, there are approximately 150 new cases of leprosy each year, but since 2010, 72 cases were reported, and 19 cases occurred in 2015. According to Department of Health in Aceh province, the number of new case detection rate in 2013 reached 9 per 100.000 inhabitants. Also, the total of newly identified cases in 2011, 2012 and 2013 subsequently were 48 cases, 51 cases, and 45 cases. Leprosy is diagnosed when at least one of the following cardinal signs is manifested as definite loss of sensation in a hypopigmented or reddish skin patch, a thickened or enlarged peripheral nerve, and the presence of acid-fast bacilli in a slit-skin smear. Variety of leprosy clinical forms related to the type and strength of the immune response, so there was the classification that most often used today is Ridley-Jopling and WHO. Based on the WHO criteria for leprosy were divided into two groups depending on the number of the skin lesions and smear examination of the skin lesions. There are Paucibacillary (PB) and Multibacillary (MB). The Ridley and Jopling classification divided into some groups based on the patient's immunity, which is indeterminate (I), tuberculoid (TT), Borderline tuberculoid (BT), mid- borderline (BB), Borderline lepromatous (BL) and lepromatous leprosy (LL). The treatment of leprosy is aimed to cure the patient, to avoid the handicap and to demolish the contagion effect. If the patients do not consume the medication regularly, the leprosy strain will be immune and causes the permanent symptom or even a new worse symptom. Three standard first-line drugs that rifampicin, clofazimine, and dapsone are available for use in multidrug therapy (MDT) of fixed duration. Leprosy reaction affects 30 to 50% of the patients that often appear at the initiation of treatment however it may occur at any time during the disease. The reaction may affect the skin, other organs like eyes and can cause permanent damage to nerves that high risk of disability. Ocular affection in leprosy which iridocyclitis is the most important, sometimes forerunner of other complications including blindness, and it is common in lepromatous leprosy. Reduced vision and lagophthalmos, visible damage and loss of tissue to the hands and feet are grade two disability also claw hand and drop foot. Management of disabilities should be an integral part of routine treatment services; it should include the provision of aids and appliances, specialist medical care, and surgical reconstruction and rehabilitation facilities.

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Figure 1. clinical manifestasion of the patient

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We diagnosed patient with lepromatous leprosy and grade two disability with suspect uveitis. The patient received a combined daily regimen of rifampicin (600 mg monthly), dapsone (100 mg daily) and clofazimine (300 mg monthly and 50 mg daily) for 12 months. He also got urea 10% that applied twice daily as topical therapy for his hyperpigmented patches and dry skin. We consulted the patient to ophthalmologist then be diagnosed as iridocyclitis and got systemic and local corticosteroids with mydriatic drops 2-3 times daily. He responded well to the therapy with no adverse events or immunologically mediated reactions. The eyes complaints improved within a week of treatment, so oral corticosteroid was considered to be tapered according to the procedure. We also encouraged the patient to see a physiotherapist regarding his disability.

DISCUSSION

Leprosy or Hansen‘s disease presents a spectrum of clinical, bacteriological, immunological, and dermatopathological characteristics. The clinical presentations are multi- various because of the combinations of any different primary skin lesions with or without signs of inflammation and sequelae of peripheral nerve damage cause lose the ability to sense touch and pain, which can lead to injuries, like cuts and burns. The affected skin changes color and either becomes lighter or darker, often dry or flaky, or reddish due to inflammation of the skin. The Ridley-Jopling classification recognizes a spectrum of cell-mediated response to M. leprae infection and thus can be used to identify patients who are considered immunologically unstable and at risk of developing leprosy reactions. Lepromatous leprosy is characterized by a much more generalized disease, diffuse involvement of the skin, thickening of many peripheral nerves, and at times involvement of other organs, such as eyes, nose, testicles, and bone. The intermediate subtypes are borderline tuberculoid, mid- borderline, and borderline lepromatous leprosy. Borderline leprosy and the subtypes are characterized by more extensive disease than polar tuberculoid, with more numerous skin lesions and more nerve involvement, but not as widespread disease as in lepromatous leprosy. Leprosy is the only disease in which there can be a massive invasion of the dermis or nasal mucosa with acid-fast bacilli (AFB). In some forms of the disease, bacilli are demonstrated in slit-skin smears or nasal mucus or scrapings. The bacilli in the skin are quantified with bacteriological index (BI). It ranges from 0 to 6+ according to some bacilli per oil immersion field at the microscopic examination. It is a late marker of the antibacterial action of chemotherapy although it is of prime importance in the diagnosis of new cases or

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The 7th Annual International Conference (AIC) Syiah Kuala University and The 6th International Conference on Multidisciplinary Research (ICMR) in conjunction with the International Conference on Electrical Engineering and Informatics (ICELTICs) 2017, October 18-20, 2017, Banda Aceh, Indonesia relapses. The Morphological Index (MI) is the percentage of solidly stained bacilli of standard size and shape. These bacilli are thought to be the viable ones, that is the ones that are alive and may infect someone. With effective treatment, the MI will go to 0% rapidly, and the BI will fall by 1+ every 1 or 2 years. In this case, we found very dry skin, numerous hyperpigmented patches and diffuse infiltrates on his face and arms with anesthetic sensation to touch, pain and heat. He also had glove and stocking anesthesia and deformities on the upper and lower extremities. On nerve examination, we found that enlargement at auricularis magnus nerve. We did slit-skin smear, and the result is 3+ for Bacteriological Index and 70 % for Morphological Index. Based on these anamneses, clinical features and laboratory finding that support our suspicion that patient has lepromatous leprosy. The eyes may become involved in leprosy in three ways as a complication of involvement of the facial and occasionally the trigeminal nerve, by the invasion of the eyeball by large numbers of AFB in lepromatous leprosy, and by participation in the generalized reaction or reactive phase. It is a leading cause of irreversible blindness. Iridocyclitis is an inflammation of the iris, and ciliary body usually occurs bilateral, has a very chronic course with acute exacerbations. Acute Iridocyclitis followed leprosy reaction and common in lepromatous type and border line cases but rare in tuberculoid leprosy. Clinically, we can see mild ciliary hyperaemia on his conjunctiva next to the limbus. With a slit lamp, the earliest signs are keratic precipitates (KP) on the back of his cornea, a flare, and cells floating in his anterior chamber; these are also the last signs to disappear. Iridocyclitis may be unilateral or bilateral and be mistaken for conjunctivitis, particularly since it may occur on its own, without any sign of reaction. If you are in doubt, and you don't have a slit lamp, dilate his pupils. Any irregularity due to synechiae will then be diagnostic, but it will not tell you if his iridocyclitis is active. If dilatation relieves his pain it probably is active. Iridocyclitis usually responds well to treatment, if untreated, it can cause permanent damage and loss of vision from the development of glaucoma, cataract, or retinal edema. The treatment usually includes prescription eye drops, which dilate the pupils, in combination with anti-inflammatory drugs that usually takes several days or in some cases, several weeks. If redness or pain persists, or if his corneaes are cloudy, he may need steroids by subconjunctival injection, or rarely by oral. The patient must be taking his leprosy drugs at the same time. If he has a chronic Type Two reaction (ENL), he may need atropine once weekly and steroid ointment daily for many months.

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Leprosy reaction is body responds to the infection by becoming inflammation of skin patch, the nerves, the eyes and the internal organs in a few cases. Inflammation of nerve is severe and can be very painful, but sometimes the inflammation can destroy nerve without the person feeling anything. The damaged nerve is a high risk of disability, so it is important to treat reactions quickly. It is beneficial to assess the disability that patient has at the start of treatment and then during the therapy. The three-grade WHO disability grading system has been the most widely in use. Each eye, hand, and foot should be assessed and graded separately. Visible deformity of hands and feet includes ulceration, shortening, disorganization, and stiffness also severe visual impairment (vision less than 6/60), lagophthalmos, corneal anesthesia and iridocyclitis are grade two disability. The regimen is being used to treat leprosy is multiple drug therapy (MDT), as advocated by the WHO. This combination prevents the development of drug-resistant bacilli and has shortened the duration of treatment to 12 months for Multibacillary leprosy with 600 mg rifampicin and 300 mg clofazimine monthly doses, also 100 mg dapsone and 50 mg clofazimine for the daily doses, however, paucibacillary leprosy treatment just six months without clofazimine. The biggest problems you may find are side effects of the drugs, sign of new nerve damage or inflammation (reaction) and new social problems related to leprosy. Serious side effects of treatment are rare. The more severe side effects are drug allergy to one of the drugs and jaundice if it happens the drug must stop. The treatment for our patient using MDT multibacillary for 12 months and 10% urea as topical therapy for moisturizer. We consulted him to an ophthalmologist and was diagnosed with iridocyclitis. He got oral and local corticosteroids as anti-inflammatory also mydriatic drops 2-3 times daily for dilating the pupils. Within a week of treatment, both of eyes were improved with reduced redness and pain. We planned to refer patient to a physiotherapist regarding his disability. We gave education to the patient about the disease, treatment plan, the importance of continuing long-term therapy until the course is completed, the side effect of drugs which is rifampicin turn the urine red, and the patient may feel nausea and vomiting during treatment. He should eat nutritious foods so that his body stronger against bacteria. We also encourage patient always use footwear and gloves to avoid injury, because of the patient`s hand and feet numbness.

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We report one case of lepromatous leprosy with iridocyclitis and grade two disability in a 31-year-old man treated with multidrug therapy for multibacillary for 12 months, oral and eyes drops of corticosteroid. The improvement of eyes showed after one week of treatment by there are no redness and pain anymore. No side effect was shown during the therapy.

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Alex K, Brad HF, Debra AG, Kleonikos AT, Russel WR. Acute anterior uveitis. American Academy of Ophthalmology. 2014. Antunes DE; Araujo S; Ferreira GP; Cunha CS; Costa AV; et al. Identification of Clinical, Epidemiology and Laboratory risk Factor for Leprosy Reaction for Leprosy Reactions during and after Multidrug Theraphy. Dermatology Sanitria, Hospital de Clinicas, Faculdade de Medicine, Universidade Federal de Uberlandia. Brasil. 2013. Vol 7. 108:901-908. Dogra S; Kumaran MS; Narang T; Radotra BD; Kumar B. Clinical characteristic and outcome in multibacilllary (MB) leprosy patients treated wiyh 12 months WHO MDT-MBR: a retrospective analysis of 730 patient from a leprosy clinic at a tertariary care hospital of Northern India. Departement of Dermatology venereology and Leprology, Department of Histopatology, Postgraduate Institue of Medical Education and Research Chandigarh 160012, India. 2013, 84:65-75. Giran RJS; Soares NVL; Pinheiro JV; Oliveira GDP; carvalho SMF; Abrue LCD; Valenti VE. Leprosy Treatment Dropout: A Systematis Review. International Archives of Medicine. Brazil. 2013. 6:34. Legendre DP; Pham D; Christiana A; Muzny MD; Swiatlo E. Hansen‘s Disease (Leprosy). Current and future Pharmecotherapy and Treatment of Disease-related Immunologic Reactions. 2012, 32(1): 27–37. Lockwood DN, Saunderson PR. Nerve damage in leprosy: a continuing challenge to scientists, clinicians, and service providers. International Health. 2012, 4: 77-85. Grzybowski A, Nita M, Virmond M. Ocular leprosy. Clinical Dermatology. 2015, 33 (1): 79- 89. Polycarpou A; Walker SL; Lockwood DNJ. New findings in the pathogenesis of leprosy and implications for the manangement of leprosy. Curr Opin Infection Disease. 2013, 26: 413-419. Pranesh K, Gururaj VW, Shreyans PK. Ocular manifestations in leprosy. International Journal of Basic and Applied Medical Sciences2014, 4 (3): 192-195. Wahyuni ET; Amiruddin MD; Vitayani. Reversal Reaction in Tuberculoid Leprosy. Departement od Dermatology Medical Faculty of Hasanuddin University/Wahidin Sudirohusodo Hospital. Makassar. 2012. Vol 1. No 3. P 68-74.

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World Health Organization. WHO multidrug therapy (MDT). World Health Organization, Geneva, Switzerland. 2013. http://www.who.int/lep/mdt/en/.

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