Dermatology Reports 2019; volume 11(s1):8057

The correlation of Ig M Mycobacterium leprae (M. leprae). Leprosy Anti PGL-1 antibody between can be cured but it still continues to cause Correspondence: Medhi Denisa Alinda, significant health problems in many parts of Dermatology Venereology Dept, Faculty of blood veins and dryed capillary the world. World Health Organization Medicine, Universitas Airlangga - DR blood on filter papers in house- (WHO) reported the number of new cases Teaching Hospital, Jl. Mayjen. Prof. Dr. , No. 47, Airlangga, Gubeng, of leprosy in Southeast Asia in 2015 as hold contact , Jawa Timur, 60286, Indonesia. of leprosy patient many as 154,834 cases where Indonesia Tel.: +6285645050746. ranks third largest after Brazil is as many as E-mail: [email protected] 1 17,025 cases. [email protected] Anisha Calista Prakoeswa,1 Leprosy is still a health problem in Bayu Bijaksana Rumondor,1 Indonesia, including . Key words: household contact of leprosy, IgM Meva Nareza Trianita,1 ,2 Transmission of leprosy is still high enough anti-PGL-1, capillary blood on filter paper, cubital vein. Fatma Rosida,3 Linda Astari,3 with the incidence rate of new leprosy in 3 2 M. Yulianto Listiawan, East Java which in 2014 found 4,114 cases. Contributions: the authors contributed equally. Indropo Agusni,3 Shinzo Izumi,2 In Dr. Soetomo General Hospital, Surabaya, Medhi Denisa Alinda3 Indonesia, there are 713 (6.49%) new lep- Conflict of interest: the authors declare no 1 rosy patients from a total of 10,970 leprosy potential conflict of interests. Faculty of Medicine Universitas patients in the Outpatient Clinic 2011- Airlangga, Dr. Soetomo Teaching 2015.3 Received for publication: 1 February 2019. 2 Hospital; Leprosy Study Group, The failure of leprosy elimination is Accepted for publication: 13 February 2019. Institute of Tropical Diseases, caused by the failure of the transmission 3 This work is licensed under a Creative Universitas Airlangga; Department of chain termination, namely the difficulty of Commons Attribution-NonCommercial 4.0 Dermatology and Venereology Faculty of early detection of Subclinical Leprosy (SL), International License (CC BY-NC 4.0). Medicine Universitas Airlangga, a condition characterized by specific anti- Dr. Soetomo Teaching Hospital, bodies against M. leprae high enough in the ©Copyrightonly A.C. Prakoeswa et al., 2019 Surabaya, Indonesia blood without any clinical symptoms. SL is Licensee PAGEPress, Italy thought to be primarily derived from house- Dermatology Reports 2019; 11(s1):8057 hold contacts of leprosy patients, because doi:10.4081/dr.2019.8057 until now it is believed that the main trans- Abstract use mission of leprosy is from Multi bacillary samples taken have to be sent from the field 4,5 Delays of leprosy detection and treat- leprosy patients. to the central laboratory at a controlled tem- ment can lead to disability and potential Early diagnosis of leprosy is an impor- perature to ensure stability; still need a cen- transmission. Serologic examination has the tant issue as late detection and treatment trifuge for serum taking; and in certain advantage in detecting Subclinical Leprosy. can result in disability as well as the psy- cases such as children and the elderly, the The procedure of serologic test, which is chosocial impacts due to the persistent process of taking blood is difficult to per- one of its main limitation, could be simpli- stigmatization of the disease in the commu- form. If the time period since the blood fied by the use filter paper. This study aims nity. In addition, it is also necessary to sample is taken until the serum is examined to assess the effectiveness of the use of cap- watch out for potential transmission of lep- in the laboratory long enough, preservatives illary blood dropped on filter paper as a sub- rosy in cases that are too late to be detected. and special storage are required. All of the stitute for venous blood in household con- Various studies on leprosy diagnostic sero- above obstacles cause leprosy serology tests tact of leprosy patients. Seventeen samples logic examination continue to be developed. are difficult to do on a large scale in leprosy of capillary blood dried on filter paper and Phenolic Glycolipid-1 (PGL-1) is a specific endemic areas. It is well known that the venous blood samples from the same indi- antigen for M. leprae which was first dis- serum of blood required for serological tests vidual were examined by ELISA method to covered in the 1980s. Ig M anti PGL-1 anti- does not need too much, just 50 µl of blood determine the levels of IgM anti-Phenolic bodies indirectly indicate the presence of M. serum can be obtained from a few drops of glycolipid-1 (PGL-1). The mean Non-commercial of anti- leprae in individuals, which may reflect blood. For that we need to develop a more PGL-1 IgM levels of filter paper samples both SL and leprosy. Seropositivity of IgM practical blood-taking method that is 163.31±126.16; whereas the mean of levels anti-PGL-1 antibodies was found to be 80- through filter paper that can absorb the from venous samples was 473.16±411.26. 100% in untreated untreated multibasilar blood and then dissolved by soaking There was significant difference and corre- (MB) leprosy patients, whereas in patients process. Blood storage to filter paper has lation between these two groups. Samples with pausibasiler (PB) type, there was a several advantages such as it does not on filter paper in household contact can be lower titer of IgM antibodies with 30-60% require complicated sample processing and 6-8 used to determine the level of anti-PGL-1 seropositivity. samples can be stored for long periods so as IgM in serum by converting on the regres- Detection of antibodies against PGL-1 to facilitate serological examination in lep- sion basis. Further study is required to eval- has been widely used for community sur- rosy endemic areas that generally have lim- uate the potency of filter paper methods to veys, early diagnosis, treatment monitoring, ited health facilities and infrastructure. In conduct large-scale serological screening. reaction monitoring and identification of addition, blood sampling could derived relapses. At this time serology was per- from an easier location such as from a fin- formed mostly using a blood serum sample gertip. from the cubital vein. The main obstacles to Prakoeswa in 2013 reported that 6% Introduction this serologic examination in addition to the subclinical leprosy with high IgM anti- relatively expensive costs are often a prob- PGL-1 titers become manifest leprosy in 4 Leprosy or Hansen’s disease is a chron- lem in the field associated with fairly com- years, that means 1.5% annually. Range of ic infectious disease caused by plex sample treatments such as: the blood Ig M anti-PGL-1 antibody titer in leprosy

[page 106] [Dermatology Reports 2019; 11(s1):8057] Article patients with seropositive range between who live in the house and close contact for tometer (Elisa reader) with Optical Density 605 units / ml (cut off for population of East at least 6 months with multibacillary lep- (OD) unit at 492 nm. Further data is stored Java) up to > 5300 units / ml. While the rosy patients (spouses, parents, or children) and processed with Biolyse / X read pro- anti-PGL-1 titer study was conducted in maximum of 3 domestic people. Blood gram. The result is quantitative data with East Java populations divided into seropos- samples were collected following local unit / mL unit. itive +1 (605-1,000 u / ml), +2 (1,000-2000 ethics committee approval from Dr. u / ml), +3 (2,000-3,000 u / ml) and +4 (> Soetomo General Hospital and after partici- 3,000 u / ml). Individuals with +3 and +4 pants signed their informed consent forms. Results seropositivity are potentially become mani- A sample of 10 µL of capillary blood fest leprosy.9 from a vaccinotile / Micro Hematocrit cap- Seventeen fingertips blood capillaries Until now there has been no research on illary tube/plain (MHC tube) wound on the samples of household contact were dried on the examination of Ig M anti-PGL-1 anti- fingertips was taken by a capillary micro filter paper and 17 cubital vein blood sam- body levels by using capillary blood on fil- tube containing heparin. The blood is ples from the same individuals were exam- ter paper in household contact of leprosy impregnated on Whatman chromatography ined by ELISA method to determine the patients in Outpatient Clinic Dermatology paper (Whatman International Ltd, levels of IgM anti PGL-1. ELISA examina- Venereology, Dr. Soetomo General Maidstone, UK) filter and dried at room tion revealed the mean of IgM anti-PGL-1 Hospital, Surabaya. The aim of this study temperature, subsequently labeled and level from capillary blood sample on filter was to evaluate the effectiveness of the use inserted in a plastic bag. After that, it was paper was 163.31±126.16; whereas the of capillary blood using filter paper as a inserted in eppendorf tube, plus 900 µl dilu- mean of IgM anti-PGL-1 levels from serum substitute for serum samples by proving the tion buffer (PBRT and Skim milk) and filter samples cubital vein was 473.16±411.26. presence of correlation between anti-PGL-1 paper for 2 hours to get optimum result, fol- There was significant difference between IgM levels in fingertip capillary blood using lowed by Vortex for 5 minutes to dissolve capillary sample and cubital vein (p=0,006). filter paper with serum IgM anti-PGL-1 serum. Dilution of 1: 300 with dilution In Figure 1, we can see the difference of serologic level from cubital vein blood of buffer is then subsequently stored at a tem- IgM anti-PGL-1 levels in capillary blood household contact. The benefits of this perature of 2-10°C until the ELISA test time samplesonly on the filter paper and cubital study were to obtain a diagnostic SL in is carried out. Furthermore, 2 cc of blood venous blood samples in each sample where household contact that was simpler and eas- samples were taken from the cubital vein of the anti-PGL-1 IgM level in capillary blood ier to perform by officers in the field, espe- household contact of leprosy patients, cen- samples on the filter paper is lower than the cially in the endemic area of leprosy. trifuged, serum was taken, preserved, stored cubital vein blood sample. at 2-10°C until the ELISA test wasuse per- Pearson correlation test showed signifi- formed. IgM anti PGL-1 serology test with cant correlation between IgM anti-PGL-1 Materials and Methods ELISA method: a technique to measure level from capillary blood sample on filter anti-PGL-1 IgM antibody level, using syn- paper and cubital vein blood samples The design of the study was observa- thetic antigen NTP-P-BSA particles which (p=0,00) with Correlation Coefficient 0,994 tional cross-sectional analytic. The sample is a combination of synthetic tricaccharides (strong correlation). Figure 2 shows that the of the study was a household contact of with BSA (Bovin Serum Albumin). This higher anti-PGL-1 IgM level in the cubital multibacillary leprosy patients who came to antigen is reacted with domestic serum vein is followed by the rise of the anti-PGL- the Outpatient Dermatology and serum with a certain dilution and is a reac- 1 IgM titres at the fingertips. In the regres- Venereology Clinic, Dr Soetomo General tion between specific antibodies (anti PGL- sion calculation, the regression equation Hospital, Surabaya that meets the inclusion 1) with synthetic antigens. The result is a was obtained: Y (IgM anti-PGL-1 blood criteria of the study: Household contact staining reaction read with a spectropho- sample from cubital vein) = -7.704+2.944 X

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Figure 1. The comparasion titer IgM anti-PGL-1 level from cap- Figure 2. The correlation between titer IgM anti-PGL-1 level illary blood sample on filter paper and cubital vein blood sam- from capillary blood sample on filter paper and cubital vein ples. blood samples.

[Dermatology Reports 2019; 11(s1):8057] [page 107] Article

(IgM anti-PGL-1 capillary blood sample on blood should be demonstrated by a strong Ilmu Kesehatan Kulit dan Kelamin FK filter paper). Using this formula we can correlation between them and followed by Unair/ RSUD Dr.Soetomo Surabaya, convert the anti-PGL-1 IgM results taken the determination of the regression equation 2013. h. 1-25. from the fingertip capillary blood using a as we have done, where a strong correlation 6. Spencer JS, Brennan PJ. The role of filter paper to determine the anti-PGL-1 with 0.941 correlation coefficient is mycobacterium leprae phenolic glycol- IgM titres in cubital venous blood. obtained. Research with different sample ipid i (pgl-i) in serodiagnosis and in the that is using patient of leprosy done by pathogenesis of leprosy. Lepr Rev 2011; Prakoeswa (2007) and Yamashita (1999) 82: 344-357. Discussion also showed the same result that is strong 7. De Moura RS, Calado KL, Oliviera correlation with correlation coefficient MLW, Bührer-Sékula S. Leprosy serol- Household contact is an important respectively that is 0.98 and 0.97.13,15 The ogy using PGL-I: a systematic review. determinant of leprosy transmission chain advantages of filter paper can be used in Rev Soc Bras Med Trop 2008; 41( Supl failure due to potential SL that would poten- areas with high temperatures and humidity 11): 11-18. tially be a source of transmission and as in the tropics, and the result is no change 8. Britton WJ, Lockwood DNJ. Leprosy. become manifest leprosy. Detection of SL in yields for long enough storage, ie 1 year The Lancet 2004; 363: 1209-1219 by using Ig M Anti PGL-1 titer with serum at -10-4 ° C. Other factors such as tempera- 9. Prakoeswa CRS, Citrashanty I, Astari sample has been done by Moura (2008) ture, humidity, length and way of storage L,Iswahyudi, Wahyuni R, Adriaty D, Furini (2011) Lobato (2011), and Cabral and sample delivery can be said not to affect Agusni I. Importance of subclinical (2013), but not much research has been the results of the inspection when per- infection in leprosy control. 12th Asia done for SL detection by using filter 15 formed in accordance with the procedure. Pasific Environmental and paper.7,10-12 In this study, it has been demon- Occupational Dermatology Symposium strated that in household contact of leprosy (APEODS) in conjuction with 13th patients, the Ig M Anti PGL-1 filter paper is Conclusions Annual Scientific Meeting of significantly lower than that of the anti- Indonesian Society of Dermatology and PGL-1 Ig M titles. This corresponds to the This study supports the detection of SL Venereologyonly (PIT - PERDOSKI).: difference in the process of both methods, by examination of fingertip capillary blood 2013. namely the use of filter paper obtained less samples on filter paper because of good 10. Bazan-Furini R, Motta ACF, Simão than directly measuring the titer of the results, relatively cheap with simpler meth- JCL, Tarquínio DC, Marques Jr W, serum. The lower yield on filter paper is ods than cubital vein blood samples. It use Barbosa MHN, et al. Early detection of caused partly by absorption of blood and could facilitate large scale serological leprosy by examination of household followed by blood dissolution, so not all screening in the field for detecting SL espe- contacts, determination of serum anti- samples can be dissolved. A study conduct- cially in household contact, early diagnosis PGL-1 antibodies and consanguinity. ed by Prakoeswa (2007) in leprosy patients of leprosy, monitoring of treatment out- Mem Inst Oswaldo Cruz 2011; 106 (5): showed similar results, Ig M Anti PGL-1 comes and identification of relapse and 536-40. filter paper was significantly lower than that reactions. of IgM Anti PGL-1 in serum (p <0.05).13 11. Lobato J, Costa MP, Reis EM, et al. Differences between Prakoeswa’s research Comparison of three immunological tests for leprosy diagnosis and subclini- (2007) and this study were the mean of anti- References PGL-1 IgM levels from capillary blood cal infection. Lepr Rev 2011; 82: 389- samples on filter paper (624,73±493,71) 1. World Health Organization. Global 401. higher than our study (163.31±126.16); Leprosy Update 2015: time for action, 12. Cabral PB, Cunha JE, Macedo AC, et similarly, the mean titer of IgM anti-PGL-1 accountability and inclusion. Weekly al. Anti-PGL1 salivary IgA/IgM, serum of the serum samples from cubital vein in Epidemiological Record 2016; 91:405- IgG/IgM, and nasal Mycobacterium the Prakoeswa’s study (1534,2±1267,95) 20. leprae DNA in individuals with house- was higher than the results we obtained 2. Kementrian Kesehatan RI Direktorat hold contact with leprosy. Int J of Inf (473.16±411.26). This correspondsNon-commercial to the Jenderal Pengendalian Penyakit Dan Dis 2013; 17: 1005-1010. difference in the number of IgM Anti PGL- Penyehatan Lingkungan. Pedoman 13. Prakoeswa CR, Listiawan MY, Adriaty 1 circulating in the patient’s blood relative Nasional Program Pengendalian D, Wahyuni R, Iswahyudi, Agusni I, et to the contact persons, which is based on the Penyakit Kusta. ; 2015. al. Korelasi kadar antibodi Ig M anti difference in the number of M. leprae in the 3. Annual report Outpatient Clinic Dr PGL-1 antara darah vena cubiti dengan individual body. Syahputra (2003) reported Soetomo General Hospital, Surabaya. darah kapiler yang dikeringkan di kertas that no significant difference in Ig M anti 2016. Surabaya: 2016. saring pada penderita kusta. Berkala PGL-1 antibody levels between blood sam- 4. Prakoeswa CRS, Agusni I, Izumi S. Ilmu Kesehatan Kulit dan Kelamin ples taken from cubital vein was then Detection of Mycobacterium leprae 2007; 19(2): 115-119. dropped onto filter paper and serum sam- DNA in blood of the subclinical leprosy. 14. Syahputra E, Matodihardjo S, Ervianti ples of leprosy patients (p=0.147).14 Folia Medica Indonesiana 2007; 43(2): E, Agusni I, Izumi S. Penggunaan Differences in the results of this study with 64-67. Kertas Saring untuk Uji Serologi Kusta. Syahputra (2003) were caused not only by 5. Prakoeswa CR, Citrashanty I, Astari L, Maj Kedok Indon 2004; 54 (12): 491- the different samples used (household con- Listiawan MY, Agusni I. Beberapa 95. tact versus patients), but also on filter paper prosedur diagnostik terkini pada 15. Yamashita T, Nguyen H, Maeda M, method, Syahputra using cubital venous penyakit kusta. Dalam: Prakoeswa CR, Flageul B. Anti PGL-1 determination blood vein, while our research using finger- Agusni I, Listiawan MY, editors. Kapita using blood collection on filter paper in tip capillaries. The use of filter paper from selekta penatalaksanaan Morbus leprosy patients. Rev. Inst. Med. Trop. fingertip capillary drop to replace venous Hansen terkini. Surabaya: Dept/SMF 1999; 41(4): 239–42.

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