05. Kiranasari.cdr Vol.15, No.3, September 2021, p 113-117 DOI: 10.5454/mi.15.3.5 Comparison of N-Acetyl-L-Cysteine-Sodium Hydroxide-Based and Modified Petroff's Decontamination Method for Mycobacterium tuberculosis Culture ARIYANI KIRANASARI MUHAMMAD RAYHAN 1* 2 AND 1Microbiology Department, Faculty of Medicine, Universitas Indonesia Jalan Pengangsaan Timur No.16, Jakarta Pusat, 10320, Indonesia; 2 Medical Science, Faculty of Medicine, Universitas Indonesia. Indonesia is one of 22 countries with a high incidence of tuberculosis in the world, particularly related to TB- HIV and MDR-TB cases. Contamination of normal flora from nasopharyngeal tract is the main problem to isolate Mycobacterium tuberculosis (MTB) from sputum. Therefore, a safe solution to decontaminate sputum without killing MTB bacilli is needed. N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH)-based method and Petroff's methods which were modified with NaOH (4%) are widely used in laboratories. In the present study, we will evaluate the different of these methods. Of the 110 sputum samples were collected from suspected cases of Pulmonary TB, and the decontamination of sputum by these methods was performed, after acid-fast bacillus (AFB) smear, then the samples were cultured in Lowensteins Jensen slant medium. The positive culture was validated by chromatography test for detecting the antigen of MPT-64 and PNB. Based on the investigation, it has been shown that neither NALC-NaOH (71%) nor modified Petroff's methods (66%) had a significant effect on the positivity rate of AFB smear. However, the contamination on culture was significantly higher in samples treated with NALC-NaOH (21%) compared to Modified Petroff methods (13%) (p< 0.05). In addition, the proportion of positive culture in NALC-NaOH was lower than Modified Petroff. In conclusion, our study proved that Modified Petroff methods are still more effective on sputum decontamination than NALC-NaOH based on the positivity rate of MTB culture. Though, not significantly different on AFB microscopic examination. Petroff's methods M NALC-NaOHKey words: decontamination, , TB, Modified Indonesia merupakan salah satu dari 22 negara dengan insiden TB tertinggi di dunia, khususnya yang berkaitan dengan kasus TB-HIV dan MDR-TB. Kontaminasi flora normal dari saluran nasofaring merupakan masalah utama mengisolasi (MTB) dari sputum. iperlukan dalam Mycobacterium tuberculosis Oleh sebab itu d solusi yang aman untuk dekontaminasi sputum tanpa membunuh MTB. melakukan sampel bakteri Untuk memberikan hasil yang lebih baik dekontaminan yang sering digunakan adalah metode berbasis N-asetil-L- sistein-natrium hidroksida (NALC-NaOH) dibandingkan dengan dari metode Petroff yang dimodifikasi dengan NaOH (4%) Sampel sputum yang digunakan untuk pengujian berasal dari 110 kasus suspek TB Paru dan . dilakukan dekontaminasi menggunakan NALC-NaOH 2% atau (4% NaOH) yang sebelumnya sudah dilakukan pemeriksaan BTA. sputum dibiakkan media Lowensteins Jensen. Setiap kelompok Selanjutnya, pada tersebut dinilai tingkat kontaminasi dan kultur positif. Kultur positif divalidasi menggunakan uji berdasarkan hasil kromatografi untuk mendeteks antigen MPT-64 dan PNB. Hasil penelitian menunjukkan bahwa NALC-i adanya NaOH dan metode Petroff termodifikasi tidak berpengaruh nyata terhadap laju kepositifan BTA, dengan nilai masing-masing sebesar 71% dan 66%. Kontaminasi pada kultur secara signifikan (p=0,034) lebih tinggi pada sampel yang diberi NALC-NaOH (21%) dibandingkan dengan metode Modified Petroff (13%). Proporsi kultur positif pada sampel yang diberi perlakuan NALC-NaOH lebih rendah dari Modified Petroff, memberikan hasil masing-masing 65% dan 70% dengan nilai p=1. Kesimpulan dari penelitian ini menunjukkan bahwa dekontaminasi sputum dengan metode Modified Petroff masih lebih efektif dibandingkan dengan NALC-NaOH untuk meningkatkan laju kepositifan kultur MTB. Namun kedua metode tersebut tidak berbeda nyata untuk mendapatkan hasil positif pada pemeriksaan mikroskopis BTA. Kata kunci: dekontaminasi metode , M , NALC-NaOH Petroff's, TBModified MICROBIOLOGY INDONESIA Available online at http://jurnal.permi.or.id/index.php/mionline ISSN 1978-3477, eISSN 2087-8575 *Corresponding author: Phone 62-21 3160491;: + - E-mail: gahariyani@gmail.com (AFB) is rapid, inexpensive, highly specific, and capable of identifying the most infectious cases of TB. The only disadvantage of this method is low sensitivity (varying from 50 to 80%) relative to culture (Forbes et al. 2010). The Gold standard for diagnosing pulmonary TB remains in culturing. Decontamination of clinical specimens such as sputum is an important and critical step in the isolation of mycobacteria, to obtain better Tuberculosis (TB) is one of the deadliest infectious disease which caused by .Mycobacterium tuberculosis TB is a major air-borne disease in human. It remains a major worldwide health problem with global mortality ranging from 1.6 to 2.2 million lives per year (WHO 2016). Direct smear microscopy for acid fast bacilli 114 KIRANASARI ET AL . Microbiol Indones results. However, bacterial and fungal contamination of the culture frequently may interfere with the interpretation data result. This condition can result the need to repeat the culture and lead to reduce the effectiveness of culture as a method of diagnosis of tuberculosis, because it will take more time for a colony to appear (Bruchfeld . 2000; Sjahrurachman . et al et al 2012; Korean CDC 2014; Gopi 2018). et al. Contamination of culture and its harmful effect can be prevented by decontamination of sputum sample. Decontamination and concentration of sputum samples by Modified Petroff's method is one of the most commonly used methods for culture. M. tuberculosis However, N-acetyl-L-cysteine-sodium hydroxide (NALC NaOH) solution may recommended as a gentle - but effective digesting and decontaminating agent. Addition of a large volume of phosphate buffer (PB) with pH 6.8 makes strong shift in pH, washes the specimen, dilutes toxic substances and decreases the specific gravity of the specimen so that centrifugation is more effective. Therefore, it is generally accepted that the NALC-NaOH method (Kent and Kubica 1985) should be given preference over the modified Petroff's methods. But, the technical and procedural factors may influence the sensitivity of each method (Peres . et al 2009). tudies in different laboratory may give different S results since the result of decontamination may be influenced by various technical factors (Forbes . et al 2010; Peres . 2009; Sjahrurachman . 2012).The et al et al aim of this study is to compare the NACL-NaOH-based and Modified Petroff methods with 4% NaOH to obtain better result on microscopic examination of AFB and MTB culture . MATERIALS AND METHODS The cross-sectional study was Sputum Samples. c o n d u c t e d i n t h e l a b o r a t o r yt u b e r c u l o s i s , Microbiology Departement, Faculty of Medicine, Universitas Indonesia from De ember 2016 Jul , c to y 2017 One hundred ten . sputum samples were isolated from . suspected case of pulmonary tuberculosis The s then be decontaminated with NALC-NaOH-amples based method or Modified Petroff's methods. Mic r os c o pi c Mi c ro sc opic Ex am inat io n. examination was performed before and after decontamination. AFB smear was conducted using Ziehl-Neelsen technique recommended by World Health Organization (WHO 2016; Tripathi . 2014; et al Burdz 2003et al. ). Specimens for digestion and decontamination were mixed well using vortex and equally divided into two parts, and each treated by NALC NaOH and modified Petroff's method before - inoculated it directly on Lowenstein-Jensen (LJ) medium (Peres et al. 2009; Tripathi . 2014).et al NALC-NaOH-based Me thod. NALC-2% sodium hydroxide-sodium citrate solution was prepared as described by Kent and Kubica (Tripathi et al. 2014). An equal volume of NALC-NaOH citrate reagent was added into 3-5 mL of seeded sputum sample, and vortexed briefly for ec. The samples 30 s then were incubated for 15 min at room temperature and added by 0.067 M of phosphate buffer (pH 6.8) until the volume reach 50 mL. After homogenization, the sputum samples were pelleted by centrifugate it at 3,000g for 15 min. The supernatant was discarded, -20 and the pellet was resuspended with 1 mL of PB . The S smear was made and a 0.5 mL of cell suspension was inoculated on LJ slopes. The culture slants were incubated at 37 C.35- o Modified Petroff's Method. In brief, 3-5 mL of sputum was homogenized for 15 min in a shaker using an equal volume of 4% NaOH. After centrifugation at 3 000 for 15 min, the were neutralized with . g -20 seeds Fig 1 Microscopic examination of acid-fast bacilli with Ziehl Neelsen staining. 20 mL of sterile distilled water . or aqua pro injection The samples were again centrifuged at 3 000 for 15 . g - 20 min. From the sediment, LJ medium was inoculated and smear was made. The culture slants were incubated at 3 7 C (Pathak . 1973; ; 5-3 o et al Tripathi . 2014et al Costa 2018 . All slopes were observed daily for et al. ) first week and weekly for 8 weeks. Some parameters such as, growth rate, optimum temperature, colony morphology and pigmentation were also observed as growth parameters. The chromatography test was using to detect the of 4 and tests. antigen MPT-6 PNB The absence of growth at the end of 8 weeks was th regarded as negative culture. Contamination, if any, was recorded separately. The number of culture failures for a certain decontamination method, included the number of specimens with negative culture as well as number of contaminated cultures. The data acquired from the study is processed using SPSS software version 20. Bivariate analysis was done using Chi-square test. RESULT Based on direct microscopy, out of samples,110 88 ( %) were smear positive and ( %) were 80 22 20 negative. The smear was carried out again after decontamination, and the result showed that 71 samples (71%) were positive by NALC-NaOH and 73 samples ( %) were positive by modified Petroff's 66 methods (Table 1). The total number of culture failures (which includes both contamination and negative cultures) were (30%) in NALC-NaOH as against ( %) in 33 38 35 modif ied Petroff's methods (Table 2) . The contamination rate was NALC NaOH higher - 23 ( %), whereas it was in modified Petroff's 21 lowest methods 14 13 ( %) (Table 2). A significant difference (p<0.05) in the proportion of contaminated and uncontaminated culture was shown between those treated with NALC-NaOH and Modified Petroff. Higher proportion of contaminated culture is observed from group treated with NALC- NaOH in comparison to Modified Petroff (Table 3). However, the proportions of uncontaminated culture with positive and negative results show no significant difference between group treated with NALC-NaOH and Modified Petroff (Table 4). DISCUSSION Sputum culture is an important tool for TB method control programs it is more sensitive than because smear microscopy in diagnosing TB. The culture also f the but sinceacilitates drug susceptibility testing, sputum samples pass through the oropharynx tract during collection, culture contamination limits the diagnostic yield of sputum culture for TB (WHO 2016; Forbes . 2010). et al Decontamination process for removing bacteria and yeast in order to isolate in the Mycobacteria sputum, unfortunately may also kill . The Mycobacteria percentage of killed organisms will vary according to the method used and also the population of mycobacteria in the specimen. In this study the use of NALC-NaOH for sputum digestion, as in Kent and Kubita method, will have final concentration about 2% NaOH. Such concentration will only give less destructive to compare to the used of 4% Mycobacteria NaOH in Petroff's method (GLI 2014; Tripathi . et al 2014; Burdz . 2003). et al In the analysis of 110 samples, the decontaminated by the NALC-NaOH method provided isolation of M. tuberculosis in a percentage ( %) compared lowest 65 to modified Petroff's method (7 %). This is in accordance 0 with the research of Sharma . in 2012 showed that et al the proportion of positive culture results was greater in samples processed with NALC-NaOH 63 7% than . samples processed with Modified Petroff 46 7%. . Meanwhile, from the research of Chatterjee . et al ( )2013 showed that the proportion of positive culture results was greater either in samples processed with NALC-NaOH was 62.7% than samples processed with Modified Petroff 58.5% Chaudhary ( 2013).et al. Meanwhile, the research results of Pathak SK . et al (1 )973 showed that the proportion of positive culture results was 79.4% for NALC-NaOH. This figure is much greater than the proportion of positive culture results for NALC-NaOH which was shown well by the study of Sharma . 2012 and Chatterjee . 2013et al et al . The probable reason are, 4% NaOH is used s maybe for modified Petroff's method as compared NALC- NaOH method which uses 2% NaOH while concentrating the sputum may kill or seriously injure few Mycobacteria Hence (Korean CDC 2014). , recovery by NALC-NaOH was faster and better than modified Petroff's method. In our study we found smear positivity higher than the culture positivity. The reason might be that microscopy sometimes gives false positive results and in our condition, it cannot distinguish between dead and live bacteria. In such cases, the patients might be treated with antitubercular drugs and in the microscopy of these samples, the AFB Volume 15, 2021 Microbiol Indones 115 116 KIRANASARI ET AL . Microbiol Indones might be dead. For these reason, the dead isolates did not grow in the L-J culture media. This also reveals that AFB microscopy does not always give accurate results for the diagnosis of TB Sharma . 2012 ( ; GLI 2014).et al The contamination rate by NALC-NaOH method was % in our study which is than that reported 21 higher by other workers. Sharma . (2012) 3.2%et al reported 1 while reported 4 Chatterjee . (2013) .98%. Several et al studies show the contamination r te by modified a Petroff's was 13% in our study which is lower than that reported by other workers. Sharma 2012 reported et al 23.1% while Chaudary in 2013 reported 8% and et al. TripathiK . in 2014 12%. Our study showed, first et al the NALC-NaOH method for AFB smear and culture improves the sensitivity when compared to modified Petroff's method. Second, Modified Petroff method has lower contamination rate than NALC-NaOH, and culture using LJ medi NALC-NaOH method is not um suitable for routine use. hird, t modified Petroff's T he method for AFB smear and culture improves the sensitivity when compared with NALC-NaOH the method. In conclusion, the Modified Petroff is methods more effective than NALC-NaOH to prevent contamination highly contaminated sputum. from However, the positivity result of microscopic examination of AFB was not significantly different between this two methods. REFERENCES Bruchfeld J, Aderaye G, Palme IB, Bjorvatn B, Källenius G, Lindquist L. 2000. Sputum concentration improves diagnosis of tuberculosis in a setting with a high prevalence of HIV. Trans R Soc Trop Med Hyg. 94: 677-80. doi: 10.1016/s0035-9203(00)90230-x. Burdz TVN, Wolfe J, Kabani A. 2003. Evaluation of sputum decontamination methods for Mycobacterium tuberculosis using viable colony counts and flow cytometry. 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