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Arlita Leniseptaria Antari, David Pakaya, Dahliatul Qosimah

INTRACELLULAR KILLING CAPACITY TO MYCOBACTERIUM TUBERCULOSIS OF MACROPHAGES WERE OBTAINED FROM PULMONARY AND HEALTHY SUBJECTS’ AT RISK FOR PULMONARY TUBERCULOSIS

ABSTRACT

Background: Macrophages are the first defense mechanism in tuberculosis. The entry of Mycobacterium tuberculosis into the host macrophages and its survival in this environment are key components of tuberculosis pathogenesis. The aims of this study was to know whether there is any difference in intracellular killing capacity of Mycobacterium tuberculosis between macrophages obtained from pulmonary tuberculosis patients’ and healthy subjects’ at risk for pulmonary tuberculosis.

Methods: PBMC were isolated from buffy coat of pulmonary tuberculosis patients’ and healthy subjects’ at risk for pulmonary tuberculosis. Monocytes (105 cell/ml) were cultured in 24–wells tissue culture plate thatplated onto glass coverslip, then added RPMI 1640 supplemented with

10% HI-PHS (Heat Inactivated Pooled Human Serum) and incubated at 37°C, 5% CO2. At the end of that incubation periods, the cultures were harvested and washed with PBS. Mycobacterium tuberculosis befreed from that macrophages are then plated on solid media Middlebrook 7H10 and incubated during 7 days, 10 days and 14 days. Viable colonies of Mycobacterium tuberculosis were counted as CFU (Colony Forming Units).

Results: The result showed that the mean of viable colonies of Mycobacterium tuberculosis on solid media Middlebrook 7H10 after killing intracellular process are greater on pulmonary tuberculosis patients’ macrophages than healthy subjects’ at risk for pulmonary tuberculosis macrophages. The result of Univariate Analysis of Variance followed by F test at the level of 5% significance showed that the difference was very significant (p < 0.01).

Conclusion: The intracellular killing capacity of Mycobacterium tuberculosis from pulmonary tuberculosis patients’ is lower than the intracellular killing capacity of Mycobacterium tuberculosis from healthy subjects’ at risk for pulmonary tuberculosis, showed by the greater of the mean of viable colonies of Mycobacterium tuberculosis on solid media Middlebrook 7H10 from pulmonary tuberculosis patients’ macrophages than healthy subjects’ at risk for pulmonary tuberculosis macrophages.

Keywords: Mycobacterium tuberculosis, macrophages, intracellular killing.

ABSTRAK

Latar belakang: Makrofag merupakan sistem pertahanan yang pertama pada infeksi tuberkulosis, dimana masuknya Mycobacterium tuberculosis ke dalam makrofag dan kemampuan bertahan hidup didalamnya merupakan elemen kunci dari patogenesis tuberculosis. Penelitian ini bertujuan untuk menguji perbedaan kemampuan intracellular killing terhadap Mycobacterium tuberculosis antara makrofag penderita dan individu sehat berisiko tuberkulosis paru.

Metode: PBMC diisolasi dari buffy coat penderita dan individu sehat berisiko tuberculosis paru. Monosit (105 cell/ml) dikultur dalam

24-wells tissue culture plate berisi coverslip, kemudian ditambahkan RPMI 1640 yang disuplementasi 10% HI–PHS (Heat Inactivated Pooled Human Serum) dan diinkubasi pada 37°C, 5 % CO2. Pada akhir periode inkubasi, kultur dipanen, dibilas PBS, Mycobacterium tuberculosis yang terbebas kemudian dikultur dalam media padat Middlebrook 7H10 dan diinkubasi selama 7 hari, 10 haridan 14 hari. Koloni Mycobacterium tuberculosis yang viabel dihitung sebagai CFU (Colony Forming Units).

Hasil: Hasil penelitianmenunjukkan bahwa jumlah koloni Mycobacterium tuberculosis yang viabel setelah intracellular killing lebih

banyak terdapat pada makrofag penderita tuberkulosis paru daripada makrofag individu sehat berisiko tuberkulosis paru. Hasil analisa dengan menggunakan uji Univariate Analysis of Variance menunjukkan adanya perbedaan yang signifikan (p < 0,01).

Simpulan: Intracellular killing makrofag penderita tuberkulosis paru lebih rendah daripada individu sehat berisiko tuberkulosis paru. Hal tersebut ditunjukkan dengan lebih banyaknya jumlah koloni Mycobacterium tuberculosis yang viabel pada media Middlebrook 7H10 yang berasal dari makrofag penderita tuberkulosis paru.

Kata kunci: Mycobacterium tuberculosis, makrofag, intracellular killing.

 

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