Evaluasi Pooling Test Real Time RT-PCR Untuk Deteksi Sars-Cov-2 Dan Varian B1.1.529 Di Laboratorium Covid-19 Univeristas Gadjah Mada Yogyakarta

M EDWIN WIDYANTO D, Prof. dr. Tri Wibawa, PhD, Sp.MK(K).; Prof.dr. Titik Nuryastuti, MSi, PhD, SpMK(K)

2022 | Tesis-Spesialis | Mikrobiologi Klinik

Pendahulan: Coronavirus Disease 2019 (COVID-19) merupakan penyakit yang saat ini menjadi ancaman dunia. COVID-19 disebabkan oleh severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Penyakit ini telah menyebar di seluruh belahan dunia dan WHO telah menyatakan status pandemik. Metode real time RT-PCR (reverse transcription polymerase chain reaction) adalah metode standar dalam penegakan diagnosis COVID-19 namun adanya peningkatan jumlah kasus positif dan meningkatnya angka kematian menjadi perhatian pemerintah dalam memberikan hasil diagnosis yang lebih efisien dan cepat. Metode pooling skrining sampel SARS-CoV-2 menjadi salah satu opsi dalam memberikan hasil yang lebih efisien dalam waktu yang relatif lebih cepat dibandingkan skinning sampel per individu. Dalam penerapan skirining sampel SARS-CoV-2 oleh laboratorium yang tersebar di Indonesia masih ada yang memiliki keterbatasan sumber daya manusia (SDM) dan distribusi bahan habis pakai (BHP) sehingga diperlukan suatu penelitian aplikasi penggunaan metode pooling untuk memberikan alternatif yang efisien dalam mendiagnosa COVID-19. Metode dan Subjek Penelitian: Penelitian ini merupakan penelitian observasional deskriptif dengan pendekatan potong lintang. Dalam desain potong lintang, observasi atau pengukuran variabel dilakukan pada waktu yang bersamaan. Sampel penelitian merupakan sisa sampel yang dan diterima Laboratorium COVID-19 Univeristas Gadjah Mada di FK-KMK UGM untuk dilakukan penapisan SARS-CoV-2 berupa sampel swab nasofaring dalam viral transport medium (VTM). Jumlah sampel yang akan diproses untuk pooling test metode NAAT/PCR SARS-Cov-2 non SGTF sebanyak 440 sampel (88 pool yang terdiri dari 5 sampel per pool) sedangkan jumlah sampel yang akan diproses untuk pooling test metode NAAT/PCR SARS-Cov-2 SGTF sebanyak 90 sampel (18 pool yang terdiri dari 5 sampel per pool) yang tersimpan di Laboratorium COVID-19 Fakultas Kedokteran, Kesehatan Masyarakat dan Keperawatan, Univeristas Gadjah Mada (FK-KMK UGM). Hasil penelitian: Sebanyak 440 sampel diskrining dengan membuat 88 pool yang masing-masing berisi 5 sampel di pooling test dengan metode NAAT/PCR SARS-Cov-2 non-SGTF, penggunaan reagen ekstraksi 1 kit (Qiagen QIAamp viral RNA mini kit) dan PCR 1 kit (Allplex seegen) lebih sedikit kit reagen yang digunakan dengan metode pooling dibandingkan per sampel masing-masing 2 kit ekstraksi dan 5 kit PCR. Dari 88 pool tersebut, 9 di antaranya positif SARS-CoV-2. Tingkat positifnya adalah 10.23%. Empat puluh lima sampel dari 9 pool ini diuji satu per satu dalam mesin RT-PCR yang sama dengan teknik yang sama dan 13 sampel dinyatakan positif SARS-CoV-2. Tingkat positif di antara 45 sampel ini adalah 28.89 %. Secara total, dari 440 sampel, 2.95 % positif. Dari data yang diperoleh dapat disimpulkan spesifitas dan sensitifitas pooling SARS-CoV-2 memiliki sensitivitas sebesar 69 % dan spesifitas sebesar 100%. Selanjutnya sebanyak 100 sampel diskrining dengan membuat 20 pool yang masing-masing berisi 5 sampel dilakukan pooling test metode NAAT/PCR SARS-Cov-2 SGTF. Dari 20 pool tersebut, semua pool terdeteksi positif sebagai varian Omicron United Kingdom (UK), South Afrika, Japan, dan Brazil. Dari 100 sampel yang setelah dilakukan identifikasi individual diperoleh 2% UK variant (B.1.1.7 lineage, 20I/501Y.V1), 90% UK variant (spike protein deletion HV69/70 del), South Africa (B.1.351 lineage, 20H/501Y.V2 dan E484K mutation), Japan, Brazil (P.1 lineage, 20J/501Y.V3) dan 8% non-variant Kesimpulan: Pooling dengan metode NAAT/PCR SARS-CoV-2 non-SGTF dan NAAT/PCR SARS-CoV-2 SGTF dapat diaplikasi untuk skrining sampel SARS-CoV-2 yang diproses di Laboratorium COVID-19 UGM dan memberikan efisensi penggunaan BHP, SDM dan mempercepat Turn Around Time (TAT). Namun Skrining SARS-CoV-2 menggunakan pooling NAAT/PCR SARS-CoV-2 SGTF tidak dapat memberikan menyederhanakan identifikasi Omicron varian UK/SA/JPN sehingga masih disarankan penggunaan identifikasi dengan WGS.

Introduction: Coronavirus Disease 2019 (COVID-19) is a disease that is currently a global threat. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This disease has spread all over the world and WHO has declared a pandemic status. The real time RT-PCR (reverse transcription polymerase chain reaction) method is the standard method in establishing the diagnosis of COVID-19, but the increase in the number of positive cases and the increasing mortality rate has become the government's concern in providing more efficient and faster diagnosis results. The pooling method for screening samples of SARS-CoV-2 is one option in providing more efficient results in a relatively faster time than skinning individual samples. In the application of SARS-CoV-2 sample screening by laboratories spread across Indonesia, there are still limited human resources (HR) and distribution of consumables so an application study using the pooling method is needed to provide an efficient alternative in diagnosing COVID -19. Methods and Subjects: This study is a descriptive observational study with a cross-sectional approach. In a cross-sectional design, observations or measurements of variables are carried out at the same time. The study sample uses remaining sample that has been sent and received by the COVID-19 Laboratory at Gadjah Mada University at FK-KMK UGM for SARS-CoV-2 in the form of a nasopharyngeal swab sample in viral transport medium (VTM). The number of samples to be processed for the non-SGTF SARS-CoV-2 NAAT/PCR pooling test is 440 samples (88 pools consisting of 5 samples per pool) while the number of samples to be processed for the SARS-Cov-2 NAAT/PCR pooling test method 2 SGTF with 90 samples (18 pools consisting of 5 samples per pool) stored in the COVID-19 Laboratory of the Faculty of Medicine, Public Health and Nursing, Gadjah Mada University (FK-KMK UGM). Results: A total of 440 samples were screened by 88 pools, each containing 5 samples in the pooling test using the non-SGTF SARS-Cov-2 NAAT/PCR method. A total of 440 samples were screened by making 88 pools, each of which contained 5 samples in the pooling test with the SARS-Cov-2 non-SGTF NAAT/PCR method, using 1 kit extraction reagent (Qiagen QIAamp viral RNA mini kit) and 1 kit PCR (Allplex seegen) fewer reagent kits used by pooling method than per sample, 2 extraction kits and 5 PCR kits respectively. Of the 88 pools, 9 of them were positive. The positive rate is 10.23%. Forty-five samples from these 9 pools were tested one by one in the same RT-PCR machine with the same technique and 13 samples were tested positive. The positivity rate among these 45 samples was 28.89%. In total, of the 440 samples, 2.95% were positive. From the data obtained, it can be concluded that the sensitivity and specificity of SARS-CoV-2 pooling has a sensitivity of 69% and a specificity of 100%. A total of 100 samples were screened by making 20 pools, each containing 5 samples, a pooling test was carried out using the SARS-Cov-2 SGTF NAAT/PCR method. Of the 20 pools, all pools were detected as positive as variants of Omicron United Kingdom (UK), South Africa, Japan, and Brazil. From 100 samples, after individual identification, obtained 2% UK variant (B.1.1.7 lineage, 20I/501Y.V1), 90% UK variant (spike protein deletion HV69/70 del), South Africa (B.1.351 lineage, 20H/501Y.V2 and E484K mutation), Japan, Brazil (P.1 lineage, 20J/501Y.V3) and 8% non-variant. Conclusion: Pooling with the SARS-CoV-2 non-SGTF NAAT/PCR method and the SARS-CoV-2 SGTF NAAT/PCR method can be applied to screen SARS-CoV-2 samples processed at the UGM COVID-19 Laboratory and provide efficient use of consumable, HR and accelerate Turn Around Time (TAT). However, SARS-CoV-2 screening using NAAT/PCR SARS-CoV-2 SGTF pooling could not simplify the identification of the UK/SA/JPN variant Omicron, therefore, it is still recommended to use WGS identification.

Kata Kunci : COVID-19, pooling test, Non-SGTF, SGTF