結核菌鑑定與藥敏試驗

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1 結核菌鑑定 -ICT method 林口長庚醫院醫檢部郭安靜 2013/9/5, 2013/9/6 1

2 大綱 Introduction Identification of Mycobacterium -- Immunochromatographic test (ICT) BD MGIT TBc Identification Test TiBilia TM (Capilia TB test) SD Bioline TB Ag 結核菌認可實驗室結核鑑定 -ICT 調查表 Conclusion 2

3 Policy Framework for Implementing New Tuberculosis Diagnostics, WHO

4 M. tuberculosis identification Morphology Positive AFB culture Molecular biology Phenotypic Approach Genotypic Approach Biochemical and growth tests (Slow) Immunochromatography (fast) DNA probes with amplification w/o amplification DNA Sequencing 4

5 Identification of M. tuberculosis Culture Conventional biochemical test High-performance liquid chromatography (HPLC) Nucleic acid amplification test (NAATs) Immunochromatographic test (ICT) 5

6 Identification procedure Phenotypic methods CLSI, M48-A 6

7 Morphology-M. tuberculosis Flocculation( 絮凝 ): granular, non-homogeneous suspension Acid fast stain from broth:cord-like appearance serpentine cords of varying length or district linear clumping 7

8 Cords Fine-striped Short bacilli Long striped bacilli Sea urchin GNB-like Long bacilli 8 Eur J Clin Microbiol Infect Dis (1998) 17:

9 Sensitivity 90%, specificity 100%, PPV 100%, NPV 83% Eur J Clin Microbiol Infect Dis (1998) 17: Sensitivity 74.5%, specificity 98%, PPV 96%, NPV 84% J Clin Microbiol 2000;38:

10 Morphology-M. tuberculosis Colony morphology: buff-coloured (never pigmented) rough waxy appearance of bread crumbs( 麵包屑 ) or cauliflower( 菜花 ). 10

11 M. fortuitum/m. chelonae rapid-growers produce visible colonies in less than a week the time of subculture Colony : R/S 11

12 M. avium-introcellular complex dispersed aspect( 分散 ) non-chromogenic mycobacteria Colony: St/R Eur J Clin Microbiol Infect Dis (1998) 17:

13 M. kansasii beaded appearance, strip Photochromogens Colony: SR/S 13

14 M. gordonae Scotochromogens Colony : S 14

15 Conventional biochemical test

16 MTB is a slow-growing microorganism: solid media cultures should be read at day 2 for contamination and then weekly for 8 weeks before being reported as negative. MTB colonies on solid media show a characteristic morphology used for presumptive identification. Presumptive identification from positive liquid culture can be performed by ZN staining (presence of cords ). Contaminated cultures showing presence of MTB could be re-decontaminated. Culture results should be recorded regularly and reported promptly. 16

17 New Laboratory Diagnostic Tools for tuberculosis Control, WHO

18 Mycobacterium Diagnosis In Clinical Lab Clinical Specimen Culture Solid media Liquid Media (MGIT TM ) Sample processing 21 Days 10 Days Days Identification Biochemical Molecular methodology Rapid Test (ICT) 18 Days 4 + hours 15 min Days Susceptibility testing Proportional Method Modified Proportional Method (MGIT TM ) 21 Days 12 Days 18

19 結核菌鑑定 -ICT method 19

20 Immunochromatographic test (ICT) A species-specific secreted antigen, MPT-64, ESAT-6/CFP10 Simple. Easy. Accurate. Results in 15 minutes. BD MGIT TBc Identification Test TiBilia TM (Capilia TB test) SD Bioline TB Ag ESAT-6/CFP-10 complex strip assay (Formosa Biomedical Technology Corp., Taiwan) 20

21 Distribution of Diagnostic Antigens in mycobacterial species The LANCET :

22 Distribution of Diagnostic Antigens in mycobacterial species The LANCET :

23 廠牌 BD TBC ID Tibilia SD Bioline TBAg Antigen MPT64 MPT64 MPT64 Time 15 min 15 min 15 min sample 需要 sample 量 Positive result Negative result Detection limit Positive MGIT liquid culture colony, liquid culture Colony, Condensation fluid(solid cultures) or Liquid cultures 100 μl 100 μl 100 μl M. tuberculosis, M. bovis, M. africanum, and M. microti NTM M.tuberculosis H37Rv, H37Ra, M.africanum, M.bovis deer, M.microti, M.bovis BCC-Tokyo, - Russia, -Moreau NTM, M.bovis BCG Glaxo, -Pasteur, - Tice M. tuberculosis complex, M. tuberculosis, M. africanum, and M. bovis NTM CFU/ml 1.2 x 10 6 CFU/mL 10 5 CFU/ml 23

24 廠牌 BD TBC ID Tibilia SD Bioline TBAg Antigen MPT64 MPT64 MPT64 Time 15 min 15 min 15 min 1. 有些 M. bovis BCG 亞 1. 有些 M. bovis BCG 亞菌株不會產生 MPT64, 造成陰性 1. 有些 M. bovis BCG 亞菌株不會產生 MPT64, 結果菌株不會產生 MPT64, 產品限制造成陰性結果 2.MPB64 gene mutation 2.MPB64 gene mutation 3. 檢體中含有可產生 protein A 菌株 (S. sureus) 造成陰性結果 2.MPB64 gene mutation 備註 25 tests/kit 20 tests/kit 25 tests/kit 必須保存在 2 35 C, 不必須保存在 2 30 C, 可冷凍使用前必須回復至室溫不可冷凍使用前必須回復至室溫 *colony:1 μl loop colonies into 200 μl extraction buffer 必須保存在 1 30 C, 不可冷凍使用前必須回復至室溫 *colony:3~4 colonies into 100 μl extraction buffer 24

TBc ID 1. Have a positive MGIT tube (manual or 960) MGIT+ AFB+ Pipette 100ul 1 step TB 2. Perform an AFB smear and see AFB in smear from + tube 3. Pipette 100 ul from MGIT tube into device 4.

25 TBc ID 1. Have a positive MGIT tube (manual or 960) MGIT+ AFB+ Pipette 100ul 1 step TB 2. Perform an AFB smear and see AFB in smear from + tube 3. Pipette 100 ul from MGIT tube into device 4. Wait 15 minutes then read results 5. If positive, report as Mtbc. If negative, report as AFB, not Mtbc Positive Test for TBc Negative Test for TBc (MPT64 antigen present) (no MPT64 antigen detected) Invalid Test 25

26 Specimen Preparation Liquid Media Solid Media Stain 1. Mix well μl 1. 1 μl loop colonies 2. Extraction Buffer 0.2ml 3. Mix well μl 26

27 Operation and Interpretation Minimum Detection Limit- 1.2 x 10 6 CFU/ml 27

28 SOP MGIT 培養管呈現陽性並進行抗酸菌染色陽性之後, 必須於 10 天內完成試劑組測試試劑組必須回溫至室溫下使用將抗酸性染色陽性的 MGIT 培養管均勻混合, 不可離心打開 MGIT 培養管蓋子, 用無菌的吸管取 100 μl 檢體加到已標示檢體編號的試劑接種區, 關上 MGIT 培養管蓋子, 開始計時 15 分鐘 15 分鐘後進行判讀, 切勿超過 60 分鐘判讀 CDC-SOP 初稿 28

29 Reading-positive 陽性 ( 具有 MPT64 抗原 ): 在 T 位置及 C 位置均呈現紅至粉紅色線條 T 位置及 C 位置的線條強度有可能不同背景應為白色或是淡粉紅色 CDC-SOP 初稿 29

30 Reading-Negative 陰性 ( 不具有 MPT64 抗原 ): 在 C 位置呈現紅至粉紅色線條此線條的顯現表示試劑的功能性正常以及操作步驟無誤當陰性試驗發生時必需使用分子鑑定以偵測陽性 MGIT 培養管是否有 MTBC 核酸, 分子鑑定未偵測到 MTBC 核酸才可以發 MTBC 陰性報告 CDC-SOP 初稿 30

31 Reading-Invalid 無效試驗 : 在 C 位置沒有紅至粉紅色線條或者背景值太深蓋過 T 位置及 C 位置判讀區域皆屬於無效試驗當無效試驗發生時必需重複試驗 CDC-SOP 初稿 31

32 Quality control 內部品管 : 每個試劑皆有內部品管, 其結果顯示在反應區的 C 位置線條此線條的顯現表示試劑的功能性正常以及操作步驟無誤外部品管 : 陽性及陰性外部品管應與檢體採取相同操作步驟取陽性 MGIT 培養管內含已知結核菌群 (MTBC) 為陽性品管液, 結果應為陽性 ; 取未接種之 MGIT 培養管為陰性品管液, 結果應為陰性 CDC-SOP 初稿 32

33 J CLIN MICROBIOL, 2011, 49:

34 Meta-analysis : 20 study (3087 Mtb, 1900 NTM) Capilia TB test : 9 BioLine SD Ag : 5 MGIT TBcID: 7 J CLIN MICROBIOL, 2011, 49:

35 1.1% The mpb64 gene was sequenced for 27 of 53 (51%) clinically independent isolates for which the MPT64 ICT gave a false-negative result, and a mutation was demonstrated in 23 (85%) of those 27 isolates 0.2% Among 10 isolates that gave a false-positive MPT64 ICT result, 7 (3 M. kansasii, 2 M. avium complex [MAC], 1 M. chelonae, and 1 M. gordonae) were identified at the species level. CDC-SOP 初稿 35

36 Limitation 本試劑無法排除細菌 - 結核菌群共同生長的干擾本試劑無法區分結核菌群 (MTBC) 中單一菌種本試劑結果應與病人臨床症狀及其他診斷方法合併考量作為判讀結果當 MPT64 抗原產生基因突變時, 本試劑陰性結果無法完全排除結核菌群 (MTBC) 的存在在結核菌群 (MTBC 中一些 M. bovis BCG 亞株因缺乏分泌 MPT64 能力, 故無法為本試劑所偵測 CDC-SOP 初稿 36

37 Limitation 檢體內有 Staphylococcus aureus 時, 可能有偽陽性結果少數文獻報告 M. marinum M. flavescens 有弱的陽性訊號,M. intracellulare M. aviumintracellulare complex 及 M. chelonae 有偽陽性結果使用免疫呈色法陽性應同時注意固態培養基菌落型態及觀察液態培養基菌液外觀,MTBC 生長通常為綿絮狀或顆粒狀, 如果混濁通常為 NTM 或細菌生長情形, 觀察抗酸菌染色抹片時, 要注意是否有非抗酸菌體如有上述情形, 免疫呈色法結果陽性, 應再進行分子鑑定確認 CDC-SOP 初稿 37

38 Limitation 免疫呈色法靈敏度與 MPT64 在檢體內的量有關, MGIT 培養管呈現陽性並進行抗酸菌染色陽性, 但免疫呈色法陰性, 可將檢體先置入培養箱培養 3 日後再重覆進行試驗 CDC-SOP 初稿 38

39 結核菌認可實驗室結核鑑定 -ICT 調查表分發 32 份回收 31 份回收率 (96.9%) 39

40 實驗室目前使用結核菌鑑定主要的方法為? ICT (28/31=90%) 其他 BluePoint TM MycoID Kit MycoCheck, CMP Probe Tec 40

41 目前實驗室使用的 ICT 試劑廠牌? BD MGIT TBc (8/28=28.5%)# Tibilia TM (11/28=39.3%)*# SD Bioline TB Ag (12/28=42.9%)* 其他 -X *# 二家使用二種廠牌如果同時使用二種廠牌, 需有 comparison 的結果 41

42 實驗室何時開始使用 ICT 作結核菌鑑定? 42

43 實驗室使用 ICT 鑑定的時機? MGIT 陽性 AFB 陽性之所有檢體 (19/28=67.9%) MGIT 陽性 AFB 陽性之檢體, 每一位病人只作一支 (5/28=17.9%) MGIT 陽性 AFB 陽性菌落疑似 MTBC 之所有檢體 (1/28=3.6%) MGIT 陽性 AFB 陽性菌落疑似 MTBC 之檢體, 每一位病人只作一支 (2/28=7.1%) 其他 :MGIT 陽性 AFB 陽性之檢體, 每位病人 3 個月內同類型檢體只作一支 (1/28=3.6%) 43

44 -1 SD median +1 SD 疾管署閾值 90% 11.1% 33.3% 陽性件數陽性人次疑似 MTBC 件數疑似 MTBC 人次非 ICT 方法 50% 操作的頻率如果一個病人只做一支, 少數 TB mix NTM 的情況下有可能遺漏 TB 44

45 實驗室發 MTBC 陽性報告的條件? ICT 陽性, 即發 MTBC -X ICT 陽性配合 AFB 抺片有 cord 的型態, 發 MTBC (14/28=50%)* ICT 陽性配合菌落疑似 MTBC 型態, 發 MTBC (19/28=67.9%)* 其他 -X * 四家使用二種方法 45

46 實驗室發 NTM 陽性報告的條件? ICT 陰性, 即發 NTM -X ICT 陰性配合 AFB 抺片有無 cord 的型態, 發 NTM (7/28=25%)* ICT 陰性配合菌落非 MTBC 型態, 發 NTM (25/28=89.3%)* 其他 ( 配合生化或分子鑑定結果 )(8/27=29.6%) # * 四家使用二種方法 # 同時配合菌落型態 46

47 當 ICT 鑑定結果與特徵形態不合時, 實驗室採用的不符合處理程序? 使用第二種鑑定方法 NAAT (25/28=89.3%)* 傳統生化鑑定 (5/28=17.9%)* * 二家使用二種方法 47

48 使用 ICT 鑑定 MTBC 之偽陽性情況? 偽陽性率 ~1.4% (5/28=17.9%) 目前為止, 曾發現偽陽性 1-12 件 (8/28=27.5%) 未曾發現偽陽性 (13/28=46.4%) 其他 - 未提供 (2/28=7.1%) 三個廠牌皆有偽陽性如菌落疑似 MTBC 之檢體作 ICT, 則無偽陽性 48

49 使用 ICT 鑑定 MTBC 之偽陰性情況? 偽陰性率 % (12/28=42.9%) 目前為止, 曾發現偽陰性 1-16 件 (10/28=35.7.0%) 未曾發現偽陰性 (4/28=14.3%) 其他 - 未提供 (2/28=7.1%) 三個廠牌皆有偽陰性如疑似 MTBC 菌落之檢體作 ICT 或同一病人只作一次, 則偽陰性偏高 49

50 其他使用經驗可與各認可驗室作分享判讀時需要與菌落型態一起確認當 band 太弱時, 須留意是否有偽陽性的情形偽陽性問題, 與試劑偶發的問題有關, 發生率很低偽陰性的問題, 可能是有 NTM 同時存在而 MTBC 菌量很少所導致 MGIT(+) AFS 染色陽性後, 放置於 37 三天後操作 ICT, 可以得到更明顯的 ICT 結果如果一個病人只做一支, 少數 TB mix NTM 的情況下有可能遺漏 TB 50

51 結論 Studies have demonstrated that the rapid speciation tests compare favorably with other established phenotypic or genotypic methods. This test provides results within 15 minutes and is highly sensitive and specific (98.6% and 97.9%, respectively). 51 New laboratory diagnostic tools for tuberculosis control, WHO, 2008

52 結論 No additional equipment or consumables are needed to perform the test and can detect TB even when mixed with nontuberculous mycobacteria. The test detects organisms belonging to M. tuberculosis complex but does not specifically identify M. tuberculosis strains. 52 New laboratory diagnostic tools for tuberculosis control, WHO, 2008

53 結論 Expired reagents affect the results of identification tests. The immunochromatographic test allows fast identification of TB complex from positive cultures. 流程上應思考配合菌落發報告結核鑑定時效 7 天達成率操作的時機點操作的頻率如何降低偽陽性及偽陰性 53

54 Thanks for your attention 54

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