探討 interferon gamma 以及 interferon gamma inducible protein-10 做為肺結核診斷以 及 治 療 的 潛 在 性 生 物 指 標 的 可 行 性

Investigate the use of interferon gamma and interferon gamma inducible protein-10 as potential biomarkers to assess tuberculosis diagnosis and treatment outcome

江雅惠(Chiang Ya Hui)

指導教授 ： 曹昌堯

中山醫學大學/醫學院/醫學研究所/碩士(2013年)

https://doi.org/10.6834/CSMU.2013.00049

摘要

目的: 目前在整個結核病治療療程，要確定並早期預測病患治療結果，只能以追蹤X光及痰液作基準來判別，但痰液的採取與保存充滿了許多不確定的因素。例如：檢體運送的過程:1.無法保持低溫，而影響檢體品質; 2.取痰方式不正確; 3.忘了繳回痰液; 4. 小孩子無法提供痰液的檢體..等等原因。都會影響診斷結果及延誤治療 , 因此新的簡單快速的檢驗方式需要被探討做為結核病的治療成效指標。近年來，IFN-γ release assays (IGRAs) 在許多國家臨床上已經拿來用於協助偵測M. tuberculosis latent infection。我們所使用的 IGRAs kits 包括: 1) T-SPOT.TB; 2) QuantiFERON-TB Gold in-tube (QFT-GIT)。
而我們在2008也使用過IGRA (QFT-GIT)來追蹤MDR-TB患者的接觸者檢查，但是IGRA無法區分active TB 或 潛伏性結核感染 (LTBI)或是成為治療成效的依據。因此許多研究結合IGRAs和其他cytokines (例如IP-10, IL-2和TNF-α)，觀察是否可以做為偵測結核菌感染或治療成效的生物指標（biomarker）。尤其是近年來許多的研究發現丙型干擾素誘導蛋白（INF-γ inducible protein-10, 簡稱IP-10）指數，有可能可作為偵測結核菌感染的生物指標（biomarker）。
方法：針對新發現一般TB個案和MDR-TB個案進行抽血檢驗, 我們主要是抽丙型干擾素 (interferon-gamma release assay, IGRA*) 及 人體干擾素誘導蛋白-10（human interferon gamma inducible protein-10, 簡稱IP - 10）。將病人群主要分成三組﹕（1）一般結核病患（2）多重抗藥結核病患（3）健康人對照組，並比較此三組細胞激素之變化。
此研究所選擇納入研究的對象: （1）新通報之一般TB個案和MDR-TB；（2）還未接受抗結核病治療, 或痰液檢體持續陽性; 而排除條件則為（1）個案為未滿18歲之兒童,（2）個案使用二線藥物之前即痰陰轉， (3）HIV測試陽性﹐ (4）血液透析（洗腎）病患等個案經評估符合上述條件則不納入研究計畫。(Table 1)
探討各因素包括:性別、年齡、參加這項研究的病人本身原本具有的疾病:例如, 糖尿病， 高血壓, 心臟方面疾病(heart disease)， 肺部疾病 (lung diseases)、癌症，以及病人的 X-ray是否有開洞； 病人是否接受第一次抗結核菌治療，或病人本身是否因結核病再次復發而接受抗結核菌治療為依變項。
在納入治療之個案進行抗結核病治療前（或仍痰持續陽性），先採血液樣本 (IP-10 and IGRA)作為baseline； 另外分別在開始治療二星期、二個月、及六個月時，各採一套血液樣本作為對照指標，進行檢驗。（Figure. 3）並根據所收集到之IGRA與IP-10指數與傳統的痰檢狀況，比較分析個案在治療前後是否和臨床變化相吻合，並藉以瞭解結核病患在治療不同時期相關數據的變化。
結果：
我們將病人群主要分成三組：（1）一般結核病患，有20位病患，（2）多重抗藥結核病患有17位病人， （3）健康人對照組則有30位 (Table 1)，並比較此三組細胞激素之變化。 在各治療階段，兩組治療組 (TB和MDR組) 無太大的差距，治療期間並未有一致性的表現。但是，比較整體的IP-10值時，control組健康人的IP-10平均值指數比TB或MDR-TB個案低： （1） TB組為458.71±301.90pg/ml，（2）MDR-TB組是513.66±279.80pg/ml，（3）健康人為131.94±102.83pg/ml。Control 組 (即健康人對照組)有少數2位IP-10值較高，其中1位受C肝治療的影響，另一位為隔離病房看護，是TB高危險群，有可能是受感染在發病者。
IP-10平均值分別在實驗組 (TB組和MDR-TB組)，在開始治療後二星期、二個月、六個月都有逐漸下降的趨勢。在治療後痰抹片與培養都陰性 (IP-10 level 為397.44 pg/ml) 時IP-10平均值相對的比痰抹片與培養都陽性(IP-10 level 為544.05 pg/ml)時低。(Figure 4.) 將IP-10值與IGRA相比較，發現其數值在接受抗結核菌藥物後都有明顯的下降，但單就個案觀察治療期間的treatment response 與 biomarker並未有一致的變化。因為， IGRA為陰性時IP-10值呈現出90.03pg/ml-1041.74pg/ml的數據，而當 IGRA為陽性時IP-10值也呈現出86.19pg/ml-1066.58pg/ml的數據。從以上這兩組的數據並未發現可以有幫助TB治療成效的指標。這結果與南非Theron 的研究相似，在不同的治療階段，IGRA與抗結核藥物並未有相關聯的固定表現模式。(Figure 5, 6-1, 6-2, 6-3, 6-4)

關鍵字

肺結核 ； IP-10 ； interferon gamma

並列摘要

Objective：
In our previous research (Latent infection among close contacts of multidrug-resistant tuberculosis patients in central Taiwain, 2008), we used an IGRA (QFT-GIT) to examine the contactors of tuberculosis. However, IGRAs cannot differentiate active or latent tuberculosis infection, nor the outcome of treatment. Therefore, may studies have been designed with a combination of IGRA examinations of IGRAs and other cytokines, such as IP-10, IL-2 and IFN-γ. Recently, the serum level of IP-10 (INF- γ inducible protein – 10 ) has been proposed a new biomarker to detect tuberculosis infection.
Methods：
We chose the new cases of pulmonary tuberculosis and MDR-TB and obtain blood samples for IGRA and IP-10. We divided these patients into the three groups: 1.those with pulmonary tuberculosis ; 2. those with MDR-TB; and 3. healthy controls. The inclusion criteria were newly diagnosed TB and MDRTB MDRTB those who had not received the anti-Tb treatment, or persistently positive findings on sputum acid – fast staining . The exclusion criteria were an age less than 18 years, negative sputum acid – -fast staining before the use of second-line anti- TB drugs, those who were HIV positive and hemodialysis patients. (Table 1)
Blood samples for IP-10 and IGRA were collected at baseline before anti-TB treatment. We also collected blood samples in the second week, second month and the six month after anti-TB treatment .(Figure 3) We compared the IGRA and IP-10 data during the anti-TB treatment course compared with conventional sputum samples. and IP-10 level data.
Results：
There were 30 healthy controls, 17 patients with MDR-TB (resistant to INH and RMP), and 20 patients with TB (Table1). During the course of the treatment, there was no significant difference in IP-10 level between the two therapy groups (the MDR –TB and TB groups). The IP-10 level were also not revealed compatible variation between the 2 therapy groups. The average IP-10 data was 131.94±102.83pg/ml in the control group (healthy persons), 458.71±301.90pg/ml in the TB group, and 513.66±279.80pg/ml in the MDR-TB group. According to these data, the IP-10 level was obviously more decreased than the therapy groups. In the control group, only 2 persons had the elevated IP-10 levels. However, one of them was also receiving hepatitis C treatment, the other was the sitter in the isolation room. In the therapy group, the average IP-10 level was declined at 2 weeks, 2 months and 6 months after treatment. Compared with positive findings of sputum smears and cultures, the average IP-10 level was 544.05pg/ml in the patients with positive sputum smears and cultures, and average IP-10 level was 397.44pg/ml in the patients with negative finding of sputum smear and culture.(Figure 4.) We found that the IGFA and IP-10 level were both declined in the treatment group when theses patients received the anti-TB drugs. However, we also observed that the treatment response was not compatible with the variation in biomarkers level. For example, when IGRA was positive , the average IP-10 level was 86.19pg/ml to 1066.58pg/ml. However, when IGRA was negative , the average IP-10 level was 90.03pg/ml to 1041.74pg/ml. This result is similar to the study by Theron in South Africa and suggest that the IGRA level did not obviously correlate with the anti-TB treatment during different stages of the treatment. (Figure 5, 6-1, 6-2, 6-3, 6-4 )
Conclusion:
The average IP-10 level in the therapy group was far lower than in the controls. The value of IP-10 was investigated as a method to detect TB infection and to enhance the screening of the TB cases. We also observed that the IP-10 values in the TB group did not correspond with those in the MDR-TB group. This may be because the presentation of antigens or antibodies different and it was therefore difficult to analyze the IP-10 data. Therefore, IP-10 was not a biomarker of the indicators of outcome of the TB treatment.

並列關鍵字

pulmonary tuberculosis ； IP-10 ； interferon gamma

參考文獻

Received 30 November 2012; accepted with revision 3 April 2013

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