生物氣膠採樣效能研究-退伍軍人菌

Study on Sampling Performance of Bioaerosols – Legionellae

洪珮瑜(Pei-Yu Hung)

指導教授 ： 張靜文

國立臺灣大學/公共衛生學院/環境衛生研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.00009

摘要

吸入嗜肺性退伍軍人菌 (Legionella pneumophila)與退伍軍人菌 (Legionella spp.)可能導致退伍軍人症的發生，因此正確的在實場環境中定量空氣中退伍軍人菌濃度是重要的環境衛生議題，而此需借重適當的採樣與分析方法，然目前對此仍欠缺完整的實場研究予以釐清。因此，本研究以安養院冷卻水塔、淋浴間及工業區污水處理廠曝氣池三類設施附近空氣環境為研究對象，在採樣5、10、15、30、60及270分鐘下，評估兩種以培養基收集的採樣器 (MAS-100 with agar及Andersen one-stage sampler)、四種以液體收集的採樣器 (BioSampler、AGI-30、SASS 2300及MAS-100 with liquid)以及三種以濾紙收集的採樣器(IOM sampler結合明膠濾紙(gelatin filter) 、濾紙匣 (Cassette)結合聚碳酸酯樹脂濾紙(polycarbonate filter)及MD8 sampler結合明膠濾紙)之採樣L. pneumophila與Legionella spp.之效能，並透過培養法、即時定量聚合酶鏈鎖反應 (Real-time quantitative polymerase chain reaction)以及結合ethidium monoazide與qPCR之EMA-qPCR分析法，進行各式採樣法偵測三種設施空氣中可培養、活性及總L. pneumophila與Legionella spp.之效能比較。另亦採取三設施之水樣，進行L. pneumophila與Legionella spp.濃度評估。
　　結果發現，空氣樣本在未經酸處理下，使用BCYEα agar及DGVP agar進行培養，L. pneumophila陽性檢出率最高可達50%及25%，然真菌污染率最高可達65% (BCYEα agar)及46.7% (DGVP agar)；經酸處理後，真菌污染率分別下降至12.9%及5.4%，而Legionella spp.陽性檢出率分別為50%及40%，顯示利用酸處理配合使用DGVP agar可有效降低樣本污染，且不影響L. pneumophila及Legionella spp.陽性檢出（p> 0.05）。比較各類採樣器發現，可檢出空氣中具培養性L. pneumophila及Legionella spp.者為MAS-100 with agar、AGI-30、BioSampler、MAS-100 with liquid、Cassette、IOM及SASS 2300，其中BioSampler為唯一在三類設施中皆有陽性檢出的採樣器；最高的陽性檢出率則出現在以BioSampler採樣60分鐘及以SASS 2300採樣270分鐘時 (66.7及50%，p<0.05)；而BioSampler可在最短的採樣時間內 (15-30分鐘)檢出空氣中可培養之L. pneumophila及Legionella spp.。
　　對於捕集空氣中活性及總L. pneumophila與Legionella spp.而言，在採樣15分鐘時，比較AGI-30、BioSampler、MAS-100 with liquid及MD8 sampler四類採樣器，在三類設施中皆以AGI-30具有最佳之採樣效能，其與MAS-100達統計顯著差異 (p<0.05 )。在採樣30、60及270分鐘下，於冷卻水塔及曝氣池評估AGI-30、BioSampler、MAS-100 with liquid、MD8 sampler、Cassette、IOM及SASS 2300七種採樣器 (其中MD8未納入60及270分鐘採樣，AGI-30、BioSampler與MAS-100 with liquid未納入270分鐘採樣)，結果顯示均以IOM結合gelatin filter分之採樣效能最高，且與除Cassette外之其他採樣器達到統計顯著差異（p<0.05)。而在淋浴間比較的AGI-30、BioSampler及Cassette採樣器中，在採樣30-60分鐘下，均以Cassette搭配PC filter擁有最佳採樣效能 (p<0.05)。
　　依據最適採樣法所得數據進行暴露評估，三設施空氣中活性及總嗜肺性退伍軍人菌濃度上以冷卻水塔結果為最高 (1.7×106 cell/m3，3.3×106 cell/m3；p<0.05)，而在水樣部分，活性及總嗜肺性退伍軍人菌濃度同樣以冷卻水塔較高 (2.3×104 cell/L，5×104 cell/L)，顯示冷卻水塔設施具有較高的暴露風險。在三設施中，其水樣可培養及總L. pneumophila與Legionella spp.未超過可能感染疾病的暴露風險，但在空氣中的總Legionella spp.濃度則已超過可能感染疾病的暴露標準，顯示雖然水體濃度未達感染疾病可能的暴露風險，但在空氣中的L.pneumophila及Legionella spp.仍有潛在暴露後感染可能，若僅針對水樣濃度進行暴露風險評估，可能會低估空氣傳播中真正造成人體吸入後感染的菌種濃度。
　　整體而言，利用AGI-30採樣器搭配液體、IOM及Cassette採樣器搭配濾紙採樣在定量活性及總退伍軍人菌及嗜肺性退伍軍人菌上有最佳的採樣效能;在檢測可培養性退伍軍人菌上，則以BioSampler於採樣60分鐘時有最高之陽性檢出率；此外，本研究證實EMA-qPCR可運用於實場樣本，定量空氣中活性退伍軍人菌及嗜肺性退伍軍人菌。本研究調查之冷卻水塔、淋浴間以及曝氣池，部分空氣中總Legionella spp.濃度已達可引發退伍軍人病或龐堤亞克熱之風險，其中尤以冷卻水塔周圍空氣具有較高的暴露風險，宜有適當的介入措施及legionellae濃度控制進行處理。

關鍵字

嗜肺性退伍軍人菌 ； 退伍軍人菌 ； 可培養性 ； 活性 ； 生物氣膠採樣器 ； 即時定量聚合酶鏈鎖反應 ； ethidium monoazide

並列摘要

Legionella pneumophila or Legionella spp. is the causative agent of Legionnaires’ disease. Airborne transmission is the exposure route of Legionnaires’ disease. Thus, quantifying airborne legionellae in the field is important to environmental health. There’s no standard method in the detection and quantification of legionellae in the fields. The accuracy should rely on the appropriate sampling and analysis methods. Therefore, in this study, evaluating the sampling performance in 5、10、15、30、60 and 270 sampling times of two agar-based samplers (Andersen one stage and MAS-100)、four liquid-based samplers with sterilized water (AGI-30, BioSampler, MAS-100 and SASS 2300) and three filter-based sampler (IOM and MD8 with gelatin filter、Cassette with polycarbonate filter) for collecting culturable、total and viable Legionella spp. and L. pneumophila cells in the cooling towers、aeration tank of wastewater and shower rooms, detected by culture method、real-time quantitative polymerase chain reaction (qPCR) and ethidium monoazide combined with qPCR (EMA-qPCR). Water samples were also colleced from the three facilities to determine the exposure assessment in addition.
　　The results showed that L. pneumophila reveal positive rate with BCYEα and DGVP agar but on acid treatment were 50% and 25% by culture method. But that samples were contaminated with fungi for 65% (BCYEα agar)and 46.7% (DGVP agar). With acid treatment, the contaminations of fungi were reduced to 12.9% and 5.4%, positive rates of Legionella spp. were 50% and 40%. It showed that acid treatment, combined with culture on DGVP agar,was most appropriate for decreacing the contaminations of fungi, but did not affect legionellae detection (p> 0.05). MAS-100 with agar、AGI-30、BioSampler、MAS-100 with liquid、Cassette、IOM and SASS 2300 samplers could detected the culturable L. pneumophila and Legionella spp. Furthermore, only BioSampler were detected the culturable legionellae in the three facilities. The performance of samplers for L. pneumophila and Legionella spp. determined by culture with positive rate showing BioSampler for 60min sampling time and SASS 2300 for 270min sampling time were the best (66.7%,50%,p<0.05) among all. And Biosampler required the shortest sampling time, which 15-30 min was able to detect culturable L. pneumophila and Legionella spp.
　　As for total and viable airborne L. pneumophila and Legionella spp., AGI-30 is with the best sampling performance in three facilities and it was significantly better than MAS-100 by sampling with AGI-30、BioSampler、MAS-100 with liquid and MD8 sampler in 15min (p<0.05). Evaluating the performance of AGI-30、BioSampler、MAS-100 with liquid、MD8 sampler、Cassette、IOM and SASS 2300 for 30、60 and 270min sampling time in cooling towers and aeration tank of wastewater (MD8 didn’t sample for 60 and 270min, AGI-30、BioSampler、MAS-100 with liquid didn’t sample for 270min), IOM sampler with gelatin filter showed the significant better sampling performance for collecting total and viable L. pneumophila and Legionella spp than the other samplers except Cassette (p<0.05). Furthermore, Cassette sampler with polycarbonate filter had the best performance for 30-60 min sampling time by AGI-30、BioSampler and Cassette in the shower rooms (p<0.05).
　　In accordance with the sampling and analysis methods appropriate for the exposure assessment. Cooling towers detected the highest airborne concentration of total and viable L. pneumophila and Legionella spp. (1.7×106 cell/m3, 3.3×106 cell/m3, p<0.05). Total and viable concentration of L. pneumophila and Legionella spp. were also higher in the water samples of cooling towers (2.3×104 cell/L, 5×104 cell/L) compared to other sampling sites. It is revealed that cooling towers is the highest exposure risk to Legionnaires’ disease. The water concentrations of total and culturable L. pneumophila and Legionella spp. among three facilities were under the infection threshold values in other countries in the water networks. But the total concentrations of the airborne Legionella spp. were out of a margin of safety for Legionnaires’ disease warrants further evaluation. As for the airborne concentration of L. pneumophila and Legionella spp., were concerning with the exposure risk to Legionnaires’ disease in spite of the cell’s concentrations were under the infection threshold value. Determine exposure assessment only by the legionellae concentrations in water samples might underestimate the cell concentration and fail to aware the chance that may result in Legionnaires’ disease by inhalation of L. pneumophila or Legionella spp..
　　In conclusion, AGI-30 with sterilized water、IOM and Cassette sampler with filter had the best performance of quantification of total and viable L. pneumophila and Legionella spp. By culture method, BioSampler for 60min sampling time had the highest positive rate. Furthermore EMA-qPCR be monitored to quantify the concentration of viable airborne L. pneumophila and Legionella spp. in the field. To the exposure assessment, A part of total concentrations of airborne Legionella spp. might in the infected risk of Legionnaires’ disease during exposure in the cooling towers、shower rooms and aeration tank of wastewater. The cooling towers in the study had a highest exposure risk since the concentration of L. pneumophila and Legionella spp. in the water and air. We suggest the facility should be periodically disinfected, cleaned, and monitored the legionellae concentrations in order to minimize the risk to Legionnaires’ disease.

並列關鍵字

Legionella pneumophila ； Legionella spp. ； culturable ； viable ； bioaerosol samplers ； real-time qPCR ； ethidium monoazide

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