大台北地區土壤中活性棘阿米巴原蟲定量研究

Abstract

棘阿米巴原蟲(Acanthamoeba spp.)為廣泛存在於土壤環境中具潛在致病性自由型阿米巴。過去文獻多屬定性棘阿米巴原蟲之資料，或僅針對農田土壤進行棘阿米巴原蟲定量，故本研究以ethidium monazide(EMA)搭配即時定量聚合酶連鎖反應(real-time quantitative polymerase chain reaction, qPCR)針對沙質土中活性棘阿米巴原蟲進行定量方法建立。並以EMA-qPCR方法定量大台北地區之玩沙區(公園、國小)、海灘(濕沙、乾沙)、水稻田等表層(0-1 cm)、深層(15-30 cm)土壤中活性棘阿米巴原蟲濃度，同時探討此些土壤之環境因子：異營性細菌濃度、總懸浮固體濃度、含水量、飽和導電度、鹽度、pH值、有機碳含量及土壤質地與活性棘阿米巴原蟲濃度關係。 沙質土中活性棘阿米巴原蟲定量部分，因文獻指出樣本之總懸浮固體濃度(Total suspended solids, TSS) < 1000 mg/L時可搭配2.3 μg/mL EMA，TSS < 6000 mg/L時可搭配23 μg/mL EMA進行活性棘阿米巴原蟲定量，故先探討海灘乾沙與玩沙區之沙土之懸浮固體濃度差異，發現海灘乾沙總懸浮固體濃度較低於玩沙區，顯示總懸浮固體濃度可能受不同地區土壤特性之影響，故每批次樣本進行活性棘阿米巴原蟲定量前須先測定總懸浮固體濃度，以確保EMA不受土壤樣本中總懸浮固體濃度干擾。此外，本研究將棘阿米巴原蟲添加至沙質土混合液中，測定震盪0、30、60、90、120、150及180秒之活性棘阿米巴原蟲回收率，結果顯示震盪60秒時回收率最高，但與震盪30秒無顯著差異，故擇定沙質土混合液震盪30秒取其上清液，視其總懸浮固體濃度進行不等倍稀釋，以添加EMA進行活性棘阿米巴原蟲濃度定量。 本研究表層土壤以海灘濕沙活性棘阿米巴原蟲陽性率(27.8%)顯著低於各類土壤(Fisher exact test, p-value=0.047)，深層土壤則以公園玩沙區之陽性率(28.6 % )低於各類土壤(Fisher exact test, p-value=0.87)。表層、深層水稻田活性棘阿米巴原蟲檢出濃度(3.99 log cell/g of dw., 4.02 log cell/g of dw. )皆顯著高於沙質土(Kruskal-Wallis test, p-value < 0.0001, p-value = 0.0009)，表層沙質土則以國小玩沙區平均檢出濃度(1.71 log cell/g of dw.)顯著高於最低平均濃度之海灘濕沙樣本(0.05 log cell/g of dw.)，深層各類沙質土活性棘阿米巴檢出濃度則無顯著差異；而於表深層皆有採集之海灘乾沙、公園玩沙區、國小玩沙區合併之沙質土及所有土壤之表深層活性棘阿米巴原蟲之陽性率與檢出濃度皆無顯著差異(McNemar’s test, p-value = 0.56, 0.20; Wilcoxon signed rank test, p-value = 0.99, 0.19)。 以複邏輯斯迴歸(multiple logistic regression with stepwise procedure)評估沙質土活性棘阿米巴原蟲檢出之影響環境因子於表層(n=52)、表/深層合併(n=84)樣本皆見異營性細菌濃度高於105 CFU/g of dw.時，活性棘阿米巴原蟲檢出情形越高(表層異營性細菌濃度大於1.1×105 CFU/g of dw. OR=33.53, p-value= 0.009；表/深層合併異營性細菌濃度大於1.7×105 CFU/g of dw. OR=7.87, p-value= 0.0007)；而以多元線性逐步迴歸(multiple linear regression with stepwise procedure)評估影響沙質土活性棘阿米巴原蟲檢出濃度，則見當土壤中總懸浮固體濃度越高時，活性棘阿米巴原蟲檢出濃度越高(表層：β=0.01，p-value=0.01；深層β=0.001，p-value=0.01；表/深層合併：β=0.002，p-value=0.01)。 所有土壤部分，也以複邏輯斯迴歸評估影響活性棘阿米巴原蟲檢出之環境因子仍見異營性細菌濃度高於105 CFU/g of dw.時，活性棘阿米巴原蟲檢出情形越高(66件表層樣本異營性細菌濃度大於1.6×105 CFU/g of dw. OR=10.11，p-value=0.0011；112件表/深層合併樣本異營性細菌濃度大於1.9×105 CFU/g of dw. OR=6.47，p-value=0.0008)。而影響陽性樣本檢出濃度則為有機碳含量，當有機碳含量越高時，活性棘阿米巴原蟲檢出濃度越高(表層：β=5522.5，p-value<0.0001；深層β=7394.9，p-value<0.0001；表/深層合併：β=3804.0，p-value=0.005)。 本研究為首篇針對沙質土類型土壤中活性棘阿米巴原蟲之定量研究，且嘗試探討土壤中活性棘阿米巴原蟲與環境因子之相關性，然僅定量土壤中活性棘阿米巴(Acanthamoeba spp.)之研究，對於其中致病性棘阿米巴原蟲所佔比例並不知，故若欲進一步了解土壤中棘阿米巴原蟲對人體致病之可能風險，將可再針對檢出濃度較高之土壤類型進行進一步研究評估。

Acanthamoebae spp. is potentially pathogenic free-living amoebae (FLA) that are widely distributed in soil. For better understanding of the risk posed to human health from Acanthamoeba, adding the quantitative data of soil Acanthamoeba is necessary. But so far, the studies with the quantitative data on environmental Acanthamoeba are very limited. Hence, this study has adopted ethidium monazide coupled with real-time quantitative polymerase chain reaction (qPCR) to quantify viable Acanthamoeba in sandy soil. Further, we quantify the concentration of viable Acanthamoeba in soil through Taipei metropolitan area (sandpits in parks and elementary schools, beach (wet sand, dry sand) and rice field). Also, we are going to discuss the environmental factors including heterotrophic plate count, total suspended solids (TSS), water content, electric conductivity, salinity, pH, organic carbon, soil texture. Quantifing viable Acanthamoeba in sandy soil , we found that the maximum recovery rate of viable Acanthamoeba when vortex 60 seconds is no significant difference when vortex 30 seconds. So we chose vortex 30 seconds and followed the suggestion of past study TSS<1000 mg/L treated 2.3 μg/mL EMA or TSS < 6000 mg/L treated 23 μg/mL. The positive rate of viable Acanthamoeba in wet sand (27.8%) was significant lower than each soil type (Fisher exact test, p-value=0.047). In addition, the positive rate of park depth sample was the lowest (Fisher exact test, p-value=0.87). No matter Either surface or depth sample of rice field, the concentrations were higher than each soil type (3.99 log cell/g of dw., 4.02 log cell/g of dw.,). Multiple logistic regression with stepwise procedure analyzed the association between soil parameter and viable Acanthamoeba presence in sandy soil. We found surface(n=52), and surface+depth (n=84) were positive with heterotrophic plate count >105 CFU/g of dw. (surface: OR=33.53, p-value= 0.009；surface+depth: OR=7.87, p-value= 0.0007). The results of multiple linear regression with stepwise procedure showed viable Acanthamoeba concentration was positive with total suspended solids(surface：β=0.01，p-value=0.01；depthβ=0.001，p-value=0.01；surface+depth：β=0.002，p-value=0.01). We also found that multiple logistic regression with stepwise procedure analyzed the association between soil parameter and viable Acanthamoeba presence in all soil showed the same results (surface: OR=10.11，p-value=0.0011；surface+depth: OR=6.47，p-value=0.0008). The results of multiple linear regression with stepwise procedure showed viable Acanthamoeba concentration was positive with organic carbon (surface:β=5522.5，p-value<0.0001；depth: β=7394.9，p-value<0.0001；surface+depth:β=3804.0，p-value=0.005).