病毒氣膠環境監測與控制技術評估

Abstract

病毒是屬於絕對寄生的微生物，經由研究指出，部分病毒已被確認是人或動物的致病菌。病毒會經由許多的傳播途徑對人體產生健康威脅，如：空氣、飛沫、水、血液以及病媒等途徑，在這些傳播途徑之中，由於病毒經由空氣或飛沫傳播是最快速的，因此對人體健康是最具威脅的傳播途徑之一。有鑑於SARS疫情爆發以及腸病毒之流行，使我們有必要針對病毒氣膠做進一步的研究，建立其採樣分析技術，並利用該技術針對高風險之環境進行監測。本研究之目的在建立病毒氣膠之採樣分析技術與評估各種控制技術的控制效率，以確實達到公共衛生上預防疾病的目的。本研究首先進行病毒氣膠之採樣及分析評估，並在實驗室反應腔產生具有不同核酸形式(dsDNA, ssDNA, dsRNA, ssRNA)以及外型之病毒氣膠，同時利用不同採樣方法(Andersen impactor, AGI-30 impingers, gelatin filter 和 Nuclepore filter)來評估採樣器對病毒氣膠之收集效率。經由結果顯示，病毒的外型構造以及是否具有套膜均會影響其採樣效率。對於親水性的病毒(不具套膜)來說，Andersen impactor, impinger, and gelatin filter 均能有效的捕集病毒氣膠，至於Nuclepore filter，由於其在採樣以及萃取的過程中會使病毒去活化，因此其採樣效率較低。另外，對於具有套膜的病毒來說，本研究中各種採樣器對其採樣效率均相當低，主要是因為套膜對於環境壓力(Environmental Stress)相當敏感，採樣過程中的所產生的採樣壓力會對病毒造成去活化的影響。 在病毒的分析方法方面，為發展快速定量病毒之方法以彌補傳統培養定量方法(plaque assay)之不足，本研究利用同步定量PCR (Real-Time Quantitative PCR)，建立空氣中重要的病毒氣膠(如腸病毒、流行性感冒病毒以及腺病毒)之最佳化採樣及分析方法，以進行病毒氣膠之環境監測。本研究成功的利用同步定量PCR監測醫院急診室小兒部門、小兒科門診間與候診區空氣中的病毒氣膠，此方法對於空氣中腸病毒的偵測極限最低可達34 copies/m3，流感病毒為160 copies/m3，而腺病毒可達10 copies/m3。結果顯示空氣中可偵測到的病毒種類與病人症狀間具有高度的相關性，同時病毒氣膠的濃度也會隨著病人的聚集而升高。在空氣中可以偵測到這些病毒代表這三種病毒是非常有可能以空氣傳播的，特別是腸病毒，在過去腸病毒的傳播途徑都是由呼吸道或是糞便是否能分離出腸病毒來做判斷，並沒有一個實際的環境監測研究來做這樣的驗證。雖然經由本研究可以在空氣中偵測到這些病毒，但直到目前為止，想要瞭解空氣中的病毒氣膠是否具感染力仍需要進一步的研究。 最後，除了建立病毒氣膠之採樣及分析方法外，本研究也評估紫外光、臭氧以及氯對病毒氣膠、表面病毒與水中病毒之控制效率。在病毒氣膠與表面病毒方面，進行的是紫外線以及臭氧的殺菌效率評估，此兩種方法都是常用且有效的微生物控制技術，結果顯示紫外線與臭氧對於病毒氣膠以及表面病毒都具有良好的去除效果，且紫外線與臭氧劑量都與病毒的存活率有著良好的劑量效應關係。在紫外線方面，結果發現單股RNA/DNA病毒對紫外線的易感受性較雙股RNA/DNA病毒高，而提高相對濕度可降低病毒對紫外線的易感受性。就臭氧而言，病毒蛋白質髓鞘的組成越簡單對臭氧的易感受性越高，而提高相對濕度反而會增加病毒對臭氧的易感受性，這與臭氧會與水氣反應產生反應性的自由基有關。而在水體病毒控制方面，則是評估常用的加氯消毒以及臭氧水對病毒感染力的影響，結果顯示病毒對氯與臭氧的易感受性與病毒蛋白質髓鞘的構造有關，同時氯與臭氧的劑量都與病毒的存活率有良好的劑量效應關係。經本研究的結果顯示，對病毒的去活化效果與不同方法的殺菌機制有關，在水中的加氯以及加臭氧消毒均能在低濃度以及短暴露時間內處理大量的病毒。雖然目前美國環保署所規定的Ct值也都能夠有效的去除本研究所選擇的四種指標病毒，但在未來的研究及應用中，不同的病毒指標，病毒濃度以及處理水的種類等都應該做進一步的探討。

Viruses are obligate parasites and are pathogenic to humans and animals. Recently, Severe Acute Respiratory Syndrome (SARS) virus and enterovirus have attracted public attention. In order to understand the health risk from virus exposure, this study was to establish of methods for sampling and analysis virus-containing aerosols. Furthermore, the effectiveness of different microbial control methods for inactivating viruses should be investigated for virus monitoring and control strategy. In this study, bacteriophages were surrogates for mammalian viruses in assessing sampling efficiency of Andersen impactor, impingers, gelatin filter and Nuclepore filter. Our results demonstrated that virus particle morphology with/without envelope could significantly affect virus sampling performance. For hydrophilic virus, Andersen impactor, impinger, and gelatin filter were likely to perform better than Nuclepore filter. The recovery of lipid-envelope virus sensitive to sampling stress was very low. In the past years, traditional cell culture was the major method for detecting viruses, however, Polymerase Chain Reaction (PCR) are considered to be more rapid, sensitive, and specific. Therefore, establishment of nonculture based method (Real-time quantitative PCR) for viruses quantification are needed. Therefore, we established the optimal sampling and analysis methods for virus monitoring in hospital. An environmental monitoring for enterovirus, influenza virus and adenovirus in hospital emergency department, consulting room and waiting room of pediatrics was conducted to investigate the major transmission route and risk factors of virus infection. From our findings, the real-time PCR method could perform lowest measurements of 34 copies/m3, 160 copies/m3 and 10 copies/m3for Enterovirus, Influenza virus and Adenovirus, respectively. The virus species in air were highly related to the patients’ syndromes. Moreover, virus concentration increased as the numbers of patient increased. The detection of influenza virus, adenovirus and enterovirus in air suggests that these viruses could be an opportunistic airborne infection. Especially for enterovirus, the potential droplet transmission route was only supposed by virus was detected from respiratory/fecal specimens or not. From our current study, the possibility of aerosol infectious droplet spreading of the enterovirus may prove for this apparent risk of transmission. Up to now, detection of viable virus in environmental air samples is problematic. Further studies are needed to modify the method for getting the viable results of virus-containing aerosol. Finally, different kinds of disinfection methods were assessed for different species of virus in either aerosol droplets, surface medium or water medium. The potential disinfection methods included UV, ozone and chlorine. Viruses in air and on surface with single-stranded nucleic acid were more susceptible to UV inactivation than were those with double-stranded ones. For all tested viruses by the same inactivation method, the UVGI dose at 85% RH was higher than that at 55% RH. For ozonation, viruses with more complex capsid architectures were observed to be less susceptible to ozone inactivation than those with simple ones. For all tested viruses at the same inactivation, the ozone concentration at 85% RH was lower than that at 55% RH. This might be related to generation of more radicals from ozone reacted with more water vapor at higher RH. For virus control in water, it was demonstrated that survival fractions of all tested phages were found to decrease exponentially with increasing chlorine and ozone doses. The results of this study indicate that the microbicidal activity of chorine and ozone is greatly affected by the applied dose, the presence of virus capsid protein and envelope. Chlorination and ozonation have the advantage of having a strong effect on some types of viruses even low treatment doses and short contact times are applied. Current Ct values supposed by USEPA are sufficient for virus inactivation by ozone and chlorine. However, virus specie, concentration and the water type should be further considered.