呼出氣體中流行性感冒病毒與腸病毒之偵測

Detection of Influenza Virus and Enterovirus in Human Exahled Air

郭婉琦(Wan-Chi Kuo)

指導教授 ： 陳培詩

高雄醫學大學/健康科學院/公共衛生學研究所/碩士(2009年)

摘要

流行性感冒病毒是常見且具有公共衛生重要性的呼吸道疾病病毒之一，即使在醫療進步的21世紀，流行性感冒病毒每年在全世界仍然造成至少五百萬人感染以及20∼50萬人死亡。本研究室過去以成功的在空氣中偵測到流行性感冒病毒，證實流行性感冒病毒會經由空氣傳播；同時也在幼稚園孩童的呼出氣體中偵測到流行性感冒病毒的存在，但病患並未經由醫師確診，僅以問卷評估是否感冒，亦無紀錄其症狀，無法做更進一步的科學探討與公共衛生應用。
腸病毒在1998年首度引發台灣78名孩童死亡，2008年再度爆發大流行，造成14名孩童死亡，十年來政府推動的勤洗手政策，卻無法減緩腸病毒的流行。在腸病毒眾多的血清型中，只有第21型克沙奇病毒（Coxsackievirus A21, CA21）確定會經由空氣傳播，其他血清型的傳播途徑並不明確。台灣常見的流行血清型別多為第16型克沙奇病毒（Coxsackievirus A16, CA16）與第71型腸病毒（Enterovirus 71, EV71），一般認為是經由糞口傳染，但由流行病學研究顯示，家庭內成員感染比例偏高，使人好奇腸病毒是否會經由其他途徑如空氣進行傳播。
因此，本研究主要分為兩部份：（一）探討乎氣體與臨床喉頭拭子中流行性感冒病毒之間的相關性，（二）偵測醫院小兒門診診間空氣的腸病毒及感染腸病毒病患呼出氣體中的腸病毒。在流行性感冒病毒方面，主要目的為了解呼出氣體樣本與臨床喉頭拭子檢體之間的相關性，在腸病毒方面，主要目的為探討腸病毒經由空氣傳播的可能性，以及呼出氣體中腸病毒定量方法的建立。
做法上，為配合此兩種病毒的流行季，流行性感冒病毒的研究在2008年11月與2009年2月間進行，共採得22個呼出氣體樣本；此外亦採檢了病患的喉頭拭子檢體與記錄病患的症狀表現並評分。腸病毒的研究是在2008年6月∼2008年8月進行，共收集14個腸病毒空氣樣本與24個呼出氣體樣本。空氣採樣以直徑37mm，孔徑1.0μm的鐵弗龍濾紙，以流速20L/min進行採樣，並記錄診間的溫度、相對溼度、一氧化碳濃度、二氧化碳濃度以及換氣率。呼出氣體採樣是以直徑37mm，孔徑2.0μm的鐵弗龍濾紙來補集病毒，採樣體積為10公升。偵測方法是以分子檢測Real-time quantitative polymerase chain reaction（qPCR）技術來偵測病毒濃度。
結果方面，首先在流行性感冒病毒上，呼吃氣體在A型流行性感冒病毒的陽性率為0%（0/22），B型流行性感冒病毒為68.2%（15/22）；喉頭拭子在A型流行性感冒病毒的陽性率為31.8%（7/22），在B型流行性感冒病毒為72.7%（16/22）。整體而言，喉頭拭子的陽性率高於呼出氣體。在感染流行性感冒病毒病患的喉頭拭子檢體中，85%（17/20）的病患只感染單一種流行性感冒病毒，但有15%（3/20）的病患同時感染A型與B型流行性感冒病毒。顯示在一般情況下，大多數人只會感染一種流行性感冒病毒，而在同時感染的病患中，可看到A型流行性感冒病毒的濃度與B型流行性感冒病毒的濃度呈顯著的正相關（p=0.019）。相關性檢定中，呼出氣體中B型流行性感冒病毒的濃度與症狀指數呈顯著正相關；另外，呼出氣體中有無偵測到B型流行性感冒病毒與鼻炎有顯著的正相關。此外也發現呼出氣體陽性的病患，其感冒症狀除了腹痛之外的其餘症狀表現比例皆高於喉頭拭子陽性的病患。
其次在腸病毒方面，在空氣樣本與呼出氣體兩者樣本中均可偵測到腸病毒，且其偵測陽性率各為64.3%（9/14）與79.2%（19/24）。顯示腸病毒可能會經由空氣傳播。再以迴歸分析後發現，呼出一氧化碳濃度增加1ppm，呼出氣體中的腸病毒濃度增加1.01x1013 copies/m3。顯示呼出一氧化碳可能是腸病毒感染的指標，但仍需要進一步的研究來探討。
本研究探討了流行性感冒病毒在呼出氣體樣本與喉頭拭子檢體的相關性，亦探討了感冒症狀表現與檢體是否偵測到病毒間的相關性。然本研究的樣本數偏小，無法進行精確的統計分析，未來若能增加樣本數，將可進行更多議題探討。在腸病毒方面，本研究為首次證實非CA21的血清型之腸病毒可能會經由空氣傳播。然而本研究為前驅性的研究，初步發現到腸病毒可能會經由空氣傳播，未來可進一步進行分型鑑定，以了解可以經由空氣傳播的血清型為何。

關鍵字

呼出氣體 ； 流行性感冒病毒 ； 腸病毒

並列摘要

Influenza virus is one of the most common types causing respiratory infections. It infects at least 5 million people and kills 250 to 500 thousand people every year. We had successfully detected influenza virus from the air and exhaled air samples in the previous study. This confirms that influenza virus can be transmitted by air. However, the patients who provided exhaled air samples were not diagnosed by doctor, and the symptoms were also not recorded in the previous study. For illustrating the relation between exhaled virus concentration and disease, more efforts might need to be done.
There were 78 children died because of the infection of enterovirus during 1998 in Taiwan. In 2008, enterovirus attacked Taiwan again and caused 14 children died. During the past ten years, the Centers for Disease Control (CDC) in Taiwan tried to carry out the “wash hand” policy, but still can’t slow down the epidemic of enterovirus. Among the various serotypes of enterovirus, only coxsackievirus A16 has been confirmed that it can be transmitted by aerosol. The transmission route of other serotypes is not clear. The most common serotypes of enterovirus in Taiwan are CA16 and EV71. It is said that the transmission route of CA16 and EV71 is fecal-oral route, but the high infections in household contacts in Taiwan made us curious about other transmission route like airborne transmission of enterovirus.
There are two main aims of the present study. One the aims of this study is to figure out the association between exhaled air and throat swab samples provided by patients who infected influenza virus. The other aim of this study is to develop the quantification method of enterovirus in both the air samples and the exhaled samples. Then, enterovirus in air samples in the pediatric department and the exhaled air samples provided by patients who infected enterovirus were quantified to figure out that if enterovirus can be transported by air.
There are two sampling period of influenza virus, 2008/11 and 2009/02. T total of 22 exhaled samples were collected during the two periods. Throat swab specimens also obtained from patients as well as the symptoms records of patients. The sampling period of enterovirus was from 2008/06 to 2008/08. A total of 14 air samples and 24 exhaled samples were obtained. We used Teflon filter coupled with real-time qPCR for virus detection. The air samples were sampled by 37 mm, 1.0 μm Teflon filters, at 20L/min. The exhaled samples were sampled by 37mm, 2.0μm Teflon filters, and the sampling volume was 10 L.
The positive rate of influenza A and B virus in exhaled sample were 0% and 68.2%, respectively. The positive rate of influenza A and B virus in throat swab specimen were 31.8% and 72.7%, respectively. 85% (17/20) of throat swab specimens detected one kind of influenza virus, 15% (3/20) detected both influenza A and B viruses. There was a positive correlation between the concentration of influenza A virus and influenza B virus in throat swab specimens detected both influenza A and B viruses. It was found that a positive correlation between the concentration of influenza B virus and symptom score, and a positive correlation between in influenza B virus and rhinitis. In regard to the flu symptoms, it was demonstrated that the symptoms percentage in positive exhaled samples was higher than that in positive throat swab samples for all symptoms expect abdominal pain.
The positive rate of enterovirus in air samples and exhaled air samples were 64.3% and 79.2%, respectively. This demonstrated that enterovirus may be transported by air. For regression analysis, the concentration of exhaled CO arised 1 ppm, the concentration of exhaled enterovirus arised 1.01×1013 copies/m3. Exhaled CO might be an indicator of respiratory infection, but further research is still needed.
In conclusion, the correlation between the concentration of influenza viruses in exhaled samples and the symptoms were investigated in the present study. A positive correlation between the concentration of influenza B virus and symptom score, and a positive correlation between in influenza B virus and rhinitis was demonstrated. In regard to enterovirus, high positive rate of enterovirus in air samples and exhaled samples demonstrated that enterovirus, caused epidemic in Taiwan, can be transported by air. To our knowledge, this is the first study to detect enterovirus in air samples around infected patients and exhaled samples from infected patients.

並列關鍵字

exhaled air ； influenza virus ； enterovirus

參考文獻

AbuBakar, S., et al. (2009). "Enterovirus 71 outbreak, Brunei." Emerg Infect Dis 15(1): 79-82.

Ahmad, K. (2000). "Hand, foot, and mouth disease outbreak reported in Singapore." Lancet 356(9238): 1338.

Alexander, J. P., Jr., et al. (1994). "Enterovirus 71 infections and neurologic disease--United States, 1977-1991." J Infect Dis 169(4): 905-8.

Amorim, L. C. and L. C. Z. de (2007). "Breath air analysis and its use as a biomarker in biological monitoring of occupational and environmental exposure to chemical agents." J Chromatogr B Analyt Technol Biomed Life Sci 853(1-2): 1-9.

Ang, L. W., et al. (2009). "Epidemiology and control of hand, foot and mouth disease in Singapore, 2001-2007." Ann Acad Med Singapore 38(2): 106-12.