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Detection of airborne viruses in a pediatrics department measured using real-time qPCR coupled to an air-sampling filter method.


Children are more vulnerable than the general population to viral infections. Recently, some clinically severe or highly transmissible pathogenic viruses, such as influenza A virus (INFAV), human adenovirus (HAdV), and enteroviruses, have attracted public attention (Heikkinen, 2006; Lin, Huang, Ning, & Tsao, 2004). For INFAV and HAdV, the World Health Organization (WHO) has suggested that aerosol exposure may be the major route for spreading viral diseases (World Health Organization [WHO], 2005). For enteroviruses, the most common mode of transmission is the fecal-oral route, although aerosol transmission has also been reported (Couch, Douglas, Lindgren, Gerone, & Knight, 1970). Generated from sneezing or coughing, viral aerosols typically range from 1 [micro]m to 100 [micro]m and evaporate to nuclei-sized aerosols about the size of individual microbes (Kowalski & Bahnfleth, 1998). Nuclei-sized aerosols can remain airborne for up to one day and can potentially be retained in the respiratory tract (Ijaz, Karim, Sattar, & Jhonson-Lussenburg, 1987).

Although unconfirmed, aerosol transmission is suspected of causing the enterovirus 71 epidemic which caused 78 deaths in Taiwan in 1998 as well as later outbreaks there (Ho et al., 1999; Lin et al., 2002). At that time, despite an intensive public program to encourage hand washing, a high rate of enterovirus transmission occurred in families, which supports the notion that aerosol transmission played a major role in the spread of disease (Chang et al., 2004). To date, no specific validated method has become available for detection of viral aerosols, nor has aerosol transmission of enteroviruses been established.

Impinger and slit samplers are the most commonly used samplers for collecting aerosol viruses (Agranovski et al., 2005; Booth et al., 2005; Hermann et al., 2006; Tseng & Li, 2005). These two samplers are thought to be able to recover more viable viruses in the liquid phase of collection medium than the filtration method (Agranovski et al., 2005; Tseng & Li, 2005). After sampling, the collected virus particles are transferred to culture medium for quantification by plaque assay or 50% tissue culture infective dose ([TCID.sub.50]) (Alexandersen & Donaldson, 2002; Hermann et al., 2006; Tseng & Li, 2005). Until now, these culture-based methods have been applied in studies of places with extremely high viral concentrations such as laboratories and farms (Donaldson, Ferris, & Gloster, 1982; Hermann et al., 2006; Hietala, Hullinger, Crossley, Kinde, & Ardans, 2005; Tseng & Li, 2005). Using these methods in field sampling is not practical because of bacterial contamination and the difficulty of cultivating some viruses.

Because the impinger or slit samplers used to collect viral aerosols are not well suited for the detection of low viral concentrations, filter sampling coupled with reverse-transcriptase polymerase chain reaction (RT-PCR) has been successfully used to measure SARS coronavirus aerosols (Booth et al., 2005). This method yields only a qualitative result, however: positive or negative. Recently, a real-time quantitative polymerase chain reaction (real-time qPCR) system has been developed and used in clinical laboratories. This system was designed to detect and quantify target sequences by using specific primers and fluorescent probes. The detection of amplified nucleic acid products is accomplished in a closed system to prevent carryover contamination (Broccolo et al., 2003). Filter sampling coupled with real-time qPCR would be a potential method to detect viruses in air.

An environmental monitoring program for detection of viral aerosols is very important to identify the concentration profiles of airborne viruses, especially among children, so that viral infections in this group can be better prevented and controlled. We used filter sampling coupled with real-time qPCR (filter/real-time qPCR) to measure INFAV, HAdV, and enterovirus in the aerosols of pediatrics department. The clinical manifestations and number of patients were also compared to the detection of the viruses and the viral load in aerosols.


Study Patients

In April and May 2006, we collected aerosol samples from the emergency room and outpatient clinic where children under age 18 were seen for infectious diseases at one medical center in Taipei, Taiwan. We categorized the doctors' diagnoses into (a) upper respiratory tract infections (URI), comprising URI, pharyngitis, and tonsillitis; (b) lower respiratory infections (LRI), including bronchiolitis, bronchitis, bronchopneumonia, or pneumonia; and (c) herpangina (if an oral ulcer over the throat or uvula was present). The patients' clinical manifestations were also recorded. This study was approved by the institutional review board of the medical center.

Aerosol Sampling

We collected 33 samples in the emergency room and 26 samples at the outpatient clinics, including the consulting room and the waiting room of the pediatric department. According to the surveillance weekly report published by Taiwan Center for Disease Control (TCDC), a peak period of enterovirus occurred during our sampling period. Physical factors such as temperature and relative humidity of the emergency room and the outpatient clinic were also recorded daily. For quality control, samples of field blanks were performed daily at the nursing office of the emergency room.

The central air-conditioning plant of the hospital was provided by Air-Handling Units (AHU). The air exchange rate of the emergency room and pediatric department was found to be in the range of 8/hr. to 10/hr. and meets the range (8-12/hr) suggested by American Society of Heating, Refrigerating, and Air-Conditioning Engineers (ASHRAE). The aerosol samples were filtered through a 37 mm-diameter Nuclepore filter with straight-through pores of uniform size (0.4 [micro]m). Supported by cellulose pads, these filters were loaded into close-face three-piece plastic cassettes and were connected by a plastic tube to a vacuum pump. The pump and filter apparatus were placed about one meter from the patient's seat in the emergency room and in the consulting room and also in the corridor in the waiting room. In addition, the sampling location was three meters from the square ceiling diffuser to avoid wind interference. The sampling height was 0.8-1 m above the floor to be within a child's breathing zone. The sampling apparatus, which had a high flow rate of 20 L/min, was operated for 24 hours daily in the emergency room. Because of shorter operating times of the outpatient clinic, we only performed eight-hour sampling in the waiting room and four-hour sampling in the consulting room.

After sampling, the Nuclepore filter was removed from the holder and placed in a test tube containing 4 mL of sterile deionized water. Then the suspension was slowly vortexed in a rotator for five minutes. After transferring viral particles from filter to eluate, the eluate was removed for DNA or RNA extraction with a MagNA Pure LC Instrument and a Pure LC Total Nucleic Acid Isolation Kit, respectively. For cDNA synthesis, a 1st Strand cDNA synthesis kit was used with AMV reverse transcriptase and random hexamers. The cDNA was stored at -70[degrees]C before real-time qPCR analysis.

Analysis for Viruses

Table 1 lists the PCR primers and fluorophore-labeled hybridization probes used to detect INFAV, HAdV, and enteroviruses. All reagents were from LightCycler[R] FastStart DNA Master Hybprobe. A sequence detector system called LightCycler[R] was used for amplification and measurement of fluorescence.

The PCR primers and fluorophore-labeled hybridization probes used for INFAV analysis were synthesized from TIB MOLBIOL. The matrix gene was shown to be highly conserved among influenza A H1 and H3 subtypes by nucleotide sequence alignments. For human adenoviruses, the primer sequences used in this study were designed and evaluated for a conserved region of adenoviruses as described by Heim and co-authors (2003). For human enteroviruses, the primers and probe sequences were based on highly conserved regions in the 5'-untranslated region of the enterovirus genome (Nijhuis et al., 2002).

Calibration Curve

For all the viruses we tested, the target cDNA from cloning plasmid was synthesized by Mission Biotech (Taipei). A calibration curve was created by serially diluting the external standard and the numbers of copies were calculated automatically by LightCycler[R] software. For all three tested viruses, the known amounts of plasmid cDNA yielded Ct values ranging between 17.9 and 38.9 cycles. The calibration curves were linear for five orders of magnitude for enteroviruses (3.0 to 3.0 x [10.sup.4] copies/[micro]l) and for six orders of magnitude for INFAV (5.4 to 6 x [10.sup.5] copies/[micro]l) and HAdV (6.0 to 6.0 x [10.sup.5] copies/[micro]l). Moreover, the correlation coefficient (r) values of the calibration curve were 1.0 for both INFAV and enteroviruses and 0.99 for HAdV. After calculating the dilution ratio, sampling time and flow rate, the detection thresholds of real-time qPCR method for per cubic meter of air were found to be 160 copies/[m.sup.3] for INFAV, 10 copies/[m.sup.3] for HAdV, and 34 copies/[m.sup.3] for enteroviruses.

Statistical Analysis

We used Spearman's rank correlation (r = Spearman's rho) in SPSS Version 10.0 to assess the association of virus concentrations with the number of patients seem during the sampling period. The differences in positive rates among different viruses were analyzed using a Chi-square test, and the differences in viral concentrations of the different viruses were assessed by Kruskal-Wallis test.


Viral Aerosols in the Emergency Room

In the emergency room, ambient temperatures ranged from 22.1[degrees]C to 26.0[degrees]C and relative humidity ranged from 59.0% to 75.1%. Within the sampling periods, 31% (469/1507) of patients were diagnosed as having an URI, while 9% (130/1507) of patients were diagnosed as having an LRI (Figure 1). Figure 1 suggests there may have been separate outbreaks of INFAV H1, H3, or adenovirus, while the enterovirus outbreak only occurred in the last week of the study. This suggests that ongoing community outbreaks were occurring at the same time that could account for the varying incidence of detection of the different agents.



For INFAV, the viral RNA was detected in eight (24%) of 33 total aerosal samples, with concentrations varying from 167.6 to 5,020 copies/[m.sup.3]. Additionally, INFAV was classified into H1 and H3 subtypes, and the positive rates of H1 and H3 were 9% and 15%, respectively. The peak in the positive influenza samples occurred during the weekend. Although no statistically significant correlation occurred between the viral concentrations and the number of URI patients (p =.06, r = .33, Figure 2a), the viral concentrations of INFAV were significantly correlated with the number of LRI patients (p = .02, r = .43, as shown in Figure 2b).

HAdV had the highest positive rate, 36% (12/33), of the three viruses we tested, with its concentrations ranging from 10.4 to 362 copies/[m.sup.3]. The viral concentrations were significantly statistically correlated with the number of URI patients (p = .005, r = .53) and the number of LRI patients (p = .02, r = .48) (Figures 3a and 3b).

The viral concentrations of enteroviruses varied from 43.6 to 30,000 copies/[m.sup.3], which was the highest aerosol viral load of all the positive aerosol samples. Although only five of the 33 (15%) samples were positive, those five samples were collected when patients with herpangina visited the emergency room. The positive samples were significantly statistically correlated with the number of cases of herpangina (p = .03, r = .373). Positive rates among INFAV, HAdV, and enterovirus were not statistically significant (p = .14), nor were their concentrations (p = .27).

Viral Aerosols in the Outpatient Clinics

In the outpatient clinic, 17% (71/416) of patients were diagnosed with URI, and 21% (86/416) were diagnosed with LRI (Figure 4). The 17% of outpatient clinic patients with URI was significantly lower than the 31% of emergency room patients with URI (p < .001). Temperatures in the outpatient clinics ranged from 22.0[degrees]C to 26.0[degrees]C, and relative humidity ranged from 55.5% to 75.0%. Only HAdV (not INFAV or enteroviruses) was detected by the filter/real-time qPCR assay from the outpatient clinics (Figure 4). In the waiting room, HAdV was detected on five days (May 16, 22, 23, 24, 25), with concentrations ranging between 43.2 to 69.1 copies/[m.sup.3]. In the consulting room, it was only detected on two days (May 24 and 25), with concentrations ranging from 128 to 238 copies/[m.sup.3]. HAdV was detected in both the waiting room (mean concentration was 91.5 copies/[m.sup.3]) and the consulting room (mean concentration was 153.7 copies/[m.sup.3]) on May 24 and 25. At the outpatient clinic, HAdV concentrations were not significantly statistically correlated with the number of URI patients (r = .01) or the number of LRI patients (0.32) (p > .05).


Characterizing the Concentration Profiles of Airborne Viruses in Hospitals

Our study found filter/real-time qPCR assay can be used to screen for viral aerosols in the field. Until now, very limited data existed regarding environmental monitoring of airborne viruses. Although area monitoring may not be as robust as personal sampling, it still could provide important information on the concentration profile of, and potential routes of exposure to, the viral agents to which children are exposed.

It is well known that the influenza virus and HAdV can be spread through coughing and sneezing. Our results showed the concentration of INFAV dose dependently correlated with the number of LRI patients. HAdV had a positive rate 1.5 times that of INFAV, and concentrations of this virus were significantly correlated with the number of URI cases and LRI cases. The higher positive rate of HAdV may be related to the lower detection thresholds applied for HAdV detection. Our data were in close accord with the viral isolation results from our medical center, where adenovirus was the most common isolate of all viral isolates from April to May 2006. Taiwan's island-wide viral surveillance also identified HAdV as the most prevalent circulating respiratory virus during our sampling period (CDC of R.O.C., 2006).

Enterovirus can be spread extensively via fecal-oral and aerosol routes. Although fecal-oral transmission predominates, respiratory excretion can also occur, especially with the coxsackieviruses (Couch et al., 1970). Until now, qualitative and quantitative data have been limited with regard to enterovirus aerosols in the environment. We found enteroviruses in concentrations similar to those of INFAV and HAdV, with the peak reaching 30,000 copies/[m.sup.3]. This high viral load could explain why, despite preventive hand washing procedures, familial and kindergarten transmission rates of enteroviruses are still high (Hatch & Warren, 1969).

We also found the positive samples to be significantly correlated with the number of patients with herpangina, although only five positive samples were found from the samples taken on the days that the patients with herpangina visited. The positive rate may be low because of not enough enterovirus RNA to detect or because certain serotypes of enteroviruses do not spread in aerosols. Another important factor related to the exhaled virus concentrations is disease severity, but we did not investigate this in the current study.


Only HAdV was detected in the air of the outpatient clinics, though viral concentrations there were not significantly correlated with the number of URI cases or LRI cases. Since there were URI cases and LRI cases, we might be able to assume that HAdV might be another major pathogen leading to URI and LRI in outpatient clinics. In this study, the virus profiles in the emergency room are different from those in the outpatient clinics. This may be related to patient profiles since the samples were taken sequentially, not simultaneously. During our sampling periods in the emergency room, the island-wide virus positive rates detected by Taiwan CDC were 4%-17% for INFAV, 4%-6% for HAdV and 3%-21% for enterovirus. The high prevalence of these three viruses may explain why we could successfully detect these viruses in air. In the outpatient clinic, the virus positive rates of HAdV and enterovirus remained the same, while the positive rate of INFAV decreased to under 2%. This could explain why only HAdV can be detectable. In addition, if the patients were sicker or stayed longer in the emergency room than in the outpatient clinic, more sick people presented at certain times which may account for the higher copy number measured at those times. For enterovirus, it cannot be detected in the air of the outpatient clinic may be related to the concentration is below the detection limit of 34 copies/[m.sup.3]. Another possible reason is that the sampling time in the waiting room and consulting room is shorter than the sampling time in the emergency room.


Health Risk from Virus Exposure

Some viral infectious doses have been documented by exposure in animal models. Guinea pigs have been found to be highly susceptible to infection with a dose of five plaque-forming units of INFAV (H3N2) (Lowen, Mubareka, Tumpey, Garcia-Sastre, & Palese, 2006). Given intra-nasally to humans, adenovirus types 1, 2, 3, 4, 5, and 7 have been to found to be infectious at doses less than 150 plaque-forming units (Health Canada, 2001a). In humans, coxsackie A21 virus has been reported to be infectious at doses less than 18 plaque-forming units by inhalation (Health Canada, 2001b). Although these viral concentrations were not transmitted through inhalation, they remain relevant to our study. Forty-three percent of the positive samples we found in the emergency room and outpatient clinic had viral concentrations higher than 100 copies/[m.sup.3], which is a level that could pose a health risk to health care workers, patients, and visitors. The high copy number in the air does indeed suggest transmission could occur, but it does not prove well because of the lack of virus culture data.

Future Direction and Challenges

Our method of detection, using a filter/real-time qPCR assay, can measure virus concentrations at extremely low levels. Previously, this method has been able to detect SARS coronavirus (Booth et al., 2005), rhinovirus (Myatt et al. 2004), varicella-zoster virus (Sawyer, Chamberlin, Wu, Aintablian, & Wallace, 1994), respiratory synctial virus (Aintablian, Walpita, & Sawyer, 1998), and bacteriophages (Tseng & Li, 2005). Personal sampling from individual subjects, however, would provide a more accurate assessment of personal exposure. Methods of personal sampling for detecting airborne viruses should be further developed.

Some cautions for airborne virus sampling should be mentioned. Only sampling using air filters for several hours that are then extracted for nucleic acid amplification can increase the sensitivity for virus detection. A major disadvantage of this method, however, is the filtration and dehydration during sampling and the extraction process may cause enough biological stress to the viruses that they lose some infectivity (Tseng & Li, 2005). Impingement could have yielded culture positives of these viruses, which are known to grow relatively well in culture. Our study had even tried to culture airborne viruses in the field by impingement; however, all samples ended up indetectable because of the limited operation time or the host cells were contaminated by bacteria. Further investigation is needed to find better ways to recover viable viruses for culture over a long sampling period.


In conclusion, a well-established filter/real-time qPCR assay was successfully applied to measure viral aerosols in a hospital. The assay is a highly sensitive (less than 160 copies/[m.sup.3]) and fast (<5 hr.) approach to detect and measure viral aerosols. In this study, we found INFAV, HAdV, and enteroviruses to be present via aerosols. Environmental monitoring of airborne viruses could provide an early indicator of dangerous viruses in the air.

Although most of the information presented in the Journal refers to situations within the United States, environmental health and protection know no boundaries. The Journal periodically runs International Perspectives to ensure that issues relevant to our international constituency, representing over 60 countries worldwide, are addressed. Our goal is to raise diverse issues of interest to all our readers, irrespective of origin.

Acknowledgements: This work was supported by grant 94-2321-B-002-026 from the National Science Council, Taipei, Taiwan, Republic of China.


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Chun-Chieh Tseng, PhD

Luan-Yin Chang, MD, PhD

Chih-Shan Li, PhD

Corresponding Author: Chun-Chieh Tseng, Assistant Professor, Department and Graduate Institute of Public Health, Tzu Chi University, Hualien, Taiwan, R.O.C. Room H409, 4F, 701 Sec. 3, Jhongyang Rd., Hualien 970, Taiwan. E-mail:
Primer and Probe Sequences Used in the Real-Time qPCR

Species   Primer or Probe   Sequence

INFA H1   Forward primer    5'-GTG-TTC-ATC-ACC-CGC-C-3'
          Reverse primer    5'-AGC-CTC-TAC-TCA-GTG-C-3'
          Probe 1           5'-LC Red- 640-CTA-TTT-CTG-GGG-
          Probe 2           5'-CCT-GAT-CTC-TTA-CTT-TGG-

INFA H3   Forward primer    5'-ACG-CAG-CAA-AGC-CTA-C-3'
          Reverse primer    5'-CCC-AAA-TGT-ACA-ATT-TGT-CAA-A-3'
          Probe 1           5'-LC Red- 640-GAG-GCA-TAA-
                            TCC-GGC-ACA-TCA-TAA -PH-3'
          Probe 2           5'-CGG-ATG-AGG-CAA-CTA-GTG-
                            ACC-TAA-G -fluorescein-3'

HAdV      Forward primer    5'-GCC-ACG-GTG-GGG-TTT-CTA-AAC-TT-3'
          Reverse primer    5'-GCC-CCA-GTG-GTC-TTA-CAT-GCA-CAT-C-3'
          Probe             5'-[FAM]-TGC-ACC-AGA-CCC-GGG-
                            CTC-AGG-TAC-TCC-GA -[TAMRA]-3'

Entero-   Forward primer    5'-TCC-TCC-GGC-CCC-TGA-3'
virus     Reverse primer    5'-AAT-TGT-CAC-CAT-AAG-CAG-CCA-3'
          Probe             5'-[FAM]-CGG-AAC-CGA-CTA-CTT-
                            TGG-GTG-TCC-GT -[TAMRA]-3'
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Author:Tseng, Chun-Chieh; Chang, Luan-Yin; Li, Chih-Shan
Publication:Journal of Environmental Health
Article Type:Report
Geographic Code:9TAIW
Date:Nov 1, 2010
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