Aerosol Science and Technology

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Quantification of Airborne Mycobacterium

tuberculosis in Health Care Setting Using Real-
Time qPCR Coupled to an Air-Sampling Filter
Method

Pei-Shih Chen & Chih-Shan Li

To cite this article: Pei-Shih Chen & Chih-Shan Li (2005) Quantification of Airborne
Mycobacterium tuberculosis in Health Care Setting Using Real-Time qPCR Coupled to
an Air-Sampling Filter Method, Aerosol Science and Technology, 39:4, 371-376, DOI:
10.1080/027868290945767

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Published online: 23 Feb 2007.

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Aerosol Science and Technology, 39:371–376, 2005
Copyright
c American Association for Aerosol Research
ISSN: 0278-6826 print / 1521-7388 online
DOI: 10.1080/027868290945767
Quantification of Airborne Mycobacterium tuberculosis
in Health Care Setting Using Real-Time qPCR Coupled
to an Air-Sampling Filter Method
Pei-Shih Chen and Chih-Shan Li
Graduate Institute of Environmental Health, College of Public Health, National Taiwan University,
Taipei, Taiwan, R.O.C.
Mycobacterium tuberculosis infection remains one of the major
public health issues worldwide. Current qualitative assays (only
positive or negative results) do not provide comprehensive informa-
tion regarding health risk of M. tuberculosis. This study attempted
to develop a quantitative assay to measure air concentration of
M. tuberculosis in a health care setting.
A total of 22 air samples were taken from the negative pres-
sure isolation rooms of tuberculosis patients. The air was filtered
through a Nuclepore filter with sampling time of 8 h. The DNA of
M. tuberculosis in these airborne samples was then analyzed by the
ABI 7700 real-time quantitative polymerase chain reaction (real-
time qPCR) system.
The real-time qPCR method could perform measurements of
counts in a dynamic range of over 6 orders with a high sensitivity.
The measured M. tuberculosis concentrations varied widely, from
1.43 × 10 copies/m3 to 2.06 × 105 copies/m3 . Comparisons among
airborne M. tuberculosis levels, sputum smear, results, and sputum
culture results showed moderate correlations.
The filter/real-time qPCR method proved extremely sensitive
and rapid for quantifying airborne M. tuberculosis. In addition,
it is a powerful sampling tool that has potential applications as
an investigational device, which might be valuable in conducting
studies that validate the efficacy of engineering controls and work
practices.
INTRODUCTION
Tuberculosis (TB) remains a major public health problem
worldwide due to its high risk of person-to-person transmission,
morbidity, and mortality (Falkinham 1996; Martin and Lazarus
2000). Currently, Mycobacterium tuberculosis infects one-third
Received 9 September 2004; accepted 24 February 2005.
The authors thank Dr. Shin-Yu Su for his assistance during this in-
vestigation. This work was supported by grant NSC91-2621-Z-002-025
from the National Science Council, Republic of China. Pei-Shih Chen
was supported by a graduate scholarship from the same grant during
part of this research effort.
Address correspondence to Chih-Shan Li, Graduate Institute of En-
vironmental Health, College of Public Health, National Taiwan Univer-
sity, Room 1449, No. 1, Jen Ai Road, 1st Section 100, Taipei, Taiwan,
R.O.C. E-mail: csli@ccms.ntu.edu.tw
of the world’s population (Dye et al. 1999), and it is the second-
most infectious cause of death worldwide (WHO 2001). More-
over, progressive increases in M. tuberculosis infections are ex-
pected, and a worldwide annual incidence of 12 million cases
by 2005 is predicted by the World Health Organization (Martin
and Lazarus 2000). A sensitive method for monitoring airborne
M. tuberculosis is therefore crucial for TB control.
In airborne M. tuberculosis analysis, identification of M. tu-
berculosis from field sampling by using a culture method is not
practical due to slow growth of M. tuberculosis and to contami-
nation by other bacteria and fungi (Loudon et al. 1976; Macher
et al. 1992; Riley et al. 1976). Therefore, filter sampling coupled
with a commercial DNA detection method based on polymerase
chain reaction (PCR) (Amplicor M. tuberculosis PCR test) was
proposed to monitor M. tuberculosis in air (Schafer et al. 1998;
Schafer and Fernback 1999). This filter-PCR method was also
recommended by the National Institute of Occupational Safety
and Health for airborne M. tuberculosis measurement (NIOSH
1998). However, this method only qualitatively determines pos-
itive or negative responses in a narrow dynamic range (less than
four orders of magnitude) (Schafer et al. 1998; Schafer and
Fernback 1999). In clinical studies, the most commonly used
methods to analyze M. tuberculosis involve using either a smear
method for acid-fast bacilli (AFB) staining samples or a culture
method. However, these two methods have disadvantages: the
AFB staining method has low sensitivity, and the culture method
is time consuming.
Rapid identification methods using molecular techniques have
been developed recently and utilized in clinical laboratories
(Broccolo et al. 2003; Cleary et al. 2003; Desjardin et al. 1998;
Kraus et al. 2001; Miller et al. 2002; Shretha et al. 2003). One
such method is the real-time quantitative PCR (real-time qPCR)
system, which is a commercially available system designed to
decrease the time required for PCR assays by using fluorescent
probes to monitor the amplification of the target sequences in
real time. One benefit of this real-time qPCR system for clini-
cal assays is that carryover contamination does not occur during
postamplification because detection of amplified nucleic acid
products is accomplished in a closed system (Broccolo et al.
371
372 P.-S. CHEN AND C.-S. LI

2003; Cleary et al. 2003; Desjardin et al. 1998; Kraus et al.
2001; Miller et al. 2002; Shretha et al. 2003). Consequently, this
molecular method for identification of cultured isolates has been
suggested and assessed (Roger van Doorn et al. 2003). However,
the use of this technology for airborne M. tuberculosis evaluation
needs to be validated.
The aim of this study is to validate this rapid and high-
throughput filter/real-time qPCR assay for directly measuring
airborne M. tuberculosis in respiratory isolation of TB patients.
Moreover, the measured airborne M. tuberculosis levels are also
compared with the TB patient status (based on sputum smear
and culture results).
MATERIALS AND METHOD
Sampling Location
The respiratory isolation (negative-pressure) rooms of TB
patients were investigated. The patients selected for this study
were identified by clinical diagnosis of M. tuberculosis pneumo-
nia based on history, symptoms, physical examination, and chest
radiographs. These patients were inpatients at the Hospital for
Chronic Disease in Taipei and had sputum samples submitted
for AFB staining and culture.
Air Sampling
Air samples were taken from the isolation rooms of the se-
lected TB patients. A total of 22 samples from the rooms of six
patients were measured from 6 November to 18 December 2003.
In this hospital, every patient stayed individually in an isolation
room. The age of the six patients ranged from 26 to 74, and
five were male and one was female (Table 1). For quality con-
trol, three samples of field blank were performed in TB patient
room. Our results demonstrated that no detectable M. tuberculo-
sis DNA was observed in field blank control (data not shown). In
addition, side-by-side duplicate field samples gave comparable
results (with relative difference 13%; data not shown).
For the filter/real-time qPCR assay, the air in each isola-
tion room was filtered through a 37 mm diameter Nuclepore
filter (Costar, Cambridge, MA, USA), which is a track-etched
polycarbonate filter consisting of a polycarbonate membrane

TABLE 1
Tuberculosis patients characteristics and airborne M. tuberculosis concentrations
Date Patient/sex/
P a : Temp.b Therapy Real-time qPCR Real-time qPCR
sampled age, (year) pa (◦ C) RH%c ACHd initiation Culturee Smear f (copies/m3 ) (CFU/M3 )
2003/11/6 A/26/M 14.0 23.6 71.3 9.1 2003/11/3 ++ − 5.88 × 102 2.32
2003/11/6 B/67/M 14.0 23.2 71.2 9.1 2003/10/8 +++ ++++ 1.20 × 103 4.75
2003/11/13 13.0 20.2 67.6 9.1 ++ +++ 1.36 × 104 53.8
2003/11/20 15.0 22.5 69.4 9.1 ++ ++++ 1.21 × 104 48
2003/12/4 18.0 20.1 70.2 9.9 ++ +++ 1.24 × 103 4.90
2003/12/11 8.0 20.6 77.8 9.3 ++ 5.21 × 103 20.6
2003/12/18 8.0 18.4 63.0 9.12 ++ ++ 1.72 × 103 6.79
2003/11/6 C/74/M 8.0 23.7 74.9 8.8 2003/10/31 ++ +++ 2.07 × 103 8.16
2003/11/13 8.0 20.3 65.7 9.1 ++ +++ 2.40 × 103 9.48
2003/11/20 8.0 26.0 61.5 8.9 + ++ 2.20 × 103 8.71
2003/12/4 11.0 19.5 71.7 9.0 + − 7.79 × 102 3.08
2003/12/11 13.7 20.8 74.9 8.9 − + 3.19 × 102 1.26
2003/12/18 13.7 19.0 60.2 8.6 + + 5.70 × 102 2.25
2003/11/13 D/74/M 9.8 21.4 61.8 10.1 2003/11/11 ++ + 1.74 × 103 6.86
2003/11/20 15.7 23.7 71.2 10.4 ++ + 1.78 × 105 703
2003/12/4 8.8 23.4 72.5 10.6 ++ ++++ 2.06 × 105 814
2003/12/11 8.8 21.0 74.0 10.0 ++ + 1.24 × 103 4.88
2003/12/18 6.9 18.9 59.8 10.4 ++ + 5.58 × 102 2.21
2003/12/4 E/50/F 7.8 20.3 68.3 9.1 2003/11/19 + + 8.55 × 102 3.38
2003/12/11 F/53M 12.0 20.7 75.3 16.2 2003/12/4 + − 6.52 × 102 2.58
2003/12/18 12.0 18.5 60.8 16.4 − − 1.43 × 101 0.06
a
Pressure difference between isolation room and outside.
b
Temperature.
c
Relative humidity.
d
Air change per hour.
e
−, no colony growth; +, <50 CFU/plate; ++, 50–100 CFU/plate; +++, 100–200 CFU/plate.
f
Smear (×400 magnification). +, 1–3 per slide; ++, 1–9 per 10 field; +++, 1–9 per field; ++++, >9 per field.
 QUANTIFICATION OF AIRBORNE MYCOBACTERIUM TUBERCULOSIS IN HEALTH CARE SETTING
with straight-through pores of uniform size (0.4 µm). The fil-
ters were supported by cellulose pads and loaded into open-face
three-piece plastic cassettes. Before sampling, the filters and
support pads were autoclaved and the plastic cassettes were ster-
ilized with ethylene oxide. The sampling flow rate was 22 l/min,
and the sampling time was 8 h. The pump and filter apparatus
were placed within 1 m from the patient’s bed on an adjacent
nightstand. The sampling height was 1.2–1.5 m above the floor,
so as to be within the human breathzone. After sampling, the
Nuclepore filter was removed from the holder and placed in a
test tube containing 4 ml of sterile deionized water. The water
and filter were then vortexed in the tube for 60 s.
DNA Extraction Method
DNA extraction of the filter elution samples was accom-
plished using a DNA extraction kit (Hexwater Inc., Germany).
First, 200 µl of sample suspension was added to 1 ml of a WB1
lysis buffer, vortexed, and then pelleted by centrifugation at
12,000 rpm (8000 × g) for 1 min. After the supernatant was
removed, the pellet was suspended in 200 µl WB2 lysis buffer,
which included the beads at the bottom of the buffer, and then in-
cubated at 55◦ C for 30 min. The pellet suspension was vortexed
for 5 s, spun down, and then boiled at 100◦ C for 8 min to release
the DNA. The samples were again pelleted by centrifugation at
12,000 rpm (8000 × g) for 3 min. Finally, 100 µl supernatant
of each sample was saved for PCR and stored at −20◦ C. All
manipulations of the samples were performed in a biological
safety cabinet.
ABI 7700 Quantification
Oligonucleotide Primers and Probes
The real-time qPCR assay was done using a forward primer
(5 -GGCTGTGGGTAGCAGACC-3 ) and reverse primer (5 -
CGGGTCCAGATGGCTTGC-3 ), which were directed at a
163 bp region of the IS6110 gene sequence (DesJardin et al.
1998). The internal oligonucleotide probe was labeled with fluo-
rescent dyes, namely, 5-carboxyfluorescein (FAM) on the 5 end
and N,N,N ,N ,-tetramethyl-6-carboxyrhodamine (TAMRA)
on the 3 end (5 -[FAM]-TGTCGACCTGGGCAGGGTTCG-
[TAMRA]-3 ). The primer and probe were synthesized using
an ABI 7700 (Applied Biosystem Inc., Foster City, CA, USA).
PCR Conditions
In the PCR assay, 50 µl of the PCR mixture solution was
placed in each well of a MicroAmp Optical 96-well reaction
plate, and then each well was capped with a MicroAmp Opti-
cal Cap (PE Biosystems, Foster City, CA, USA). All reagents
were from a TaqMan Core PCR Reagent Kit (PE Biosystems,
Foster City, CA, USA). The PCR mixture solution consisted of
5 µl of extracted DNA solution, 25 µl of 2 × TaqMan universal
Master Mix, 300 nM forward primer, 300 nM reverse primer, and
250 nM probe. A sequence detector system (ABI Prism 7700;
Applied Biosystem, Foster City, CA, USA) was used for ampli-
373
fication and fluorescence measurement. All cycles began with
2 min at 50◦ C for UNG enzyme incubation and then 10 min at
95◦ C for AmpliTaq Gold activation. The subsequent PCR assay
consisted of 50 cycles, where each cycle involved denaturation
at 95◦ C for 20 s and then annealing and extension at 60◦ C for
1 min. All samples analyzed using the real-time qPCR were
done in triplicate (CV% < 5%).
Standard Curve and Inhibitory Effect
The target DNA standard solution from cloning plasmid was
purchased from Mission Biotech (Taipei, Taiwan, R.O.C.). The
threshold was set at 10 times the standard deviation of the mean
baseline emission calculated for PCR cycles 3–10. The amount
of product in a particular reaction mixture solution was deter-
mined by interpolation from a standard curve of cycle threshold
(Ct) values generated from a known starting concentration of
DNA at the same run. Positive (purified dilutions of plasmid
DNA) and negative controls were analyzed for each run. The
inhibitory effect of the collected air samples on real-time qPCR
assay was evaluated by diluting method with 1/10 and 1/100
dilutions of two samples, 2.07 × 103 copies/m3 and 1.78 ×
105 copies/m3 . The results confirmed no inhibitory effect of PCR
assay in the collected airborne samples (results not shown).
RESULTS AND DISCUSSION
The aim of this study was to validate the filter/real-time PCR
assay for quantitative evaluation of airborne M. tuberculosis in
hospital rooms of TB patients. Correlations among airborne M.
tuberculosis levels, smear results of sputum samples, and sputum
culture results were also assessed.
Dynamic Range and Analytical Sensitivity of
Real-Time qPCR Assay
The ABI 7700 TaqMan system has been used to quantity M.
tuberculosis DNA in sputum during the treatment of TB patients
by using a probe specific for the IS6110 gene region (Desjardin
et al. 1998). The IS6110 is a multicopy insertion element found
in M. tuberculosis complex organisms and has been the target of
numerous diagnostic assays. The region targeted for amplifica-
tion has been shown to be specific for M. tuberculosis (Hellyer
et al. 1996). Therefore, the IS6110 was also used as a target in
our current study.
A quantitative assay was developed for IS6110 using the
ABI Prism 7700 Sequence Detection System (TaqMan).
Figure 1 shows the calibration curve of known M. tuberculo-
sis DNA concentrations and threshold cycle (Ct) by real-time
qPCR. The known amounts of M. tuberculosis plasmid DNA
yielded Ct values ranging from 20–40 cycles (triplicate stan-
dards, with coefficients of variation were less than 1%). The
standard curves were linear for over 6 orders of magnitude, rang-
ing from 48 copies/µl to 4.8 × 107 copies/µl, with a correlation
coefficient (r) value of 0.992. Based on our results, 1 CFU was
approximately equal to 253 copies in the real-time qPCR assay
374 P.-S. CHEN AND C.-S. LI
FIG. 1. Calibration curve of known M. tuberculosis DNA concentrations and threshold cycle (Ct) by real-time qPCR assay.
by analyzing the fresh cultured M. tuberculosis. The tenfold se-
rial dilutions subjected to the real-time qPCR assay revealed that
the sensitivity was less than 48 copies/µl (240 copies/reaction).
These results confirm that less than 1 CFU of M. tuberculosis (10
genomes of M. tuberculosis) could be detected by the real-time
qPCR assay with high sensitivity, similar to previously reported
sensitivity (Schafer et al. 1998; Schafer and Fernback 1999).
In conclusion, the filter/real-time qPCR assay developed here
could prove useful for rapid identification and quantification of
airborne M. tuberculosis.
Airborne M. tuberculosis in TB Patient Rooms
Physical factors of each respiratory isolation room was mea-
sured, namely, airflow rate, temperature, relative humidity, and
air pressure difference (
P). Table 1 summarizes these measured
factors. All six of the patient rooms met the recommended values
of air change per hour (ACH; ACH > 8–12) and wind velocities,
although two did not meet the recommended pressure difference
(>8 pa).
In our study, the filter/real-time qPCR assay was able to de-
tect and measure the concentration of airborne M. tuberculosis
in the TB patient rooms. To our knowledge, this is the first
report describing the quantification of airborne M. tuberculo-
sis in field samples. It was found that airborne M. tuberculo-
sis concentration varied widely, from 1.43 × 10 copies/m3 to
2.06 × 105 copies/m3 (equal to 1.26 × 101 tubercle bacilli /m3
to 8.14 × 103 CFU/m3 ) (as shown in Table 1). Because infec-
tious dose of M. tuberculosis is as low as 1–5 tubercle bacilli
(Balasubramanian et al. 1994), according to our data airborne M.
tuberculosis levels in the TB patient rooms studied here might
pose a health risk to hospital workers and to families of the
patients.
Qualitative filter-PCR assay previously detected M. tuber-
culosis in the isolation rooms of 6 out of 7 patients studied
(Mastorides et al. 1999). In addition, 60% of the isolation rooms
and the outpatient department area in a TB center contained air-
borne M. tuberculosis (Wan et al. 2004). These findings clearly
demonstrate that routine monitoring of airborne M. tuberculosis
is crucial in TB centers to ensure the safety of the public and
health care personnel. Qualitative assays, however, cannot pro-
vide precise information about the health risk of M. tuberculosis.
The filter/real-time qPCR assay can provide deeper insight
into hospital epidemiology and infection control, as well as M.
tuberculosis transmissibility. This system can be used to de-
termine the background M. tuberculosis levels within isolation
rooms, outpatient department areas, microbiology laboratories,
and tuberculosis clinics, and to assess the potentially infectious
M. tuberculosis dose in room air. Our findings demonstrated
that sputum smear results and sputum culture results were mod-
erately correlated with measured airborne M. tuberculosis lev-
els (Figure 2), and that sputum smear and culture results were
moderately correlated (r = 0.57, data not shown). Moreover, 19
of 21 smear samples and 18 of 21 culture samples were posi-
tive (Table 1). The M. tuberculosis concentrations in the rooms
of patients with negative smear and culture results were lower
than those with positive smear and culture results (Table 1). In
general, the airborne M. tuberculosis level in each room was
correlated with the clinical TB status of patient.
Based on sputum smear and culture results, patient B seemed
more seriously ill than patient D. However, airborne M. tubercu-
losis levels in the room of patient D were the highest among all
 QUANTIFICATION OF AIRBORNE MYCOBACTERIUM TUBERCULOSIS IN HEALTH CARE SETTING
FIG. 2. (a) Correlation between airborne M. tuberculosis levels and sputum
smear results. −, No colony growth; +, <50 CFU/plate; ++, 50–100 CFU/plate;
+++, 100–200 CFU/plate. (b). Correlation between airborne M. tuberculosis
levels and sputum culture results. +, 1–3 per slide; ++, 1–9 per 10 field; +++,
1–9 per field; ++++.
samples. From our observation and surveillance, patient D never
wore a mask and frequently spit sputum (in the first three sam-
pling dates, 13 November, 20 November, and 4 December). After
patient D wore a mask and covered his mouth while coughing
and speaking, airborne M. tuberculosis level dramatically de-
creased (11 December and 18 December). The hygienic prac-
tice of this patient might play an important role in preventing
the spread of M. tuberculosis.
In conclusion, the filter/real-time qPCR assay is highly sen-
sitive (less than 1 CFU of M. tuberculosis) and fast (<5 h) for
375
quantifying airborne M. tuberculosis. In addition, this quanti-
tative method has potential applications as an investigational
device that may be valuable in conducting studies that validate
the efficacy of engineering controls and work practices.
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