Variability of Infectious Aerosols Produced during
Coughing by Patients with Pulmonary Tuberculosis
Kevin P. Fennelly1, Edward C. Jones-Lo ´pez2,3,4, Irene Ayakaka3, Soyeon Kim5, Harriet Menyha6,
Bruce Kirenga7, Christopher Muchwa6, Moses Joloba6,8, Scott Dryden-Peterson9, Nancy Reilly4,
Alphonse Okwera10, Alison M. Elliott7,11, Peter G. Smith12, Roy D. Mugerwa3,7, Kathleen D. Eisenach6,13,
and Jerrold J. Ellner2,3
1Southeastern National Tuberculosis Center and Emerging Pathogens Institute, Department of Medicine, University of Florida, Gainesville, Florida;
2Section of Infectious Diseases, Department of Medicine, Boston Medical Center and Boston University School of Medicine, Boston, Massachusetts;
3Makerere University—University of Medicine and Dentistry of New Jersey Research Collaboration, Kampala, Uganda;4Division of Infectious
Diseases, Department of Medicine, and5Department of Preventive Medicine and Community Health, New Jersey Medical School—University
of Medicine and Dentistry of New Jersey, Newark, New Jersey;6Mycobacteriology Laboratory, Joint Clinical Research Center, Kampala, Uganda;
7Department of Medicine and8Department of Microbiology, Makerere University College of Health Sciences, Kampala, Uganda;9Division of
Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts;10Mulago
Hospital Tuberculosis Clinic, Mulago Hospital, Kampala, Uganda;11Department of Clinical Research and12Department of Infectious Disease
Epidemiology, London School of Hygiene and Tropical Medicine, London, United Kingdom; and13Department of Pathology, University of Arkansas
for Medical Sciences, Little Rock, Arkansas
Rationale: Mycobacterium tuberculosis is transmitted by infectious
aerosols, but assessing infectiousness currently relies on spu-
tum microscopy that does not accurately predict the variability
Objectives: To evaluate the feasibility of collecting cough aerosols
and the risk factors for infectious aerosol production from patients
with pulmonary tuberculosis (TB) in a resource-limited setting.
Methods: We enrolled subjects with suspected TB in Kampala,
Uganda and collected clinical, radiographic, and microbiological
data in addition to cough aerosol cultures. A subset of 38 subjects
was studied on 2 or 3 consecutive days to assess reproducibility.
Measurements and Main Results: M. tuberculosis was cultured from
cough aerosols of 28 of 101 (27.7%; 95% confidence interval [CI],
19.9–37.1%) subjects with culture-confirmed TB, with a median 16
aerosol cfu (range, 1–701) in 10 minutes of coughing. Nearly all
(96.4%) cultivable particles were 0.65 to 4.7 mm in size. Positive
scores(P ¼ 0.016), higher sputum acid-fast bacilli smearmicroscopy
grades (P ¼ 0.007), lower days to positive in liquid culture (P ¼
0.004), stronger cough (P ¼ 0.016), andfewer days onTB treatment
(P ¼ 0.047). In multivariable analyses, cough aerosol cultures were
associated with a salivary/mucosalivary (compared with purulent/
mucopurulent) appearance of sputum (odds ratio, 4.42; 95% CI,
1.23–21.43) and low days to positive (per 1-d decrease; odds ratio,
0.94) and interday test (kappa, 0.62; 95% CI, 0.43–0.82) reproduc-
ibility were high.
Conclusions: A minority of patients with TB (28%) produced cultur-
able cough aerosols. Collection of cough aerosol cultures is feasible
and reproducible in a resource-limited setting.
Keywords: tuberculosis; cough; air microbiology; infectious disease
transmission; infection control
Tuberculosis (TB) continues to be a major cause of global
morbidity and mortality, especially among those infected with
HIV (1). The spread of multidrug-resistant (MDR) and exten-
sively drug-resistant TB has highlighted the importance of pre-
venting transmission of TB, both in the community and within
healthcare facilities (2, 3). Mycobacterium tuberculosis is trans-
mitted by fine aerosols (i.e., via the airborne route in infectious
droplet nuclei , 5 mm in diameter), yet assessment of infectious-
ness has been based on microscopic examination of sputum
for more than a century. Both experimental and epidemiolog-
ical data suggest that sputum examination for acid-fast bacilli
(AFB) is neither a sensitive nor a specific indicator of infec-
tiousness (4–7). Moreover, although the size of aerosol particles
is a critical determinant of aerosol deposition in the lungs and
a factor that impacts infection control measures, the magnitude
and size distribution of aerosols generated by patients with TB
(Received in original form March 10, 2012; accepted in final form June 21, 2012)
Supported by the Wellcome Trust—Burroughs Wellcome Fund Infectious Dis-
eases Initiative grant 063410/ABC/00/Z, National Institute of Health Career De-
velopment Award #1K23 AI01676 (K.P.F.), and the American Society for Tropical
Medicine and Research (K.P.F.).
The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Author Contributions: Conception and design: K.P.F., E.C.J.-L., A.M.E., P.G.S.,
R.D.M., K.D.E., J.J.E.; acquisition of data: K.P.F., E.C.J.-L., I.A., H.M., B.K., C.M.,
M.J., S.D.-P., N.R., A.O., A.M.E., R.D.M.; analysis and interpretation: K.P.F.,
E.C.J.-L., S.K., P.G.S., J.J.E. All authors contributed to either drafting or revising
this manuscript and gave final approval.
Correspondence and requests for reprints should be addressed to Kevin P. Fennelly,
M.D., M.P.H., Associate Professor of Medicine, Division of Mycobacteriology,
Department of Medicine, Southeastern National Tuberculosis Center, Emerging
Pathogens Institute, Room 257, University of Florida, Gainesville, FL 32610.
Am J Respir Crit Care Med
Copyright ª 2012 by the American Thoracic Society
Originally Published in Press as DOI: 10.1164/rccm.201203-0444OC on July 12, 2012
Internet address: www.atsjournals.org
Vol 186, Iss. 5, pp 450–457, Sep 1, 2012
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject
There is no definitive method to determine which patients
with tuberculosis (TB) are most infectious, and there is con-
siderable variability of infectiousness among patients based
on both epidemiological and experimental studies.
What This Study Adds to the Field
We report on the measurement of infectious aerosols from
This study demonstrates the feasibility of collecting cough
aerosols from patients as a way of detecting infectiousness.
As part of a study of nosocomial transmission of TB in
Uganda, we modified the previously described Cough Aerosol
Sampling System (CASS) (8) to collect, quantify, and size aero-
sol particles containing culturable M. tuberculosis produced by
voluntary coughing in patients with active pulmonary TB. We
also evaluated factors associated with cough aerosol production.
Some of the results from this study have been previously re-
ported in the form of abstracts (9, 10).
Study Population and Measurements
From November 5, 2002 to December 14, 2004, we recruited subjects
with suspected TB attending the National Tuberculosis and Leprosy
Program Chemotherapy Centre at Mulago Hospital in Kampala,
Uganda. Patients were recruited if they had a recent diagnosis (past
7 d) of sputum AFB-positive pulmonary TB from any laboratory. Sub-
jects were included in the final analysis if their sputum was confirmed
to be culture-positive for M. tuberculosis. Patients with hemoptysis,
pneumothorax, or other serious comorbid conditions, and patients
who were unable to walk to the procedure room were ineligible. We
used a standardized questionnaire to collect demographic and clinical
information. The extent of disease on a chest radiograph at baseline
was graded on an ordinal scale by an experienced clinician. Subjects
were offered HIV testing and a CD41lymphocyte cell count was mea-
sured in HIV-infected patients; patients with a CD41cell count less
than 200 cells/ml were referred for antiretroviral treatment according to
existing national guidelines (11). All patients were offered TB treatment
according to Ugandan National Tuberculosis and Leprosy Program
treatment guidelines. Patients found to have MDR-TB were treated
when medications became available, as explained elsewhere (12).
Sputa specimens were processed with the standard digestion and de-
contamination method using N-acetylcysteine-sodium hydroxide (13). Cen-
trifugates were used to prepare smears and cultures on 7H10 agar and in
the BACTEC 460 liquid culture system (Becton Dickinson, Franklin
Lakes, NJ) according to the manufacturer’s recommendations (14). Spu-
tum smear microscopy was performed using auramine O fluorescent stain
and reported according to the CDC microscopy grading scheme (15).
Confirmation of M. tuberculosis complex was determined by the BACTEC
NAP test (Becton Dickinson). Drug susceptibility testing for first-line TB
drugs was done on isolates from sputum using BACTEC 460.
We used the CASS method previously described (8) with minor mod-
ifications. Briefly, the CASS consists of a custom-built stainless steel
cylindrical chamber with noncompressible tubing connecting the inlet
to a disposable mouthpiece (Figure 1). The chamber holds two Ander-
sen six-stage cascade impactors for viable bioaerosol sampling
(Thermo Scientific, Inc., Rockford, IL), each with six plastic Petri
plates (Fisher Scientific, Inc., Hanover Park, IL) holding selective
7H11 agar that were loaded in a class II biological safety cabinet. A
vacuum pump (GAST, Inc., Benton Harbor, MI) connects the air sam-
plers by tubing (Tygon, Cole Parmer, Inc., Vernon Hills, IL) to fittings
that pass through the wall of the chamber. In-line 47-mm filter holders
(Cole Parmer, Inc.) loaded with high-efficiency particle air filters (EPM
2000; Whatman, Inc., Piscataway Township, NJ) are placed between
the chamber and the vacuum pump for biosafety.
A single-stage impactor (SKC, Inc., Eighty-Four, PA) loaded with the
same7H11 agar was used to sample ambient room air. One settle plateof
sample large aerosol particles. A timer (GraLab, Inc., Centerville, OH)
connected to the vacuum pump was set for 5 minutes. The vacuum pump
was calibrated using a primary flow meter (DryCal DC Lite; BIOS, Inc.,
Butler, NJ), and calibrations were rechecked every 6 months.
CASS Study Protocol
All studies were performed before the morning meal by one of two
trained technicians. During each study, the windows in the study room
were open and a fan used to direct airflow from behind the technician,
past the subject, and out through the windows. All study personnel wore
fit-tested N95 respirators. Before the subject entered the study room,
we recorded ambient temperature and relative humidity, and the room
air was sampled for 5 minutes to determine if airborne M. tuberculosis
was present. Subjects were instructed to cough into the CASS mouth-
piece as much and as frequently as was comfortable for two 5-minute
sessions separated by a rest of approximately 5 minutes. No sputum
induction was used. The technician subjectively assessed the cough
strength as weak, moderate, or strong. Flow rates through the samplers
were recorded. Sputum specimens were collected if produced. The
CASS chamber was autoclaved and other components were disinfected
after each study.
After the study, the aerosol samplers were removed and trans-
ported to the laboratory, where they were unloaded in the biological
safety cabinet. Plates were incubated at 378C. They were read at 1 week
to detect any rapidly growing contaminants and then at 3, 6, and
9 weeks to record cfu of M. tuberculosis; as there were rarely new
cfu at 9 weeks, we used the 6-week count as the outcome measure.
Confirmation of M. tuberculosis complex was determined by BACTEC
NAP. The appearance of sputa specimens expectorated during studies
was classified as purulent, mucopurulent, mucosalivary, salivary, or
bloody by the microbiology technicians according to laboratory gui-
delines (16). These data were dichotomized into two groups for anal-
ysis: purulent/mucopurulent or salivary/mucosalivary, with two bloody
specimens excluded. To assess reproducibility of the CASS method,
the last 40 subjects were asked to return (without interrupting TB
treatment) for two additional studies on consecutive days, with a goal
of three cough aerosol studies per subject.
Participating patients provided written informed consent in their native
of the Uganda National Council of Science and Technology, the Insti-
tutional Review Boards at the University of Medicine and Dentistry of
New Jersey, and the London School of Hygiene and Tropical Medicine.
Differences in the production of positive cough aerosol cultures were
assessed in unadjusted analyses using Fisher exact (categorical data),
Wilcoxon rank sum (continuous measures), and Cochran-Armitage
trend (ordinal measures) tests. We used Spearman rank correlation
to evaluate combinations of continuous or ordinal measures. Variables
that were positively associated with cough aerosol cultures in univariate
analyses at P less than or equal to 0.2 were included in a stepwise
logistic regression model to identify independent predictors of cough
aerosol production. Reproducibility between cough aerosol cultures
collected during the first and second sessions was assessed using intra-
class correlation coefficient (ICC) on log-transformed cfu11 values
and when dichotomized (any versus no cfu) using McNemar test. Con-
cordance of aerosol production between studies on the same patient
was assessed using Cohen kappa when there were two measurements
per patient and Fleiss kappa when more than two measurements, and
the ICC on log-transformed cfu11 values. Statistical analyses were
conducted using SAS 9.1 (Cary, NC). All tests were two-tailed and
conducted at the 5% significance level.
Characteristics of Patients with TB
We evaluated 112 patients with suspected TB; 101 (90%) had
confirmed culture-positive sputum and were further analyzed.
Most subjects were men (70%), had advanced radiographic dis-
ease (63%), and had high bacillary load as assessed by sputum
smear (74% with > 31 AFB) (Table 1). Nearly all (99%) sub-
jects presented with chronic cough. MDR-TB was isolated from
eight (8%) participants. Of the 84 (83%) subjects with HIV
results, 49 (58%) were HIV infected and had a median CD4
cell count of 112 cells/ml (interquartile range, 33–274).
Fennelly, Jones-Lo ´pez, Ayakaka, et al: Cough Aerosols of M. tuberculosis451
Cough Aerosol Cultures
Of the 101 subjects with positive sputum cultures, 28 (27.7%;
95% confidence interval [CI], 19.9–37.1%) produced culture-
positive cough aerosols from the first CASS study (first 5-min
cough period). Among the positive aerosols, the median was 16
CFU (interquartile range, 5–30) with a range of 1 to 710 cfu
(Table 2); 16 (57%) of these subjects produced 10 or more cfu in
aerosols. The proportion of patients who generated culture-
positive aerosols increased as sputum smear microscopy grade
increased (Spearman correlation, 0.40; P ¼ 0.033; Figure 2) and
as sputum BACTEC days to positive (DTP) decreased (Spear-
man correlation, 20.31; P ¼ 0.001). Although all CASS-positive
patients were sputum AFB smear positive, the majority of spu-
tum AFB smear–positive subjects (62 of 90, 69%) did not pro-
duce culturable cough aerosols. Conversely, none of the 11
sputum AFB-negative/culture-positive subjects produced cough
Tuberculous Aerosol Particle Size Distribution
The mode of the particle size distribution of culturable aerosols
was on stage 5 (1.1–2.0 mm), and nearly all (96.4%) particles
collected measured between 0.65 and 4.7 mm in aerodynamic
diameter (i.e., deposited in stages 3 to 6 of the Andersen cascade
impactors) (Figure 3). Of the 74 settle plates inside the chamber,
only 8 (11%) had positive growth. No M. tuberculosis was cultured
from ambient air, but 45% of these plates were contaminated with
Factors Associated with Cough Aerosol Cultures
In unadjusted analyses (Table 1), the production of culturable
aerosols during the first 5-minute cough period was associated
with a higher Karnofsky performance score (P ¼ 0.016), higher
sputum AFB smear microscopy grade (P ¼ 0.007), lower
BACTEC DTP (P ¼ 0.004), strong cough (P ¼ 0.016), and
fewer days on TB treatment before enrollment (P ¼ 0.047).
Other variables marginally associated with aerosol production
were a higher CD4 cell count (P ¼ 0.11), a salivary or mucosa-
livary appearance of sputum (P ¼ 0.077), and a higher ambient
relative humidity at the time of testing (P ¼ 0.068). Of 14 sub-
jects with resistance to isoniazid and/or rifampicin, 7 (50%) had
culturable aerosols (P ¼ 0.090). Factors not associated with
cough-generated aerosols were HIV status, number of days of
cough before enrollment, extent of disease on chest radiograph,
or cavitary disease.
In multivariable analyses (Table 3), only a salivary or muco-
salivary appearance of the sputum (odds ratio, 4.42; 95% CI,
1.23–21.4) and lower sputum BACTEC DTP (per 1-d decrease;
odds ratio, 1.17; 95% CI, 1.05–1.33) were independently associ-
ated with cough aerosols of M. tuberculosis. The exclusion of
HIV-infected subjects did not significantly change these results
There was excellent agreement in the log-cfu11 between the
two 5-minute sessions of coughing in the same session (ICC,
0.83; 95% CI, 0.56–0.88). However, participants were more
Figure 1. Cough Aerosol Sampling System. View inside of chamber with two Andersen cascade impactors and settle plate (left) and set up in
procedure room ready for use (right).
452AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 186 2012
TABLE 1. CHARACTERISTICS OF PARTICIPANTS AT ENROLLMENT ACCORDING TO COUGH AEROSOL SAMPLING
Characteristic*All†(N ¼ 101)
Aerosol Negative‡(N ¼ 73)
Aerosol Positive‡(N ¼ 28)
Body mass index, kg/m2
CD4 count (HIV1 only), cells/ml
TB treatment category
Any smoking history
Days of cough before enrollment
Chest X-ray findings
Extent of disease
Acid-fast bacilli smear
Middlebrook 7H10 agar culture, cfu
Days to positive BACTEC 460 culture
Drug susceptibility testing
Sensitive to I and R
Resistant to I or R
Resistant to I and R (MDR)
Relative humidity, %
Cough assessment (subjective)
Days on TB treatment before enrollment
Non-MDR only (n ¼ 75)
MDR only (n ¼ 6)
7.5 (4–12) 6.5 (4–12) 9 (5–12.5)0.32W
Definition of abbreviations: CASS ¼ Cough Aerosol Sampling System; cfu ¼ colony forming units of Mycobacterium tuberculosis; I ¼ isoniazid;
IQR ¼ interquartile range; MDR ¼ multidrug resistant; R ¼ rifampicin; TB ¼ tuberculosis.
*Missing data as follows: age (n ¼ 4), BMI (19), Karnofsky score (14), HIV status (17), CD4 (2), TB status (4), smoking history (19), days of cough
before enrollment (19), days on TB treatment before enrollment (20), albumin (24), chest X-ray extent of disease (20), cavitations (5), drug
susceptibility testing (3), cough assessment (1). Middlebrook culture results were missing (1) or contaminated (2).
yValues are n (column %) or median (IQR).
zValues are n (row %) or median (IQR).
xCategorical variables compared using exact (E) or Cochran Armitage trend (T) tests and continuous variables using a Wilcoxon (W) test.
Fennelly, Jones-Lo ´pez, Ayakaka, et al: Cough Aerosols of M. tuberculosis 453
likely to produce culturable aerosol in the first versus the second
5-minute period of coughing (McNemar P ¼ 0.008). None of the
subjects without cfu in the first period produced aerosols in the
Of the 40 subjects recruited for the assessment of day-to-day
reproducibility, 38 were sputum culture positive; of these, 34
(89%) completed all three studies, and 4 (11%) completed
two studies. Of the 38 subjects, 14 (36%) generated cultivable
aerosols in at least one of the three sampling periods: 8 (57%)
on the second, and another 2 (14%) in the third study. Of those
participating in all three studies, 26 of 34 (76%) were concordant
were no significant differences in the aerosol cfu between the
three cough aerosol studies (P ¼ 0.67), and the ICC was 0.62
(95% CI, 0.46–0.76). The pattern of discordance appeared to be
random, and discordance mostly involved subjects with less than
10 aerosol cfu, as observed with subjects with same-day discor-
Of the 1,344 solid culture plates used for cough aerosol cultiva-
tion in the 112 patients screened, 161 (12%) were contaminated
associated with mold isolation from ambient air (P , 0.001).
Mold collection was positively associated with relative humidity
(P ¼ 0.004) and negatively associated with temperature (P ,
One subject vomited during the procedure; at the time, study
personnel were not aware the subject had eaten breakfast before
the cough study. Otherwise, the procedure was well tolerated.
Figure 2. Aerosol cfu by sputum acid-fast bacilli (AFB)
TABLE 2. COUGH-GENERATED AEROSOL PRODUCTION ACCORDING TO SPUTUM ACID-FAST BACILLI AND CULTURE RESULTS
CharacteristicLevel Statistic All Sputum AFB Negative Sputum AFB 11 Sputum AFB 21 Sputum AFB 31 Sputum AFB 41 P Value*
7H10 agar culture, cfu
n (row %)
n (col %)
5 (5)11 (11) 24 (24) 50 (49)
Sputum BACTEC 460
n (col %)
Aerosol cfu, (all) No
Aerosol cfu (CASS
Definition of abbreviations: AFB ¼ acid-fast bacilli; CASS ¼ Cough Aerosol Sampling System; cfu ¼ colony forming units of Mycobacterium tuberculosis; DTP ¼ days to
Missing results were excluded.
*Cochran Armitage trend test (T), testing whether Spearman Correlation is zero (S), or Wilcoxon rank sum test (W).
454 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 186 2012
ticle size distributions of aerosols with culturable M. tuberculosis
from patients with pulmonary TB during voluntary coughing.
Most of the subjects in the initial report were studied during
sputum induction procedures and had MDR-TB (8). In this
larger study, we confirmed the original observation that fewer
than one-third of patients with sputum culture–positive TB gen-
erate viable tuberculous aerosols during voluntary coughing, and
we demonstrated that cough aerosol sampling is feasible in a re-
source-limited, high-burden setting.
Cough aerosol cultures were best predicted by the sputum
bacterial load measured by DTP in liquid culture and by the ap-
pearance of the sputum. However, these factors explained only
15% of the variability in culturable cough aerosol production.
Thus, it is clear that cough aerosol cultures are not simply a re-
flection of the sputum bacillary load, even though DTP was the
strongest predictor of cough aerosol cultures. DTP may not only
reflect bacillary concentration but likely also measures the met-
abolic capacity or “vitality” of the bacilli. Sputum with a salivary
appearance was more highly associated with cough aerosols
than purulent sputum. Sputum appearance is most likely a sur-
rogate for viscosity and other rheological, or flow, properties of
respiratory secretions. These properties have long been sus-
pected as determinants of infectiousness (17), and experimental
data using mucus stimulants suggest that aerosolization is in-
versely associated with “cohesivity” (18).
Most of the culturable cough aerosols were less than 5 mm, an
aerodynamic size range that can be inhaled and deposited in the
lower respiratory tract or can remain suspended in room air
indefinitely. These data, obtained by direct measurement, are
consistent with the early theoretical estimates by Wells that
airborne M. tuberculosis is transmitted in infectious droplet nu-
clei of this size range (19). The size distribution of these tuber-
culous aerosols is similar to that observed in cough aerosols of
gram-negative bacteria from patients with cystic fibrosis (20).
The positive association of cough aerosol cultures with cough
strength, Karnofsky performance status, and CD4 counts among
HIV-infected patients in univariate analyses suggests that hea-
lthier ambulatory patients may be more infectious than very ill
bedridden patients. Other studies will be needed to confirm this,
especially as these associationswerenot significant inthe adjusted
model. Similarly, the association of cough aerosol cultures with
fewer days of TB treatment and with INH resistance in univariate
analyses may have implications for prevention of TB transmission
in both healthcare facilities and the community. Isoniazid has the
best early bactericidal activity among TB drugs, and further re-
search will be needed to determine if early bactericidal activity
is associated with decreased infectiousness.
Our findings are consistent with the concept that sputum
smear status should only be considered a risk factor (not the
Figure 3. Mean percentage of aerosol cfu on each Andersen
stage in subjects producing at least one aerosol cfu.
TABLE 3. RESULTS OF MULTIPLE LOGISTIC REGRESSION TO PREDICT AEROSOL PRODUCTION
Characteristic Level*Odds Ratio (95% CI)P Value†
All subjects (N ¼ 101)
Sputum appearance Purulent/mucopurulent
Per 1-d decrease
0.0014 BACTEC 460 culture
HIV-uninfected only (N ¼ 35)
Per 1-d decrease
0.0012 BACTEC 460 culture
Definition of abbreviation: CI ¼ confidence interval.
*Participants with missing data for dependent or independent variables and bloody sputum type (2 subjects) were excluded
yLikelihood ratio tests.
Fennelly, Jones-Lo ´pez, Ayakaka, et al: Cough Aerosols of M. tuberculosis 455
sine qua non) for infectiousness, as suggested by others (21).
There is experimental (22–24), classic epidemiologic (25, 26),
and molecular epidemiologic (27–30) evidence of considerable
variability of infectiousness among sputum smear–positive
patients (6). In addition, cough aerosols may provide a better
estimate of inhaled dose than the sputum AFB smear and, thus,
may help provide insights into TB pathogenesis. In animal mod-
els, the inhaled dose of tuberculous aerosols predicts infectivity,
severity of disease, and mortality (31).
Although it seems logical that individuals who have cough aero-
sols of M. tuberculosis in a transmissible size are more likely to
be infectious than those who do not, this study was not designed
to directly measure transmission. In addition, as patients with
smear-negative specimens were excluded in our screening, our
study cannot estimate the frequency of culturable cough aero-
sols among these patients.
Although it may have been scientifically preferable to study
all patients with TB off therapy, ethically we could not delay
treatment of these patients with a high rate of TB-HIV coinfec-
tion and on open wards, so most patients were studied after ini-
tiating treatment for TB. Thus, it is possible that our data may
have underestimated the infectiousness of patients with un-
treated TB due to an early effect of treatment on infectiousness.
However, these dataare probably a reasonableestimateof infec-
tiousness of patients with TB who are newly diagnosed and just
started on treatment. We chose an exclusion criterion of 7 days
of treatment based on the initial data from patients in the United
States, most of whom had MDR-TB (8), but there may be
differences in the rate at which infectious aerosols decrease
after treatment with first-line versus second-line antituberculous
drugs. The rate at which patients become noninfectious is un-
known, although in our first study in the United States, the
cough aerosol cultures of four patients with MDR-TB treated
with effective drugs decreased exponentially over a 3-week pe-
riod (8). Although a review in 1976 suggested that most patients
probably become noninfectious within 2 weeks (32), subsequent
authors argued against that conclusion (33, 34). The earlier re-
view cited data from household contact studies that did not find
additional infections among household contacts after the index
TB case was placed on effective treatment. However, such anal-
yses are limited by considerable selection bias, as the contacts
who were susceptible or exposed had been infected before the
case was treated, removing them from the pool of subjects under
subsequent study. In addition, there is experimental evidence that
tubercle bacilli remain viable and potentially infectious during
early treatment, as guinea pigs were infected by injection with
bacilli from the washed sputum from patients treated for 3 to 7
weeks (35, 36). The uncertainty about when patients on treat-
ment become noninfectious is reflected in current guidelines that
recommend a conservative approach to removal from respiratory
isolation (37). We anticipate that future developments of cough
aerosol measurement could provide data to help reduce this un-
certainty about when patients become noninfectious.
Another potential limitation of our study is that there may be
bacilli in cough-generated aerosols that are viable but not cultur-
able, such as the recently identified lipid-laden bacilli that may
be associated with nonreplicating persistence (38). Mold con-
tamination of culture plates was more common in this tropical
setting than it was in the high desert of Denver, Colorado (8).
In Kampala, 12% of the cough aerosol plates were contaminated
with mold compared with only 0.06% in Denver. Although mold
contamination did not appear to impair our ability to identify
M. tuberculosis aerosol production, it might have decreased our
total cfu counts in some subjects. As in many other tests of pul-
monary function (39, 40), cough-generated aerosol production is
effort dependent and probably varies with motivation, strength,
sense of well-being, and other factors. However, collection of
sputum specimens is also limited by similar issues (41, 42).
The nearly 3-log range of cough aerosol cultures suggests that
a minority of patients are more highly infectious than others,
consistent with both older (24) and more recent (22) findings
of disseminators of TB. In the near future, it may be possible to
identify the minority of patients with TB who are most likely
infectious using cough aerosol collections with point-of-care
devices. Identification of the most highly infectious patients
could allow for more cost-effective use of resources, both for
infection control in hospitals (e.g., isolation rooms) and for
public health control of TB (e.g., active case finding with tar-
geted treatment of contacts exposed to the most highly infec-
tious cases). Such targeted treatment around “superspreaders”
of disease is theoretically more efficient in controlling epidemics
(43) and might improve TB control. In addition, improved iden-
tification of infectious cases may decrease exposure misclassifi-
cation and improve the precision of future drug and vaccines
studies that depend on accurate ascertainment of exposed
household contacts. As a major goal of drug therapy is to render
patients noninfectious to halt transmission, an improved and
validated method of measuring infectiousness could also offer
a novel outcome measure used in the evaluation of new treatment
regimens. Knowing when patients become noninfectious could also
allay concerns about hospital discharges and community-based
treatment, especially for patients with MDR-TB or extensively
Author disclosures are available with the text of this article at www.atsjournals.org.
Acknowledgment: The authors thank Dr. William Worodria (medical officer), Ms.
Helen Nabanjja and Ms. Grace Nyakoojo (home health visitors), Mr. Haruna
Butunzi (driver), the Mulago TB ward nurses, and project administrator Ms.
Annette Mugenyi for their invaluable contributions. They also thank the following
persons for their expert advice and support for this project: Robert Wallis, M.D.
and Ruth McNerney, Ph.D. (coinvestigators), Beth Temple, M.Sc. and Susan
Nakubulwa (data managers), Leigh Anne Shafer, Ph.D. and Jonathan Levin,
Ph.D. (biostatisticians), Susan Kayes, B.S. and Karen Morgan, B.S. (laboratory
supervisors at the Joint Clinical Research Center), David Hom, M.S. (data analysis),
and Dr. Francis Adatu-Engwau (Head of Uganda National Tuberculosis Leprosy
Programme). The authors also thank the patients and staff of the Mulago Hospital
TB Wards for their participation, without which this study could not have
1. Corbett EL, Watt CJ, Walker N, Maher D, Williams BG, Raviglione
MC, Dye C. The growing burden of tuberculosis: global trends and
interactions with the HIV epidemic. Arch Intern Med 2003;163:1009–
2. Basu S, Andrews JR, Poolman EM, Gandhi NR, Shah NS, Moll A,
Moodley P, Galvani AP, Friedland GH. Prevention of nosocomial
transmission of extensively drug-resistant tuberculosis in rural South
African district hospitals: an epidemiological modelling study. Lancet
3. Gandhi NR,Moll A, Sturm AW, Pawinski R,Govender T,Lalloo U, Zeller
K, Andrews J, Friedland G. Extensively drug-resistant tuberculosis as
a cause of death in patients co-infected with tuberculosis and HIV in
a rural area of South Africa. Lancet 2006;368:1575–1580.
4. Behr MA, Warren SA, Salamon H, Hopewell PC, Ponce de Leon A,
Daley CL, Small PM. Transmission of Mycobacterium tuberculosis from
patients smear-negative for acid-fast bacilli. Lancet 1999;353:444–449.
5. Elwood RK, Cook VJ, Hernandez-Garduno E. Risk of tuberculosis in
children from smear-negative source cases. Int J Tuberc Lung Dis
456 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 186 2012
6. Fennelly KP. Variability of airborne transmission of mycobacterium Download full-text
tuberculosis: implications for control of tuberculosis in the HIV era.
Clin Infect Dis 2007;44:1358–1360.
7. Hernandez-Garduno E, Cook V, Kunimoto D, Elwood RK, Black WA,
FitzGerald JM. Transmission of tuberculosis from smear negative
patients: a molecular epidemiology study. Thorax 2004;59:286–290.
8. Fennelly KP, Martyny JW, Fulton KE, Orme IM, Cave DM, Heifets LB.
Cough-generated aerosols of Mycobacterium tuberculosis: a new
method to study infectiousness. Am J Respir Crit Care Med 2004;169:
9. Fennelly KP, Jones EC, Menyha H, Peterson SD, Eisenach KD, Okwera
A, Mugerwa RD, Ellner JJ. Variability of cough-generated aerosols
of Mycobacterium tuberculosis in Kampala, Uganda [abstract]. Am
J Resp Crit Care Med 2004;169:A532.
10. Fennelly KP, Jones-Lopez EC, Menyha H, Ayakaka I, Muchwa C,
Joloba M, Okwera A, Mugerwa RD, Eisenach K, Ellner JJ. Repro-
ducibility of sampling cough-generated aerosols of Mycobacterium
tuberculosis [abstract]. Proc Am Thorac Soc 2005;2:A552.
11. van Oosterhout JJ, Laufer MK, Graham SM, Thumba F, Perez MA,
Chimbiya N, Wilson L, Chagomerana M, Molyneux ME, Zijlstra EE,
et al. A community-based study of the incidence of trimethoprim-
sulfamethoxazole-preventable infections in Malawian adults living
with HIV. J Acquir Immune Defic Syndr 2005;39:626–631.
12. Jones-Lopez EC, Ayakaka I, Levin J, Reilly N, Mumbowa F, Dryden-
Peterson S, Nyakoojo G, Fennelly K, Temple B, Nakubulwa S, et al.
Effectiveness of the standard who recommended retreatment regimen
(category ii) for tuberculosis in Kampala, Uganda: a prospective co-
hort study. PLoS Med 2011;8:e1000427.
13. Kent PT, Kubica GP. Public health mycobacteriology—a guide for the
level III laboratory. Atlanta, GA: US Department of Health and
Human Services, Public Health Service, Centers for Disease Control;
14. Bactec 460 system: product and procedure manual. Sparks, MD: Becton,
Dickinson and Company; 1996.
15. ATS/CDC/IDSA. Diagnostic standards and classification of tuberculosis in
adults and children. Am J Respir Crit Care Med 2000;161:1376–1395.
16. Rieder HL, Chonde TM, Myking H, Urbanczik R, Laszlo A, Kim SJ,
Van Deun A, Trebucq A. The public health service national tuber-
culosis reference laboratory and the national laboratory network:
minimum requirements, role and operation in a low-income country.
Paris, France: International Union Against Tuberculosis and Lung
17. Bates JH, Stead WW. Effect of chemotherapy on infectiousness of tu-
berculosis. N Engl J Med 1974;290:459–460.
18. Zayas G, Dimitry J, Zayas A, O’Brien D, King M. A new paradigm in
respiratory hygiene: increasing the cohesivity of airway secretions to
improve cough interaction and reduce aerosol dispersion. BMC Pulm
19. Wells WF. Airborne contagion and hygiene. Cambridge, MA: Harvard
University Press; 1955.
20. Wainwright CE, France MW, O’Rourke P, Anuj S, Kidd TJ, Nissen MD,
Sloots TP, Coulter C, Ristovski Z, Hargreaves M, et al. Cough-
generated aerosols of pseudomonas aeruginosa and other gram-
negative bacteria from patients with cystic fibrosis. Thorax 2009;64:
21. Snider DE Jr, Kelly GD, Cauthen GM, Thompson NJ, Kilburn JO. In-
fection and disease among contacts of tuberculosis cases with drug-
resistant and drug-susceptible bacilli. Am Rev Respir Dis 1985;132:
22. Escombe AR, Moore DA, Gilman RH, Pan W, Navincopa M, Ticona
E, Martinez C, Caviedes L, Sheen P, Gonzalez A, et al. The infec-
tiousness of tuberculosis patients coinfected with HIV. PLoS Med
23. Riley RL, Mills CC, O’Grady F, Sultan LU, Wittestadt F, Shivipuri DN.
Infectiousness of air from a tuberculosis ward-ultraviolet irradiation
of infected air: comparative infectiousness of different patients. Am
Rev Respir Dis 1962;85:511–525.
24. Sultan L, Nyka W, Mills C, O’Grady F, Wells W, Riley RL. Tuberculosis
disseminators: a study of the variability of aerial infectivity of tuber-
culous patients. Am Rev Respir Dis 1960;82:358–369.
25. Brooks SM, Lassiter NL, Young EC. A pilot study concerning the in-
fection risk of sputum positive tuberculosis patients on chemotherapy.
Am Rev Respir Dis 1973;108:799–804.
26. van Geuns HA, Meijer J, Styblo K. Results of contact examination in
Rotterdam, 1967–1969. Bull Int Union Tuberc 1975;50:107–121.
27. Alland D, Kalkut GE, Moss AR, McAdam RA, Hahn JA, Bosworth W,
Drucker E, Bloom BR. Transmission of tuberculosis in New York
City: an analysis by DNA fingerprinting and conventional epidemio-
logic methods. N Engl J Med 1994;16:1710–1716.
28. Borgdorff MW, Nagelkerke NJ, de Haas PE, van Soolingen D. Trans-
mission of Mycobacterium tuberculosis depending on the age and sex
of source cases. Am J Epidemiol 2001;154:934–943.
29. Hamburg MA, Frieden TR. Tuberculosis transmission in the 1990s. N Engl
J Med 1994;330:1750–1751.
30. Small PM, Hopewell PC, Singh SP, Paz A, Parsonnet J, Ruston DC,
Schecter GF, Daley CL, Schoolnik GK. The epidemiology of tuber-
culosis in San Francisco: a population-based study using conventional
and molecular methods. N Engl J Med 1994;16:1703–1709.
31. Glover RE. Infection of mice with Mycobact. tuberculosis (bovis) by the
respiratory route. Br J Exp Pathol 1944;25:141–149.
32. Rouillion A, Perdrizet S, Parrot R. Transmission of tubercle bacilli: the
effects of chemotherapy. Tubercle 1976;57:275–299.
33. Menzies D. Effect of treatment on contagiousness of patients with active
pulmonary tuberculosis. Infect Control Hosp Epidemiol 1997;18:582–
34. Noble RC. Infectiousness of pulmonary tuberculosis after starting che-
motherapy: review of the available data on an unresolved question.
Am J Infect Control 1981;9:6–10.
35. Cassidy JT. Tubercle bacilli retain pathogenicity after seven weeks
chemotherapy. Med J Aust 1981;1:588–589.
36. Clancy LJ, Kelly P, O’Reilly L, Byrne C, Costello E. The pathogenicity of
Mycobacterium tuberculosis during chemotherapy. Eur Respir J 1990;3:
37. Jensen PA, Lambert LA, Iademarco MF, Ridzon R. Guidelines for
preventing the transmission of Mycobacterium tuberculosis in health-
care settings, 2005. MMWR Recomm Rep 2005;54:1–141.
38. Garton NJ, Christensen H, Minnikin DE, Adegbola RA, Barer MR.
Intracellular lipophilic inclusions of mycobacteria in vitro and in
sputum. Microbiology 2002;148:2951–2958.
39. Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A,
Crapo R, Enright P, van der Grinten CP, Gustafsson P, et al. Stand-
ardisation of spirometry. Eur Respir J 2005;26:319–338.
40. Pellegrino R, Viegi G, Brusasco V, Crapo RO, Burgos F, Casaburi R,
Coates A, van der Grinten CP, Gustafsson P, Hankinson J, et al.
Interpretative strategies for lung function tests. Eur Respir J 2005;26:
41. Sakundarno M, Nurjazuli N, Jati SP, Sariningdyah R, Purwadi S,
Alisjahbana B, van der Werf MJ. Insufficient quality of sputum sub-
mitted for tuberculosis diagnosis and associated factors, in Klaten
district, Indonesia. BMC Pulm Med 2009;9:16.
42. Tenover FC. Developing molecular amplification methods for rapid di-
agnosis of respiratory tract infections caused by bacterial pathogens.
Clin Infect Dis 2011;52:S338–S345.
43. Woolhouse ME, Dye C, Etard JF, Smith T, Charlwood JD, Garnett GP,
Hagan P, Hii JL, Ndhlovu PD, Quinnell RJ, et al. Heterogeneities in
the transmission of infectious agents: implications for the design of
control programs. Proc Natl Acad Sci USA 1997;94:338–342.
Fennelly, Jones-Lo ´pez, Ayakaka, et al: Cough Aerosols of M. tuberculosis 457