The Infectiousness of Tuberculosis Patients
Coinfected with HIV
A. Roderick Escombe1,2,3*, David A. J. Moore1,2,3,4, Robert H. Gilman3,4,5, William Pan5, Marcos Navincopa6,7,
Eduardo Ticona6, Carlos Martı ´nez8, Luz Caviedes4, Patricia Sheen4, Armando Gonzalez7, Catherine J. Noakes9,
Jon S. Friedland1,2, Carlton A. Evans1,2,3,4,5
1 Department of Infectious Diseases and Immunity, Imperial College London, United Kingdom, 2 Wellcome Trust Centre for Clinical Tropical Medicine, Imperial College
London, United Kingdom 3 Asociacio ´n Bene ´fica PRISMA, Lima, Peru ´, 4 Facultad de Ciencias, Universidad Peruana Cayetano Heredia, Lima, Peru ´, 5 Johns Hopkins Bloomberg
School of Public Health, Baltimore, Maryland, United States of America, 6 Servicio de Enfermedades Tropicales y Infecciosas, Hospital Dos de Mayo, Lima, Peru ´, 7 Universidad
Nacional Mayor San Marcos, Lima, Peru ´, 8 Servicio de Neumologı ´a, Hospital Nacional Dos de Mayo, Lima, Peru ´, 9 School of Civil Engineering, University of Leeds, Leeds,
Funding: This research was funded
by the Sir Halley Stewart Trust and
the Sir Samuel Scott of Yews Trust,
the charity Innovation for Health and
Development (IFHAD), and the
Wellcome Trust. ARE, DAJM, RHG,
JSF, and CAE are funded by the
Wellcome Trust, UK; and ARE, DAJM,
and CAE had Wellcome Trust Clinical
Tropical Medicine Research
Fellowships. RHG is supported by
USAID award #HRN-5986-A-00-6006-
00, GHS-A-00-03-00019–00 and
Global Research Activity Cooperative
(T35A107646). These funding
agencies had no role in study
design, data collection and analysis,
decision to publish, or preparation
of the manuscript.
Competing Interests: The authors
have declared that no competing
Academic Editor: Peter Wilson,
University College London, United
Citation: Escombe AR, Moore DAJ,
Gilman RH, Pan W, Navincopa M, et
al. (2008) The infectiousness of
tuberculosis patients coinfected with
HIV. PLoS Med 5(9): e188. doi:10.
Received: May 29, 2007
Accepted: July 7, 2008
Published: September 16, 2008
Copyright: ? 2008 Escombe et al.
This is an open-access article
distributed under the terms of the
Creative Commons Attribution
License, which permits unrestricted
use, distribution, and reproduction
in any medium, provided the
original author and source are
Abbreviations: IQR, interquartile
range; MDR, multidrug-resistant; SD,
standard deviation; TB, tuberculosis;
XDR, extensively drug-resistant
* To whom correspondence should
be addressed. E-mail: rod.escombe@
A B S T R A C T
The current understanding of airborne tuberculosis (TB) transmission is based on classic
1950s studies in which guinea pigs were exposed to air from a tuberculosis ward. Recently we
recreated this model in Lima, Peru ´, and in this paper we report the use of molecular
fingerprinting to investigate patient infectiousness in the current era of HIV infection and
multidrug-resistant (MDR) TB.
Methods and Findings
All air from a mechanically ventilated negative-pressure HIV-TB ward was exhausted over
guinea pigs housed in an airborne transmission study facility on the roof. Animals had monthly
tuberculin skin tests, and positive reactors were removed for autopsy and organ culture for M.
tuberculosis. Temporal exposure patterns, drug susceptibility testing, and DNA fingerprinting of
patient and animal TB strains defined infectious TB patients. Relative patient infectiousness was
calculated using the Wells-Riley model of airborne infection. Over 505 study days there were
118 ward admissions of 97 HIV-positive pulmonary TB patients. Of 292 exposed guinea pigs,
144 had evidence of TB disease; a further 30 were tuberculin skin test positive only. There was
marked variability in patient infectiousness; only 8.5% of 118 ward admissions by TB patients
were shown by DNA fingerprinting to have caused 98% of the 125 characterised cases of
secondary animal TB. 90% of TB transmission occurred from inadequately treated MDR TB
patients. Three highly infectious MDR TB patients produced 226, 52, and 40 airborne infectious
units (quanta) per hour.
A small number of inadequately treated MDR TB patients coinfected with HIV were
responsible for almost all TB transmission, and some patients were highly infectious. This result
highlights the importance of rapid TB drug-susceptibility testing to allow prompt initiation of
effective treatment, and environmental control measures to reduce ongoing TB transmission in
crowded health care settings. TB infection control must be prioritized in order to prevent health
care facilities from disseminating the drug-resistant TB that they are attempting to treat.
The Editors’ Summary of this article follows the references.
PLoS Medicine | www.plosmedicine.org September 2008 | Volume 5 | Issue 9 | e1881387
P PL Lo oS S MEDICINE
Seminal experiments demonstrating airborne tuberculosis
(TB) transmission by droplet nuclei were performed by Riley
and coworkers in the 1950s–1960s [1,2]. Guinea pigs acquired
TB by breathing exhaust air from a TB ward. The studies
demonstrated TB transmission from a minority of patients,
marked variability in patient infectiousness, and reduced
infectiousness following initiation of effective chemotherapy
[1–3]. These classic studies were recently recreated in Lima,
Peru ´, in the modern era of HIV infection and multidrug-
resistant (MDR) TB, and again showed striking variability in
patient infectiousness .
The strongest predictor of TB patient infectiousness is
sputum smear status [5–7]. However, considerable variation
exists in TB prevalence amongst contacts of smear-positive
patients , and the importance of smear-negative trans-
mission has also been demonstrated . Other determinants
of infectiousness include lung cavitation and cough frequency
, and cough-inducing procedures have been associated with
extensive transmission [10–12]. Additional factors are likely
and may include sputum volume or consistency, and TB
strain variables such as ability to survive in the airborne state.
Recent studies detected culture-positive cough-generated
aerosols in only 25% of new TB patients .
The relative infectiousness of MDR versus drug-susceptible
TB remains controversial. The patients with drug-susceptible
TB studied by Riley were four to eight times more likely to
infect guinea pigs than were those with drug-resistant disease
[1–3]. Epidemiological studies of contacts of both drug-
resistant and drug-susceptible TB infected patients have
shown no difference in relative transmissibility [14–16], whilst
studies utilising molecular epidemiology have had conflicting
results [17–20]. Similarly, the effect of HIV infection on TB
infectiousness remains disputed . The beneficial effect of
anti-TB chemotherapy on reducing TB infectiousness is well
known, but this presupposes that the treatment administered
is effective. The inadequate treatment of MDR TB and the
emergence of extensively drug-resistant (XDR) TB therefore
have important consequences for hospital TB infection
In this paper we report the results of molecular finger-
printing to determine which patients had infected which
guinea pigs in our in vivo air sampling model above a TB
ward. We investigate factors associated with TB transmission
from this group of HIV-coinfected TB patients with a high
prevalence of MDR TB.
An airborne infection study facility was constructed on the
roof of a TB-HIV ward at Hospital Nacional Dos de Mayo,
Lima, Peru ´, as previously described . All air from two 4-
bedded negative-pressure patient rooms passed over guinea
pigs in the study facility before being exhausted into the
atmosphere. Airflow from the ward and into the animal
facility was measured using a capture hood (Alnor, Shoreview,
United States) at air injection and extraction vents.
The ward operated as the hospital TB-HIV service, and
patient admission, management, and duration of stay were
not influenced by the study. All patients were invited to join
the study through written consent. An admission question-
naire recorded daily symptoms including cough, haemoptysis,
and fever. Twenty-four-hour sputum collections were made
daily for auramine microscopy  and TB culture using
MODS . For nonconsenting patients, anonymised un-
linked information routinely available for ward infection
control, including sputum microscopy and treatment, was
Outbred Peruvian guinea pigs were maintained in quar-
antine for 1–3 mo. Animals were skin tested monthly as
described  and induration measured 48 h later. Animals
were transferred to the hospital following at least two
negative skin tests to ensure freedom from TB. In May
2002, 144 animals began ward air exposure, and 6 mo later,
148 animals were added. Monthly skin tests continued
throughout 505 d exposure to ward air, and positive reactors
were removed for humane killing and autopsy. Evidence of
TB infection was sought in lungs, mediastinal lymph nodes,
spleen, and liver. Tissues were homogenised and cultured for
TB as described . Forty negative control guinea pigs were
maintained separately, breathing fresh air.
Determination of Patient Infectiousness
TB drug-susceptibility testing of animal and patient strains
for susceptibility to isoniazid, rifampicin, streptomycin,
ethambutol, capreomycin, and ciprofloxacin was performed
using the tetrazolium microplate assay  and DNA finger-
printing performed using spoligotyping . Linkage of
source patient with infected guinea pigs relied upon
genotypic (identical spoligotype) and phenotypic (drug-
susceptibility pattern) concordance and patient ward occu-
pancy 3–9 wk prior to animal PPD conversion, reflecting TB
incubation in these guinea pigs . Individual patient
infectiousness was determined using the Wells-Riley airborne
infection equation (see Text S1) . Determinants of patient
infectiousness were assessed by univariate and multiple
logistic regression in which nonsignificant factors were
sequentially dropped from the model using SPSS and SAS
statistical software .
The study was approved by the Institutional Review Boards
at Hospital Dos de Mayo, Asociacio ´n Bene ´fica PRISMA, Peru ´,
and Imperial College London, Hammersmith Hospital
Campus, UK. Animal ethical approval was obtained from
the Veterinary Medicine Faculty, Universidad Nacional
Mayor San Marcos, Lima, Peru ´, which supervised all animal
There were 185 ward admissions by 161 HIV-positive
patients, resulting in 2,667 patient days, comprising 118
admissions of 97 pulmonary TB patients (1,798 [67%] patient
days), 33 admissions of 30 extrapulmonary TB patients (609
[23%] patient days), and 34 admissions of 34 TB suspects who
subsequently had no laboratory evidence of TB (260 [10%]
patient days). Of the 64 extrapulmonary disease patients or
TB suspects, 59 able to produce sputum were acid-fast-
PLoS Medicine | www.plosmedicine.org September 2008 | Volume 5 | Issue 9 | e1881388
bacillus smear and TB culture negative. Median length of stay
was 11 d (interquartile range [IQR] 6–21 d). Monthly
variations in pulmonary TB patient days according to sputum
status are shown in Figure 1A. Of 66 sputum culture–positive
pulmonary TB admissions, 35 (53%) were sputum smear
positive and 31 (47%) were sputum smear negative.
Pulmonary TB patients composed a heterogeneous group
of new and existing TB diagnoses, admitted for diagnosis and
treatment, adverse treatment effects, or other complications
of TB or HIV infection. All patients were HIV positive, none
were taking combination antiretroviral therapy, and CD4þT
cell counts were not available. Twelve pulmonary TB patients
had isoniazid- or rifampicin-monoresistant strains (251 [14%]
pulmonary TB patient days); 21 patients had confirmed MDR
TB (434 [24%] pulmonary TB patient days); and 11 patients
had presumed MDR TB (treated empirically for drug-
resistant disease due to treatment failure; 143 [8.0%]
pulmonary TB patient days). No patients had XDR TB.
Thirty-four patients had confirmed drug-susceptible TB (687
[38%] pulmonary TB patient days); and 20 patients had
presumed drug-susceptible TB treated empirically without
drug-susceptibility results (275 [15%] patient days). Two
patients with drug-susceptible TB acquired MDR TB with a
new spoligotype pattern, which was not demonstrated to have
been acquired on the ward. For MDR TB patients, signifi-
cantly more patient bed days were accounted for by sputum
culture-positive patients than by sputum culture-negative
patients; in contrast, for non-MDR TB patients, significantly
more patient bed days were accounted for by sputum culture-
negative patients than by sputum culture-positive patients
(both p , 0.0001; Figure 2).
The treatment status of pulmonary TB patients is shown in
Table 1. ‘‘Optimal’’ signifies an antibiotic regimen suitable
for the TB strain drug resistance pattern, whilst ‘‘suboptimal’’
signifies a regimen less likely to result in cure. The suboptimal
treatment category included standard first-line therapy (4 mo
of isoniazid, rifampicin, pyrazinamide, and ethambutol
followed by 2 mo of rifampicin and isoniazid ) adminis-
tered to MDR TB cases whilst indirect drug susceptibility
results were awaited, a process commonly taking 2–3 mo. The
suboptimal treatment group also included patients with drug-
resistant strains given the now abandoned second-line
regimen  containing standard first-line drugs with the
addition of a single drug, streptomycin, and any first-line or
second-line therapy that was delayed because of adverse drug
reactions or drug shortages.
Spoligotype results were available for 49 (42%) pulmonary
TB admissions, corresponding to 39 (40%) of 97 patients. The
remaining admissions were predominantly patients with
culture-negative sputum, or those unable to produce sputum.
Sixteen culture-positive patients, of whom four were smear
positive, had no spoligotyping result, due to laboratory
contamination or unwillingness to consent. For the 49
admissions with spoligotyping results, 25 different patterns
were observed, none of which corresponded to Beijing strains
of M. tuberculosis. Figure 1B shows the temporal distribution of
patient days accounted for by patients with different
Detection of TB Transmission to Guinea Pigs
No positive PPD skin tests were seen in quarantine (760
tests) or unexposed negative control animals (287 tests).
During 505 study days, 292 animals were exposed to ward air,
an average of 92 each month. A total of 159 animals
developed positive tests (? 7.5 mm), 124 were TB culture
positive, and a further five had autopsy evidence of
pulmonary TB . Nine ward air-exposed PPD-negative
animals had evidence of TB, as did six of 25 intercurrent
deaths between skin tests . In total, cultures were positive
in 135 (46%) of 292 animals. No evidence of TB was found in
unexposed negative controls.
Of 135 culture-positive guinea pigs, drug susceptibility and
spoligotyping results were available for 125. Of this group 121
(97%) were multidrug resistant, one was isoniazid mono-
resistant, and three were fully drug susceptible. Eight differ-
ent spoligotype patterns were observed. Figure 1C shows the
distribution of guinea pig TB infections and corresponding
spoligotype patterns according to the monthly skin test when
TB was diagnosed. Data from intercurrent deaths are
included in the subsequent skin test. There was a large
monoclonal TB outbreak in month 10 (spoligotype pattern
#7), which continued into months 11 and 12. This strain
reappeared in months 14 and 15, but was not seen in month
In ten guinea pigs, spoligotyping was performed on
positive cultures from three separately dissected tissues
(lymph nodes, lung, spleen). Spoligotype patterns were
identical in all tissues. In nine animals with multiple
pulmonary primary foci, two foci were dissected individually
from different lung lobes with separate sets of instruments.
All primary foci yielded identical spoligotype patterns.
Distribution of infection according to animal cage location
was random, consistent with airborne transmission from the
ward, not horizontal spread between animals.
Infectiousness of Patients
Of 125 guinea pigs with spoligotype results, 122 (98%) were
matched with a patient with an identical TB drug-suscepti-
bility and spoligotype pattern who had resided on the ward
3–9 wk prior to the guinea pig skin test conversion. Ten
different infectious patients were identified, who had seven
spoligotype patterns. Of these, one was unique to one patient,
and the remainder were seen in multiple patients. However,
in the 3–9 wk preceding the infection of each guinea pig
cluster, only one patient was ever identified with such a
pattern. The characteristics of the ten identified infectious
patients are shown in Table 2. Six had MDR, one had
monoresistant, and three had drug-susceptible TB. Amongst
the six patients with MDR TB strains, five strains were
additionally resistant to ethambutol, of which two strains
were also resistant to streptomycin. Two of the three patients
with drug-susceptible TB were newly started on treatment
and hence they had spent time on the ward untreated, and
the third had stopped treatment. All identified patients with
infectious drug-resistant TB had been suboptimally treated:
six were on inadequate treatment regimens, and one had had
the initiation of correct treatment delayed for 11 d.
There were at least two further infectious patients. One
infected two animals with an MDR TB strain not seen
amongst patients’ spoligotyping results. During the 3–9 wk
prior to the animals’ infection there were three sputum
culture–positive MDR TB (one smear-positive) patients with-
out spoligotyping results on the ward, any of whom could
have infected these animals. A second unidentified infectious
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PLoS Medicine | www.plosmedicine.org September 2008 | Volume 5 | Issue 9 | e1881390
patient (potentially one of the three above) resulted in a
guinea pig PPD conversion seen in month 5, with an MDR TB
strain with spoligotype pattern #1.
Table 2 also shows patient infectiousness, q, the number of
infectious quanta produced per hour. For absolute ventila-
tion in the Wells-Riley equation, mean total air entering the
animal enclosure was used: 28 m3/min (standard deviation
[SD] 0.9 m3/min). This comprised ward air (21 m3/min), air
from a small room used in the mornings for non-TB patients
(3.4 m3/min), and outside air infiltration into ducts between
ward and animal house (3.4 m3/min). The calculation
methodology and a worked example for infectious patient
number 6 are provided in Text S1. Average patient
infectiousness over the entire study was 8.2 infectious
quanta/h (using 174 total animal infections) or 6.7 (using
144 total animal infections with evidence of disease, exclud-
ing PPD-positive animals without autopsy or culture evidence
Determinants of Infectiousness
Regression analysis was performed for putative determi-
nants of infectiousness for pulmonary TB admissions with
spoligotype results. Admissions rather than patients were
analysed, because patients with multiple admissions changed
sputum smear, drug-susceptibility, or treatment status. Forty-
nine admissions (39 patients) were included. Ten were classed
as infectious resulting in TB transmission to guinea pigs, and
39 as noninfectious. In univariate analysis, the same patient
characteristics were significantly associated with both
whether or not TB transmission occurred (Table 3) and with
the degree of patient infectiousness (q, see Text S1; Table 4):
sputum smear-positivity, MDR TB versus non-MDR TB, and
suboptimal treatment. Multiple regression analysis of
whether or not a patient admission caused TB transmission
to guinea pig(s) showed an independent statistically signifi-
cant association only with MDR status (p ¼ 0.02); TB
transmission to guinea pigs was also observed more
frequently in admissions of sputum smear-positive (versus
smear-negative) patients, but this association was not statisti-
cally significant (p ¼ 0.08; Table 3). Predicted probabilities
were estimated from an exact multiple logistic regression: the
predicted probability of infectiousness was 3% for smear-
negative non-MDR TB patients; 18% for patients who were
either smear positive or had MDR TB; and 59% for smear-
positive MDR TB patients. Further univariate analyses were
performed on a subset of 33 consenting patient admissions
for which symptom information such as cough frequency was
available, but no variable approached significance (all p .
0.6), which may reflect low power due to the small sample.
Multiple regression analysis of data from all patients
identified as having caused TB transmission demonstrated
that the degree of patient infectiousness was independently
significantly associated with MDR TB; patient infectiousness
appeared possibly to increase with sputum-smear positivity
and days on the ward without treatment, but these
associations were not found to be statistically significant
(Table 4). This statistical model allowed patient character-
Figure 2. Pulmonary TB Patient Bed Days According to Sputum Smear Status and TB Drug Susceptibility
The numbers of patient bed days accounted for by patients with MDR TB, or with non-MDR TB, are shown. Results are subdivided into sputum-smear
positive and culture positive (white shading); sputum-smear negative and culture positive (dark grey shading); and culture negative (black shading).
Two patients are not included in this figure: one culture-positive patient with drug-susceptible TB (accounting for 6 patient days) with an unavailable
smear result; and one smear-negative, culture-positive patient (accounting for 8 patient days) with no TB drug-susceptibility information. The drug-
resistant category included 32 MDR TB cases and 12 isoniazid or rifampicin monoresistant cases. In non-MDR TB admissions, 34 smear-negative culture-
positive and 241 culture-negative patient bed days were accounted for by patients treated empirically, without confirmation of drug-susceptibility
status in our laboratory. In MDR TB admissions, 45 smear-negative culture-positive and 98 culture-negative patient days were accounted for by such
patients. Comparisons between groups were made using the two-sample z-test of proportions.
Figure 1. Pulmonary TB Patient Bed Days and TB Strain Spoligotype Pattern Compared with TB Infection in Guinea Pigs by Study Month
(A) Number of bed days in each study month resulting from pulmonary TB patients, who were either smear positive (patterned bars, ‘‘þve’’), or smear
negative (white bars, ‘‘?ve’’) at the time of admission.
(B) Number of bed days in each study month resulting from pulmonary TB patients for whom a TB strain spoligotype pattern was available. Each block
of colour corresponds to one patient, and each colour to one of the eight spoligotype patterns observed in the guinea pigs. Pale yellow represents the
remaining 19 patterns observed in patients whose TB was not seen in the guinea pigs. If a patient resided on the ward for a period spanning more than
one study month, that patient is included in the month where they accounted for more smear positive patient bed days. The coloured blocks
containing numbers correspond to the ten identified infectious patients, numbered 1 to 10 in Table 2.
(C) Percentage of animal colony skin tested each study month that were PPD positive. Each colour represents one spoligotype pattern, except for pale
blue, which represents ten guinea pigs culture positive for TB but for which spoligotype patterns were unavailable. White represents animals that were
PPD positive but TB culture negative. Animals culture positive for TB that were PPD false negatives or that died between skin tests were included in the
subsequent month’s skin test.
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istics to be used to predict the number of infectious quanta
produced per hour (q). The regression model (Table 4)
indicated that: non-MDR TB, smear-negative patients with
undelayed treatment had a predicted infectiousness q ¼ 0.3;
patients with MDR TB or sputum-smear positivity without
treatment delay had a predicted q ¼ 3.9; more than 2 d of
treatment delay resulted in an increase in q of 1.7; sputum-
smear positive MDR TB had a predicted q¼24; MDR TB and
(more than 2 d of delayed treatment or sputum-smear
positivity) had predicted q ¼ 14; and sputum-smear positive
MDR TB with more than 2 d of delayed treatment had a
predicted q ¼ 54.
This study provides novel characterization of the hetero-
geneity and determinants of infectiousness of HIV-positive
TB patients by applying molecular strain characterization to
track airborne TB transmission to guinea pigs. This research
has for the first time (to our knowledge) demonstrated that
amongst HIV-positive patients TB infectiousness is extremely
variable, that a few HIV-positive patients were highly
infectious, and that inadequately treated MDR TB patients
accounted for the great majority of TB transmission. In
contrast to seminal studies of TB transmission using a similar
guinea-pig method of detection half a century ago, this study
was conducted in a real-life busy ward in a low-resource
setting with unselected patients, composed of a heteroge-
neous mix of new and established diagnoses of drug-
susceptible and drug-resistant TB, with varying treatment
regimens. These results therefore have important implica-
tions for TB infection control, especially in the era of
increasingly integrated TB and HIV care and the emergence
of XDR TB strains.
Average patient infectiousness over the whole study period
for these HIV-positive TB patients with high rates of MDR TB
was up to six times greater than that calculated for the
heterogeneous mix of patients in the 1950s studies (q ¼ 1.25)
[4,27]. However, this average masks considerable variability in
infectiousness between patients. Three highly infectious
patients were observed, all with MDR TB, with q-values of
40, 52, and 226. It should be noted that these q-values reflect
TB transmission from humans to guinea pigs. The infectious
dose of M. tuberculosis for humans is unknown; hence the
concept of infectious quanta used in airborne infection
models . For fully virulent M. tuberculosis strains just one
droplet nucleus can establish infection and disease in guinea
pigs, but for strains of reduced virulence for guinea pigs, up
to four aerosolised colony-forming units may be required to
establish a single pulmonary primary focus . Thus some
caution is needed in comparing the infectiousness of patients
in this study with published q-values calculated for human-to-
human transmission, such as q ¼ 13 for an untreated office
worker who infected 27 coworkers over 4 wk prior to
diagnosis , and q ¼ 250 for an outbreak associated with
intubation and bronchoscopy of a TB patient [11,27].
However, comparisons can be drawn with q-values calculated
for Riley’s study: q ¼ 1.25 average for all patients; q ¼ 60 for
the most infectious case, with laryngeal TB . Direct
comparisons should, however, be made cautiously owing to
methodological differences between the studies such as the
type of guinea pig used and the cutoff for a positive skin test
. This model for identifying infectious patients required
TB strains to be not only transmissible to guinea pigs, but also
sufficiently virulent to cause disease from which a strain
could be recovered with corresponding spoligotype. Animals
with positive PPD skin test conversions but culture negative
for TB were observed throughout the study. Due to the
continuous exposure to ward air, mixed infections might be
expected in the animals, but in fact were not observed despite
the culture of separately dissected lung foci. Because they are
relatively uncommon, mixed infections were not specifically
sought in patients .
Table 1. Antituberculosis Chemotherapy Treatment Status of Confirmed and Presumed Drug-Susceptible and Drug-Resistant
Pulmonary TB Admissions to the Ward
TreatmentCategory Drug-Susceptible TB
All Pulmonary TB
Numbers of days on the ward are shown, with percentage of total pulmonary TB days (1,798 d) in parenthesis. ‘‘Optimal’’ indicates an antibiotic regimen suitable for the TB strain drug
resistance pattern; ‘‘suboptimal’’ indicates a regimen less likely to result in cure.
aOne patient who resided on the ward for 8 d and for whom no drug-susceptibility information is available has been excluded from this table.
b‘‘Established’’ treatment denotes treatment of ? 2 wk duration.
c‘‘Recent’’ treatment denotes treatment commenced within the previous 2 wk.
d‘‘New’’ treatment denotes treatment commenced during the study hospital admission.
e‘‘None’’ denotes abandoned treatment, or treatment withdrawn due to adverse effects.
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The relative infectiousness of patients in this study was
highly variable, and a patient with MDR TB was found to be
highly infectious, producing 226 infectious quanta/h. The TB
strain responsible, not a Beijing strain, was also seen in a
second highly infectious MDR TB patient, producing 52
infectious quanta/h. This observation suggests a potentially
strain-related factor involved in transmissibility, perhaps an
enhanced ability to survive aerosolisation and the physical
stresses of being airborne. An alternative explanation might
be an effect on disease phenotype. It is interesting to note
that both patients had fever and cough and produced large
volumes of sputum, between 10 and 90 ml daily. Neither
patient had cavitation on chest X-ray. Although no formal
ear-nose-throat assessment was documented, neither was
diagnosed with laryngeal TB. Both patients also spent time
on the ward untreated. The first was untreated for the first 11
d of a 32 d admission before second-line drugs were
commenced, due to difficulties in access to medications.
The second patient had recently been commenced on
suboptimal treatment (standard first-line therapy plus strep-
tomycin) and this was suspended for ten of 26 ward days
because of adverse effects.
The finding that sputum smear positivity was associated
with TB transmission concurs with previous studies [5–7]. The
effect of treatment on the infectiousness of TB patients is also
well known [2,5], with numbers of viable bacteria falling
precipitously following initiation of effective chemotherapy
[34,35]. Whilst some data suggest that apparent cure of MDR
TB may be achieved with first-line drugs [36,37], other studies
have shown poor outcomes for such patients . The
current study shows how suboptimal treatment of MDR TB
patients is likely to facilitate ongoing TB transmission. There
is conflicting evidence concerning the relative transmissibil-
ity of MDR versus drug-susceptible TB strains [14–20]. In this
study, patients with MDR TB were significantly more likely
than those without MDR TB to transmit TB to guinea pigs.
However, this finding should be interpreted with caution
because of colinearity with suboptimal treatment (5 of 6
identified infectious MDR TB patients were on suboptimal
regimens, and the other had treatment initiation delayed for
11 days whilst suitable medications were acquired). Regard-
less, the high relative infectiousness of inadequately treated
MDR TB patients demonstrated in this study underscores the
importance of prompt specific treatment guided by rapid
drug-susceptibility testing, rather than restricting MDR TB
testing and specific therapy to patients who survive failing
empiric first-line therapy, as currently happens in most low-
resource settings. It also has important implications for
hospital policies that allow suboptimally treated MDR TB
cases to be cared for in multi-bedded rooms.
The highly infectious nature of some of the HIV-positive
MDR TB patients identified in this study has important
implications for TB infection control. Administrative control
measures that facilitate the rapid diagnosis, isolation, and
prompt treatment of such patients are paramount. With
increasing congregation of infectious and susceptible indi-
viduals not only in hospitals but also in such settings as
antiretroviral therapy roll-out, HIV antenatal care, and
voluntary counselling and testing facilities , environ-
mental control measures are also of great importance. As the
infectiousness of a TB source increases, the relative protec-
tion provided by dilutional room ventilation decreases 
Table 2. Characteristics of Identified Infectious Patients
Quanta per Hour
(6 2 SD)
New correct, but delayed
Characteristics of the ten infectious patients identified by matching TB spoligotype pattern and temporal association with a guinea pig infection are shown.
aMaximum daily sputum volume during ward admission is recorded.
bPatients 3 and 9 did not consent and therefore symptom details are unavailable, denoted n/a.
cInfectious patients 11 and 12, both with MDR strains, remained unidentified.
PLoS Medicine | www.plosmedicine.org September 2008 | Volume 5 | Issue 9 | e1881393
and may become inadequate at the relatively low levels of air
exchange usually provided by mechanical ventilation. High
rates of ventilation would be required to provide protection
from the extremely infectious newly diagnosed MDR TB case
observed in this study. Achieving this through mechanical
means is an expensive solution for much of the world where
TB is most prevalent. In contrast, well-designed natural
ventilation  provides high ventilation rates for little cost,
and furthermore is highly applicable to areas such as crowded
waiting rooms where infectious, untreated TB patients are
most likely to be encountered. TB infection control must be a
priority in the current roll-out of enhanced HIV care, and
should be carefully considered in the design and construction
of any new infrastructure for such programmes.
The need for strengthened TB infection control is also
highlighted by the recent outbreak of XDR TB amongst HIV-
coinfected patients in South Africa; this outbreak was
predominantly nosocomial and resulted in extremely high
mortality . The variability of infectiousness of patients
demonstrated in this study highlights the usefulness of a
potential test for TB infectiousness that would allow targeted
isolation of the most infectious patients in the settings where
isolation facilities are sparse, as is unfortunately the case in
much of the world where TB is most prevalent. One patient in
our study, with MDR TB, infected over half of the guinea pig
colony. The development of tests that allow early identifica-
tion and isolation of such patients in a clinical setting is a
There are some limitations to this study. The first is the
incomplete set of spoligotyping data, with results in only 49
of 118 pulmonary TB admissions. Despite this deficiency, ten
of at least 12 infectious patients were identified. It is possible
that transmission occurred from other patients for whom
spoligotyping was unavailable, but certain factors suggest that
this is not the case. Most patients without spoligotype results
had negative sputum cultures, and whilst smear-negative TB
transmission occurs , smear-positive patients account for
the majority [5–7]. In this study, 16 culture-positive patients,
of whom four were smear positive, had no spoligotype result.
Fortuitously, these patients were either temporally or
phenotypically (drug susceptibility pattern) unrelated to the
ten guinea pig clusters linked with infectious patients,
Table 3. Determinants of Patient Infectiousness: Analysis of Infectious Versus Noninfectious Patients
n ¼ 10
n ¼ 39
Simple Clustered ExactSimpleExact
OR p-Value p-ValueOR p-Value ORp-Value ORp-Value
Days on ward, all
.12 d on ward
.2 d on ward
Age in years, all
.30 y of age
?40 y of age
?40 y of age
9 (90%)20 (51%) 8.5 (1.0–74) 0.050.059 8.3 (1.0–394) 0.05 7.4 (0.8–70) 0.086.7 (0.7–335) 0.1
6 (60%)6 (15%) 8.2 (1.8–38)0.0070.008 7.8 (1.4–51)0.027.3 (1.4–37) 0.02 6.6 (1.1–48)0.04
8 (80%) 12 (31%) 9.0 (1.7–49)0.01 0.017 8.6 (1.4–95)0.01————
1/4 (25%)8/33 (24%) 1.0 (0.1–12)1.01.0 1.0 (0.02–15) 1.0————
1.0 (1.0–1.1) 0.4
1.0 (0.9–1.1) 0.7
——2.2 (0.5–9.6) 0.3 0.3 2.2 (0.4–12) 0.5————
1.0 (1.0–1.1) 0.4
0.8 (0.2–3.1) 0.7
0.8 (0.1–4.4) 0.8
——0.6 (0.1–3.6) 0.6 0.60.6 (0.1–5.6) 0.9————
9 (90%)29 (74%)3.1 (0.4–28) 0.3 0.3 3.0 (0.4–149) 0.6————
aFor categorical independent variables, the number of cases (% of total in parentheses) is shown, and for continuous independent variables, the median (IQR in parentheses) is shown. The
primary analysis treated the independent variables ‘‘Days on Ward’’ and ‘‘Days on Ward without Treatment’’ and ‘‘Age’’ as raw, continuous variables. These primary analyses are
complemented by secondary analyses (in parentheses) of each of these variables divided into categories around cutoff points chosen to maximize the likelihood of the outcome. Note that
the analysis of these independent variables as continuous or categorical data gave similar results.
bRegression analysis was performed on putative determinants of infectiousness for the 49 ward admissions by pulmonary TB patients for which spoligotype patterns were available. Four
patients accounted for the ten multiple admissions in this group. The first had MDR TB and was infectious on one admission when inadequately treated, and non-infectious on a
subsequent admission when adequately treated. The second had four admissions: three were noninfectious with drug-susceptible TB that was first smear-positive and then smear-
negative. The fourth admission was infectious, with acquired MDR-TB. The third patient had two noninfectious admissions separated by 4 mo with smear-positive inadequately treated
monoresistant TB. The fourth patient had two noninfectious admissions separated by two months with smear-negative drug-susceptible TB.
cThe simple univariate regressions are shown with robust clustered SE estimated (to account for repeated hospitalizations by some patients) and with exact logistic regression (to account
for the sample size). Note that simple, clustered, and exact univariate logistic regressions gave similar results. ORs (95% confidence intervals) are included.
dBecause of the sample size, only the three independent variables with clear associations with infectiousness (sputum-smear, MDR TB and suboptimal treatment) were considered for
multiple regression analysis. In both simple and exact multiple logistic regression, the best model by AIC criteria included only sputum-smear positivity and MDR TB (exact multiple logistic
regression AIC ¼ 43.8). ORs (95% confidence intervals) are included.
OR, odds ratio.
PLoS Medicine | www.plosmedicine.orgSeptember 2008 | Volume 5 | Issue 9 | e188 1394
excluding them as coinfectors. Indeed the two clusters of
MDR TB guinea pigs with unidentified infectious sources
became infected at times corresponding to the ward
residency of smear-positive MDR TB patients without
spoligotype results. We cannot exclude the possibility that
guinea pig infections occurred from staff or visitors with TB,
however all staff and visitors wore particulate respirators. It is
possible that the large monoclonal outbreak observed in the
guinea pigs was in fact made up of more than one strain.
However, the concordant drug susceptibility data and
epidemiological match with a patient on the ward at an
appropriate time prior to the infections obviates the need for
secondary typing of strains. The Wells-Riley model has
inherent limitations , but these do not influence evalua-
tions of relative infectiousness, and it allows comparison with
published values of TB infectiousness calculated using the
same model. The design of this study did not permit
determination of the duration of patient infectiousness
because of the interval of one month between skin tests,
and the variability in the period required for these guinea
pigs to become PPD positive following TB infection. It is
possible that values for patient infectiousness are under-
estimates, because the entire period of a patient’s hospital
admission was used for the exposure duration variable in
calculations, and it would normally be expected for patient
infectiousness to tail off once treatment was initiated,
although this would not be the case with suboptimal treat-
ment. However, in univariate analyses patient days on the
ward was not significantly associated with TB transmission.
The relatively narrow age range and the small number of
women amongst the patients is a further limitation of this
study, because both young age and male sex have been
associated with TB transmission to contacts [15,43]. Because
all patients were HIV positive, our study was unable to yield
evidence concerning the infectiousness of HIV-positive
versus HIV-negative MDR TB patients, and this could be a
future area of study using our airborne infection facility.
In conclusion, this study has demonstrated the potential of
HIV-positive patients with MDR TB to be highly infectious.
With the great majority of TB transmission in this study
occurring from inadequately treated MDR TB patients, these
results identify the importance of early drug susceptibility
testing and initiation of effective chemotherapy for drug-
resistant TB to prevent ongoing transmission and facilitate
TB control. Furthermore, this study highlights the impor-
tance of environmental control measures to prevent airborne
TB transmission in crowded health care settings, especially in
areas with a high prevalence of HIV infection and drug-
resistant TB, and in today’s era of emerging XDR TB. HIV-
positive patients with unrecognised or inadequately treated
MDR TB coinfection may be highly infectious, and effective
TB infection control measures are essential to prevent health
care facilities from disseminating drug-resistant TB.
Text S1. (A) Patient infectiousness using the Wells-Riley equation.
(B) Estimating predicted degree of infectiousness, q, from Tobit
Found at doi:10.1371/journal.pmed.0050188.sd001 (45 KB DOC).
Table 4. Determinants of Patient Infectiousness: Analysis of the Degree of Infectiousness
Independent VariableData for
n ¼ 49
Predicted qp-Value Coefficientp-Value Predicted q
Days on ward
.12 d on ward
Days on ward
.2 d on ward
Age in years
.30 y of age
20–29 versus ?40 y of age
30–39 versus ?40 y of age
—— 0.30.2 180.4 4.30.090.15
The infectiousness of individual patients (q, see Text S1) was tested for associations with the independent variables listed in the table. The p-value refers to the univariate associations
assessed with the Mann-Whitney U test for categorical independent variables and the nonparametric Spearman correlation coefficient for continuous independent variables. The p-value is
also shown for equivalent exact statistical testing. The Tobit (censored) regression analysis allows for the censoring of data from patients who did not infect any guinea pigs and who
therefore had an undetectable degree of infectiousness (see Text S1). After an initial model fit for all covariates individually, the variable that entered the model based on the highest
pseudo-R2was MDR TB. Each additional variable added would be based on the lowest p-value derived from likelihood ratio statistic provided that the p-value was , 0.2. The second
variable selected was sputum-smear positivity and the third variable entered was the categorical variable of more than 2 d on the ward without treatment. No additional variables entered
the model. The final model has a pseudo-R2of 0.10.
PLoS Medicine | www.plosmedicine.orgSeptember 2008 | Volume 5 | Issue 9 | e188 1395
The authors would like to thank the patients and nursing and medical
staff at the Servicio de Enfermedades Infecciosas y Tropicales at
Hospital Nacional Dos de Mayo, Lima, Peru ´, for their invaluable
support in this and continuing studies. The authors thank Patricia
Fuentes, Pilar Navarro, Jorge Coronel, and the staff of the
Laboratorio de Investigacio ´n y Desarrollo at Universidad Peruana
Cayetano Heredia, Lima, Peru ´ for their assistance in processing of
animal specimens, and veterinary surgeon Miguel Gil Saavedra for
supervising animal care.
Author contributions. All authors contributed to the design or data
analysis of the study, the writing of the article and approved the final
version to be published. ARE had full access to all the data in the
study and had final responsibility for the decision to submit for
publication and is the guarantor.
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Editors’ Summary Download full-text
Background. Every year, more than nine million people develop
tuberculosis—a contagious infection usually of the lungs—and nearly
two million people die from the disease. Tuberculosis is caused by
Mycobacterium tuberculosis. These bacteria are spread in airborne
droplets when people with the disease cough or sneeze. Most people
infected with M. tuberculosis never become ill—their immune system
contains the infection. However, the bacteria remain dormant within the
body and can cause tuberculosis years later if host immunity declines.
The symptoms of tuberculosis include a persistent cough, weight loss,
and night sweats. Diagnostic tests for the disease include chest X-rays,
the tuberculin skin test, and sputum cultures (in which bacteriologists try
to grow M. tuberculosis from mucus brought up from the lungs by
coughing). Tuberculosis can usually be cured by taking several powerful
antibiotics daily for several months.
Why Was This Study Done? Scientists performed definitive experiments
on airborne tuberculosis transmission in the 1950s by exposing guinea
pigs to the air from a tuberculosis ward. They found that a minority of
patients actually transmit tuberculosis, that the infectiousness of
transmitters varies greatly, and that effective antibiotic treatment
decreases infectiousness. Since the 1950s, however, multidrug-resistant
(MDR) and more recently extensively drug-resistant (XDR) strains of M.
tuberculosis have become widespread. Treatment of drug-resistant
tuberculosis is much more difficult than normal tuberculosis, requiring
even more antibiotics, and for long periods, up to 2 years and beyond. In
addition, HIV (the virus that causes AIDS) has emerged. HIV weakens the
immune system so HIV-positive people are much more likely to develop
active tuberculosis (and to die from the disease, which also speeds the
development of HIV/AIDS) than people with a healthy immune system.
Have these changes altered tuberculosis transmission between people?
The answer to this question might help to optimize the control of
tuberculosis infection, particularly in hospitals. In this study, the
researchers investigate current patterns of tuberculosis infectiousness
among HIV-positive patients by recreating the 1950s guinea pig model
for tuberculosis transmission in a hospital in Lima, Peru ´.
What Did the Researchers Do and Find? The researchers passed all the
air from an HIV–tuberculosis ward over guinea pigs housed in an animal
facility on the hospital’s roof. The guinea pigs were tested monthly with
tuberculin skin tests, and tissues from positive animals were examined
for infection with M. tuberculosis. Sputum was also collected daily from
the patients on the ward. The researchers then used the timing of
patient admissions and guinea pig infections, together with the drug
susceptibility patterns and DNA fingerprints of the M. tuberculosis strains
isolated from the animals and the patients, to identify which patients
had infected which guinea pigs. Finally, they used a mathematical
equation to calculate the relative infectiousness of each patient in
airborne infectious units (‘‘quanta’’) per hour. During the 505 study days,
although 97 HIV-positive patients with tuberculosis were admitted to the
ward, just ten patients were responsible for virtually all the characterized
cases of tuberculosis among the guinea pigs. Six of these patients had
MDR tuberculosis that had been suboptimally treated. The average
patient infectiousness over the entire study period was 8.2 quanta per
hour—six times greater than the average infectiousness recorded in the
1950s. Finally, the three most infectious patients (all of whom had
suboptimally treated MDR tuberculosis) produced 226, 52, and 40 quanta
What Do These Findings Mean? These findings show that a few
inadequately treated HIV-positive patients with MDR tuberculosis caused
nearly all the tuberculosis transmission to guinea pigs during this study.
They also show for the first time that tuberculosis infectiousness among
HIV-positive patients is very variable. The increase in the average patient
infectiousness in this study compared to that seen in the 1950s hints at
the possibility that HIV infection might increase tuberculosis infectious-
ness. However, studies that directly compare the tuberculosis infectious-
ness of HIV-positive and HIV-negative patients are needed to test this
possibility. More importantly, this study demonstrates the potentially
high infectiousness of inadequately treated MDR TB patients and their
importance in ongoing TB transmission. These findings suggest that
rapid, routine testing of antibiotic susceptibility should improve tuber-
culosis control by ensuring that patients with MDR TB are identified and
treated effectively and quickly. Finally, they re-emphasize the importance
of implementing environmental control measures (for example, ad-
equate natural or mechanical ventilation of tuberculosis wards, or
crowded waiting rooms or emergency departments where tuberculosis
patients may be found) to prevent airborne tuberculosis transmission in
health-care facilities, particularly in areas where many patients are HIV
positive and/or where MDR tuberculosis is common.
Additional Information. Please access these Web sites via the online
version of this summary at http://dx.doi.org/10.1371/journal.pmed.
? The US National Institute of Allergy and Infectious Diseases provides
information on all aspects of tuberculosis, including multidrug-
resistance tuberculosis, and on tuberculosis and HIV
? The US Centers for Disease Control and Prevention provide several fact
sheets and other information resources about all aspects of
tuberculosis (in English and Spanish)
? The World Health Organization’s 2008 report on global tuberculosis
control—surveillance, planning, financing provides a snapshot of the
current state of the global tuberculosis epidemic and links to
information about all aspects of tuberculosis and its control (in several
? HIVInsite provides detailed information about coinfection with HIV
? Avert, an international AIDS charity, also provides information about
the interaction between HIV and tuberculosis
? Tuberculosis Infection-Control in the Era of Expanding HIV Care and
Treatment is a report from the World Health Organization
PLoS Medicine | www.plosmedicine.orgSeptember 2008 | Volume 5 | Issue 9 | e1881397