Comparison of macroscopic and microscopic assessment of specimens collected for the diagnosis of tuberculosis

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The Open Infectious Diseases Journal, 2012, 6, 1-4 1

1874-2793/12 2012 Bentham Open
Open Access
Comparison of Macroscopic and Microscopic Assessment of Specimens
Collected for the Diagnosis of Tuberculosis
Sayera Banu1, Shahed Hossain1, Mohammad Khaja Mafij Uddin1, Md. Toufiq Rahman1, Razia
Khatun1, K Zaman1, M A Quaiyum1 and Frank van Leth*,2,3
1International Centre for Diarrhoeal Disease Research, Bangladesh
2KNCV Tuberculosis Foundation, The Hague, The Netherlands
3Department of Global Health, Academic Medical Centre, University of Amsterdam, Amsterdam Institute for Global
Health and Development, The Netherlands
Abstract: Diagnosis of tuberculosis in a field setting depends on the quality of specimens submitted for smear-
microscopy. Macroscopic assessment (sputum or saliva) of the specimen for suitability for further examination is common
practice in routine care. We examined whether macroscopic assessment could correctly identify sputum specimens based
on four published algorithms using microscopic features in the setting of active case finding in a community survey.
The study included 901 randomly selected adults who reported cough for 3 weeks or more in the national tuberculosis
prevalence survey in Bangladesh. A single specimen of each was assessed with microscopy and microscopy (Gram-stain)
to classify it as either sputum or saliva. The primary outcome was the agreement between the two assessment methods
(Kappa statistic)
From 901 specimens, 561 (62%) were macroscopically classified as saliva and 340 (38%) as sputum. From these, 888
Gram-stained slides could be examined for microscopic features. The agreement between the macroscopic assessment
with any of the four microscopy algorithms for sputum was very poor (all Kappa’s below 0.1).
While macroscopic assessment of submitted specimens might be of value in routine care, it is not warranted in a setting of
active case finding in a community survey. Submitting a specimen in the first place should be the primary goal in this
setting.
Keywords: Macroscopy, microscopy, sputum quality, survey, tuberculosis.
INTRODUCTION

One-third of the world's population is estimated to have been
infected with Mycobacterium tuberculosis and eight million
new cases of tuberculosis arise each year [1]. TB diagnosis
can only be made reliably by demonstrating the presence of
tubercle bacilli in the sputum by means of microscopy and/or
culture in the laboratory. The gold standard for diagnosing
pulmonary tuberculosis is culture of a sputum specimen.
However, due to lack of access to culture facilities and the
long turn-around times involved with sputum culture, the
cornerstone of the diagnosis of tuberculosis is still the
microscopic examination of sputum specimens for acid-fast
bacilli (AFB) in many developing countries including
Bangladesh.
Tuberculosis (TB) is a disease of global importance.

quality sputum specimen is available for microscopic
examination in order to diagnose TB accurately. Patients
frequently provide pure saliva or very small amounts of
sputum in saliva instead of an appropriate, purulent, sputum

Under this condition it is very important that a high

Address correspondence to this author at the Amsterdam Institute for Global
Health and Development, Pietersbergweg 17, 1105BM Amsterdam, The
Netherlands; Tel: +31 20 566 1593; E-mail: f.vanleth@aighd.org
specimen. Saliva smears are in general more likely to be
negative or have an AFB density below the threshold of
microscopy detection than sputum smears [2]. A negative
result issued on examination of such a specimen is
misleading, since it implies that a correct sputum specimen
has been examined. This holds particularly true in settings
where AFB microscopy is used in the context of large-scale
TB prevalence surveys, where participants are less likely to
be able to produce an adequate specimen.

implemented in a large number in countries in an attempt to
provide data for the assessment of the progress on goal 6 of
the Millennium Development Goals [3].
These surveys are currently being prepared or

recently-discharged material from the bronchial tree, with
minimum amounts of oral or nasal material. Satisfactory
quality implies the presence of mucoid or mucopurulent
material and is of greater significance than volume. Poor
quality specimens are thin and watery or composed largely
of bubbles. It is common practice in many TB diagnostic
centres that sputum provided for AFB examination is first
assessed macroscopically by laboratory staff. If the quality
of the sputum is deemed inadequate, the specimen is rejected
and the TB suspect asked to provide a new specimen.
Providing sequential sputum specimens in a short period of
Macroscopically, a good sputum specimen consists of

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2 The Open Infectious Diseases Journal, 2012, Volume 6 Banu et al.
time is considered difficult by many TB suspects. The risk
exists that the TB suspect will not return at a later stage to
provide a sputum specimen leading to a missed opportunity
for TB case finding.

done by skilled laboratory staff involved but can be
subjective.
A macroscopic assessment of specimen quality must be

examined and graded according to an algorithm, has been
proposed as a means to ensure sufficient specimen quality.
Further specimens can be then requested if quality is
inadequate [4]. However, this is much more time consuming
for the laboratory. There are several published algorithms for
microscopic specimen assessment. Among the defined
criteria, Murray & Washington [5] and Van Scoy [6]
consider the specimens as saliva or sputum based on only the
number of white blood cell (WBC) or epithelial cells (EPI)
per low powered field (LPF). However, variations in the
thickness of material in different areas of the slide may
require extensive examination to obtain an overall average
for each slide. To minimize the variability, the other two
criteria (Barry [7] and Gal-Oz [8]) involve assessment of the
ratio of WBC to EPI in several areas of the slide. It has been
shown earlier that in the context of diagnosing respiratory
infections the different microscopic criteria performed in a
comparable manner [4].
Microscopic specimen assessment, in which smears are

worthwhile to culture a respiratory specimen. Using these
algorithms for assessing the possibility for adequate AFB
microscopy has been much less examined. A previous study
on the usefulness of macroscopic assessment of respiratory
specimens for AFB diagnosis was carried out in a routine TB
diagnostic setting [9]. This implies a passive case finding
strategy in which individuals report themselves to a health
facility because of symptoms. The current study is part of a
TB prevalence survey which, by definition, entails an active
case finding strategy in which individuals are approached
and asked for symptoms. Investigators do not have the
possibility to return to the individual at a later stage in such a
setting. It is therefore pertinent that any decision on rejecting
a specimen for investigation is made on solid arguments in
this brief encounter. The objective of this study was to assess
if these arguments should be based on macroscopic or
microscopic assessment of respiratory specimens. Several
countries will be embarking on similar large-scale TB
prevalence surveys in the near future. The result of the
present study will be able to guide field activities in these
surveys in relation to the handling of submitted specimens.
These algorithms were developed to assess if it was
METHODS
Setting and Population

during 2007-09 in which a representative sample size of
52,089 adults ? 15 years were divided over 40 (20 rural and
20 urban) randomly selected clusters [10]. Each participating
adult (15 years or older) provided two sputum specimens
(one spot and one morning) regardless of symptoms during a
household survey. The specimens were kept cool until
transport to the field laboratory. Specimens were collected
from several locations at the same time during the survey
The nation-wide TB prevalence survey was implemented
and arrived in a random fashion in the field laboratory. In
this sub-study all survey participants who reported cough for
3 weeks or more were eligible for inclusion. The study was
restricted to the last 21 clusters of the total 40 clusters for
logistical reasons.
Field Activities

saliva or sputum by the trained field laboratory technicians at
the time of arrival of the specimen in the field laboratory.
Based on National Tuberculosis Control Programmme
guidelines, a specimen was considered saliva on visual
assessment if it had a clear, watery appearance and contained
no purulent material [11]. Two smears from each of the fresh
specimens (uncentrifuged) were prepared at the field
laboratories, one for AFB and another for Gram staining.
The slides prepared for Gram staining were heat fixed at the
field laboratories and transported to the laboratory of the
International Centre for Diarrhoeal Disease Research,
Bangladesh (ICDDR, B). Specimens were refrigerated at the
field sites and transported to the central laboratory for
culture using conventional method within 48 hours of
specimen collection. No preservatives were added.
Each collected specimen was scored macroscopically as
Laboratory Activities

enrolled individual in the sub-study was Gram stained. If
there was no morning specimen available, the prepared slide
of the spot specimen was used for Gram staining. Slides
were stained with crystal violet and then treated with an I2-
KI mixture (mordant) to fix the stain, washed briefly with
95% alcohol (destained) and finally counterstained with
carbol fuchsin. The stained slides were examined under low-
power (X100) magnification by the microscopist on duty.
After examining 10 fields, two parameters were recorded:
the average number of polymorphonuclear neutrophils
(PMNs) and squamous epithelial cells (SEC) per LPF. Four
different algorithms (Table 1) were used for classifying a
specimen as sputum or saliva based on microscopic features;
(1) Murray & Washington: based on the average number of
SECs; (2) Van Scoy: based on polymorphic cells; (3) Barry:
based on epithelial and polymorphic cells; (4) Gal-Oz:
Sputum quality was assessed by the presence of PMNs and
SECs per LPF.
The prepared slide of the morning specimen of each
Statistical Analysis

agreement between the scoring algorithms and macroscopic
assessment was done by using the ? (kappa) statistic, where
the ? value of > 0.4 was considered to be adequate
concordance.
The data were analyzed using SPSS v 17.0. The
RESULTS

able to obtain complete macroscopic and microscopic
assessment data from 888 (98.56%). The quality of the
remaining 13 slides was inadequate for proper reading.
Of the 901 specimens available in the study, we were

282 (51.4%) and 299 (54.5%) were classified as sputum by
Murray & Wahington and Gal-Oz respectively. Among 340
(37%) specimens classified macroscopically as sputum, 187
(55.2%) and 198 (58.4%) were also sputum by both of the
Among the 561 (63%) macroscopically saliva specimens,

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Assessment of Sputum Quality The Open Infectious Diseases Journal, 2012, Volume 6 3
criteria. There was much poorer agreement between
macroscopic assessment and the algorithms of Van Scoy and
Barry (Table 2). All kappa statistics were below 0.1
indicating a very poor agreement between macroscopic and
microscopic assessment of the specimens.
Table 2. Agreement Between Macroscopic and Microscopic
Assessment of Sputum Specimens

Sputum According to Saliva (n=561) Sputum (n=340) Kappa
Murray & Washington 282 (51.4%) 187 (55.2%) 0.04
Van Scoy 2 (0.4%) 7 (2.1%) 0.02
Barry 126 (23.0%) 98 (28.9%) 0.06
Gal-Oz 299 (54.5%) 198 (58.4%) 0.04


negative for AFB, but five of them were found to be positive
on conventional culture. If culture of the specimens was
conditional of the screening criterion used, then macroscopic
assessment of the specimen would have missed one culture
positive for TB, the microscopic criterion of Murray &
Washington, Barry, and Gal-Oz two, and the criterion of
Scoy all five.
All specimens in this sub-study were microscopically
DISCUSSION

sputum specimens has received considerable attention as a
means for improving the reliability of sputum microscopy as
well as sputum culture for tuberculosis diagnosis [12]. The
evaluation of the quality of sputum using cytological
parameters is very important but requires experience and
qualification and is also time and resource-consuming.
The use of Gram-stained smears to assess the quality of

macroscopic and microscopic assessment of sputum
specimens was poor in the setting of an active case-finding
strategy of a TB prevalence survey. Assuming the
microscopic classification as a gold standard, this indicates
that macroscopic assessment of provided specimens could
not properly distinguish between saliva and sputum. In the
national TB prevalence survey a total of 33 new smear-
positive TB cases were detected among 52,089 population
[10]. Out of these 33 cases the morning sputum specimens
collected from 19 (57.6%) were macroscopically classified
This study showed that the agreement between
as sputum whereas the remaining 14 (42.4%) were saliva.
These two observations make that rejecting specimens based
on the macroscopic assessment in this setting is not
recommended since it will underestimate the prevalence of
smear-positive specimens.

assessment as a procedure did not contribute to the
identification of specimens that were culture positive.
Apparently it is more important that an actual specimen is
submitted for sputum examination than that the submitted
specimen is meeting specific macroscopic or microscopic
qualities. From this it follows that survey personnel should
be trained in obtaining specimens for subsequent
microscopy, especially in patients who have difficulties in
coughing up material. Recent studies showed that instruction
given for a good quality sputum specimen increased both the
quality and quantity of the sputum specimen with an
increased positivity rate [13].
This study also showed that microscopic sputum

in our study differs from the observation by Kahn et al., who
stated that macroscopic assessment was a valid approach for
identification of smear-positive respiratory specimens [9].
This conclusion was based on the fact that macroscopic
assessment rejected the least number of specimens in this
study. As mentioned earlier, the main difference between the
two studies is the setting, which indicates that operating
procedures should be tested for validity in the setting they
are being used rather than taken as face-value. Since the
physicians are primarily responsible for submitting proper
specimens to a laboratory, the use of the gram stain might be
of some value in isolated instances where one needs to know
whether specimens from patients suspected of having
mycobacterial disease, but who have consistently negative
smears and cultures, are of lower respiratory origin. In all
other situations, including that of active case finding in a TB
prevalence survey, efforts should be directed to obtain a
specimen for processing.
The poor performance of macroscopic assessment found

large-scale national TB prevalence surveys in other
countries. Survey protocols tend to incorporate procedures
derived from routine patient care. Without careful
considering the different setting of the survey, this might
lead to biased measurements in the field and the reporting of
invalid prevalence estimates. This observation does not hold
true only for TB prevalence surveys but can be considered
These findings should be considered in the design of
Table 1.

Summary of Four Published Criteria for Judging Acceptability of Sputum Specimens
Author Method Criteria for Acceptability
Murray & Washington Average no. of EPI/LPF <10 EPI/LPF
Van Scoy Average no. of WBC/LPF >25 WBC/LPF
Barry
Assign + and – values, 3+ if > 150 WBC/LPF;
2+ if 76-150 WBC/LPF; 1+ if 1-75 WBC/LPF;
-3 if >25 EPI/LPF; -2 if 16-25 EPI/LPF;
-1 if 5-15 EPI/LPF
Any positive score
(sum of + and – values)
Gal-oz
Informative: <10 SEC/LPF & >25 PMNs/LPF
Semi-informative: <10 SEC/LPF or >25 PMNs/LPF
Uninformative: >10 SEC/LPF & <25 PMNs/LPF
(Semi) informative considered to be Sputum

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for ongoing community surveillance systems in the field of
respiratory disease in general.
ACKNOWLEDGEMENTS

Tuberculosis Foundation, The Netherlands, World Health
Organization, and the International Centre for Diarrhoeal
Disease and Research, Bangladesh (ICDDR, B).
This work was supported by grants from the KNCV
CONFLICT OF INTEREST
None of the authors reports a conflict of interest.
ABBREVIATIONS
AFB = Acid-fast Bacilli
EPI = Epithelial cell
LPF = Low power field
PMN = Polymorphonuclear neurtrophil
SEC = Squamous epithelial cell
TB = Tuberculosis
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Received: December 12, 2011

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Revised: January 6, 2012 Accepted: January 6, 2012